Download Eucalyptus grandis cellulose synthase 1 Arabidopsis thaliana MARJA M. O’NEILL

Functional genetic analysis of the Eucalyptus
grandis cellulose synthase 1 (EgCesA1) gene in
Arabidopsis thaliana
by
MARJA M. O’NEILL
Submitted in partial fulfilment of the requirements for the degree
Magister Scientiae
In the Faculty of Natural and Agricultural Sciences
Department of Genetics
University of Pretoria
Pretoria
July 2009
Under the supervision of Prof. Alexander A. Myburg
and co-supervision of Dr. Sanushka Naidoo
© University of Pretoria
DECLARATION
I, the undersigned, hereby declare that the dissertation submitted herewith for the degree
M.Sc. to the University of Pretoria, contains my own independent work and has not been
submitted for any degree at any other university.
Marja M. O’Neill
July 2009
ii
PREFACE
The multi-billion dollar world-wide forest products industry utilizes wood as raw substrate.
Wood is comprised of a complex network of cellulose, hemicellulose and lignin that are
mostly contained in secondary cell walls, with cellulose forming the most important
component for paper and pulp manufacturers. Cellulose is a cell wall polymer of chains of β1,4-linked glucose molecules that crystallize to form cellulose microfibrils in plant cell walls.
Biosynthesis of cellulose in plants occurs at the plasma membrane through the action of
rosette-shaped cellulose synthase (CESA) protein complexes. The rosette complexes are
embedded in the plasma membrane and utilize UDP-glucose monomers in the cytoplasm to
polymerize growing glucan chains in the cell wall. Each rosette is hypothesised to consist of
six subunits each containing six CESA proteins. This hypothesis is mostly based on structural
features of rosettes observed using freeze-fracture microscopy.
CesA genes have been
identified in many plant species including Arabidopsis thaliana and Eucalyptus grandis.
Arabidopsis thaliana has proven to be especially useful in studying the function of CESA
proteins by analysing plant development and cell wall chemistry in plants with mutated CesA
genes. Although Arabidopsis does not form large quantities of secondary xylem, it has been
shown to undergo secondary growth under certain conditions. Availability of the full genome
sequence, ease of transformation and access to mutant plant lines, makes Arabidopsis an
attractive model plant in which to study xylogenesis (wood biosynthesis). Arabidopsis has
the potential to be used in elucidating the function of heterologous CesA genes from
important tree species like Eucalyptus spp. Two general scientific questions were addressed
in this study: (1) Is cellulose production regulated at the transcriptional level and (2) can
cellulose biosynthesis be increased by over-expression of a single heterologous CESA
protein? The aim of this M.Sc study was to functionally characterise the Eucalyptus CesA1
gene, involved in cellulose deposition in secondary cell walls of Eucalyptus xylem tissues, in
Arabidopsis thaliana plants.
iii
Literature pertaining to the biosynthesis of cellulose in plants is reviewed in Chapter
1. The structure of primary and secondary plant cell walls and the genes that affect the
synthesis of the various cell wall polymers are discussed and a description of cellulose
structure in plant cell walls is provided. Thereafter, the review is focused on cellulose
biosynthesis. The CesA gene family is discussed, with special attention to Arabidopsis and
Eucalyptus CesA homologues. A model for cellulose synthesis is proposed, highlighting
possible functions of other proteins believed to be involved in the process. Finally, support
for the use of Arabidopsis to study xylogenesis is presented and potential problems in
transgenic gene expression studies are considered.
Three different CESA proteins are believed to comprise each rosette involved in
primary cell wall cellulose synthesis and three other CESA proteins form rosette complexes
for cellulose synthesis during secondary cell wall deposition. Current knowledge on the
organization of proteins within rosettes is shortcoming. Modes of transcriptional and posttranscriptional regulation of CesA genes are still unknown.
It seems prudent to gain
knowledge on these processes in order to increase cellulose biosynthesis in commercially
important trees. Because of the difficulties involved with functional analysis of genes in
trees, this study aimed to functionally characterise a Eucalyptus CesA gene, EgCesA1, in
Arabidopsis. In Chapter 2 of this dissertation, questions regarding the effect of overexpressing EgCesA1 in Arabidopsis plants are addressed. The questions included: How will
over-expression of EgCesA1 affect growth and development of Arabidopsis? Will cell wall
chemistry be affected in stem tissues?
Will expression of endogenous CesA genes be
affected? Will other cell wall related biosynthetic pathways be affected? The final question
that is addressed asks whether EgCesA1 over-expression is sufficient to affect cellulose
synthesis or whether it will be necessary to express all three secondary cell wall CESA
proteins. General findings, conclusions and implications of this M.Sc study are summarized
in the final section of this dissertation titled Concluding Remarks.
iv
Outcomes from a study undertaken March 2006 to February 2009 in the Department
of Genetics, University of Pretoria, under the supervision of Prof. A.A. Myburg are presented
in this dissertation. Cell wall chemistry analysis, described in Chapter 2, was carried out at
the University of British Columbia, Vancouver, Canada by Prof. S.D. Mansfield. In order to
confirm EgCesA1 as a functional orthologue of the homologous Arabidopsis CesA gene,
AtCesA8, the study initially also involved mutant complementation of an Arabidopsis plant
containing a mutated AtCesA8 gene (irx1), with EgCesA1.
However, difficulties with
transformation of cell wall mutant plants of the Landsberg erecta background hampered
progress and prevented us from completing this aspect of the study. Therefore, only the overexpression of the EgCesA1 gene in wild-type Arabidopsis plants is reported in this
dissertation. The following posters were generated based on the preliminary results obtained
in this M.Sc study:
O’Neill, M., Ranik, M. and Myburg, A.A. 2007. Functional analysis of the secondary cell
wall-associated cellulose synthase (CesA) genes of Eucalyptus grandis.
IUFRO Tree
Biotechnology Meeting, June 3-8, Ponta Delgada, Azores, Portugal.
O’Neill, M. and Myburg, A.A. 2008.
Functional analysis of the Eucalyptus grandis
secondary cell wall associated cellulose synthase 1 (EgCesA1) gene in Arabidopsis thaliana.
SAGS Congress, March 26-29, Pretoria, South Africa.
v
ACKNOWLEDGEMENTS
I would like to convey my gratitude to the following people, organizations and institutes for
assisting me in the completion of this study:

Prof. A.A. Myburg for his valued supervision and leadership on this project and his
patience and guidance in reviewing the dissertation.

Dr. S. Naidoo for her advice on statistical analysis and for her contribution to the
review of the dissertation.

Mr. E. Mizrachi for his motivational guidance and insight on the project and valued
review of the dissertation.

Prof. S.D. Mansfield for chemical analysis of Arabidopsis inflorescence stems.

Mr. M. Mphahlele for much appreciated assistance in microscopy and Arabidopsis
transformation.

Ms. M.H. de Castro for her advice, friendship and unwavering willingness to assist in
this project.

Mr. M. Ranik for providing Eucalyptus grandis cDNA and his valued advice.

All my Forest Molecular Genetics (FMG) colleagues for support and friendship
throughout the project.

Sappi and Mondi for funding of the project.

The National Research Foundation of South Africa (NRF) for funding of the project
and the awarded scholarship.

The Human Resources and Technology for Industry Programme (THRIP) for
financial contributions to the Wood and Fibre Molecular Genetics Programme
(WFMG).

The Department of Genetics of the University of Pretoria and the Forestry and
Agricultural Biotechnology Institute (FABI) for providing facilities and stimulating
an academic environment.
vi

Finally, my husband and family for their encouragement, patience and support
throughout the project.
vii
THESIS SUMMARY
Functional genetic analysis of the Eucalyptus grandis cellulose
synthase 1 (EgCesA1) gene in Arabidopsis thaliana
Marja M. O’Neill
Supervised by Prof. A.A. Myburg
Co-supervised by Dr. S. Naidoo
Submitted in partial fulfilment of the requirements for the degree Magister Scientiae
Department of Genetics
University of Pretoria
Cellulose is the most important component of paper and pulp products and increased cellulose
biosynthesis in commercially important trees like Eucalyptus spp. could greatly benefit paper
and pulp industries. Cellulose in plants occurs mostly in the secondary cell walls together
with lignin and hemicellulose.
It is biosynthesised by membrane-bound rosette-shaped
protein complexes. The rosette complexes are believed to be comprised of six sub-units each
containing six cellulose synthase (CESA) proteins. The CESA proteins utilise UDP-glucose
to polymerize growing glucan chains that coalesce to form cellulose microfibrils. Three
distinct CESA proteins form the rosette complexes during primary cell wall formation and
three different CESA proteins form complexes during secondary cell wall deposition. The
exact means by which the CESA proteins interact within a rosette complex remains unknown.
Elucidating rosette protein complex assembly and better characterization of CESA protein
activity is required in order to increase cellulose biosynthesis in commercially important trees.
Because of the difficulties to characterise genes and proteins in tree species, Arabidopsis
viii
thaliana has been used to study xylogenesis. Although it is a herbaceous weed, Arabidopsis
has been shown to undergo secondary growth under certain conditions.
A literature study of cellulose biosynthesis in plants has highlighted several scientific
questions: Will over-expression of a heterologous secondary cell wall CESA protein in
Arabidopsis lead to increased cellulose biosynthesis? What effect will the over-expression of
a heterologous protein have on the growth and development of Arabidopsis? Will it have an
effect on cell wall chemistry in stem tissues? What effect will expression of the transgene
have on endogenous Arabidopsis gene expression? The aim of this M.Sc study was to
functionally characterise the Eucalyptus secondary cell wall associated cellulose synthase
gene, EgCesA1, in Arabidopsis.
The EgCesA1 coding sequence was constitutively expressed in wild-type Arabidopsis
plants.
Three transgenic lines expressing EgCesA1 was generated.
Hypocotyl and
inflorescence vascular cell wall phenotypes were compared between transgenic and wild-type
plants. Chemical analysis of inflorescence tissues were performed to detect changes in
monosaccharide and lignin content of transgenic plants compared to wild-type plants.
Transcript levels of EgCesA1 and endogenous Arabidopsis genes involved in cell wall
biogenesis were quantified and compared between wild-type and transgenic lines.
No
significant changes in cell wall morphology could be detected, despite small alterations in
inflorescence cell wall chemical composition in transgenic plants. Expression of EgCesA1
did not appear to have a statistically significant effect on endogenous gene transcript levels.
It was concluded that constitutive expression of a single transgenic CesA gene is insufficient
to increase cellulose biosynthesis.
ix
TABLE OF CONTENTS
DECLARATION …………………………………………………... ii
PREFACE ………………………………………………………….. iii
ACKNOWLEDGEMENTS ……………………………………….. vi
THESIS SUMMARY …………………………………………..….. viii
TABLE OF CONTENTS ………………………………………..… x
LIST OF FIGURES …………...…………………………………… xiii
LIST OF TABLES ……………………………………………..…... xvi
LIST OF ABBREVIATIONS ……………………………............... xvii
CHAPTER 1 ………………………………………………………... 1
LITERATURE REVIEW – MOLECULAR BIOLOGY OF CELLULOSE
BIOSYNTHESIS IN PLANTS ….……………………………………………….......... 1
1.1
Introduction ………………………………………………………………........... 2
1.2
Plant cell wall structure and the genes that affect it ..……………………...........
5
1.2.1
Primary cell walls ……………..………………………………............... 5
1.2.2
Secondary cell walls ……………………………………..………........... 8
1.3
Cellulose structure …………………………………………………………......... 11
1.4
Cellulose biosynthesis ……………………………………………………........... 12
1.4.1
Cellulose microfibril synthesis ……………………………...……......... 14
1.4.2
The cellulose synthase superfamily ………..………..……………......... 15
1.4.3
Cellulose synthases of the primary cell wall ………………………........ 16
1.4.4
Cellulose synthases of the secondary cell wall ……………………........ 17
1.4.5
Eucalyptus CesA homologues ……….……………………….……....... 17
1.4.6
Other genes involved in cellulose biosynthesis ……..……………......... 18
1.5
Arabidopsis as a model for xylogenesis …………………………………............ 23
x
1.5.1
Positional and silencing effects on transgene expression ….................... 24
1.5.2
Secondary growth in Arabidopsis …………………………………........ 25
1.6
Conclusions ………………………………………………………………........... 27
1.7
References ……………………………………………………………….…........ 28
CHAPTER 2 ..………………………………………………………. 44
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN
ARABIDOPSIS THALIANA ……………………………………………………............ 44
2.1
Abstract ……………………………………………………………………......... 45
2.2
Introduction …………………………………………………...…………............ 45
2.3
Materials and Methods …………………………………………...………........... 48
2.3.1
Biological material and growth conditions ..……………………............ 48
2.3.2
Vector construction ………………..……………………………............ 49
2.3.3
Transformation of competent Agrobacterium tumefaciens cells ............. 52
2.3.4
Transformation of Arabidopsis thaliana plants …………………........... 52
2.3.5
Selective screening of transgenic Arabidopsis plants …...………........... 53
2.3.6
PCR analysis of transgenic plants …………………………………....... 54
2.3.7
Reverse transcription PCR analysis ………………………………........
54
2.3.8
Microscope analysis of cell wall phenotype ….…………………..........
56
2.3.9
Cell wall chemistry analysis of Arabidopsis stems ………………........
57
2.3.10
Quantitative real-time PCR analysis ……...………………………........
57
2.4
Results ………………………………………………...…………………........... 59
2.4.1
Vector construction ……………………………………………….........
59
2.4.2
Selective screening and PCR of transgenic plants ………………..........
62
2.4.3
RT-PCR of transgenic plants ……………………………………..........
63
2.4.4
Microscopy of stem cell walls ……………………………………........
65
2.4.5
Cell wall chemistry of transgenic plant stems …………………............
66
xi
2.4.6
Expression analysis of EgCesA1 and other cell wall related genes in
wild-type and transgenic Arabidopsis plants ........................................... 67
2.5
Discussion ………………………………………………………………............
77
2.6
References ………………………………………………………………............ 86
2.7
Supplementary material …………………………………………………...........
92
CONCLUDING REMARKS …………………………...……......... 122
References ………………………………………………………….…………….......... 126
xii
LIST OF FIGURES
Figure 1.1 β (1-4) glycosidic bond between glucose monomers in cellulose …….........
11
Figure 1.2 Hydrogen bonds in type Iβ crystalline cellulose …………............................
12
Figure 1.3 Diagrams of rosette structures ………………………………………... ........
14
Figure 1.4 Model of cellulose biosynthesis ……...………………………………. ........
19
Figure 2.1 Schematic representation of the LR cloning reaction ……………………....
51
Figure 2.2 Illustration of tissues sampled for RT-PCR and qRT-PCR …………...........
55
Figure 2.3 1% EtBr/Agarose gel electrophoresis of EgCesA1 CDS PCR products
amplified from cDNA .......................................................................................................
60
Figure 2.4 1% EtBr/Agarose gel electrophoresis of colony PCR products amplified from
pTOPO-EgCesA1 and pMDC32-EgCesA1………………………………………...........
61
Figure 2.5 Hygromycin selection of T1 seeds ...……………..........................................
62
Figure 2.6 1% EtBr/Agarose gel electrophoresis of the amplified EgCesA1 PCR products
from leaf genomic DNA ……………………………………...........................................
63
Figure 2.7 1% EtBr/Agarose gel electrophoresis of extracted total RNA and PCR
products amplified from cDNA using UBQ10 primers flanking an intron ………..........
64
Figure 2.8 1% EtBr/Agarose gel electrophoresis of RT-PCR products …………...........
65
Figure 2.9 Images of inflorescence stem and hypocotyl cross sections of wild-type (WT)
and transgenic plant lines (MO1, MO2 and MO3) as observed using light microscopy .
66
Figure 2.10 Chemical composition of stems from wild-type (WT) and transgenic insertion
lines (MO1, MO2 and MO3) transformed with EgCesA1…...…..........….......................
68
Figure 2.11 Relative transcript abundance levels of EF1 and UBQ10 in leaves, upper- and
lower stems of wild-type (WT) and transgenic (MO1, MO2 and MO3) plants ...............
69
Figure 2.12 Relative transcript abundance of EgCesA1 in wild-type and transgenic plant
lines, MO1 and MO3 ....................................................................…………………........
72
Figure 2.13 Relative transcript abundance levels of Arabidopsis primary cell wall associated
CesA genes ……...........................................................................………...….................
xiii
73
Figure 2.14 Relative transcript abundance levels of Arabidopsis secondary cell wall
associated CesA genes …………………………………………………………..............
74
Figure 2.15 Relative transcript abundance levels of CYT1, KOBITO 1, KORRIGAN and
COBRA ..............................................................................................................................
75
Figure 2.16 Relative transcript abundance levels of SPP, SPS, SuSy1 and SuSy4…........ 76
Figure 2.17 Relative transcript abundance levels of AtCAD4, AtCAD5 and THESEUS 1 77
Figure S2.1 Vector map of pTOPO-EgCesA1 ……………………………….................. 92
Figure S2.2 Vector maps of the empty and recombinant destination vector,
pMDC32 and pMDC32-EgCesA1 …………………………………………………......... 93
Figure S2.3 Alignment of predicted amino acid sequences of four pTOPO-EgCesA1
clones ................................................................................................................................. 94
Figure S2.4 Melting curves of the qRT-PCR products for each gene specific primer
pair ....................................................................................................................................
98
Figure S2.5 1% EtBr/Agarose gel electrophoresis of bulked qRT-PCR products for 22
gene-specific primer pairs …………………………………………………….................
99
Figure S2.6 Relative expression levels of EgCesA1 and Arabidopsis cell wall related
genes ……………………………………………………………………………… .........
100
Figure S2.7 Regression analysis of relative transcript levels of all genes compared
between biological replicates across all tissues ………………...……………….... .........
105
Figure S2.8 Regression analysis of relative transcript levels compared between biological
replicates of wild-type plants …………………………………………............................
106
Figure S2.9 Regression analysis of relative transcript levels compared between biological
replicates of MO1 transgenic plants ………………………………….............................. 107
Figure S2.10 Regression analysis of relative transcript levels compared between biological
replicates of MO2 transgenic plants ………………………………….............................. 108
Figure S2.11 Regression analysis of relative transcript levels compared between biological
replicates of MO3 transgenic plants ………………………………….............................. 109
Figure S2.12 Transcript levels of UBQ10 and EF1 before normalization ………. .........
xiv
110
Figure S2.13 Sequence alignment of AtCesA8 and EgCesA1 cDNA ……………. .........
xv
111
LIST OF TABLES
Table 2.1 Oligonucleotide primers used for vector construction, DNA sequencing and
RNA analysis ..................................................................................................................... 50
Table 2.2 RNA samples obtained from transgenic and wild-type plants .........................
56
Table 2.3 R2-values for biological replicates of wild-type, MO1, MO2 and MO3 plants
70
Table 2.4 Slope-values of the regression lines for biological replicates of wild-type, MO1,
MO2 and MO3 plants .......................................................................................................
71
Table S2.1 Oligonucleotide primers used for qRT-PCR analysis of the EgCesA1 transgene
and endogenous Arabidopsis genes …………..................................………..……........... 119
Table S2.2 Oligonucleotide primers used for amplification of putative housekeeping
genes ……………………………………………………………………...……..…........
120
Table S2.3 Sequence results of bulked qRT-PCR products analysed using BLAST …..
121
xvi
LIST OF ABBREVIATIONS
ACT2
ACTIN 2
AGP
Arabinogalactan protein
cDNA
Complementary deoxyribonucleic acid
CAD
Cinnamyl alcohol dehydrogenase
CDS
Coding sequence
CesA
Cellulose synthase
CMV
Cauliflower mosaic virus
Col-0
Columbia ecotype 0
CYT1
CYTOKINESIS DEFECTIVE 1
D
Aspartate
dNTP
Deoxyribonucleotide-triphosphate
EF1
ELONGATION FACTOR 1α
EtBr
Ethidium bromide
GPI
Glycosylphosphatidylinositol
GX
Glucuronoxylan
MAP
Microtubule associated protein
NCBI
National Center for Biotechnology Information
PCR
Polymerase chain reaction
PME
Pectin methyl esterase
PTGS
Post-transcriptional gene silencing
qRT-PCR
Quantitative real-time polymerase chain reaction
RISC
RNA-induced silencing complex
RT-PCR
Reverse transcriptase polymerase chain reaction
SPP
Sucrose-phosphate phosphatase
SPS
Sucrose-phosphate synthase
SuSy
Sucrose synthase
xvii
T-DNA
Transferred DNA
TGS
Transcriptional gene silencing
TMH
Trans-membrane helix
TUB
TUBULIN
UBQ
UBIQUITIN
UGPase
Uridine diphosphate-glucose pyrophosphorylase
YEP
Yeast extract peptone
XET
Xyloglucan endotransglycosylase
xviii
CHAPTER 1
LITERATURE REVIEW
MOLECULAR BIOLOGY OF CELLULOSE
BIOSYNTHESIS IN PLANTS
LITERATURE REVIEW
1.1
Introduction
There is no denying the importance of wood and wood products in the lives of people across the globe.
