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Plant Cell Advance Publication. Published on March 2, 2017, doi:10.1105/tpc.16.00583
RESEARCH ARTICLE
Cytokinin-Mediated Regulation of Reactive Oxygen Species Homeostasis
Modulates Stomatal Immunity in Arabidopsis
Dominique Arnaud,a,1,4 Seungchul Lee,b,4 Yumiko Takebayashi,c Daeseok Choi,b Jaemyung Choi,a,2 Hitoshi
Sakakibarac and Ildoo Hwanga,3
a
Department of Life Sciences, POSTECH Biotech Center, Pohang University of Science and Technology, Pohang 790784, Korea
b
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang
790-784, Korea
c
RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan
1
Current address: College of Life and Environmental Sciences, University of Exeter, Exeter, UK
Current address: Department of Plant and Microbial Biology, University of California, Berkeley, USA.
3
Address correspondence to [email protected].
4
These authors contributed equally to this work.
2
Short title: Cytokinins regulate stomatal immunity
One-sentence summary:
Cytokinins stimulate stomatal immunity and plant resistance to bacteria by the ARR2-mediated transcriptional
regulation of apoplastic peroxidase genes, which control ROS homeostasis in guard cells.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with
the policy described in the Instructions for Authors (www.plantcell.org) is: Ildoo Hwang ([email protected])
Abstract
Stomata play an important role in pre-invasive defense responses by limiting pathogen entry into leaves. Although the
stress hormones salicylic acid (SA) and abscisic acid (ABA) are known to regulate stomatal immunity, the role of
growth promoting hormones is far from understood. Here, we show that in Arabidopsis, cytokinins (CKs) function in
stomatal defense responses. The cytokinin receptor HISTIDINE KINASE3 (AHK3) and the RESPONSE
REGULATOR2 (ARR2) promote stomatal closure triggered by pathogen-associated molecular pattern (PAMP) and
resistance to Pseudomonas syringae pv tomato bacteria. Importantly, the cytokinin trans-zeatin induces stomatal closure
and accumulation of reactive oxygen species (ROS) in guard cells through AHK3 and ARR2 in an SA-dependent and
ABA-independent manner. Using pharmacological and reverse genetics approaches, we found that CK-mediated
stomatal responses involve the apoplastic peroxidases PRX4, PRX33, PRX34, and PRX71, but not the NADPH
oxidases RBOHD and RBOHF. Moreover, ARR2 directly activates the expression of PRX33 and PRX34, which are
required for SA- and PAMP-triggered ROS production. Thus, the CK signaling pathway regulates ROS homeostasis in
guard cells, which leads to enhanced stomatal immunity and plant resistance to bacteria.
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©2017 American Society of Plant Biologists. All Rights Reserved
INTRODUCTION
Guard cells are specialized cells that form stomatal pores at the leaf epidermis and mediate gas exchange between the
plant and the environment. Environmental and internal signals such as light, plant hormones, biotic, and abiotic stresses
regulate stomatal opening and closing (Melotto et al., 2008; Acharya and Assmann, 2009; Sirichandra et al., 2009). As a
part of the innate immune response, stomata play a critical role in plant defense (Melotto et al., 2008). Upon contact
with pathogens or pathogen-associated molecular patterns (PAMPs), plants actively close their stomata to prevent
pathogen invasion in the apoplast and subsequent colonization of host tissues (Lee et al., 1999; Melotto et al., 2006). In
turn, pathogens have evolved virulence factors, such as the phytotoxin coronatine (COR) secreted by the bacterial
pathogen Pseudomonas syringae pv tomato (Pst) strain DC3000, to counteract host stomatal defenses by promoting
stomatal reopening (Melotto et al., 2006).
