Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis.

Abstract

Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.

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Dual-Level Regulation of ACC Synthase Activity by
MPK3/MPK6 Cascade and Its Downstream WRKY
Transcription Factor during Ethylene Induction in
Arabidopsis
Guojing Li1,2., Xiangzong Meng2., Ruigang Wang1., Guohong Mao2¤, Ling Han2, Yidong Liu2,
Shuqun Zhang2*
1College of Life Sciences, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China, 2Division of Biochemistry, Interdisciplinary Plant Group, and Bond Life
Sciences Center, University of Missouri, Columbia, Missouri, United States of America
Abstract
Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we
reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea–induced ethylene biosynthesis and
their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated
protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of
additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants
under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not
clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a
Type II ACS isoform, also contribute to the B. cinerea–induced ethylene production. In addition to post-translational
regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction.
Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation
but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/
MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay
reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that
WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response
to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a
key role in determining the kinetics and magnitude of ethylene induction.
Citation: Li G, Meng X, Wang R, Mao G, Han L, et al. (2012) Dual-Level Regulation of ACC Synthase Activity by MPK3/MPK6 Cascade and Its Downstream WRKY
Transcription Factor during Ethylene Induction in Arabidopsis. PLoS Genet 8(6): e1002767. doi:10.1371/journal.pgen.1002767
Editor: Joseph Kieber, The University of North Carolina at Chapel Hill, United States of America
Received January 6, 2012; Accepted April 26, 2012; Published June 28, 2012
Copyright: ? 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NSF grants MCB-0543109 and IOS-0743957 to SZ. GL was supported by the Innovative Research Group Fund
NDPYTD2010-3 from Inner Mongolia Agricultural University and a fellowship award from the China Scholarship Council. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: zhangsh@missouri.edu
¤ Current address: Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America
. These authors contributed equally to this work.
Introduction
The gaseous phytohormone ethylene profoundly impacts plant
growth, development, and response to environmental stimuli [1–
7]. Studies from a number of labs have defined a signaling
pathway—from ethylene receptors to downstream signaling
components to transcription factors—that alters gene expression
and leads toethylene-induced
[1,2,5,8,9]). Ethylene-regulated responses are suppressed in the
absence of ethylene, and such suppression is released upon plant
sensing of ethylene. As a result, all ethylene-regulated processes
begin with the induction of ethylene biosynthesis [10]. Plants
under stress, including wounding, flooding, drought, osmotic
shock, ozone, and pathogen/insect invasion, produce elevated
levels of ethylene [1,6,7,11]. For this reason, ethylene is also
known as a plant stress hormone. The biosynthetic pathway of
phenotypes(reviewedin
ethylene has been fully elucidated for over two decades. Two
enzymatic steps are unique to ethylene biosynthesis: conversion of
S-adenosyl-methionine (SAM), a common metabolic precursor, to
1-amino-cyclopropane-1-carboxylic acid (ACC) by ACC synthase
(ACS) and oxidative cleavage of ACC to form ethylene by ACC
oxidase (ACO) [1,4,12]. ACS activity is very low in tissues that do
not produce a large amount of ethylene and is enhanced under
conditions that promote ethylene formation [1,4,12–14]. In
contrast, ACO is constitutively present in most vegetative tissues.
As a result, ACS is believed to be the committing and generally
rate-limiting enzyme in ethylene biosynthesis.
ACS is encoded by a small gene family in plants. In Arabidopsis,
there are nine ACS members. Based on the presence/absence of
phosphorylation sites in their C-termini, ACS isoforms are
classified into three types [15]. Type I ACS isoforms, which
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include Arabidopsis ACS1, ACS2, and ACS6, have phosphory-
lation sites by both mitogen-activated protein kinases (MAPKs)
and calcium-dependent protein kinases (CDPKs) [16,17]. Type II
ACS isoforms, which include Arabidopsis ACS4, ACS5, ACS8,
and ACS9, only have putative CDPK phosphorylation sites. In
contrast, Type III ACS isoforms have shorter C-terminal
extension and lack both phosphorylation sites. ACS7 and
ACS11 are the two Type III ACS isoforms in Arabidopsis.
ACS1 has a short deletion with the highly conserved tripeptide
Thr-Asn-Pro (TNP) missing. It is enzymatically inactive as a
homodimer, but can form functional heterodimers with other
Type I isoforms and may contribute to ethylene biosynthesis
[18,19]. ACS isoforms show cell- and tissue-specific expression and
are developmentally regulated. In addition, expression of some
members is highly responsive to extracellular stimuli [1,20].
More recent studies have highlighted the importance of ACS
protein stability regulation by protein phosphorylation and
dephosphorylation. MAPK cascades are signaling modules down-
stream of sensors/receptors that transduce extracellular stimuli
into intracellular responses in eukaryotes. A basic MAPK cascade
is composed of three interconnected kinases. MAPKs function at
the bottom of the three-kinase cascade and are activated by
MAPK kinases (MAPKKs) through phosphorylation on the Thr
and Tyr residues in their activation motif between the kinase
subdomain VII and VIII. The activity of MAPKKs is, in turn,
regulated by MAPKK kinases (MAPKKKs) via phosphorylation
of two Ser/Thr residues in the activation loop of MAPKKs.
MAPKKKs receive signals from upstream receptors/sensors, most
of the time indirectly with additional components involved
[21,22]. The outputs of a MAPK cascade are dependent on the
substrates of the MAPK(s) in the cascade. A subset of MAPKs in
plants, represented by tobacco SIPK/Ntf4/WIPK and Arabidop-
sis MPK3/MPK6, is activated under various stress conditions that
elevate ethylene production (reviewed in [21,23–26]). A gain-
of-function analysis in tobacco revealed that activation of
SIPK/WIPK induces high levels of ethylene production [27].
More detailed analyses in Arabidopsis have demonstrated that
ACS2 and ACS6, two Type I ACS isoforms, are substrates of
MPK3 and MPK6 [16,28]. Phosphorylation of ACS2/ACS6 by
MPK3 and MPK6 stabilizes the ACS protein in vivo, resulting in
increases in cellular ACS activity and in ethylene production. The
degradation machinery targets the C-terminal, non-catalytic
domain of ACS6 and possibly ACS2 because of their sequence
similarity [29]. Phosphorylation of ACS6 introduces negative
charges to its C-terminus, which reduces the turnover of ACS6 by
the ubiquitin-proteasome degradation machinery.
In addition to protein phosphorylation, protein dephosphory-
lation also plays critical role in ACS stability regulation. Recently,
it was demonstrated that protein phosphatase 2A dephosphory-
lates ACS2/ACS6 and destabilizes them, a critical process that
counteracts with MAPK phosphorylation [30]. Members of the
Type II group, including ACS5 and ACS9, are also regulated at
protein stability levels, possibly by protein phosphorylation as well
[15,31–33]. However, the kinase(s) involved remain unidentified.
Because of the complex regulation of ACS protein/activity at
multiple levels, many details about the up-regulation of ethylene
biosynthesis remain unclear, including the specific ACS isoforms
involved in the ethylene induction in response to a specific
stimulus, the regulatory pathways that control the expression of
ACS genes, and the components involved in the regulation of ACS
protein stability. It has been known for decades that a subset of
ACS genes, including Arabidopsis ACS6, is transcriptionally
activated in plants under stress or pathogen attack. However,
the importance of this transcriptional activation and the under-
lying regulatory mechanism are not known. Furthermore, ethylene
induction by different stimuli exhibits different kinetics and
magnitude. The underlying molecular mechanism of such
differential induction is also unclear.
We are interested in the regulation of ethylene biosynthesis in
plants infected by pathogens. ACS2 and ACS6, two Type I ACS
isoforms, are involved in Botrytis-induced ethylene production [28].
