Download Effect of ABA signalling in primary cell wall Minor Thesis Report

Effect of ABA signalling in primary cell wall
Minor Thesis Report
Charles Somi
Supervisor: Bas Dekkers
Effect of ABA signalling in primary cell wall
Minor Thesis Report
Charles Josephat SOMI
Registration number: 850906785040
Supervisor: Bas Dekkers
Examiners: Bas Dekkers
Leonie Bentsink
Chair group: Plant Physiology
Msc thesis plant physiology
Course code: 80424
August, 2015
Minor thesis research submitted as fulfilment of the requirement of the degree of Masters of
Plant Sciences at Wageningen University
Table of Contents
List of figures ......................................................................................................................... iv
List of tables ........................................................................................................................... v
Abstract .................................................................................................................................. vi
Acknowledgement .................................................................................................................... vii
1.
Introduction ................................................................................................................... 1
1.1 ABA signalling pathway ....................................................................................................... 1
1.2 The role of xyloglucan in the primary cell wall ........................................................................ 3
1.4 The mechanism of ABA action on primary cell wall .................................................................. 4
1.6 Goal and specific objectives ................................................................................................. 6
2.
Materials and Methods ..................................................................................................... 7
2.1 Plant material .................................................................................................................... 7
2.2 Methods ........................................................................................................................... 8
2.2.1 Uptake of compound in double xxt1 xxt2 mutants lines ...................................................... 8
2.3 Genotyping of single xth mutant lines ................................................................................ 9
2.3.1 DNA extraction............................................................................................................. 9
2.4 Germination assay of xth mutant lines in ABA ................................................................... 10
Assessment of loss and re-establishment of DT in mutant seeds ................................................ 10
3.
Results ........................................................................................................................ 11
3.1 Literature study ............................................................................................................... 11
3.1.1 Xyloglucan structure modification .................................................................................... 11
3.2 Uptake of compounds in double xxt1 xxt2 mutant lines ......................................................... 12
3.2.1 Germination assay under pacrobutrazol treatment........................................................... 12
3.2.2 Staining double xxt1 xxt2 mutant in Triphenyl chloride tetrazolium solution ........................ 12
3.3 Phenotyping the sensitivity of xth mutant lines in ABA ........................................................... 14
3.4 Assessment of GBF4 gene in re-establishment of desiccation tolerance (DT) ............................. 16
3.5 Genotyping of xth mutants lines......................................................................................... 17
4.
Discussion .................................................................................................................... 23
4.1 Confirmation of compound uptake ...................................................................................... 23
4.2 Confirmation of T-DNA insertion of xth mutant gene Arabidopsis ............................................. 23
4.3 Modification of xyloglucan structure .................................................................................... 24
4.4 Phenotyping the ABA sensitivity of xth mutants .................................................................... 25
4.5 Role of GBF4 gene in re-establishment of desiccation tolerance (DT) ....................................... 27
5.
Reference ..................................................................................................................... 29
iii
List of figures
Figure 1. Core ABA signalling pathway interactions across a broad range of ABA
concentrations.........................................................................................................................................2
Figure 2. Seedling establishment and germination performance of different mutants in two ABA
concentrations.........................................................................................................................................3
Figure 3. Germination response of abi5, xxt1, xxt2, xxt1 xxt2 and Col WT in different ABA
concentrations.........................................................................................................................................5
Figure 4. Schematic representation of different types of XyG and known proteins involved in its
synthesis and modification....................................................................................................................12
Figure 5. Predicted T-DNA insertion sites in singleaxy4, mur2, mur3, bgal10, xyl1, bgal10 and axy8
mutants.................................................................................................................................................13
Figure 6. Germination performance of double xxt1 xxt2 mutant lines and Col-0 in various
concentration of pacrobutrazol……….....................................................................................................14
Figure 7. Visual representation of seedling staining Columbia wildtype and double xxt1 xxt2
mutant…….............................................................................................................................................14
Figure 8. Seedling establishment and germination performance of different xth mutant and Col-0 in
ABA........................................................................................................................................................15
Figure 9. Comparison of statistical difference between xth mutant lines and Col-0 wildtype..............16
Figure 10. Re-induction of DT of germinated mutants lines in stage I and stage II..............................17
Figure 11. Predicted T-DNA insertion sites in single xth24, xth16, xth8 and xth15 mutants.................19
Figure 12-18. Gel images for xth mutant lines with forward and reverse (F+R) primers only and
including border primer (F+R+Bp) ...................................................................................................19-22
iv
List of tables
Table 1. List of single xth mutant genes that used for genotyping..........................................................7
Table 2. List of homozygous single xth mutants lines that were crossed to obtain homozygous double
xth mutants lines.....................................................................................................................................7
Table 3. List of gbf4 mutant alleles used for investigating role of gbf4 gene in re-induction of DT in
germinated Arabidopsis..........................................................................................................................8
Table 4. List of xth mutant lines used for ABA sensitivity test.................................................................8
Table 5. List of crossed homozygous xth mutant lines identified..........................................................18
v
Abstract
Abscisic acid (ABA) is one of the classical plant hormones that regulate many aspects of plant growth
and development. ABA affects plant growth and development through inhibition of cell elongation
and expansion which involve alteration of cell wall structure. However the interaction between ABA
and cell wall during growth is still unclear. Also ABA signalling involve regulation of bZIP transcription
factors that conferring expression of ABA responsive gene. To confirm the effect of ABA we tested
the two aspect; the possible link between ABA and xyglucan and role of GBF4 in re-establishment of
desiccation tolerance in germinated seeds. Our findings suggest that ABA might have possible link
with xyloglucan but to confirm the interaction between ABA and xyloglucan different enzymes that
modifying xyloglucan-cellulose network structure should be taken into account rather than XTH
family. Whereas the sensitivity of xth mutants to ABA, tested in this study was not significant differ
from Col-0 in wide range of ABA concentration. However the results of gbf4 mutant suggest that the
GBF4 do not play important role in re-establishment of desiccation tolerance because of all
parameters used scored more than 50% of desiccation tolerance after (-2.5 MPa) PEG treatment.
vi
Acknowledgement
I would like to thank my supervisor Dr. Bas Dekkers for his close supervision and inspiration during
the whole period I conduct this research study. He provided to me the guidance and ideas in both
practical experiment and report writing but also I appreciate his positive criticism towards my
mistakes. I would like to express my gratitude to Dr. Wilco, Ligterink and Professor Leonie, Bentsink
for giving me opportunity to work on this project, their technical support towards this study and their
encouragement. Final I would like to thank all Seed lab and PPH members for their support during
the whole period of this research study.
vii
1. Introduction
Abscisic acid (ABA) is a product of carotenoid that contains 15 carbon atoms structure and belongs to
the class of metabolites known as isoprenoid. ABA is derived from the oxidative cleavage of a 9-cisepoxy-carotenoid to form C15 apo-aldehydes and xanthoxin, a precursor of ABA biosynthesis in plants
(Nambara and Marion-Poll 2005; Qin and Zeevaart 1999). ABA is known to regulate diverse aspects
of plant growth and development such as embryo and seed development, dormancy, germination
inhibition, cell division and elongation and adaptation to environmental stresses like drought, salinity,
desiccation and UV radiation (Vieira et al. 2010; Finkelstein 2013). These physiological responses to
ABA occur in large part of plant phenotype and influenced by changes in gene expression. Moreover,
genetic analysis of ABA mutants presented in many studies has identified some three classes of the
ABA response genotypes. These classes are ABA deficient (aba), ABA hypersensitive and ABA
insensitive (abi) mutants. Several Arabidopsis mutants that displaying ABA hypersensitivity resulting
in diminished germination rates at low ABA concentrations and reduced water loss due to enhanced
ABA-induced stomata closure (Hugouvieux et al. 2001; Steber and McCourt 2001). While ABAdeficient mutants exhibit wilting and stunted growth under water stress due to impaired of ABA
regulated stomata closure and rate of water loss (Xiong et al. 2002; Seo et al. 2000). In spite of
phenotype and physiological change of ABA mutants, exogenous application of ABA to aba mutant
plants restore normal cell expansion and growth (Finkelstein 2013).
