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Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar pyrophosphorylase (USPase)
from Kelampayan (Neolamarckia cadamba)
Lee Vivian
QK 495 US L477 2f)13 Bachelor of Science with Honours
(Resource Biotechnology) 2013
- ----~-----__-
Pusat Khidmat MakJumat Akademik lINJVIRSm MALAYSIA SARAWAK
PKHIDMAT MAKLUMAT AKADEMIK
1llIllIlIlfi~illllllllll 1000246611
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
A Thesis Submitted in Partial Fulfillment of The Requirement ofThe Degree of Bachelor of Science with Honours (Resource Biotechnology)
Supervisor Dr Ho Wei Seng Co-supervisor Dr Pang Shek Ling
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
2013
ACKNOWLEDGEMENT
First and foremost I would like to express my profoundest and warmest gratitude to my
project supervisor Dr Ho Wei Seng for the continuous support of my study and research
for his enthusiasm patience motivation and immense knowledge along with his
invaluable advices and guidance His guidance helped me in all the time of this research
and writing of this thesis whilst allowing me the room to work in my own way
My sincere thanks also go to Dr Pang Shek Ling my co-supervisor for her time
and effort devoted in providing warm encouragement and insightful comments while
leading me on this research project Under her guidance I have successfully overcome
many difficulties and learned a lot
I would also like to acknowledge with much appreciation my fellow lab mates in
Forest Genomics and Infonnatics Lab (GiL) for the stimulating discussions and their
constant support and help throughout this research
Last but not least I would like to extend huge heartiest thanks to my family and
friends who provide a carefree environment and cheering me up through the good times
and bad with their love care and moral support
DECLARATION
I hereby declare that this thesis is of my original work except for quotations and citations
all of which have been duly acknowledged I also declare that it has not been previously
or concurrently submitted for any other degree at UNIMAS or any other institutions
Lee Vivian
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
Pusat Khidmat Maklumat Akademik I middot -~~Tf ~HIAYSlt SARlWl K
T ABLE OF CONTENTS
ACKNOWLEDGEMENT I
DECLARATION II
TABLE OF CONTENTS III
LIST OF ABBREVIATIONS VI
LIST OF TABLES VIII
LIST OF FIGURES IX
ABSTRACT 1
10 INTRODUCTION 2
20 LITERATURE REVIEW 5
21 Neolamarckia cadamba 5
22 Wood Formation in Forest Trees 6
221 Overview of the Development of Woody Stem 6
222 Chemical Composition of Woody Cell Walls 7
23 UDP-sugars 8
231 Biosynthesis and Biochemical Role ofUDP-sugars 8
24 UDP-sugar Pyrophosphorylase (USPase) II
241 Metabolic Role ofUSPase 11
242 USPase Protein 12
30 MATERIALS AND METHODS 16
31 Materials 16
311 Plant Materials 16
32 Methods 16
III
321 Primer Design 16
322 Collection of Plant Material 20
3221 Developing Xylem Tissues 20
3222 Leaf Tissues 20
323 Nucleic Acids Extraction 21
3231 Total RNA Isolation 21
3232 Genomic DNA Extraction and Purification 23
324 Assessment of Nucleic Acid Integrity by Agarose Gel Electrophoresis 24
3241 Assessment of Total RNA Integrity 24
3242 Assessment of gDNA Integrity 25
325 Nucleic Acids Quantification 26
326 Reverse Transcription 27
327 Polymerase Chain Reaction (PCR) 28
3271 PCR of cDNA 28
3272 PCR ofgDNA 31
328 PCR Product Purification via Gel Extraction 33
329 DNA Sequencing and Sequence Data Analysis 35
40 RESULTS 36
41 Nucleic Acids Integrity and Quality 36
411 Total RNA from Developing Xylem Tissues 36
412 Genomic DNA from Leaf Tissues 37
IV
42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39
43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40
44 PCR Product Purification via Gel Extraction 41
45 Purified PCR Products 42
46 DNA Sequencing and Data Analysis 43
50 DISCUSSIONS
51 Isolation ofTotal RNA from Developing Xylem Tissues 44
52 Nucleic Acids Integrity and Quality 45
53 Reverse Transcription-PCR (RT-PCR) 47
54 PCRofgDNA 48
55 PCR Product Purification via Gel Extraction 49
56 DNA Sequencing and Data Analysis 49
60 CONCLUSIONS AND RECOMMENDATIONS 51
REFERENCES 52
v
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
- ----~-----__-
Pusat Khidmat MakJumat Akademik lINJVIRSm MALAYSIA SARAWAK
PKHIDMAT MAKLUMAT AKADEMIK
1llIllIlIlfi~illllllllll 1000246611
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
A Thesis Submitted in Partial Fulfillment of The Requirement ofThe Degree of Bachelor of Science with Honours (Resource Biotechnology)
Supervisor Dr Ho Wei Seng Co-supervisor Dr Pang Shek Ling
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
2013
ACKNOWLEDGEMENT
First and foremost I would like to express my profoundest and warmest gratitude to my
project supervisor Dr Ho Wei Seng for the continuous support of my study and research
for his enthusiasm patience motivation and immense knowledge along with his
invaluable advices and guidance His guidance helped me in all the time of this research
and writing of this thesis whilst allowing me the room to work in my own way
My sincere thanks also go to Dr Pang Shek Ling my co-supervisor for her time
and effort devoted in providing warm encouragement and insightful comments while