The residual levels of ethylene induction in the acs2/acs6 double
mutant suggest involvement of additional ACS isoforms. In this
study, we investigated (1) the potential involvement of all ACS
isoforms in ethylene induction triggered by B. cinerea infection, (2)
the importance of transcriptional activation of ACS gene expres-
sion, (3) the signaling pathways involved in the ACS gene
activation, and (4) the molecular mechanism underlying the
differential kinetics and magnitude of ethylene induction by
different stimuli. We found that members in all three ACS groups
are involved in pathogen-induced ethylene production, with
ACS2, ACS6, and ACS7 contributing the most to B. cinerea-
induced ethylene production. Based on analyses of an ACS6
knockdown mutant and of conditional gain-of-function ACS6
transgenic lines, we also can conclude that the transcriptional
activation of the ACS6 gene plays a critical role in sustaining high
levels of ethylene induction. Interestingly, MPK3 and MPK6 not
only function in the phosphorylation-induced stabilization of
ACS2/ACS6 proteins, but also signal the ACS2 and ACS6 gene
activation after B. cinerea infection. WRKY33, a MPK3/MPK6
substrate that regulates camalexin biosynthesis [34], is also
responsible for turning on ACS2/ACS6 expression downstream of
MPK3/MPK6 cascade. WRKY33 binds to the W-boxes in the
ACS2/ACS6 promoters in vivo and is directly involved in MPK3/
MPK6-induced ACS2/ACS6 gene expression. The duration and
magnitude of MPK3/MPK6 activation vary with different stimuli
and correlate well with the duration and magnitude of ethylene
induction. Regulation of ACS activity at multiple levels by the
MPK3/MPK6 cascade is an important mechanism by which the
Author Summary
Plant immunity, similar to that in animals, also involves
mitogen-activated protein kinase (MAPK) cascades. How-
ever, plants use unique MAPK substrates and secondary
signaling molecules in the process. Among them, ethylene,
a gaseous plant hormone, plays critical roles. Ethylene-
regulated responses begin with the induction of ethylene
biosynthesis. 1-amino-cyclopropane-1-carboxylic acid syn-
thase (ACS) catalyzes the committing and rate-limiting
step in ethylene biosynthetic pathway. The Arabidopsis
genome encodes nine different ACS isoforms. Two of
them, ACS2 and ACS6, were previously shown to be
phosphorylated and stabilized by MPK3 and MPK6, two
Arabidopsis pathogen-responsive MAPKs. Using a genetic
approach, we identified additional ACS isoforms including
ACS7, ACS8, and ACS11 that also contribute to pathogen-
induced ethylene production. In addition to direct phos-
phorylation modification and stabilization of ACS2 and
ACS6 proteins, MPK3 and MPK6 also regulate the
expression of ACS2 and ACS6 genes through another
MPK3/MPK6 substrate, WRKY33, a member of the plant-
specific WRKY transcription factor family. Regulations of
ACS isoforms at both transcriptional and post-translational
levels contribute to the high-level ethylene production in
plants challenged by invading pathogens. These findings
shed light on our understanding of the regulation of the
kinetics and magnitude of ethylene induction under
different stress conditions.
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levels/kinetics of ethylene production are regulated during plant
stress/defense response.
Results
Activation of ACS gene expression in B. cinerea–infected
Arabidopsis
Our previous research demonstrated involvement of ACS2 and
ACS6 in ethylene induction in B. cinerea-infected Arabidopsis [28].
This research also implicated the involvement of additional ACS
genes since there was still an approximately 25% residual level of
ethylene induction in the acs2/acs6 double mutant. To identify the
ACS isoforms involved, we profiled the expression of all nine ACS
genes in Arabidopsis infected with B. cinerea. As shown in
Figure 1A, transcripts of ACS2, ACS6, ACS7, and ACS8 accumu-
lated approximately 1600, 200, 50, and 1200 fold, respectively,
over their basal levels. ACS11 transcript also accumulated about 6
fold. ACS5 and ACS9 transcripts could be reliably detected, but no
increases were observed. In contrast, ACS1 and ACS4 transcripts
were not detectable. To better assess the potential contribution of
each ACS gene to ethylene production, we also calculated their
expression levels relative to that of EF1a (Figure 1B). This
calculation allowed us to compare the relative levels of expression
between different ACS genes. From this dataset, we found that the
expression levels of ACS2, ACS6, and ACS7 were among the
highest. ACS8 and ACS11 had lower levels of expression after
induction, while ACS5 and ACS9 expression remained very low.
The expression of ACS8 increased more than 1200 fold relative to
its basal level (Figure 1A). However, because of its low basal level
expression, the induced level of ACS8 transcript was still much
lower than those of ACS2, ACS6, and ACS7 (Figure 1B). If
regulation at other levels is the same, ACS8 is likely a minor
contributor despite the high-fold induction. In contrast, levels of
ACS7 transcript were considerably elevated (Figure 1B), despite a
relatively low fold induction (Figure 1A), a result of a relatively
high basal level. Based on these results, we speculated that ACS7
might be a major contributor to ethylene induction after plant
sensing of pathogen invasion besides ACS2 and ACS6.
B. cinerea–induced ethylene production involves ACS
isoforms in all three groups
To establish the involvement of ACS7, we identified two null
mutant alleles of ACS7, acs7-1 (FLAG_431D05, in Ws-0
background) and acs7-2 (CSHL_ET5768, in Ler-0 background).
In both mutant alleles, B. cinerea-induced ethylene production is
slightly reduced (Figure S1), similar to that in the acs2 or acs6 single
mutant [28]. This result suggests that ACS7 also contributes to B.
cinerea-induced ethylene production. In our previous publications
[16,28], we did not assign allele numbers to the acs2 and acs6
mutants. To be consistent with the nomenclature used in Dr.
Theologis’s lab [35], the acs6 allele (Salk_090423) was given an
allele number of acs6-2. This allele turned out to be a knockdown
mutant (more discussion later). In contrast, the acs6-1 mutant allele
(SALK_025672) in the study by Tsuchisaka et al. (2009) is a null
mutant with a T-DNA insertion in the open reading frame (ORF)
[35]. We failed to identify any plant with a T-DNA insertion when
we initially ordered this line from the Arabidopsis Biological
Resource Center (ABRC) in 2003. The acs2 and acs7-1 mutant
alleles we used [16,28](Figure S1) are the same as the acs2-1 and
acs7-1 alleles, respectively, in Tsuchisaka et al. (2009) report [35].
Figure 1. Activation of ACS gene expression in Arabidopsis after B. cinerea infection. (A) Twelve-day-old Arabidopsis seedlings grown in GC
vials were inoculated with B. cinerea spores. Samples were collected at indicated times for total RNAs isolation. Expressions of all nine ACS genes were
quantified by real-time PCR. Induction of ACS gene expression (fold of induction relative to the level before inoculation) was calculated by the double
DCt method. (B) ACS transcript levels are expressed as percentage of EF1a transcript, which allows comparison of expression levels between different
ACS genes. In both calculations, the expression of EF1a was used as a reference. Error bars indicate standard deviations (n=3). ND, not detectable.
doi:10.1371/journal.pgen.1002767.g001
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We then crossed acs7-1 into the acs2-1/acs6-2 double mutant
background and identified an acs2-1/acs6-2/acs7-1 triple mutant
in the F3 generation. As shown in Figure 2, only about 10%
residual ethylene production was observed in the acs2-1/acs6-2/
acs7-1 triple mutant, confirming the importance of ACS7 in B.
cinerea-induced ethylene production. Residual ethylene induction
in the acs2-1/acs6-2/acs7-1 triple mutant again points to involve-
ment of additional ACS members. To identify them, we utilized the
high-order acs mutants generated in Dr. Theologis’ lab [35]. We
found that acs2-1/acs6-1 seedlings produced a lower level of
ethylene than our acs2-1/acs6-2 double mutant after challenged
with B. cinerea (Figure 3 and Figure S2), which is consistent with the
knockdown nature of our acs6-2 allele. Additional mutation of
ACS4, ACS5, and ACS9 genes, either one at a time or all three at
once, in the acs2-1/acs6-1 background did not further reduce the
ethylene induction. This finding is consistent with our previous
conclusion that ACS5 and ACS9 are not involved in the ethylene
induction triggered by B. cinerea infection [28]. In contrast,
mutation of the ACS7 gene in the acs2-1/acs6-1/acs4-1/acs5-2/
acs9-1 background resulted in further reduction in the ethylene
induction. In this sextuple mutant (acs2-1/acs6-1/acs4-1/acs5-2/
acs9-1/acs7-1) background, mutation of ACS11, but not ACS1,
slightly reduced the ethylene production. The very low level of
ethylene induction in the acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9-
1/acs7-1/acs11-1 plants also implicates the involvement of ACS8.
In the absence of B. cinerea infection, seedlings of all genotypes
produced less than 15 nL ethylene per gram of seedlings within
24 hours, a very low level in comparison to the B. cinerea-induced
ethylene production (Figure S3). From this dataset, we can also
conclude that ACS7 contributed the most to the basal level
ethylene production. Seedlings without a functional ACS7 gene
including the acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9-1/acs7-1/
acs11-1 failed to produce a detectable level of ethylene. As a result,
the low-level ethylene production in this octuple acs mutant in
response to B. cincerea infection is indeed a contribution of ACS8
gene.