Wood can be utilized for an array of products ranging from toilet paper to mining timber. It is a
renewable and vital source of carbon to households and industry where different wood characteristics
can be exploited to meet the demands of consumers.
Understanding the biological processes
underlying wood development is becoming increasingly important as the demand for bio-fuels and
other commercial applications of wood increases.
The process of xylogenesis (wood formation) occurs in five major steps. First, xylem mother
cells originate from dividing fusiform and ray initials in the vascular cambium. Secondly, the xylem
mother cells divide and cells that originated from fusiform initials elongate longitudinally. The third
step involves the deposition of the secondary cell wall which mainly consists of cellulose and lignin.
After the secondary cell wall has been deposited, the now mature xylem cells undergo programmed
cell death (PCD, Kozela and Regan, 2003). The final step in xylogenesis is heartwood formation
(Plomion et al., 2001). The main difference between heartwood and sapwood, that lies between the
vascular cambium and heartwood, is that the vertical and ray parenchyma cells in sapwood are still
metabolically active. Rays are parenchyma cells that facilitate horizontal channelling of nutrients and
water across the stem. They have also been proposed to be involved in storage of metabolic waste
substances secreted from cells. Increased levels of excretory substances could lead to cell death of the
vertical and ray parenchyma cells and the formation of heartwood (Stewart, 1966; Mauseth, 1988).
Tylose formation involves blocking or sealing of vessel cells by adjacent parenchyma cells that push
part of their cytoplasm through pit apertures into the lumen of the vessel cell. The outgrowths of
cytoplasm contained within the vessel cell wall are tyloses. Several tyloses can fill the cavity of a
vessel cell. Tylose formation is the final step in the conversion of sapwood to heartwood. (Stewart,
1966).
Cellulose, the predominant polymer in wood, is made up of β-1,4-linked glucan molecules that
crystallize to form cellulose microfibrils. It is synthesised at the cell membrane by protein complexes
2
LITERATURE REVIEW
(Brown Jr and Montezinos, 1976). The first cellulose synthase (CESA) protein was discovered in
1982 in Acetobacter xylinum (Aloni et al., 1982) and the gene sequence was determined in 1990
(Saxena et al., 1990). Since then several other CesA genes have been identified in a range of species
including plants like cotton (Pear et al., 1996), Arabidopsis thaliana (Arioli et al., 1998), maize
(Appenzeller et al., 2004), barley (Burton et al., 2004), rice (Tanaka et al., 2003), poplar (Joshi et al.,
2004) and Eucalyptus (Ranik and Myburg, 2006). The CesA gene family forms part of a larger
cellulose synthase superfamily consisting of one CesA and eight CesA-like (Csl) sub-families
(Richmond and Somerville, 2000; Lerouxel et al., 2006). Cellulose synthase (CESA) proteins are
UDP-glycosyltransferases that form terminal complexes in the cell membrane. In plants, the CESA
proteins form a membrane-bound terminal complex structure that resembles a rosette that was first
observed in Micrasterias denticulata (Kiermayer and Sleytr, 1979) and later observed in higher plants
(Mueller and Brown, 1980). It is thought to consist of six subunits that each contains six CESA
proteins (Herth, 1983). It has been suggested that homo- and heterodimerization of CESA proteins
occur prior to rosette assembly (Doblin et al., 2002; Timmers et al., 2009). The CESA proteins are
believed to add UDP-glucose monomers to growing β-1,4-glucan chains. Although more than two
decades have passed since the first cellulose synthase gene was discovered, it is still not known how
the CESA proteins assemble to form active complexes that synthesise cellulose.
Several other proteins involved in cellulose deposition have been identified. They include
KORRIGAN (Szyjanowicz et al., 2004), KOBITO 1 (Pagant et al., 2002), COBRA (Wakabayashi et
al., 2005) and CYTOKINESIS DEFECTIVE 1 (CYT1, Lukowitz et al., 2001). The precise functions
of these proteins remain elusive, but plants mutated for the genes encoding these proteins all show a
level of cellulose deficiency. The deficiencies varied between 33% in kobito 1 (Pagant et al., 2002)
and cobra (Schindelman et al., 2001) mutant plants, 30% to 60% in korrigan mutants (Sato et al.,
2001; Szyjanowicz et al., 2004) and up to a five-fold decrease in cyt1 mutant plants (Lukowitz et al.,
2001).
Studying the process of xylogenesis and the roles the aforementioned genes play in trees could
be problematic due to the long generation times and low efficiency of tree-transformation. This
3
LITERATURE REVIEW
creates a need for a model plant in which xylogenesis can be examined. Even though the well
established model plant, Arabidopsis thaliana, is a herbaceous weed, it has been proven to be a
valuable model for xylogenesis (Lev-Yadun, 1994; Chaffey et al., 2002). Arabidopsis meets the
requirements for a good model organism. The full genome sequence is publicly available (The
Arabidopsis Genome Initiative 2000).
Genome-, proteomic- and metabolomic resources can be
accessed through the online Arabidopsis Information Resource, TAIR (www.arabidopsis.org). Results
from a large number of microarray studies are obtainable through GENEVESTIGATOR
(www.genevestigator.com) to allow comparative gene expression analysis (Zimmermann et al., 2004).
Mutant strains of Arabidopsis are easily obtainable for knock-out studies (abrc.arabidopsis.org) and
Arabidopsis has high transformability (Nieminen et al., 2004). Because Arabidopsis is widely used as
a model for transgenic and promoter activity studies, new and improved methods are continuously
being developed to increase transformation efficiency (Davis et al., 2009). Despite these improved
techniques, there are aspects that need to be considered when embarking on transgene expression
studies. The genomic region where the transgene inserts and the effects of post-transcriptional gene
silencing (PTGS) are some of the concerns addressed later in the review.
A better understanding of the processes that underlie xylogenesis is necessary to fully utilize
the potential of wood and wood products. Many genes have been shown to be involved in wood
formation, but many remain to be characterized. In this review, the assembly of plant cell walls and
our current knowledge on the interaction of cell wall polymers and the genes underlying their
biosynthesis are discussed. Special attention is given to the structure of cellulose and the genes
expressing proteins that play a role in cellulose deposition. A revised model for cellulose biosynthesis
is proposed highlighting areas where current knowledge on cellulose synthesis is still shortcoming.
Finally, the review addresses the use of Arabidopsis to study secondary cell wall formation and
aspects that need to be considered when using transgenics to study gene function.
4
LITERATURE REVIEW
1.2
Plant cell wall structure and genes that affect it
1.2.1 Primary cell walls
Primary cell walls consist of a framework of cellulose microfibrils with hemicellulose, pectin and
proteins assembled in between. Cellulose is synthesised by CESA protein rosette complexes that are
embedded in the plasma membrane. (Details of cellulose biosynthesis are discussed in section 1.4)
The cellulose microfibrils are connected via single-chain polysaccharides such as xyloglucans which
form hydrogen bonds between their neutral backbones and the cellulose microfibril surface (McCann
et al., 1990). Non-cellulosic polymers are soluble before they are linked to other molecules and are
capable of moving in the cell wall environment to their destinations between the cellulose microfibrils
(Reviewed in Somerville et al., 2004). These include three types of pectin, namely homogalacturonan,
rhamnogalacturonan I, and rhamnogalacturonan II as well as glucuronoarabinoxylan, callose and
xyloglucan, which is the most abundant hemicellulose in the primary cell walls of angiosperms
(Richmond and Somerville, 2000; Sterling et al., 2006). The most abundant pectic polysaccharide is
homogalacturonan. It is a linear polymer of α-1,4-linked galacturonic acid. Rhamnogalacturonan II is
a substituted homogalacturonan and rhamnogalacturonan I is the most variable pectic polysaccharide
with a complex backbone composition (Reviewed in Mohnen, 2008).
The primary cell wall plays a vital role in cell and organ growth. This is also true for
xylogenesis where the formation and expansion of the primary cell wall form part of the early events
in wood development. Xyloglucan cross-links cellulose microfibrils and, by doing so, limits cell wall
extensibility (Hayashi et al., 1987).
Xyloglucan networks can be relaxed by xyloglucan
endotransglycosylases (XETs) that cleave and rejoin xyloglucan chains to loosen the cell wall matrix
and allow cell expansion (Fry et al., 1992). The primary cell wall can also be loosened by expansins.
Expansins are proteins that facilitate wall loosening by weakening non-covalent bonds between the
polysaccharides in the wall. Turgor pressure in the cell then pushes the cell wall to expand where the
cell wall is loosened (McQueen-Mason, 1995; Cosgrove, 2000). Xylem cell-specific expansins have
been identified in maize (Im et al., 2000) and poplar (Gray-Mitsumune et al., 2004). These expansins
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LITERATURE REVIEW
are believed to be involved in intrusive growth of the primary cell walls in differentiating xylem cells.
Intrusive growth occurs when one cell outgrows the cells around it by passing between the walls of
several adjacent cells (Lev-Yadun, 2001; Chernova and Gorshkova, 2007).
The orientation in which cellulose microfibrils are deposited contribute to directing cell and
organ growth. Kerstens and Verbelen (2003) observed that elongating cells in Arabidopsis hypocotyls
have a mostly transverse mean microfibril orientation.
This orientation is believed to enhance
longitudinal growth while restricting girth expansion (Kerstens and Verbelen, 2003). Pectin forms a
gel with varying mechanical strength between the cellulose-xyloglucan scaffold and can be modified
to facilitate cell expansion. Pectin methyl-esterases (PMEs) remove the methyl-esters from pectin
galacturonic acid and influence the gelling properties of pectin (Micheli, 2001). The degree of methylesterification has been shown to influence cell elongation in Arabidopsis hypocotyls (Derbyshire et
al., 2007).
Microtubules have been shown to play a role in the deposition of cellulose microfibrils
(Green, 1962; Oda et al., 2005; Paredez et al., 2006; Wasteneys and Fujita, 2006). The interaction of
cellulose with microtubules have long been apparent (Green, 1962), but the mechanism by which
microtubules direct cellulose deposition still remains unknown. Although microtubule altering drugs
have been shown to drastically affect cellulose microfibril orientation (Green, 1962; Quader, 1986;
Oda et al., 2005), the most convincing evidence for the interaction of microtubules with microfibrils
was provided in a study by Paredez et al. (2006). By means of spinning disk microscopy and a
fluorescent protein fusion with Arabidopsis thaliana cellulose synthase 6 (AtCESA6), the authors
were able to track CESA protein complexes in living cells and show that these complexes follow a
path aligned with cortical microtubules during primary cell wall formation (Paredez et al., 2006). It is
not clear whether this model pertains to secondary cell wall formation.
The interaction between microtubules and emerging cellulose microfibrils have been proposed
to be facilitated by proteoglycans called arabinogalactan proteins (AGPs, Baskin, 2001; Driouich and
Baskin, 2008). Baskin (2001) proposed a model for cellulose deposition where a scaffold of proteins
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LITERATURE REVIEW
in the cell wall, connected to the plasma membrane, form a template wherein the nascent cellulose
microfibril is orientated. The protein template responds to microtubule orientation and must therefore
have some interaction with the cytosol. AGPs are proteins in the cell wall anchored to the plasma
membrane. Many AGPs have glycosylphosphatidylinositol (GPI)-anchoring domains (Orlean and
Menon, 2007). Driouich and Baskin (2008) suggested that AGPs could form part of this protein
scaffold. Another key protein suggested to be involved is COBRA. It is also a GPI-anchored wall
protein (Schindelman et al. 2001). The COBRA gene is expressed during rapid organ expansion and is
suggested to be involved in the interactions between microtubules and cellulose microfibrils
(Wasteneys and Fujita, 2006). The cobra mutant plants exhibit radial swelling in the roots. It has
been observed that COBRA has a polar distribution in elongating root cells (Roudier et al. 2005). The
means by which COBRA influences cellulose biosynthesis remains to be proven. Driouich and
Baskin (2008) suggested that newly synthesised microfibrils interact with AGPs which interacts with
COBRA. COBRA is proposed to be the physical link with microtubules to direct the orientation of
cellulose deposition.
Proteins that affect cellulose synthesis and associate with microtubules have also been
identified. A microtubule associated protein (MAP) indentified in poplar, PttMAP20 (Rajangam et
al., 2008) was shown to localise to microtubules and bind as a target to 2,6-dichlorobenzonitrile
herbicide (a known inhibitor of cellulose synthesis). FRAGILE FIBRE 1 (FRA1) is a kinesin-like
putative microtubule binding protein (Zhong et al., 2002). The fra1 mutant plants have normal
cellulose content and microtubule organization, but altered cellulose microfibril orientation resulting
in weakened fibre cell walls. MAPs, COBRA and AGPs could interact to facilitate the orientation of
cellulose microfibrils in relation to microtubule organization, but the exact means by which this occurs
remains unknown.
In summary, microtubules are hypothesised to contribute to the orientation in which cellulose
is deposited. The deposited cellulose microfibril framework is reinforced by cross-linking polymers
like xyloglucan and pectins. Cross-links can be loosened through the actions of molecules like XETs,
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LITERATURE REVIEW
expansins and PMEs which enables the primary cell wall to still undergo expansion and allow cellular
growth.
1.2.2 Secondary cell walls
The secondary cell wall is deposited between the primary cell wall and the plasma membrane. It
provides structural support against gravitational forces and is essential to cells involved in water
transport. The secondary cell wall consists of three layers, S1, S2 and S3 (Cote Jr et al., 1968). S1 is
deposited first and occurs just inside of the primary cell wall, while S3 is deposited last and is the
closest to the plasma membrane. S2 occurs between S1 and S3. The angle at which cellulose
microfibrils are deposited is different in each layer. S1, which is the thinnest of the three layers, has
microfibrils at an angle of 60° to 80° to the cell axis. S2 microfibrils are deposited 5° to 30° relative to
the cell axis. S2 is also the thickest of the three layers comprising 75% to 85% of the secondary cell
wall. The inner most layer, S3, has cellulose microfibrils deposited in a parallel arrangement at an
angle 60° to 90° to the cell axis (Plomion et al., 2001). Because S2 is the thickest, the angle of the
cellulose microfibrils in this layer greatly influences the overall strength of the cell wall. The greater
the microfibril angle relative to the cell axis, the less rigid the wall becomes as is observed in juvenile
wood (Plomion et al., 2001).
Another contributing factor involved in secondary cell wall reinforcement is lignification
(Reiter, 2002). Lignin accumulates in cell walls of mature xylem and schlerenchyma cells. It is
mainly a coniferyl and sinapyl alcohol polymer that causes the cell walls to become impermeable to
gas and water. It also strengthens and rigidifies cell walls (Mauseth, 1988). The genes involved in
monolignol biosynthesis have been extensively reviewed (Boudet, 2000; Boerjan et al., 2003; Li et al.,
2006), but the exact mode of interaction of lignin with other cell wall polymers is not certain. One
model proposed suggests that highly condensed lignin polymers interact with xylans that coat the
cellulose microfibrils in secondary cell walls (Ruel et al. 2006). Ectopic lignification has been
observed in plants with reduced cellulose content (Cano-Delgado et al., 2003). Ectopic lignification
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LITERATURE REVIEW
could be a defence response in plants since lignin is resistant to degradation by microorganisms
(Campbell and Sederoff, 1996).
The deposition of cell wall carbohydrates involves two groups of genes. The first group is
involved in the linkage of mono- and disaccharides to form the backbone of a polymer. These genes
encode synthases. The second group encode glycosyltransferases that add specific sugars to the
polymer backbone (Carpita et al., 2001). The genes encoding the cellulose synthases (CesAs) form
part of a super gene family called the cellulose synthase superfamily, which include one CesA gene
family (see section 1.4.2) and eight CesA-like gene sub-families (CslA-H, Richmond and Somerville,
2000; Lerouxel et al., 2006).
Genes in the CesA-like (Csl) gene sub-families are believed to encode proteins responsible for
hemicellulose biosynthesis. However, it has been difficult to assign a specific gene to the assembly of
a particular hemicellulose, because of genetic redundancy and the great complexity of hemicellulose
(Richmond and Somerville, 2000; Bonetta et al., 2002). β-mannan synthase was the first member of
the CesA-like A (CslA) gene family to have been assigned a function. It was found to encode the
protein that synthesises the backbone of the hemicellulose mannan in guar seeds (Dhugga et al., 2004).
It was later discovered that a CSLA protein was capable of not only synthesising mannan backbones
(made of mannose monomers), but also the backbones of glucomannan (made of mannose and glucose
monomers, Liepman et al., 2005). The backbones of mixed-linkage glucans and xyloglucans were
later revealed to be synthesised by proteins transcribed by members of the CslF and CslC gene
families, respectively (Persson et al., 2005; Burton et al., 2006).
Xylan, the most abundant hemicellulose in secondary cell walls, is comprised of backbones of
1,4-linked β-xylosyl residues that are substituted with α–D-glucuronic acid, arabinose or 4-O-methylα-D-glucuronic acid.
Xylan is classified under (methyl)glucuronoxylan (GX), arabinoxylan and
glucuronoarabinoxylan. The classification of xylan is based on the nature of the side-chains. GX also
have a characteristic glycosyl sequence at the reducing end of the polymer (Lee et al., 2007a). Genes
involved in the synthesis of GX backbones were identified by the phenotypes of mutant plants. The
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LITERATURE REVIEW
mutants had abnormal xylem cell walls and some were therefore classified as irregular xylem (irx)
mutants. These include irx8 (Persson et al., 2005), irx9 (Brown et al., 2005), irx14 (Brown et al.,
2007), irx10 and irx10-like (irx10-L) (Brown et al., 2009). FRAGILE FIBRE 8 (FRA8) and PARVUS
were also shown to be involved in GX synthesis (Zhong et al., 2005; Lee et al., 2007b). fra8 mutants
(irx7) have reduced xylan and cellulose compared to wild-type plants. Mutations in the PARVUS gene
lead to reduced GX content. PARVUS, FRA8 and IRX8 have been shown to be required for the
synthesis of the characteristic glycosyl sequence on the end of the GX chains, while IRX9, IRX10,
IRX10-L and IRX14 are involved in the synthesis of the GX backbone(Brown et al., 2007; Lee et al.,
2007a; Pena et al., 2007; Brown et al., 2009).
The side-chains of hemicellulose are added by glycosyltransferases. Xyloglucan has, among
others, xylose-galactose-fucose sidechains.
The addition of fucose is mediated by xyloglucan
fucosyltransferase encoded by the MUR2 gene (Vanzin et al., 2002). Pea and Arabidopsis plants
mutated for this gene showed a deficiency in fucose. Another mutant, mur3, also exhibited fucose
deficiency, but this was due to a mutated gene that encodes xyloglucan galactosyltransferase. Since
the galactose is attached before the fucose residue, mur3 mutants are deficient in galactose and fucose
(Madson et al., 2003).
The secondary cell wall is a complex system of cellulose microfibril frameworks
interconnected with hemicellulose and fortifying lignin. Together, the components serve to withstand
turgor and gravitational forces imposed on secondary cell walls. Better understanding of the synthesis
of the various components may elucidate the exact means of interaction between them. It is clear that
all components are essential for correct functioning of the secondary cell wall, since a reduction in any
one of the components weakens secondary cell walls and causes the cells to collapse and exhibit the
irx phenotype.
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LITERATURE REVIEW
1.3
Cellulose structure
Cellulose consists of glucose monomers which are connected by covalent bonds to form β-1,4-glucan
chains. The monomers are joined at a 180° angle to each other (Figure 1.1). The chains are aligned
and crystallization (the formation of hydrogen bonds and Van der Waals forces between glucan
chains) can occur, resulting in a cellulose microfibril with a diameter of approximately 3 nm
(Muhlethaler, 1967; Herth, 1983). These microfibrils can further crystallize to form fibrils.
Figure 1.1 β (1-4) glycosidic bond between glucose monomers in cellulose.
