Download 1 Cell wall integrity controls root elongation via ACC Corresponding

Cell wall integrity controls root elongation via ACC
Corresponding author:
Thomas S. Nühse
University of Manchester
Faculty of Life Sciences
Oxford Road
Manchester M13 9PT
United Kingdom
Office phone +44 161 2755545
[email protected]
Research category: Signal transduction and hormone action
1
Cell Wall Integrity Controls Root Elongation
via a General 1-Aminocyclopropane-1-carboxylic Acid-Dependent,
Ethylene-Independent Pathway
Dat Luen Tsang, Clare Edmond, Jennifer Louise Harrington and Thomas Sebastian Nühse
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT,
United Kingdom
2
Footnotes
Research was funded by a Biotechnology and Biological Sciences Research Council
Doctoral Training grant and a David Phillips fellowship to TSN.
Corresponding author e-mail: Thomas S. Nühse ([email protected])
3
Abstract
Cell expansion in plants requires cell wall biosynthesis and rearrangement. During periods of
rapid elongation, such as during growth of etiolated hypocotyls and primary root tips, cell
respond dramatically to perturbation of either of these processes. There is growing evidence
that this response is initiated by a cell wall integrity-sensing mechanism and dedicated
signalling pathway rather than being an inevitable consequence of lost structural integrity.
However, the existence of such a pathway in root tissue and its function in a broader
developmental context has remained largely unknown. Here we show that various types of
cell wall stress rapidly reduce primary root elongation in Arabidopsis thaliana. This
response depended on biosynthesis of 1-Aminocyclopropane-1-carboxylic acid (ACC). In
agreement with the established ethylene signalling pathway in roots, auxin signalling and
superoxide production are required downstream of ACC to reduce elongation. However, this
cell wall stress response unexpectedly does not depend on perception of ethylene. We show
that the short-term effect of ACC on roots is partially independent of its conversion to
ethylene or ethylene signalling and that this ACC-dependent pathway is also responsible for
the rapid reduction of root elongation in response to pathogen-associated molecular patterns
(PAMPs). This acute response to internal and external stress thus represents a novel,
noncanonical signalling function of ACC.
4
Introduction
Cell proliferation and cell expansion are the two aspects of growth that determine cell, tissue
and ultimately organ size in multicellular organisms. Unlike in metazoans, cell expansion in
plants is an important contributor to organ size. An osmolyte-filled vacuole exerts hydraulic
pressure against a mechanically strong wall. Controlled relaxation of these walls allows for
cell expansion via water influx into the vacuole. Many factors, including environmental
conditions and the physical stability of the wall, determine how far cell wall polymer
remodelling can proceed and thus how big the cell can become (De Cnodder et al., 2005).
Cell elongation in roots is negatively controlled by ethylene, which in turn requires auxin
biosynthesis and -transport (Ruzicka et al., 2007; Stepanova et al., 2007; Swarup et al.,
2007) as well as the production of reactive oxygen species (ROS) (De Cnodder et al., 2005).
ROS have both a signalling function and a direct effect on cell wall elasticity; apoplastic
peroxidases can crosslink hydroxyproline-rich glycoproteins in the presence of ROS to
stiffen walls (Passardi et al., 2004), while hydroxyl radicals can sever polysaccharide chains
(Mueller et al., 2009).
Plant tissues undergoing rapid expansion are particularly vulnerable to cell wall defects.
Etiolated hypocotyls and root tips have therefore been used in forward genetic screens to
identify genes with a role in cell wall biogenesis (Baskin et al., 1992; Hauser et al., 1995;
Desnos et al., 1996). Reduced elongation and a loss of growth anisotropy in roots that
manifests as radial swelling are hallmarks of cell wall-deficient mutants. Other characteristic
phenotypes particularly of cellulose-deficient mutants are ethylene- and jasmonic aciddependent defence responses such as ectopic lignification (Cano-Delgado et al., 2000; Ellis
et al., 2002). These responses are phenocopied by inhibitors of cellulose biosynthesis like
isoxaben and dichlobenil (2,6 dichlorobenzonitrile, DCB). It could be argued that reduced
cell expansion is an inevitable consequence of loss of cell wall integrity. However, the
characteristic defence-like responses that occur in addition to growth defects suggest a more
complex situation and a dedicated signalling pathway. In order to take external factors into
account, the regulatory circuit that controls cell expansion must include a mechanism to feed
back information about cell wall integrity into the cytoplasm. Early evidence for such a
pathway came from the observation that in dark-grown Arabidopsis seedlings , commitment
to rapid elongation (up to 48 hours after imbibition) but not elongation itself was sensitive to
the cellulose synthase inhibitor isoxaben (Refregier et al., 2004).
5
Our knowledge of this postulated signalling pathway is limited. Fungal cell walls, despite
their different chemical composition, have a mechanical function very similar to that of plant
cell walls. In yeast, a cell wall integrity signalling pathway has been characterised in detail
(Levin, 2005) that requires a family of highly glycosylated transmembrane sensors (WSC13, MID2, MTL1). No obvious plant orthologue of the WSC family sensor proteins can be
identified because of their low sequence complexity. Several families of plant receptor-like
kinases have been proposed to perform cell wall sensing function and/or mediate responses
to cell wall damage (Humphrey et al., 2007; Seifert and Blaukopf, 2010; Steinwand and
Kieber, 2010). Mutations in some of these genes cause reduced cell expansion up to severe
dwarfism and other phenotypes typical for cell wall defects, but evidence for direct binding
of the extracellular domain of RLKs to cell wall polysaccharides remains limited (Kohorn et
al., 2009). The clearest genetic evidence for an active signalling mechanism that
communicates cell wall status and controls growth comes from the theseus mutant. Loss of
the CrRLK-family member THE1 partially suppresses the short hypocotyl phenotype of a
weak cellulose-deficient mutant, procuste 1-1 (Hematy et al., 2007), without restoring its cell
wall defect.