In summary, we can conclude that ACS2 and ACS6 are major
contributors of ethylene induction and that ACS7, ACS8, and
ACS11 contribute less, with a total of ,15% of the ethylene
induction in B. cinerea-infected Arabidopsis. Among the minor
contributors, the role of ACS7 and ACS8 in B. cinerea-induced
ethylene production is clear. In contrast, the contribution of ACS11
is somewhat uncertain because of the conclusion of its involvement
is based on a small quantitative difference. One potential
mechanism underlying each ACS isoform’s contribution to
ethylene induction is through the up-regulation of their gene
expression (Figure 1). There is a good correlation between the
transcriptional activation of ACS gene expression (Figure 1) and
involvement in B. cinerea-induced ethylene production (Figure 3).
Induction of ACS2 and ACS6 gene expression is
dependent on MPK3/MPK6 pathway
Previously, we demonstrated the importance of phosphorylation
regulation of ACS2 and ACS6 by MPK3 and MPK6 in
Arabidopsis in response to pathogens/pathogen-associated molec-
ular patterns (PAMPs) [16,28,29]. It is well known that the ACS6
gene is highly induced by stress, including wounding and pathogen
infection [1,4,6,11,36,37]. However, the importance of ACS gene
activation in pathogen-induced ethylene production remains
unclear. After our discovery that phosphorylation of ACS2 and
ACS6 proteins by MPK3/MPK6 is required for ACS2/ACS6
protein stabilization and accumulation, we started to explore the
potential contribution of ACS gene activation. Theoretically, an
increase in ACS transcript levels is likely to increase the rate of de
novo ACS protein synthesis, which, in turn, will increase the net
ACS protein/activity after MAPK phosphorylation and protein
stabilization.
To determine whether ethylene induction in the conditional
gain-of-function GVG-NtMEK2DD(DD, for short) plants [16,28] is
associated with ACS gene activation, we profiled the expression of
ACS genes in DD plants after dexamethasone (DEX) treatment. As
shown in Figure 4, the expressions of ACS2 and ACS6 were highly
induced. Different from B. cinerea-infected seedlings, no induction
in ACS7, ACS8, and ACS11 expression levels were observed. In
addition, we noticed the kinetics of ACS6 induction in the DD
plants were different from that in B. cinerea-infected plants (Figure 4
versus Figure 1). This difference is likely a result of the
Figure 2. ACS7 also contributes to B. cinerea-induced ethylene
production in Arabidopsis. Twelve-day-old wild type (Col-0), acs2-1/
acs6-2 double mutant, and acs2-1/acs6-2/acs7-1 triple mutant Arabi-
dopsis seedlings grown in GC vials were inoculated with B. cinerea.
Ethylene levels in the headspace were determined at indicated times.
Error bars indicate standard deviations (n=3).
doi:10.1371/journal.pgen.1002767.g002
Figure 3. Ethylene induction in high-order acs mutants after B.
cinerea infection. Twelve-day-old wild type (Col-0) and acs mutants
generated in Dr. Theologis’ lab were inoculated with B. cinerea. Ethylene
levels in the headspace were determined at 24 hrs after spore
inoculation. Error bars indicate standard deviations (n=3). The allele
numbers are omitted for easy labeling. They are acs1-1, acs2-1, acs4-1,
acs5-2, acs6-1, acs7-1, acs9-1, and acs11-1.
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synchronous response in DD plants. In contrast, the infection
process of B. cinerea is progressive. As more cells sensed the growing
hyphae, higher levels of ACS6 transcript were induced at later time
points.
To confirm that MPK3 and MPK6 are responsible for
induction of ACS2 and ACS6 expression levels in the gain-of-
function DD plants, we examined the expression of ACS2 and
ACS6 in DD/mpk3 and DD/mpk6 plants. As shown in Figure 5,
induction of ACS2 and ACS6 was compromised in both the mpk3
and mpk6 single mutant backgrounds. This finding demonstrates
that induction of ACS2 and ACS6 in DD seedlings after DEX
treatment is indeed a result of MPK3/MPK6 activation. Based on
the fact that MPK3 and MPK6 are highly activated after B. cinerea
infection [28,38], we speculate that the MPK3/MPK6 cascade is
involved in regulating the B. cinerea-induced ACS2/ACS6 gene
expression and that induction of ACS7, ACS8, and ACS11
expression is regulated by pathway(s) other than MPK3/MPK6
cascade.
To provide loss-of-function evidence to support the role of the
MPK3/MPK6 cascade in B. cinerea-induced ACS2/ACS6 gene
activation, we compared the induction of ACS2 and ACS6 gene
expressions in wild type, mpk3 single mutant, mpk6 single mutant,
and rescued mpk3/mpk6 double mutant. The rescued mpk3/mpk6
double mutant was obtained by transforming a DEX-inducible
promoter-driven MPK6 (GVG-MPK6) into mpk32/2/mpk6+/2
plants. When the T3 mpk32/2/mpk6+/2/GVG-MPK6+/+plants
began to flower, DEX was sprayed every other day to rescue the
embryo lethality of the mpk32/2/mpk62/2/GVG-MPK6+/+zygotes.
Progenies with mpk32/2/mpk62/2/GVG-MPK6+/+genotype were
called rescued mpk3/mpk6 double mutants [39], and were used for
this experiment. As shown in Figure 6, B. cinerea-induced ACS2 and
ACS6 expressions were little affected in either the mpk3 or mpk6
single mutant. In the rescued mpk3/mpk6 double mutant, the
induction of both genes was dramatically reduced, which supports
the conclusion that MPK3 and MPK6 regulate expressions of ACS2
and ACS6 based on the gain-of-function analysis.
Different from B. cinerea-induced ACS2/ACS6 gene activation,
gain-of-function DD-induced ACS2/ACS6 gene activation was
compromised in either mpk3 or mpk6 single mutant background
(Figure 5 and Figure 6). There are several potential explanations
for this seemingly contradictory observation. First of all, MAPK-
phosphorylation regulation of ACS2/ACS6 gene activation will be
affected by both the phosphorylation of the downstream
transcription factor(s) such as WRKY33 (more discussion below),
and their dephosphorylation by the unidentified phosphatase(s). It
is possible that in the gain-of-function DD plants, both MPK3 and
MPK6 are needed to overcome the action of the phosphatase(s) to
maintain the phosphorylation of there transcription factor(s) and
the subsequent up-regulation of ACS2/ACS6 expression. In the
absence of either MAPK, the signaling strength is below the
threshold to counteract the phosphatases and the activation of
ACS2/ACS6 expression is severely compromised. It is possible that,
in addition to the activation of MPK3/MPK6 cascade, pathogen
infection may also inactivate the dephosphorylation process as a
mechanism to promote higher levels of ethylene production. In
this situation, the absence of only one MAPK may not be sufficient
Figure 4. Activation of ACS gene expression after MPK3/MPK6 activation in the gain-of-function DD Arabidopsis seedlings. (A)
Twelve-day-old conditional gain-of-function DD Arabidopsis seedlings grown in GC vials were treated with 1-mM DEX. Samples were collected at
indicated times for total RNA preparation. After reverse transcription, expressions of all nine ACS genes were quantified by real-time PCR. Induction of
ACS gene expression (fold of induction relative to the level before inoculation) was calculated by the double DCt method. (B) ACS transcript levels
expressed as percentage of EF1a transcript, which allows comparison of expression levels among different ACS genes. In both calculations, the
expression of EF1a was used as a reference. Error bars indicate standard deviations (n=3). ND, not detectable.
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to block ACS2/ACS6 activation. Alternatively, it is possible that the
activation of pathways other than MAPK cascade can compensate
the weakened MAPK pathway in the single mpk3 or mpk6 mutant,
making it necessary to mutate both MPK3 and MPK6 to see the loss-
of-function phenotype in response to B. cinerea infection. Similar
phenomenon was also observed in MPK3/MPK6-mediated
camalexin induction in response to B. cinerea infection [38].
