The Plant Cell Wall Integrity
Sensing Mechanism
A thesis submitted to The University of Manchester for the
degree of
Doctor of Philosophy in the Faculty of Life Science
2013
Dat Luen Tsang
Table of Contents
Table of Figures ..................................................................................................................................... 2
Abstract ................................................................................................................................................ 3
Declaration ........................................................................................................................................... 4
Copyright Statement ............................................................................................................................. 5
List of Abbreviation ............................................................................................................................... 6
Chapter 1: Introduction ........................................................................................................................ 8
1.1 Plant Growth: Cell Division, Cell Expansion and Hormonal Control ........................................... 8
1.2 Cell Wall Biosynthesis............................................................................................................... 14
1.3 The Consequences of Cell Wall Deficiency ............................................................................... 17
1.4 The Cell Wall Integrity Pathway ............................................................................................... 19
1.5 Candidate Sensors of Cell Wall Integrity in Arabidopsis ........................................................... 24
Aims and Objectives ........................................................................................................................... 28
Chapter 2: Results ............................................................................................................................... 30
Cell Wall Integrity Signalling via an ACC Dependent, Ethylene-Independent Pathway ....................... 30
2.1 Inhibitors of Cellulose Biosynthesis or Crystallization Reduces Root Cell Growth .................... 30
2.2 Short Term Responses to General Stress is ACC Dependent but Ethylene Independent ......... 33
2.3 Signalling Downstream of ACC in Short Term Stress Responses .............................................. 45
Screening for Loci Involved in Perceiving Damage to Cell Wall Integrity ............................................ 50
2.4 Transcriptional Changes in Roots 1 hour After Cell Wall Stress ............................................... 50
2.5 An S-Locus Receptor Kinase is Required for the Early Response to Isoxaben .......................... 54
Chapter 3: Discussion and Conclusion ................................................................................................ 60
3.1 Early Responses to Cell Wall Stress .......................................................................................... 60
3.2 ACC Signals the Rapid Response to General Stress .................................................................. 61
3.3 S-Locus Receptor Kinase Plays a Role in Responding to Cell Wall Integrity Damage. ............... 66
3.4 Conclusion ............................................................................................................................... 68
Materials and Methods....................................................................................................................... 71
Acknowledgement .............................................................................................................................. 78
References .......................................................................................................................................... 79
Supplemental Data ............................................................................................................................. 80
Word Count = 22,744
1
Table of Figures
Figure 1. Left-Longitudinal section of the Arabidopsis root. ..................................... 10
Figure 2. Hormonal cross-talk involved in the regulation of root growth and
development. .......................................................................................................... 11
Figure 3. Illustrations of the cellulose synthase complex composed of thirty-six CesA
subunits. ................................................................................................................. 15
Figure 4 CWI signaling pathway in yeast ................................................................ 21
Figure 5. The short term response of Arabidopsis roots to isoxaben. ...................... 32
Figure 6. Compounds that binds to cell wall polysaccharides and inhibit their
crystallization leads to cell wall stress. .................................................................... 33
Figure 7. Inhibitors of ACC synthase or ACC oxidase restore root cell elongation in
the presence of isoxaben. ....................................................................................... 35
Figure 8. Blocking ethylene perception have no effect on the short term isoxabeninduced growth arrest. ............................................................................................ 36
Figure 9. Controls for the blocking of ethylene responses in roots with silver
thiosulfate. .............................................................................................................. 37
Figure 10. The etr1-3 response to ethephon and isoxaben ..................................... 38
Figure 11. The effects of ACS inhibitors on ACC-induced root growth arrest .......... 40
Figure 12. Novel ACS inhibitors. ............................................................................. 42
Figure 13. An ACC-dependent pathway regulates responses to both cell wall
damage and PAMPs. .............................................................................................. 44
Figure 14. Isoxaben-mediated root growth reduction requires both auxin and ROS. 46
Figure 15. DELLA is not involved in regulating growth in early responses. .............. 47
Figure 16. MAPK activation by isoxaben, flagellin22 and Congo red. ...................... 49
Figure 17. The early transcriptional response to cell wall stress. ............................. 52
Figure 18. Comparison of Isoxaben induced LEH in 4-day old Col-0 and T-DNA
knock-out seedlings. ............................................................................................... 55
Figure 19. A- Domain organisation of At1g61390 .................................................... 57
Figure 20. The transcriptional and LEH responses in At1g61390 related genes. .... 58
Figure 21. A working model for the regulation of cell wall homeostasis. ................. 70
Figure S1. Two clusters of isoxaben specifically induced genes ............................. 80
2
Abstract
The Plant Cell Wall Integrity Sensing Mechanism
The University of Manchester
Dat Luen Tsang
The Degree of Doctor of Philosophy in the Faculty of Life Science
Plant cell walls are dynamic and responsive structures rather than rigid cages.
Disruption of cell wall integrity prevents cell expansion: for example, inhibition
of cellulose biosynthesis with isoxaben rapidly reduces root elongation. This
is not a passive consequence of mechanical failure but an active response
regulated by cell wall integrity (CWI) sensing pathway. Interestingly, the plant
response to pathogen-associated molecular patterns (PAMPs) like flagellin
shares many similarities with the CWI pathway response; such as the
production of reactive oxygen species (ROS) and lignin. While root elongation
is also reduced by flagellin, microarrays show that the two treatments lead to
a very different expression profile. Little is known about the mechanisms and
components of the CWI pathway.
Here we show that inhibiting the biosynthesis of 1-aminocyclopropane-1carboxylic acid (ACC), the immediate precursor of the plant hormones
ethylene, restores elongation in roots treated with isoxaben but exacerbates
the extent of the cell wall damage. Unexpectedly, ethylene itself is not
required for the process. Further experiments show that the response to flg22
in roots also requires this putative signalling pathway. Auxin appears to work
downstream of ACC since inhibiting auxin activity prevents growth reduction in
roots treated with ACC, isoxaben or flg22
We have also identified a receptor-like kinase (SRK) which is required for
growth repression in root cells that are experiencing cell wall damage.
Expression of this gene is rapidly and transiently induced by isoxaben. SRK
(S-locus RLK) is part of a highly expanded locus of RLKs related to selfincompatibility proteins in Brassica. However, while other genes of this family
are induced by cell wall stress, only SRK mutants are insensitive to acute cell
wall damage. Further characterisation of the SRK is under way to confirm its
role in the CWI pathway.
3
Declaration
No portion of the work referred to in the thesis has been submitted in support
of an application for another degree or qualification of this or any other
university or other institute of learning
4
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5
List of Abbreviation
ACC
-
1-aminocyclopropane-1-carboxylic-acid
Ag
-
Silver Thiosulfate
AIB
-
a-Aminoisobutyric acid
AOA
-
aminooxyacetic acid
BR
-
Brassinosteroid
CBI
-
Cellulose biosynthesis inhibition
CEV
-
Constitutive expression of vegaetative storage protein
COB
-
Cobra
CORE -
Conditional root expansion
CSI
-
Cellulose synthase interactive protein
CUD
-
Cudgel
CWI
-
Cell wall integrity
DAMP -
Damage associated molecular pattern
DPI
-
Diphenylene iodonium
ECM
-
Extracellular matrix
ELI
-
Ectopic lignin
GA
-
Gibberellic acid
GEF
-
Guanine exchange factor
GPI
-
Glycosylphosphatidylinositol
HCE
-
Hierarchical Clustering Explorer
HPRG -
Hydroxproline-rich glycoproteins
IAA
-
Indole-3-acetic acid
JA
-
Jasmonic acid
KOR
-
Korrigan
LIT
-
Lion’s tail
LRR
-
Leucine-rich repeat
MAPK -
Mitogen-activated protein kinase
NBD
-
2,5-norbornadiene
OG
-
Oligogalacturonides
PAMP -
Pathogen associated molecular pattern
PEO-IAA
α-(phenylethyl-2-oxo)-indole acetic acid
6
PDF
-
Plant Defensin
PKC
-
Protein Kinase C
PLT
-
Plethora
PPB
-
Preprophase band
PRC
-
Procuste
QUI
-
Quill
RAM
-
Root apical meristem
RLK
-
Receptor-like kinase
S4B
-
Pontamine fast scarlet 4B
SCR
-
Scarecrow
SDS-PAGE
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
SHR
-
Short-root
SOS
-
Salt overly sensitive
THE
-
Theseus
VSP
-
Vegetative storage protein
WAK
-
Wall associated kinases
7
Chapter 1: Introduction
1.1 Plant Growth: Cell Division, Cell Expansion and Hormonal Control
Plants cells lack the ability to migrate during development, a characteristic
feature of animal embryogenesis. Therefore, the shape and size of plant
organs is determined by proliferation and cell expansion alone. Overall control
of these processes is exerted by plant hormones, which in turn respond to
genetic programmes and environmental signals. I will discuss these
processes mainly in the context of root growth, the experimental system used
in this thesis.
Cell division is dependent on the formation of a cytoskeletal structure called
the phragmoplast (Smith, 2001). The growth of the phragmoplast is driven
towards a set of cortical microtubules in belt-like arrangement located at the
division plane called the preprophase band (PPB) (Smith, 2001). The
alignment of the phragmoplast with the PPB is required for correct cell wall
formation at the division site (Smith, 2001). Cell wall materials are transported
along microtubules associated with the phragmoplast via golgi-derived
vesicles (Smith, 2001). The decision to divide at the right time and location is
required for proper organogenesis; the plant does this by intercellular
communication (Dupuy et al., 2010). Auxin is required for division, as seen in
cells lacking auxin are unable to divide until auxin is reinstated (Tréhin et al.,
1998). Auxin mediates cell division by promoting the cell cycle progression
within a cell (Himanen et al., 2002; Magyar et al., 2005; Harashima et al.,
2007). Plant cells may undergo symmetrical or asymmetrical division.
Asymmetrical cell division plays a central role in the development of plants
8
(ten Hove and Heidstra, 2008). It occurs during the first zygotic division,
vascular development, the formation of guard cells and the generation of the
cell lineages found in roots (Abrash and Bergmann, 2009; Rasmussen et al.,
2011). The coordinated division and expansion of cells from the root apical
meristem (RAM) provides cells for the root cap below and all other cell types
of the growing root above: outer epidermal layer, cortex and endodermis, and
the central stele that later differentiates into phloem and xylem (figure 1). The
cortex and endodermal cell columns initiates from a group of stem cells called
the cortex-endodermal initials (Dolan et al., 1993; Scheres et al., 2002). These
cells undergo an anticlinal division producing a daughter cell which is identical
to the parent and another cell which forms the cortex and endodermis cells by
periclinal division (Dolan et al., 1993; Scheres et al., 2002).
Following several rounds of cell division, these cells reorganise the
cytoskeleton, deposit cell wall material and develop a large central vacuole in
preparation for rapid cell expansion in the elongation zone of the root
(Verbelen et al., 2006). This elongation increases cell size by an order of
magnitude and happens over just six to eight hours for each cell (Beemster
and Baskin, 1998).
9
Figure 1. Left-Longitudinal section of the Arabidopsis root. The colours represent the
different cell types. Right- The cortex/ endodermis initial and its daughter undergo two
divisions to form the cortex/endodermis cell columns (Scheres et al., 2002).
Multiple hormonal pathways are involved during the development of roots.
Hormones often work antagonistically or synergistically to regulate cell
proliferation and elongation during development. The complexity of hormone
interactions during root growth is reviewed in Benkova & Hejatko (2009)
(figure 2).
The concentration gradient of auxin plays a vital role in root development and
it is actively maintained by auxin transporters (Sabatini et al., 1999). The auxin
gradient determines the location of the quiescent centre (Sabatini et al., 1999).
Auxin induces the expression of the AP2 class of transcription factors,
PLETHORA1 (PLT1) and PLT2 (Aida et al., 2004). In turn, PLT1 and PLT2
promote the expression of the GRAS transcription factors SCARECROW
(SCR) and SHORT-ROOT (SHR), which is required for the asymmetrical
10
division of the cortex-endodermal initials (Benfey et al., 1993; Di Laurenzio et
al., 1996; Pysh et al., 1999; Sabatini et al., 2003; Heidstra et al., 2004;
Levesque et al., 2006; Scheres, 2007). Rapidly elongating root cells are also
Figure 2. Hormonal cross-talk involved in the regulation of root growth and
development. Dashed lines correspond to not completely clear or mostly indirect
regulations. c.d. is transition zone where differentiation starts (Benkova and Hejatko,
2009)
sensitive to ethylene, as seen in seedlings grown with the ethylene precursor,
ACC (Le et al., 2001; Alonso et al., 2003). Ethylene interacts with several
other hormones in the root, in particular auxin (Ivanchenko et al., 2008).