1.1 ABA signalling pathway
Numerous studies of molecular, biochemical and genetic research describe three main components
involves in ABA signalling pathway; type 2C protein phosphatase (PP2C), SNF1 related protein kinase
(SnRKs) and soluble ABA receptors Pyrabactin resistant (PYR/PYL) and regulatory components of ABA
receptor (RCARs). PP2Cs have a protein dephosphorylation feature and function as a negative
regulators of ABA signalling, while SnRKs have a protein phosphorylation features and act as a
positive regulator of ABA signalling (Cutler et al. 2010; Miyakawa et al. 2013). The Pyrabactin family
proteins contain a total of thirteen members which act as ABA receptors (Fujii et al. 2009). These
members are categorized as either monomeric or dimeric receptors depending on their affinity of
binding to PP2C in response to ABA (Zhang et al. 2012; Hao et al. 2011). The model (Fig. 1)
demonstrates the core pathway of ABA signalling. It shows that in absence of ABA, the monomeric
members PYL 4-10 only weakly interact with PP2C and therefore inactivate SnRKs hence ABA
signalling blocked. However, in the presence of ABA (1 µM), the same monomeric members PYL4-10
strongly interact with PP2C and releasing the SnRKs from their inhibitory complex. Therefore the
SnRKs become active and start downstream ABA signalling e.g. phosphorylation of bZIP transcription
1
factors. Dimeric members PYR1/PYL1-3 remain aggregated in both absence and at low concentration
of ABA and only dissociate and strongly interact with PP2C at higher concentrations of ABA. However,
both monomeric and dimeric receptors increase their affinity to interact with PP2C at high
concentration of ABA.
Figure 1. Core ABA signalling pathway interactions across a broad range of ABA concentrations. Monomeric
receptors (PYL4-10) weakly interact with PP2C in the absence of ABA and strong interact in low ABA
concentrations when ABA binding change their conformation. Dimeric receptors (PYR1/PYL1-3) have a lower
affinity for ABA, so remain aggregated in the absence of ABA and dissociate at high ABA concentration in order
to interact with PP2C. All receptor interactions with PP2Cs result in inactivation of the PP2Cs and derepression
of the SnRK2s, which then phosphorylate numerous proteins involved in ABA response. Green arrows indicate
activation and red bars indicate repression figure from (Finkelstein 2013)
In stress conditions such as drought or high salinity, the plant hormone ABA play important role in
stress responsive gene-expression through mainly through basic leucine zipper (bZIP) transcription
factor. The bZIP transcription factors thought to have a role in mediating ABA signalling for gene
expression (Fig. 1). A group of bZIP transcription designated as ABRE-binding (AREB) proteins or ABRE
binding factor (ABFs). The AREB/ABFs encode bZIP transcription factors and belong to the group A
subfamily, which is composed of thirteen homologs in the Arabidopsis genome (Jakoby et al. 2002).
In contrast to ABI5, other members are suggested to be involved in ABA signalling in response to
osmotic stresses (Jakoby et al. 2002; Fujita et al. 2013). However, some studies of higher order
mutants in Arabidopsis genome have shown substantial functional overlap among these bZIP factors,
2
and some cross-regulation of family members (Yoshida et al. 2010; Umezawa et al. 2010). Moreover,
(Uno et al. 2000; Choi et al. 2000) discuss widely the biochemical and genetic data of ABI5 compared
to other bZIP family members of clade A. Lack of adequate genetic data for some members of clade A
family pause the confirmation whether they perform unique or act redundantly to ABI5 or each other
in expression of responsive gene. G-BOX BINDING FACTOR (GBF) 4 is such clade A bZIP transcription
factor. Preliminary experiments that are presented in Fig. 2 show that gbf4 mutants have an ABA
insensitive phenotype. Previous studies suggest ABA induce desiccation tolerance in germinating
seeds ((Maia et al. 2014) and based on our result ABA effect can be regulated by bZIP transcription
factors. Thus, in this study we discuss the correlation between GBF4, ABI5 and the combination of
these two bZIP transcription factors in re-establishment of desiccation tolerance (DT) in germinated
seeds.
120
germination
Percentage
100
seedling
80
60
40
20
0
WT a5-7 g4-1 g4-2 DM1 DM2 WT a5-7 g4-1 g4-2 DM1 DM2
2.5 ABA
5ABA
Figure 2. Seedling establishment and germination performance of different mutants in two ABA concentrations.
Green bars represent percentage of seedling established and grey bars represent percentage of germination.
Results are expressed as average of three replicates. The abbreviations presented in the graph are genotypes of
Col-0 (WT), single mutant abi5-7 (a5-7), gbf4-1(g4-1), gbf4-2(g4-2), double mutant abi5-7 gbf4-1 (DM1), abi5-7
gbf4-2 (DM2)
1.2 The role of xyloglucan in the primary cell wall
One of the major components of the plant primary cell wall is hemicelluloses which are composed of
a heterogeneous group of polysaccharides formed by different biosynthetic routes from cellulose.
Most of hemicelluloses are functioning like cellulose, to support the material within the plant
primary cell wall which together forming complex and dynamic structure controlling cell shape and
plant morphology. The key interactions in the primary wall of dicotyledons are formed between
cellulose and the hemicellulose xyloglucan, together typically comprise about two thirds of the dry
wall mass (Fry 2004; Rose et al. 2002). During cell wall elongation and expansion the deposition of
3
new wall materials is required in order to maintain the spacing between cellulose microfibrils and
irreversibly extension of wall that make wall compartment to become large (Obel et al. 2007;
Thompson 2005). Xyloglucan (XyG) is thought to play an important role in cell wall
elongation/expansion and consequently the morphology of whole plant. XyG is also suggested to
cross-link adjacent cellulose microfibrils to form a XyG-cellulose network that create load bearing
structure of the primary cell wall. The formation of load bearing structure in primary cell wall enable
XyG to tethering cell wall matrix components thereby preventing cellulose microfibrils from
aggregating (Cavalier et al. 2008; Thompson 2005). Once deposited into the wall, XyG can undergo
metabolism and turnover, which is thought to play a role in cell elongation. Although, the correlation
of XyG metabolism turnover and biochemical changes in wall extensibility are still remain unclear but
the metabolic enzymatic activity that controlled loosening XyG-cellulose network has been suggested
to play part in cell wall elongation.
Studies of XyG in Arabidopsis plants describe that, XyG synthesis require the genes that express
the activity of xylosyltransferases enzymes (XXT1 and XXT2) and these genes may have a significant
effect on XyG biosynthesis when knocked out. Furthermore, XyG metabolism, turnover and
modification are thought to play an important role in a cell elongation. From such a role one would
predict that a plant lacking XyG should have severe cellular and tissue defects which resulting in
abnormal growth (Takeda et al. 2002; Pauly et al. 2001). Surprisingly, a double xxt1 xxt2 knockout
mutant that completely lacking detectable XyG, exhibited abnormal root hairs but relatively normal
growth and development compared to wild type plants (Cavalier et al. 2008; Zabotina et al. 2012).
This observation indicates that XyG role in wall structure and function is more subtle than previous
thought. Moreover, models of the plant cell wall postulate that XyG functions to cross-link adjacent
cellulose micro-fibrils to form a cellulose-XyG network that constitutes the major load-bearing
structure of the primary cell wall (O’Neill and York 2003; Obel et al. 2007; Albersheim et al. 2010).