leading me on this research project Under her guidance I have successfully overcome
many difficulties and learned a lot
I would also like to acknowledge with much appreciation my fellow lab mates in
Forest Genomics and Infonnatics Lab (GiL) for the stimulating discussions and their
constant support and help throughout this research
Last but not least I would like to extend huge heartiest thanks to my family and
friends who provide a carefree environment and cheering me up through the good times
and bad with their love care and moral support
DECLARATION
I hereby declare that this thesis is of my original work except for quotations and citations
all of which have been duly acknowledged I also declare that it has not been previously
or concurrently submitted for any other degree at UNIMAS or any other institutions
Lee Vivian
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
Pusat Khidmat Maklumat Akademik I middot -~~Tf ~HIAYSlt SARlWl K
T ABLE OF CONTENTS
ACKNOWLEDGEMENT I
DECLARATION II
TABLE OF CONTENTS III
LIST OF ABBREVIATIONS VI
LIST OF TABLES VIII
LIST OF FIGURES IX
ABSTRACT 1
10 INTRODUCTION 2
20 LITERATURE REVIEW 5
21 Neolamarckia cadamba 5
22 Wood Formation in Forest Trees 6
221 Overview of the Development of Woody Stem 6
222 Chemical Composition of Woody Cell Walls 7
23 UDP-sugars 8
231 Biosynthesis and Biochemical Role ofUDP-sugars 8
24 UDP-sugar Pyrophosphorylase (USPase) II
241 Metabolic Role ofUSPase 11
242 USPase Protein 12
30 MATERIALS AND METHODS 16
31 Materials 16
311 Plant Materials 16
32 Methods 16
III
321 Primer Design 16
322 Collection of Plant Material 20
3221 Developing Xylem Tissues 20
3222 Leaf Tissues 20
323 Nucleic Acids Extraction 21
3231 Total RNA Isolation 21
3232 Genomic DNA Extraction and Purification 23
324 Assessment of Nucleic Acid Integrity by Agarose Gel Electrophoresis 24
3241 Assessment of Total RNA Integrity 24
3242 Assessment of gDNA Integrity 25
325 Nucleic Acids Quantification 26
326 Reverse Transcription 27
327 Polymerase Chain Reaction (PCR) 28
3271 PCR of cDNA 28
3272 PCR ofgDNA 31
328 PCR Product Purification via Gel Extraction 33
329 DNA Sequencing and Sequence Data Analysis 35
40 RESULTS 36
41 Nucleic Acids Integrity and Quality 36
411 Total RNA from Developing Xylem Tissues 36
412 Genomic DNA from Leaf Tissues 37
IV
42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39
43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40
44 PCR Product Purification via Gel Extraction 41
45 Purified PCR Products 42
46 DNA Sequencing and Data Analysis 43
50 DISCUSSIONS
51 Isolation ofTotal RNA from Developing Xylem Tissues 44
52 Nucleic Acids Integrity and Quality 45
53 Reverse Transcription-PCR (RT-PCR) 47
54 PCRofgDNA 48
55 PCR Product Purification via Gel Extraction 49
56 DNA Sequencing and Data Analysis 49
60 CONCLUSIONS AND RECOMMENDATIONS 51
REFERENCES 52
v
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
ACKNOWLEDGEMENT
First and foremost I would like to express my profoundest and warmest gratitude to my
project supervisor Dr Ho Wei Seng for the continuous support of my study and research
for his enthusiasm patience motivation and immense knowledge along with his
invaluable advices and guidance His guidance helped me in all the time of this research
and writing of this thesis whilst allowing me the room to work in my own way
My sincere thanks also go to Dr Pang Shek Ling my co-supervisor for her time
and effort devoted in providing warm encouragement and insightful comments while
leading me on this research project Under her guidance I have successfully overcome
many difficulties and learned a lot
I would also like to acknowledge with much appreciation my fellow lab mates in
Forest Genomics and Infonnatics Lab (GiL) for the stimulating discussions and their
constant support and help throughout this research
Last but not least I would like to extend huge heartiest thanks to my family and
friends who provide a carefree environment and cheering me up through the good times
and bad with their love care and moral support
DECLARATION
I hereby declare that this thesis is of my original work except for quotations and citations
all of which have been duly acknowledged I also declare that it has not been previously
or concurrently submitted for any other degree at UNIMAS or any other institutions
Lee Vivian
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
Pusat Khidmat Maklumat Akademik I middot -~~Tf ~HIAYSlt SARlWl K
T ABLE OF CONTENTS
ACKNOWLEDGEMENT I
DECLARATION II
TABLE OF CONTENTS III
LIST OF ABBREVIATIONS VI
LIST OF TABLES VIII
LIST OF FIGURES IX
ABSTRACT 1
10 INTRODUCTION 2
20 LITERATURE REVIEW 5
21 Neolamarckia cadamba 5
22 Wood Formation in Forest Trees 6
221 Overview of the Development of Woody Stem 6
222 Chemical Composition of Woody Cell Walls 7
23 UDP-sugars 8
231 Biosynthesis and Biochemical Role ofUDP-sugars 8
24 UDP-sugar Pyrophosphorylase (USPase) II
241 Metabolic Role ofUSPase 11
242 USPase Protein 12
30 MATERIALS AND METHODS 16
31 Materials 16
311 Plant Materials 16
32 Methods 16
III
321 Primer Design 16
322 Collection of Plant Material 20
3221 Developing Xylem Tissues 20
3222 Leaf Tissues 20
323 Nucleic Acids Extraction 21
3231 Total RNA Isolation 21