Cellulose occurs as crystalline and non-crystalline cellulose in plant cell walls. Crystalline
cellulose can be further classified as naturally occurring crystalline cellulose type I and processed
cellulose type II, III and IV (Davis et al., 1943). Type II cellulose occurs due to chain folding and
results in glucan chains that are in anti-parallel configuration (Kolpak and Blackwell, 1976). This is
thermodynamically the most stable form of cellulose. Cellulose type I, however, is the most abundant
allomorph in nature. It has parallel glucan chains (Jarvis 2003) and is sub-divided into two suballomorphs, type Iα and type Iβ (Atalla and VanderHart, 1984; Nishiyama et al., 2003). Although
combinations of the two sub-allomorphs occur in organisms, type Iα make up the bulk of crystalline
cellulose in algae and bacteria, whereas wood, cotton and tunicates are type Iβ rich (Sugiyama et al.,
1991). Type Iα is meta-stable and can, by means of annealing, be converted to the more stable type Iβ
(Atalla and VanderHart, 1984). Crystallization does not occur immediately after the glucan chains are
formed. Saxena and Brown (2005) suggested that there are several stages in this process. The first
step involves the assembly of glucan chain sheets by Van der Waals forces followed by hydrogen
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LITERATURE REVIEW
bonding to make a cellulose I microfibril (Cousins and Brown, 1997). Proteins that are directly
involved in cellulose crystallization in plants have not been identified, but it is believed that the rate of
crystallization influences the rate at which cellulose is synthesised (Emons and Mulder, 2000; Mulder
et al., 2004). The intra-molecular hydrogen bonds that occur in cellulose are illustrated in Figure 1.2
as blue lines. The hydrogen bonds that occur between two chains in the type Iβ sub-allomorph are
shown as red lines.
Figure 1.2 Hydrogen bonds in type Iβ crystalline cellulose. Red lines indicate hydrogen bonding between two
parallel chains and blue lines indicate intra-molecular bonds (Heiner et al., 1995).
1.4
Cellulose biosynthesis
The biosynthesis of cellulose was first studied in Acetobacter xylinum (Glaser, 1958; Ohad et al.,
1962). This gram-negative bacterium was used to uncover the biosynthetic enzymes responsible for
the production of cellulose.
Cellulose was produced in vitro using isolated membranes from
Acetobacter in 1982 (Aloni et al., 1982). From this study came the first purified cyclic diguanylic acid
(c-di-GMP) dependent cellulose synthase. The gene encoding the catalytic subunit of the synthase
was later identified as AcsA (Saxena et al., 1990). This gene formed part of an operon consisting of
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LITERATURE REVIEW
four genes of which the first three were essential for cellulose production in the bacterium (Saxena et
al., 1990).
Since then several cellulose synthases have been characterized in among others
Agrobacterium tumefaciens (Matthysse et al., 1995), Gossypium hirsutum (Pear et al., 1996) and
Arabidopsis thaliana (Arioli et al. 1998). The first plant cellulose synthases were discovered in cotton
and rice cDNA (Pear et al., 1996). In 1999, Delmer proposed a new nomenclature for the cellulose
synthase genes and the name “CesA” originated (Delmer, 1999). Originally the abbreviation CelA
was used to describe a gene homologous to the catalytic subunit of the gene first identified to encode
the cellulose synthase protein in Acetobacter xylinum. Since Cel was also used to designate cellulases,
Delmer proposed using “CeS” for cellulose synthase followed by “A” to show it is homologous to the
bacterial catalytic subunit A.
The biosynthesis of cellulose in plants occurs at the plasma membrane. Here β-1,4-glucan
chains are synthesised by hexameric protein complexes (rosettes) embedded in the plasma membrane
using UDP-glucose as substrate (Mueller and Brown, 1980). The rosette complexes are believed to be
assembled in the Golgi and transported to the plasma membrane through the vesicle shuttle (Haigler
and Brown, 1986). It consists of six sub-units each containing six CESA proteins (Figure 1.3). Three
distinct CESA proteins are assembled into a sub-unit of a rosette (see sections 1.4.3 and 1.4.4). The
CESA proteins have been hypothesised to interact in a protein specific manner so that each CESA
protein occupies one or more specific positions in a sub-unit (Arioli et al., 1998). It has been shown
that three distinctive CESA proteins are required to form cellulose in the primary cell wall (Persson et
al., 2007) and a different set of CESA proteins are required for secondary cell wall cellulose
deposition (Taylor et al., 2003). Different models for rosette complex assembly have been proposed
based on the formation of homo- and heterodimers between CESA proteins using yeast-two-hybrid
analysis and bimolecular fluorescence complementation (Doblin et al., 2002; Timmers et al., 2009).
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LITERATURE REVIEW
Figure 1.3 Diagrams of rosette structures. Each rosette is believed to consist of six sub-units, each comprised
of six CESA proteins. Three different CESA proteins comprise a rosette. Each sub-unit can consist of a single
kind of CESA protein (A) or it could be comprised of different CESA proteins (B). The possible compositions
of the sub-units are illustrated in C. The sub-unit on the left contains six of the same CESA protein, the sub-unit
in the middle contains three different CESA proteins in equal number (Timmers et al., 2009) and the sub-unit on
the right contains three different CESA proteins in a 1:2:3 ratio (Doblin et al., 2002).
1.4.1 Cellulose microfibril synthesis
From observing microfibril diameters and freeze-fractured images of the cellulose synthesising
complexes it is inferred that rosettes in higher plants each synthesise 36 cellulose chains (Herth, 1983).
This corresponds with the hypothesis that each rosette is made up of six subunits of six CESA proteins
which each synthesise a single glucan chain. These glucan chains subsequently coalesce to form a
cellulose microfibril. Williamson et al. (2002) reviewed two hypotheses for the assembly of the
glucan chains. The “direct assembly” hypothesis suggests that the glucose residues are attached to the
glucan chain directly by the glycosyltransferases. The “indirect assembly” mechanism involves the
formation of short glucan chains attached to lipid or protein primers that later become incorporated
into a long glucan polymer. Cellulose synthesis in Agrobacterium tumefaciens (Matthysse et al.,
1995) is believed to occur by indirect assembly, while Acetobacter xylinum (Kawagoe and Delmer,
1997) uses a direct assembly mechanism. Higher plants appear to be capable of both methods of
assembly (Williamson et al., 2002). The fact that the glucose monomers are attached at a 180° angle
to each other raises a problem for the direct assembly mechanism. A single enzyme with only one
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LITERATURE REVIEW
active site will most likely be unable to add residues in opposite orientations. The most plausible
explanation is that the enzyme has two substrate-binding sites that pre-align the monomers
(Williamson et al., 2001).
1.4.2 The cellulose synthase superfamily
The cellulose synthase superfamily consists of eight CesA-like (Csl) sub-families and one CesA
family (Richmond and Somerville, 2000). All of the genes in this superfamily are believed to be
integral membrane proteins. It has been proposed that each Csl sub-family is responsible for the
biosynthesis of a different non-cellulosic polysaccharide, while the CESA proteins synthesise cellulose
microfibrils in primary and secondary cell walls.
The CesA gene family has ten members in Arabidopsis and seven putative expressed
orthologues in Eucalyptus (Richmond and Somerville, 2000; Ranik and Myburg, 2006 and
unpublished data). Pear et al. (1996) described two main domains that exist in all CESA proteins.
The first is the domain around two highly conserved aspartate (D) residues. The second domain
includes another highly conserved D residue and a conserved QxxRW motif. These domains are in
CESA proteins in plants and bacteria, and in CESA-like proteins. Plant CESA proteins also have
plant-specific conserved insertions. These include two trans-membrane helices (TMH) in the Nterminal region and six in the C-terminal region. All of the conserved regions (D,D,D and QxxRW)
occur in the cytoplasm and are believed to comprise the catalytic domain. A domain with high
homology to the RING-finger domain at the N-terminus of plant CESA proteins is believed to be
involved in homo- and hetero-dimerization between CESA proteins (Kurek et al., 2002). There are
also two class-specific regions, CSRI and CSRII (Vergara and Carpita, 2001). These regions are
highly conserved between CesA homologues of different species but not between CESA proteins
within the same species.
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LITERATURE REVIEW
1.4.3 Cellulose synthases of the primary cell wall
As mentioned above, three CesA genes have been confirmed to have specific roles in the formation of
primary cell walls in Arabidopsis. AtCesA1, AtCesA3 and AtCesA6 are required for correct primary
cell wall assembly (Arioli et al., 1998; Dhugga, 2001; Desprez et al., 2002). The AtCesA1 mutant is
known as radial swelling 1 (rsw1), (Arioli et al., 1998). The rsw1 mutants have a reduction in cell and
organ length. This is due to cell wall abnormalities throughout the plant. The first mutants of
AtCesA3 and AtCesA6 identified were isoxaben resistant 1 (ixr1) and ixr2, respectively. These
mutants are isoxaben (a cellulose synthase inhibitor) resistant, have reduced cellulose and exhibit
radial swelling of the hypocotyls and roots (Dhugga, 2001; Desprez et al., 2002). Null mutants of
AtCesA1 and AtCesA3 are lethal, while AtCesA6 null mutants exhibit a milder phenotype. This is
attributed to three other CesA genes that are closely related to AtCesA6. AtCesA2, AtCesA5 and
AtCesA9 have been shown to be at least partially redundant to AtCesA6 (Desprez et al., 2007; Persson
et al., 2007). It has been proposed that the AtCesA6-related gene products could occupy the same
position in the rosette subunit during primary cell wall cellulose biosynthesis. The AtCesA6-related
genes have different expression profiles across different plant developmental stages. AtCesA2 gene
expression seems to have an influence on inter-node length (Burn et al., 2002), while AtCESA9
protein appears to be specifically required for cell wall deposition during pollen formation (Persson et
al., 2007). What these results indicate is that AtCESA6 and related proteins function differently at
different developmental stages. This probably contributes to fine-tuning of cellulose production and
possibly the characteristics of the cellulose produced and it suggests variable rosette assembly in
different tissues or conditions.
Unlike the expression profiles observed for AtCesA2, AtCesA5 and AtCesA9, AtCesA1,
AtCesA3 and AtCesA6 are expressed in most tissues throughout Arabidopsis (Cousins and Brown,
1997; Hamann et al., 2004). The expression levels of these genes have been proposed to be at a
constant ratio to each other. This was discovered when the expression of the barley homologues of the
CesA genes were analysed (Burton et al., 2004). The barley homologue of AtCesA3 (HvCesA1) had a
higher expression than the homologues for AtCesA1 and AtCesA6 (HvCesA6 and HvCesA2,
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LITERATURE REVIEW
respectively). The homologue of AtCesA1 had the lowest expression in all the tissues analysed. Eventhough the same homologues are involved in primary cell wall formation in different plant species
(Burton et al., 2004), it remains to be shown whether this expression ratio is constant in other plants.
1.4.4 Cellulose synthases of the secondary cell wall
Just as in the primary cell wall, three different CESA proteins are required for correct assembly of the
rosette structures that synthesise cellulose in the secondary cell wall (Taylor et al., 2003). The
mutants of these CesA genes were identified to have decreased cellulose in the secondary cell walls
that caused the xylem cell walls to collapse, hence the name “irregular xylem” (irx). AtCesA7 (IRX3)
and AtCesA8 (IRX1) were the first genes identified to be involved in the deposition of cellulose in the
secondary cell wall (Turner and Somerville, 1997). The third, AtCesA4 (IRX5), was only discovered
to play a role in the secondary cell wall six years later (Taylor et al., 2003). The expression of the
barley homologues of these three genes also exhibited a constant ratio to each other as in the primary
cell wall.
The AtCesA7 homologue (HvCesA8) showed higher expression, and the AtCesA4
homologue (HvCesA7) showed the lowest expression levels (Burton et al., 2004). This might explain
why the function of AtCESA4 in the secondary cell wall had eluded researchers for so long.
1.4.5 Eucalyptus CesA homologues
Seven Eucalyptus CesA genes have been isolated to date (Ranik and Myburg, 2006 and unpublished
data). Once again, three CesA genes appear to have a specific role in the formation of the primary cell
wall, while another three function in the assembly of secondary cell walls. EgCesA1 shows high
homology to AtCesA8 (Figure 2 of Ranik and Myburg 2006), EgCesA2 shows homology to AtCesA4
and EgCesA3 shows homology to AtCesA7. As expected, these genes were mostly expressed in stems
including xylem and immature xylem as well as in internodes. This correlates with the expected roles
of these genes during the deposition of secondary cell walls. The Eucalyptus CesA genes involved in
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LITERATURE REVIEW
primary cell wall cellulose biosynthesis are EgCesA4 (homologous to AtCesA3), EgCesA5
(homologous to AtCesA1) and EgCesA7 (homologous to AtCesA6). These genes showed highest
expression in flowers, internodes and young leaves (Burton et al. 2004; Ranik and Myburg 2006 and
unpublished data).
EgCesA6 was shown to be weakly expressed in all tissues analysed.
The
EgCESA6 protein had highest homology to monocot CESA proteins (Ranik and Myburg, 2006).
1.4.6 Other genes involved in cellulose biosynthesis
Many proteins have been identified to play a role in the biosynthesis of cellulose even though they
appear not to be incorporated into the cellulose synthase terminal complexes. Some of these proteins
have been proposed to be involved in carbon partitioning towards cellulose synthesis, cellulose chain
modification or N-glycosylation of the CESA proteins before they are incorporated into the cell
membrane. However, the proposed functions have yet to be confirmed. Most of these proteins have
been identified to have an influence on cellulose production based on their mutant phenotypes. Figure
1.4 is a model of cellulose biosynthesis where the different proteins and the proposed roles they play
in cellulose deposition are illustrated.
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LITERATURE REVIEW
Figure 1.4 Model of cellulose biosynthesis in plants. The terminal rosette complexes embedded in the
membrane incorporate UDP-glucose into the growing cellulose chains. SUSY (SuSy) converts sucrose into
UDP-glucose. THESEUS (THESEUS 1) is hypothesised to detect faulty rosette structures and direct carbon
flow towards lignification. COBRA is believed to facilitate the interaction of the terminal complexes with
microtubules. CYT1 could be involved in two processes, the GPI-anchoring of COBRA and the N-glycosylation
of the CESA proteins.
KORRIGAN has been proposed to be involved in chain editing of the cellulose
microfibrils and removal of the sitosterol primer in the indirect assembly model for cellulose biosynthesis.
Sucrose synthase (SUSY) is an enzyme involved in partitioning carbon to cellulose
biosynthesis. It is responsible for the conversion of sucrose into UDP-glucose and fructose. Sucrose
originates at the leaves and is translocated to sink tissues like xylem fibres via the phloem (Mauseth,
1988). Two forms of SUSY have been described, soluble (S-SUSY) and particulate (P-SUSY, Haigler
et al., 2001). S-SUSY occurs abundantly in the cytoplasm whereas P-SUSY is associated with the
membrane. It is believed that P-SUSY, specifically, is responsible for channelling UDP-glucose to
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LITERATURE REVIEW
cellulose production (Winter et al., 1997; Haigler et al., 2001; Delmer and Haigler, 2002). The
proposed function of SUSY raises an interesting question. Will an increase in SUSY protein result in
an increase in cellulose biosynthesis? Up-regulation of SUSY and Acetobacter xylinum UDP-glucose
pyrophosphorylase (UGPase) in tobacco resulted in increased total biomass due to increased height,
but cellulose content remained unchanged (Coleman et al., 2006). Expressing only Acetobacter
xylinum UGPase in transgenic poplar plants resulted in an increase in soluble sugars, cellulose content
and starch, but transgenic plants had reduced height and stem diameters (Coleman et al., 2007).
Coleman et al. (2007) also observed an increase in defence metabolites and concluded that cellulose
content can be altered but at the cost of growth across the whole plant. Although SUSY appears to be
a key player in carbon partitioning, it is by far not the only protein involved with carbon cycling in the
cell. When sucrose is converted to glucose, fructose is also produced. Sucrose-P synthase (SPS) and
sucrose-P phosphatase (SPP) are two enzymes involved in recycling fructose back to sucrose. The
complex pathway of carbon allocation to cellulose is reviewed by Delmer and Haigler (2002).
KORRIGAN is a β-1,4 glucanase (a cellulase) gene that encodes a protein active in primary
and secondary cell walls. The korrigan mutant, irx2 (Sato et al., 2001; Szyjanowicz et al., 2004;
Bhandari et al., 2006) in Arabidopsis has a cellulose deficiency. When KORRIGAN expression was
compared with secondary cell wall associated CesA gene expression in aspen trees, it was found to be
up-regulated in tension wood and down-regulated in opposite wood (tension wood forms at the top
side of a bended stem and opposite wood is on the compressed side), similar to the CesA gene
expression profiles (Szyjanowicz et al., 2004; Bhandari et al., 2006). Although it is apparent that
KORRIGAN plays a role in cellulose synthesis, there is no evidence for a direct interaction of
KORRIGAN with the CESA complexes in the cell membrane. Bhandari et al. (2006) hypothesised
that KORRIGAN might be necessary for later stages of cellulose production such as editing of the
cellulose chains or terminating microfibril synthesis.
KORRIGAN has also been implicated in
cellulose synthesis in the indirect assembly method discussed in section 1.4.1. When UDP-glucose is
first conjugated to a sitosterol primer, KORRIGAN may remove the glucan from the sitosterol-β-
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LITERATURE REVIEW
glucoside allowing the glucan to be incorporated into the glucan chain by cellulose synthases (Peng et
al., 2002).
KOBITO 1 is a plasma membrane protein associated with cellulose deposition during cell
elongation specifically (Pagant et al., 2002).
kobito 1 mutants have a dwarfed appearance and
randomized microfibrils in elongated cells. The KOBITO 1 gene appears to be only expressed during
primary cell wall formation after cell division ceases. The protein has been shown to localise to the
cell wall or extracellular matrix (Lertpiriyapong and Sung, 2003). KOBITO 1 was hypothesised to
coordinate cellulose synthesis during cell elongation, but the mechanism by which it works has yet to
be described (Pagant et al., 2002).
CYT1 is an enzyme that catalyzes the production of GDP-mannose required for Nglycosylation and GPI membrane-anchoring. Mutations in the CYT1 gene results in disruption of
cytokinesis and failure to synthesise a normal cell wall (Nickle and Meinke, 1998).
The cyt1
Arabidopsis mutant plants are five-fold decreased in cellulose content and this is hypothesised to be
due to a lack of glycosylation of the CESA proteins, since the mutant phenotype could be mimicked in
wild-type plants by tunicamycin, an N-glycosylation inhibitor, treatment (Lukowitz et al., 2001).
Considering that CYT 1 is required for GPI membrane-anchoring and that CYT1 could be responsible
for the anchoring of COBRA, perhaps some of the reduction in cellulose observed in cyt1 mutants is
due to a lack of interaction of cellulose microfibrils and microtubules.
THESEUS 1 is a protein described by Hematy et al. (2007). This protein is not directly
involved in cellulose biosynthesis, but it appears to play a crucial role when cellulose deposition is
altered or inhibited. It is a receptor-like kinase that is only expressed when cellulose synthesis is
inhibited. When the THESEUS 1 gene was mutated in a wild-type Arabidopsis background, no
phenotypic changes were observed. When THESEUS 1 was mutated in plants already mutated for the
AtCesA6 gene, the phenotype of the mutant seemed to be at least partially restored to the wild-type
phenotype (Hematy et al. 2007). Over-expression of THESEUS 1 in the Atcesa6 mutant background,
resulted in a more severe mutant phenotype. These mutants showed severe ectopic lignification and
21
LITERATURE REVIEW
dwarfed phenotypes. The effect of THESEUS 1 was not the same for all cellulose related mutations.
Only plants with mutated CesA genes could be partially rescued by mutating THESEUS 1 (Hematy et
al., 2007). This raises more interesting questions: Is THESEUS 1 involved in detecting faulty rosette
structures? It has been observed that lignin increases when cellulose decreases (Desprez et al., 2002).
Is THESEUS 1 responsible for signalling or facilitating an increase in lignin production to compensate
for decreased cellulose? Could THESEUS 1 expression be used to evaluate rosette integrity? One
way to answer some of these questions would be to study THESEUS 1 expression levels in a CesA
mutant that has a mutant CESA protein that still incorporates into the rosette, but does not function
correctly. One such mutant is the original irx1 mutant with a point mutation that only affects the
catalytic region of the protein (Turner and Somerville, 1997). This mutant has severely reduced
cellulose biosynthesis, but the protein is believed to still be incorporated into the cellulose synthase
complex.
To summarise, three distinctive CESA proteins assemble to form a rosette in the Golgi. The
rosettes are transported to the plasma membrane by means of exocytosis. At the plasma membrane the
rosettes interact with the microtubules, possibly through the activity of COBRA and AGPs. CYT1
could function in the GPI-anchoring of COBRA or the N-glucosylation of the CESA proteins before
the rosette is embedded in the membrane. SUSY converts sucrose to UDP-glucose that is then utilised
by the CESA proteins and added to the growing cellulose molecules. KORRIGAN could be involved
in subsequent editing of the glucose chains while THESEUS 1 may detect faulty rosette complexes or
be involved in signalling that induces lignifications when cellulose deficiencies are detected. The
function of KOBITO 1 remains elusive, but as in the case of the CESA proteins, KORRIGAN,
COBRA and CYT1, it is necessary for normal deposition of cellulose in Arabidopsis.