Also, XyG may have a role as a spacer-molecule by preventing the formation of micro-fibrils cellulosic
aggregates (Thompson 2005; Anderson et al. 2010) or an adapter-molecule, which enables cellulose
to interact with other cell wall matrix components (Cavalier et al. 2008)
1.4 The mechanism of ABA action on primary cell wall
The control of elongation growth in plants is associated with primary cell wall modification (Taiz
1984). Controlled, turgor-driven extension of the cell requires breakage and reformation of linkages
within the cell wall matrix (Fanutti et al. 1993). Studies with embryos of Brassica napus have
provided convincing evidence that the inhibitory effect of ABA on embryo cell elongation is
associated with its suppression of cell wall loosening (Schopfer and Plachy 1985). This theory was
confirmed by measurements of in vitro extensibilities of maize coleoptile segments treated with ABA
4
and indole-3-acetic acid (IAA). In a treatment of high IAA and low ABA the growth rate of segment
was increasing without affecting the osmotic potential, hence this results suggest that a regulation
of turgor was occurred by means of modulation of the cell wall extensibility (Kutschera and Schopfer
1986). Cell wall extensibility depends on cell wall properties which are function of linkage between
polysaccharide chains in the primary cell wall (Carpita and Gibeaut 1993).
In addition, double xxt1 xxt2 mutant of Arabidopsis that completely lack of XyG shows an insensitive
phenotype to ABA nearly comparable to that of abi5-7 (ABA insensitive positive control) while the
single mutant xxt1 and xxt2 show sensitivity to small change of ABA concentration the same as
Columbia (Col-0) wild type (Fig.3 Bas Dekkers (unpublished)). These findings suggest that there is
interaction between ABA signalling and XyG.
Col WT
100
xxt1
Germination (%)
80
xxt2
xxt1 xxt2
60
abi5
40
20
0
0
0.5
1
3
5
ABA concentration (µM)
Figure 3. Germination response of abi5, xxt1, xxt2, xxt1 xxt2 and Col WT in different ABA concentrations. Lines
represented by black (Col WT), light grey (xxt1) and grey (xxt2) have similar response to small change of ABA
concentration (0-0.75 µM) while lines represented by red (xxt1 xxt2) and blue (abi5) exhibit high germination
performance at intermediate ABA concentrations (1-3 µM).
5
1.6 Goal and specific objectives
The goal of this research is to investigate two aspects related to ABA signalling:
1) Investigate the possible role of xyloglucan in ABA signalling. Previous research indicated that, the
double xxt1 xxt2 mutants that lack detectable xyloglucan exhibit insensitivity to ABA while single
xxt1 and xxt2 mutants were sensitive to ABA as Col-0 WT. Therefore the previous findings
suggest a possible link between ABA signalling and xyloglucan.
Specific objectives
•
Confirm that lack of xyloglucan in double xxt1 xxt2 mutant is capable to uptake an exogenous
applied compound.
•
Literature study on how xyloglucan structure is modified.
•
Genotyping six putative xth mutant lines to confirm the insertion of mutant.
•
To test the sensitivity of xth mutant lines to ABA
2) The role of GBF4 transcriptional factor in ABA signalling during re-establishment of desiccation
tolerance. The results obtained from the study of (Maia et al. 2014) and preliminary results
presented in Fig. 2 suggest that bZIP transcription factors ABI5 and GBF4 have significant role in
ABA signalling. Thus in this study we investigated the role of GBF4 gene on re-establishment of
DT in germinated seeds.
Specific objectives
• To investigate the role of GBF4 transcription factor in re-establishment of desiccation tolerance in
germinated seeds.
6
2. Materials and Methods
2.1 Plant material
A total of six xth single T-DNA insertion lines were used as a source of DNA samples for genotyping
xth mutant lines to identify homozygous xth mutants (Table 1). Three identified homozygous lines
were used for crossing to produce double xth mutants (Table 2). However, apart from six mentioned
xth mutant lines, two different single xth (xth4 and xth5) and the double mutant were used to
examine ABA sensitivity. Double xxt1 xxt2 mutants and Columbia wild type (Col-0 WT) were used for
testing the uptake of compound in modified/lack of xyloglucan mutants lines. In addition, single abi5,
gbf4 and double abi5-7 gbf4 mutant lines were used for examining the role of GBF4 transcriptional
factor in re-establishment of DT in germinated seeds (Table 3). In this study Col-0 and abi5-7 mutant
were used as a positive control of re-induction of DT in germinated seeds as well as ABA sensitive
and insensitive irrespectively. All single mutants lines used in this study were obtained from
Nottingham Arabidopsis Stock Centre (NASC).
Table 1. List of single xth mutant genes that used for genotyping
No.
Name of mutant xth genes
1
SAIL-692-F08
xth16-1
2
SAIL-094-H03
xth24-2
3
SAIL-693-B08
xth16-2
4
SALK-099891
xth15-2
5
SALK-065941
xth24-1
6
SALK-021499
xth8-1
Table 2. List of homozygous single xth mutants lines that were crossed to obtain homozygous double
xth mutants lines.
No.
Cross
1
xth15-2 SALK 099891 x xth24-1 SALK 065941
2
xth8-1 SALK 021499 x xth24-1 SALK 065941
3
xth8-1 SALK 021499 x xth15-2 SALK 099891
7
Table 3. List of gbf4 mutant alleles used for investigating role of gbf4 gene in re-induction of DT in
germinated Arabidopsis
No.
List gbf4 mutants alleles
Remark
1
Columbia(Col-0)
Wild type
2
gbf4-1
3
gbf4-2
4
dm gbf4-1 abi5-7
5
dm gbf4-2 abi5-7
6
abi5-7
Table 4. List of xth mutant lines used for ABA sensitivity test
No. List xth mutant lines
Remark
1
Columbia (Col-0)
Wild type
2
xth4
3
xth5-1
4
xth5-16
5
dm xth4 xth5-1
2.2 Methods
2.2.1 Uptake of compound in double xxt1 xxt2 mutants lines
Two compounds, paclobutrazol and tetrazolium were used to test the uptake of double xxt1 xxt2
mutants which completely lack of xyloglucan with comparing to Columbia (Col-0) wild type.
2.2.1.1 Germination assay in Paclobutrazol
Seeds of Col WT and double xxt1 xxt2 mutant lines were sown in trays containing two layers of blue
paper with different concentration of paclobutrazol solution, and then the seeds were stratified
three days in cold (4°C) to obtain uniform germination. After three days of stratification, the trays
containing seeds were transferred to germination chamber in 22°C with continuous white light.
Germination was scored after seven days. Percentage of germination and standard deviation
calculated based on the average of three biological replicates of both genotypes.
8
2.2.1.2 Tetrazolium staining of germinated seeds
Stratified seeds of Col-0 and double xxt1 xxt2 mutants were germinated in distilled water under
normal germination conditions of 22°C. After 48 hours of germination, the germinated seeds placed
in 1% of triphenyl tetrazolium chloride solution which was dissolved in 10mM of MES solution at pH
7.1 and incubated in dark condition at room temperature. The observation of red staining was made
twice after 4 and 24 hours of incubation.