3232 Genomic DNA Extraction and Purification 23
324 Assessment of Nucleic Acid Integrity by Agarose Gel Electrophoresis 24
3241 Assessment of Total RNA Integrity 24
3242 Assessment of gDNA Integrity 25
325 Nucleic Acids Quantification 26
326 Reverse Transcription 27
327 Polymerase Chain Reaction (PCR) 28
3271 PCR of cDNA 28
3272 PCR ofgDNA 31
328 PCR Product Purification via Gel Extraction 33
329 DNA Sequencing and Sequence Data Analysis 35
40 RESULTS 36
41 Nucleic Acids Integrity and Quality 36
411 Total RNA from Developing Xylem Tissues 36
412 Genomic DNA from Leaf Tissues 37
IV
42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39
43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40
44 PCR Product Purification via Gel Extraction 41
45 Purified PCR Products 42
46 DNA Sequencing and Data Analysis 43
50 DISCUSSIONS
51 Isolation ofTotal RNA from Developing Xylem Tissues 44
52 Nucleic Acids Integrity and Quality 45
53 Reverse Transcription-PCR (RT-PCR) 47
54 PCRofgDNA 48
55 PCR Product Purification via Gel Extraction 49
56 DNA Sequencing and Data Analysis 49
60 CONCLUSIONS AND RECOMMENDATIONS 51
REFERENCES 52
v
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
DECLARATION
I hereby declare that this thesis is of my original work except for quotations and citations
all of which have been duly acknowledged I also declare that it has not been previously
or concurrently submitted for any other degree at UNIMAS or any other institutions
Lee Vivian
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
Pusat Khidmat Maklumat Akademik I middot -~~Tf ~HIAYSlt SARlWl K
T ABLE OF CONTENTS
ACKNOWLEDGEMENT I
DECLARATION II
TABLE OF CONTENTS III
LIST OF ABBREVIATIONS VI
LIST OF TABLES VIII
LIST OF FIGURES IX
ABSTRACT 1
10 INTRODUCTION 2
20 LITERATURE REVIEW 5
21 Neolamarckia cadamba 5
22 Wood Formation in Forest Trees 6
221 Overview of the Development of Woody Stem 6
222 Chemical Composition of Woody Cell Walls 7
23 UDP-sugars 8
231 Biosynthesis and Biochemical Role ofUDP-sugars 8
24 UDP-sugar Pyrophosphorylase (USPase) II
241 Metabolic Role ofUSPase 11
242 USPase Protein 12
30 MATERIALS AND METHODS 16
31 Materials 16
311 Plant Materials 16
32 Methods 16
III
321 Primer Design 16
322 Collection of Plant Material 20
3221 Developing Xylem Tissues 20
3222 Leaf Tissues 20
323 Nucleic Acids Extraction 21
3231 Total RNA Isolation 21
3232 Genomic DNA Extraction and Purification 23
324 Assessment of Nucleic Acid Integrity by Agarose Gel Electrophoresis 24
3241 Assessment of Total RNA Integrity 24
3242 Assessment of gDNA Integrity 25
325 Nucleic Acids Quantification 26
326 Reverse Transcription 27
327 Polymerase Chain Reaction (PCR) 28
3271 PCR of cDNA 28
3272 PCR ofgDNA 31
328 PCR Product Purification via Gel Extraction 33
329 DNA Sequencing and Sequence Data Analysis 35
40 RESULTS 36
41 Nucleic Acids Integrity and Quality 36
411 Total RNA from Developing Xylem Tissues 36
412 Genomic DNA from Leaf Tissues 37
IV
42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39
43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40
44 PCR Product Purification via Gel Extraction 41
45 Purified PCR Products 42
46 DNA Sequencing and Data Analysis 43
50 DISCUSSIONS
51 Isolation ofTotal RNA from Developing Xylem Tissues 44
52 Nucleic Acids Integrity and Quality 45
53 Reverse Transcription-PCR (RT-PCR) 47
54 PCRofgDNA 48
55 PCR Product Purification via Gel Extraction 49
56 DNA Sequencing and Data Analysis 49
60 CONCLUSIONS AND RECOMMENDATIONS 51
REFERENCES 52
v
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
Pusat Khidmat Maklumat Akademik I middot -~~Tf ~HIAYSlt SARlWl K
T ABLE OF CONTENTS
ACKNOWLEDGEMENT I
DECLARATION II
TABLE OF CONTENTS III
LIST OF ABBREVIATIONS VI
LIST OF TABLES VIII
LIST OF FIGURES IX
ABSTRACT 1
10 INTRODUCTION 2
20 LITERATURE REVIEW 5
21 Neolamarckia cadamba 5
22 Wood Formation in Forest Trees 6
221 Overview of the Development of Woody Stem 6
222 Chemical Composition of Woody Cell Walls 7
23 UDP-sugars 8
231 Biosynthesis and Biochemical Role ofUDP-sugars 8
24 UDP-sugar Pyrophosphorylase (USPase) II
241 Metabolic Role ofUSPase 11
242 USPase Protein 12
30 MATERIALS AND METHODS 16
31 Materials 16
311 Plant Materials 16
32 Methods 16
III
321 Primer Design 16
322 Collection of Plant Material 20
3221 Developing Xylem Tissues 20
3222 Leaf Tissues 20
323 Nucleic Acids Extraction 21
3231 Total RNA Isolation 21
3232 Genomic DNA Extraction and Purification 23
324 Assessment of Nucleic Acid Integrity by Agarose Gel Electrophoresis 24
3241 Assessment of Total RNA Integrity 24
3242 Assessment of gDNA Integrity 25
325 Nucleic Acids Quantification 26
326 Reverse Transcription 27
327 Polymerase Chain Reaction (PCR) 28
3271 PCR of cDNA 28
3272 PCR ofgDNA 31
328 PCR Product Purification via Gel Extraction 33
329 DNA Sequencing and Sequence Data Analysis 35
40 RESULTS 36
41 Nucleic Acids Integrity and Quality 36
411 Total RNA from Developing Xylem Tissues 36
412 Genomic DNA from Leaf Tissues 37
IV
42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39
43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40
44 PCR Product Purification via Gel Extraction 41
45 Purified PCR Products 42
46 DNA Sequencing and Data Analysis 43
50 DISCUSSIONS