22
LITERATURE REVIEW
1.5
Arabidopsis as model for xylogenesis and cellulose biosynthesis
Arabidopsis thaliana has proven to be a valuable model for plant biology research. The full genome
sequence is available (The Arabidopsis Genome Initiative 2000) (Kaul et al., 2000), over 27 000
annotated genes have been identified as protein coding (Rhee et al., 2003; Swarbreck et al., 2008),
complete transcriptome microarray chips are available for gene expression studies and mutant lines are
obtainable from several seed stock centres for reverse genetics purposes (Feng and Mundy, 2006).
These tools are easily accessible via the on-line Arabidopsis Information Resource, TAIR (TAIR;
http://www.arabidopsis.com).
Arabidopsis has been extensively used for functional analysis of genes discovered in other
plant species.
Once a specific gene of interest has been identified in one species, the on-line
Arabidopsis sequence resources can be used to identify an Arabidopsis orthologue of the gene. The
various seed databases can then be explored to find a plant line with a mutation of the Arabidopsis
gene. The mutant phenotype could reveal possible functions of the gene. Mutant complementation
can then be used to confirm that the two orthologous genes are functionally identical. For example,
the fab1 Arabidopsis mutant (containing a single missense mutation) was functionally complemented
by the constitutive expression of KAS2 cDNA from Brassica napus revealing the underlying cause of
the mutant phenotype and providing support for the proposed function of KAS2 (Carlsson et al., 2002).
This strategy has also been used to identify the function of a gene involved in nitrate uptake. The Nrt2
gene from Nicotiana plumbaginifolia, under the expression control of a double 35S cauliflower mosaic
virus (CMV) promoter, was used to complement the corresponding mutant in Arabidopsis (Filleur et
al., 2001). The same approach was employed to compare gymnosperm and angiosperm transcription
factors involved in ABA responsive gene regulation. The gymnosperm CnAB13 gene was used to
transform Arabidopsis abi3-6 mutant plants. Again, the CnABI3 gene was under the expression
control of the constitutive 35S CMV promoter. In this study, the gymnosperm gene could restore most
of the wild-type phenotypic characteristics in the mutant. The mutant complementation also revealed
functional distinctions between the gymnosperm and angiosperm orthologues (Zeng and Kermode,
23
LITERATURE REVIEW
2004). Observed differences in the functions of orthologues can be used to characterise the functions
of domains that differ between the orthologues.
1.5.1 Positional and silencing effects on transgene expression
Two aspects that need to be considered when studying the effects of a transgene in a model system
such as Arabidopsis are positional effects and silencing. Positional effects concern the physical place
where the transgene inserts into the genome of the transgenic plant (Francis and Spiker, 2005). It is
usually independent of sequence identity. Silencing, on the other hand, is sequence dependent and
occurs due to sequence homology between the transgene and endogenous genes or between multiple
copies of the transgene (Que et al., 1997).
The position within the genome of a transgenic plant where the transgene inserts could affect
the expression of the transgene in at least two ways. Firstly, the transgene could insert into a region of
heterochromatin. Because of the conformation of chromatin in these regions, the transgene is likely to
be inaccessible to transcription factors and other enzymes necessary for transcription. However, the
reporter gene within the transgenic construct will also fall within heterochromatin. The reporter gene
would not be expressed and could not confer the selectable marker trait for selection of positive
transformants. A transgenic plant line that has the transgene inserted into a region of heterochromatin
would, therefore, not be identified as transgenic (Francis and Spiker, 2005). The second positional
effect concerns positioning of the transgene in cis near a strong enhancer or suppressor thus affecting
the level of expression (So et al., 2005).
Silencing of transgene expression is triggered by interaction between homologous sequences
that are either in cis or trans orientation to the transgene. The interaction could be between the
transgene and endogenous gene sequences, between multiple copies of the transgene or between
transcripts of the same copy of the transgene (Que et al., 1997).
Silencing can occur post-
transcriptionally in the cytoplasm, known as post-transcriptional gene silencing (PTGS), when short
24
LITERATURE REVIEW
double stranded RNA incorporates into the RNA-induced silencing complex (RISC) and lead to the
degradation of homologous transgene mRNA (Baulcombe, 1996; Hammond et al., 2000).
Transcriptional gene silencing (TGS) can also affect the expression levels of transgenes.
TGS
involves a similar process to that of PTGS. Short double stranded RNA can be incorporated into the
RNA-induced transcriptional gene-silencing complex (RITS). RITS is hypothesised to attract histones
and other DNA modifying enzymes to the site of silencing, which leads to a more condensed form of
DNA (Schramke and Allshire, 2003). This form of silencing inhibits mRNA accumulation and
transcription of the transgene.
The effects of silencing and the position of the transgene within the transgenic plant genome
support the use of more than one transgenic plant line during analysis of transgene expression. By
examining multiple transgenic lines, it becomes possible to discern between effects attributed to
position and silencing and effects observed due to the expression of the transgene.
1.5.2 Secondary growth in Arabidopsis
Though there is no denying that Arabidopsis is a useful tool for studying basic plant cell biology,
biochemistry and physiology, the question remains whether Arabidopsis, a weed, can function as a
model for xylogenesis in woody plants? A comparative genomics study found that 90% of expressed
sequence tags obtained from loblolly pine wood forming tissues, have homologues in the Arabidopsis
genome (Kirst et al., 2003). This suggests that the processes underlying xylogenesis are conserved in
herbaceous and woody plants (Nieminen et al., 2004). Lev-Yadun (1994) first demonstrated that
Arabidopsis can be induced to form increased amounts of secondary xylem by enhancing cambial
activity. Secondary growth of the Arabidopsis hypocotyl resembles that of woody angiosperms
(Chaffey et al., 2002). Induction of secondary growth was accomplished by continuously removing
the primary inflorescence (Lev-Yadun, 1997).
Other methods to induce secondary growth in
Arabidopsis include growing the plants under short-day conditions or under hypergravity stimulus
(Lev-Yadun and Flaishman, 2001). It takes Arabidopsis three to four months to develop secondary
25
LITERATURE REVIEW
growth tissue under short-day conditions (8 hours light, 16 hours dark). The resulting cell types are
similar to that of woody angiosperms, but lack the characteristic radial files of parenchyma cells (rays)
found in angiosperm wood (Chaffey et al., 2002). One obvious disadvantage of this induction of
secondary growth is that it takes a long time to develop.
Ko et al. (2004) proposed a solution to avoid the wound responses from continuous
inflorescence removal, but still induce secondary growth in less time than under short-day conditions.
This was accomplished by increasing the weight of the stem. It was found that the weight of the plant
triggers secondary growth. Hypergravity has the same effect as increased weight and has also been
used to induce secondary growth. When Arabidopsis plants were centrifuged at 300 x g, tubulin genes
were activated in the hypocotyls. The activation of these genes led to increased amounts of cortical
microtubules (Yoshioka et al., 2003). As mentioned in section 1.2.1, these microtubules are involved
in cellulose deposition in plant cell walls. Arabidopsis callus cultures were also analysed under 10 x g
hypergravity. The result was that starch was no longer synthesised, instead it was degraded. At the
same time cell wall modification pathways were switched on (Martzivanou and Hampp, 2003).
Mechanoreceptors seem to be important for hypergravity perception.
The inhibition of
mechanosensitive ion channels by gadolinium ions resulted in the effects of hypergravity being
suppressed (Nakabayashi et al., 2006).
Arabidopsis can, therefore, be used to study certain aspects of wood development, but its
shortcomings include that it cannot be used to investigate perennial growth and seasonal effects. For
this scientists will have to rely on studies from the model tree, Populus. Resources like the annotated
genome sequence (Tuskan et al., 2006) and a whole genome oligonucleotide microarray are available
for poplar and can be accessed via an online resource (http://www.jgi.doe.gov/poplar).
26
LITERATURE REVIEW
1.6
Conclusion
Three CESA proteins are believed to incorporate into cellulose synthase rosette complexes. This
observation is conserved across plant species including Arabidopsis, Populus and Eucalyptus. One
hypothesis that could be proposed in light of this conserved observation is that CesA genes that are
homologous based on sequence similarity are also functional homologues. This implies that a CesA
gene from one species can functionally complement the orthologue in another. The availability of
Arabidopsis irx mutants for all three secondary cell wall associated CesA genes, creates an
opportunity to test this hypothesis.
Complementation of an irx mutant (E.g. irx1) with the
corresponding Eucalyptus CesA gene (EgCesA1) and failure to complement with non-homologous
Eucalyptus secondary cell wall CesA genes (EgCesA2 and EgCesA3) would provide strong support for
the hypothesis that CesA gene orthologues are functionally conserved across species and co-assemble
into heterospecific rosette complexes.
Considering that mutations in genes involved in hemicellulose biosynthesis (E.g. IRX8, IRX9,
FRA8 and others) and other cell wall molecules (AGPs) result in a change in cellulose content, it is
evident that some form of signalling is involved in the regulation of polymer synthesis in cell walls.
The trigger for such signalling and which molecules serve as detectors for deviations in normal cell
wall biogenesis remain to be identified. A hypothesis can be formulated stating that the constitutive
expression of a secondary cell wall associated Eucalyptus CesA gene will affect the expression levels
of endogenous cell wall associated genes in wild-type Arabidopsis plants. The presence of high levels
of transcripts of one CesA gene could result in increased expression of endogenous CesA genes.
Alternatively, the abnormal levels of a CesA gene transcript could lead to reduced transcription of
endogenous CesA genes and a reduction in cellulose biosynthesis. If the expression of a transgenic
CesA gene increases or decreases endogenous CesA gene expression, it could also affect the
expression levels of other genes with cell wall-related functions.
By addressing the above mentioned hypotheses, insight could be obtained on the conservation
of CesA genes across species. This is an important step in establishing Arabidopsis as a model
27
LITERATURE REVIEW
organism in which to study xylogenesis, a complex process that is still poorly understood. By using
Arabidopsis, elucidation could be obtained on issues regarding the synthesis and assembly of cell wall
polymers and the functions of proteins like KOBITO 1 and THESEUS 1 that play important, but not
clearly identified, roles in cell wall biogenesis. It could provide clarity on the exact means by which
cell wall polymers interact and how the interactions influence the deposition of cellulose. Clarifying
these issues is necessary to find a means to increase and optimise cellulose biosynthesis without
disrupting normal plant development. Coleman et al. (2007) have shown that increased carbon
allocated to cellulose biosynthesis can increase cellulose content, but it resulted in altered plant
growth. This could indicate that multiple pathways involved in cell wall biogenesis need to be altered
in order to effectively increase cellulose without impacting general plant growth. Arabidopsis could
prove to be an ideal candidate to study the alteration of cell wall biogenesis.
The aim of this M.Sc study is to functionally characterize a Eucalyptus CesA gene in
Arabidopsis by constitutively expressing EgCesA1 in wild-type Arabidopsis plants, analysing putative
changes in cell wall morphology and comparing the gene expression levels of endogenous Arabidopsis
genes involved in cell wall biogenesis between wild-type and transgenic plants. This is part of a wider
effort to evaluate strategies to alter cellulose biosynthesis in fast growing plantation trees such as
Eucalyptus.
1.7
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43
CHAPTER 2
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS
EgCesA1 IN ARABIDOPSIS THALIANA
Marja M. O’Neill1, Shawn D. Mansfield2, Sanushka Naidoo1 and Alexander A. Myburg1
1
Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI),
University of Pretoria, Pretoria, 0002, South Africa
2
Department of Wood Science, University of British Columbia, Vancouver, BC,
V6T 1Z4, Canada
This chapter has been prepared in the format of a manuscript for a peer-reviewed research journal, as
is the standard procedure of our research group. Cell wall chemistry was determined at the University
of British Columbia, Vancouver, Canada by Prof. S.D. Mansfield. I performed all other laboratory
work and all analyses. Prof. A.A. Myburg provided funding, facilities and infrastructure at the
University of Pretoria, Pretoria, South Africa. I wrote the manuscript with extensive suggestions on
organization and content from Dr. S. Naidoo and Prof. A.A. Myburg.
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
2.1
Abstract
Cellulose biosynthesis occurs through the activity of membrane-embedded cellulose synthase (CESA)
protein complexes in plants. Cellulose synthase (CesA) genes encode the proteins that interact to
form these rosette-shaped complexes. CesA genes have been discovered in many plant species,
including Arabidopsis thaliana, Populus trichocarpa and Eucalyptus grandis.
However, the
organization and means of interactions of CESA proteins in rosette complexes are still unknown.
Better understanding of the assembly of CESA protein complexes is required to enable manipulation
of cellulose biosynthesis in commercially important crops like Eucalyptus tree species. Difficulty
with functional genetic analysis of genes in trees has lead to the use of Arabidopsis as a model to
study gene expression in fibre cells. The aim of this study was to functionally characterise the
Eucalyptus CesA gene, EgCesA1, in wild-type Arabidopsis plants.
Constitutive expression of
EgCesA1 was achieved through a Gateway®-enabled vector transformed into wild-type Arabidopsis
Col-0 plants using the floral dip method. Phenotypic comparisons were made between plant cell
walls of transgenic and wild-type plants. Chemical composition of the plant cell walls were also
compared between wild-type and transgenic plants. Quantitative comparisons between expression
levels of endogenous genes involved in cellulose biosynthesis in wild-type and transgenic lines were
performed, based on quantitative real-time PCR analysis.
EgCesA1 expression did not have a
significant effect on cell wall morphology, despite some changes observed in the chemistry of cell
walls.
No consistent parallel could be drawn between EgCesA1 expression levels and relative
expression levels of endogenous Arabidopsis genes. This research suggests that it may not be
sufficient or possible to alter cellulose biosynthesis in Arabidopsis by heterologous expression of a
single CesA gene of Eucalyptus.
2.2
Introduction
Cellulose is the most important polymer in the paper and pulp industry. Manipulation of cellulose
biosynthesis in commercially important trees like Eucalyptus spp., holds lucrative rewards for the
45
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
industry.
This, however, requires a more comprehensive understanding of the biosynthesis of
cellulose and the genes that underlie it. Most of the cellulose in wood occurs in fibre secondary cell
walls together with lignin and hemicellulose. In plants it is biosynthesised by plasma membraneembedded rosette-shaped cellulose synthase (CESA) protein complexes proposed to consist of six
subunits, each in turn, comprised of six CESA proteins (Mueller and Brown, 1980). Phenotypic
analysis of Arabidopsis plants with mutated CesA genes (Turner and Somerville, 1997; Arioli et al.,
1998; Dhugga, 2001; Burn et al., 2002; Desprez et al., 2002; Taylor et al., 2003) revealed that three
different CESA proteins assemble to form rosette complexes involved in primary cell wall deposition
and three other CESA proteins are required for secondary cell wall formation. It was recently
suggested that the secondary cell wall associated CESA proteins form homodimers before rosette
complexes assemble (Atanassov et al., 2009), but a subsequent study revealed that only one of the
CESA proteins form homodimers in vitro (Timmers et al., 2009). The organization of CESA proteins
within rosettes therefore remains unknown. The rosette complexes utilize UDP-glucose monomers
from the cytoplasm to polymerize growing β-1,4-glucan chains that coalesce and crystallize to form
cellulose microfibrils (Perrin, 2001).
Other factors that influence cellulose biosynthesis include the availability of glucose
monomers in the cytoplasm and interaction of rosette complexes or cellulose microfibrils with cortical
microtubules that orientate cellulose deposition (Quader, 1986). Sucrose synthase (SUSY) is an
enzyme that allocates carbon resources within the cell towards cellulose production by converting
sucrose to UDP-glucose and fructose (Winter et al., 1997; Haigler et al., 2001). Manipulation of
SuSy gene expression has been shown to influence plant growth (Coleman et al., 2006) possibly
through increased availability of glucose for cell wall polymer synthesis.
Sucrose-phosphate
phosphatase (SPP) and Sucrose-phosphate synthase (SPS) are enzymes involved in recycling glucose
from fructose and may also influence the availability of glucose monomers for cell wall polymer
biosynthesis (Delmer and Haigler, 2002). Cortical microtubules are believed to guide the orientation
of cellulose deposition (Green, 1962; Quader, 1986; Oda et al., 2005; Paredez et al., 2008). It has
been proposed that cell wall anchored arabinogalactan proteins (AGPs) are involved in the interaction
46
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
between microtubules and cellulose microfibrils (Driouich and Baskin, 2008) through the activity of
another protein, COBRA (Wakabayashi et al., 2005; Driouich and Baskin, 2008). The molecular
action of COBRA has yet to be characterized.
Many other proteins have been shown to influence cellulose biosynthesis based on the
phenotypes of mutant plants. KORRIGAN is a β-1,4 glucanase that is possibly involved in editing of
the glucan chains before crystallization of the cellulose microfibril (Szyjanowicz et al., 2004;
Bhandari et al., 2006). KOBITO 1 is a plasma membrane protein involved in cellulose deposition
during cell elongation, with an unknown functional mechanism (Pagant et al., 2002) and
CYTOKINESIS DEFECTIVE 1 (CYT1) is believed to be required for N-glycosylation of CESA
proteins before rosette complexes assemble in the Golgi-apparatus (Nickle and Meinke, 1998;
Lukowitz et al., 2001).
There remains much to be learned about cellulose biosynthesis, including the means of
interaction of CESA proteins within rosettes, involvement of non-cellulose synthase proteins in
cellulose deposition, factors that influence carbon availability for cellulose biosynthesis and the effect
of increased cellulose biosynthesis on other cell wall polymers like lignin and hemicellulose.
Clarifying these issues in commercially important tree species can prove to be problematic due to the
long generation times of trees and difficulty to transform trees. Although Arabidopsis is a herbaceous
plant, it has been shown to undergo secondary growth under certain conditions (Lev-Yadun, 1997).
The availability of Arabidopsis plants with mutated CesA genes, access to the full genome sequence
(The Arabidopsis Genome Initiative 2000) and ease of transformation makes Arabidopsis a good
model system in which to study the biosynthesis of cellulose. Transgenic studies in Arabidopsis is
further simplified by the availability of Gateway®-compatible vectors for transgene expression
analysis in plants (Curtis and Grossniklaus, 2003). Gateway® technology (Invitrogen, Carisbad,
California, USA) eliminates the time-consuming processes of restriction enzyme digestion and
ligation during vector construction (Walhout et al., 2000; Hartley, 2003).
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
The aim of this study was to functionally characterise a Eucalyptus CesA gene, EgCesA1, in
wild-type Arabidopsis plants. EgCESA1 is one of the three CESA proteins that synthesise cellulose
in secondary cell walls of xylem tissues in Eucalyptus trees (Ranik and Myburg, 2006). To determine
if expression of EgCesA1 in Arabidopsis influences cellulose biosynthesis, secondary cell wall
morphology is analysed and inflorescence cell wall chemical composition is determined. Quantitative
real-time PCR is performed to assess putative effects of EgCesA1 expression on relative expression
levels of endogenous genes involved in cell wall related biosynthetic pathways.
2.3
Materials and Methods
2.3.1 Biological material and growth conditions
Arabidopsis thaliana L. Heynh. ecotype Columbia (Col-0, Nottingham Arabidopsis Stock Centre)
seeds were surface sterilized in 70% ethanol for five minutes followed by incubation in 10% (v/v)
bleach solution containing 0.1% (v/v) TritonX for 15 minutes. The seeds were washed with sterile
distilled water before being re-suspended in 0.1% (w/v) agar and spread out on a 90 mm Petri dish
containing 0.8% (w/v) agar and 1 mM KNO3. The seeds were incubated at 4°C for two days to break
dormancy before the seeds were germinated at 22°C under a 16/8 hour day/night cycle of 16 hour
light (1000 lum/sqf) and 8 hour dark (2 lum/sqf) with relative humidity of 25 % in the light and 45 %
in the dark period for one to two weeks. Thereafter, the seedlings were transferred to soil medium
(Jiffy® Products International AS, Stange, Norway) and grown at 22°C and a 16 hour photoperiod to
induce flowering.
Plants were grown in a controlled environment under the same conditions
mentioned above for six weeks for stem harvesting.
Two Escherichia coli cell lines were used during vector construction. DB3.1 was used to
propagate the empty destination vector, pMDC32 (Curtis and Grossniklaus, 2003), while recombinant
entry and destination vectors were transferred into One Shot® chemically competent E.coli TOP10
cells (Invitrogen, Carisbad, California, USA). E.coli cell lines were cultured at 37°C for 16 hours in
48
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
yeast extract peptone (YEP) medium consisting of 1% yeast extract, 1% bacto-peptone and 0.5%
(w/v) sodium chloride (Sigma, St. Louis, Missouri, USA). For propagation of pMDC32 in DB3.1
cells, 20 μg/ml chloramphenicol and 50 μg/ml kanamycin were added to the YEP medium.