11
Interestingly,
the
interaction
between
the
two
hormones
can
work
antagonistically, as in the abscission of fruits and flowers (Brown, 1997), or
synergistically, as in root elongation (Swarup et al., 2002). It has been shown
that ethylene activity in roots requires auxin as mutations in the auxin
transporters (AUX1 and PIN2) (Wilson et al., 1990; Roman et al., 1995;
Luschnig et al., 1998) lead to ethylene insensitivity which can be restored by
the addition of auxin (Stepanova et al., 2005). Furthermore, mutations in auxin
receptor (TIR1) (Alonso et al., 2003) and the AUX/IAA regulators axr2/iaa7
(Wilson et al., 1990) and axr3/iaa17 (Leyser et al., 1996; Swarup et al., 2007)
all confer ethylene insensitivity. Auxin upregulates the ethylene biosynthetic
pathway (Yang and Hoffman, 1984; Bleecker and Kende, 2000) via its rate
limiting enzyme, 1-aminocyclopropane-1-carboxylic-acid (ACC) synthase
(Tsuchisaka and Theologis, 2004). Ethylene in turn promotes auxin
biosynthesis by upregulating genes involved in auxin biosynthesis, such as
TAA1 (Stepanova et al., 2008; Tao et al., 2008), ASA1 and ASB1 (Stepanova
et al., 2005). This positive feedback loop is then fine-tuned and negatively
regulated by POLARIS (PLS) (Casson et al., 2002; Chilley et al., 2006). The
PLS gene is induced at the root tip where auxin accumulation is high and it
acts as an ethylene biosynthesis repressor, but the PLS gene itself is also
downregulated by ethylene (Chilley et al., 2006). Ethylene negatively
regulates root growth by inducing auxin biosynthesis and the production of the
auxin transportation components, PIN1, PIN2 and AUX1 (Ruzicka et al., 2007).
Auxin is then transported down the roots and inhibit cell elongation and
therefore growth in elongation zone (Ruzicka et al., 2007).
12
Brassinosteroids (BRs) also affect root growth in a concentration dependent
manner. At lower concentration, BRs have stimulatory effects on root growth,
but inhibitory effects at higher concentration (Müssig et al., 2003). Such
mechanisms may be used to generate a specific localised response. While
several studies have shown examples where BR works independently of
auxin and ethylene (Clouse et al., 1992; Clouse et al., 1993; Arteca and
Arteca, 2001), part of the BR effects on root growth involve the ethylene and
the auxin pathways (Benkova and Hejatko, 2009). BRs have been shown to
induce genes involved in ethylene biosynthesis and response in roots (Arteca
and Arteca, 2001). It has also been shown that the expression of auxin
response genes is affected in the BR deficient mutant brx which can be
restored to the wild type level by adding brassinolide (Mouchel et al., 2006).
Ethylene also works antagonistically with gibberellic acid (GA). GA promotes
root growth by destabilizing DELLA proteins, a family of growth repressors (Fu
et al., 2002). Ethylene counteracts GA-induced root growth by stabilising
DELLAs (Achard et al., 2003; Guo and Ecker, 2004).
While hormonal control of cell division in the root acts on the cell cycle and
stem cell maintenance (Himanen et al., 2002; Aida et al., 2004; Magyar et al.,
2005; Harashima et al., 2007). The wall undergoes extensive restructuring
during cell expansion (Carpita and Gibeaut, 1993; Cosgrove, 1993; Refregier
et al., 2004; Cosgrove, 2005). This requires the controlled loosening of the cell
wall to promote anisotropic growth driven by the increase in turgor pressure,
as well as the rigidification at the end of cell elongation (Carpita and Gibeaut,
1993; Cosgrove, 1993; Refregier et al., 2004). Several families of proteins are
in place to regulate this physical restructuring of the cell wall during different
13
stages of development. Among them are the expansins (Cosgrove, 2000),
which are small secreted proteins that bind directly to cellulose and induce the
loosening of the cell wall prior to rapid cell elongation. Expansins disrupt the
non-covalent adhesion between cellulose and hemicelluloses (Cosgrove,
2000). They are activated in acidic conditions which are generated by
brassinosteroid and auxin activated ATPase proton pumps at the plasma
membrane
(Cosgrove,
2000).
The
efflux
of
H+
also
causes
the
hyperpolarization of the plasma membrane and leads to the activation of a
voltage-dependent K+ channel (Philippar et al., 1999; Philippar et al., 2004).
The influx of K+ ions promotes the intake of water and generates hydrostatic
pressure which will lead to cell expansion. At the end of cell elongation, cell
wall integrity is re-established by a process of rigidification. Extensins, a family
of rod-like, hydroxproline-rich glycoproteins (HPRGs) are responsible for this
step (Baumberger et al., 2001; Baumberger et al., 2003). They are found
within the cell wall and can form covalently crosslinked networks with other
extensins at tyrosines residues that are evenly distributed along the protein
(Baumberger et al., 2001).
1.2 Cell Wall Biosynthesis
Cellulose is produced in vivo from UDP-glucose by the cellulose synthase
family (CesA1-10) in Arabidopsis; out of which CesA1,2,3,5 and 6 are
associated with primary cell wall production (Fagard et al., 2000; Burn et al.,
2002; Desprez et al., 2007). These CesA are found in large multimeric protein
complexes arranged as a hexameric rosette structure (figure 3A) observed by
freeze-fracture electron microscopy on the cell surface (Mueller and Brown,
1980; Ding and Himmel, 2006). Each of these rosettes comprises six globular
14
domains which always hold CesA1 and CesA3 in combination with one other
type of primary CesA subunits (Persson et al., 2007). This arrangement
suggests that thirty six
Figure 3. Illustrations of the cellulose synthase complex composed of thirty-six CesA
subunits. A- Proposed arrangement of a CesA subunit hexamer. B- Six hexamers form
a rosette-like array (Ding and Himmel, 2006) that synthesizes thirty six glucan chain
along the plasma membrane (C).
individual glucan chains are simultaneously synthesized and extruded into
extracellular space (figure 3C; Delmer, 1999; Richmond, 2000). This
assumption is compatible with the size of the microfibrils found in primary cell
walls. However, other data suggest that 18 or fewer individual glucan chains
form microfibrils (Chanzy et al., 1978; Ha et al., 1998; Kennedy et al., 2007).
The correct formation of the cell wall requires KORRIGAN, endo-1,4-ß -dGlucanase, also known as cellulase (Nicol et al., 1998). While kor mutant
does not affect the cellulose content within the cell wall, the structure of the
15
cell wall is altered [92]. The kor mutation does affect the rate at which CesA
complexes migrate across the cell surface (Paredez et al., 2008). Together,
these findings indicate that KORRIGAN is required for proper cellulose
microfibril arrangement (Hayashi et al., 2005).
The cellulose microfibril is generally deposited transversely to the axis of
elongation. This arrangement promotes longitudinal expansion by restricting
radial expansion (Refregier et al., 2004). Cortical microtubules were frequently
observed to lie parallel to cellulose microfibrils; this give rise to the alignment
hypothesis (Baskin, 2001). Combined imaging of fluorescently tagged CesA6
and tubulin has shown that cellulose synthase particles move bidirectionally
along a linear array at an steady velocity of 330nm/min at the plasma
membrane, and that their trajectories do indeed align with microtubules
(Paradez et al., 2006; Paredez et al., 2006). Cellulose microfibril deposition
can occur without associated microtubules (Sugimoto et al., 2003); treatment
with microtubule depolymerising drugs does not alter the CesA6 trajectories,
indicating CesA mobility is independent of intact cortical microfibrils (Paredez
et al., 2006). Instead, cellulose synthase mobility requires its enzyme activity,
as it has been shown that CesA6 mobility can be stopped by the cellulose
synthase inhibitor, isoxaben (Paredez et al., 2006). A model elaborated by
stochastic simulations suggests that cellulose synthase complex is propelled
by the force of cellulose microfibril generation (Diotallevi and Mulder, 2007).
These observations conclude that cortical microtubules are not required for
CesA mobility but rather serve as a guide to the direction of CesA movements
which is motored by the polymerization of glucose residues. Supporting this
model is the discovery of the cellulose synthase interacting protein (CSI1) as
16
a likely physical linkage between the CesA complex and the cortical
microtubules (Conti et al., 1998; Hatzfeld, 1999; Gu et al., 2010; Li et al.,
2012). Although cellulose biosynthesis is not affected in the csi1 mutation,
microfibril alignment is less uniform specifically in the cell wall of root
elongation and mature zones (Gu et al., 2010).
1.3 The Consequences of Cell Wall Deficiency
Many defects in cell wall biosynthesis lead to reduced cell elongation (Desnos
et al., 1996; Fagard et al., 2000; Gu et al., 2010). This phenotype has allowed
the identification of cell wall mutants based on their cell expansion phenotype
when grown in conditions where root growth is maximized (Conditional Root
Expansion (CORE) mutants; Hauser et al., 1995). The lion’s tail-1 (lit1) mutant,
later identified as allelic to Korrigan (Nicol et al., 1998; Scheres et al., 2002),
is the only CORE mutant showing root cells with significantly smaller volume
than the wild type. In the cobra (cob) mutant, the polarity but not the extent of
cell expansion was affected (Schindelman et al., 2001; Roudier et al., 2002;
Roudier et al., 2005). COBRA is a GPI-anchored protein which is polarly
localized at the longitudinal cell surface of the roots and may regulate oriented
cell expansion (Schindelman et al., 2001). A group of CORE mutants that
have lost the ability to regulate the extent of cell expansion includes quill (qui),
pom-pom1 (pom1), pom-pom2 (pom2), and cudgel (cud). Quill was identified
as an allele of procuste1 (prc1), a mutation in the cellulose synthase, CesA6
(Fagard et al., 2000). Both qui and prc1 exhibit a strong radial expansion
phenotype when grown in a high sucrose medium (Fagard et al., 2000).
Interestingly, the combination of two or more CORE mutations causes root
17
growth impairment even in permissive conditions where no phenotype were
observed in the individual CORE mutants (Hauser et al., 1995). CORE genes
seem partially redundant in normal but not maximal root expansion (Hauser et
al., 1995).
Cell wall damage has also been shown to induce ethylene mediated defense
responses in some mutants (Ellis and Turner, 2001). Several CesA mutants
with defects in cellulose biosynthesis have been shown to induce the ethylene
and jasmonic acid (JA) signaling pathway (Desnos et al., 1996; Ellis and
Turner, 2001; Sugimoto et al., 2001). Two allelic CesA3 mutations, cev1 and
eli1, that were identified by the constitutive expression of VSP1 and ectopic
lignin deposition, respectively, displayed ethylene dependent enhanced
resistance to powdery mildew (Ellis and Turner, 2001; Ellis et al., 2002; CañoDelgado et al., 2003). Genes related to abiotic and biotic stress, such as
VSP1 (Berger et al., 1995) and PLANT DEFENSIN 1.2 (PDF1.2) (Penninckx
et al., 1998; Caño-Delgado et al., 2003) were significantly upregulated (CañoDelgado et al., 2003). Inhibition of cellulose biosynthesis with isoxaben in the
wild type phenocopies this effect (Hamann et al., 2009; Denness et al., 2011).
Several cell wall mutants also display sugar sensitive phenotypes. The link
between sugar metabolism and cell walls is evident in a mutation of
MUR4/HSR8, a UDP-D-xylose-4-epimerase (Li et al., 2007). A recent report
has shown that short term isoxaben treatment affects carbohydrate
metabolism, and that this effect is osmosensitive (Wormit et al., 2012).
Together, the effects of cell wall damage have shown that cell wall integrity is
closely linked with multiple processes in plants. It is likely that a signalling
18
pathway monitoring cell wall integrity (CWI) is in place to regulate the plant
throughout its development.