Cell wall defects typically lead to altered wall composition and transcriptional changes
which resemble those triggered in defence responses (Humphrey et al., 2007). Turgor
pressure and sugars are required for at least some of the transcriptional response to inhibition
of cellulose biosynthesis (Hamann et al., 2009); the ectopic lignification is additionally
modulated by the NADPH oxidase, RbohD, and jasmonate. The cross-wiring of apoplastic
surveillance for pathogens and cell wall integrity is so strong that genetic screens for altered
pathogen resistance have uncovered cell wall biosynthetic genes (Nishimura et al., 2003;
Vogel et al., 2004) and vice versa, many mutants identified as cell wall deficient are more
resistant to pathogens (Hernandez-Blanco et al., 2007). This overlap is perhaps not
surprising because for plant pathogenic fungi, oomycetes and some bacteria, access to host
resources requires breaking of the cell wall barrier. During cell expansion, however,
feedback control of cell wall integrity should act rapidly and before the manifestation of
large-scale structural damage.
We are interested in the acute response to perturbation of cell wall integrity. The yeast cell
wall integrity pathway is triggered by inhibitors of glucan and chitin synthases (e.g.
echinocandin and nikkomycin, respectively) and by compounds that bind to cell wall
polysaccharides and inhibit their higher-order assembly (Congo Red and Calcofluor
White)(Levin, 2005). We have used analogous tools to trigger cell wall damage in plants
6
including inhibitors of cellulose biosynthesis (isoxaben, thaxtomin A and dichlobenil) and a
cellulose-binding dye (Congo Red). We show that the accelerated elongation phase of root
cells is rapidly inhibited by both types of cell wall damaging agents. This inhibition is ACCdependent but strikingly, is not dependent on ethylene perception. It represents a general
rapid-response pathway of root growth control that also underlies at least the initial response
to microbial PAMPs (pathogen-associated molecular patterns). Auxin signalling and
superoxide production are required downstream of ACC to reduce elongation, in agreement
with the previously described effect of ethylene on root elongation.
7
Results
It takes several days for newly produced root cells to traverse the cell division zone. In
contrast, expansion to their mature size in the elongation zone only takes six to eight hours
(Beemster and Baskin, 1998). This tenfold or higher increase in length and thus cell surface
area places a great demand on cell wall biosynthesis. In 4d old seedlings, expression of the
primary wall cellulose synthase genes CesA1, CesA3 and CesA6 is strongest around the
transition zone and continues into the elongation zone (Fig. 1a) (Scheible et al., 2001;
Desprez et al., 2007). We have used the herbicide isoxaben to inhibit cellulose biosynthesis
(Heim et al., 1990) and so phenocopy the type of cell wall damage seen in cellulose-deficient
mutants. CesA3 and 6 are targets of isoxaben (Scheible et al., 2001; Desprez et al., 2002);
CesA6-YFP labelled particles disappear from the cell surface within 20 minutes of isoxaben
treatment (Paredez et al., 2006). Over 16h, isoxaben induces root swelling and strongly
reduced elongation, as evident in the “crowding” of root hairs due to shortening of
trichoblasts (Fig. 1b). To analyse more acute effects of cellulose biosynthesis inhibition, we
used a simple proxy for the complex spatial variation of elongation rates along the root. The
length of the first epidermal cell with a visible root hair bulge (LEH, Fig, 1C) (Le et al.,
2001; De Cnodder et al., 2005) is a parameter that reflects rapid effects on elongation much
more sensitively than macroscopic root length measurements. Isoxaben reduced the LEH to
about 35% of control within 8h; a significant effect is evident from about one hour (Figure
1D). For the following experiments we chose 3h treatments which led to a robust response.
With this time window, the differentiating trichoblast cells measured for the LEH parameter
would have already committed to rapid elongation at the time of treatment (Beemster and
Baskin, 1998). The fact that the LEH drops continuously over several hours shows that root
cells are sensitive to relevant environmental signals throughout elongation, in contrast to
cells in etiolating hypocotyls which lose sensitivity to cell wall damage once committed to
rapid elongation (Refregier et al., 2004). Although the study of root growth is complicated
by the contribution of both cell division and expansion, the short term experiments shown
here are unlikely to be affected by changes in cell division.
Many plant hormones negatively affect root growth, including ABA, auxin and jasmonate,
but the best-characterised effect specifically on elongation is exerted by ethylene (Benkova
and Hejatko, 2009). To find out if the isoxaben effect is mediated by ethylene, we applied
8
isoxaben
together
with
a
chemical
inhibitor
of
ethylene
biosynthesis,
Aminoethoxyvinylglycine (AVG), or with silver ions (as silver thiosulfate) to block ethylene
perception. While silver had no significant effect on cell length in control or isoxaben-treated
roots, AVG fully restored elongation in the presence of isoxaben (Fig. 2A). Often roots
treated with both isoxaben and AVG showed dramatic symptoms of cell wall defects such as
blebbing (Fig. 2B) while this was practically never seen when isoxaben alone was applied.
These observations demonstrate that cell expansion can proceed despite cell wall defects.
The reduction of elongation triggered by blocking cellulose biosynthesis is an active, AVGsensitive process rather than a passive biomechanical consequence of weakened walls.
The results of the inhibitor treatments were surprising because they suggested that ethylene
perception was not required for this response while AVG blocks it. In a canonical ethylenedependent pathway, inhibiting any step between ACC biosynthesis and ethylene perception
should block the response. An alternative ethylene receptor blocker, norbornadiene, did not
restore elongation either (Fig. S2A). To assess the efficiency of silver treatment, we treated
Col-0 seedlings with ACC or with ethephon, a compound that hydrolyses to ethylene above
pH 3.5 (such as in plant cell cytoplasm). Both compounds reduced the LEH but only the
response to ethephon-released ethylene could be completely reversed with 10µM silver
thiosulfate (Fig. 2C). In some cases, the effect of ethephon has been shown to be
independent of ethylene generation (Lawton et al., 1994). However, a control experiment
showed that buffered phosphoric acid plus hydrochloric acid (the other hydrolysis products
of ethephone) at the equivalent concentration did not affect LEH (Fig. S2B).
A “side effect” of silver treatment, the promotion of indole-3-acetic acid efflux, has recently
been described (Strader et al., 2009). We therefore sought additional confirmation of
ethylene-independent short-term effects of ACC on root elongation. Externally applied ACC
reduced root elongation in the same timescale as isoxaben, with a maximal response above
ca 100nM ((Le et al., 2001) and Fig. S1). The ein3 eil1 mutant is completely ethyleneinsensitive; hypocotyls of dark-grown ein3 eil1 seedlings show no response to ACC
((Alonso et al., 2003) and Fig. S3), and long-term root growth (4-5 days) is insensitive to
both ACC and ethephon (data not shown). While the LEH in Col-0 seedlings was reduced by
ethylene (Fig. 2D; here applied as gas generated from ethephon without direct contact
(Zhang and Wen, 2010)), ein3 eil1 seedlings were insensitive. In contrast, isoxaben and
ACC significantly reduced the LEH both in mutant and wild type. ACC thus appears to have
a short-term influence on root cell elongation that is independent of the canonical ethylene
signalling pathway.