Propagation of the recombinant entry vector in TOP10 cells (Invitrogen) required the addition of 100
μg/ml spectinomycin to the YEP medium. 50 μg/ml kanamycin was added to the medium for
propagation of the recombinant destination vector. Agrobacterium tumefaciens strain LBA4404 was
cultured at 28°C in YEP medium, containing 50 μg/ml rifampicin and 30 μg/ml streptomycin for two
days in preparation for transformation with the recombinant destination vector. For Arabidopsis
transformation, clones containing the recombinant destination vector were propagated in the same
medium with 50 μg/ml kanamycin added.
2.3.2 Vector construction
Cloning of the EgCesA1 coding sequence into the entry vector
Gateway® enabled vectors (Curtis and Grossniklaus, 2003) were used to construct the binary vector
containing the EgCesA1 gene. RNA was extracted and used to synthesise cDNA from Eucalyptus
grandis xylem (Ranik and Myburg, 2006). The full-length EgCesA1 coding sequence (CDS), (Ranik
and Myburg, 2006) was amplified from the xylem cDNA using EgCesA_gb_5'UTR+28 and
EgCesA_3'UTR-120 as forward and reverse PCR primers, respectively (Table 2.1). The primers
flank the CDS of EgCesA1 (Figure S2.1). PCR conditions were: one cycle of denaturation for one
minute at 94°C, 25 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 55°C
and elongation for three minutes at 72°C and a final cycle of elongation for 15 minutes at 72°C. All
PCR and RT-PCR were carried out in a BioRad iCycler thermocycler (Bio-Rad, Hercules, California,
USA). The EgCesA1 CDS PCR product was purified using the PCR Purification Kit (Qiagen, Hilden,
Germany) and cloned into the pCR®8/GW/TOPO® TA Cloning® (Invitrogen) entry vector according
to the manufacturer’s instructions.
The resulting pTOPO-EgCesA1 vector (Figure S2.1) was
49
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
transformed into One Shot® chemically competent E.coli TOP10 cells (Invitrogen), according to
manufacturer’s guidelines.
Positive colonies were picked for colony PCR to confirm that the EgCesA1 CDS was cloned
and to determine the orientation of the cloned fragment. A vector-specific M13-29-F primer (Figure
S2.1) in combination with the gene-specific EgCesA_3'UTR-120 primer was used to determine the
fragment’s orientation. PCR conditions were: denaturation at 94°C for two minutes followed by 30
cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and elongation at
72°C for three minutes. A final elongation step at 72°C for five minutes was also included. Both
fragments of the full-length EgCesA1 CDS fragments were then sequenced from four clones to
determine if any base pair changes occurred during PCR amplification and cloning. Primers used for
the sequencing reaction are listed in Table 2.1. Recombinant pTOPO-EgCesA1 plasmid DNA was
extracted from One Shot® cells using the GeneJET™ Plasmid Miniprep Kit (Fermentas, Ontario,
Canada) as described by the manufacturer. The purified plasmid DNA was used as template for DNA
sequencing (Macrogen, Rockville, Maryland, USA). All DNA sequences were analysed using Vector
NTI Advance™ 9.1 software (Invitrogen).
Table 2.1 Oligonucleotide primers used for vector construction, DNA sequencing and RNA analysis
Primer name
Primer sequence (5’→3’)
EgCesA_gb_5’UTR+281,2
CTCATTGGGTCGCGAGAAGAT
1,2
EgCesA_3'UTR-120
CAACACTGGCGCAATTCAAC
1,2
M13-29-F
CACGACGTTGTAAAACGAC
M13-narrow-R1,2
GGAAACAGCTATGACCATG
1
M13-40-F
GTTTTCCCAGTCACGAC
M13-wide-R1
GGATAACAATTTCACACAGG
1,2
EgCesA1_seq_F
GGTGCTGCGATGCTCTCATT
EgCesA1_seq_R1,2
GTGTGGCTGCTGCTCTTGTT
UBQ10-F
ATTCTCAAAATCTTAAAAACTT
UBQ10-R
TGATAGTTTTCCCAGTCAAC
DNA-QC-F
GTCAATCTTGGCCATGGACT
DNA-QC-R
GGTGGTCCTTGGTGTTGTTC
1. Primer positions are given in Figure S2.1 and Figure S2.2.
2. Primers used for sequencing.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Transferring EgCesA1 to the destination vector
The EgCesA1 CDS was transferred into the pMDC32 (Curtis and Grossniklaus, 2003) destination
vector (Figure S2.2) using the Gateway® LR Clonase™ II Enzyme Mix (Invitrogen). The LR cloning
reaction is illustrated in Figure 2.1. The LR Clonase™ II reaction was transformed into chemically
competent TOP10 One Shot® cells (Invitrogen). Orientation of the EgCesA1 CDS in pMDC32 was
determined by colony PCR (PCR cycle and conditions as described previously) using a vectorspecific M13-40-F primer and gene-specific EgCesA_3'UTR-120 primer. The entire cloned coding
sequence was analysed in four clones as described above to confirm that no base pair changes
occurred during cloning (results not shown). The pMDC32-EgCesA1 recombinant plasmid DNA was
extracted from E.coli as described above and transformed into competent Agrobacterium tumefaciens
cells.
Figure 2.1 Schematic representation of the LR cloning reaction. The diagram illustrates the expression
cassette of pMDC32 before and after LR recombination and the region between the attL and attP sites of the
pTOPO-EgCesA1 vector (Figure S2.1). Positive recombinants were selected based on resistance to kanamycin,
since cells transformed with non-recombinant pMDC32 plasmid DNA would die due to the expression of the
ccdB gene and cells that do not contain recombinant or non-recombinant pMDC32 plasmid DNA would not be
resistant to kanamycin (Figure S2.2).
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
2.3.3 Transformation of competent Agrobacterium tumefaciens cells
Competent Agrobacterium tumefaciens LBA4404 cells were prepared by first inoculating 5 ml YEP
medium containing 50 μg/ml rifampicin and 30 μg/ml streptomycin with LBA4404 cells. This was
incubated until the mid-log phase at 28°C for two days. A larger volume (50 ml) YEP containing the
same antibiotics was inoculated with 2 ml of the 5 ml culture and incubated at 28°C for two to three
days with shaking at 200 rpm. The culture was then centrifuged for 15 minutes at 3000 x g and the
pellet resuspended in 1 ml ice-cold CaCl2. The solution was aliquoted in 100 μl volumes and rapidly
frozen in liquid nitrogen.
To transform Agrobacterium tumefaciens with the recombinant pMDC32-EgCesA1 vector, 3
μg of vector DNA was pipetted onto 100 μl of frozen competent LBA4404 cells. The mixture was
exposed to a heat shock at 37°C for five minutes and transferred back to ice before 900 μl of YEP
(containing 50 μg/ml rifampicin and 30 μg/ml streptomycin) was added. The culture was incubated at
28°C with shaking at 200 rpm for four hours. Thereafter the culture was centrifuged at 12 000 x g for
one minute and the resulting pellet was resuspended in 50 μl YEP medium. The resuspended cells
were streaked out on 0.8% (w/v) agar-YEP plates containing 50 μg/ml rifampicin, 30 μg/ml
streptomycin and 50 μg/ml kanamycin. The plates were incubated at 28°C for three days. Colony
PCR (cycles and conditions described in section 2.3.2) with gene and vector-specific primers,
EgCesA_3'UTR-120 and M13-40-F, was used to confirm that positive Agrobacterium colonies were
transformed with pMDC32-EgCesA1.
2.3.4 Transformation of Arabidopsis thaliana plants
To prepare the transformation culture, one colony of Agrobacterium containing pMDC32-EgCesA1
was used to inoculate 5 ml of YEP medium containing rifampicin (50 μg/ml), streptomycin (30
μg/ml) and kanamycin (50 μg/ml). The medium was incubated at 28°C for two days until mid-log
phase was reached. Two millilitres of the culture was used to inoculate a larger volume of 200 ml
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
YEP containing the same antibiotics and the larger volume was incubated for two days before it was
centrifuged at 2 300 g for five minutes. The resulting pellet was resuspended in 5 ml 5% (w/v)
sucrose solution. The resuspended culture was then adjusted to OD600 of 0.8 to 1.0 with 5% (w/v)
sucrose. The surfactant, Silwett L-77 (LETHEL, Round Rock, Texas, USA), was added to the
transformation culture to a concentration of 0.5% (v/v).
The floral dip method described by Clough and Bent (1998) was used to transform wild-type
Arabidopsis thaliana Col-0 plants. Primary inflorescence stems of two-week-old plants were cut at
the base to induce the development of secondary inflorescence stems. The plants were ready for
transformation when several immature flowers were visible and few fertilized siliques could be seen.
The Jiffy® peat soil pots in which the plants were growing were thoroughly hydrated one day before
transformation.
The transformation medium described above was prepared on the day of
transformation. As described in the floral dip method, the inflorescence stems of the plants were
immersed in the transformation medium for approximately 30 seconds with gentle agitation. The
excess medium was shaken off the stems, the plants were laid horizontally and the tray containing the
plants was covered overnight. The following day the plants were placed upright again. The floral dip
procedure was repeated after one week. The plants were grown until T1 seeds could be harvested.
2.3.5 Selective screening of transgenic Arabidopsis plants
T1 seed harvested and dried for one week at room temperature were selected on 0.8% (w/v) agar
plates containing 1 mM KNO3 and 20 µg/ml hygromycin B. The seed were surface sterilized and
dispersed on the agar plates as described in section 2.3.1. Hygromycin B resistant seedlings (all
viable seeds germinated, but only resistant plants rooted and grew taller than the non-resistant
germinated plants) were transferred to hydrated Jiffy® pots after five days. The seedlings were
allowed to self-pollinate and T2 seed were collected and selected in the same way as T1 seed.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
2.3.6 PCR analysis of transgenic plants
Genomic DNA was extracted from antibiotic resistant T1 plants using the NucleoSpin III Plant DNA
Extraction Kit (Machery-Nagel, Düren, Germany) following the manufacturer’s instructions. PCR
was performed using the EgCesA_gb_5’UTR+28 (1 μM) and EgCesA_3'UTR-120 (1 μM) primers, 1
x PCR buffer, 0.20 mM dNTPs and 0.2 U Excel Taq DNA polymerase. The PCR had an initial
denaturation step of 94°C for one minute followed by 35 cycles of denaturation for 20 seconds at
94°C, annealing for 20 seconds at 55°C and elongation for three minutes at 72°C. A final elongation
step of 10 minutes at 72°C was performed.
2.3.7 Reverse transcription PCR analysis
Four six-week-old plants of wild-type Arabidopsis Col-0 and four of each of the three transgenic T3
lines were used to prepare samples for RT-PCR (Figure 2.2 B). Total RNA was isolated from upper
stems, lower stems and leaves (Figure 2.2 A) using the Concert™ Plant RNA Reagent (Invitrogen)
following the manufacturer’s guidelines. All isolations were done in duplicate to generate two
biological replicates (Table 2.2). Tissues were collected and immediately ground to a powder in
liquid nitrogen using a cold pestle and mortar before cold Plant RNA Reagent was added. The total
RNA samples were treated with RNase-free DNaseI (Roche, Basel, Switzerland) to remove genomic
DNA. One to two units of DNaseI, 20 U of RNasin RNase inhibitor (Promega, Madison, Wisconsin,
USA), 5 μl reaction buffer (Promega) and 5 μg of RNA sample were mixed and incubated at 37°C for
30 minutes. The DNaseI digested samples were further purified using the RNeasy Plant Mini Kit
(Qiagen) according to the instructions of the manufacturer. First-strand cDNA was synthesised from
each total RNA sample using Improm-II™ Reverse Transcriptase (Invitrogen) according to the
manufacturer’s instructions. The samples were tested for genomic DNA contamination using the
primer pair, UBQ10-F and UBQ10-R (Table 2.1), that flanks the first intron of the UBIQUITIN 10
(UBQ10) gene. PCR amplification of an 850 bp fragment indicated the presence of genomic DNA
54
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
and amplification of a 545 bp fragment indicated the presence of cDNA.
The PCR reaction
conditions included an initial denaturation step at 94°C for one minute followed by 30 cycles of
denaturation for 30 seconds at 94°C, primer annealing for 30 seconds at 55°C and elongation for one
minute at 72°C. A final elongation step of five minutes at 72°C was also performed. If any genomic
DNA was detected, digestion with DNaseI was repeated followed by first-strand cDNA synthesis.
The cDNA was diluted 1:10 before RT-PCR and qRT-PCR was carried out.
RT-PCR was carried out in a BioRad iCycler thermocycler (Bio-Rad) using
EgCesA_gb_5'UTR+28 and EgCesA_3'UTR-120 primers (Table 2.1) to amplify the full-length
EgCesA1 gene transcript from cDNA. The PCR conditions included a two-minute denaturation step
at 94°C followed by 30 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at
55°C and elongation for three minutes at 72°C. This was followed by a final elongation step of five
minutes at 72°C.
Figure 2.2 Illustration of tissues sampled for RT-PCR and qRT-PCR. A. The upper stem tissues were taken
from above the lowest leaf on the inflorescence stem to just below the first flower. Lower stem tissues were
from below the first leaf on the inflorescence stem to just above the rosette. Leaves were taken from the
rosettes. A diagram illustrating the biological repeats of the transgenic and wild-type lines are given in B.
MO1, MO2 and MO3 are T3 transgenic lines. Tissues (leaves, upper stems and lower stems, respectively) from
four plants were combined to form one biological repeat. Two biological repeats were prepared per line.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Table 2.2 RNA samples obtained from transgenic and wild-type plants
Sample number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Biological replicate name
MO1 A
MO1 A
MO1 A
MO1 B
MO1 B
MO1 B
MO2 A
MO2 A
MO2 A
MO2 B
MO2 B
MO2 B
MO3 A
MO3 A
MO3 A
MO3 B
MO3 B
MO3 B
WT A
WT A
WT A
WT B
WT B
WT B
Tissue
Upper stem
Lower stem
Leaves
Upper stem
Lower stem
Leaves
Upper stem
Lower stem
Leaves
Upper stem
Lower stem
Leaves
Upper stem
Lower stem
Leaves
Upper stem
Lower stem
Leaves
Upper stem
Lower stem
Leaves
Upper stem
Lower stem
Leaves
2.3.8 Microscope analysis of cell wall phenotype
The cell wall phenotype of transgenic and wild-type Arabidopsis Col-0 plant stems were compared
using a light microscope (Axon, Sunnyvale, California, USA). The inflorescence stems of the plants
were removed repeatedly to induce the development of more stems until the plants were five weeks
old. New inflorescence stems were then collected and sectioned transversely to 11 μm thickness
using a Leica micro-cryotome (model CM1100, Leica, Nussloch, Germany). The sections were then
stained to visualise lignified cell walls using phloroglucinol-HCl (Speer, 1987), or to visualise
cellulose and lignin using toluidine blue (Turner and Somerville, 1997). For phloroglucinol-HCl
staining, the sections were immersed in 1% phloroglucinol (Sigma) (w/v) in 92% ethanol solution
followed by immediate immersion in 25% hydrochloric acid. A cover slide was placed over the
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
section and the section was viewed directly after staining. For toluidine blue staining, the sections
were immersed in a drop of toluidine blue (Sigma) followed by a brief wash step with a drop of
distilled water before a cover slide was placed over it.
2.3.9 Cell wall chemistry analysis of Arabidopsis stems
A modified micro-Klason analysis (Huntley et al., 2003) was used to analyse the chemical
composition of the stems from transformed and wild-type Arabidopsis plants. Stems from six-weekold T3 plants were bulked and dried. Approximately 0.1 gram dry stems were ground to pass a 40mesh screen using a Wiley mill. The tissue was soxhlet-extracted with acetone for six hours and
digested with 72% H2SO4 for two hours. The extracted tissue was further hydrolysed in 4% H2SO4
for one hour at 121°C in an autoclave. The total weight of non-hydrolysed, extracted components was
gravimetrically determined (this represented the acid-insoluble lignin component), while the filtrate
was analysed for acid-soluble lignin by measuring absorbance at 205 nm according to TAPPI Useful
Method UM 250 (TAPPI, South Norcross, Georgia, USA). The composition of the carbohydrates in
the filtrates, expressed in the anhydro-form, was determined by high performance anion-exchange
chromatography on a CarboPac PA-1 column using a Dionex High Pressure Liquid Chromatography
(HPLC) system (Dionex, Sunnyvale, CA, USA) equipped with a pulsed amperometric detector.
Carbohydrates were expressed as a percentage of the dry weight of the sample. Volumes were scaled
to accommodate 20 mg of starting material. The analysis were performed twice for each bulked
sample to give an indication of technical variation.
2.3.10 Quantitative real-time PCR analysis
Sample preparation for quantitative real-time PCR was performed as described previously (Section
2.3.7). The optimal cDNA concentration was determined for each sample by performing a PCR using
a serial dilution of each cDNA sample. All samples were subsequently diluted 1:10.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
qRT-PCR was performed using the LightCycler® 480 Real-Time PCR System (Roche
Diagnostics, GmbH, Basel, Switzerland). One microlitre of 1:10 diluted cDNA was used in each 11μl PCR reaction with 5 μl of 2 x LightCycler® SYBR Green I PCR master mix (Roche) and 0.5 μM
each of the forward and reverse primers (Table S2.1 for qRT-PCR). Thermal cycling conditions
included an enzyme activation step at 95°C for five minutes and a quantification step with 45 cycles
of 95°C for ten seconds, 60°C for ten seconds and 72°C for 15 seconds with single quantification at
the end of each cycle. This was followed by a melting curve generating step with one cycle of 95°C
for five seconds, 65°C for one minute and 95°C continuously. All reactions were performed in
triplicate. Tubulin 6 (Table S2.2) amplification of cDNA bulked from all samples was used as an
inter-plate calibrator.
Primers used for qRT-PCR were designed using Primer Designer 4 v4.20 (Sci Ed Central,
Cary, North Carolina, USA) and synthesised by Inqaba Biotech (Pretoria, South Africa). Internal
standard curves were generated for each of the primers used. This was done by performing PCR
reactions using serial dilutions of cDNA bulked from the different samples for each of the primer
pairs. Crossing point data was exported and analysed using the qBASEplus v1.0 (Biogazelle, Ghent,
Belgium) software.
Seven putative housekeeping genes, Actin 2 (ACT2), Ubiquitin 5 (UBQ5), UBQ10, 18S rRNA
(18S), Elongation factor 1α (EF1), Tubulin 4 (TUB4) and Tubulin 6 (TUB6), were tested to determine
which were best suited as endogenous reference genes for normalisation of the relative expression
levels of EgCesA1 and other genes tested in this experiment. Standard curves were generated for each
primer pair (Table S2.2) by using a serial dilution of bulked cDNA of the different samples. The
crossing point data was exported to qBASEplus v1.0 software to generate standard curves. Transcript
levels of the housekeeping genes were determined in all of the samples and compared using
qBASEplus v1.0.
The relative transcript abundance levels of EgCesA1, the endogenous Arabidopsis CesA
genes and other cell wall related genes were determined using the above mentioned protocol.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Crossing point data was exported to qBASEplus v1.0 and normalised to the expression levels of
UBQ10 and EF1. The normalised data was exported to Microsoft® Office Excel 2007 for further
analysis. A type two (two-sample equal variance) double-tail Student’s t-test was performed to
determine whether the variation observed could be attributed to variation among the biological and
technical repeats of the same line or whether the variation was due to the expression of EgCesA1 in
the transgenic lines compared to the wild-type line. Conclusions were drawn at a significance level of
α = 0.05. The null hypothesis was that the expression of the tested gene was the same in the wild-type
and transgenic plants. Where the null hypothesis was not rejected, it was assumed that variance
observed was due to technical and/or biological variance within a sample or line.
2.4
Results
2.4.1 Vector construction
Cloning EgCesA1 into the entry vector
The EgCesA1 CDS was amplified from xylem cDNA and the PCR products were analysed using
agarose gel electrophoresis (Figure 2.3).
After purification, EgCesA1 was cloned into the
pCR®8/GW/TOPO® (Invitrogen) entry vector and TOP10 E.coli cells (Invitrogen) were transformed
with the recombinant vector. Seven colonies containing pTOPO-EgCesA1 recombinant vector were
analysed to determine the orientation of the EgCesA1 CDS in the vector by performing a colony PCR.