1.4 The Cell Wall Integrity Pathway
The concept of a pathway which monitors the performance and integrity of the
plant cell wall, with input from developmental processes (cell cycle, expansion)
and external signals (cell-cell contacts, mechanical stress, pathogens) was
first proposed by Somerville et al. in 2004 (Somerville et al., 2004). The plant
CWI pathway model has been updated in recent reviews (Seifert and Blaukopf,
2010; Wolf et al., 2012). In addition to the mutant and inhibitor data described
above, clear evidence for a cell wall integrity regulating pathway came from
observation of the effect of cell wall damage on dark grown hypocotyls
(Refregier et al., 2004). The cell wall of etiolated Arabidopsis hypocotyls
becomes considerably thinner during the rapid growth phrase, and the original
thickness of the cell wall is only restored after cells have reached their
maximal length. Before the transition to rapid expansion, deposition of a large
amount of newly synthesised cell wall material is necessary to maintain a safe
thickness of the wall during elongation, as reflected in the high expression
levels of primary wall CesA genes (Caño-Delgado et al., 2003; Desprez et al.,
2007; Persson et al., 2007). As mutants with impaired cellulose biosynthesis
show (Hauser et al., 1995; Fagard et al., 2000), inability to keep up with the
demand for cellulose production significantly affects growth. When etiolated
seedlings are transferred to medium containing isoxaben within 48h after
germination –but not later-, they show severely inhibited hypocotyl elongation
(Refregier et al., 2004). Refregier et. al. hypothesized that cell wall integrity is
required before commitment to growth acceleration into hypocotyl (Refregier
19
et al., 2004). However, once accelerated growth has initiated, disrupting cell
wall integrity no longer affects hypocotyl length (Refregier et al., 2004). This
finding suggests that the stunted growth triggered by cellulose deficiency was
not due to structural weakness but rather a response to a regulatory system
monitoring cell wall integrity. Similarly, the short hypocotyl phenotype of the
prc1 mutant, caused by cellulose deficiency, can be partially restored by a
suppressor mutation in the theseus1 (the1) locus (see below) without
restoring cellulose biosynthesis; providing yet more evidence for a cell wall
integrity (CWI) sensing system (Hématy et al., 2007; Hématy and Höfte, 2008).
Many aspects of the plant CWI pathway have yet to be uncovered. The well
understood yeast cell wall integrity pathway (figure 4) is often used for
comparison with the plant’s CWI pathway (Somerville et al., 2004; Humphrey
et al., 2007; Hamann and Denness, 2011). Cell wall damage in yeast is
detected by several mechanosensors at the cell surface; these include Mid1p
(Locke et al., 2000), Mid2p (Ono et al., 1994), the cell Wall integrity and
Stress response Component Wsc1p (Gray et al., 1997; Verna et al., 1997;
Jacoby et al., 1998) and their homologues Mid Two-Like Mtl1p and Wsc2-4p
(Verna and Ballester, 1999). These mechano-sensors contain antenna-like
structures formed by o-mannosylation, which are essential for their function
(Lodder et al., 1999; Philip and Levin, 2001), and detect plasma membrane
stretch caused by the increased turgor pressure after cell wall damage has
been sustained. Upon receiving cell wall damage, Mid1p activates a stretchactivated calcium channel, Cch1p (Paidhungat and Garrett, 1997; Kanzaki et
al., 1999; Locke et al., 2000). The resulting calcium influx activates calcineurin,
which switches on genes associated with cell wall integrity (Batiza et al.,
20
1996). The activated mechano-sensors also initiate a protein kinase C (PKC)
– mitogen-activated protein kinase (MAPK) cascade (Levin, 2005).
Figure 4 CWI signaling pathway in yeast proposed by Levin et al. (2005). Changes in
the state of the cell wall are detected by sensor proteins (Mid1,2 and Wsc1,2,3) at the
plasma membrane (PM). These proteins activate a central regulator, the small GTPase
Rho1, via Rom1/2. Rho1 in turn activates a number of effectors such as the glucan
synthase FKS1/2, the exocyst component Sec3 and the actin-organizing formin, Bni1.
Protein kinase C (Pkc1) activates the downstream components of the CWI pathway
(Rlm1, Swi4/6 and Skn7) to promote transcription of e.g. Fks2.
The cascade begins by the binding of the activated mechano-sensor, such as
Wsc1p and Mid2p, to Rom2 (Philip and Levin, 2001). Rom2 is a guanine
nucleotide exchange factor (GEF) of Rho1. The GTP bound Rho1 then
activates PKC1 in the CWI pathway and eventually activates a number of
21
effectors involved in the response to cell wall damage, including the FKS1, the
ß 1,3-glucan synthase(Levin, 2005).
The role of this pathway in maintaining cell integrity is evident in yeast
mutants with defects in CWI components. These typically show altered cell
morphology, misplaced and disoriented cell walls or even cell lysis (Lee et al.,
1993; Martin et al., 1993; Lodder et al., 1999; Philip and Levin, 2001; Tobias
et al., 2005). Although there are no obvious plant orthologs to many of the
proteins involved in the yeast CWI pathway (in particular, no comparable
sensors), the role of such a pathway and the consequences of defects should
be similar. For example, the mutation in FKS1, which encodes a 1,3-ß -Dglucan synthase that produces that the major structural component of the
yeast cell wall, leads to the constitutive activation of the pathway (de Nobel et
al., 2000); comparable to the effects observed in the CesA mutants (Desnos
et al., 1996; Fagard et al., 2000; Ellis and Turner, 2001; Ellis et al., 2002).
Reduced elongation and a loss of growth anisotropy in roots that manifests as
radial swelling are hallmarks of cell wall deficient mutants in Arabidopsis
(Tsang et al., 2011). In plants as in yeast, cell wall damage will cause plasma
membrane stretch due to turgor pressure. Mechanosensors at the cell surface
may play the role of monitoring cell wall damage. Such similarities have
enabled the isolation and characterization of functional plant orthologs by
using yeast complementation assays. For example, the Arabidopsis
mechanosensor, MCA1, was isolated and characterised by its ability to
complement the lethal phenotype of the mid1 mutant in S. cerevisiae
(Nakagawa et al., 2007). MCA1 and the intracellular turgor pressure are
required for several responses to isoxaben (Hamann et al., 2009; Denness et
22
al., 2011; Wormit et al., 2012). Consistent with the observations in cev1, a
dramatic increase in the JA concentration was observed in seedlings treated
with isoxaben (Hamann et al., 2009; Denness et al., 2011). Treatment with
sorbitol neutralizes the isoxaben effects on JA production and the expression
of JA responsive genes (Hamann et al., 2009).
In plants, damaged cell walls release wall-derived signaling molecules which
trigger specific cellular responses (Aziz et al., 2007; Galletti et al., 2009; Wolf
et al., 2012). These endogenous molecules with elicitor activity released
during biotic and abiotic stress are termed damage-associated molecular
patterns (DAMPs) (Zipfel, 2009). The pectin derived oligogalacturonides (OGs)
are the best characterized DAMPs in plant (Ferrari et al., 2013). OGs are
elicitors of defense response (Cervone et al., 1989) and are speculated to be
involved in the regulation of development due to their antagonistic effect on
auxin (Bellincampi et al., 1993; Savatin et al., 2011). The biological activity of
OGs require the formation of a Ca 2+- mediated intermolecular cross-linking
structure resembling egg boxes (Braccini and Pérez, 2001). The maturation of
the egg box conformation is required for binding to its receptor, a family of
Wall-Associated Kinases or WAKs (see below) (Cabrera et al., 2008).
Perturbation of CWI in plants may be sensed via the release of cell wall
fragments or the resulting mechanical stress on the plasma membrane (Zipfel,
2009; Denness et al., 2011; Wormit et al., 2012; Ferrari et al., 2013).
Plant, animal, fungal, and algal systems share similarities in that the
communication and physical linkage between the extracellular matrix (ECM)
and the cell plays a fundamental role in cell growth and division (Fowler and
Quatrano, 1997; Lukashev and Werb, 1998; Trotochaud et al., 1999; Li et al.,
23
2012).Proteins found on the cell wall or at the cell surface may function as a
sensor for any of these stimuli. In the recent years, several receptor kinases
are found and speculated as CWI sensors (Humphrey et al., 2007; Seifert and
Blaukopf, 2010; Steinwand and Kieber, 2010).
1.5 Candidate Sensors of Cell Wall Integrity in Arabidopsis
There is strong evidence that WAKs are cell wall integrity sensors (Wagner
and Kohorn, 2001). WAKs contain an extracellular domain that binds very
tightly to pectin (Wagner and Kohorn, 2001). WAK expression is strongly
induced in response to pathogens invasion, aluminum toxicity and other
stresses (He et al., 1998; Wagner and Kohorn, 2001; Sivaguru et al., 2003).
The non-lethal WAK2 null mutant (wak2-1) shows a decreased rate of cell
expansion (but mature cell lengths like the wt) in conditions where
endogenous carbon supplies are limited (Kohorn et al., 2006). This phenotype
can be rescued by the addition of sucrose, fructose or glucose, but not nonmetabolisable sugars (Kohorn et al., 2006), suggesting that wak2-1 have a
defective sugar metabolism. The expression of the vacuolar invertase
(AtvacINV1), which regulates cell expansion by controlling the turgor pressure,
in wak2-1 roots was reduced by 60% (Kohorn et al., 2006). WAK2 is likely to
be involved in sensing cell wall integrity and regulating cell expansion during
development, wounding and changes to the balance of carbohydrates
(Kohorn et al., 2006). WAKs may monitor cell wall integrity via their tight
interaction with pectin and signal their response via the intracellular kinase
domain.
24
Several members of the Catharanthus roseus-like RLKs (CrRLK1L) have
been implicated to be CWI sensors (Hématy et al., 2007; Hématy and Höfte,
2008). Loss-of-function mutations of theseus (the1) partially suppress the
short hypocotyl phenotype of prc1 (Hématy et al., 2007). Overexpression of
THE1 in prc1-1/the1-1 exacerbates the prc1-1 phenotype while it has no
effect on the wild type (Hématy et al., 2007). The prc phenotype is caused by
cellulose deficiency (Fagard et al., 2000), and the1 interestingly restores
hypocotyl growth in prc1-1 without restoring cell wall integrity, indicating that
the role of THESEUS is in signaling, not cell wall repair (Hématy et al., 2007).
In vitro autophosphorylation of THE1 confirmed that it is an active kinase
(Hématy et al., 2007). THE1 is unlikely to be activated in response to general
cell wall damage since the THE1 mutation failed to restore the hypocotyl
growth in kor1 (Hématy and Höfte, 2008). The actual stimulus for THE1
activation may be the defects in cellulose synthesis or the loss of intact CesA
complex. It was concluded that THE1 actively inhibits cell elongation upon
perturbation of cellulose synthesis and induces ectopic lignin accumulation
(Hématy and Höfte, 2008). FERONIA (FER), HERKULES1 and HERKULES2
(HERK) of the CrRLK1L family also regulate cell elongation (Guo et al., 2009).
Furthermore, these CrRLK1Ls are induced by BR, indicating their role in BR
regulated cell growth (Guo et al., 2009).
Recently two leucine-rich repeat receptor-like kinases (LRR-RLK) with a role
in regulating cell wall function were identified in Arabidopsis (Xu et al., 2008).
Disruption of both FEI1 (At1g31420) and FEI2 (At2g35620) (but not either
single mutant) causes a conditional swollen-root phenotype on high sucrose
medium (Xu et al., 2008). The kinase activity of FEI is required for its optimal
25
activity but not essential for its function (Xu et al., 2008). The fei1fei2 double
mutation renders the root hypersensitive to isoxaben; similar to what can be
seen in wild type plants grown in a high sucrose medium or in the prc1-1 (Xu
et al., 2008). Furthermore, cellulose biosynthesis is affected in fei1fei2 in nonpermissive conditions (Humphrey et al., 2007; Xu et al., 2008). FEI1 and FEI2
appear to work independently from the pathway triggered by the cobra and
procuste mutations as their defects are additive to those observed in fei1fei2
(Xu et al., 2008). In contrast, FEI1 and FEI2 are epistatic to Salt Overly
Sensitive5 (SOS5), a putative cell surface adhesion protein with AGP-like and
fasciclin-like domain that is required for normal cell expansion (Shi et al.,
2002). Interestingly, FEI1FEI2 function appears to be dependent on ACC, the
precursor of ethylene, but independent of ethylene signaling (Xu et al., 2008).
Yeast two-hybrid assays showed that the intracellular kinase domain of FEI1
and FEI2 interacts with ACS5 and ACS9 independently of kinase activity (Xu
et al., 2008). Xu et al. (2008) proposed that FEI kinase play a role in
regulating cell wall architecture by recruiting ACS or complex ACS with other
proteins such as SOS5 (Xu et al., 2008).
Protein phosphorylation plays a vital role in signalling; often activated by
receptor kinases initiating a phosphorylation cascade. Kinase activity has
been shown to be required for optimal function of some putative cell wall
sensor kinases (Wagner and Kohorn, 2001; Xu et al., 2008) but substrates
have yet to be found for most of them. Previous studies have shown that
many processes (Jonak et al., 1999; Bent, 2001) such as touch (Mizoguchi et
al., 1996), stress (Nakagami et al., 2005b), plant defence (Frye et al., 2001;
Pitzschke et al., 2009a) and the ethylene signalling pathway (Yoo and Sheen,
26
2008), in plants are signalled via mitogen-activated protein kinases (MAPKs).