WRKY33 is involved in the MPK3/MPK6-regulated ACS2/
ACS6 gene activation
WRKY33 is a substrate of MPK3/MPK6 in regulating the
pathogen-induced phytoalexin biosynthesis [34]. WRKY33 func-
tions as a transcriptional activator downstream of MPK3 and
MPK6 in promoting the expression of camalexin biosynthetic
genes. To determine whether WRKY33 also is involved in
activation of ACS2 and ACS6 genes downstream of the MPK3/
MPK6 cascade, we quantified the expression of these two genes in
DD and DD/wrky33 plants. As shown in Figure 7B, the induction
of ACS2 and ACS6 mRNA by the gain-of-function DD transgene
was compromised in wrky33 mutant background. Associated with
this, the induction of ethylene biosynthesis was mostly inhibited
(Figure 7A). Previously, we showed that DD protein induction and
MPK3/MPK6activationinDD/wrky33plantsareindistinguishable
from those in DD plants [34], which strongly supports the
conclusion that WRKY33 also functions downstream of MPK3/
MPK6 in promoting the expression of ACS2 and ACS6 genes. The
low residual levels of ACS2 and ACS6 gene activation is likely to be a
result of other WRKY transcription factors that can partially
substitute for the loss of WRKY33.
In contrast to the gain-of-function DD plants, mutation of the
WRKY33 gene had a minor impact on ethylene induction
triggered by B. cinerea infection. As shown in Figure 8A, we
observed only about a 20% decrease in ethylene production in
both alleles of the wrky33 mutant. A comparison of ACS2 and ACS6
gene expressions in the wild type and in the wrky33 mutant
revealed that about one-third of the induction in ACS2/ACS6
expression remained in the wrky33 mutant (Figure 8B). This
suggests that ACS2 and ACS6 still could be partially activated in the
absence of WRKY33. Because of the low residual ACS2/ACS6 gene
activation in the mpk3/mpk6 mutant (Figure 6), we speculate that
the residual levels seen in the wrky33 mutant are MPK3/MPK6-
dependent but WRKY33-independent, again pointing to addi-
tional transcription factors, possibly WRKY33 homologs that
partially replace WRKY33 in its absence.
No major reductions were observed in the induction of ACS7,
ACS8, and ACS11 expression in wrky33 infected with B. cinerea
(Figure S4), which is consistent with the conclusion that their
activation is MPK3/MPK6 and WRKY33 independent. The
normal activation of ACS7, ACS8, and ACS11 expressions, together
with the residual level of ACS2/ACS6 gene activation and protein
phosphorylation stabilization, may explain the observation that the
induction of ethylene in the wrky33 mutant was reduced by only
about 20% after B. cinerea infection (Figure 8A). In contrast, the
wrky33 mutation almost completely blocked induction of ethylene
biosynthesis in the gain-of-function DD plants (Figure 7A). B.
cinerea can activate multiple signaling pathways in plants. It is
possible that pathway(s) other than MPK3/MPK6 cascade are
Figure 5. Activation of ACS2 and ACS6 gene expressions in gain-
of-function DD is dependent on downstream MPK3 and MPK6.
Twelve-day-old DD, DD/mpk3, and DD/mpk6 seedlings grown in GC
vials were treated with 1-mM DEX. Samples were collected at indicated
times. Total RNAs were extracted and treated with DNase to remove
trace genomic DNA contamination. After reverse transcription, expres-
sions of ACS2 (A) and ACS6 (B) genes were quantified by real-time PCR.
ACS transcript levels were calculated as a percentage of the EF1a
transcript. Error bars indicate standard deviations (n=3).
doi:10.1371/journal.pgen.1002767.g005
Figure 6. B. cinerea induced ACS2 and ACS6 gene activation is
dependent on functional MPK3 and MPK6. Twelve-day-old wild
type (Col-0), mpk3, mpk6, and rescued mpk3/mpk6 double mutant
seedlings grown in GC vials were inoculated with B. cinerea spores.
Samples were collected at indicated times. Total RNAs were extracted
and treated with DNase to remove trace genomic DNA contamination.
After reverse transcription, expressions of ACS2 (A) and ACS6 (B) genes
were quantified by real-time PCR. ACS transcript levels were calculated
as a percentage of the EF1a transcript. Error bars indicate standard
deviations (n=3).
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able to partially compensate the loss of WRKY33. It is known that
pathogen infection induces a large number of WRKY genes
[40,41], some of which might be able to partially compensate the
loss of WRKY33 gene in activating the expression of ACS2/ACS6.
WRKY33 binds to the W-boxes in the promoters of ACS2
and ACS6 genes in vivo
Genetic analysis revealed that WRKY33 is essential for gain-of-
function MPK3/MPK6-induced ACS2/ACS6 gene expression
(Figure 7B). Examination of the ACS2 and ACS6 promoters
revealed the presence of eight and seven W-boxes, respectively
(Figure 9A). To further substantiate the role of WRKY33 in the
activation of ACS2 and ACS6 gene expression, we performed
chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR)
analysis to determine whether ACS2 and ACS6 genes are direct
targets of the WRKY33 transcription factor. For this experiment,
we used DD/wrky33 mutant plants complemented with a 35S
promoter-driven WRKY33 transgene, which contains a four-
copy myc epitope tag at the N-terminus (DD/wrky33/35S:4myc-
WRKY33) [34]. The presence of the myc tag allowed us to
immunoprecipitate the WRKY33-DNA complex by using a commer-
cial anti-myc antibody. As shown in Figure 9B, immunoprecipitation
Figure 7. WRKY33 functions downstream of the MPK3/MPK6
cascade in inducing the expression of ACS2 and ACS6 genes in
the gain-of-function DD seedlings. (A) Mutation of WRKY33
compromises ethylene induction in DD seedlings. Twelve-day-old DD
and DD/wrky33 seedlings grown in GC vials were treated with 1-mM
DEX. Ethylene accumulation in GC vials was monitored at indicated
times, and then seedlings were collected for gene expression analysis.
Error bars indicate standard deviations (n=3). (B) MPK3/MPK6-induced
ACS2 and ACS6 gene expression in DD plants is dependent on WRKY33.
Total RNA was extracted from seedlings collected in (A). Expressions of
ACS2 (upper panel) and ACS6 (lower panel) genes were quantified by
real-time PCR. ACS transcript levels were calculated as a percentage of
the EF1a transcript. Error bars indicate standard deviations (n=3).
doi:10.1371/journal.pgen.1002767.g007
Figure 8. Induction of ACS2 and ACS6 gene expression after B.
cinerea infection was partially inhibited in wrky33 mutant. (A)
Mutation of WRKY33 partially blocks ethylene induction in Arabidopsis
infected by B. cinerea. Twelve-day-old wild type (Col-0) and wrky33
seedlings grown in GC vials were inoculated with B. cinerea spores.
Ethylene accumulation in GC vials was monitored at indicated times,
and seedlings were collected for gene expression analysis. Error bars
indicate standard deviations (n=3). (B) B. cinerea-induced ACS2 and
ACS6 gene expression is compromised in the wrky33 mutant. Total RNA
was isolated from the seedlings collected in (A). Expressions of ACS2
(upper panel) and ACS6 (lower panel) genes were quantified by real-
time PCR. ACS transcript levels were calculated as a percentage of the
EF1a transcript. Error bars indicate standard deviations (n=3).
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with the anti-myc antibody greatly enriched ACS2 and ACS6 promoter
regions containing the W-boxes. In contrast, the IgG control antibody
failed to enrich either gene promoter. This result demonstrates that
WRKY33 directly binds to the promoters of ACS2 and ACS6 in vivo,
suggesting that WRKY33 is the transcription factor downstreamofthe
MPK3/MPK6 cascade involved in the activation of ACS2 and ACS6
expression.
High levels of ACS6 gene expression elevate B. cinerea–
induced ethylene production
To provide further direct evidence in support of the role of
ACS6 gene activation in B. cinerea-induced ethylene production, we
transformed a DEX-inducible promoter-driven ACS6 (GVG-ACS6)
construct into the acs2-1/acs6-2/acs7-1 mutant background and
then compared the ethylene induction in acs2-1/acs6-2/acs7-1/
GVG-ACS6 plants with and without DEX treatment. Two
independent lines (#5 and #12) with different levels of ACS6
transgene induction after DEX treatment were used for this
experiment to establish a correlation between the levels of ACS6
gene expression and the levels of ethylene induction. As shown in
Figure 10A, without DEX treatment, both GVG-ACS6 transgenic
lines produced about the same levels of ethylene after B. cinerea
treatment in comparison to the acs2-1/acs6-2/acs7-1 triple mutant.