Agarose gel electrophoresis was performed to analyse the PCR products (Figure 2.4 A). A 3086 bp
sized PCR product was expected if the EgCesA1 gene was in the sense orientation and 105 bp product
if in the antisense orientation. All the selected colonies contained vectors with the EgCesA1 CDS in
sense orientation, except for samples 4 and 5 that contained vectors with EgCesA1 CDS in antisense
orientation (vector map not shown). Sequencing of the full-length EgCesA1 CDS in pTOPO-EgCesA1
vectors from four colonies revealed several base-pair changes caused by PCR amplification of the
EgCesA1 CDS before cloning. Not all changes resulted in altered amino acid sequences. One clone,
59
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
EgCesA1 D, had only one PCR-induced base-pair change that resulted in a change from a glutamate
to a lysine residue. This clone was used for subsequent procedures (Figure S2.3).
Figure 2.3 1% (w/v) EtBr/Agarose gel electrophoresis of EgCesA1 CDS PCR products amplified from cDNA
using EgCesA_gb_5'UTR+28 and EgCesA_3'UTR-120 primers (Figure S2.1). The expected fragment size was
2.98 kb. Lane M is a 1 kb DNA size standard (GeneRuler™ 1 kb DNA Ladder, Fermentas), lanes 1, 2 and 3
contain amplified EgCesA1 CDS PCR products.
Transferring EgCesA1 to the destination vector
The pTOPO-EgCesA1 plasmid (Figure S2.1) contained attL recombination sites on either side of the
EgCesA1 gene and a gene that confers resistance to spectinomycin, aadA1 (Liebert et al 1999). (A
schematic outline of the pMDC32 destination vector before and after recombination with the pTOPOEgCesA1 vector is provided in Figure 2.1.) The pMDC32 vector contained the ccdB gene and a
chloramphenicol resistance gene flanked by attR sites for recombination. The T-DNA region of the
vector also contained a gene that provides resistance to the herbicide hygromycin (HYGROMYCIN
PHOSPHOTRANSFERASE), a double 35S CMV promoter and a Nos terminator.
NEOMYCIN
PHOSPHOTRANSFERASE II, a gene that confers resistance to kanamycin in bacteria, was located
outside the T-DNA region.
The recombinant pMDC32-EgCesA1 vector was transformed into
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Agrobacterium LBA4404 cells for use in plant transformation and confirmed by colony PCR (Figure
2.4 B).
Figure 2.4 1% (w/v) EtBr/Agarose gel electrophoresis of colony PCR products amplified from pTOPOEgCesA1 and pMDC32-EgCesA1. Gene-specific EgCesA_3'UTR-120 and vector-specific M13-29-F primers
(Figure S2.1) were used to determine the orientation of the EgCesA1 CDS in the pTOPO-EgCesA1 vector
isolated from TOP10 E.coli cells (A). Amplification of the fragment in sense orientation yielded a 3086 bp PCR
product. PCR products 105 bp in size were obtained from amplification of fragments in antisense orientation.
Lane M is a 1 kb DNA size standard (GeneRuler™ 1 kb DNA Ladder, Fermentas) and lanes 1 to 7 contain
amplified colony PCR products. Colony PCR products amplified from pMDC32-EgCesA1 in Agrobacterium
LBA4404 cells using EgCesA_gb_5’UTR+28 and EgCesA_3'UTR-120 primers are shown in B. Lane M is a 1
kb DNA size standard (GeneRuler™ 1 kb DNA Ladder, Fermentas) and lanes 1 to 3 contain amplified colony
PCR products.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
2.4.2 Selective screening and PCR of transgenic plants
After hygromycin selection (Figure 2.5), resistant T1 plants were transferred to Jiffy® pots and
allowed to self-pollinate. Three T2 lines derived from three different T1 plants, MO1, MO2 and
MO3, each containing a single pMDC32-EgCesA1 T-DNA insert were identified using segregation
analysis. The ratio of hygromycin-resistant to non-resistant germinated T2 plants were 3:1, indicating
that the T1 plants were hemizygous for the hygromycin resistance gene and that the gene occurred as
a single copy in the genome. T3 seed from individual T2 plants were germinated separately and nonsegregating (all germinated seeds resistant to hygromycin) lines were advanced as T3 homozygous
lines.
Figure 2.5 Hygromycin selection of T1 seeds. Seeds obtained from plants that were dipped in Agrobacterium
dip medium were sown on agar-plates containing hygromycin B.
All of the seeds germinated, but only
transgenic T1 plants (indicated by arrows) survived after five days of incubation and were taller than the
untransformed germinated plants.
Genomic DNA was extracted from the leaves of the T1 plants and PCR was performed using
EgCesA1-specific primers to confirm that the transgenic plants contained the EgCesA1 gene. The
PCR products were analysed using EtBr/agarose gel electrophoresis (Figure 2.6). Correctly sized
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
PCR products were obtained for each of the T1 lines (lanes 4, 5 and 6) and no product was present in
the DNA sample from the wild-type Col-0 control plant (lane 3).
Figure 2.6 1% (w/v) EtBr/Agarose gel electrophoresis of the amplified EgCesA1 PCR products from leaf
genomic DNA. Lane M1 is a 1 kb DNA size standard (GeneRuler™ 1 kb DNA Ladder, Fermentas), lane M2
contains a 100 bp DNA size standard (GeneRuler™ 100 bp Ladder Plus, Fermentas), lane 1 contains PCR
product amplified from non-recombinant pMDC32 vector (negative control), lane 2 contains PCR products
amplified from pMDC32-EgCesA1 plasmid (positive control), lane 3 contains PCR products from wild-type
Arabidopsis genomic DNA (negative control) and lanes 4 to 6 contain PCR products amplified from genomic
DNA of the three transgenic lines (MO1, MO2 and MO3, respectively). EtBr/Agarose gel A contains PCR
products obtained using EgCesA_gb_5'UTR+28 forward and EgCesA_3'UTR-120 (Table 2.1) reverse primers
and the expected fragment length was 2981 bp. EtBr/Agarose gel B contains PCR products amplified using
primers that target a 452 bp segment of the endogenous AtCesA7 gene (DNA-QC-F and DNA-QC-R).
2.4.3 RT-PCR
Total RNA was extracted from T3 plants of the three transgenic lines (MO1, MO2 and MO3) and
wild-type plants and analysed using EtBr/agarose gel electrophoresis (Figure 2.7 A). Concentrations
of the isolated RNA ranged between 0.7 μg/μl and 1.2 μg/μl. The RNA samples were retreated with
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
RNase-free DNaseI (Roche). After treatment with DNaseI, first strand cDNA was synthesised from
total RNA. cDNA samples were tested for the presence of an intron in the UBQ10 gene to detect the
presence of residual genomic DNA. EtBr/agarose gel electrophoresis of the PCR products revealed
that three samples (sample 7, 8 and 10) still contained traces of genomic DNA (Figure 2.7 B). Total
RNA of these samples were again treated with DNaseI and first strand cDNA was synthesised, after
which no genomic DNA was detected (results not shown).
Figure 2.7 1% (w/v) EtBr/Agarose gel electrophoresis of extracted total RNA and PCR products amplified
from cDNA using UBQ10 primers flanking an intron. Lane M is a 100 bp DNA size standard (GeneRuler™
100 bp Ladder Plus, Fermentas). Total RNA was extracted from MO1, MO2, MO3 and wild-type Col-0 (A).
Lanes 1 to 24 are RNA samples corresponding to the cDNA samples in Table 2.4. Sample 19 was negative and
RNA was successfully re-extracted (results not shown). B: 1% (w/v) EtBr/Agarose gel electrophoresis of PCR
products amplified using UBQ10 primers flanking an intron. Samples seven, eight and ten had genomic DNA
contamination evidenced by the presence of an 850 bp band along with the expected 545 bp band amplified
from cDNA.
RT-PCR
using
EgCesA1-specific
primers
(EgCesA_gb_5'_UTR+28
forward
and
EgCesA_3'_UTR-120 reverse) was performed to establish whether EgCesA1 was transcribed in the
three transgenic Arabidopsis lines. The full-length EgCesA1 RT-PCR products were analysed using
EtBr/agarose gel electrophoresis (Figure 2.8). EgCesA1 transcripts were detected in the upper stem
tissue cDNA from all three transgenic lines, but not in wild-type Col-0 control plant cDNA.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.8 1% (w/v) EtBr/Agarose gel electrophoresis of RT-PCR products. Lane M1 is a 1 kb DNA size
standard (GeneRuler™ 1 kb DNA Ladder, Fermentas), lane 1 contains PCR product amplified from empty
pMDC32 vector (negative control), lane 2 contains PCR products amplified from pMDC32-EgCesA1 plasmid
(positive control), lane 3 have PCR products from wild-type Arabidopsis upper stem cDNA (negative control),
lanes 4 to 6 contain PCR products amplified from upper stem cDNA of the three transgenic lines (MO1, MO2
and MO3, respectively) and lane M2 contains a 100 bp DNA size standard (GeneRuler™ 100 bp DNA Ladder
Plus, Fermentas).
PCR was performed using EgCesA_gb_5'_UTR+28 forward and EgCesA_3'_UTR-120
reverse primers and the expected band size was 2.98 kb (A). EtBr/Agarose gel B contains PCR products
amplified using primers that target a 452 bp segment of the endogenous AtCesA7 gene (DNA-QC-F and DNAQC-R).
2.4.4 Microscopy of stem cell walls
Light microscopy analysis was done to observe any changes in cell wall morphology in the transgenic
lines compared to the wild-type plants. Two sections, lower inflorescence stem and hypocotyls, were
taken from each line and stained with phloroglucinol-HCl to observe the morphology of secondary
cell walls. Sections were also stained with toluidine blue to observe cellulose content of the cell
walls. The morphological features that were focused on were cell shape, cell wall thickness and
cellulose content in vascular bundles. No obvious differences were observed between the transgenic
and wild-type lines (Figure 2.9).
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.9 Images of inflorescence stem and hypocotyl cross sections of wild-type (WT) and transgenic plant
lines (MO1, MO2 and MO3) as observed using light microscopy. Sections were stained for lignin using
phloroglucinol-HCl to detect changes in cell wall shape and cell shape and cell wall thickness (A). Toluidine
blue staining was used to detect cellulose (B). Scale bars indicate 100 μm.
2.4.5 Cell wall chemistry of transgenic plant stems
Inflorescence cell wall chemistry was analysed to determine if the heterologous expression of the
EgCesA1 CDS had an influence on the chemical composition of Arabidopsis cell walls.
The
procedure was repeated once to determine technical variability. A Student’s t-test was used to test for
significant changes in cell wall chemistry between wild-type and transgenic plants (Figure 2.10). The
percentage total and insoluble lignin fractions were decreased by 2-18% and 15-31%, respectively, in
the transgenic lines when compared to that of the wild-type, with MO3 having the most significant
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
decrease. However, the percentage soluble lignin was increased in the transgenic lines. Wild-type
plants had 15-21% less soluble lignin than transgenic lines. Levels of glucose and xylose were also
decreased in the transgenic lines when compared to that of the wild-type (glucose levels were 24%
less in MO1, 14% less in MO2 and 8% less in MO3, while xylose levels were 28% less in MO1, 17%
less in MO2 and 8% less in MO3). Arabinose was observed at decreased levels in transgenic line
MO1 and MO2 (18% decreased), but not in MO3 as compared to the wild-type plant line.
2.4.6 Expression analysis of EgCesA1 and other cell wall related genes in
wild-type and transgenic Arabidopsis plants
Transcript abundance levels of several selected cell wall related Arabidopsis genes were analysed to
determine if they were influenced by the heterologous expression of EgCesA1. Quantitative real-time
PCR (qRT-PCR) analysis of gene transcripts in the leaves and upper- and lower inflorescence stems
of the three transgenic lines were compared to that in the wild-type Col-0 control plants. The gene
expression levels of EgCesA1 and Arabidopsis cell wall related genes were normalised to the relative
expression levels of the control genes, UBQ10 and EF1 (Figure 2.11). Raw crossing-point data for
potential normalization genes were analysed using qBASEplus v1.0.
Normalization factors were
calculated based on the expression levels of the control genes (Table S2.2) with the most constant
expression, EF1 and UBQ10.
A melting curve analysis was performed to confirm that each gene-specific primer pair only
amplified a single product (Figure S2.4). The qRT-PCR products for each individual primer pair were
bulked and analysed using agarose gel electrophoresis to ensure that single fragments of the expected
size were produced (Figure S2.5). Each primer pair tested yielded a single melting peak. To confirm
the identities of the qRT-PCR products for each gene-specific primer pair, PCR purified amplification
products were sequenced and analysed using BLAST (NCBI, Table S2.3).
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.10 Chemical composition of stems from wild-type (WT) and transgenic insertion lines (MO1, MO2
and MO3) transformed with EgCesA1. The y-axis indicates the amount of monomers observed as a percentage
of the total dry weight analysed. Error bars indicate technical variability based on two technical replicates. The
* indicates that the difference observed between the transgenic line and the wild-type stem composition is
statistically significant (α = 0.05).
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.11 Relative transcript abundance levels of EF1 and UBQ10 in leaves, upper- and lower stems of
wild-type (WT) and transgenic (MO1, MO2 and MO3) plants. The graphs were derived from qBASEplus v1.0.
The y-axis indicates the relative expression levels in arbitrary units (A.U.) of the genes. The x-axis indicates
samples analysed. Bulk cDNA was obtained from all samples and used to generate internal standard curves for
the primer pairs. The green segments of the bars indicate technical variability among three technical replicates
(Figure S2.12)
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
The expression levels of EgCesA1 and Arabidopsis cell wall related genes were normalized
using qBASEplus v1.0 software and the results were exported to Microsoft® Excel 2007 for further
analysis.
The expression levels of biological replicates of transgenic lines were compared to
biological replicates of the wild-type using a Student’s t-test. Comparisons were made between the
same tissue in wild-type and transgenic plants. The relative expression levels of the genes analysed
are illustrated in Figure S2.6. Variation among biological replicates of the same plant and tissue were
evaluated by regression analysis (Table 2.3, Table 2.4, Figures S2.7 to S2.11). Slope-values of
regression trends were analysed to determine the level of similarity between biological replicates
(Table 2.4), while R2-values were analysed to determine the level of correlation between the data
points and the regression trend (Table 2.3). Transgenic line MO2 showed the highest degree of
variation between biological replicates when all tissues were analysed (Figure S2.7) and was
subsequently excluded from further analysis. Wild-type plants also had a high degree of variation,
which was mostly observed in lower stem tissues (Figure S2.8). Upon closer examination it was
observed that variation between biological replicates of wild-type plant lower stem tissues were
mostly observed for gene transcript levels of AtCesA3, secondary cell wall CesA genes, KORRIGAN
and COBRA (Figure S2.6).
Table 2.3 R2-values for biological replicates of wild-type, MO1, MO2 and MO3 plants1
Wild-type
MO1
MO2
MO3
All tissues
0.19
0.58
0.17
0.83
Upper stem
0.86
0.63
0.49
0.72
Lower stem
0.03
0.11
0.49
0.94
Leaves
0.46
0.95
0.93
Regression lines indicated in Figures S2.7 to S2.11.
0.99
1
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Table 2.4 Slope-values of the regression lines for biological replicates of wild-type, MO1, MO2 and
MO3 plants1
Wild-type
MO1
MO2
MO3
All tissues
0.32
0.74
0.20
0.84
Upper stem
0.89
0.72
0.42
0.52
Lower stem
-0.06
0.36
0.14
1.06
Leaves
0.53
1.28
1.48
Regression lines indicated in Figures S2.7 to S2.11.
1.08
1
As expected for 35S promoted expression, EgCesA1 transcripts were observed in all three
tissues of all three transgenic lines, but not in wild-type plants (Figure 2.12). Of the three transgenic
lines, relative expression of EgCesA1 was highest in MO3.
Relative transcript levels of the
endogenous primary cell wall associated CesA genes, AtCesA1, AtCesA2 (partially redundant to
AtCesA6) and AtCesA3, in the transgenic plant lines did not differ significantly from that observed in
the wild-type plants (Figure 2.13). The only difference between wild-type and transgenic lines that
could not be attributed to technical and biological variation only, was that observed for AtCesA3 in the
upper stems of transgenic line MO1. The transcript abundance levels of primary cell wall associated
genes, CYT1 and KOBITO 1, also appeared the same in the transgenic lines as in wild-type plants
(Figure 2.15).
The upper stem tissues of MO1 showed significantly higher transcript levels for the secondary
cell wall associated CesA genes, AtCesA4 and AtCesA8, however, the expression levels of AtCesA7
showed significant biological variation (Figure 2.14). In contrast, the relative expression levels of
AtCesA4 and AtCesA7 were significantly lower in MO3 upper stems compared to that observed in
wild-type plants.
The lower stem tissues in wild-type plants showed high variation between
biological replicates (Table 2.3 and Table 2.4) when relative expression levels of the three secondary
cell wall associated CesA genes were analysed (Figure 2.14). This was also observed for the relative
expression levels of AtCesA3 (Figure 2.13), KORRIGAN and COBRA (Figure 2.15).
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.12 Relative transcript abundance of EgCesA1 in wild-type and transgenic plant lines, MO1 and MO3.
Error bars indicate technical variation. The y-axis and x-axis indicate relative transcript abundance and tissues
analysed, respectively. A and B designate biological replicates.
All the genes involved in carbon allocation and the lignin biosynthetic pathway (Figure 2.16
and Figure 2.17) analysed appeared unaffected by the expression of EgCesA1, as no significant
differences were observed when transcript levels of the genes were compared between wild-type and
MO1 and MO3 plants, respectively. Of the genes involved in carbon allocation analysed, SPP had the
most unvaried transcript abundance levels (Figure 2.16). SuSy1, SuSy4 and SPS had similar transcript
abundance profiles.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.13 Relative transcript abundance levels of Arabidopsis primary cell wall associated CesA genes.
Error bars indicate technical variation of three replicates. * indicates that the expression level difference
observed between wild-type and the transgenic plants could not be attributed to technical and biological
variation alone at a significance of α = 0.05. The y-axis and x-axis indicate relative transcript abundance and
tissues analysed, respectively. A and B designate biological replicates.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.14 Relative transcript abundance levels of Arabidopsis secondary cell wall associated CesA genes.
Error bars indicate technical variation of three replicates. * indicates that the expression level difference
observed between wild-type and the transgenic plants could not be attributed to technical and biological
variation alone at a significance of α = 0.05. The y-axis and x-axis indicate relative transcript abundance and
tissues analysed, respectively. A and B designate biological replicates.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.15 Relative transcript abundance levels of CYT1, KOBITO 1, KORRIGAN and COBRA. Error bars
indicate technical variation of three replicates. The y-axis and x-axis indicate relative transcript abundance and
tissues analysed, respectively. A and B designate biological replicates.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.16 Relative transcript abundance levels of SPP, SPS, SuSy1 and SuSy4. Error bars indicate technical
variation of three replicates. The y-axis and x-axis indicate relative transcript abundance and tissues analysed,
respectively. A and B designate biological replicates.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure 2.17 Relative transcript abundance levels of AtCAD4, AtCAD5 and THESEUS 1. Error bars indicate
technical variation of three replicates. The y-axis and x-axis indicate relative transcript abundance and tissues
analysed, respectively. A and B designate biological replicates.
2.5
Discussion
The effect of constitutively expressing the Eucalyptus secondary cell wall associated CesA gene,
EgCesA1 (Ranik and Myburg, 2006), in wild-type Arabidopsis was assessed by transforming Col-0
plants with a vector containing the EgCesA1 CDS under expression control of a double 35S CMV
promoter (Kay et al., 1987).
Using the Gateway® (Invitrogen) system for constructing plant
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
expression vectors (Curtis and Grossniklaus, 2003) and the floral dip method (Clough and Bent, 1998)
for Agrobacterium mediated Arabidopsis transformation, three transgenic lines were generated (MO1,
MO2 and MO3). Cell wall phenotypes of the three transgenic lines were compared with that of wildtype Col-0 plants using light microscopy and modified micro-Klason (Huntley et al., 2003) chemical
analysis. The relative transcript abundance levels of endogenous Arabidopsis CesA and other cell
wall related genes were also compared between transgenic and wild-type lines.
Constitutive expression of EgCesA1 and its effect on cell wall morphology and chemistry
The EgCesA1 transgene was shown to be expressed in all three transgenic plant lines (Figure 2.8).
Relative transcript levels varied between the three lines (Figure S2.6), with MO3 showing the highest
relative expression in all three tissues (upper stem, lower stem and leaves) analysed. This could be
due to the position within the genome where the transgene construct inserted (Francis and Spiker,
2005). These results provide evidence that EgCesA1 was transcribed in the transgenic lines, but
EgCESA1 protein levels still need to be analysed to establish if translation of EgCesA1 mRNA
occurred. This could be achieved using Western blot analysis. No antibody for the EgCESA1 protein
was available; therefore, Western blot analysis could not be performed for this study.