Evidence suggests that MAPKs signalling also plays a role in CWI signalling;
similar to the CWI pathway observed in yeast (Levin, 2005; Kohorn et al.,
2009). Pectin fragments activate MAPKs and induce multiple genes
associated with cell wall biogenesis and pathogen response in a WAK2
dependent manner (Kohorn et al., 2009). MAPKs also play a major role in
reactive oxygen species (ROS) signaling in that production of ROS is both a
trigger and consequence of the activation of MAPKs signaling pathway
(Nakagami et al., 2004; Nakagami et al., 2005a; Pitzschke et al., 2009b). ROS
production is required for root growth regulation (Kwak et al., 2003). Plant
respond to cellulase treatment with ROS production, leading to OXI1dependent
activation
of
MPK3
and
MPK6,
indicating
that
protein
phosphorylation and ROS are involved in the CWI signaling/ response (Rentel
et al., 2004). It is likely that different types of cell wall sensors are in place to
monitor different types of cell wall damage and initiate the appropriate
response via a complex network of ROS, protein phosphorylation and
hormones. Chronic cell wall deficiency, as cause by genetic defects in cell
wall biosynthesis and maintenance, leads to an exaggerated response to cell
wall damage. It is likely that the role of the CWI pathway in structural cell wall
homoeostasis during normal development is much more subtle, which makes
its study more challenging.
27
Aims and Objectives
The current understanding of the plant cell wall is no longer that of a dead,
rigid structure whose sole purpose is to provide mechanical support to the
plant cell. Similar to extracellular matrix signaling in animals and fungi, plant
cells respond to signals from the cell wall generated during development or by
the environment. In the past decade, more and more information about this
CWI pathway has been revealed. Several putative CWI sensors have been
identified based on their role in regulating cell expansion (Wagner and Kohorn,
2001; Hématy et al., 2007; Xu et al., 2008). Changes to cell expansion in
response to cell wall deficiency had been linked with protein phosphorylation
cascades, hormonal and ROS signaling, but the actual mechanism linking the
perception of cell wall damage to the activation of these signaling pathways
has yet to be determined (Ellis and Turner, 2001; Rentel et al., 2004; Kohorn
et al., 2009).
The aim of this project is to study short-term responses to acute cell wall
damage and to identify molecular components of the CWI signalling pathway.
Objective 1: Establishment of an experimental system for the study of
acute cell wall damage responses. So far, the majority of what is known
about the response to cell wall damage is based on plants that have
developed in the presence of permanent cell wall damage, leading to extreme
responses such as the severe growth defects seen in rsw1 (Baskin et al.,
1992; Peng et al., 2000).It is likely that the original purpose of the CWI
response is to closely regulate or to rapidly restore cell wall integrity during
development, biotic and abiotic stress. Roots are excellent models for
analyzing the CWI response due to their high demand for cellulose
28
biosynthesis during rapid elongation (Beemster and Baskin, 1998). Le et al.
have developed a system to record fast responses to ethylene in root cell
elongation (Le et al., 2001). This experimental approach will be utilized to
study root responses to small molecules that block cellulose biosynthesis or
interfere with cell wall assembly.
Objective 2: Links between CWI signalling and development. Root
expansion is controlled by a complex interaction of hormones. Inhibitors and
hormone signalling mutants will be used to test if CWI acts upstream,
downstream or independently of known hormone signalling pathways in the
root.
Objective 3: Identification of components of the CWI pathway by
candidate and systematic approaches. As described in the introduction,
MAP kinases are involved in the yeast CWI pathway and have been
implicated in plant cell wall signalling as well. An experimental system will be
developed to harvest young root tips of Arabidopsis seedlings in bulk and
analyse MAPK activity in response to cell wall stress.
Proteins involved in a signalling pathway (including receptors) are often
transcriptionally induced in addition to activation at the protein level in
response to the same signal. This approach had in at least one case allowed
the identification of a peptide receptor (Zipfel et al., 2006). Using the same
bulk system as before, transcriptional responses to cell wall damage and
related stress signals will be analyzed. Genes responding to cell wall damage
specifically will be identified from this approach. KO mutants of such genes,
where available, will be tested for their involvement in the CWI pathway.
29
Chapter 2: Results
Cell Wall Integrity Signalling via an ACC
Dependent, Ethylene-Independent Pathway
2.1 Inhibitors of Cellulose Biosynthesis or Crystallization Reduces Root
Cell Growth
Cellulose deficient mutants generally exhibit a reduction in growth (Desnos et
al., 1996; Nicol et al., 1998; Schindelman et al., 2001). Treatment with
inhibitors of cellulose synthesis phenocopies these cell wall mutants (Heim et
al., 1990; Himmelspach et al., 2003; Bischoff et al., 2009). Previous work has
indicated that responses to cell wall stress occur within hours (Paradez et al.,
2006; Hamann et al., 2009). Here, I attempt to identify the earliest observable
response to isoxaben induced cell wall damage in roots. The majority of the
work described in this chapter is documented in Tsang et al. (2011). We are
interested in finding out how quickly Arabidopsis roots respond to cell wall
damage, and more importantly, how soon this response can be detected in
order to identify a suitable time point for further experiments.
Le et al. (2001) have transferred Arabidopsis seedlings to agar-coated
microscope slides supplemented with drugs or hormones to study the shortterm effect of ethylene on root elongation. Instead of complex kinematic
analyses of strain rates resolved in space and time (Beemster and Baskin,
1998), Le et al. (2001) measured the length of epidermal trichoblast cells at
the point where they were just beginning to develop root hairs. This happens
at the end of the rapid elongation phase when cells have reached 70-80% of
their final length (Le et al., 2001). LEH (length of the first epidermal cell with a
visible root hair bulge) is thus a useful parameter that reflects root elongation
30
in the hours before measurement. To study the effect of cellulose biosynthesis
inhibition (CBI), 4-day old Arabidopsis wild type (Col-0) seedlings were
transferred to ½ MS agar medium supplemented with various concentrations
of isoxaben, from 1.5 nM to 500 nM, and returned to the growth chamber for 3
hours. The LEH was measured after 3 hours to determine the extent of the
isoxaben-induced growth reduction. Continued growth of the roots was
strongly dependent on sufficient humidity, but short handling times and the
use of high humidity chambers to hold the slides with seedlings ensured
reproducible results (data not shown). Treatment with isoxaben induced
“crowding” of root hairs within a few hours (figure 5B), similar to that observed
in ACC treated seedlings (Le et al., 2001). This was not due to ectopic root
hair production (Tanimoto et al., 1995) but shortening of trichoblast cells. A
significant reduction in LEH was observed within 3 hours in seedlings treated
with 1.5 nM isoxaben (9% reduction). In a steep dose response curve, the
reduction in elongation continues until the effect saturates at 150 nM isoxaben
(33% reduction; figure 5A). A time-course experiment was conducted using
this just-saturating concentration of isoxaben. As early as 1 hour after transfer
of the seedlings to isoxaben-supplemented medium, a 15% LEH reduction
was observed. The LEH continues to decrease at a steady rate, and at the
last measured time point (8 hours), cell length was reduced by 65% (figure 5C;
Tsang et al., 2011).
31
Figure 5. The short term response of Arabidopsis roots to isoxaben. Each point
represents the average LEH taken from 15 seedlings. The error bars represents the
standard error. A- Dose response curve for seedlings treated with isoxaben for 3 hours.
B- Roots treated with solvent or 150 nM isoxaben viewed at 20x magnification. Bar =
10032µm C- Time course of treatment with 150 nM isoxaben for 1 to 8 hours (Tsang et
al., 2011).
For the following experiments, 3 hour treatments, which led to a robust
response, were used. To test LEH reduction was an effect specific to inhibition
of cellulose biosynthesis or a more general response to cell wall perturbation,
the experiment was repeated using the dyes Congo red and pontamine
scarlet 4B (S4B). Congo red is a direct dye that is known to damage yeast cell
32
walls by binding to newly synthesized (1, 3)-β-d-glucan and preventing
crystallization (Kopecka and Gabriel, 1992).
Figure 6. Compounds that binds to cell wall polysaccharides and inhibit their
crystallization leads to cell wall stress. Here we show that the 3 hours treatment with
either of the two compounds, Congo red and pontamine scarlet 4B, triggers growth
arrest in 4-day old Arabidopsis root cells. The average LEH was taken from 15
seedlings and the standard error represented by error bars. *** indicates a significant
reduction in LEH versus the mock treatment (P < 0.01)
S4B is a dye that binds specifically to cellulose (Hoch et al., 2005). S4B has
been used to visualise cellulose microfibril reorientation in the cell wall in vivo
(Anderson et al., 2009). Treatment with either 5 mg/L Congo red or 0.25%
(w/v) S4B for 3 hours reduced LEH to a similar extent as isoxaben treatments
(33% reduction; figure 6). The effect of isoxaben, Congo red and S4B on root
expansion was similar in timing and amplitude.
2.2 Short Term Responses to General Stress is ACC Dependent but
Ethylene Independent
Short term treatment with isoxaben, Congo red or S4B reduces the LEH in a
similar time scale as treatment with ethylene or its precursor ACC (Le et al.,
2001). To test if cell wall perturbation reduces LEH via ethylene, isoxaben was
33
applied together with chemical inhibitors of ethylene biosynthesis or
perception. When applied alone, 10µM of the ACC synthase inhibitors
aminooxyacetic
acid
aminoethoxyvinylglycine
(AOA)
(AVG)
(Broun
or
the
and
Mayak,
1981),
1-
ACC
oxidase
inhibitor,
-
aminoisobutyric acid (AIB) did not affect the LEH or slightly increased it in
roots of 4-day old seedlings (figure 7A; Tsang et al., 2011). However, all of
these three inhibitors completely restore cell elongation in seedlings treated
with 150 nM of isoxaben (figure 7A; Tsang et al., 2011). It is noteworthy that
roots that are “forced” to elongate despite blocked cellulose biosynthesis often
show dramatic symptoms of apparent cell wall weakness and/or loss of
growth anisotropy such as blebbing (figure 7B; Tsang et al., 2011).
The result above seems to show that the reduction of cell expansion triggered
by cell wall perturbation requires the established ethylene signalling pathway.
However, treatment with 0.001 % (v/v) 2,5-norbornadiene (NBD) or 20 µM
silver thiosulfate, both blockers of ethylene perception, surprisingly did not
restore cell elongation in isoxaben treated seedlings (figure 8; Tsang et al.,
2011). Although the treatment with NBD alone reduced root growth by 16%
(figure 8; Tsang et al., 2011); it is unclear what caused this reduction. ACC
and ethephon, a compound that hydrolyzes to ethylene above pH 3.5 (Yang,
1969), were used to assess the efficiency of silver thiosulfate in blocking
ethylene perception. Seedlings were transferred to agar containing 200 µM
ethephon with or without 10 µM silver thiosulfate.
34
Figure 7. Inhibitors of ACC synthase or ACC oxidase restore root cell elongation in the
presence of isoxaben. A- Effect of ACC biosynthesis or oxidation inhibitors on LEH in
isoxaben (150 nM) treated roots. n.s. indicates no significant difference relative to the
mock treatment (P > 0.05). Error bars and *** are as in figure 6. B- Environmental
scanning electron microscopy (ESEM) images of elongation and differentiation zone of
roots treated for 4 hour with isoxaben, AVG, or both. Epidermal cells of the elongation
zone show swelling/ blebbing when isoxaben is supplied together with AVG, an effect
that is not seen when either treatment is applied alone (Tsang et al., 2011).
35
Figure 8. Blocking ethylene perception have no effect on the short term isoxabeninduced growth arrest. Error bars and *** are as in figure 6 (Tsang et al., 2011).
Ethephon reduces the LEH by 44% within 3 hours. This effect is completely
restored by the addition of silver thiosulfate (figure 9A; Tsang et al., 2011). In
some cases, the effect of ethephon has been shown to be independent of
ethylene generation (Lawton et al., 1994). The process of ethephon
conversion to ethylene generates as by-products hydrochloric acid and
phosphoric acid. To confirm that the root response to ethephon is based on
ethylene, the LEH in roots treated with ethylene gas was measured (from here
on referred to as ethylene treatment, generated from ethephon without
physical contact with roots) or the acidic hydrolysis products alone. Ethylene
gas concentrations could not easily be controlled because the speed of
chemical hydrolysis was unknown, but were set up to reach a maximum of
500ppm in the humidity chamber after full hydrolysis. Hydrochloric acid and
phosphoric acid were buffered with HEPES and used at equimolar
concentrations to ethephon in the “direct contact” experiment. Treatment with
36
the acid by-products alone did not have a significant impact on the LEH
whereas ethephon as well as ethylene gas strongly reduced LEH (figure 9B).