9
To further analyse the AVG-silver discrepancy, we also applied Aminoxyacetic acid (AOA),
another inhibitor of ACC synthase (Yu et al., 1979), and α-aminoisobutyric acid (AIB), an
inhibitor of ACC oxidase (Satoh and Esashi, 1983). AIB or AOA had no effect on LEH but
all three inhibitors fully restored LEH in isoxaben-treated roots. (Fig. 3A, light grey bars). In
batches of seedlings with lower initial LEH, AVG increased elongation relative to the
control. Chemical inhibitors of ACC biosynthesis are widely used because ACC synthases
(ACS) are encoded by a multigene family and complete elimination of all nine members
causes embryonic lethality (Tsuchisaka et al., 2009). However, AVG and AOA not only
inhibit ACS but all pyridoxal phosphate requiring enzymes, including tryptophan
aminotransferase in the auxin biosynthetic pathway (Soeno et al., 2010). To test their
specificity in our assay, we applied ACC together with either inhibitor. If AVG and AOA act
upstream of ACC by inhibiting ACS activity, root elongation should still be reduced by
ACC. Instead, both compounds prevented most of the ACC-induced reduction of LEH (Fig.
3A, dark grey bars). This indicates that in our experimental system AVG and AOA inhibit
processes that are downstream or independent of ACC biosynthesis so are not suitable
inhibitors. Conversely, AIB should act downstream of ACC in the ethylene biosynthesis
pathway but did not affect the ACC response (Fig. 3A). It is possible that AIB inhibits
isoxaben-induced processes that are upstream or independent of ACC. Another not mutually
exclusive explanation is that the 10µM dose of ACC used here is too high for ethylene
generation to be efficiently inhibited by AIB, a very weak competitive inhibitor of ACC
oxidase. To reverse most of the root response to 1µM external ACC, the highest tested dose
of 1mM AIB was required (Fig. S1). Our finding that 10µM 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, novel inhibitors of ACC synthase have been identified from a chemical screen (Lin
et al., 2010). To obtain additional evidence that ACC biosynthesis is required to block root
elongation in response to isoxaben, we tested the compound that was most active as
suppressor of the constitutive triple response phenotype of the eto1-4 mutant (Lin et al.,
2010), 2-anilino-7-(4-methoxyphenyl)-7,8-dihydro-5(6H)-quinazolinone (7303). At 5µM,
the compound did not affect elongation on its own, but completely inhibited the acute
response to isoxaben (Fig. 3A). In contrast, externally added ACC reduced elongation as
much as in the untreated control. Since this compound is an uncompetitive inhibitor of ACC
synthase that is structurally and mechanistically unrelated to the more established AVG,
10
these results independently corroborate ACC biosynthesis as a necessary component of cell
wall integrity signalling.
Other cell wall stress inducers like the cell wall-binding dye Congo Red reduced root
elongation on a similar time scale, and the two ACC synthase inhibitors restored most or all
of this inhibition (Fig. 3B). Stress factors other than cell wall defects are also known to
reduce plant growth in general and/or root elongation in particular, including microbial/
pathogen-associated molecular patterns (MAMPs) or elicitors (Gomez-Gomez 1999). We
tested the short-term effect of flagellin-22 which is known to trigger defence responses in
roots (Millet 2010). Flagellin reduced the LEH within 5h to about the same level as isoxaben
at 3h. This reduction, too, was sensitive to AIB and 7303 (Fig. 3B). These results show that
root elongation is rapidly controlled via ACC biosynthesis in response to a wide range of
stress triggers.
Regulation
of ACC
synthase activity occurs
at
both the
transcriptional and
posttranscriptional level (Argueso et al., 2007). To test whether ACC synthase expression
was induced in the elongation zone in response to isoxaben, we analysed all available
pACS::GUS lines. ACS isoforms 2,4,6 and 8 showed strong (and ACS5 weak) constitutive
expression while ACS9 was not detected. In contrast, ACS11 was induced in the root
elongation zone within a few hours of isoxaben treatment (Fig. S4).
A complex interplay between hormones regulates root growth (Benkova and Hejatko, 2009).
Several recent studies have shown that auxin biosynthesis and transport are required to
mediate ethylene responses in roots (Ruzicka et al., 2007; Stepanova et al., 2007; Swarup et
al., 2007): ACC/ ethylene activates auxin synthesis via a root tip-specific pathway involving
anthranilate synthase and tyrosine aminotransferase (Stepanova et al., 2005; Stepanova et al.,
2008). In addition, basipetal auxin transport via AUX1 and EIR1/PIN2 (but not acropetal
transport via PIN1 and PIN4) is required. We have analysed the isoxaben- induced LEH
reduction in these mutant backgrounds. pin4-3 responded like the wild type; the aux1-T,
eir1-1 and tir1-1 mutants showed a response that was slightly smaller in amplitude than the
wt, but the reduction in these cases was from a considerably bigger cell size down to the
level of untreated wild type roots (Fig. 4A and S5). To phenocopy the effect of strong auxinresistant mutants, we used a synthetic antagonist of TIR1 receptor function, α-(phenyl ethyl2-oxo)-indole acetic acid (PEO-IAA, (Hayashi et al., 2008)) as well as its inactive 5-methyl
derivative. The antagonist PEO-IAA, but not 5-methyl-PEO-IAA, completely abrogated the
11
effect of isoxaben or ACC on LEH (Fig. 4B and S6). Therefore, reduced elongation
triggered by stress/ ACC requires auxin signalling but is only partially dependent on
basipetal auxin transport via AUX1 and PIN2.
De Cnodder et al have shown (De Cnodder et al., 2005) that the effect of ACC or ethylene
on root elongation is mediated by extracellular events that affect cell wall crosslinking.
Specifically, the production of reactive oxygen species and crosslinking of hydroxyprolinerich glycoproteins (HRGPs) 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 diphenylene iodonium (DPI, Fig. 4C),
an inhibitor of flavin-containing enzymes including NADPH oxidases.