51 Isolation ofTotal RNA from Developing Xylem Tissues 44
52 Nucleic Acids Integrity and Quality 45
53 Reverse Transcription-PCR (RT-PCR) 47
54 PCRofgDNA 48
55 PCR Product Purification via Gel Extraction 49
56 DNA Sequencing and Data Analysis 49
60 CONCLUSIONS AND RECOMMENDATIONS 51
REFERENCES 52
v
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
321 Primer Design 16
322 Collection of Plant Material 20
3221 Developing Xylem Tissues 20
3222 Leaf Tissues 20
323 Nucleic Acids Extraction 21
3231 Total RNA Isolation 21
3232 Genomic DNA Extraction and Purification 23
324 Assessment of Nucleic Acid Integrity by Agarose Gel Electrophoresis 24
3241 Assessment of Total RNA Integrity 24
3242 Assessment of gDNA Integrity 25
325 Nucleic Acids Quantification 26
326 Reverse Transcription 27
327 Polymerase Chain Reaction (PCR) 28
3271 PCR of cDNA 28
3272 PCR ofgDNA 31
328 PCR Product Purification via Gel Extraction 33
329 DNA Sequencing and Sequence Data Analysis 35
40 RESULTS 36
41 Nucleic Acids Integrity and Quality 36
411 Total RNA from Developing Xylem Tissues 36
412 Genomic DNA from Leaf Tissues 37
IV
42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39
43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40
44 PCR Product Purification via Gel Extraction 41
45 Purified PCR Products 42
46 DNA Sequencing and Data Analysis 43
50 DISCUSSIONS
51 Isolation ofTotal RNA from Developing Xylem Tissues 44
52 Nucleic Acids Integrity and Quality 45
53 Reverse Transcription-PCR (RT-PCR) 47
54 PCRofgDNA 48
55 PCR Product Purification via Gel Extraction 49
56 DNA Sequencing and Data Analysis 49
60 CONCLUSIONS AND RECOMMENDATIONS 51
REFERENCES 52
v
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
42 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 39
43 Polymerase Chain Reaction (PCR) of Genomic DNA (gDNA) 40
44 PCR Product Purification via Gel Extraction 41
45 Purified PCR Products 42
46 DNA Sequencing and Data Analysis 43
50 DISCUSSIONS
51 Isolation ofTotal RNA from Developing Xylem Tissues 44
52 Nucleic Acids Integrity and Quality 45
53 Reverse Transcription-PCR (RT-PCR) 47
54 PCRofgDNA 48
55 PCR Product Purification via Gel Extraction 49
56 DNA Sequencing and Data Analysis 49
60 CONCLUSIONS AND RECOMMENDATIONS 51
REFERENCES 52
v
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
LIST OF ABBREVIATIONS
A
BLAST
BLASTn
bp
cDNA
CG
CTAB
cm
DEPC
DNA
DNase
dNTP
EMBL-EBI
g
gDNA
MgCh
min(s)
ml
mM
NCBI
ng
Ampere
Basic Alignment Search Tool
Basic Alignment Search Tool for nuc1eotides
Base pair
Complementary deoxyribonucleic acid
Cytosine guanine
Cetyltrimethylarnmonium bromide
Centimeter
Double-distilled water
Dietylpyrocarbonate
Deoxyribonucleic acid
Deoxyribonucleic acid-ase
Deoxyribonucleotide triphosphate
European Molecular Biology Laboratory-European Bioinformatics Institute
gram
Genomic DNA
Magnesium chloride
Minute(s)
Mi1i1itre
Milimolar
National Centre for Biotechnology Information
Nanogram
VI
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
V
PCR
PPi
RNA
RNAse
rpm
rRNA
RT-PCR
sec(s)
TAE
UDP
JlI
USPase
UTP
UV
Polymerase Chain Reaction
Pyrophosphate
Ribonucleic acid
Ribonucleic acid-ase
Revolution per minute
Ribosomal RNA
Reverse Transcription-Polymerase Chain Reaction
Second(s)
Tris-Acetate EDT A
Uridine diphosphate
Microlitre
UDP-sugar pyrophosphorylase
U ridine-5 -triphosphate
Ultraviolet
Volt
Degree Celcius
VII
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
LIST OF TABLES
Page Table 31 Composition of reaction mixture for first-strand
cDNA synthesis 28
Table 32 Composition ofPCR mixture for USPase partial cDNA amplification 29
Table 33 Composition ofPCR mixture for [primers] optimization of USPase partial cDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 30
Table 34 Composition of PCR mixture for USPase partial gDNA amplification 31
Table 35 Composition ofPCR mixture for [MgCh] and [gDNA] optimization of USPase partial gDNA amplification (Numbers in parentheses denote corresponding concentrations and volumes used for optimization) 33
Table 41 Spectrophotometric readings of total RNA isolated from developing xylem tissues ofN cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 37
Table 42 Spectrophotometric readings of purified genomic DNA extracted from leaf tissues of N cadamba (Kelampayan) measured with NanoDrop 2000 Spectrophotometer 38
Table 43 BLASTn output for partial gDNA sequence of Kelampayan USPase 43
VIII
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
LIST OF FIGURES
Figure 21 The role of products of the enzymatic reaction ofUSPase
Page
10
Figure 22 Biochemical reaction catalyzed by UDP-sugar
pyrophosphorylase (USPase) 11
Figure 23 Evolutionary tree of USPase generated based on amino acid sequence 13
Figure 24 Cryastallized structure ofUSPase from Leishmania 14
Figure 31 Partial result of mUltiple alignment between nucleotide
sequences ofArabidopsis thaliana Glycine max (soybean)
and Populus trichocarpa (poplar) showing the most conserved regions of USPase between the three species 17
Figure 32 Output information of primer search using Primer Premier 60 19
Figure 41 Gel electrophoresis of total RNA isolated from developing
Figure 42 Gel electrophoresis of genomic DNA extracted from leaf