The effect of ethylene gas on root elongation is also completely blocked by
silver thiosulfate (data not shown). Together, the results confirm that silver
ions completely block ethylene effects on roots and suggest that the isoxaben
induced LEH reduction did not require ethylene perception.
Figure 9. Controls for the blocking of ethylene responses in roots with silver
thiosulfate. A- Ethephon treatment reduces LEH. This growth reduction can be restored
by blocking ethylene perception with silver thiosulfate (Ag). B- Ethylene is solely
responsible for the ethephon effect on growth. The acid by-products generated from
the hydrolysis of ethephon into ethylene have little effect on LEH. n.s. indicates no
significant difference between mock and isoxaben treatment. Error bars and *** are as
in figure 6 (Tsang et al., 2011).
To clarify this further, etr1-3, a dominant negative ethylene receptor mutant
was used to test this hypothesis. As expected, etr1-3 is completely insensitive
to the ethylene generated by buffered ethephon whereas the LEH in wild type
was reduced by 26% (figure 10A). In contrast, the LEH of etr1-3 was fully
37
sensitive to isoxaben, and the reduction in LEH could be restored by AVG as
in the wild type (figure 7 & 10B). These data show that the isoxaben induced
growth reduction is ACC dependent but ethylene independent.
Figure 10. The etr1-3 response to ethephon and isoxaben A- While ethephon reduces
the LEH in wild type roots (Col-0), ethephon has no effect on the LEH of etr1-3 a
dominant negative ethylene receptor mutant. B- Isoxaben treatment reduces growth in
etr1-3 indicating that ethylene is not required for isoxaben effects. However, blocking
ACC synthesis with AVG completely restored the LEH in etr1-3 as it did with the wild
type. n.s. are as in figure 7. Error bars and *** are as in figure 6 (Tsang et al., 2011).
However, AVG and AOA do not only inhibit ACC synthase but all pyridoxal
phosphate requiring enzymes, including Trp aminotransferase in the auxin
biosynthetic pathway (Soeno et al., 2010).To test the specificity of these two
inhibitors for the assay used in this report, seedlings were treated with ACC in
combination with either of the ACS inhibitors. Specific ACC synthase inhibitors
should not affect the reduction of root elongation by externally added ACC
because they act upstream of ACC. Treatment with 10µM ACC reduces the
38
LEH by approximately 40%. Both AVG and AOA (10µM) partially restore
elongation in the presence of ACC (figure 11A; Tsang et al., 2011), indicating
drug targets downstream or independent of ACC biosynthesis. AVG and AOA
are therefore not suitable for investigating an ACC dependent pathway. The
mechanistically differently acting AIB did not affect the ACC-induced growth
reduction. This is surprising since AIB is a competitive inhibitor
39
Figure 11. The effects of ACS inhibitors on ACC-induced root growth arrest. AAVG/AOA (10µM) which inhibit pyridoxal phosphate requiring enzyme, partially restore
the LEH of ACC treated (10µM) roots. The competitive ACC oxidase inhibitor, AIB
(10µM), had no effect on the ACC-induced LEH. Error bars and *** are as in figure 6. BDose-response curve of LEH reduction by ACC in the presence and absence of AIB
(Tsang et al., 2011).
of ACC. It is possible that the 10 µM dose of ACC used here is too high to be
efficiently inhibited by AIB, a very weak competitive inhibitor of ACC oxidase.
40
To test this, we have compared the effect of increasing concentrations of ACC
with and without a high concentration of AIB (1 mM; figure 11B; Tsang et al.,
2011). Increasing the concentration of ACC treatment in the absence of AIB
gradually reduces the LEH. This effect saturates at 0.3 µM ACC with a 54%
LEH reduction. ACC-induced growth reduction was still observed in the
presence of AIB, however, the effect was significantly reduced (figure 11B;
Tsang et al., 2011). The fact that 10 μM AIB reverses the isoxaben response
suggests that only small amounts of ACC are generated and/or that the
inhibitor acts on a different target.
Recently, several novel inhibitors of ACC synthase have been identifed from a
chemical screen (Lin et al., 2010), some with an inhibitory mechanism distinct
from AVG and AOA. To obtain additional evidence that ACC biosynthesis is
required to block root elongation in response to isoxaben, the efficiency of
these inhibitors in restoring root growth in isoxaben treated seedlings was
tested. Out of the four inhibitors, 2-anilino-7-(4-methoxypheny)-7,8-dihydro5(6H)-quinazolinone (7303) was the most efficient in restoring root growth in
the presence of isoxaben, although it appeared to have a negative effect on
the LEH on its own (figure 12A; Tsang et al., 2011). To separate these
negative effects from the restoration of elongation, various concentration of
7303 with or without isoxaben were tested. While 25 µM 7303 reduced LEH
on its own, the lower dose of 5 µM 7303 restored root growth in the presence
of isoxaben without affecting growth in mock-treated control seedlings (figure
12B; Tsang et al., 2011).
41
Figure 12. Novel ACS inhibitors. A- Four
novel ACS inhibitors (10 µM) were
screened for the ability to restore root
growth in isoxaben (150 nM) treated roots.
2-anilino-7-(4-methoxypheny)-7,8-dihydro5(6H)-quinazolinone (7303) was the most
efficient at inhibiting the isoxaben
response but negatively affects the LEH on
its own. B- At 5 µM, 7303 fully blocks the
isoxaben response with no effect on LEH
on its own. C- 7303 has no effect on the
ACC-induced reduction in LEH. n.s. are as
in figure 7. Error bars and *** are as in
42
figure 6 (Tsang et al., 2011).
To confirm that 7303 acts specifically on ACC synthase, seedlings were
treated with 5 µM 7303 and 10µM ACC. Unlike the other ACC synthase
inhibitors, 7303 did not affect ACC action (figure 12C; Tsang et al., 2011).
This is in accordance with the original characterisation of 7303 as an
uncompetitive inhibitor of ACC synthase that acts mechanistically in a different
way from AVG.Our results show that inhibition to ACC synthase is sufficient to
prevent growth reduction in roots in the presence of cellulose biosynthesis
inhibitors, and that this response acts independently of ethylene perception.
This ACC mediated growth reduction is a response to general cell wall stress
since both AIB and 7303 restore the LEH in seedlings treated with 5 mg/L
Congo red (figure 13A; Tsang et al., 2011). Stress factors other than cell wall
defects are also known to reduce root elongation, including microbial PAMPs
or elicitors (Gómez-Gómez et al., 1999). Flagellin22 (Flg22) is a 22aa peptide
derived from the bacterial flagellin protein known to trigger the plant’s immune
response (Millet et al., 2010). Treatment with 50µM of flg22 for 5 hours
reduces the LEH of 4-day old Col-0 seedlings by 40% (figure 13B; Tsang et
al., 2011). Similar to what was observed in cell wall inhibitors treatment, both
7303 and AIB completely restored root growth in flg22 treated seedlings
(figure 13B; Tsang et al., 2011). These results show that root elongation is
rapidly controlled via ACC biosynthesis in response to a wide range of stress
triggers.
43
Figure 13. An ACC-dependent pathway regulates responses to both cell wall damage
and PAMPs. Addition of either 7303 or AIB restores root elongation in Congo Red and
flagellin-22 treated roots. n.s. are as in figure 7. Error bars and *** are as in figure 6
(Tsang et al., 2011).
44
2.3 Signalling Downstream of ACC in Short Term Stress Responses
As discussed earlier, a complex interplay between hormones regulates root
growth (Benkova and Hejatko, 2009). Recent studies have shown that auxin
biosynthesis and transport are required to mediate ethylene responses in
roots (Ruzicka et al., 2007; Swarup et al., 2007). ACC/ethylene have been
shown to activate auxin biosynthesis (Stepanova et al., 2005; Stepanova et
al., 2008). To test whether auxin biosynthesis is required for ACC-mediated
growth regulation, we have analyzed the isoxaben-induced LEH reduction in
the presence of a synthetic antagonist of TIR1 receptor function, α(phenylethyl-2-oxo)-indole acetic acid (PEO-IAA) as well as its inactive 5methyl derivative (Hayashi et al., 2008). Treatment with 25 µM antagonist
PEO-IAA, but not 5-methyl-PEO-IAA, completely restored the LEH in
isoxaben treated seedlings (figure 14A; Tsang et al., 2011) indicating that the
ACC mediated reduced elongation requires auxin signalling.
De Cnodder et al. (2005) have shown that the effect of ACC or ethylene on
root elongation is mediated by extracellular events that affect cell wall crosslinking. Specifically, the production of ROS and cross-linking of Hyp-rich
glycoproteins was linked with the reduction of root elongation. As was
previously shown for the response to ACC (De Cnodder et al., 2005), root
elongation in the presence of isoxaben could be completely restored by 10 µM
diphenylene iodonium (figure 14B; Tsang et al., 2011), an inhibitor of flavincontaining enzymes, including NADPH oxidases.
45
Figure 14. Isoxaben-mediated root growth reduction requires both auxin and ROS. AInhibition of TIR receptors with the antagonist PEO-IAA and B- Diphenylene iodonium
(DPI), an inhibitor of NADPH oxidase, completely suppresses the isoxaben-induced
root growth reduction. n.s. are as in figure 7. Error bars and *** are as in figure 6
(Tsang et al., 2011).
46
Figure 15. DELLA is not involved in regulating growth in early responses. The LEH had
been reduced in the wild type (Landsberg erecta) and in the quintuple della knock-outs
in the presence of isoxaben, ACC or ethylene. Error bars and *** are as in figure 6.
Previous studies had shown that DELLA mutants are more resistant to ACC
induced growth reduction than the wild type (Achard et al., 2003). GA and
ethylene regulates growth antagonistically via the DELLA proteins (Fu et al.,
2002; Achard et al., 2003). ACC dependent growth reduction in response to
short term stress may work via DELLA. If this is true, plants with the loss-offunction mutations in the DELLA proteins would be more resistant to the
isoxaben induced growth arrest. To test this, seedlings of a quintuple-DELLA
mutant were treated with 150nM isoxaben for 3 hours. The isoxaben induced
LEH reduction was identical in quintuple-DELLA and in the wild type, Ler
(figure 15). This indicates that DELLA may not be involved in regulating the
short term growth reduction. To clarify this, the response to a 3 hour ACC and
ethylene (generated from ethephon) treatment was compared between
quintuple DELLA and Ler (figure 15). The quintuple DELLA mutation leads to
47
partial resistance to ACC. However, no significant difference in response to
ethylene was observed between the mutant and the wild type.
As previously discussed, mitogen-activated protein kinases (MAPKs) are
involved in many biotic and abiotic processes (Mizoguchi et al., 1996; Jonak
et al., 1999; Bent, 2001; Frye et al., 2001; Nakagami et al., 2005b; Pitzschke
et al., 2009a) and ethylene signalling pathway (Yoo and Sheen, 2008). A
MAPK is required in the well-established yeast CWI pathway (Levin, 2005).
Furthermore, a recent report has shown the involvement of MAPK signalling in
the plant CWI signalling pathway (Kohorn et al., 2009). The activation of the
MAPK pathway upon detecting cell wall damage is likely to be fast consdiering
the rapid subcellular effects of isoxaben (Paradez et al., 2006). That the
pathogen-associated molecular patterns (PAMPs) Flg22 (Asai et al., 2002)
and chitin (Wan et al., 2004) induce dual-phosphorylation in MAPK6 in
Arabidopsis within minutes. To clarify the involvement of MAPK in the short
term response to isoxaben, MAPK activation was analysed in root tissues
isolated from hydroponic culture after a 10 min treatment with 150 nM
isoxaben. Active MAPKs were detected using an antibody against the dual
phosphorylation motif pTEpY. Two bands at approximately 42 kDa and
44 kDa were detected in isoxaben-treated but not control extracts (figure 16).
The lower band was observable in all extracts including the mock treatment,
although the signal is notably weaker (figure 16). The larger 44 kDa band was
only observable in response to isoxaben treatment.
48
Figure 16. MAPK activation by isoxaben, flagellin22
and Congo red. Doubly phosphorylated MAPKs were
detected in root protein extracts with an antiphospho-MAPK antibody.