2.3 Genotyping of single xth mutant lines
2.3.1 DNA extraction
Six single xth mutant lines grown in a rock wool block inside of a climate room at 22°C, 70% relative
humidity and a light period of 16h light and 8h dark. After 3-4 weeks a single leaf sample was picked
from each mutant plant for DNA isolation. Every collected leaf sample was placed in a 2 ml
eppendorf tube containing metal bead. Then 200 µl of DNA extraction buffer was added to each tube
and ground for 90 seconds at 2700 rpm in a dismembrator (Sanbio, Germany) and the mixture was
incubated for about 60 minutes at 60°C. The DNA extraction buffer consisted of 2M NaCl, 200 mM
Tris-HCL pH 8.0, 70 mM EDTA and 20 mM Na2S2O5. During incubation period 30 µl of 10M NH4Ac and
75 µl of isopropanol were mixed together in a 1.5 ml eppendorf tube with respect to the number of
samples incubated. After 60 minutes of incubation the samples were centrifuged at 2800 rpm for 5
minutes to separate supernatant from leaf debris. 75 µl supernatant of each sample was transferred
to new 1.5 ml tube containing NH4Ac/isopropanol mixture and then mixed thoroughly. The new
mixture kept for about 15 minutes at room temperature before centrifugation at maximum speed
(13000 rpm) for 5 minutes in order to precipitate the DNA. Then, the supernatant of new mixture
decanted and the remains of liquid drained above the tissue paper. Furthermore 300 µl of 70%
ethanol was added in order to wash the DNA pellet followed by centrifugation at maximum speed
(13000 rpm) for 3 minutes. After centrifugation the ethanol was poured out and tubes drained above
the tissue and were then dried for about 30 minutes. Finally, the pellet dissolved in 60 µl of distilled
water for overnight at 4°C.
2.3.2 Polymerase Chain Reaction (PCR) and gel electrophoresis
To screen the gene of interest of isolated DNA in section 2.3.1, the PCR reaction mix was conducted.
A total volume of 15 µl of PCR mix per sample was prepared in PCR tube by using the following
components; 0.15 µl Firepol (Solis BioDyne), 0.3 µl dNTPs, 1.5 µl of 10x buffer, 1.5 µl of MgCl2, 2 µl of
sample 9.15 µl of distilled water and 0.4 µl of either two or three primers. The PCR protocol program
was running as following; initial heating at 94°C for 4 minutes followed by 30 cycles consisting 30 sec
at 94°C, 30 sec at 60°C and 1.5 minutes at 72°C and final extension at 72°C for 10 minutes. 3 µl of
9
loading buffer were added to the obtained PCR products and then 7-10 µl of mixture was analysed
on a 1% agarose containing 20 µl ethidium bromides. The agarose placed in a gel solution with a
running electricity system. After 30 minutes running of the gel, the agarose containing DNA samples
were placed in EUROGENTEC machine and the Image was taken by using Image lab software. The
primers for different T-DNA insertion lines were designed using a web tool available at
http://signal.salk.edu/tdnaprimers.2.html
2.4 Germination assay of xth mutant lines in ABA
Seeds of the single mutant xth4, xth5-1, xth5-16, double xth4 xth5-1 mutant lines and Col-0 were
sown in trays containing two layers of blue paper under various concentrations of ABA. The seeds
were stratified at 4°C for three days then transferred to germination in continuous white light
condition and at a temperature of 22°C. After 10 days of germination, seed germination and seedling
establishment was scored. The seeds were counted to be germinated when they showed radicle
protrusion and counted as seedling when showed both root formation and green, expanded
cotyledons. The data of germinated seeds and seedling formation expressed as an average of
percentage of three biological replicates.
Assessment of loss and re-establishment of DT in mutant seeds
To assess the re-establishment of DT in germinated seeds, the two developmental stages (stage I and
II) of Col-0, single gbf4, gbf4-2, abi5-7 mutant and double abi5-7 gbf4, abi5-7 gbf4-2 mutant seeds
were selected by using a stereomicroscope and each stage were incubated in 1.2 mL of PEG solution
(-2.5 MPa) at temperature of 22°C in dark for three days according to Maia et al. (2011). Then the
germinated seeds washed thoroughly by using distilled water and transferred to new petri dishes
containing one layer of dry filter paper followed with 3 days of drying at 22°C, 30% RH with air
circulation in a closed humidity cabinet. After three days of drying the dried germinated seeds
allowed to grow in trays containing two layers of blue paper and 50 mL of distilled water at 22°C.
After 10 days, primary roots survival, cotyledon survival and seedling formation were counted and
expressed in average of percentage of three replicates from each sample.
10
3. Results
3.1 Literature study
3.1.1 Xyloglucan structure modification
XyG is made up by β-1, 4 glucan backbone that is partially substituted with xylosyl substituents at the
O-6 position and combination of side chains with various lengths (Fig. 4). These side chains generally
have one to three sugars, starting with D-Xyl then D-Gal and L-Fuc; a single-letter nomenclature (X, L,
F) corresponding to the last substituted sugar designates each type of side chain (Fry et al. 1993).
Theoretically, increase in the number of XyG tethers the cellulose micro-fibrils increasing the rigidity
of cell walls, while the modification of XyG structure may change the cell function and properties.
Modification
of
XyG
structure
can
occur
through
the
activity
of
Xyloglucan
endotransglycosylase/hydrolases (XTHs) which has been considered as an important enzyme of cell
wall-loosening (Hayashi and Kaida 2011). The XTHs exhibit xyloglucan endotransglycosidase (XET)
and/or xyloglucan endohydrolase (XEH) activities which are responsible for both the integration of
newly synthesized XyG and for polysaccharide remodelling (Rose et al. 2002; Baumann et al. 2007).
The enzyme (XET) mediates the cleavage and re-synthesis the linkages within XyG backbones. The
breaking and re-joining of XyG has been proposed as a mechanism of achieving cell wall loosening in
elongating tissue, without compromising strength (Hayashi and Kaida 2011). The enzymes XEH
modify the structure of XyG by hydrolysing and cleavage the glucan backbone and leave the short
pieces of XyG structure without re-joining them. XEH enzyme regarded as destructive enzyme of XyG
structure.
Corresponding to XTHs enzyme activities, Arabidopsis plants also have a set of glycosidase that
modify xyloglucan side chains after deposition to the primary cell wall (Sampedro et al. 2010).
Removal of XyG side chains alters the properties of both XyG polymers and cross-linking with
cellulose microfibrills (Cosgrove 2005; Park and Cosgrove 2012; Wolf et al. 2012). As depicted in (Fig.
4), XyG side chains structure can be modified by a large set of apoplastic enzymes of glycosidase or
trans-glycosidase (β-glucosidases, α-xylosidases, β-galactosidases and α-fucosidases) which act
specifically on XyG side chains group and modulating the length of side chains consequently, change
their susceptibility to XTH activity and binding properties to cellulose microfibrils (Schultink et al.
2014; Günl et al. 2011). Some of the enzymes that act on side chains are Xylosidase 1 (XYL1), Altered
Xyloglucan 8 (AXY8) and Beta-galactosidase 10 (BGAL10). For future investigation on role of side
chain group that involve in modification of XyG structure we used web tool (www.arabidopsis.org)
to predict the insertion site of T-DNA encoding mutation of activity of trans-glycosidase hydrolase
side chain enzymes . The predicted insertion sites and mutational gene are illustrated in Fig. 5.
11
3.2 Uptake of compounds in double xxt1 xxt2 mutant lines
3.2.1 Germination assay under pacrobutrazol treatment
Pacrobutrazol is a compound that functions as an inhibitor of gibberellin synthesis. This compound
inhibits cell expansion and plant growth, in mature seeds it block the germination. To test the uptake
of compound of the double xxt1 xxt2 mutant, the particular cell wall mutant together with Columbia
(Col-0) were germinated in various concentrations of pacrobutrazol for 7 days. Both Col-0 and dm
xxt1 xxt2 exhibit insensitivity to pacrobutrazol at the concentration below 25 µM whereas the
germination was approximately 100%. From 25 µM an abrupt drop in germination decreasing to 45%
for dm xxt1 xxt2, 63% for Col-0 and final reach to less than 10% at concentration of 75 µM and 150
µM. Both Col-0 and double xxt1 xxt2 mutant has the same trends of germination performance with
regards to the pacrobutrazol treatment.