Discussion
Growing primary roots are vulnerable to cell wall damage; the rapid elongation phase
increases the cell surface area by an order of magnitude. This requires massive cell wall
rearrangement and greatly increased polysaccharide biosynthesis. We have shown that
inhibition of cellulose biosynthesis or interference with cell wall assembly rapidly reduces
elongation. Understanding the molecular mechanisms of cell wall integrity control poses a
dilemma: Cell wall damage, be it genetically or pharmacologically induced, will ultimately
lead to structural problems that are incompatible with growth. It is conceivable that growing
cells with too weak walls simply burst and die. Growth arrest, as observed in isoxabentreated seedlings, might be due to osmotic stress, plasma membrane stretch and/or general
stress, rather than a dedicated system of cell wall integrity surveillance like in yeast and
other fungi. However, two major lines of evidence have supported the existence of such a
monitoring system so far: First, receptor-like kinases without obvious cell wall biosynthetic
capacity have been found (e.g. FEI1 and FEI2, FERONIA, THESEUS and HERCULES1
(Xu et al., 2008; Guo et al., 2009)) whose absence causes cell wall defects similar to mutants
in cell wall biosynthetic genes. This suggests control of wall biogenesis by an integrated
signalling pathway. Second, if a signalling process lies between cell wall defects and growth
reduction or other compensatory responses, it should be possible to restore growth by
disrupting signal transduction without restoring the cell wall defect. The identification of the
12
receptor kinase THE1 (Hematy et al., 2007) provided clear genetic evidence that this is the
case in hypocotyls. Dwarfism and reduced elongation of etiolated hypocotyls in several
moderately cellulose-deficient mutants is much less pronounced in the absence of THE1. In
this study we have demonstrated that an active signalling process reduces root elongation
when cell wall biosynthesis is impaired. Disrupting this signalling process at any one of
three different steps (ACC biosynthesis, auxin signalling, superoxide production) fully
restores elongation in the short term despite clearly visible cell wall damage. We have thus
confirmed the existence of a cell wall integrity signalling pathway for roots. Interestingly,
neither of the abovementioned receptor kinase mutants with a cell wall integrity phenotype
(fei1 fei2 and the1) was affected in the rapid response to isoxaben in our assays (Fig. S5 and
data not shown). The original sensor(s) which communicate(s) deficient cell wall structure or
biosynthesis to the cytoplasm thus also remain to be identified. The cell wall integrity
pathway merges with a general rapid stress response pathway that requires ACC biosynthesis
but not ethylene perception to control root elongation.
Ethylene reduces root elongation via auxin biosynthesis in the root tip (Stepanova et al.,
2005; Stepanova et al., 2008) and basipetal auxin transport (Ruzicka et al., 2007; Stepanova
et al., 2007; Swarup et al., 2007). Further down in the pathway, NADPH oxidase-dependent
superoxide production and cell wall protein maturation and crosslinking are required (De
Cnodder et al., 2005). Our experiments first seemed to show that the cell wall damage
response (as well as the response to microbial PAMPs) follows this established pathway, but
surprisingly ethylene perception was not required for this response. As summarised in Fig. 5,
in the rapid control of root cell elongation, both ACC and ethylene can act via what appears
to be the same pathway. For long-term growth responses, such as those shown in Fig. S3,
conversion of ACC to ethylene is required. We have also found that the expression of some
rapidly isoxaben-induced genes requires ethylene perception (data not shown). The “short
cut” pathway from ACC may be limited to short-term responses including reduced
elongation. We have not formally established that auxin is upstream of ROS production in
this context. While this order is best supported by the literature (Joo et al., 2001; Schopfer et
al., 2002), auxin-ROS interactions are not one-way, and both have independent functions in
root growth control (Potters et al., 2007; Tsukagoshi et al., 2010). The exact process
inhibited by DPI –ROS signalling or direct oxidative remodelling of cell walls- remains
open.
One puzzling fact remains. Externally added ACC acts on root elongation both as ACC itself
and after conversion to ethylene, as shown by the partial sensitivity to silver. Why then is the
13
short-term response to cell wall damage or PAMPs completely ethylene-independent? If
ACC biosynthesis is activated, how could it not be converted to ethylene and thus act on
elongation? One explanation could be that either ethylene formation or ethylene signalling is
(temporarily) suppressed. An alternative, perhaps more likely working hypothesis is that
ACC concentrations induced by cell wall or biotic stress are initially low and that ACC binds
to a dedicated receptor or binding protein with higher affinity than to ACC oxidase,
triggering a reduction in elongation. As ACC accumulates, this system is saturated and ACC
oxidation to ethylene begins. Externally added ACC would practically always exceed the
capacity of this system and exert its effects at least partially via ethylene. Low
concentrations of AIB efficiently inhibit the postulated ACC-binding protein, as proposed by
(Xu et al., 2008), while millimolar concentrations are required to block ACC oxidase. It
remains unclear exactly how ACC biosynthesis is controlled in the cell wall damage
response. Although expression of ACS11 is induced rapidly by isoxaben, several other
isoforms are constitutively expressed. Posttranslational mechanisms plays an important role
in the regulation of ACS stability (Argueso et al., 2007) and might explain the discrepancy.
The idea that in some developmental pathways ACC might act as a signal in its own right,
independently of ethylene receptors or the canonical downstream pathway, has been
proposed before (Xu et al., 2008; Tsuchisaka et al., 2009). Multiple knockouts of ACC
synthase genes have increasingly severe developmental defects (Tsuchisaka et al., 2009)
while completely ethylene-insensitive mutants like ein2 and ein3 eil1 are relatively healthy
(Alonso et al., 1999; Alonso et al., 2003). Currently we do not know to what degree the
responses to ACC and to ethylene overlap. ACC could trigger distinct responses or “just” be
a shortcut to ethylene responses. The advantage of an “ACC shortcut” to at least some
ethylene responses could be that they can be triggered earlier in a cell-autonomous way
before threshold concentrations of the easily dissipating gaseous ethylene are reached. We
note that for the short-term response to isoxaben or ACC, auxin signalling but not auxin
transport is required (Fig. 4B and S5), while the long-term growth response requires both
(Ruzicka et al., 2007; Strader et al., 2010). This suggests that for auxin, too, only cellautonomous functions may be required in the hypothetical “ACC shortcut”. Which of the
established later elements of the ethylene pathway (if any) are required to transmit this rapid
ACC-dependent signal remains the subject of further research.