Figure 43 Gel electrophoresis of amplicons from gradient PCR
Figure 44 Gel electrophoresis of amplicons from gradient PCR
xylem tissues ofN cadamba (Kelampayan) on 1 (wv) gel 36
tissues ofN cadamba (Kelampayan) on 08 (wv) gel 38
using cDNA as template 39
using gDNA as template 40
Figure 45 Gel electrophoresis of pooled PCR products for gel extraction 41
Figure 46 Purified DNA from purification ofPCR products with
Wizardreg SV Gel and PCR Clean-Up System (Promega USA) 42
Figure 47 Electropherogram of USPase S3 showing row signal without recognizable sequence generated 43
IX
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
Isolation and Characterization of Partial Gene Sequence Encoding for UDP-sugar
pyrophosphorylase (USPase) from Kelampayan (Neolamarckia cadamba)
Lee Vivian
Resource Biotechnology Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Neolamarckia cadamba or locally known as Kelampayan has emerged as an important tree species in plantation forestry as it is believed to hold the promise for sustainable harvesting of forest in the future due to its fill t-growing property and its ability to produce wood for various economic uses UDP-sugar pyrophosphorylase (USPase) also known as UTP-monosaccharide-l-phosphate uridyltransferase is believed to playa role in Kelampayan wood fonnation due to its enzymatic function in plant carbohydrate metaboli m which is involved in cell wall synthesis The aim of this study is to isolate and characterize partial USPase gene of Kelampayan Total RNA was isolated from developing xylem of Kelampayan and then reverse transcribed to cDNA which was amplified using reverse transcription-PCR (RT-PCR) approach Genomic DNA was also extracted from leaf tissues to isolate the gene The isolated partial genes from both cDNA and gDNA were sequenced and subsequently subjected to in-silico characterization Sequence homology search at nucleotide level showed no matching identity between partial gene sequence ofKelampayan USPase and USPase characterized in other plant species
Keywords Neolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) wood formation gene isolation sequence homology
ABSTRAK
Neolamarckia cadamba atau nama tempatannya Kelampayan telah munclll sebagai spesies pokok yang
penting dalam bidang perhutanan tanaman kerana ia dipercayai memegang janji untuk penuaian hutan yang mampan pada masa hadapan kerana kecepatan tumbesarannya dan keupayaannya dalam penghasilan kayu untuk pelbagai kegunaan ekomomik UDP-sugar pyrophosphorylase (USPase) juga dikellali sebagai UTP-monosaccharide-l -phosphate uridyltransferase dipercayai berperanan dalam pembetltukan kayu Kelampayan kerana fungsi enzimnya dalam metabolisme karbohidrat tumblhan yang terlibal dalam sintesis din ding sel Objektij kajian ini adalah untuk mengekstrak dan mencirikan gell separa USPase daripada Kelampayan RNA daripada developing xylem pokok Kelampayan diekstrak dan ditranskripsi terbalikkan ke cDNA yang diamplifikasikan dengall RT-PCR DNA genomik tunll diekstrak daripada lisu daun untuk memperoleh gen tersebut Gen separa yang diperoleh daripada cDNA dan gDNA
dijujukkatl dan tertakluk kepada pencirian in-silico Pencarian homologi jujkan pada tahap nukleotida menunjlikaan tiada padanan identiti dalam jujukan gen USPase an tara Kelampayan dan spesies tumbuhan yang laill
Kata klinci Nolamarckia cadamba (Kelampayan) UDP-sugar pyrophosohorylase (USPase) pembentukan kayu pengasingan gen homologijujukan
1
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
10 INTRODUCTION
Locally known as Kelampayan in Malaysia Neotamarckia cadamba is a deciduous tree
that is being cultivated widely in recent years It belongs to the family of Rubiaceae and
nonnally grows up to 45 metres tall with tnmk diameter of about 100 cm to 160 cm
(Joker 2000) This tree is naturally distributed in India China Thailand Indonesia
Malaysia Papua New Guinea Philippines Singapore and Vietnam (Gaumat et at 2012
Joker 2000) Kelampayan is also cultivated worldwide to complement the impact of nonshy
sustainable harvesting of forest trees and to cater the need for commercial productions
Besides it is frequently grown as an ornamental plant and shade tree in plantations (Patel
2011) Kelampayan wood is light and hard thus it has emerged as a commercial timber
providing the source for plywood and used for lightweight construction works Besides
Kelampayan wood is also a source of pulp for paper production (Joker 2000)
Apart from these commercial productions Kelampayan possess a wide range of
pharmacological properties The therapeutic properties are mostly found in its bark and
leaves The leaves have been used as folk remedies to pacify a wide range of such
illnesses as burning sensation urinary retention fever diarrhea menorrhagia and ulcers
(Gautam el at 2012) Additionally it is useful in the treatment of snake-bite (Dubey et at
2011) While Collins et at (as cited in Richter and Dallwitz 2000) stated that the leaf
material of Kelampayan is active against some tumors another common medicinal belief
is that the leaves of this species are antidiabetic agents and studies have been conducted
to validate thi therapeutic property of Kelampayan (Ahmed et at 2010) In addition the