49
Screening for Loci Involved in Perceiving
Damage to Cell Wall Integrity
2.4 Transcriptional Changes in Roots 1 hour After Cell Wall Stress
Cellular signalling typically involves cytoplasmic events such as protein
phosphorylation, small G protein activation and second messenger production,
as well as changes in gene expression downstream (Okamoto et al., 2001;
Rentel et al., 2004; Nakagawa et al., 2007; Pitzschke et al., 2009a). Rapid
cytoplasmic events do not generally require transcriptional upregulation of the
participating proteins. For example, the oxidative burst in plant innate
immunity is insensitive to blocking transcription or translation with actinomycin
or cycloheximide, respectively (Brisson et al., 1994). However, in Arabidopsis
immunity, for example, increased expression of signalling proteins such as
MAPKs is an important part of stress response priming (Beckers et al., 2009),
and the receptor for bacterial EF-Tu peptide (a PAMP) has been identified
based on transcriptional upregulation in response to its ligand (Zipfel et al.,
2006). Therefore, DNA microarray expression profiling may identify genes that
play a role in the response to cell wall damage, either at the level of signalling
or in repairing damage. A preliminary experiment to identify the earliest time
point where transcriptional changes can be detected after isoxaben treatment
was setup to understand the kinetics of the transcriptional response to cell
wall damage. It is likely that the “early induced genes” would include
signalling-related genes because structural damage (and consequently,
unspecific stress) are still limited. Previous studies have indicated that
changes in the transcription of several hundred genes can be detected 4
hours after Arabidopsis seedlings had been exposed to isoxaben (Hamann et
50
al., 2009). As previously observed, changes in root cell elongation occur as
early as 1 hour after isoxaben treatment (figure 5); it is likely that
transcriptional responses would also be seen at this time point. RNA samples
from root tips were used for this experiment due to their sensitivity and
responses to isoxaben seen earlier in this report. A hydroponic culture setup
was used for this experiment, where sterilised seeds were spread on a 300
µm mesh nylon sieve and allowed to grow their roots into liquid medium below.
Isoxaben and other chemicals could be added to this medium for short term
exposure of roots, and roots were harvested by freezing the drained sieves in
liquid nitrogen, followed by scraping off of the root tips.
The following genes based on experiments with isoxaben-treated etiolated
seedlings (Herman Höfte, personal communication to Thomas Nühse) after 1
and 3 hours of 150 nM isoxaben treatment were used to test the
transcriptional responses to isoxaben:
At2g26530 – Small basic protein of unknown function (DUF1645), potentially
involved in jasmonic acid signalling.
At2g26560 – Encodes patatin-related phospholipase A II, involved in various
processes including promoting jasmonic acid production (La Camera et al.,
2005; Yang et al., 2007; Yang et al., 2012).
At5g57220 – Encodes a Cytochrome P450 enzyme involved in glucosinolate
metabolism and defence; required for MAMP-induced callose deposition
(Clay et al., 2009; O'Brien et al., 2012).
At5g61160 – Encodes the agmatine coumaroyltransferase (AtACT) which
catalyzes the last reaction in the hydroxycinnamic acid amides (HCAAs),
secondary metabolites in the pathogen response (Muroi et al., 2009).
51
At3g59220 – Encodes Pirin1 which interacts with G protein a-subunit to
regulate seed germination and early seedling development (Lapik and
Kaufman, 2003).
At5g19110 – Encodes an aspartyl protease. It was found during screening for
effective myristoylation in G proteins (Boisson et al. 2003, supplemental data),
although the function of this protein is not yet clear, it was assumed that the
role could be in development (Boisson et al., 2003).
Figure 17. The early transcriptional response to cell wall stress. A-RT-PCR bands from
the roots of 4-day old seedling after a 3 hour isoxaben treatment. B- Repeat of the
experiment shown in A with 1 hour isoxaben or Congo red treatment. Actin2 primers
were used as a cDNA load control.
These genes were tested for isoxaben induction in 4-day old Col-0 roots by
RT-PCR. Of this set, only At5g19110 and At2g26530 showed transcriptional
induction by isoxaben (figure 17A). In a second experiment with shorter
treatment, transcript levels of both genes were induced as early as 1 hour
after disruption of cell wall synthesis or assembly with isoxaben or Congo Red
treatment, respectively (figure 17B).
52
The overlap of the responses to biotic and cell wall stress are well
documented (Nishimura et al., 2003; Vogel et al., 2004; Hernández-Blanco et
al., 2007). Therefore it is likely that a significant amount of genes activated by
cell wall perturbation would also be activated in response to other types of
stress. The transcriptional responses to interference with cell wall structure
were compared with those induced by other types of stress to get a clear
overview of the genes specifically responsive to cell wall signals. The
following treatments were used to perturb cell wall integrity:
Isoxaben – a herbicide acting as specific inhibitor of cellulose synthases CesA
3 and the closely related CesA2, 5 and 6 (Scheible et al., 2001; Desprez et al.,
2002; Desprez et al., 2007).
Thaxtomin A – a toxin produced by Streptomyces scabies that inhibits
cellulose biosynthesis and although structurally unrelated produces many of
the same responses as isoxaben (Bischoff et al., 2009).
Congo red – a dye that binds to cell wall polysaccharides (most linear glucans)
and inhibits their higher order assembly (Levin, 2005).
We have also included reagents and treatments that trigger other types of
responses that may be related to cell wall stress:
Mechanical stress –Stirring of the hydroponic culture with a magnetic stirring
bar exposes the developing roots to a continuous shearing stress.
Flagellin22 – a 22 aa peptide and PAMP derived from bacterial flagella that
known to trigger defense responses in roots (Millet et al., 2010).
53
Yariv reagent – specifically binds to and crosslinks AGPs located at the
plasma membrane/ cell wall interface. Clustering of AGPs triggers
programmed cell death in suspension cultured cells (Gao and Showalter,
1999) and disrupts the organization of cortical microtubules in Arabidopsis
roots (Nguema-Ona et al., 2007).
Four day old Col-0 seedlings grown in hydroponic culture were treated for 1
hour and RNA extracted for microarray analysis. After normalising and
background correction, transcriptional changes induced by each type of
treatment were calculated as “fold change” of transcript levels relative to mock
treatment. For hierarchical clustering, genes whose expression was
significantly changed in any one of the treatments were chosen and the fold
change tables were entered into Hierarchical Clustering Explorer (HCE) 1.0.
The data was clustered using the hierarchical clustering setting, the data was
normalised by standardization and the distance was measured using
Euclidean distance. Different treatments induced a specific set of genes,
although there was little overlap (data not shown). Two clusters of genes are
activated more specifically by isoxaben (designated cluster isoxaben A and
cluster isoxaben B). Cluster A included 87 genes with specific response to
isoxaben. Isoxaben specific activation of several genes involved in GA and JA
biosynthesis were detected (figure S1). Cluster isoxaben B contain 67 genes
that respond similarly to both isoxaben and flg22.
2.5 An S-Locus Receptor Kinase is Required for the Early Response to
Isoxaben
The putative members of the CWI pathway were identified by screening for
genes that are required for the isoxaben induced reduction of root cell
54
expansion. T-DNA knock-outs of several of these isoxaben specifically
induced genes were obtained from the Nottingham Arabidopsis Stock Centre
(NASC). The lines that were already available as homozygous mutants were
screened for reduced sensitivity or resistance to 150 nM isoxaben in the root
elongation assay (LEH measurement) described above. An S-locus receptor
kinase knock-out was found to be resistant to isoxaben induced growth
reduction. Disruption of At1g61390 restores the LEH in isoxaben treated
seedlings from 58% of control in the wt to 91% of control (figure 18).
Figure 18. Comparison of Isoxaben induced LEH in 4-day old Col-0 and T-DNA knockout seedlings. At1g61390- encodes an S-locus receptor kinase. The S-locus region in
Brassica is known to be required for self-incompatibility. At5g40430- encodes a
putative transcription factor, MYB22. At5g44400- encodes a FAD-linked oxidase located
at the cell wall. At5g60500- encodes an undecaprenyl diphosphate synthase. The
seedlings were given 3 hour mock/ isoxaben (150nM) treatment. The deletion of
At1g61390 suppresses the isoxaben effect on LEH. *** and n.s. are as in figure 6 and 7.
The At1G61390 locus gene contains a 3.3 kb gene encoding an 831aa protein.
It is annotated as a member of the S-locus receptor kinase family, a central
component of self-incompatibility in Brassica, based on its domain
55
composition and sequence similarity (Takasaki et al., 2000). The predicted
extracellular part of the protein C-terminal of the signal peptide contains
(figure 19): a bulb-type lectin domain which is known to have mannose
binding properties (Barre et al., 1997), an epidermal growth factor like domain
of unknown function which is found in the extracellular domain of membranebound proteins (Davis, 1990) and a PAN domain which is present in S-locus
glycoprotein and S-receptor kinases. The PAN domain is predicted to mediate
protein-protein and protein-carbohydrate interactions (McMullen et al., 1991).
The protein also contains a cytoplasmic Ser/Thr protein kinase. Loss of
function of this gene causes short-term isoxaben resistance suggesting that
the S-locus receptor kinase might be involved in the responses to cell wall
damage. The S-locus receptor kinases form a large family in the Arabidopsis
genome, including twelve genes located very closely to the genomic
At1g61390 locus. Sequence analysis with ClustalW2 showed that there are
four other S-locus receptor kinases which are particularly closely related to
At1g61390. These are At1g11280, At1g61360, At1g61370 and At1g61380
(figure 19B). T-DNA knock-outs of these genes were screened for isoxaben
insensitivity to test if these S-locus receptor kinases are required for the short
term CWI response (figure 20A).
56
Figure 19. A- Domain organisation of At1g61390 based on SMART sequence analysis
(http://smart.embl-heidelberg.de/). B Lectin, bulb type lectin domain; EGF, epidermal
growth factor domain; PAN_AP, divergent subfamily of apple domain; the blue bar
represents a transmembrane domain; STYKc, Ser/Thr kinase. B- Phylogenetic tree of
the 30 proteins most similar to At1g61390 The tree was prepared using the ClustalW2
algorithm (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Sequences closely related to
At1g61390 that are referred to in the text are highlighted in the red box.
57
Figure 20. The transcriptional and LEH responses in At1g61390 related genes. A- TDNA knock-outs of four S-locus receptor kinase closely related to At1g61390, were
screened for responses to a 3 hour isoxaben (150nM) treatment. The error bars and ***
are as in figure 6. B- Gene induction by 1 and 4 hour isoxaben treatment (150nM)
expressed as fold change versus mock treatment. The error bars represents the
standard error calculated from Ct values of the control treatment.
While the At1g61390 knock-out was virtually resistant to isoxaben in the short
term root elongation assay, sensitivity of the other S-locus receptor kinase
knock-outs was identical to the wild type (figure 20A). The microarray data
showed that At1g61390 was induced 3 fold within 1 hour of treatment with
58
isoxaben (supplementary material). This induction was confirmed by using
qPCR. Transcripts for At1g61390 were induced 6-fold in roots treated with
isoxaben for 1 hour (figure 20B). In contrast, transcripts for the closely related
At1g61360 and At1g11280 remained unchanged (figure 20B). At 4 hours after
addition of isoxaben. transcript level of At1g61390 returns to normal while
At1g61360 was induced 4 fold relative to mock treatment (figure 20B).
59
Chapter 3: Discussion and Conclusion
3.1 Early Responses to Cell Wall Stress
Plant development is heavily dependent on cell elongation and division. The
synthesis of new cell wall material is in demand throughout development but
especially during the rapid cell elongation phase in hypocotyls and roots.
Plants with impaired cell wall synthesis show reduced growth, as seen in the
root swelling and short hypocotyls of CORE mutants and CesA mutants
(Hauser et al., 1995; Fagard et al., 2000; Ellis et al., 2002). These phenotypes
reflect long term cell wall stress in plants that go through all of their
development with a cell wall deficiency. However, the maintenance of
structural stability during normal development, for example in cell expansion,
should require responses to cell wall damage to be triggered very quickly
before the manifestation of large-scale structural damage. As Somerville et al.
postulated (Somerville et al., 2004), the cyclic process of cell wall production,
modification and relaxation that underlies growth must have a mechanism of
feeding back information on the mechanical performance and integrity of the
wall into the cytoplasm.
In this thesis, the acute response of rapidly expanding root cells to cell wall
damage was analysed. Inhibition of cellulose synthesis by the herbicide
isoxaben rapidly reduces cell expansion in the elongation zone of young roots.
This growth reduction could be observed as early as 1 hour after treatment in
the form of “premature” differentiation of shorter trichoblast cells. Increased
expression of a specific set of genes occurred on the same time scale. Even
before these responses, MAPK activation was detected 20 min after
60
chemically induced cell wall damage comparable with the speed of cellulose
synthase depletion from the plasma membrane in isoxaben-treated GFPCesA6 reporter lines (Paradez et al., 2006). It is likely that the very earliest
responses to cellulose biosynthesis inhibition on the cellular level are at least
as fast as that, but we lack a suitable readout. In this thesis, phenotypic
changes on the microscopic level were detected after 1 hour. The short term
response to isoxaben had been studied in the past, although on a time scale
of 4 – 7 hours of isoxaben treatment. Transcriptional changes (Hamann et al.,
2009), JA and ligin production (Denness et al., 2011) and changes in
carbohydrate metabolism (Wormit et al., 2012) were reported in young
seedlings. The latter three responses were not analysed in this thesis.