3.2.2 Staining double xxt1 xxt2 mutant in Triphenyl chloride tetrazolium solution
Early stage seedlings of Col-0 and double xxt1 xxt2 mutant lines were collected and incubated in 1%
of triphenyl chloride tetrazolium solution for about 24 hours in dark condition at room temperature.
Before incubation germinated seed from all genotype has white root and green cotyledons. In
general, seedlings of both genotypes stained red after 24 hours of incubation in tetrazolium solution.
Despite the fact that all genotypes staining red, the Col-0 stained darker red compared to double
xxt1 xxt2 mutant lines (Fig. 7). Moreover, cotyledons of all seedlings were staining deep red colour
than roots and hypocotyl.
Figure 4. Schematic representation of different types of XyG and known proteins involved in its synthesis and
modification. Arrows and scissors indicate the site of glycosyltransferase or glycosyl hydrolase activity,
respectively.
12
3’
5’
XYL1
SALK 034796.55
SAIL 735-F06
5’
SALK 039200
3’
BGAL10
SAIL 129169
SALK 150946
5’
3’
AXY8
SALK 132995
SALK 150930
SAIL 314-D02
XYL2
SAIL 151-A07
SALK 139678
3’
5’
MUR2
SALK 1178
SAIL 143-A03
5’
3’
MUR3
SAIL 8-B06
SALK 034613
SAIL 716-E10
5’
3’
AXY4
SAIL 800-CO4
SALK 044972
Figure 5. Predicted T-DNA insertion sites in singleaxy4, mur2, mur3, bgal10, xyl1, bgal10 and axy8 mutants. The
insertion of T-DNA marked inside the boxes; white boxes represent insertion sites either in coding or noncoding region while yellow boxes represent the insertion is the promoter. Non-coding region represented by
light blue boxes, coding regions represented by deep blue boxes and Introns are represented by blue lines.
13
120
% germination
100
80
60
Col-0
dm xxt1xxt2
40
20
0
0
5
25
75
150
Pacrobutrazol concentration (µM)
Figure 6. Germination performance of double xxt1 xxt2 mutant lines and Col-0 in various concentration of
pacrobutrazol. White and black bars represent percentage of germination of Col-0 and dm xxt1 xxt2 mutant
lines, respectively. The data was obtained after seven days of germination and expressed as average of
percentage of germination from three replicates ± SD
Col-0
dm xxt1 xxt2
Figure 7. Visual representation of seedling staining red colour Col-0 (left) and dm xxt1 xxt2 (right) after
incubated in 1% triphenyl chloride tetrazolium solution for about 24 hours in dark at room temperature.
3.3 Phenotyping the sensitivity of xth mutant lines in ABA
ABA can block both seed germination and seedling formation. In study (Hilhorst 1995) suggested that
ABA may suppress the enzymatic hydrolysis of load bearing bond (XTH activity) in the cell wall. In our
studytwe tested the ABA sensitivity of single xth4, xth5-1, xth5-16 mutant and double xth4 xth5-1,
xth4 xth5-16 mutant lines with comparison to Col-0 (ABA sensitive genotype). Three replicates of
each xth mutants lines and Col-0 grown in different ABA concentration. After 10 days we scored
14
radicle emergence and seedling formation as germinated seeds while the presence of both roots and
cotyledon scored as seedling formation. In general, the increase of ABA phenotype sensitivity of
both xth mutant lines and Col-0 were depending with d the increase of ABA concentration. However,
single xth5-1 mutant show high sensitivity phenotype at 1 µM ABA (whereas, xth5-1 has less than 50%
of seedling formation compared to other genotypes) (Fig. 8a). On the other hand dm xth4 xth5-1 and
xth5-16 exhibit germination insensitivity to ABA even at 2 µM ABA whereas have higher than 50% of
germination compared to other genotypes. Although both xth mutants lines and Col-0 had similar
trend of phenotype sensitivity to ABA but all of them display zero percentage of seedling formation
at 3 µM ABA.
The analysis of statistical difference was done based on the overview trends of seedling
establishment and germinated seeds with respect to various ABA concentrations. The trends in Fig.
8a and 8b show that two concentration of ABA (1 µM and 2 µM) exhibit the variation of seedling
formation and germination between genotypes irrespectively. At 1 µM ABA, only seedling formation
of xth5-1 displayed significant difference to Col-0. However the seedling formation of other xth
mutant were not significant differ from each other but also with comparison to xth5-1 and Col-0 (Fig.
9a). Comparison of percentage of germination, interesting is the xth5-1 has lowest percentage of
germination and were significant differ compared to all genotypes at concentration of 2 µM ABA
while dm xth4 xth5-1 and xth5-16 were significant differ compared to Col-0 at the same
concentration (2 µM ABA) (Fig. 9b).
120
120
100
100
Percentage (%)
Percentage (%)
Germinated seeds
b)
Seedling formation
a)
80
60
40
20
80
60
40
20
0
0
0
1
2
ABA conc (µM)
3
0
1
2
ABA conc (µM)
Col-0
xth4
Col-0
xth4
xth5-1
xth5-16
xth5-1
xth5-16
dm xth4 xth5-1
3
dm xth4 xth5-1
Figure 8. Seedling establishment (a) and germination performance (b) of different xth mutant and Col-0 in
various ABA concentrations obtained after 10 days of germination. Lines represented by black (Col-0), red
15
(xth4), green (xth5-1), purple (xth5-16), and blue (dm xth4 xth5-1). All data expressed as average of percentage
from three replicates
b)
1 µM ABA
100
90
80
70
60
50
40
30
20
10
0
ab
ab
ab
b
a
% germination
% seedling formation
a)
2 µM ABA
100
90
80
70
60
50
40
30
20
10
0
cd
c
b
bd
a
Figure 9. Comparison of statistical difference between xth mutant lines and Col-0. Graph (a) represents
percentage of seedling establishments in 1 µM ABA while graph (b) represent percentage of germination in 2
µM ABA. All data are expressed as average of percentage from three replicates and standard deviation
expressed in error bars. The same letter means no significant difference. dm represent xth4 xth5-1 mutant.
3.4 Assessment of GBF4 gene in re-establishment of desiccation tolerance (DT)
Previous studies have shown that DT can be fully re-established in germinated seeds of Arabidopsis
(Vieira et al. 2010; Maia et al. 2011; Maia et al. 2014). ABA has been shown to be important for the
induction of DT in germinated seeds. Based on results in Fig. 2 the sensitivity variation of gbf4 mutant
in two concentration of ABA led to question whether the GBF4 gene has function in other
mechanism related to ABA. Therefore, we tested whether DT could be re-induced in gbf4 mutants
and the abi5-7 gbf4 double mutant by treating them in a PEG (-2.5 MPa) solution for three days in
dark at room temperature. Based on reports (Maia et al. 2014) re-induction of DT in germinated
mutants seeds depend on development stages. Therefore, we defined two clearly distinct
developmental stages to assess the re-induction of DT which are stage I (testa rupture), stage II
(seeds at radicle protrusion).
To assess the re-induction of DT we used three parameters; survival of the cotyledon, survival of the
primary root and seedling formation. Single gbf4, gbf4-2, abi5-7 mutant, double abi5-7 gbf4, abi5-7
gbf4-2 mutants and Col-0 lines were used in this study. In overall more than 50% of all parameters
(primary root, cotyledon and seedling formation) from both stages I and II were being rescued after
desiccation due to PEG treatment. In stage I, double mutant abi5-7 gbf4, abi5-7 gbf4-2 have lower
percentage of primary root compared to single mutant and Col-0 while the percentage of cotyledon
16
survival and seedling formation of all mutant were almost similar to Col-0 at the same stage (Fig. 10a).