bark of the plant is reported to exhibit tonic anti-inflammatory digestive diuretic
2
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
constipating and antiemetic properties and is given to treat the fever and inflammation of
eyes (Dubey et al 2011)
Despite the many usable values of this tree species knowledge on the structural
and regulatory genes that govern wood fonnation of Kelampayan has not been
established to a comprehensive extent as compared with other higher plant species such
as Populus and Eucalyptus trees Thus knowing that most of the production values of
Kelampayan arise from the usage of its wood it is essential to study specific genes that
contribute significance to the plant development especially in wood fonnation
UDP-sugar pyrophosphorylase (USPase) also annotated as UTP-
monosaccharide-I-phosphate uridyltransferase is believed to be an important wood
formation gene in woody trees It is one of the key enzymes in plant carbohydrate
metabolism that catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5shy
triphosphate) to sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) The
product UDP-sugar is the most prominent nucleotide sugars in plant physiology which in
turn acts as a precursor for the fonnation of plant metabolites and more importantly
structural components of the cell wall which may play a significant role in wood
formation in Kelampayan
However at present the only woody plant of which its USPase gene
characterized is Populus trichocarpa Although this gene has been studied in a variety of
agricultural plant species which mostly are herbaceous it is not known whether this gene
in Kelampayan displays significant sequence and functional homologies with other
characterized USPases Although this is advantageous in a way that the sequences of the
3
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
t
characterized plant genomes are available one limiting factor of using these herbaceous
plants as model species is the fact that many genes expressed during wood formation in
woody trees do not exhibit homology with the herbaceous crop genes (Ranik 2005)
Furthermore since there is only one woody tree species with its USPase characterized its
role in wood formation requires further validation
To address these problems this study aims to isolate and amplify the partial
cDNA and gDNA encoding for USPase in Kelampayan and subsequently characterize it
by performing in-silico analyses with reference to currently available data in the public
domain The study was done at both levels of gDNA and eDNA as comparison between
them could suggest the location of transcribed region in the genome
4
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
Pusat Khidmat MakJumat Ak d k rlVERSm MALAYSIA S~~~
20 LITERATURE REVIEW
21 Neolamarckia cadamba
Neolamarckia cadamba (Roxb) Bosser of the family Rubiaceae is conunonly known as
Kelampayan in the Malay language It is cultivated worldwide in tropical regions with
geographical distribution covering India Pakistan Sri Lanka Thailand Indochina
eastward in the Malaysian Archipelago and Papua New Guinea (Joker 2000 Richter and
Dallwitz 2009) Kelampayan is an evergreen tropical tree typically found in secondary
rainforests It is light-demanding and is not frost hardy Abundant rainfall (1500 nun rain
year) favours its growth but this tree can as well tolerate dry climate (200 mm rain year)
(Joker 2000)
Due to its special properties Kelampayan has been propagated for a wide variety
of uses Of ecological role this tree species is suitable for reforestation because it is fast-
growing With umbrella-shaped crown it is useful as a shade tree for dipterocarp line
planting (Joker 2000) In term of wood production the wood is light and hard but with
poor durability Thus it is mainly used to produce plywood and for lightweight
construction besides as a source of pulp producing low- and medium- quality paper
(Joker 2000) In addition N cadamba tree exhibits therapeutic properties that make it
useful remedies in the indigenous system of medicine A wide range of medicinal
activities in various parts of Kelampayan were reported by Gautam et al (2012) in their
pharmacological studies Leaf extracts of Kelampayan were shown to possess most
therapeutic values including analgesic anti-flammatory anti-pyretic antioxidant
antihepatotoxic antifungal antimicrobial and wound healing activities Besides Ahmed
5
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
et al (2010) have carried out a study to evaluate the possible glucose tolerance efficacy
of methanolic extract ofN cadamba leaf and have validated that N cadamba leaves has
antidiabetic property In addition to its medicinal property Collins et al (as cited in
Richter and Dallwitz 2000) also reported that leaf material of N cadamba is active
against some tumors The bark of Kelampayan also shows some similar activities found
in the leaves such as analgesic and anti-flammatory activities with addition of diuretic
and laxative activity Even the roots display medicinal property which is hypolipidemic
activity (Gaumat et al 2012)
22 Wood Formation in Forest Trees
221 Overview of the Development of Woody Stem
Wood is an irreplaceable natural product which holds a massive prospect lD global