Conclusion
14
We have established that a rapid reduction in root cell elongation is an acute response to
perturbation of cell wall integrity. We have shown that this is a response mediated by an
active signalling pathway rather than a simple consequence of cell wall failure. For at least a
few hours, elongation can proceed despite cell wall damage if signalling is blocked. This
means that the short term LEH assay can be used as a tool to separate cell wall signalling
from broader structural damage induced by isoxaben. We are currently analysing a range of
mutants in candidate cell wall receptors and other signalling proteins for loss of short-term
responsiveness in this assay.
It is intriguing that ACC biosynthesis but not canonical ethylene signalling are required for
this stress-induced morphogenic response (Potters et al., 2007). Having established the root
elongation zone as a target of an “ACC shortcut”, it would be very interesting to dissect the
transcriptional response to ACC versus ethylene in roots of wild type and ethyleneinsensitive mutants. The relative contribution of canonical and ethylene-independent
pathways remains to be established; unique genetic components of ACC signalling have yet
to be identified.
Materials and methods
Plant material and growth conditions
The following mutants and transgenic lines were obtained from the Nottingham Arabidopsis
stock centre: pin4-3 (N9368), aux1-T (N657534), eir1-1 (N8058), tir1-1 (N3798), Theo-AtACS1-GUS/GFP (N31379), Theo-At-ACS2-GUS/GFP (N31380), Theo-At-ACS4-GUS
(N31381), Theo-At-ACS5-GUS (N31382), Theo-At-ACS6-GUS/GFP (N31383), Theo-AtACS8-GUS/GFP
(N31385);
Theo-At-ACS9-GUS/GFP
(N31386),
Theo-At-ACS11-
GUS/GFP (N31387), ProCESA1:GUS (N70755) and ProCESA6:GUS (N70760). Seeds of
ein3-1 eil1-1, the1-3 and fei1 fei2 mutants and the CEV1::GUS line were kindly provided by
Joseph Ecker, Herman Hofte, Joseph Kieber and John Turner, respectively. Seeds of
Arabidopsis thaliana were surface-sterilised with 70% ethanol (5min), then 15% household
bleach (Parozone) for 30min and washed 6 times with sterile water. After at least 2 days at
4˚C, seeds were sown onto square plates with ½ Murashige and Skoog minimal salts
(Melford, Chelsworth, UK), 2g/L sucrose and 0.8% (w/v) agar. Seedlings were grown
15
vertically in a Sanyo MLR-351 growth chamber for 4 days at a 16h light (60% of full output
or ca. 100µmol m-2 s-1, 24˚C), 8h dark (21˚C) cycle.
Root treatments and LEH measurements
At least 20 four day old seedlings were carefully transferred onto microscopic slides with a
“cushion” of ½ MS agar containing the treatment. The slides were kept in a petri dish with a
wet filter paper, sealed with surgical tape and returned to the growth chamber for typically
3h. The following final concentrations of reagents were used: 150nM isoxaben,
Aminoxyacetic acid (AOA), Aminoethoxyvinylglycine (AVG) and α-aminoisobutyric acid
(AIB) all at 10µM, 50µM flg-22 peptide (Eurogentech, Seraing, Belgium), 5mg/L Congo
Red, 10µM silver thiosulfate (a 20mM stock was freshly prepared by mixing 1 volume
100mM silver nitrate with 4 volumes 100mM sodium thiosulfate), 10µM 2-anilino-7-(4methoxyphenyl)-7,8-dihydro-5(6H)-quinazolinone (compound # 9127303 or short “7303”,
Hit2Lead/ Chembridge Corp., San Diego, USA), 200µM ethephon (from 5mM stock mixed
with an equal volume 15mM HEPES/KOH pH 6.5) for direct contact or 200µl of 5mM
ethephon added to a filter paper wetted with 1mM K2HPO4 pH 9 for ethylene gas generation
(resulting in a maximal concentration of 500µL L-1 ethylene gas); 25µM α-(phenyl ethyl-2oxo)-indole acetic acid (PEO-IAA) and 5-methyl PEO (kind gift of Ken-ichiro Hayashi,
Okayama University of Science, Japan), 10µM Diphenylene iodonium. All chemicals were
from Sigma/ Aldrich unless noted otherwise.
After 3h of treatment, roots were analysed directly (i.e. without a coverslip) with a
microscope (DMR, Leica) fitted with a 20x objective (HL PL Fluotar; Leica, n.a. = 0.50).
Images were taken with a SPOT Xplorer 4Mp camera (Diagnostic Instruments) and the
length of the first epidermal cell with a visible root hair bulge (LEH, (Le et al., 2001))
measured with the SPOT software or ImageJ. On each root, one to three cells could be
measured with confidence, resulting in 30-50 measurements per treatment. Each result is
representative of at least three independent experiments.
GUS staining
Seedlings were transferred to fresh control or isoxaben plates for 3hours. For staining,
seedlings were transferred to chilled 90% acetone for 20min and then incubated with
staining buffer (50mM sodium phosphate pH7.2; 0.2% Triton X-100; 1 mM each
K3[Fe(CN)6] and K4[Fe(CN)6], 1mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) for 3
16
hours (proACS::GUS lines) or overnight (pCesA::GUS lines). Staining was stopped with
100% ethanol and seedlings were imaged as described above.
Environmental Scanning Electron Microscopy
The microscope images were taken using a FEI Quanta 200 scanning electron microscope
(Philips) in ESEM mode (Gaseous secondary electron detector, 30 kV, 3.5-5.5 Torr, sample
cooled to 5˚C). The apical 5-6mm of root tips were detached with a razor blade, transferred
to a drop of water onto the sample stub and inserted into the chamber. Images were taken
just before all water had evaporated from the sample stub.
Acknowledgements
We would like to thank Raymond Wightman and Patrick Hill for help with light and electron
microscopy, respectively; John G. Turner (University of East Anglia), Herman Höfte (INRA
Versailles), Joseph J. Kieber (University of North Carolina) and Joseph R. Ecker (Salk
Insitute) for transgenic/ mutant seeds and Ken-ichiro Hayashi (Okayama University of
Science) for a generous gift of auxin inhibitors.