Predicting the fate of the EgCESA1 protein, if it is translated in Arabidopsis, could prove to
be a challenge. Over-expression of a mutated AtCesA7 gene, fragile fibre 5 (fra5) that had a missense
mutation in the second cytoplasmic domain in wild-type Arabidopsis plants had a dominant negative
effect on cellulose biosynthesis in secondary and primary cell walls causing xylem cell walls to
collapse and affecting cell elongation. Incorporation of the mutated CESA protein into the rosette
subunits was hypothesised to interfere with endogenous CESA protein activity. In contrast, overexpression of a missense mutated AtCesA8 gene in wild-type Arabidopsis apparently did not alter
cellulose biosynthesis. However, it should be noted that data to support this observation was not
presented (Zhong et al., 2003). Protein levels of AtCESA7 in transgenic lines over-expressing wildtype AtCesA7 were regulated through phosphorylation. When transgene protein levels exceeded wildtype levels, excess protein was phosphorylated and degraded. Phosphorylation was hypothesised to
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
be a key process for regulating relative CESA protein levels (Taylor, 2007). If translated, EgCesA1
expressed in Arabidopsis could result in the incorporation of EgCESA1 proteins into Arabidopsis
cellulose synthase complexes. If the protein does not function correctly, it could affect the activity of
endogenous CESA proteins resulting in reduced cellulose and collapsed xylem cell walls, or it could
have no affect as observed for mutated AtCesA8 over-expression (Zhong et al., 2003). Alternatively,
over-expression of EgCesA1 could result in phosphorylation and subsequent degradation of proteins
in excess of wild-type levels of the endogenous orthologue, AtCESA8. Analysis of AtCESA8 protein
levels could reveal if phosphorylation and degradation occurs.
Light microscopy was performed to determine if over-expression of EgCesA1 altered the
phenotype of cell walls in stems and hypocotyls. Phloroglucinol-HCl stains lignin red and was useful
to analyse secondary cell wall morphology. Because phloroglucinol-HCl does not stain cellulose,
toluidine blue, which stains cellulose blue and lignin blue-green, was also used. These stains enabled
visualisation of vascular cell walls and comparisons could be made between cell walls of the three
transgenic lines and wild-type Arabidopsis plants (Figure 2.9). No distinguishable differences were
observed in any of the analysed tissues. Cell shape and cell wall thickness appeared similar among
the transgenic and wild-type plants. The amount of cellulose and lignin stained with toluidine blue
also did not appear to differ between transgenic and wild-type sections. Therefore, the expression of
EgCesA1 did not visibly affect cell wall morphology in transgenic plants.
Altered cellulose biosynthesis and cell wall morphology have been shown to be accompanied
by drastic changes in the chemical composition of cell walls (Lane et al., 2001; Taylor et al., 2003;
Zhong et al., 2003; Brown et al., 2005; Coleman et al., 2007). Analysis of the sugar composition of
cell walls enables detection of changes due to altered cellulose biosynthesis and changes in
hemicellulose content. However, not all changes in cell wall associated chemistry lead to visibly
altered cell wall morphology (Coleman et al., 2006).
Cell wall chemistry was analysed in
inflorescence stems of transgenic and wild-type lines (Figure 2.10). Although some differences
between transgenic and wild-type plants that could not be attributed to technical variability were
observed (lignin, glucose, xylose and arabinose), the differences in polysaccharide content were not as
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
severe as those observed in other studies (Taylor et al., 2003; Zhong et al., 2003; Brown et al., 2005).
There was also greater variation among the three transgenic lines than between transgenic and wildtype plants. This could explain why no phenotypic changes were detected when xylem cell walls
were examined under light microscopy.
Relative expression analysis of Arabidopsis cell wall related genes
The plant cell wall is a complex network of cellulose intertwined with hemicellulose, pectin (in
primary cell walls), lignin (in secondary cell walls) and cell wall proteins. Some genes involved in
the biosynthesis and deposition of the various components of cell walls have been shown to be coexpressed and possibly co-regulated (Brown et al., 2005; Persson et al., 2005). Changes in cellulose
biosynthesis can influence monosaccharide levels in cell walls (Zhong et al., 2003; Brown et al.,
2005) indicating that changes in cellulose deposition can influence hemicellulose content. Defects in
cellulose biosynthesis have also been shown to cause ectopic lignification (His et al., 2001; Desprez et
al., 2002; Cano-Delgado et al., 2003). It has been proposed that some form of cell wall integrity
sensing leads to alterations in non-cellulose components of the cell wall in reaction to altered cellulose
content (Hematy et al., 2007). A question that was posed in this study is; would constitutive
expression of EgCesA1 in wild-type Arabidopsis plants influence the expression of endogenous genes
involved in cellulose, lignin or hemicellulose biosynthesis?
qRT-PCR was used to quantify the relative expression levels of EgCesA1, endogenous CesA
genes and other Arabidopsis genes known to be involved in cell wall deposition. qRT-PCR provides
an accurate means to study transcript abundance levels, as it is sensitive enough to detect small
changes in mRNA abundance (Rockett and Hellmann, 2004; Steibel et al., 2009). To correct for
technical variability three technical replicates of each reaction was done, inter-run calibrators were
included and gene expression levels were normalised to the relative expression levels of two control
genes, EF1 and UBQ10 (Figure 2.11). cDNA quality was assessed by PCR amplification of a
segment of the UBQ10 gene (Figure 2.7) and ideal concentrations of cDNA to be used for qRT-PCR
was determined by testing the amplification efficiencies of a serial dilution of each cDNA sample.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Plant material from four plants in each line was combined to represent one biological
replicate (Figure 2.2 B). Despite this bulking of tissues, considerable variation in gene expression
levels was observed between some biological replicates. MO2 had the highest biological variation
across all tissues (Table 2.3 and Table 2.4). Normalization of transcript levels to the transcript
abundance of the control genes UBQ10 and EF1 aimed to correct for variations in cDNA
concentration. There appeared to be no correlation between the transcript abundance levels of control
genes and biological variation observed among MO2 biological replicates (Figure S2.12). The
variation could rather be attributed to cDNA quality and concentration. High biological variation was
also observed in wild-type lower stem tissues (Table 2.3 and Table 2.4). The variation was not
observed for all genes analysed. Expression levels of AtCesA3 (Figure 2.13), secondary cell wall
associated CesA genes (Figure 2.14), KORRIGAN and COBRA (Figure 2.15) varied between lower
stem tissues of the two wild-type biological replicates. Replicate A had higher transcript abundance
for these genes than replicate B. SuSy4 and SPS transcript levels also showed high biological
variation among lower stem tissues of wild-type plants with replicate B having higher transcript levels
than replicate A (Figure 2.16). Such inherent biological variability in an experiment could influence
data to appear statistically non-significant, resulting in a Type II error (Willems et al., 2008). A
method for standardizing qRT-PCR data from independent biological replicates have been proposed
by Willems et al. (2008). It involves three steps; log transformation of normalized relative expression
levels to limit the effect of outliers, mean-centring to correct for differences between control levels in
biological replicates and autoscaling that corrects differences in fold change between experimental
conditions. The authors show that standardizing the data with these three steps does not increase the
rate of false positives. This method can be used when expression levels of three or more biological
replicates are quantified. These considerations should be kept in mind for future studies of this
nature.
Arabidopsis primary cell wall associated CesA genes (AtCesA1, AtCesA3, AtCesA6 and
AtCesA6-related genes, AtCesA2 and AtCesA5) are involved in cellulose deposition in primary cell
walls (Arioli et al., 1998; Burn et al., 2002; Desprez et al., 2002). Expression analysis of AtCesA1,
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
AtCesA2 and AtCesA3 revealed that all three genes were expressed at constant levels in leaves, upper
and lower stems of MO1, MO3 and wild-type plants (Figure 2.13). When transcript levels were
compared between transgenic and wild-type plant lines, the only difference that could not be
attributed to biological and technical variation alone was the increased transcript abundance of
AtCesA3 in upper stems of MO1 (Figure 2.13). The difference was small and relative transcript
abundance of AtCesA3 did not differ significantly in upper stems of MO3 compared to that in the
wild-type. The difference in relative expression levels of AtCesA3 in upper stems of MO1 was likely
not a response to EgCesA1 transgene expression. To correct for multiple testing, the single testing
significance should also be considered at α = 0.05/n where n is the number of independent tests
analysed. The relative transcript abundance of each gene was independently tested in two transgenic
lines. At a significance of α = 0.025 the difference between AtCesA3 transcript levels in MO1 and
wild-type upper stems is no longer statistically significant. Two other genes with proposed functions
in cellulose deposition in primary cell walls are CYT1 (Nickle and Meinke, 1998) and KOBITO 1
(Pagant et al., 2002). CYT1 is believed to be required for N-glycosylation of CESA proteins. The
cyt1 mutant plants exhibit severe changes in primary cell wall morphology due to decreased cellulose
content (Lukowitz et al., 2001). KOBITO 1 occurs in the extracellular matrix and is required for
normal cellulose biosynthesis during cell elongation (Lertpiriyapong and Sung, 2003). Like the
relative expression levels of AtCesA1, AtCesA2 and AtCesA3, expression levels of CYT1 and KOBITO
1 appeared unaffected by the expression of EgCesA1 in transgenic plant lines (Figure 2.15). The
genes were expressed in constant levels across the three tissues analysed corresponding with their
proposed function in primary cell wall deposition (Figure 2.15).
Relative transcript abundance of the secondary cell wall associated Arabidopsis CesA genes,
KORRIGAN and COBRA showed similar profiles (Figures 2.14 and 2.15).
These genes have
previously been shown to be co-expressed (Brown et al., 2005; Persson et al., 2005). Transcript
levels of these genes were on average at least three-fold higher in stems than in leaves of wild-type
and MO1 plants. High variation in expression levels of these genes in the lower stem tissues of wildtype biological replicates was observed, as mentioned previously.
82
Because of this, statistical
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
significance could not be given to differences observed between lower stem tissues of wild-type and
transgenic plants. Transcripts of secondary cell wall genes were on average more abundant in stem
tissues than in leaves. This agrees with their functions during secondary cell wall deposition (Taylor
et al., 2003).
AtCesA4 and AtCesA8 expression levels in upper stem tissues of MO1 were
significantly higher than the wild-type. AtCesA7 also appeared to be expressed at higher levels in
upper stems of MO1, but biological variation was too high to give statistical significance to the
observation. Interestingly, secondary cell wall associated CesA genes had lower relative expression
in transgenic line MO3 than in the wild-type and MO1, in contrast to the higher relative expression of
EgCesA1 in MO3 (Figure 2.12). Expression of EgCesA1 could have a negative effect when transcript
levels exceed a certain threshold. High levels of EgCesA1 could cause post-transcriptional gene
silencing (PTGS) of endogenous genes that have homologous gene sequences to EgCesA1 (Hammond
et al., 2000). PTGS could occur if gene sequences share a 21 bp uninterrupted homologous cDNA
sequence. When AtCesA8 and EgCesA1 cDNA sequences were aligned one uninterrupted segment of
23 bp of homologous sequence was observed (Figure S2.14). Down-regulation of the endogenous
genes could subsequently cause changes in expression levels of co-expressed genes. Down-regulation
of the secondary cell wall genes was not enough to cause changes in the cell wall morphology of
MO3 plants (Figure 2.9) or chemistry of the cell walls (Figure 2.10). Since changes in transcript
levels of secondary cell wall associated genes were not consistent between MO1 and MO3, it could
not be positively correlated with the expression levels of the EgCesA1 transgene. Nevertheless, it
would be interesting to probe for the presence of a small interfering RNA molecule corresponding to
the 23 bp conserved region in transgenic plants.
Genes involved in carbon partitioning and cycling, SuSy1, SuSy4, SPP and SPS, have been
suggested to control the flux of UDP-glucose towards cellulose biosynthesis (Delmer and Haigler,
2002). An increase in SPS protein activity has been correlated with increased cellulose biosynthesis
(Babb and Haigler, 2001). Relative transcript abundance observed for SPS appeared unaffected by
EgCesA1 expression (Figure 2.16). Gene expression levels of SuSy1, SuSy4 and SPP were also
unaffected by the expression of EgCesA1 (Figure 2.16). A recent study by Barratt et al. (2009)
83
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
suggested that cytosolic invertase was required for normal carbon partitioning in Arabidopsis plants
rather than SuSy.
It therefore seems prudent to characterise transcript abundance levels of
CYTOSOLIC INVERTASE genes, CINV1 and CINV2, in future studies of this nature.
As mentioned previously, ectopic lignification has been observed in plants with decreased
cellulose biosynthesis (Desprez et al., 2002; Cano-Delgado et al., 2003).
Cinnamyl alcohol
dehydrogenase (CAD) is an enzyme involved in monolignol biosynthesis. CAD enzymes catalyse the
conversion of cinnamyl aldehydes to coniferyl and sinapyl alcohols (Walter et al., 1988).
In
Arabidopsis two CAD genes, AtCAD4 and AtCAD5, were found to be expressed in, among others, the
vascular tissues of stems and leaves (Kim et al., 2007). The relative expression levels of AtCAD4 and
AtCAD5 were quantified to determine if constitutive expression of EgCesA1 had an effect on
endogenous CAD gene expression. Again, no significant changes in gene transcript abundance levels
could be detected when transgenic and wild-type plants were compared (Figure 2.17) despite variation
observed in lignin content (Figure 2.10).
Ectopic lignification is believed to be a defence response to a decrease in cellulose (CanoDelgado et al., 2003) and THESEUS 1 has been proposed to be involved in signalling the response.
THESEUS 1 is a membrane-bound receptor-like kinase. THESEUS 1 expression has been shown to
be increased in plants with mutated CesA genes (Hematy et al., 2007). A mutation of THESEUS 1 in
the CesA gene mutant background, results in a decrease in ectopic lignification possibly through the
production of reactive oxygen species (Hematy and Hofte, 2008). Conversely, over-expression of
THESEUS 1 in plants with a mutated CesA gene results in increased amounts of ectopic lignin
(Hematy et al., 2007). Microscopy and chemical analysis did not reveal any evidence for ectopic
lignification (Figure 2.9 and Figure 2.10) and relative transcript abundance levels of THESEUS 1 did
not differ significantly between wild-type and transgenic lines (Figure 2.17).
Constitutive expression of the EgCesA1 gene in wild-type Arabidopsis was successfully
achieved (Figure 2.12). The presence of high levels of secondary cell wall associated CesA gene
transcripts did not have any noticeable effect on cell wall morphology (Figure 2.9). Despite changes
84
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
in cell wall chemistry (Figure 2.10) and significant changes in endogenous secondary cell wall CesA
gene transcript abundance in MO1 (Figure 2.14), significant correlations could not be drawn between
EgCesA1 expression and relative expression levels of endogenous cell wall related genes (Figures
2.13 to 2.17). Transgenic line MO2 had especially high variation between biological replicates for
gene expression in stem tissues. cDNA concentration and quality of MO2 upper and lower stem
samples could have been insufficient for the experiment. Because no significant differences in cell
wall chemistry and morphology could be detected in MO2 compared to MO1 and MO3, MO2 was
excluded from analysis of relative gene expression levels. In order to increase statistical power, at
least three biological replicates should in future be analysed when quantifying relative expression
levels of genes using qRT-PCR. The variation in gene expression observed between transgenic lines
provides support for the use of multiple transgenic events when analysing the effects of transgene
expression. It enables one to discern between effects that are caused by expression of the transgene
and those that occur due to positional effects.
Manipulation of cellulose biosynthesis will greatly benefit the wood products industry. Better
understanding of the assembly of rosette protein complexes and the interaction of cellulose
microfibrils and other cell wall polymers will facilitate attempts to increase cellulose biosynthesis in
commercially important tree species. Constitutive expression of the EgCesA1 transgene did not have
a noticeable effect on cellulose biosynthesis. This study does not provide support for the hypothesis
that expression of a single secondary cell wall associated Eucalyptus CesA gene will lead to increased
cellulose production and affect the expression levels of endogenous cell wall associated genes in wildtype Arabidopsis. However, it does not provide support against the alternative hypothesis that
expression of all three secondary cell wall CesA genes need to be increased to increase cellulose
biosynthesis. Hypotheses that could explain the observations made in this study are proposed in the
Concluding Remarks section of the dissertation.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
2.6
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2.7
Supplementary material
Figure S2.1 Vector map of pTOPO-EgCesA1. The EgCesA1 CDS (EgCesA1) was inserted between the attL1
and attL2 Gateway® recombination sites in sense orientation. The vector had a pUC origin of replication (pUC
ori) and a gene that conferred resistance to spectinomycin. Primer binding sites for vector-specific primers
(M13-29-F and M13-narrow-R), gene-specific primers (EgCesA_gb_5’UTR+28 and EgCesA_3’UTR-120) and
internal sequencing primers (EgCesA1_seq_F and EgCesA1_seq_R) are indicated by black arrows.
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Figure S2.2 Vector maps of the empty and recombinant destination vectors, pMDC32 and pMDC32-EgCesA1.
pMDC32 has a pBR322 origin of replication and a gene that conferred bacterial resistance to kanamycin. The
vector also contained a gene that conferred resistance to hygromycin as plant selectable marker, a double 35S
CMV promoter and Nos terminator between the left and right borders (LB and RB, respectively) of the
expression cassette that would incorporate into the genome of transgenic plants. Vector-specific primer binding
sites, M13-40-F and M13-wide-R, are indicated as black arrows. The EgCesA1 CDS was transferred to
pMDC32 from pTOPO-EgCesA1 by recombination between the attR1 and attR2 sites of pMDC32 (A) and the
attL1 and attL2 sites of pTOPO-EgCesA1 (Figure S2.1) resulting in the formation of attB1 and attB2 sites in the
recombinant vector (B). Gene-specific primer binding sites for EgCesA_gb_5’UTR+28 and EgCesA_3’UTR120 primers are indicated as black arrows in B.
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EgCESA1 A, B, C and D predicted proteins
Figure S2.3 Alignment of predicted amino acid sequences of four pTOPO-EgCesA1clones. Positions 1 to 301.
The full-length EgCesA1 CDS was sequenced. EgCesA1D was used for subsequent procedures. Darker gray
shading indicates similarity between two or more of the sequences analysed. Light grey shading indicates
complete consensus.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
EgCESA1 A, B, C and D predicted proteins
Figure S2.3 (Cont.) Alignment of predicted amino acid sequences of four pTOPO-EgCesA1clones. Positions
302 to 602. The full-length EgCesA1 CDS was sequenced. EgCesA1D was used for subsequent procedures.
Darker gray shading indicates similarity between two or more of the sequences analysed. Light grey shading
indicates complete consensus.
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CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
EgCESA1 A, B, C and D predicted proteins
Figure S2.3 (Cont.) Alignment of predicted amino acid sequences of four pTOPO-EgCesA1clones. Positions
603 to 903. The full-length EgCesA1 CDS was sequenced. EgCesA1D was used for subsequent procedures.
Darker gray shading indicates similarity between two or more of the sequences analysed. Light grey shading
indicates complete consensus.
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EgCESA1 A, B, C and D predicted proteins
Figure S2.3 (Cont.) Alignment of predicted amino acid sequences of four pTOPO-EgCesA1clones. Positions
904 to 978. The full-length EgCesA1 CDS was sequenced. EgCesA1D was used for subsequent procedures.
Darker gray shading indicates similarity between two or more of the sequences analysed. Light grey shading
indicates complete consensus.
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Figure S2.4 Melting curves of the qRT-PCR products for each gene-specific primer pair. The y-axis indicates
(d/dT) fluorescence (483-533) and the x-axis indicates temperature (°C).
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Figure S2.5 1% (w/v) EtBr/Agarose gel electrophoresis of bulked qRT-PCR products for 22 gene-specific
primer pairs. Lane M contains a 100 bp DNA size standard (GeneRuler™ 100 bp DNA Ladder Plus, Fermentas)
and lanes 1 to 22 contain 5 μl of bulked qRT-PCR products including the products from the amplification of the
control genes EF1 and UBQ10 in lanes 19 to 22.
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Figure S2.6 Relative transcript levels of EgCesA1 and endogenous genes involved in cell wall biosynthesis
pathways. The y-axis indicates relative transcripts levels. The x-axis indicates tissues from wild-type (WT) and
transgenic lines (MO1, MO2 and MO3) for two biological replicates, A and B. Error bars indicate technical
variation among three technical replicates.
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Figure S2.6 (Cont.) Relative transcript levels of EgCesA1 and endogenous genes involved in cell wall
biosynthesis pathways. The y-axis indicates relative transcripts levels. The x-axis indicates tissues from wildtype (WT) and transgenic lines (MO1, MO2 and MO3) for two biological replicates, A and B. Error bars
indicate technical variation among three technical replicates.