industry with a multitude of applications Despite the fact that wood is an important
natural product knowledge about the structural and regulatory genes that govern its
fonnation in forest trees is relatively insufficient (Ranik 2005) A thorough
understanding of the molecular biology of wood development therefore is imperative for
improvement of wood and fiber quality of forest trees
Despite the importance of the forest biome currently majority of wood is
harvested from natural forests destructively In addition the facts that forest trees require
naturally long generation times and lack of mutant lines have become obstacles for them
to un4ergo agricultural evolution of creating varieties of desirable traits like that lD
6
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
cultivation and domestication of crop species such as rice and soybean Therefore
improving the chemical composition of wood of forest trees becomes one of the main
applications of genes characterized in synthesis of traits superior to their wild ancestors
Wood formation has been focused on the anatomical level for decades According
to Ranik (2005) focus of wood fonnation studies have shifted away from morphology to
genetic mechanisms that govern wood development and properties For example the
completed sequencing of tree genomes including that of Populus trichocarpa
(Wullschleger et al 2002) has made a significant impact on forest tree genomics
222 Chemical Composition of Woody Cell Walls
The process of wood fonnation also known as xylogenesis as described by Plomion et al
(2001) encompasses at least five major steps cell division (cambium cells divide to fonn
xylem and phloem) cell elongation cell wall thickening programmed cell death and
heartwood formation
The structure and composition of wood are influenced by cellular and
biochemical processes occurring in each of these steps The structure and composition of
wood in tum impact in the processing of wood In the development of woody stem of
trees of other wood-forming species the development of xylem and phloem from the
vascular cambium is expanded to a secondary level where they function to support and
transport Besides the secondary thickening of the cell wall is also one of the major
factQrs that determine the structure and composition of wood Secondary cell walls are
7
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
composed of cellulose lignin hemicellulose and proteins (Hu et al 1999)
Hemicellulose and lignin are heteropolymeric compounds with variable compositions
(Ranik 2005) This gives rise to the variability of cell wall components between different
types of wood and wood from different species (Mellerowicz et al 2001) On the other
hand each phase of xylogenesis is regulated by the interaction of the differentiating cells
by honnonal signaling and cell-ceU interactions (Kuriyama and Fukuda 2002) In
addition Friml (2003) has described that wood fonnation is also regulated by the plants
adaptability to the environmental changes
23 UDP-sugars
231 Biosynth esis and Biochemical Role of UDP-sugars
By synthesizing carbohydrates by photosynthesis or other anabolic pathways plants
convert light energy to chemical energy which is stored in the bonds of sugar in forms of
monosaccharides disaccharides and polysaccharides (Meng 2008) Monosaccharides are
building blocks of disaccharides and polysaccharides To form disaccharides and
polysaccharides a monosaccharide needs to be activated which is by the addition of a
nucleoside-diphosphate group to the sugar resulting in the formation of a nucleotide
sugar Nucleotide sugars are the universal sugar donors for the formation of
polysaccharides glycoproteins proteoglycans glycolipids and glycosylated secondary
metabolites (Bar-Peled amp ONeill 2011)
8
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
The sugar component in nucleotide sugars are derived from a variety of sources
including the carbohydrate derived from photosynthesis the sugar generated by
hydrolysis of translocated sucrose the sugars released from storage carbohydrates the
salvage of sugars from glycoproteins and glycolipids the recycling of sugars released
during primary and secondary cell wall restructuring and the sugar generated during
plant-microbe interactions (Bar-Peled amp ONeill 2011)
Among all uridyl diphosphate-sugars (UDP-sugars) are the most prominent
nucleotide sugars which constituents include a monosaccharide and a nucleotide
Biosynthesis ofUDP-sugars occurs through both de novo and salvage pathways in higher
plants (Kotake et ai 2004) In the de novo pathway UDP-glucose (UDP-Glc) acts as the
starting substrate that is sequentially converted to UDP-sugars On the other hand in the
salvage pathway glycosidases remove polysaccharides glycoproteins and glycolipids
from cell wall These compounds then are incorporated into the cells and then converted
to UDP-sugars via monosaccharide I-phosphates (Kotake et ai 2007)
With the action of a variety of glycosyltransferases the sugar residue of
ucleotide sugars can be linked to other compounds such as carbohydrate protein and
pid (Kleczkowski et ai 2011) Particularly uridine diphosphate glycosyltransferases