References
Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional
transducer of ethylene and stress responses in Arabidopsis. Science 284: 2148-2152
Alonso JM, Stepanova AN, Solano R, Wisman E, Ferrari S, Ausubel FM, Ecker JR
(2003) Five components of the ethylene-response pathway identified in a screen for
weak ethylene-insensitive mutants in Arabidopsis. Proceedings of the National
Academy of Sciences of the United States of America 100: 2992-2997
Argueso CT, Hansen M, Kieber JJ (2007) Regulation of ethylene biosynthesis. Journal of
Plant Growth Regulation 26: 92-105
Baskin TI, Betzner AS, Hoggart R, Cork A, Williamson RE (1992) Root Morphology
Mutants in Arabidopsis thaliana. Australian Journal of Plant Physiology 19: 427-437
Beemster GTS, Baskin TI (1998) Analysis of cell division and elongation underlying the
developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiology
116: 1515-1526
Benkova E, Hejatko J (2009) Hormone interactions at the root apical meristem. Plant
Molecular Biology 69: 383-396
Cano-Delgado AI, Metzlaff K, Bevan MW (2000) The eli1 mutation reveals a link
between cell expansion and secondary cell wall formation in Arabidopsis thaliana.
Development 127: 3395-3405
De Cnodder T, Vissenberg K, Van Der Straeten D, Verbelen JP (2005) Regulation of
cell length in the Arabidopsis thaliana root by the ethylene precursor 1-
17
aminocyclopropane-1-carboxylic acid: a matter of apoplastic reactions. New
Phytologist 168: 541-550
Desnos T, Orbovic V, Bellini C, Kronenberger J, Caboche M, Traas J, Hofte H (1996)
Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell
elongation, respectively in dark and light-grown Arabidopsis seedlings. Development
122: 683-693
Desprez T, Juraniec M, Crowell EF, Jouy H, Pochylova Z, Parcy F, Hofte H, Gonneau
M, Vernhettes S (2007) Organization of cellulose synthase complexes involved in
primary cell wall synthesis in Arabidopsis thaliana. Proceedings of the National
Academy of Sciences of the United States of America 104: 15572-15577
Desprez T, Vernhettes S, Fagard M, Refregier G, Desnos T, Aletti E, Py N, Pelletier S,
Hofte H (2002) Resistance against herbicide isoxaben and cellulose deficiency
caused by distinct mutations in same cellulose synthase isoform CESA6. Plant
Physiology 128: 482-490
Ellis C, Karafyllidis I, Wasternack C, Turner JG (2002) The Arabidopsis mutant cev1
links cell wall signaling to jasmonate and ethylene responses. Plant Cell 14: 15571566
Guo HQ, Li L, Ye HX, Yu XF, Algreen A, Yin YH (2009) Three related receptor-like
kinases are required for optimal cell elongation in Arabidopsis thaliana. Proceedings
of the National Academy of Sciences of the United States of America 106: 76487653
Hamann T, Bennett M, Mansfield J, Somerville C (2009) Identification of cell-wall stress
as a hexose-dependent and osmosensitive regulator of plant responses. Plant Journal
57: 1015-1026
Hauser MT, Morikami A, Benfey PN (1995) Conditional Root Expansion Mutants of
Arabidopsis. Development 121: 1237-1252
Hayashi K, Hatate T, Kepinski S, Nozaki H (2008) Design and synthesis of auxin probes
specific to TIR1, auxin receptor. Regulation of Plant Growth and Development 43
(suppl.): 95
Heim DR, Skomp JR, Tschabold EE, Larrinua IM (1990) Isoxaben inhibits the synthesis
of acid-insoluble cell wall materials in Arabidopsis thaliana. Plant Physiology 93:
695-700
Hematy K, Sado PE, Van Tuinen A, Rochange S, Desnos T, Balzergue S, Pelletier S,
Renou JP, Hofte H (2007) A receptor-like kinase mediates the response of
Arabidopsis cells to the inhibition of cellulose synthesis. Current Biology 17: 922931
Hernandez-Blanco C, Feng DX, Hu J, Sanchez-Vallet A, Deslandes L, Llorente F,
Berrocal-Lobo M, Keller H, Barlet X, Sanchez-Rodriguez C, Anderson LK,
Somerville S, Marco Y, Molina A (2007) Impairment of cellulose synthases
required for Arabidopsis secondary cell wall formation enhances disease resistance.
Plant Cell 19: 890-903
Humphrey TV, Bonetta DT, Goring DR (2007) Sentinels at the wall: cell wall receptors
and sensors. New Phytologist 176: 7-21
Joo JH, Bae YS, Lee JS (2001) Role of auxin-induced reactive oxygen species in root
gravitropism. Plant Physiology 126: 1055-1060
Kohorn BD, Johansen S, Shishido A, Todorova T, Martinez R, Defeo E, Obregon P
(2009) Pectin activation of MAP kinase and gene expression is WAK2 dependent.