3.2 ACC Signals the Rapid Response to General Stress
It is conceivable that growth arrest in isoxaben treated root cells is due to
osmotic stress or plasma membrane stretch caused by the weakened cell wall,
rather than a dedicated system of cell wall integrity surveillance. However, it is
possible to restore elongation without restoring cell wall integrity. A loss-offunction mutation of the receptor kinase THE1 partially restores elongation of
etiolated hypocotyls in several cellulose deficient mutants (Hématy et al.,
2007). The results in this thesis have shown that in roots, too, growth arrest
can be separated from cell wall damage by blocking specific signalling
pathways, thus providing evidence for cell wall integrity signalling. Inhibition of
ACC biosynthesis restores elongation in isoxaben treated roots despite clearly
visible cell wall damage. Le et al. (2001) have shown that ethylene treatment
specifies a maximal cell length for elongating root cells. Any cells longer than
the specified length at the time of treatment will immediately stop elongating
61
and differentiate, while younger cells expand until they reach the newly set
limit (Le et al., 2001). Given that the isoxaben-induced growth reduction
reported here is ACC-dependent, the LEH observed is likely to be specified by
the ACC mediated pathway. This is consistent with the fact that the
constitutive ACC/ethylene production in the cev1 allele of CesA3 leads to
stunted growth (Ellis and Turner, 2001; Ellis et al., 2002). The effect of
ethylene on root growth in wt seedlings depends on auxin biosynthesis and
transport (Stepanova et al., 2005; Ruzicka et al., 2007; Swarup et al., 2007;
Stepanova et al., 2008). This thesis has shown that the short term isoxaben
induced LEH reduction does not strictly follow this established pathway.
Surprisingly, while inhibitors of ACC biosynthesis or a non-functional structural
analog of ACC, are able to restore root growth in isoxaben-treated seedlings
(figure 7A), growth reduction triggered by cell wall perturbation still occurs in
ethylene insensitive mutants or when ethylene receptors are blocked;
indicating an ACC-dependent, ethylene-independent signalling pathway.
Furthermore, rapid growth reduction in response to PAMPs is also signalled
via this pathway. The existence of this postulated ACC mediated pathway is
supported by the fact that ethylene insensitive mutants are relatively healthy
whereas the multiple knock-outs of ACS genes have increasingly severe
developmental defects (Alonso et al., 1999; Alonso et al., 2003; Tsuchisaka et
al., 2009). As a signalling molecule in its own right, ACC appears to have
developmental functions that are nonredundant with ethylene. There are
already indications of an ethylene-independent signalling pathway that uses
ACC (Xu et al., 2008). Similar to our findings, the conditional cell wall
deficiency phenotype of the fei1fei2 mutant can be completely rescued by
62
blocking ACC biosynthesis or action, but not by blocking ethylene or in
ethylene insensitive mutants (Xu et al., 2008).
The results have shown that both ROS and auxin biosynthesis are required to
regulate the rapid ACC mediated response to isoxaben; similar to the
established ethylene signalling pathway (De Cnodder et al., 2005; Stepanova
et al., 2005; Stepanova et al., 2008). However, while basipetal auxin transport
is required in the established ethylene signalling pathway (Ruzicka et al., 2007;
Strader et al., 2010), Tsang et al. (2011) showed that auxin transport is not
required in this hypothetical ACC pathway that serves as a shortcut which
bypasses ethylene signalling to generate a rapid response. In seedlings
treated with ACC, Silver thiosulfate –which completely blocks the effect of
saturating concentrations of ethylene gas in the same experimental setup- can
only partially restore root elongation. This indicates that the externally applied
ACC acts on root elongation both as ACC itself and after conversion to
ethylene. If this is true, it is unclear why the short term responses to isoxaben
and other types of cell wall stress completely ethylene independent. It seems
that ACC generated in response to these stimuli is fed directly (and
exclusively) into the ACC-dependent pathway and not converted to ethylene.
It remains unclear why or how this happens. A plausible hypothesis is that the
ACC concentration induced by cell wall stress is initially low and ACC binds to
a receptor or binding protein with higher affinity than to ACC oxidase. ACC
applied externally to roots in this report would almost invariably reach much
higher intracellular concentrations than the capacity of this system, and the
excess is converted into ethylene; hence the partial restoration of elongation
by silver. In this model, ACC triggers rapid responses in a cell-autonomous
63
way that share at least some similarities with ethylene responses before
threshold concentrations of the easily dissipating gaseous ethylene are
reached. However, ACC was reported to work differently in the SOS5/FEIs
pathway. Xu et al (2008) have postulated that the FEI1 and 2 receptor kinases
act as a scaffold for ACS5 and ACS9 to generate a localized ACC signal.
This report and other recent studies have identified several differences
between the responses to short term and long term cell wall damage. DELLA
is another component which mediates root growth downstream of ethylene
(Achard et al., 2003).
Given that growth arrest triggered by long term
ethylene/ACC treatment can be restored partially by the loss-of-function
mutation in gai-t6 and/or rga-24 (Achard et al., 2003), it was surprising that the
quintuple-DELLA behaved like the wild type in terms of reduced root growth
triggered by the short term ACC, ethylene or isoxaben treatment. These
results indicate that unlike ethylene, ACC does not stabilize DELLA, even
though the signalling pathway downstream of ACC shares many similarities
with ethylene. ACC has been used to show ethylene insensitivity in many
mutants (Alonso et al., 1999; Alonso et al., 2003; Lohar et al., 2009).
Insensitivity to long term ACC treatment was observed in the etiolated
hypocotyl of ein3eil1 (Supplemental figure; Tsang et al., 2011). Interestingly,
root elongation in etr1-3 and ein3eil1 (as well as ein2, personal
communication Thomas Nühse) displayed some sensitivity to ACC but not to
ethylene in a 3 hour treatment. Following the model hypothesized in Tsang et
al. (2011), this means that the control of root growth via stabilization of DELLA
proteins plays a role in the long term response downstream of ethylene but
64
not in the “ACC shortcut”(Tsang et al., 2011). However, the shortcut would
continue to be activated in a prolonged treatment.
In the response to cell wall stress and PAMPs, the ACC shortcut pathway may
be regulated by transcriptional and/or posttranscriptional regulation of ACS
levels and activity. Tsang et al. (2011) have observed a transcriptional
induction of ACS11 during isoxaben treatment. In ACS11 knockout mutants,
root elongation is less sensitive to isoxaben (T. Nühse, personal
communication). Expression of other ACS isoforms is not strongly changed by
isoxaben within one hour based on our microarray data, but it is possible that
posttranslational modification additionally regulates ACS11 or other isoforms
in response to inhibition of cellulose biosynthesis. More work is necessary to
clarify the relationship between cell wall perturbation and ACC biosynthesis.
The role of ACC as a signalling molecule in plants has being reviewed
recently (Yoon and Kieber, 2012). Octuple ACS mutants show some
responses that resemble ethylene insensitive mutants but also novel
phenotypes which further implicate a role for ACC independently of ethylene
signalling (Tsuchisaka et al., 2009). The exact mechanism and targets of ACC
in this pathway remain unclear. It has been hypothesized that AIB targets
ACC binding proteins other than ACC oxidase (Xu et al., 2008; Tsang et al.,
2011). One important question is whether ACC itself or some ACC derivative
acts as the signalling molecule in this pathway (Yoon and Kieber, 2012). The
identification of the fate of ACC in this postulated pathway is a major question
to be answered.
65
3.3 S-Locus Receptor Kinase Plays a Role in Responding to Cell Wall
Integrity Damage.
A putative S-locus receptor kinase (SRK) was one of a small number of genes
induced specifically in response to isoxaben and not other types of cell wall
damage or stress. In a screen of KO mutants of several of these genes, a TDNA mutant of the SRK At1g61390 was resistant to the short term response
to isoxaben. S-locus receptor kinases were originally identified to mediate
self-incompatibility in Brassica (Stein et al., 1991). Self-incompatibility is
regulated by the interaction between the two protein products encoded by the
S-locus: the plasma membrane anchored S-locus receptor kinase (Stein et al.,
1991; Stein et al., 1996) and its ligand, S-locus cysteine-rich protein (Takasaki
et al., 2000), which is located in the pollen coat. Given that Arabidopsis
thaliana, a related plant from the Brassicaceae family is highly self-fertile
(Kusaba et al., 2001), the S-locus found in Arabidopsis thaliana is likely to
serve a different role. The fact that the loss of function of this gene leads to
apparent short term insensitivity to isoxaben suggests a role for this SRK in
the CWI pathway in roots. Its predicted protein domains would equip it for cell
wall signalling (figure 19A). The predicted mannose binding lectin domain may
act as a receptor for DAMPs or cell wall polysaccharides, or interact with an
S-locus cysteine-rich protein in a similar manner to interactions regulating selfincompatibility. The low levels of expression of At1g61390 make it difficult to
predict with confidence if any such genes are co-expressed with the SRK.
Alternatively, the putative S-locus receptor kinase may respond to changes in
carbohydrate metabolism triggered by isoxaben (Wormit et al., 2012). Apart
from extracellular domains that could plausibly interact with cell wall
polysaccharides, the predicted Ser/Thr kinase domain marks SRK as a
66
candidate cell wall sensor and signalling protein. However, the requirement for
kinase activity for SRK function was not tested in this thesis.
It is noteworthy that the root cells of the isoxaben treated At1g61390 T-DNA
knockout mutants, although elongating to the levels of untreated wt cells,
appeared to be relatively intact compared with those observed in the
AVG+Isoxaben treatment (data not shown, figure 7B; Tsang et al., 2011). This
suggests less cell wall damage in these root cells despite the presence of
isoxaben. Analysing the effects of the SRK KO on CesA activity would provide
further explanation. The role of the putative S-locus receptor kinase in cell wall
integrity signalling is likely to be restricted to short term response. Firstly, the
knock-out mutant appeared to be completely resistant to isoxaben during our
short-term LEH experiments (figure 18) but behaved like the wild type when
grown in isoxaben (results not shown). Secondly, the transcriptional induction
of At1g61390 by isoxaben was only transient (figure 20), suggesting a role in
acute signalling rather than long-term tolerance or repair. Work on At1g61390
is still at an early stage and many questions are yet to be answered. Is the
predicted S-locus receptor kinase required for other stress response (such as
PAMPs, Congo red, DCB, thaxtomin A etc.)? Where is this protein localized?
What are the ligands or binding partners to this protein? Is the kinase domain
active, and if so, required for its function? Does this putative S-locus receptor
kinase play a part in the ACC shortcut pathway proposed here (Tsang et al.,
2011)?
67
3.4 Conclusion
In this report, several components required for the rapid response to inhibition
of cellulose biosynthesis and possibly cell wall maintenance in vivo were
identified. This work and other reports have indicated several independent
pathways to regulate cell wall homeostasis and/or cell expansion (Wagner
and Kohorn, 2001; Xu et al., 2008; Tsang et al., 2011). For example, the FEI
pathway was shown to work independently from the pathways regulated by
WAK and THE (Xu et al., 2008). My transcriptional profiling experiment has
shown that different mechanisms of cell wall perturbation produce a distinct
set of transcriptional responses (data not shown). The plant is likely to
produce specific sets of responses to each type of stimulus, such as: CBI,
DAMPs, hyperosmotic stress, stretch/ mechanical stress and cell wall
deficiency. The term “cell wall damage” has become too simplistic to be used
to describe a stimulus which activates plant responses. It is likely that any one
defect causes multiple different triggers and response pathways. The CWI
pathway in plants seems to form a network of sensory/response mechanism
between the plant ECM and the cell. Work by Xu et al. (2008) and this report
have indicated that the different signalling pathways converge at ACS (Xu et
al., 2008; Tsang et al., 2011; Yoon and Kieber, 2012). There are eight
functional ACS in the Arabidopsis genome (Chae and Kieber, 2005) which
may be used to trigger specific responses. In agreement with this hypothesis,
there are two classes of ACS, regulated by either calcium-dependent kinase
or MAPK. Different ACS isoforms are involved in the FEI pathway (ACS5) and
in the response to isoxaben (ACS11; Chae and Kieber, 2005; Xu et al., 2008;
68
Tsang et al., 2011). The hypothetical ACC pathway presented in this report
was triggered by various stimuli (Tsang et al., 2011). Given the early time
point at which this pathway is activated, it is likely that this pathway may serve
as an initial response to general stress (Tsang et al., 2011). Figure 21 shows
a hypothetical overview of the CWI signalling in plants. In this model, external
stimuli are detected by specific receptors. The response to different stimulus
would be signalled via the transcriptional to posttranscriptional regulation of
ACS. The hypothetical pathway proposed in (Tsang et al., 2011), is activated
in this model as the rapid response to general stress that is dependent on
auxin biosynthesis and ROS signalling. Prolonged exposure to the stimulus
would trigger a specific set of response. Further research should reveal novel
components to the CWI pathways and provide further insight in how the CWI
pathways interact with one another to regulate cell wall homeostasis.