In the presence of DEX, which induced ACS6 expression
(Figure 10B), the ethylene production was greatly enhanced. The
higher level of ACS6 induction in line #5 in the presence of DEX
correlated with a higher level of ethylene induction than that in
line #12. Furthermore, ethylene induction in Line #5 was higher
than that in the wild type, indicating that transgene induction after
DEX treatment not only complements the loss of ACS6, but also
compensates the loss of ACS2 and ACS7 genes. In the absence of B.
cinerea infection, DEX treatment only slightly elevated the ethylene
production (Figure S5) to a level similar to the basal level ethylene
production of Col-0 (,10 nL/g FW in 24 hrs). This low-level
ethylene production is likely a result of high-level ACS6 gene
induction after DEX treatment (and associated higher-level of de
novo ACS6 protein synthesis) in combination with the basal level
activity of MPK6, which can phosphorylate and stabilize ACS6
protein. MPK6 has very low basal activity even in the absence of
stress/pathogen infection [16]. This is consistent with our previous
conclusion that the overexpression of ACS6 gene in the absence of
MPK3/MPK6 activation is not sufficient to induce ethylene
production due to the lack of phosphorylation stabilization [16]. As a
result, we can conclude that the high level of ethylene production seen
in acs2-1/acs6-2/acs7-1/GVG-ACS6 lines after DEX and B. cinerea
treatment is a combination of high level of gene expression (as a result
of DEX treatment), and phosphorylation stabilization due to MPK3/
MPK6 activation by B. cinerea infection.
Our attempts to identify the T-DNA insertion line in the coding
region of the ACS6 gene (SALK_025672, acs6-1) failed to reveal a true
mutant plant from the seeds received. As a result, we have been using
the SALK_090423 line (acs6-2), which has a T-DNA insertion 170 bp
upstream of the ATG start codon (Figure 9A)[16,28]. In the past, we
routinely used the double DCt method to quantify gene expression in
real-time PCR analysis, which indicated the acs6-2 mutant allele as a
knockout mutant (Figure 11B, upper panel). However, a more careful
analysisperformedinthisstudyrevealedthatitisactuallyaknockdown
mutantwithanelevatedbasallevelexpression.AsshowninFigure11B
(lower panel), acs6-2 seedlings showed a higher basal level of ACS6
expression, but no increase in its transcripts was detected after B. cinerea
infection. In contrast, no transcript was detected in the acs6-1 mutant
before and after B. cinerea infection. Side-by-side comparison demon-
strated that ethylene production levels in acs6-2 and acs6-1 single
mutants after B. cinerea inoculation were similar (Figure 11A). In
contrast, acs2-1/acs6-2 seedlings produce higher levels of ethylene than
acs2-1/acs6-1 (both have the same acs2 mutant allele)(Figure S2). The
observable difference between acs6-2 and acs6-1 alleles in the acs2-1
mutant background could be due to the reduction of total ethylene
production in the absence of ACS2 gene, which makes it possible to
observe a small difference. These results suggest that acs6-2 mutant
allele is not a null mutant as acs6-1 allele, and that the high level
induction of ACS6 is important to pathogen-induced ethylene
production. Together with the gain-of-function evidence shown in
Figure10,wecanconcludethatACS6geneactivationplaysanessential
role in promoting ethylene production in plants challenged by
pathogens.
Discussion
Plants challenged by pathogens, especially necrotrophs such as
B. cinerea, produce very high levels of ethylene, a critical event in
Figure 9. WRKY33 transcription factor binds to the promoter of
ACS2 and ACS6 genes in vivo. (A) The promoters of ACS2 and ACS6
genes are rich in W-boxes, the cis-element binding sites of the WRKY
transcription factor. A diagram indicates the number and relative
position of the W-boxes in the promoters of the ACS2 and ACS6 genes.
Line arrows indicate the position of primers used for qPCR after
chromatin immunoprecipitation (ChIP). Positions of the predicted
transcriptional starting sites are indicated by arrows with turning lines
and negative numbers. The T-DNA insertion site in the SALK_090423
acs6-2 allele, which locates in the promoter region of ACS6 gene, is also
indicated. (B) ChIP-qPCR analysis was performed using DD/4myc-
WRKY33WTplants generated from the cross of wrky33/4myc-WRKY33WT
with DD lines. Input chromatin was isolated from two-week-old
seedlings 12 hr after DEX treatment. Epitope-tagged WRKY33-chroma-
tin complex was immunoprecipitated with an anti-myc antibody. A
control reaction was processed side-by-side using mouse IgG. ChIP- and
input-DNA samples were quantified by real-time qPCR using primers
specific to the promoters of ACS2 (left panel) and ACS6 (right panel)
genes. ChIP results are presented as percentage of input DNA. Error
bars indicate standard deviations (n=3).
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plant disease resistance [6,7,35]. In contrast, other stress stimuli,
including wounding, only trigger a transient and low-level ethylene
biosynthesis. Previously, we reported that MPK3/MPK6 phos-
phorylation-induced stabilization of ACS2 and ACS6 proteins is
an important mechanism in promoting ethylene induction in
Arabidopsis [16,28,29]. It is also clear from data based on acs2-1/
acs6-2 double mutant that ACS2 and ACS6 are not the only
contributors in pathogen-induced ethylene production [28]. In this
report, we demonstrate the involvement of three additional
members of the ACS gene family. Mutation of ACS2 and ACS6,
two Type I ACS members, abolishes ,85% of the ethylene
production induced by B. cinerea (Figure 2 and Figure 3) [28]. We
failed to detect the transcript from ACS1, the third member of the
Arabidopsis Type I ACS isoforms, in the seedlings, and its mutation
does not reduce pathogen-triggered ethylene production (Figure 3)
[28]. As a result, we believe that ACS1 contributes little, if any, to
pathogen-triggered ethylene production. ACS7, a member of the
Type III ACS, plays an intermediate role. Its mutation in the acs2-
1/acs6-2 background reduces ethylene induction to less than 5% of
that observed in the wild type (Figure 2 and Figure 3). This isoform
is also the major contributor of the basal level ethylene production
in the absence of pathogen infection (Figure S3). The transcripts of
ACS2, ACS6, and ACS7 are among the most abundant in B. cinerea-
infected Arabidopsis (Figure 1B). The residual levels of ethylene
induction in acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9-1/acs7-1 mu-
tant are likely from ACS8, a Type II ACS isoform, and ACS11, a
Type III ACS isoform.
Dual-level regulation of ACS2 and ACS6 by the MPK3/
MPK6 cascade in plant stress/defense response
In addition to post-translational regulation, we found that
transcriptional activation of the ACS genes is also critical to the
high-level of ethylene induction, as depicted in our working model
(Figure 12). Stress- and pathogen-activation of ACS genes, such as
Arabidopsis ACS6, is well established [1,36,42]. In this report, we
delineated a signaling pathway involved in the transcriptional
activation of ACS2/ACS6 in Arabidopsis after pathogen infection. It
is interesting to find that MPK3 and MPK6 not only function in the
phosphorylation-induced stabilization of ACS2/ACS6 proteins, but
also regulate the expression of ACS2 and ACS6 genes. The MPK3/
MPK6 cascade-induced ACS2/ACS6 gene activation is mediated by
WRKY33, another MPK3/MPK6 substrate [34]. WRKY33 binds
to the W-boxes in the promoters of the ACS2 and ACS6 genes
directly in vivo (Figure 9) and is involved in the MPK3/MPK6-
induced ACS2/ACS6 gene expression (Figure 7). Mutation of
WRKY33 resulted in a smaller reduction (,60%) in ACS2/ACS6
gene activation in response to B. cinerea infection (Figure 8), possibly
duetothepresenceofotherWRKY(s)thatcanpartiallycompensate
the loss of WRKY33. Conditional overexpression of ACS6 in the
acs2-1/acs6-2/acs7-1 mutant background greatly enhances the
ethylene induction (Figure 10). Furthermore, reduction in ethylene
induction in the acs6-2 allele, a knockdown mutant (Figure 11),
providesloss-of-functionevidencethatdemonstratestheimportance
of ACS6 gene activation during pathogen invasion. Transcriptional
activation of ACS2 likely has a similar role.
Induction of ACS6 expression is associated with stress-induced
ethylene production [1,20,36,42]. However, direct evidence
supporting the role of ACS gene activation has been lacking. In
Arabidopsis, overexpression of wild type ACS6 genes is not
sufficient to elevate ethylene production because of the require-
ment of protein phosphorylation and stabilization [16]. In
addition, overexpression of the ACS6 gene in the wild type
background fails to enhance ethylene production upon B. cinerea
inoculation (Li, G., Liu, Y., and Zhang, S., unpublished data), a
result of the high-level gene activation of the endogenous ACS
genes (Figure 1). In this study, we expressed the ACS6 gene in an
acs2-1/acs6-2/acs7-1 mutant background. The use of a DEX-
inducible promoter and two independent lines with different levels
of ACS6 gene induction after DEX treatment allowed us to
demonstrate the importance of ACS6 gene activation (Figure 10).