Plant Journal 60: 974-982
Lawton KA, Potter SL, Uknes S, Ryals J (1994) Acquired-Resistance Signal Transduction
in Arabidopsis is Ethylene Independent. Plant Cell 6: 581-588
18
Le J, Vandenbussche F, Van der Straeten D, Verbelen JP (2001) In the early response of
arabidopsis roots to ethylene, cell elongation is up- and down-regulated and
uncoupled from differentiation. Plant Physiology 125: 519-522
Levin DE (2005) Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiology
and Molecular Biology Reviews 69: 262-291
Lin LC, Hsu JH, Wang LC (2010) Identification of Novel Inhibitors of 1Aminocyclopropane-1-carboxylic Acid Synthase by Chemical Screening in
Arabidopsis thaliana. Journal of Biological Chemistry 285: 33445-33456
Mueller K, Linkies A, Vreeburg RAM, Fry SC, Krieger-Liszkay A, Leubner-Metzger
G (2009) In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed
Germination and Elongation Growth. Plant Physiology (Rockville) 150: 1855-1865
Nishimura MT, Stein M, Hou BH, Vogel JP, Edwards H, Somerville SC (2003) Loss of
a callose synthase results in salicylic acid-dependent disease resistance. Science 301:
969-972
Paredez AR, Somerville CR, Ehrhardt DW (2006) Visualization of cellulose synthase
demonstrates functional association with microtubules. Science 312: 1491-1495
Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how plant peroxidases
modify the cell wall. Trends in Plant Science 9: 534-540
Potters G, Pasternak TP, Guisez Y, Palme KJ, Jansen MAK (2007) Stress-induced
morphogenic responses: growing out of trouble? Trends in Plant Science 12: 98-105
Refregier G, Pelletier S, Jaillard D, Hofte H (2004) Interaction between wall deposition
and cell elongation in dark-grown hypocotyl cells in Arabidopsis. Plant Physiology
135: 959-968
Ruzicka K, Ljung K, Vanneste S, Podhorska R, Beeckman T, Friml J, Benkova E
(2007) Ethylene regulates root growth through effects on auxin biosynthesis and
transport-dependent auxin distribution. Plant Cell 19: 2197-2212
Satoh S, Esashi Y (1983) α-Aminoisobutyric acid, Propyl Gallate and Cobalt Ion and the
Mode of Inhibition of Ethylene Production by Cotyledonary Segments of Cocklebur
Seeds. Physiologia Plantarum 57: 521-526
Scheible WR, Eshed R, Richmond T, Delmer D, Somerville C (2001) Modifications of
cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in
Arabidopsis Ixr1 mutants. Proceedings of the National Academy of Sciences of the
United States of America 98: 10079-10084
Schopfer P, Liszkay A, Bechtold M, Frahry G, Wagner A (2002) Evidence that hydroxyl
radicals mediate auxin-induced extension growth. Planta 214: 821-828
Seifert GJ, Blaukopf C (2010) Irritable Walls: The Plant Extracellular Matrix and
Signaling. Plant Physiology 153: 467-478
Soeno K, Goda H, Ishii T, Ogura T, Tachikawa T, Sasaki E, Yoshida S, Fujioka S,
Asami T, Shimada Y (2010) Auxin Biosynthesis Inhibitors, Identified by a
Genomics-Based Approach, Provide Insights into Auxin Biosynthesis. Plant and Cell
Physiology 51: 524-536
Steinwand BJ, Kieber JJ (2010) The Role of Receptor-Like Kinases in Regulating Cell
Wall Function. Plant Physiology 153: 479-484
Stepanova AN, Hoyt JM, Hamilton AA, Alonso JM (2005) A link between ethylene and
auxin uncovered by the characterization of two root-specific ethylene-insensitive
mutants in Arabidopsis. Plant Cell 17: 2230-2242
Stepanova AN, Robertson-Hoyt J, Yun J, Benavente LM, Xie DY, DoleZal K, Schlereth
A, Jurgens G, Alonso JM (2008) TAA1-mediated auxin biosynthesis is essential for
hormone crosstalk and plant development. Cell 133: 177-191
19
Stepanova AN, Yun J, Likhacheva AV, Alonso JM (2007) Multilevel interactions
between ethylene and auxin in Arabidopsis roots. Plant Cell 19: 2169-2185
Strader LC, Beisner ER, Bartel B (2009) Silver Ions Increase Auxin Efflux Independently
of Effects on Ethylene Response. Plant Cell 21: 3585-3590
Strader LC, Chen GL, Bartel B (2010) Ethylene directs auxin to control root cell
expansion. Plant Journal 64: 874-884
Swarup R, Perry P, Hagenbeek D, Van Der Straeten D, Beemster GTS, Sandberg G,
Bhalerao R, Ljung K, Bennett MJ (2007) Ethylene upregulates auxin biosynthesis
in Arabidopsis seedlings to enhance inhibition of root cell elongation. Plant Cell 19:
2186-2196
Tsuchisaka A, Yu GX, Jin HL, Alonso JM, Ecker JR, Zhang XM, Gao S, Theologis A
(2009) A Combinatorial Interplay Among the 1-Aminocyclopropane-1-Carboxylate
Isoforms Regulates Ethylene Biosynthesis in Arabidopsis thaliana. Genetics 183:
979-1003
Tsukagoshi H, Busch W, Benfey PN (2010) Transcriptional Regulation of ROS Controls
Transition from Proliferation to Differentiation in the Root. Cell 143: 606-616
Vogel JP, Raab TK, Somerville CR, Somerville SC (2004) Mutations in PMR5 result in
powdery mildew resistance and altered cell wall composition. Plant Journal 40: 968978
Xu SL, Rahman A, Baskin TI, Kieber JJ (2008) Two Leucine-Rich Repeat Receptor
Kinases Mediate Signaling, Linking Cell Wall Biosynthesis and ACC Synthase in
Arabidopsis. Plant Cell 20: 3065-3079
Yu YB, Adams DO, Yang SF (1979) 1-Aminocyclopropanecarboxylate Synthase, A Key
Enzyme in Ethylene Biosynthesis. Archives of Biochemistry and Biophysics 198:
280-286
Zhang W, Wen CK (2010) Preparation of ethylene gas and comparison of ethylene
responses induced by ethylene, ACC, and ethephon. Plant Physiology and
Biochemistry 48: 45-53
20
Figure legends
Fig. 1 Isoxaben rapidly reduces root cell elongation. A, Histochemical assay showing GUS
expression in the root tip of proCesA1::GUS, proCesA3::GUS and proCesA6::GUS
transgenic Arabidopsis seedlings. B, Root tips 16h after control or isoxaben treatment
(150nM). C, The length of the first epidermal cell with a visible root hair bulge (LEH) was
measured as the distance from the first visible root hair bulge to the next more differentiated
root hair in the same trichoblast cell file. D, Time course of LEH reduction by treatment with
150nM isoxaben.
Fig. 2 Fig. 2 Cell wall stress reduces root elongation via an AVG-sensitive, ethyleneindependent pathway. A, Inhibition of ACC biosynthesis but not ethylene action restores
isoxaben-induced root growth inhibition. *** and n.s., LEH difference significant
(p<0.0001) or not significant (p>0.05, 2-tailed t-test) B, Elongation and differentiation zone
of roots treated for 4h with isoxaben, AVG or both (ESEM images). Scale bar, 200µm. Note
the severe bulging of roots treated with both compounds. C, The effect of ACC on root
elongation is partially ethylene-independent.
Symbols a, b, significantly (p<0.0001)
+
different from control or Ag -only treated roots, respectively, or n.s., not significantly
different (p>0.05, 2-tailed t-test). D, The ethylene-insensitive mutant ein3eil1 responds to
ACC and isoxaben.