In stage II, the double mutant abi5-7 gbf4-2 has the lowest percentage primary root survival
compared to other mutant lines and Col-0. As well as the percentage of cotyledon survival and
seedling formation were similar across all mutants and Col-0 (Fig. 10b).
Stage I
Stage II
b)
120
120
100
100
Desiccation tolerance(%)
Desiccation tolerance (%)
a)
80
60
40
20
0
80
60
40
20
0
P. Root
Cotyledon
seedling
P. Root
Cotyledon
seedling
Col-0
abi5-7
Col-0
abi5-7
gbf4
gbf4-2
gbf4
gbf4-2
dm abi5-7 xgbf4
dm abi5-7 x gbf4-2
dm abi5-7 xgbf4
dm abi5-7 x gbf4-2
Figure 10. Re-induction of desiccation tolerance of germinated mutants lines in stage I (a) and stage II (b).
Desiccation tolerance was determined after drying the germinated seeds with previous 3d PEG treatment,
followed by rehydration. Survival of cotyledons, primary roots (P.root) and seedlings formation was scored 10 d
after rehydration. Each data point is the average percentage of two replicates. Bars represent standard error.
3.5 Genotyping of xth mutants lines
A total of six single xth mutant lines (Table 1) were genotyped to test their of T-DNA insertion gene
with comparison to Col-0 wild type gene. The T-DNA insertion was determined by using
Polymerization Chain Reaction (PCR) method and verified in comparison of theibands size with DNA
fromCol-0 wild type. We used both two (F+R) primers and three (F+R+Bp) primers to test the
homozygosity of mutant lines as instructed on http://signal.salk.edu/tdnaprimers.2.html. By using
reaction of either two or three primers, the Col-0 DNA (no insertion) should give a product of about
17
900-1100 basepairs (bPs). For DNA of xth homozygous mutant should give a blank in the reaction of
two primers while can give the product lower than 900 bPs in the reaction of three primers. However,
the DNA of xth heterozygous mutantive a product (900-1100 bPs) in the reaction of two primers and
the same sample give lower than 900 bPs in the reaction of three primers. In this study, we used
several independent plants from each single xth mutant lines as a source of DNA material to screen
for the T-DNA insertion. Before genotyping, firstly we determined sequence flanking insertion site of
the T-DNA xth mutant. Different mutant genes have shown to be inserted at different region (Fig. 12).
Genotyping was done in two rounds; first was done with large number of samples to get an overview
of band size of T-DNA inserted xth mutant compared to Col-0 DNA. Out of six xth mutant tested only
three lines were identified to be homozygous due to having lower bands of DNA fragments
compared to Col-0 in the reaction of three (F+R+Bp) primers and no bands at all in the reaction of
two (F+R) primers (Fig. 13, 14, 15, 16, 17 and 18) and these three lines were confirmed to be
homozygous in second screening of few samples (Fig. 19)
The confirmed xth homozygous mutant lines were used to develop double xth mutants. All three
single xth mutant lines were crossed each other in multiple flowers of plants (Table 5), however two
mutants (xth15-2 SALK 099891, xth24-1 SALK 005941) were used both as male and female genotype
while xth8-1 SALK 021499 used as a male genotype only. The double xth mutant generated from
crossing of these three genotypes will be used for further research in determining their sensitivity to
ABA and other secondary factors.
Table 5. List of homozygous xth mutant lines identified and crossed to generate double xth mutant
No.
Cross
1
xth15-2 SALK 099891 x
xth24-1 SALK 005941
2
xth8-1 SALK 021499 x
xth24-1 SALK 005941
3
xth8-1 SALK 021499 x
xth15-2 SALK 099891
18
3’
SALK
005941
5’
XTH24
SAIL 692 F08
5’
3’
XTH16
SALK 021499
3’
5’
XTH8
3’
5’
XTH15
SALK 099891
Figure 11. Predicted T-DNA insertion sites in single xth24, xth16, xth8 and xth15 mutants. The insertion of TDNA marked inside
the boxes;
white boxes represent insertion sites either in coding or non-coding region while
xth16-1
SAIL-692-F08
yellow boxes represent the insertion is the promoter. Non-coding region represented by light blue boxes,
1
2
3
4
5
6
WT
(-)
F+R+Bp primers
F+R primers
coding regions represented by deep blue boxes and Introns are represented by blue lines.
Figure 12. Gel image for xth16-1 SAIL-692-F08 mutant lines with forward and reverse (F+R) primers only (above)
and including border primer (F+R+Bp) (below). From left to light: samples 1-6 are single xth16-1 mutant lines,
WT (Col-0), (-) is the water and smart ladder marker. The short bold lines are the bands of DNA fragments
illustrate the presence of the gene. All DNA fragments of xth16-1 have the same band size as Col-0 (WT) in all
primers reactions.
19
1
2
3
4
5
6
7
8
9
10
11
WT
WT
(-)
F+R+Bp primers
F+R primers
xth16-2 SAIL-693-B08
Figure 13. Gel image for xth16-2 SAIL-693-B08 mutant lines with forward and reverse (F+R) primers only
(above) and including border primer (F+R+Bp) (below). From left to light: samples 1-11 are single xth1612mutant lines, WT (Col-0), (-) is the water and smart ladder marker. The short bold lines are the bands of
DNA fragments illustrate the presence of the gene. All DNA fragments of xth16-2 have the same band size as
Col-0 (WT) in all primers reaction.
1
2
3
4
5
6
7
8
9
10
11
12
13 WT WT (-)
F+R+Bp primers
F+R primers
xth24-1 SALK-065941
Figure 14. Gel image for xth24-1 SAIL-06941 mutant lines with forward and reverse (F+R) primers only (above)
and including border primer (F+R+Bp) (below). From left to light: samples 1-13 are single xth24-1 mutant
lines, WT (Col-0), (-) is the water and smart ladder marker. The short bold lines are the bands of DNA
fragments illustrate the presence of the gene. All DNA fragments of xth24-1 have no band in (F+R) primers
and lower band size in (F+R+Bp) primers compared to Col-0 (WT).
20
1
2
3
4
WT
(-)
F+R+Bp primers
F+R primers
xth24-2 SAIL-94-H03
Figure 15. Gel image for xth24-2 SAIL-94-H03 mutant lines with forward and reverse (F+R) primers only
(above) and including border primer (F+R+Bp) (below). From left to light: samples 1-4 are single xth16-1
mutant lines, WT (Col-0), (-) is the water and smart ladder marker. The short bold lines are the bands of
DNA fragments illustrate the presence of the gene. All DNA fragments of xth24-2 has the same band size as
Col-0 (WT)
1
2
3
4
5
6
7
8
9
10
11
WT
WT
(-)
F+R+Bp primers
F+R primers
xth15-2 SALK-099891
Figure 16. Gel image for xth15-2 SAIL-099891 mutant lines with forward and reverse (F+R) primers only
(above) and including border primer (F+R+Bp) (below). From left to light: samples 1-13 are single xth15-2
mutant lines, WT (Col-0), (-) is the water and smart ladder marker. The short bold lines are the bands of DNA
fragments illustrate the presence of the gene. All DNA fragments of xth15-2 have no band in (F+R) primers
and lower band size in (F+R+Bp) primers compared to Col-0 (WT).