Our acs2-1/acs6-2 double mutant produces about 25% of the
wild type level of ethylene after B. cinerea infection (Figure 2) [28].
In contrast, the acs2-1/acs6-1 line only produces ,15% of the wild
type ethylene (Figure 3). The difference between these two double
mutants is likely a result of different acs6 mutant alleles since
both have the same acs2-1 mutant allele. The difference between
Figure 10. Conditional expression of the ACS6 gene in the acs2-1/acs6-2/acs7-1 mutant background restores ethylene induction
triggered by B. cinerea infection. (A) Induction of ACS6 gene expression in the acs2-1/acs6-2/acs7-1/GVG-ACS6 seedlings restores B. cinerea-
induced ethylene production. Twelve-day-old seedlings of wild type, acs2-1/acs6-2/acs7-1, and two independent lines of the GVG-ACS6 transgene in
the acs2-1/acs6-2/acs7-1 background (#5 and #12) were inoculated with B. cinerea spores. Two groups of acs2-1/acs6-2/acs7-1/GVG-ACS6 seedlings
were included with one treated with 1-mM DEX and the other treated with an equal volume of ethanol, the solvent for the DEX stock solution, at the
time of B. cinerea spore inoculation. Ethylene accumulation in GC vials was monitored at indicated times. Error bars indicate standard deviations
(n=3). (B) Induction of ACS6 transgene expression in acs2-1/acs6-2/acs7-1/GVG-ACS6 seedlings after DEX treatment. Seedlings were collected before
(0 hr) and 6 hr after DEX treatment. Induction of the ACS6 from the GVG-ACS6 transgene was quantified by real-time PCR. ACS6 transcript levels were
calculated as a percentage of the EF1a transcript. Error bars indicate standard deviations (n=3).
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acs2-1/acs6-2 and acs2-1/acs6-1 also suggests that the acs6-2 allele
is not a complete null mutant, which is supported by the presence
of ACS6 transcript in acs6-2 allele (Figure 11B). Since the T-DNA
insertion in this allele is in front of the transcriptional starting site
(Figure 9A), functional transcript is likely to be produced in this
mutant allele. Nonetheless, the reduction of ethylene induction in
the acs6-2 single mutant or in the acs2-1/acs6-2 double mutant
[28](Figure 2) demonstrates the importance of high-level
induction of ACS6 expression in pathogen-induced ethylene
production. Interestingly, the reduction in ethylene induction in
the acs2/acs6 double mutant is always more than the sum of the
reduction in each single mutant (Figure 3) [28]. It is possible that
the heterodimers of ACS2 and ACS6 are less active than each of
homodimer. In the absence of one isoform, only the homodimer
can be formed, which partially compensates for the loss of the
other isoform.
Importance of dual-level regulation ACS isoforms in
determining the levels and kinetics of ethylene induction
Although very inefficient energy-wise, regulation of the
ACS protein at the protein stability level by phosphorylation/
dephosphorylation allows rapid induction of ethylene biosynthesis,
which can occur within minutes after plant sensing of external
stimuli [43–45]. Such rapid response could be important to plant
response to the stress/pathogen stimuli. However, even after
phosphorylation stabilization, the ACS6 protein may not be very
stable. The half-life of the phospho-mimicking ACS6DDDis only
,3 hours [29]. In the meantime, protein phosphatase 2A will
counteract with the MAPKs by dephosphorylating the phospho-
ACS protein [30]. Under this circumstance, transcriptional
activation can provide another mechanism to further enhance
the ethylene induction in response to pathogen infection. It is well
known that stress-induced ethylene production follows different
kinetics depending on the stimuli. However, the molecular
mechanism underlying this difference is unclear. A good
correlation exists between the kinetics of MPK3/MPK6 activation
and ethylene induction. For instance, wounding induces a
transient ethylene production, which is associated with a transient
activation of MAPKs [46,47]. In contrast, infection of plants by
pathogens, especially necrotrophic fungal pathogens, triggers a
long-lasting and high-level induction of ethylene biosynthesis,
which correlates with a long-lasting and high-magnitude activation
of MPK3/MPK6 [28].
As depicted in Figure 12, MPK3 and MPK6 regulate ethylene
induction via two different mechanisms: by direct phosphorylation
and stabilization of ACS2 and ACS6 proteins [16,28,29] and by
activation of ACS2 and ACS6 gene expression (this study). Transient
activation of MPK3/MPK6 by wounding is also associated with the
activation of ACS2/ACS6 gene expression [36]. However, due to the
transient nature of MAPK activation, which returns to basal level
within ,0.5 hr to 1 hr [47], the de novo synthesized ACS protein
maynot havethe chanceto be phosphorylatedand will be degraded
quickly in the absence of MAPK phosphorylation. In B. cinerea-
infectedplants,theinductionofACS2andACS6gene expressionwill
result in high rates of de novo protein synthesis. On top of this, the
high-level and long-lasting activation of MPK3/MPK6 [28,38] can
maintain de novo synthesized ACS2 and ACS6 proteins in a
phosphorylated state and thereby stabilize the protein against
proteasome-mediated degradation [16,29]. This dual-level regula-
tory mechanism can maintain a greatly enhanced level of cellular
ACS activity and ethylene production in pathogen-infected plants.
Recently, it was shown that a PP2A protein phosphatase can
counteractwithMPK3/MPK6bydephosphorylatingACS2/ACS6
and can destabilize the ACS protein [30]. In this situation, it is even
more important to have the high-level, long-lasting activation of
MPK3/MPK6 in order to maintain the ACS2/ACS6 protein in a
phosphorylated state to ensure the high rate ethylene biosynthesis
observed in plants challenged by pathogens.
WRKY33 is a key transcriptional regulator downstream of
MPK3/MPK6 in regulating gene expression in multiple
pathways
Activation of MPK3/MPK6 and their orthologs in other plant
species induces the expression of large number of stress/defense
related genes [48,49], suggesting the involvement of downstream
transcription factors. ERF104 is a substrate of MPK6. Phosphor-
ylation of ERF104 by MPK6 results in release of ERF104 from the
Figure 11. The acs6-2 mutant allele is a knockdown mutant. (A)
B. cinerea-induced ethylene production in wild type, acs6-2
(SALK_090423), and acs6-1 (SALK_025672) plants. Twelve-day-old
seedlings grown in GC vials were inoculated with B. cinerea spores.
Ethylene accumulation in GC vials was monitored at indicated times,
and seedlings were collected for gene expression analysis. Error bars
indicate standard deviations (n=3). (B) Induction of ACS6 expression in
wild type (Col-0), acs6-2, and acs6-1 seedlings after B. cinerea
inoculation. Total RNA was isolated from the seedlings collected in
(A). Expression of the ACS6 gene was quantified by real-time PCR. ACS6
transcript levels were expressed as fold of induction relative to the zero
time point (upper panel) and as a percentage of the EF1a transcript
(lower panel). Error bars indicate standard deviations (n=3).
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complex, which allows ERF104 to activate the expression of genes
further downstream [50]. Recently, we identified the transcription
factor WRKY33 as the substrate of MPK3 and MPK6 [34].
WRKY33 is involved in the induction of camalexin biosynthesis
by promoting the expression of camalexin biosynthetic genes
[34,51]. In this report, we demonstrate that WRKY33 is involved
also in activation of ACS2 and ACS6 gene expression and ethylene
induction. In the wrky33 mutant background, gain-of-function DD-
induced ACS2 and ACS6 gene activation is essentially abolished.
Furthermore, a ChIP-qPCR analysis demonstrated that WRKY33
directly binds to the promoters of ACS2 and ACS6 genes (Figure 9).
These results reveal that WRKY33 regulates gene expression in
multiple stress/defense responses and may function as a master
transcriptional regulator downstream of the MPK3/MPK6
cascade.
The expression of both ACS2 and ACS6 genes are regulated by
the MPK3/MPK6 cascade and its downstream WRKY33.