Fig. 3 Inhibition of ACC biosynthesis restores root elongation in the presence of cell wall
damage and other stress. A, LEH of roots treated for 3h with isoxaben or ACC in the
presence or absence of AIB, AVG, AOA or 7303. A separate set of controls minus 7303 is
indicated with grey dashed lines. Symbols a, b, c, e, LEH is significantly (p<0.0001) reduced
vs roots treated with vehicle, AIB, AVG or 7303 only, respectively (2-tailed t-test). Symbol
(d), significantly reduced (p<0.01) vs roots treated with AOA only. “n.s.”, not significantly
different (p>0.05) from appropriate inhibitor-only value. B, Inhibition of ACC synthase or
ACC action restores elongation in the presence of a cell wall binding dye (Congo Red) or a
microbial elicitor (flg22). *** and n.s. as before.
Fig. 4 The repression of root elongation by cell wall stress requires auxin signalling and
reactive oxygen species. A, Isoxaben reduces root elongation in the auxin receptor mutant,
21
tir1-1. B, The auxin response inhibitor PEO-IAA, but not its inactive 5-methyl derivative,
restores elongation in the presence of isoxaben. C, Inhibition of NADPH oxidases restores
root elongation in the presence of isoxaben. Indicators of t-test results as in Fig. 2.
Fig. 5 A working model for the control of root elongation by ACC or ethylene. Short-term
effects of ACC on root elongation are partially ethylene-independent. Note that we have not
formally established whether auxin is upstream of ROS production or acts independently.
SAM, S-adenosyl methionine.
22
A
B
C
D
140
120
100
H
E
L
80
)
m
(µ 60
H
E
L
40
20
0
CesA1
CesA3
CesA6
co
isox
0
2
4
6
Time (hours)
8
Fig. 1
Fig. 1 Isoxaben rapidly reduces root cell elongation. A, Histochemical assay showing GUS
expression in the root tip of proCesA1::GUS, proCesA3::GUS and proCesA6::GUS
transgenic Arabidopsis seedlings. B, Root tips 16h after control or isoxaben treatment
(150nM). C, The length of the first epidermal cell with a visible root hair bulge (LEH) was
measured as the distance from the first visible root hair bulge to the next more
differentiated root hair in the same trichoblast cell file. D, Time course of LEH reduction
by treatment with 150nM isoxaben.
A
***
B
n.s.
***
120
100
)
m80
(µ
H 60
E
L
40
20
isox
-
+
Ag
+
+
AVG
+
C
n.s.
120
100
)
m80
µ
(
H60
E
L
D
b
100
)
m80
µ
(
H60
E
L
40
20
20
-
+
AVG
Isoxaben
+AVG
n.s.
40
ACC
+
ethephon
isoxaben
120
a
a
control
+
Ag
+
+
isoxaben
ACC
ethylene
a
a
a
a
a
+
+
Col - 0
+
+
+
+
ein3 eil1
Fig. 2
Fig. 2 Cell wall stress reduces root elongation via an AVG-sensitive, ethyleneindependent pathway. A, Inhibition of ACC biosynthesis but not ethylene action
restores isoxaben-induced root growth inhibition. *** and n.s., LEH difference
significant (p<0.0001) or not significant (p>0.05, 2-tailed t-test) B, Elongation and
differentiation zone of roots treated for 4h with isoxaben, AVG or both (ESEM
images). Scale bar, 200µm. Note the severe bulging of roots treated with both
compounds. C, The effect of ACC on root elongation is partially ethylene-independent.
Symbols a, b, significantly (p<0.0001) different from control or Ag+-only treated roots,
respectively, or n.s., not significantly different (p>0.05, 2-tailed t-test). D, The
ethylene-insensitive mutant ein3eil1 responds to ACC and isoxaben.
A
n.s.
n.s.
120
c
120
n.s.
(d)
100
)
m80
µ
(
H
60
E
L
100
a
80
b
a
60
40
40
20
20
isox
ACC
+
+
+
AIB
-
B
+
+
***
n.s.
+
AVG
***
120
120
100
100
a
+
7303
n.s.
n.s.
80
60
40
40
20
20
Congo
Red
e
a
+
+
AOA
n.s.
)
m80
µ
(
H
60
E
L
+
n.s.
+
+
AIB
+
flg22
7303
+
+
AIB
+
7303
Fig. 3
Fig. 3 Inhibition of ACC biosynthesis restores root elongation in the presence of cell wall
damage and other stress. A, LEH of roots treated for 3h with isoxaben or ACC in the presence
or absence of AIB, AVG, AOA or 7303. A separate set of controls minus 7303 is indicated
with grey dashed lines. Symbols a, b, c, e, LEH is significantly (p<0.0001) reduced vs roots
treated with vehicle, AIB, AVG or 7303 only, respectively (2-tailed t-test). Symbol (d),
significantly reduced (p<0.01) vs roots treated with AOA only. “n.s.”, not significantly
different (p>0.05) from appropriate inhibitor-only value. B, Inhibition of ACC synthase or
ACC action restores elongation in the presence of a cell wall binding dye (Congo Red) or a
microbial elicitor (flg22). *** and n.s. as before.
A
140
***
n.s.
140
***
120
B ***
***
120
100
100
)
m 80
(µ
H 60
E
L
80
60
40
40
20
20
+
isox
+
Col - 0
+
-
tir1-1
+
PEO-IAA
+
5-methylPEO-IAA
C
140
***
n.s.
120
100
80
60
40
20
+
-
+
DPI
Fig. 4
Fig. 4 The repression of root elongation by cell wall stress requires auxin signalling and
reactive oxygen species. A, Isoxaben reduces root elongation in the auxin receptor
mutant, tir1-1. B, The auxin response inhibitor PEO-IAA, but not its inactive 5-methyl
derivative, restores elongation in the presence of isoxaben. C, Inhibition of NADPH
oxidases restores root elongation in the presence of isoxaben. Indicators of t-test results
as in Fig. 2.
PAMPs
Cell Wall Stress
?
SAM
ACS
ACC
Ethylene
Auxin
ROS
Long-term
responses
Elongation
Fig. 5
Fig. 5 A working model for the control of root elongation by ACC or ethylene. Shortterm effects of ACC on root elongation are partially ethylene-independent. Note that
we have not formally established whether auxin is upstream of ROS production or acts
independently. SAM, S-adenosyl methionine.