21
xth8-1 SALK 021499
2
3
4
5
6
7
8
9
10
WT
WT
-
F+R+Bp primers
F+R primers
1
Figure 17. Gel image for xth8-1 SALK-021499 mutant lines with forward and reverse (F+R) primers only (above)
and including border primer (F+R+Bp) (below). From left to light: samples 1-10 are single xth8-1 mutant lines,
WT (Col-0), (-) is the water and smart ladder marker. The short bold lines are the bands of DNA fragments
illustrate the presence of the gene. All DNA fragments of xth8-1 have similar band size in all primers reactions
except sample 1 which has no band in F+R and lower band size in (F+R+Bp) primers compared to Col-0 (WT).
xth15-2 SALK099891
2 3
WT (-)
1
2
3
WT (-)
xth8-1 SALK021499
1
WT
(-)
F+R+Bp primers
F+R Primers
1
xth24-1 SALK 065941
Figure 18. Confirmation gel image for single xth15-2, xth24-1 and xth8-1 mutant lines with forward and reverse
(F+R) primers only (above) and including border primer (F+R+Bp) (below). From left to light: numbers are
samples of xth mutant lines, WT (Col-0), (-) is the water sample and smart ladder marker. The short bold lines
are the bands of DNA fragments illustrate the presence of the gene. All DNA fragments of xth have no band in
(F+R) primers and lower band size in (F+R+Bp) primers compared to Col-0 (WT).
22
4. Discussion
In previous studies, it has been demonstrated that the double xxt1 xxt2 mutant which completely
lack detectable XyG were insensitive to ABA compared to Col-0 Wt. The insensitivity of double xxt1
xxt2 mutant to ABA indicate that ABA signalling and XyG may have interaction during plant growth
and development. ABA is phytohormone that regulate diverse aspect of plant physiology particularly
adaptation to environmental stress (Umezawa et al. 2010). As shown in (Fig. 2) the sensitivity to ABA
shown by gbf4 mutant suggests that bZIP transcription factor might have a role in response to
adaptation to various stress condition. Therefore we investigated two aspects of ABA signalling in
this study.
•
To investigate possible link between ABA and XyG.
•
To determine the role of GBF4 gene in the re-induction of desiccation tolerance.
4.1 Confirmation of compound uptake
The double xxt1 xxt2 mutants that completely lack detectable XyG were highly insensitive to ABA but
the permeability of this mutant was questionable due to change of the properties of cell walls. As
result depicted the double xxt1 xxt2 mutant exhibit the same response to pacrobutrazol as Col-0
whereas both genotypes were highly sensitive to pacrobutrazol from concentrations of 75-150 µM
(Fig. 6). Furthermore, the germinated seed of both Col-0 and double xxt1 xxt2 mutant incubated in
tetrazolium solution under dark condition at room temperature. After 24 hours both genotypes staining
to red colour (Fig. 7). Both observation confirm that the double xxt1 xxt2 mutant were able to uptake
the compound. Therefore the phenotype insensitivity of double xxt1 xxt2 mutants to ABA was caused
by change in response to ABA signalling and not cell wall permeability. In addition, the change in
response also confirmed in reaction of tetrazolium with germinated seeds whereas the Col-0 WT
exhibit deep red colour compared to double xxt1 xxt2 mutants.
4.2 Confirmation of T-DNA insertion of xth mutant gene Arabidopsis
Insertional mutagenesis is an alternative means of disrupting gene function and is based on the
insertion of foreign DNA into the gene of interest. The use of either transposable elements or T-DNA
is the mutual technique used in Arabidopsis mutagenesis. The T-DNA mutagenesis also acts as a
marker for subsequent identification of the mutation. The preference of using T-DNAs as the
insertional mutagen rather than transposable element is that T-DNA insertions will not transpose
subsequent integration within the genome and are therefore chemically and physically remain stable
over multiple generations (Martienssen 1998; Wisman et al. 1998). .
23
In our study we tested the homozygosity of six xth mutant lines. We used PCR method to determine
the T-DNA insertion in the Arabidopsis. We found that only three xth mutant were homozygous (Fig.
18). The confirmation were done based on size of the product after reaction in both two (F+R) and
three (F+R+Bp) primers. Other mutant lines were not homozygous although before claimed that
have been inserted mutation.
4.3 Modification of xyloglucan structure
The polysaccharide XyG consists of a β-1, 4 glucan chain that is attached with a certain pattern of
glycosyl groups (Fig. 4). The structure of XyG can be modified through the mechanism of metabolic
enzymes which either cleavage the backbone of XyG or modulate the lengths of side chain. XTH
activity play a role in modification and newly synthesized xyloglucan molecules into the cell wall
and/or the tethering of newly deposited microfibrils by xyloglucans and/or restructuring of the
existing cellulose/xyloglucan network. furthermore (Miedes et al. 2011) suggests that some XTH
isoenzymes use cellulose-xyloglucan complexes as substrates, and that they gradually trim the
xyloglucans that extend from the microfibril surface, a feature that should favour wall loosening.
However, unexpectedly results observed in previous studies of XTH family of rice, thereby xth mutant
exhibit negligible growth phenotype (Kaewthai et al. 2013; Hara et al. 2013). One of possible reason
raised for this apparent lack of phenotype in XTH transgenic lines within the rice XTH family is
functional genetic redundancy, as was shown in the Arabidopsis XTH family. Therefore the effect of
ABA in XTH activity may not be seen due to redudancy effect of XTH family members therefore we
recommend to analyze the change of gene expression in order to have cruel the response of
particular gene.
Additionally, side chains group also may affect interactions of XyG with cellulose and possibly
interaction with other matrix polymers of the cell wall. Several glycosyltransferase and exoglycosidases found in plant cell walls have shown to be able to modify xyloglucan and/or xyloglucan
oligosaccharides. Glycosidase enzyme activities have been found in the apoplast and possibly also
present within the endomembrane system during polysaccharide biosynthesis and transport
(Vissenberg et al. 2000; Iglesias et al. 2006). They modulate the side chain group which lead to
change in XyG binding properties and structure. Three classes of apoplastic glycosidase enzymes
were identified to act on Arabidopsis XyG; the xylosidase Altered Xyloglucan 3 (AXY3)/Xylosidase 1
(XYL1), the fucosidase Altered Xyloglucan 8 (AXY8), and the galactosidase Beta-galactosidase 10
(BGAL10) (Eklöf and Brumer 2010; Günl et al. 2011). These glycosidase enzymes acted cooperatively
on xyloglucan oligosaccharides (XLFG), leading to the sequential formation of XXFG, XXLG, XXXG,
GXXG and XXG (Schultink et al. 2014; Tuomivaara et al. 2015). In previous studies of Arabidopsis XyG
24
found that the axy8 mutant have a higher degree of fucosylation than that of wild type plants,
suggesting AXY8 is active on XyG both in the apoplast and in the endomembrane system (Günl et al.
2011). Furthermore, the Arabidopsis xyl1 mutant exhibit higher abundance of XyG xylosylation, while
the abundance of galactosylated XyG structures was increased in the bgal10 mutant. Therefore both
glycosidases seem to affect plant development as siliques are dwarfed in these mutants (Günl and
Pauly 2011; Sampedro et al. 2010; Sampedro et al. 2012). Although, the increased in abundance of
fucosylated XyG oligosaccharides observed in Arabidopsis axy8 mutant but this mutant exhibit
unexpectedly results in XyG, that contains unusual XyG oligosaccharides such as XFG, GFGXXXG, and
GFGXXFG (Günl et al. 2011). In addition, the double axy8 axy3 mutant display the XyG structure
consists of a 1:1 ratio of the XyG oligosaccharide motifs XXXG and XXFG in contrast to XLFG, XXFG,
XXLG, XLXG and XXXG motifs usually found in Arabidopsis tissues. This data provides evidence that
apoplastic glycosidases also play an important role in structurally modification of XyG oligosaccharide
motifs found in the wall.