However, the induction kinetics of ACS2 and ACS6 genes are
different in both gain-of-function DD transgenic plants (Figure 4)
and wild type plants after pathogen treatment (Figure 1). One
possibility is that one or more transcription factors, other than
WRKY33, are involved. The differential involvement of these
unknown transcription factors could result in the different kinetics
observed in the induction of ACS2 and ACS6 genes. These
transcription factors may or may not be regulated by the MPK3/
MPK6 cascade. The activation of MPK3 and MPK6 proteins in
the gain-of-function DD plants is sufficient to induce the expression
of ACS2 and ACS6 genes to levels similar to those observed in B.
cinerea-infected plants (Figure 1 and Figure 4), suggesting that the
transcriptional machinery controlling expression of ACS2 and
ACS6 genes is fully turned on in DD plants. On the other hand,
mutation of WRKY33 essentially blocks DD-induced ACS2/ACS6
gene activation (Figure 7B) but only partially blocks the induction
of ACS2 and ACS6 genes in B. cinerea-infected plants (Figure 8B),
suggesting the activation of additional components by B. cinerea
that cannot be activated by MPK3/MPK6 cascade alone, possibly
homologs of WRKY33 that can partially replace the function of
WRKY33 in its absence.
Contribution of MPK3/MPK6-independent pathway(s) to
stress-/pathogen-induced ethylene biosynthesis
The aforementioned discussions are focused on the role of the
MPK3/MPK6 cascade in regulating ACS2 and ACS6, two major
contributors of pathogen-induced ethylene production, as depicted
in our working model (Figure 12). A similar level of reduction in
ethylene induction in the mpk3/mpk6 and acs2/acs6 double mutants
(,85%) is consistent with our conclusion that the MPK3/MPK6
signaling cascade only controls ACS2 and ACS6. Genetic
evidence also supports the involvement of three additional ACS
isoforms, ACS7, ACS8, and ACS11, in B. cinerea-induced ethylene
production (Figure 2 and Figure 3). ACS7 and ACS11 are the two
members in the Type III ACS group in Arabidopsis. ACS8
belongs to the Type II ACS group. Based on mutant analyses,
these three ACS genes contribute about 15% of the total ethylene
produced in B. cinerea-infected plants (Figure 3) [28]. Transcrip-
tional activation of ACS7, ACS8, and ACS11 is not regulated by the
MPK3/MPK6 cascade (Figure 4); the signaling pathway(s)
involved in the activation of their expression is unknown. It is
also unclear whether ACS7, ACS8, and ACS11 are regulated at
the protein stability level. Since ACS7 and ACS11 do not have a
typical putative phosphorylation site in their C-termini, they are
likely to be regulated at the transcriptional level only. ACS8,
similar to ACS5 and ACS9, has a putative CDPK phosphorylation
site in its C-terminus. It is possible that phosphorylation by
CDPK(s) is involved in its protein stability regulation, similar to
ACS5 and ACS9 [32,33].
Figure 12. A model depicting the dual-level regulation of ACS activity by MPK3/MPK6-dependent and independent pathways
during pathogen-induced ethylene production. Members of all three types of ACS isoforms are involved in pathogen-induced ethylene
production. In B. cinerea-infected plants, Type I (ACS2/ACS6) isoforms contribute the most (,85%). ACS2 and ACS6 are regulated by the MPK3/MPK6
cascade at both transcriptional and protein stability levels. The transcriptional up-regulation is mediated by WRKY33, a MPK3/MPK6 substrate. Type II
(ACS8 and ACS11) and Type III (ACS7) isoforms are activated at the transcriptional level although the regulatory pathway(s) involved is not clear at
present. Increase in total cellular ACS activity drives the elevated ethylene production, which triggers downstream responses.
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Ethylene plays an important role in plant disease resistance.
Using a high-order acs mutant, Tsuchisaka et al. (2009)
demonstrated that ethylene production is essential to plant
resistance against B. cinerea. However, ethylene induction was not
examined in the study. In this report, we demonstrate that
ethylene induction in acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/acs9-1/
acs7-1/acs11-1 mutant plants after B. cinerea infection is only at
,2% of that in the wild type (Figure 3). Plant sensing of abiotic
stress stimuli or invading pathogens triggers a number of signaling
events. Among them, the activation of MAPK cascades and
calcium influx are two of the earliest [21,24,52]. Our research
demonstrates the regulation of ACS2 and ACS6 by a specific
MAPK cascade at both transcriptional and post-translational
levels. This pathway contributes ,85% of the total ethylene
induction in plants challenged by pathogens. Regulation of the
remaining ACS isoforms is unclear at present. Additional studies,
including identification of the signaling pathway(s) involved in
regulation of ACS7, ACS8, and ACS11 expressions, protein
phosphorylation, and protein stability, are needed to further our
understanding of the complex regulation of ethylene induction
during the plant stress/defense response.
Materials and Methods
Plant growth conditions and treatments
Soil-grown plants were maintained at 22uC in a growth
chamber with a 14-hr light cycle (100 mE/m22sec21). For
experiments, seeds were surface-sterilized. After imbibition at
4uC for 3–5 days, seeds were sown in petri dishes with liquid
half-strength Murashige and Skoog (MS) medium and grown in a
growth chamber at 22uC with continuous light (70 mE/
m22sec21). Five-day-old seedlings were transferred to 20-ml
GC vials with 6 ml of liquid half-strength MS medium (10
seedlings per vial) and maintained under the same growth
conditions. Twelve- to fourteen-day-old seedlings grown in GC
vials were used for experiments.
Procedures for Botrytis cinerea (Strain: DSM 4709) maintenance
and spore preparation were described previously [28]. Twelve-
day-old seedlings grown in GC vials were inoculated with B. cinerea
spores at a final concentration of 4.06105spores/vial. Induction of
DD and ACS6 expressions in GVG-NtMEK2DDand GVG-ACS6
transgenic plants was performed by the addition of DEX stock
solution (5 mM in ethanol) to a final concentration of 1 mM. An
equal volume of ethanol was used as a negative (2DEX) control.
At least two independent repetitions were performed with
similar results for experiments with multiple time points. For single
time-point experiments, at least three independent repetitions
were performed.
Mutant lines and generation of transgenic plants
Arabidopsis thaliana Columbia (Col-0) ecotype was used as the
wild-type control, unless stated otherwise. T-DNA insertion
mutant alleles of MPK3 (At3g45640), MPK6 (At2g43790), ACS1
(At3g61510), ACS2 (At1g01480), and ACS6 (At4g11280) were
described previously [16,28,39]. The two ACS7 (At4g26200)
mutantalleles,
acs7-1
(FLAG_431D05)
(CSHL_ET5768), were obtained from INRA and Cold Spring
Harbor Laboratory, respectively. High-order acs mutants gener-
ated in Dr. Athanasios Theologis’ laboratory [35] were obtained
from the Arabidopsis Biological Resource Center (ABRC). The
stock numbers are CS16564 (acs2-1), CS16569 (acs6-1), CS16581
(acs2-1/acs6-1), CS16603 (acs2-1/acs6-1/acs4-1), CS16607 (acs2-1/
acs6-1/acs5-2), CS16609 (acs2-1/acs6-1/acs9-1), CS16644 (acs2-1/
acs6-1/acs4-1/acs5-2/acs9-1), CS16649 (acs2-1/acs6-1/acs4-1/acs5-
and
acs7-2
2/acs9-1/acs7-1), CS16650 (acs1-1/acs2-1/acs6-1/acs4-1/acs5-2/
acs9-1/acs7-1), and CS16651 (acs1-1/acs2-1/acs6-1/acs4-1/acs5-
2/acs9-1/acs7-1/acs11-1). Conditionally rescued mpk3/mpk6 dou-
ble mutant was generated by transformation of DEX-inducible
promoterdriven MPK6cDNA (GVG-MPK6) into mpk32/2/mpk6+/2
plants [39]. Double mutant seedlings were recovered from seeds
of mpk32/2/mpk6+/2/GVG-MPK6 plants sprayed with 30 mM
DEX during the flowering stage. GVG-NtMEK2DD(abbreviated as
DD), DD/mpk3, and DD/mpk6 lines were previously described
[28,38].
The DEX-inducible promoter driven ACS6 construct (GVG-
ACS6) was generated by cloning the ACS6 ORF with a 4xmyc tag
[29] into the Xho I/Spe I sites of the pTA7002 vector [53]. The
binary vector was transformed into Agrobacterium tumefaciens strain
GV3101. Arabidopsis transformation was performed by the floral
dip procedure [54], and transformants were identified by
screening for hygromycin resistance. Independent lines with
ACS6 transgene induction were identified based on real-time
qPCR analysis.