0 1s) mediate the transfer of glycosyl residues from activated nucleotide sugars to
tor molecules (aglycones) The conjugation leads to the formation of a range of
sylated molecules (Ross et ai 2001) Thus in plants being key precursors for
fJJCOSylation reactions UDP-sugars serve as precursors to many primary metabolites
as sucrose structural components such as celulose hemicellulose and pectin as
as glycoproteins and glycolipids (Figure 21)
9
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
SUCROSE
GLYCOLIPIDS
GLYCOPROTEINS
TREHALOSE CELLULOSE CALLOSE
RAFFINOSE STACHYOSE
HEMICELLULOSE
PECTIN I
Figure 21 The role of products of the enzymatic reaction ofUSPase Green boxes represent products of the USPase reaction (Kleczkowski et ai 2011)
An important product ofUSPase reaction UDP-Glc can be used in the formation
of disaccharides such as sucrose and trehalose as well as polysaccharides such as
cellulose and callose Plant UDP-Gal is also essential for the synthesis of raffinose and
stachyose which are the main carbon-transporting compounds In addition several other
UDP-sugars such as UDP-Gal UDP-GlcA UDP-Ara and UDP-Xyl are also synthesized
by mechanisms involving USPase These UDP-sugars take part in the formation of pectin
and hemicellulose two of the most abundant biomolecules in nature Besides they are
also required for the glycosylation of proteins and lipids (Karr et al as cited in
Kleczkowski et al 2011) Thus UDP-sugars are the main precursors for the biomass
production in plants (Kotake 2010)
10
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
24 UDP-sugar Pyrophosphorylase (USPase)
241 Metabolic Role of USPase
UDP-sugar pyrophosphorylase (USPase) (EC 27764) is synonymous to UTP-
monosaccharide-I-phosphate uridyltransferase As one of the key enzymes of the
carbohydrate metabolism in plants (Kotake et al 2007) UDP-sugar pyrophosphorylase
catalyzes a reversible transfer of the uridyl group from UTP (Uridine-5 -triphosphate) to
sugar-I-phosphate producing UDP-sugar and pyrophosphate (PPi) (Kleczkowski et aI
2011)
o 0 II IImonosaccharide UDP- +
~ O-p-Qr-p-O -I-phosphate monosaccharide I I
o 0
Diphosphate PPi UTP
Figure22 Biochemical reaction catalyzed by UDP-sugar pyrophosphorylase (USPase)
It catalyzes the converSIOn of various monosaccharide I-phosphates to the
respective UDP-sogars in the salvage pathway In the salvage pathway monosaccharides
released during hydrolytic reactions involving polysaccharides and other glycoconjugates
(gIycoproteins glycolipids) are converted to nucleotide sugars In studies done by Carpita
IIId McCann (2000) Gibeaut (2000) and Gibeaut and Carp ita (1991) there is evidence
that the salvage pathway plays a role in recycling monosaccharides released from
lysaccharides during cell wall synthesis and turnover
USPase has broad substrate specificity Besides UDP-glucose it also catalyzes the
ible formation of various sugar-I-phosphates such as UDP-galactose UDPshy
11
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
glucuronic acid UDP-l-arabinose and UDP-xylose (Meng 2008) Among these
substrates Kleczkowski et al (2011) found that hexose-I-phosphates have a higher
affinity towards USPase than pentose-l-phosphates
Previous studies in Arabidopsis have shown that USPase is essential in plant
reproductive processes USPase-knocked out plants show phenotype of pollen sterility
disabling transmission of the loss-of-function mutation through male gametophyte thus
Wlable to produce homozygous mutant In separate studies Litterer et al (2005) and
Kotake et al (2007) reported that pollen produced by USPase deficient plant lacks the bull
pectocellulosic inner layer in the cell wall and has a shrunken shape
142 USPase Protein
Based on online databases NCBI (httpwwwncbinlmnihgov) UniProtKB
(bttpllwwwuniprotorg) and EMBL-EBI (httpwwwebiacuk) there is no gene or
tein of UDP-sugar pyrophosphorylase been characterized from any plant species of
In a study done by Kleczkowski et at (2011) they found that the USPase proteins
different plants share at least 60 identity at their amino acid sequence Based on
acid sequence identity of the derived proteins a comprehensive phylogenetic tree
ase has been constructed as shown in Figure 23
12
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13
EucaryotllEUClll)OtiI
JliridiplantlleChlorophyta
krllpl_ _
~II(Jtllftm fAIsII_IIill_jor
Eucaryota rP- cruz EucaryotaEuglenozOfl
PItuIllOdi wwu Aiveolllta
Figure 23 Evolutionary tree ofUSPase generated based on amino acid sequence (Kleczkowski et aI 2011 )
As presented in the phylogenetic tree above in the Viridiplantae family to which
lampayan belongs only Populus trichocarpa is a woody plant and belongs to the
fimily Rubiaceae making it the only species closely related to Kelampayan for the
Characterization of wood formation gene
Although USPase has overlapping activities with some other UTP-dependent
aoptlOsphorylases it does not share significant homology at the amino acid sequence
with other plant UDP-sugar-producing pyrophosphorylases However they have
lIihnilar structural pattern which is inferred based on the only crystallized structure of
IJUMe protein from Leishmania a protozoa (Dickmanns et al 2011) This protein
13