69
Figure 21. A working model for the regulation of cell wall homeostasis. Different types
of stimuli are detected by different receptors. Specific responses are signalled by the
differential transcriptional and posttranscriptional regulation of the eight functional
ACS in Arabidopsis. A general short-term set of responses are signalled via ACC. The
requirement for auxin biosynthesis and ROS for reducing root growth is confirmed
(Tsang et al., 2011). The long term responses specific to the stimulus are signalled via
ACC and ethylene. Note that ACC works partially independently of ethylene in the long
term response (Xu et al., 2008).
70
Materials and Methods
Plant Material and Growth Conditions
The
following
T-DNA
knock-out
At1g61380-SALK_020650,
SAIL_1272_A05,
mutants:
At1g61390-SALK_131780C,
At1g61370-SALK_126675C,
At1g61360-GK-901G09,
At1g11280-
At5g40430-SALK_063356C,
At5g60500-SALK_072476, At5g44400-SALK_069865C and the etr1-3 mutant
were obtained from the Nottingham Arabidopsis Stock Centre. Seeds of the
ein3-1 eil1-1 mutant were kindly provided by Joseph Ecker. The sterilization of
the seeds and the growth condition of the seedlings used for LEH
measurements were performed as described in Tsang et al. (2011). For RNA
and protein extractions, 0.5g of sterilized Arabidopsis Col-0 seeds was grown
in a hydroponic culture. The hydroponic culture was setup by evenly
distributing the sterilized seeds on a 300 µm mesh nylon sieve (Wilson sieves)
using 0.1% (w/v) agarose and grown on the surface of the liquid medium
(2 gL-1 Sucrose and ½ Murashige and Skoog basal salts; Melford).
Root Cell Elongation Measurements
LEH measurements were performed as described in Le et al. (2001) and
Tsang et al. (2011). The concentrations of isoxaben, AOA, AVG, AIB, flg-22,
Congo red, silver thiosulfate, 9127303 (“7303”; Hit2Lead; Chembridge Corp.),
ethephon, PEO-IAA, 5-methyl PEO (a kind gift of Ken-ichiro Hayashi) and
diphenylene iodonium were as described in Tsang et al. (2011) . The standard
71
error was represented by error bars. Two-tailed T-test were performed for
every LEH experiment using SPSS (IBM Corporation).
GUS
Staining
and
Environmental
Scanning
Electron
Microscopy
The procedures for GUS staining of the pCesA::GUS lines and the
environmental scanning electron microscopy were described in Tsang et al.
(2011).
Extraction of RNA and Reverse Transcriptase Reaction
RNA samples were collected from the roots of 4-day old hydroponic cultures.
Treatments were applied by mixing into the liquid medium. RNA was extracted
from roots using TRI reagent (Sigma-Aldrich). RNase Zap (Sigma-Aldrich)
was used for RNase decontamination. The extracted RNA is then either
stored at -80
o
C or immediately treated with DNAse (Invitrogen). RT
Superscript II (Invitrogen) and oligo(dT) primers were used for reverse
transcription. For microarray experiments, RNA quality was checked using the
RNA 6000 Nano Assay, and analyzed on an Agilent 2100 Bioanalyser (Agilent
Technologies). RNA was quantified using a Nanodrop ultra-low-volume
spectrophotometer (Nanodrop Technologies). Arabidopsis ATH1-121501
Affymetrix GeneChips were run according to manufacturer’s instructions.
RT-PCR
72
RT-PCR were performed using Taq Polymerase (Roche). The PCR conditions
were performed exactly as recommended in the manufacturer’s guide. The
PCR mix recipe consist of the following, for each PCR reaction: 12.375 μl of
dH2O, variable amount of 1/10 diluted cDNA (to make 25 μl final volume),
3.25 μl of reaction buffer with Mg2+, 2 μl of each of the 10mM primers, 0.25 μl
of dNTP and 0.125 μl of Taq Polymerase was mixed. At the end of the PCR,
the total amount of product was loaded into a 1% agarose gel mixed with
SafeView stain SYBR green (Invitrogen). Actin2 primers were used as load
control. The gene-specific primers used were as follow:

At2G26530 5′-GGGCGGAGACCGATGATGA-3′
5′-GAGGCCGCAACAAGAAACTT-3′

At2G26560 5′-AGCCATGCTCACCGCGCC-3′
5′-CCAGATTCTCGACGGTAGCG-3′

At3G59220 5′-GGACCCGTTCGTGTTGCTA-3′
5′-GCCGATATTGCGGAAGAGTT-3′

At5G19110 5′-CGCCACCGATGGAACCTTA-3′
5′-CGTCCGCCACTTCACATTCT-3′

At5G57220 5′-CACCAGGACCAACTCCGTT-3′
5′-GCAGCTGGACACAACCGAA-3′

At5G61160 5′-CCTCCGTCGACCCGCTCATT-3′
5′-GGCTAAGACACAAGTCCCGAA-3′

Actin2
5’-CTGGTGATGG TGTGTCT-3’
5’-GTAACATTGT GCTCAGT-3’
73
Real-Time quantitative PCR - qPCR
All qPCR were performed using the EXPRESS SYBR® qPCR SuperMixes
(Invitrogen) and the ABI PRISM® 7000 Sequence Detection System. The
reaction mixtures were setup according to the manufacturer’s guideline for the
EXPRESS SYBR® qPCR SuperMixes. The cycling program was set to the
following:

50 oC ---------------- 2 mins

95 oC ---------------- 2 mins

40 cycles of :
o
95 oC-------- 15 s
o
60 oC -------- 1 min
The gene-specific primers that were used for analysing the transcript level of
S-locus receptor kinase genes were:

At1g11280 5’-TGGGCTGTGTGTGACGTCCAATCCCACAA-3’
5’-TCATGTTCCCGCGCTTCCACTCCTCCTTG-3’

At1g61360 5’-TGAGACCGATCCATCGCCTGGGGAGTTTG-3’
5’-TGTTCCCGCCCATGGACCGCTTCTCCAA-3’

At1g61390 5’-TGGGGAATCTCTCTCCCTTCGGCTTGC-3’
5’-TCGCCCATGCATCTTGTGAACTGTGGA-3’
The following primers were used to normalize the result:

At5g12240 5’-AGCGGCTGCTGAGAAGAAGT-3’
5’-TCT4CGAAAGCCTTGCAAAATCT-3’
74
The relative quantity was calculated using the following equations:
dCt = CtGOI – Ctnorm
ddCt = dCtTreated – dCtMock
Relative quantity = 2-ddCT
Three biological replicates were performed for each experiment. Three
technical replicates were performed for each sample. The standard error was
calculated from the biological replicate of the untreated.
Microarray and Bioinformatics Analysis
For the comparison of cell wall damage and other stress, hydroponic seedling
cultures were exposed to the following reagents or treatments for 1h: 500 nM
isoxaben, 500 nM thaxtomin A (Enzo Life Sciences), 10 mg/L Congo Red,
1 µM flagellin-22, 5 µM beta-D-glucosyl Yariv reagent (Biosupplies Australia)
or mechanical stress (stirring at 300rpm with a 2in magnetic bar). RNA was
harvested from the roots of seedlings grown in hydroponic culture. Three
replicate was performed for each experiment. Technical quality control and
outlier analysis was performed with dChip (V2005) (www.dchip.org, Cheng Li
and Wing Hung Wong (2001)) using the default settings.
Normalisation and expression analysis was done using multi-mgmos (Liu et
al., 2005). Differential expression between sample A and the control sample B
was assessed with a Bayesian method which includes probe-level
measurement error when assessing statistical significance (Liu et al., 2006).
Analysis was performed with the PUMA package in R (Gentleman et al.,
2004). Using the PUMA method a statistic is generated called a PPLR.
75
Probesets were considered differentially expressed when they had a PPLR
value <0.55 or >0.45.
The transcript level was averaged and the fold change between the treated
and the mock treatment was calculated. Hierarchical Clustering Explorer
(HCE) 1.0. was used to cluster the genes base on its fold change in response
to each type of treatment. The clustering was performed using the hierachial
clustering setting.
The phylogenetic tree of the 30 proteins most similar to At1g61390 was made
using Clustalw2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) following the
instructions given. The list of related proteins was identified using Wu-blast
(http://www.arabidopsis.org/wublast/index2.jsp)
following
the
instructions
given.
Soluble Protein Extraction
Proteins samples were extracted by acetone precipitation. Harvested
roots/cells were frozen in liquid nitrogen and homogenized in homogenizing
buffer (0.25 M sucrose, 2 mM EGTA, 10% (v/v) glycerol, 0.5% (w/v) BSA,
50 mM Bis Tris Propane (BTP)-2-(N-morpholine)-ethanesulphonic acid (MES),
pH 7.8, 0.25 M KI, 2 mM DTT, 1 mM phenylmethanesulphonyl fluoride (PMSF)
and 5 mM β-mercaptoethanol). The homogenate was centrifuged at 11500g
for 10 min and the supernatant was collected. Four times the supernatant
volume of acetone was added to the supernatant and kept at -20oC overnight.
The sample was centrifuged again at 11500g for 10 min. The supernatant was
carefully removed and the samples were left open for the remaining acetone
to evaporate.
76
SDS PAGE
Bio-Rad mini protean II system was used to carry out SDS-PAGE. The twophase gel contains 10% (v/v) acrylamide, 0.1% (v/v) SDS, 0.05% (v/v)
ammonium persulfate (APS), 10 µl (v/v) TEMED and 0.375 M Tris, pH8.8 for
the resolving gel and 4.5% (v/v) acrylamide, 0.1% (v/v) SDS, 0.08% (v/v) APS,
8µl (v/v) TEMED and 0.125 M Tris, pH 6.8 for the stacking gel. All protein
samples were incubated at 65 oC with the SDS sample loading buffer for 5
minutes before loading into the gel. Gels were run at 100 V until the dye front
moved into the separating gel layer, after which the voltage was increased to
200 V until the dye front reach the end of the gel.
Western Blot
The acrylamide gel containing the separated protein was removed from the
gel system and placed into a blotting cassette containing a nitrocellulose
membrane. The separated proteins were transferred to the nitrocellulose
membrane using a BioRad Mini Protean 3 Western transblot system filled with
ice cold Towbin buffer (20% (v/v) methanol, 0.2 M glycine and 25 mM Tris)
under a constant current of 400 mA for 2 hours. The membrane was
incubated on a shaker in 30 ml of blocking solution (5% (w/v) nonfat dry milk,
25 mM Tris, pH7.4, 0.15 M NaCl, 0.1% (v/v) Tween 20) for 1 hour at room
temperature. Anti-Phospho-ERK1/ERK2 antibody (R&D SYSTEMS) was used
as the primary antibody. The Primary antibody was diluted in 30 ml of blocking
solution. The membrane is then incubated at 4oC on a shaker overnight. The
membrane was washed four times with blotting buffer (25 mM Tris, pH7.4,
0.15 M NaCl, 0.1% (v/v) Tween 20) each time for at least 5 mins. IRDye
77
800CW antibodies (LI-COR) were used as the secondary antibody. The
membrane was then incubated in darkness at room temperature for 1 hour
with the secondary antibody diluted in blotting buffer. The membrane was then
washed for another four times with blotting buffer. Bands was visualised using
the Odyssey® Infrared Imaging System.
Acknowledgement
I thank my supervisor Dr Thomas Nuhse and advisor Professor Simon Turner
for supervising this project. I thank the Leo Zeef and Andy Hayes of the
Bioinformatics and Genomic Technologies Core Facilities at the University of
Manchester for providing support with regard to microarrays. Finally I like to
thank my funding agency Biotechnology and Biological Sciences Research
Council (BBSRC), whom funded my training here in the University of
Manchester.
78
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Supplemental Data
A
91
B
-ACC
Figure S1. Two clusters of isoxaben specifically induced genes were identified using
Hierachical Clustering Explorer (HCE) 1.0. A- Isoxaben Specific Cluster 1 B- Isoxaben
Specific Cluster 2. Y- Yariv reagent, F- Flagellin22,
C- Congo red, I- Isoxaben, T- Thaxtomin
92
A and M- Mechanical stress induced by stirring of the liquid medium in the hydroponic
culture. The fold change induced by each of these treatments are listed.