Ethylene measurement
GC vials with Arabidopsis seedlings were flushed and capped
immediately after treatment. At indicated times, ethylene levels in
the headspace of the GC vials were measured by gas chroma-
tography as previously described [16]. Seedlings were then
collected, weighed, frozen in liquid nitrogen, and stored at
280uC for future analysis.
RNA extraction and real-time PCR analysis
Total RNA was extracted using the Trizol reagent (Invitrogen).
After DNase treatment, RNA (2 mg) was used for reverse
transcription. Real-time PCR analysis was performed using an
OpticonTM2 real-time PCR machine as described previously [38].
The transcript of the EF1a gene was used to normalize the
samples. Relative gene expression was calculated using two
different methods. The first method is the commonly used double
DCt method, which gives fold of gene induction relative to basal
level before treatment (0 hr time point). The second method
expresses the transcript level relative to that of the EF1a gene in
the same sample, which is a better method when comparison of
expression levels of different genes is necessary. The primers used
for real-time PCR were ACS1 (At3g61510, 59-ACGCTTT
TCTCGTCCCTACTC-39 and 59-GGCCTTAAGGTACGCT-
GATTC-39), ACS2 (At1g01480, 59-GGATGGTTTAGGATTT
GCTTTG-39 and 59-GCACTCTTGTTCTGGATTACCTG-
39), ACS4 (At2g22810, 59-AACAACCTTGTGCTCACTGCT-39
and59-AGATCCCTATCAAACCCTGGA-39),
65800, 59-GACTCTCATGTTTTGCCTTGC-39 and 59-TTGG
AAGCCATTAGAGCTTGA-39), ACS6 (At4g11280, 59-GTTC
CAACCCCTTATTATCC-39 and 59-CCGTAATCTTGAACC-
CATTA-39), ACS7 (At4g26200, 59-ACGGTACGATACCATTG
TGGA-39 and 59-GCTCGCCGTCTTTAGTTTTCT-39), ACS8
(At4g37770, 59-CCTTCCTTCCTTCAAGAATGC-39 and 59-
GAGAGTCTCGTTAGCCGGAGT-39), ACS9 (At3g49700, 59-
CATACCTCGACGAAAACCAGA-39 and 59-TCATGTCAA
CCCAACAGAACA-39), ACS11 (At4g08040, 59-CAAACGATG-
GAGGTTGCTATG-39 and 59-TTGGAGACCCATTTGTTGA
TAAG-39),and
EF1a
(At5g60390,
TCTTGCTTTCA-39 and 59-GGTGGTGGCATCCATCTTGT
TACA-39).
ACS5
(At5g
59-TGAGCACGCTCT
ChIP–qPCR analysis
F1 plants generated from the cross of wrky33/4myc-WRKY33
and DD lines were used for the ChIP assay. Two-week-old
Dual-Level Regulation of Ethylene Biosynthesis
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Page 13

seedlings treated with 1-mM DEX for 12 hr were processed as
previously described [55]. Briefly, chromatin was isolated from
0.8 g of frozen tissue and sonicated with a Bioruptor sonicator
(15 s on and 15 s off cycles, medium-energy settings) for 6 min.
Immunoprecipitation was performed by incubating chromatin
with 2 mg of anti-myc antibody (Millipore) or mouse IgG (negative
control) for 1 hr at 4uC. The protein-chromatin immunocomplex
was captured using Protein G-Dynal magnetic beads (Invitrogen).
After Proteinase K digestion, the immunoprecipitated DNA was
purified using a ChIP DNA Clean and Concentrator kit (Zymo
Research Corporation). Immunoprecipitated DNA and input
DNA were analyzed by qPCR using primers specific for the
promoter regions of PAD3 and WRKY33. The primer pairs
(forward and backward) used for ChIP-qPCR were ACS2 (59-
AGGCCATAAGCCCATTCAAA-39 and 59-GCCTACAGTG-
CACGACTTCA-39)and
ACS6
TGTGTTGG-39 and 59-TGGCAGCCTTAAAGACCAGT-39),
which are in proximity of the W-boxes in the promoters. ChIP
results are presented as percentage of input DNA.
(59-AAAGTCGTTGAGAT
Accession numbers
Sequence data for this article can be found in the Arabidopsis
Genome Initiative or GenBank/EMBL databases under the
following accession numbers: MPK3 (At3g45640), MPK6 (At2g
43790), EF1a (At5g60390), ACS1 (At3g61510), ACS2 (At1g01480),
ACS4 (At2g22810), ACS5 (At5g65800), ACS6 (At4g11280), ACS7
(At4g26200), ACS8 (At4g37770), ACS9 (At3g49700), ACS11
(At4g08040), and WRKY33 (At2g38470).
Supporting Information
Figure S1
reduced B. cinerea-induced ethylene production. Two-week-old
acs7-1 and acs7-2 as well as their respective wild-type controls, Ws-
0 and Ler-0, grown in GC vials were inoculated with B. cinerea
spores. Ethylene accumulation in the headspace was determined at
the indicated times. Error bars indicate standard deviations (n=3).
(TIF)
Mutation in ACS7, a Type III ACS isoform, slightly
Figure S2
ethylene production in acs2-1/acs6-2 and acs2-1/acs6-1 double
mutants. (A) B. cinerea-induced ethylene production in wild type,
acs2-1/acs6-2, and acs2-1/acs6-1 plants. Twelve-day-old seedlings
grown in GC vials were inoculated with B. cinerea spores. Ethylene
accumulation in GC vials was monitored at indicated times. Error
bars indicate standard deviations (n=3). (B) Basal level ethylene
production in wild type, acs2-1/acs6-2, and acs2-1/acs6-1 seedlings.
Twelve-day-old seedlings grown in GC vials were mock inoculat-
Comparison of basal-level and B. cinerea-induced
ed. Ethylene accumulation in GC vials was measured after
24 hours. Error bars indicate standard deviations (n=3).
(TIF)
Figure S3
(A)Basallevelethylene productioninwild type(Col-0,acs2-1/acs6-2
double and acs2-1/6-2/7-1 triple mutant. Twelve-day-old seedlings
grown in GC vials were mock inoculated. Ethylene accumulation in
GC vials was measured 24 hours later. Error bars indicate standard
deviations (n=3). (B) Basal level ethylene production in the high-
order acs mutants generated in Dr. Athanasios Theologis’ lab.
Twelve-day-old seedlings grown in GC vials were mock inoculated.
Ethylene accumulation in GC vials was measured 24 hours later.
Error bars indicate standard deviations (n=3). The allele numbers
are omitted for easy labeling. They are acs1-1, acs2-1, acs4-1, acs5-2,
acs6-1, acs7-1, acs9-1, and acs11-1.
(TIF)
Basal level ethylene production in various acs mutants.
Figure S4
mutant after B. cinerea inoculation. B. cinerea-induced ACS7, ACS8,
and ACS11 expression is not compromised in wrky33 mutant. Total
RNA from the experiment shown in Figure 8 was reverse
transcribed. Expressions of ACS7 (A), ACS8 (B), and ACS11 (C)
genes were quantified by real-time PCR. ACS transcript levels
were calculated as percentage of EF1a transcript. Error bars
indicate standard deviations (n=3).
(TIF)
Activation of ACS7, ACS8, and ACS11 in the wrky33
Figure S5
ACS6 transgenic seedlings after DEX treatment. Twelve-day-old
wild-type (Col-0), acs2-1/acs6-2/acs7-1, and acs2-1/acs6-2/acs7-1/
GVG-ACS6 transgenic seedlings (line #5 and #12) grown in GC
vials were treated with DEX (+DEX, final concentration of 1 mM)
or ethanol solvent control (2DEX), but without B. cinerea
inoculation. Ethylene accumulation in GC vials was measured
after 24 hours. Error bars indicate standard deviations (n=3).
(TIF)
Ethylene production in acs2-1/acs6-2/acs7-1/GVG-
Acknowledgments
We thank Dr. Theologis for the high-order acs mutant seeds donated to
Arabidopsis Biological Resource Center, Melody Kroll for proofreadingof the
manuscript, and the Arabidopsis Biological Resource Center for seed stocks.
Author Contributions
Conceived and designed the experiments: SZ. Performed the experiments:
GL XM RW GM LH YL. Analyzed the data: GL XM RW GM SZ.
Contributed reagents/materials/analysis tools: GL XM RW GM LH YL.
Wrote the paper: SZ.
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