The structure of XyG can also be modified through the xylosyl substituents that most commonly
with a galactosyl-residue. Glycosyltransferase enzymes MURUS 2(MUR2), MURUS 3 (MUR3),
Xyloglucan L-sidechain Galactosyltransferase Position 2 (XLT2), and XyG O-acetyltransferase
identified as Altered Xyloglucan 4 (AXY4) were suggested to regulate the galactosyl-residue that
responsible for XyG xylosyl substituents. MUR2, MUR3 and XLT2, all act to transfer glycosyl-moieties
to the same O-2 position of the XyG xylosyl-residue (Jensen et al. 2012). AXY4 is responsible Oacetylated the galactosyl-residue in Arabidopsis. Although absence of the O-acetyl substituent on
XyG in the various tissues was not reported to affect tissue growth or morphology but may influence
polymer solubility as well as enzymatic accessibility so that facilitate modification of XyG structure
(Gille et al. 2011).
Based on different side chain group that seems to alter the structure of XyG, It is possible the
enzymes acting in a side chain group might have interaction with ABA during growth and
development of plant. Therefore we suggest further research on testing the ABA effect on knock out
mutant that lack these enzymes. In addition, to generate the exo-glycosidase and glycosyltransferase
mutant the predicted insertion site Fig. 5 can be used to design the mutant lines.
4.4 Phenotyping the ABA sensitivity of xth mutants
XTH family is the important enzymes which reported to play a great role in cell elongation and
expansion. Previous studies suggest that expression of XTH activity can be enhanced by different
plant hormone such as gibberellin acid (GA), auxin and brassinosteroid (Uozu et al. 2000; Catalá et al.
2000). Therefore one may assume that ABA supresses the enzymatic hydrolysis of load bearing
25
structure (XTH activity) in the primary cell wall because ABA act antagonistic to GA as well as based
on result in Fig. 3 the mutant line that lack XyG were insensitive to ABA. In this study we used single
xth4, xth5-1, xth5-16 mutant and double xth4 xth5-1 mutant lines to test the sensitivity of xth mutant
lines on ABA. The observation suggest that the phenotype sensitivity of xth5-1 mutant were
significant differ from that of Col-0 at 1 µM ABA. While other mutants have the same response to
ABA as Col-0 as well as xth5-1. On the other hand the percentage of germination of all mutants
except xth4 were significant different from Col-0 only at 2 µM ABA. The seedling formation of other
mutants was not significant different from Col-0 at all ranges of ABA concentration.
These observations does not support with the early assumption that ABA suppresses the XTH
activity rather than it proof the results obtained by Kaewthai et al. (2013) whereas no phenotypic
difference between xth mutant and Col-0. Basically the response of all mutants genotypes was
similar to Col-0 toward the increase of ABA concentration (Fig. 9a, 9b). However single xth5-1 and
xth5-16 mutant lines expected to have the same response to ABA because have the same insertion
gene type and only differ in alleles position, surprising is significant difference observed in xth5-1
when compared to Col-0 while xth5-16 were not (Fig. 9a). The possible reason can be due to large
variation in seedling formation within the replicates of xth5-16 mutant lines. In general xth5-1
mutant result the lowest percentage of both seedling formation and germination compared to other
genotypes tested together. Moreover the same response of mutant lines as exhibited by xth5-1 can
be shown by lines that having multiple knock out of genes (Krysan et al. 1999). This observation
confirms that xth5-1 mutant respond different to ABA compared to other xth mutant genotypes.
Double xth4 xth5-16 and xth5-16 mutant show slightly insensitivity to ABA at 2 µM while other
mutants and Col-0 have lower than 50% germination (Fig. 9b). This observation suggest that xth4 has
functional redundancy to xth5-16 in response to ABA thus double xth4 xth5-16 mutant exhibit
significant higher germination percentage than xth4 but not significant differ to xth5-16 at the same
ABA concentration.
Furthermore, the study of (Park and Cosgrove 2015; Miedes et al. 2011) question the validity of
XTH activity in loosening the cell wall during cell elongation and expansion. XyG characteristically
tethers the cellulose microfibrils and constitute XyG-cellulose network in the form of load bearing
structure. This XyG property suggested to strengthen the wall of the cell and for cell to elongate the
load bearing should be broken and resynthesized again. To confirm XTH activity (Park and Cosgrove
2012) investigated the function of three endoglucanases from glycosyl hydrolase family 12
(www.cazy.org): Xyloglucan-specific endo-β-1,4-glucanase (XEG), which digests xyloglucan but not
cellulose; cellulose-specific endoglucanase (CEG), which cuts unbranched glucans (cellulose) but not
xyloglucan, and cellulose and xyloglucan hydrolyzing endoglucanase (CXEG) which cuts both
26
xyloglucan and cellulose. Of the three enzymes only CXEG loosened the cell wall, assessed by cell wall
creep and wall compliances. These results showed that XEG-accessible xyloglucan did not strengthen
the wall. A combined treatment with CEG and XEG might be expected to mimic the effect of CXEG,
but unexpectedly such treatment did not loosen the wall. Therefore efficient digestion of load
bearing structure would require an enzyme with both xyloglucanase and cellulase activity. This
requirement could explain why enzymes from the XTH family do not cause cell wall creep (McQueenMason et al. 1993; Saladié et al. 2006) and why XTH mutants exhibit negligible growth phenotype
compared to Col-0.
4.5 Role of GBF4 gene in re-establishment of desiccation tolerance (DT)
Arabidopsis seeds like other seeds have a tendency to become desiccation sensitive and lose their DT
immediate after germination. Germinated Arabidopsis seeds when subjected to extreme drying, they
loss ability to form seedling. However, DT of Arabidopsis seeds can be full re-established in a cleardefined developmental window when submitted to a mild osmotic stress (Maia et al. 2011). Reinduction of DT in Arabidopsis germinated seed is also concomitant depends on regulation of some
gene that encodes bZIP transcription factor. According to (Maia et al. 2014) the DT of germinated
Col-0 can re-induced in stage I (testa rupture), stage II (radicle protrusion) and stage III (root hair)
when submitted to PEG (-2.5 MPa) solution for three days. In contrast to abi5-7 mutant whereas DT
can be full re-induced in stage I and II while only cotyledon survival observed in stage III (Maia et al.
2014). In our study we investigated the role of GBF4 gene in re-induction of DT in germinated seeds
under treatment of PEG (-2.5 MPa) solution for three days. In stage I, we obtained similar DT of
primary roots in single abi5-7 and gbf4, slightly low in gbf4-2 and apparent lower DT of primary roots
in double abi5-7 gbf4-2 mutant (Fig. 10a). The same lower DT of primary roots and seedling
formation have shown by double abi5-7 gbf4-2 mutant in stage II (Fig. 10b) Furthermore, the
cotyledon survival of all mutant genotype was almost similar to Col-0 in both stage I and II. In study
of Maia et al. (2014) abi5 mutant showed to re-induce full DT of primary roots, cotyledon and
seedling in both stage I and II under treatment of PEG (-2.5 MPa). In our experiments the
performance of single abi5-7 and gbf4 mutants seems to be similar in all aspects of parameters.
Although GBF4 and ABI5 both encode the bZIP transcription factor that regulated by stress and
belong to the same clade A group which comprises 13 members of transcription factors (Jakoby et al.
2002; Yoshida et al. 2010) but our result suggest that these particular genes are not functional
redundantly each other. If the GBF4 and ABI5 were function redundantly, the DT of double knockout
mutant of these genes would be higher than the single mutant gene. This was not observed in our
result rather than double abi5-7 gbf4-2 mutant has lower DT in primary root and seedling formation
compared to single mutants in stage II. It is possible that the ability of double abi5-7 gbf4-2 mutant
27
to re-establish DT is limited to certain development stage after germination. Based on the results
observed we conclude that GBF4 does not play important role in re-establishment of DT in both
stage I and II of germinated seeds and may be GBF4 is not regulated by endogenous ABA synthesized
within plant. However, in order to confirm the role of GBF4 gene in re-induction of DT at
development stages, further investigation on the change in expression of GBF4 genes during reinduction of DT is required.
28
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