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Figure S2.6 (cont.) Relative transcript levels of EgCesA1 and genes involved in carbon flux in plant cell walls.
The y-axis indicates relative transcripts levels. The x-axis indicates tissues from wild-type (WT) and transgenic
lines (MO1, MO2 and MO3) for two biological replicates, A and B. Error bars indicate technical variation
among three technical replicates.
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Figure S2.6 (Cont.) Relative transcript levels of EgCesA1 and endogenous genes involved in cell wall
biosynthesis pathways. The y-axis indicates relative transcripts levels. The x-axis indicates tissues from wildtype (WT) and transgenic lines (MO1, MO2 and MO3) for two biological replicates, A and B. Error bars
indicate technical variation among three technical replicates.
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Figure S2.6 (Cont.) Relative transcript levels of EgCesA1 and endogenous genes involved in cell wall
biosynthesis pathways. The y-axis indicates relative transcripts levels. The x-axis indicates tissues from wildtype (WT) and transgenic lines (MO1, MO2 and MO3) for two biological replicates, A and B. Error bars
indicate technical variation among three technical replicates.
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Figure S2.7 Regression analysis of relative transcript levels of all genes compared between biological
replicates across all tissues. The x-axis indicates transcript levels in replicate A and the y-axis indicates
transcript levels in replicate B. Wild-type (WT) and transgenic lines, MO1, MO2 and MO3.
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Figure S2.8 Regression analysis of relative transcript levels compared between biological replicates of wildtype plants.
The x-axis indicates transcript levels in replicate A and the y-axis indicates transcript levels in
replicate B. ALL, all tissues; US, upper stem tissues; LS, lower stem tissues; L, leaf tissues.
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Figure S2.9 Regression analysis of relative transcript levels compared between biological replicates of MO1
transgenic plants.
The x-axis indicates transcript levels in replicate A and the y-axis indicates transcript levels
in replicate B. ALL, all tissues; US, upper stem tissues; LS, lower stem tissues; L, leaf tissues.
107
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.10 Regression analysis of relative transcript levels compared between biological replicates of MO2
transgenic plants.
The x-axis indicates transcript levels in replicate A and the y-axis indicates transcript levels
in replicate B. ALL, all tissues; US, upper stem tissues; LS, lower stem tissues; L, leaf tissues.
108
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.11 Regression analysis of relative transcript levels compared between biological replicates of MO3
transgenic plants.
The x-axis indicates transcript levels in replicate A and the y-axis indicates transcript levels
in replicate B. ALL, all tissues; US, upper stem tissues; LS, lower stem tissues; L, leaf tissues.
109
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.12 Transcript levels of UBQ10 and EF1 before normalization. The y-axis indicates transcript levels
and the x-axis indicates the plant lines, wild-type (WT), MO1, MO2 and MO3, biological replicates A and B and
three replicates analysed in each tissue, upper stem, lower stem and leaves.
110
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 1 to 432. Dark grey
shading indicates complete consensus between sequences. Light grey shading indicates similarity between two
of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23 bp
homologous sequence was observed from position 1087 to 1109.
111
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 (Cont.) DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 433 to 864. Dark
grey shading indicates complete consensus between sequences. Light grey shading indicates similarity between
two of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23
bp homologous sequence was observed from position 1087 to 1109.
112
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 (Cont.) DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 865 to 1296. Dark
grey shading indicates complete consensus between sequences. Light grey shading indicates similarity between
two of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23
bp homologous sequence was observed from position 1087 to 1109.
113
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 (Cont.) DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 1297 to 1728. Dark
grey shading indicates complete consensus between sequences. Light grey shading indicates similarity between
two of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23
bp homologous sequence was observed from position 1087 to 1109.
114
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 (Cont.) DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 1729 to 2160. Dark
grey shading indicates complete consensus between sequences. Light grey shading indicates similarity between
two of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23
bp homologous sequence was observed from position 1087 to 1109.
115
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 (Cont.) DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 2161 to 2592. Dark
grey shading indicates complete consensus between sequences. Light grey shading indicates similarity between
two of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23
bp homologous sequence was observed from position 1087 to 1109.
116
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 (Cont.) DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 2593 to 3024. Dark
grey shading indicates complete consensus between sequences. Light grey shading indicates similarity between
two of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23
bp homologous sequence was observed from position 1087 to 1109.
117
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Figure S2.13 (Cont.) DNA sequence alignment of AtCesA8 and EgCesA1 cDNA. Position 3025 to 3391. Dark
grey shading indicates complete consensus between sequences. Light grey shading indicates similarity between
two of the sequences. EgCesA1D is the sequence of the EgCesA1 CDS used in this study. One segment of 23
bp homologous sequence was observed from position 1087 to 1109.
118
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Table S2.1 Oligonucleotide primers used for qRT-PCR analysis of the EgCesA1 transgene and
endogenous Arabidopsis genes
Primer name
EgCesA1_qRT-PCR_B_Forward
EgCesA1_qRT-PCR_B_Reverse
AtCesA1_qRT-PCR_Forward
AtCesA1_qRT-PCR_Reverse
AtCesA2_qRT-PCR_B_Forward
AtCesA2_qRT-PCR_B_Reverse
AtCesA3_qRT-PCR_Forward
AtCesA3_qRT-PCR_Reverse
AtCesA4_qRT-PCR_Forward
AtCesA4_qRT-PCR_Reverse
AtCesA7_qRT-PCR_Forward
AtCesA7_qRT-PCR_Reverse
AtCesA8_qRT-PCR_ Forward
AtCesA8_qRT-PCR_Reverse
CYT1_qRT-PCR_Forward
CYT1_qRT-PCR_Reverse
KORRIGAN_qRT-PCR_Forward
KORRIGAN_qRT-PCR_Reverse
COBRA_qRT-PCR_Forward
COBRA_qRT-PCR_Reverse
KOBITO_qRT-PCR_Forward
KOBITO_qRT-PCR_Reverse
THESEUS_qRT-PCR_Forward
THESEUS_qRT-PCR_Reverse
AtCAD4_qRT-PCR_Forward
AtCAD4_qRT-PCR_Reverse
AtCAD5_qRT-PCR_Forward
AtCAD5_qRT-PCR_Reverse
FRA8_qRT-PCR_Forward
FRA8_qRT-PCR_Reverse
SUS1_qRT-PCR_Forward
SUS1_qRT-PCR_Reverse
SUS4_qRT-PCR_Forward
SUS4_qRT-PCR_Reverse
SPS_qRT-PCR_Forward
SPS_qRT-PCR_Reverse
SPP_qRT-PCR_Forward
SPP_qRT-PCR_Reverse
At. number
AT4G32410
AT4G32410
AT4G39350
AT4G39350
AT5G05170
AT5G05170
AT5G44030
AT5G44030
AT5G17420
AT5G17420
AT4G18780
AT4G18780
AT2G39770
AT2G39770
AT5G49720
AT5G49720
AT5G60920
AT5G60920
AT3G08550
AT3G08550
AT5G54380
AT5G54380
AT3G19450
AT3G19450
AT4G34230
AT4G34230
AT2G28110
AT2G28110
AT5G20830
AT5G20830
AT3G43190.1
AT3G43190.1
AT1G04920.1
AT1G04920.1
AT2G35840.1
AT2G35840.1
119
Primer sequence (5’→3’)
CTGCGATCTGCCTCCTTACT
TGGTGTCTAGGCCAGCTAAC
GTTCTTCTCGCCTCCATCTT
TTCACCTTCTCTGGCTACTC
CGTTGCCTCTGCTGTTCTAC
TGCACGCTTAGGCATACAGT
GAGTTGTTGCAGGAGTCTCT
CAGTGACTCGGCTAGTGAAG
TTGGTGTTGTTGCCGGAGTT
AACAGTCGACGCCACATTGC
CGTTGTTGCAGGCATCTCAG
AGCAGTTGATGCCACACTTG
TGGAGGTGTCTCAGCTCATC
CGACGACTCCGACCAAGTTA
TTGGACCAGACGTTGCCATT
TCCTCACCGAGGATCGTCAT
ACCGACTCTTGCAGGCAATG
TTGGCAGCAGCGACAGAATC
TAGAGTCCACTGGCACGTTA
CCTGAAGGTCCAGCTTCCAT
AGCAACTCATCCAGGTCTAC
ATGCATCAGCTCCGTGCTAA
TGGAGTACGCGTTACAGCTA
TCTACCTTCCACGAGGATGA
ATTGACACCGTACCGGTGTT
ATCGTTCTTGCGAAGCCTCT
AATTGACACGGTGCCTGTTC
AACGCACATCGTTCTTCTCG
GGTGGCTCTTGGCTGTGTTC
CCGCCTAACAGACGGATCCT
GGTGATCAGGCTGCTGATAC
CCTCAAGACGGTCAAGGTTC
ATGGTGACAAGGCAGCTGAG
AGCAAGAGGAACAGCTTGAG
ATGTGGCGAACATGTACGTG
ACCGGTGAATCAACCTTGAG
GATCAAGTCCTGGCAACAGA
CTCAGATGATCCAGCTGCTA
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Table S2.2 Oligonucleotide primers used for amplification of putative housekeeping genes
Primer name
ACT2_qRT-PCR_Forward
ACT2_qRT-PCR_Reverse
UBQ5_qRT-PCR_Forward
UBQ5_qRT-PCR_Reverse
UBQ10_qRT-PCR_Forward
UBQ10_qRT-PCR_Reverse
UBQ10_qRT-PCR_B_Forward
UBQ10_qRT-PCR_B_Reverse
18S_qRT-PCR_Forward
18S_qRT-PCR_Reverse
EF1_qRT-PCR_Forward
EF1_qRT-PCR_Reverse
EF1_qRT-PCR_B_Forward
EF1_qRT-PCR_B_Reverse
TUB4_qRT-PCR_Forward
TUB4_qRT-PCR_Reverse
TUB6_qRT-PCR_Forward
TUB6_qRT-PCR_Reverse
At. number
AT3G18780
AT3G18780
AT3G62250.1
AT3G62250.1
AT4G05320
AT4G05320
AT4G05320
AT4G05320
18S RRNA
18S RRNA
AT1G07920.1
AT1G07920.1
AT1G07920.1
AT1G07920.1
AT5G44340.1
AT5G44340.1
AT5G12250
AT5G12250
120
Primer sequence (5’→3’)
TGGAATCCACGAGACAACCT
TGGACCTGCCTCATCATACT
GGTGGTGCTAAGAAGAGGAA
TCGATCTACCGCTACAACAG
CTGCGTGGAGGTATGCAGAT
CGCAGGACCAAGTGAAGAGT
GACCAGCAGAGGTTGATCTT
CCGGAGGAATACCTTCCTTG
AGTAAGCGCGAGTCATCAGC
TCCTTCCGCAGGTTCACCTA
ACAGGCGTTCTGGTAAGGAG
CCTTCTTGACGGCAGCCTTG
GTTGAGATGCACCACGAGTC
TCCTTACCAGAACGCCTGTC
GAGCGAACAGTTCACAGCTA
GCTGCTTGCTTACACAGCTT
GTCAAGCGTCTGTGACATAG
TCATACTCGCCTTCGTCATC
CONSTITUTIVE EXPRESSION OF EUCALYPTUS GRANDIS EgCesA1 IN ARABIDOPSIS THALIANA
Table S2.3 Sequence results of bulked qRT-PCR products analysed using BLAST
PCR product
BLAST hit
Score (Bits)
E value
AtCesA1
AtCesA1
324
4.00E-88
AtCesA2
AtCesA2
250
1.00E-61
AtCesA3
AtCesA3
350
8.00E-96
AtCesA4
AtCesA4
414
4.00E-115
AtCesA7
AtCesA7
172
4.00E-42
AtCesA8
AtCesA8
268
2.00E-71
CYT1
CYT1
267
1.00E-70
KORRIGAN
KORRIGAN
283
7.00E-76
COBRA
IRX6 / COBL4
532
1.00E-150
KOBITO
KOBITO
257
4.00E-68
THESEUS
THESEUS
355
2.00E-97
AtCAD4
AtCAD4
379
1.00E-104
AtCAD5
AtCAD5
237
1.00E-61
FRA8
FRA8
291
6.00E-78
SUS1
SUS1
154
9.00E-37
SUS4
SUS4
300
2.00E-42
SPS
SPS3F
302
3.00E-81
SPP
SPP
268
4.00E-71
EF1 A
EF1α
331
3.00E-90
EF1 B
EF1α
320
3.00E-42
UBQ10 A
UBQ10
475
3.00E-133
UBQ10 B
UBQ10
360
2.00E-37
121
CONCLUDING REMARKS
CONCLUDING REMARKS
In order to alter cellulose biosynthesis in plant cell walls certain questions need to be addressed: How
do cellulose microfibrils and other cell wall polymers interact? How do CESA proteins assemble to
form rosette complexes? Can cellulose biosynthesis be increased by over-expressing a single CesA
gene? Will constitutive expression of a secondary cell wall Eucalyptus CesA gene in Arabidopsis
affect xylem cell wall morphology, cell wall chemistry and expression levels of endogenous genes
involved in cell wall biosynthesis pathways? Should the transgenic CesA gene be expressed in a
tissue specific manner using a xylem specific promoter?
This M.Sc study has succeeded in
constitutively expressing a Eucalyptus CesA gene, EgCesA1, in wild-type Arabidopsis thaliana plants.
Three transgenic lines were generated, but microscopic analysis of the cell wall phenotype of
transgenic plants expressing EgCesA1 in comparison to wild-type control plants did not reveal
significant morphological changes. Analysis of the chemical composition of inflorescence stem cell
walls revealed decreased levels of glucose, xylose and lignin in all three transgenic lines compared to
wild-type control plants. However, the observed differences in cell wall chemistry were not as severe
as those detected in plants with mutated CesA genes (Taylor et al., 2003; Zhong et al., 2003), or plants
with defective hemicellulose synthesis (Brown et al., 2005). Constitutive expression of EgCesA1 did,
therefore, not significantly affect cell wall chemistry in transgenic lines.
Quantitative gene expression analysis revealed high levels of variation among biological
replicates of wild-type control and transgenic plants. Transgenic line MO2 had especially high
biological variation in expression levels of all the genes analysed. Chemical composition of MO2 cell
walls did not differ significantly from the other transgenic lines. The quality or concentration of first
strand cDNA used for one biological replicate of MO2 could have been insufficient for the
experiment. Therefore, MO2 was excluded when comparisons were drawn between transgenic and
wild-type gene expression levels.
In order to reduce the incidence of Type II errors, future
quantitative real-time PCR analysis should include at least three biological replicates of transgenic and
wild-type control lines (Willems et al., 2008).
123
CONCLUDING REMARKS
Relative expression levels of endogenous secondary cell wall associated CesA genes
(AtCesA4, AtCesA7 and AtCesA8, Taylor et al., 2003) in upper and lower stems of MO3 were lower
than in the wild-type control plants. This is in contrast to EgCesA1 relative expression levels that were
highest in MO3. One possible explanation for this observation could be post-transcriptional gene
silencing (PTGS) (Hammond et al., 2000). Expression levels above a threshold value could trigger
PTGS of gene transcripts with gene sequences homologous to EgCesA1. EgCesA1 expression levels
in MO1 could have been below such a threshold and could, therefore, not have induced PTGS.
The observed increase in expression of AtCesA4 and AtCesA8 in upper stems (inflorescence
stem above lowest leaf) of MO1 could reflect a reaction of MO1 plants to the expression of EgCesA1
when primary and secondary cell walls are actively deposited. However, transcript abundance profiles
of endogenous secondary cell wall CesA genes and profiles of EgCesA1 transcript abundance did not
reveal any consistent correlations. Taking into account the high biological variability, it cannot be
concluded that EgCesA1 expression significantly influenced endogenous gene expression levels.
Four possible hypotheses exist for why EgCesA1 expression did not influence cellulose
biosynthesis.
First, it is possible that EgCesA1 was successfully transcribed but not translated.
Although every measure was taken to ensure that functional protein could form from the EgCesA1
coding sequence (CDS) transcript, Western blot analysis would have had to be done to confirm that
EgCESA1 protein formed. Since no antibody for EgCESA1 was available, Western blot analysis
could not be performed.
Another hypothesis assumes that EgCESA1 protein formed, but that it did not incorporate into
the endogenous cellulose synthase rosette complexes. This would occur if EgCESA1 did not interact
correctly with endogenous CESA proteins. The amino acid change from a glutamate to a lysine
residue could have affected incorporation of EgCESA1 protein into rosettes. The residue is conserved
between homologues of EgCesA1, but this could be because it is situated next to a highly conserved
aspartate residue. Protein-tagging of EgCESA1 with a fluorescent reporter protein could elucidate the
sub-cellular localization of EgCESA1. Fluorescent protein-tagging of CESA proteins have been
124
CONCLUDING REMARKS
shown to result in functioning CESA proteins (Gardiner et al., 2003; Taylor et al., 2004). Since
rosettes are assembled at the Golgi-apparatus and then transported intact to the plasma membrane
(Haigler and Brown, 1986), it could be argued that localization of the fluorescent tag to the Golgiapparatus only would indicate that EgCESA1 is not successfully incorporated into the rosette, whereas
localization of the fluorescent tag to the plasma membrane and the Golgi-apparatus could indicate that
EgCESA1 is incorporated into rosettes.
The third hypothesis assumes that EgCESA1 protein forms and incorporates into the
endogenous rosette complexes, but does not function correctly. In a similar study, over-expression of
AtCesA7 with a missense mutation (fra5) in wild-type Arabidopsis resulted in severely decreased
cellulose biosynthesis. This was hypothesised to be caused by the competitive incorporation of the
mutated AtCESA7 instead of the endogenous protein (Zhong et al., 2003). In the same study overexpression of a missense mutated AtCesA8 gene was tested to determine if it had the same effect, but
no severe phenotype was observed.
The authors did, however, not provide evidence for this
observation. AtCesA8 is the Arabidopsis orthologue of EgCesA1 (Ranik and Myburg, 2006). This
could explain why no severe phenotypic changes were observed when EgCesA1 was over-expressed.
Alternatively, the lack of phenotypic change could be explained by the fourth hypothesis; again it is
assumed that EgCESA1 protein forms and incorporates into the rosettes, but the EgCESA1 protein is
assumed to function correctly in conjunction with the endogenous CESA proteins. The reason why no
change in cellulose synthesis is observed is because up-regulation of one CesA gene is insufficient to
induce assembly of more rosettes. Mutant complementation could be used to test the third and fourth
hypotheses. Complementation of the corresponding Arabidopsis mutant (irx1) by over-expressing
EgCesA1 would provide support for the fourth hypothesis, while lack of complementation of EgCesA1
in irx1 would provide support for the first three hypotheses.
Although future work is required to further elucidate the function of EgCesA1 in Arabidopsis,
this study does not support the hypothesis that cellulose biosynthesis can be manipulated by overexpression of a single CesA gene. CESA protein phosphorylation and subsequent degradation has
been shown to occur when CesA protein levels exceed normal wild-type levels (Taylor, 2007). Thus,
125
CONCLUDING REMARKS
even over-expression of multiple CesA genes might not result in increased cellulose biosynthesis.
Another limitation may be the lack of available UDP-glucose monomers. It stands to reason that
multiple genes involved in cellulose biosynthesis, not just the CesA genes, may need to be
manipulated in order to increase the cellulose content in xylem cell walls.
Manipulation of expression levels of transcription factors that induce genes involved in
cellulose biosynthesis could result in increased cellulose content (Grotewold, 2008). MYB46 is a
transcription factor that increases expression of AtCesA7 and AtCesA8 when over-expressed (Zhong et
al., 2007a).
Other transcription factors that induce expression of genes involved in cellulose
biosynthesis include NAC transcription factors, NST1 and SND1 (Zhong et al., 2006; Zhong et al.,
2007b), but these transcription factors also induce expression of genes involved in lignin- and xylan
biosynthesis. SND2, SND3 and MYB103 have recently been shown to induce cellulose biosynthetic
pathway genes in cells undergoing secondary cell wall deposition (Zhong et al., 2008).
Over-
expression of the individual genes that transcribe these transcription factors resulted in increased
cellulose content in secondary cell walls of Arabidopsis. All three of the transcription factors were
shown to increase AtCesA8 expression when over-expressed. Manipulation of expression levels of
three CesA genes, carbon flux regulatory genes or transcription factors like SND2, SND3, MYB103
and MYB46 could result in increased cellulose biosynthesis. Future research will aim to determine
which of these strategies is best for increased cellulose biosynthesis and by how much can cellulose
biosynthesis increase before normal plant development is affected.
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126
CONCLUDING REMARKS
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128