The earliest event in stomatal immunity is the recognition of PAMPs by plasma membrane pattern recognition
receptors (Arnaud and Hwang, 2015). In Arabidopsis thaliana (Arabidopsis), the receptor FLAGELLIN-SENSITIVE2
(FLS2) that recognizes the peptide flg22, the biologically active epitope of the bacterial PAMP flagellin, plays a
prominent role in stomatal immunity (Melotto et al., 2006; Zeng and He, 2010). Downstream of PAMP perception,
reactive oxygen species (ROS) function as secondary messengers to promote stomatal closure. Both the NADPH
oxidase RESPIRATORY BURST OXIDASE HOMOLOGUED (RBOHD) and apoplastic peroxidases (PRXs) have
been proposed to mediate PAMP-triggered ROS production in guard cells (Mersmann et al., 2010; Khokon et al. 2010a;
Khokon et al., 2010b; Kadota et al., 2014; Li et al., 2014). Still, the identity of these PRXs and the mechanistic details
of their transcriptional regulation remain largely unknown.
Stomatal immunity was also shown to be regulated by hormonal pathways, mainly the abscisic acid (ABA) and
salicylic acid (SA) signaling pathways (Melotto et al., 2008; Arnaud and Hwang, 2015). Particularly, the SAbiosynthesis enzyme SALICYLIC ACID INDUCTION DEFICIENT2 (SID2) and the SA-signaling regulator
NONEXPRESSER OF PR GENES1 (NPR1) are required for bacterium-induced stomatal closure (Melotto et al., 2006;
Zeng and He, 2010). Moreover, COR induces the expression of NAC transcription factors that contribute to CORmediated stomatal reopening and activate the expression of SA metabolism genes (Zheng et al., 2012; Du et al., 2014).
Several components of the core ABA pathway including OPEN STOMATA1 (OST1) activate stomatal defense
responses to bacteria (Melotto et al., 2006; Zeng and He, 2010; Du et al., 2014; Guzel Deger et al., 2015). However, the
contribution of the ABA pathway to stomatal immunity was recently questioned (Montillet et al., 2013). Indeed, it was
shown that ost1 and aba2 mutants were only partially defective in PAMP-triggered stomatal closure (Montillet et al.,
2013; Issak et al., 2013). Moreover, to our knowledge, the role of cytokinin (CK) phytohormones in the regulation of
stomatal immunity has not been investigated. Although CKs are considered to promote stomatal opening, the stomatal
response to exogenously applied CKs depends on the concentration and CK species (Acharya and Assmann, 2009).
Growing evidence support a role of the plant growth promoting hormones CKs in regulating plant-microbe
interactions (Choi et al., 2011; Argueso et al., 2012; Hann et al., 2013). It has been reported that CK stimulates the plant
defense response to Pst DC3000 bacteria through the Arabidopsis type-B RESPONSE REGULATOR2 (ARR2), which
acts downstream of the CK receptor HISTIDINE KINASE3 (AHK3) in phosphorelay signaling (Kim et al., 2006; Choi
et al., 2010). In particular, CK activates the SA signaling pathway in a NPR1-dependent manner and ARR2 interacts
with the transcription factor TGA1A-RELATED GENE3 (TGA3) to directly activate PR1 expression (Choi et al.,
2010). However, until now, most of these studies have focused on post-invasive defense responses in the apoplast, such
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as callose deposition, PTI/defense marker gene expression, or phytoalexin production (Choi et al., 2010; Großkinsky et
al., 2011; Hann et al., 2013).
In this report, we have uncovered a new function of CK in promoting stomatal immunity through a signaling
cascade involving the CK components AHK3 and ARR2. Our data show that CK induces ROS production and stomatal
closure in an SA-dependent and ABA-independent manner. Importantly, we found that ARR2 directly activates the
expression of the apoplastic peroxidases PRX33 and PRX34, which play a crucial role in stomatal defense via the SAsignaling pathway.
RESULTS
Cytokinin induces stomatal closure through AHK3 and ARR2
To investigate the role of the CK signaling pathway in stomatal immunity, we first examined the effect of the
biologically active CK t-zeatin (t-Z) on stomatal aperture in Arabidopsis Col-0 wild-type (WT) epidermal peels.
Application of t-Z from 10 nM to 1 µM gradually induced stomatal closure (Figure 1A), indicating that physiological
concentrations of exogenous CK promote stomatal closure. We then chose the t-Z concentration of 1 µM for further
stomatal assay. In a time course experiment, significant stomatal closure was observed as early as 30 min after t-Z
treatment and stomatal aperture continued to decrease up to 2 h after t-Z treatment (Supplemental Figure 1A). We then
examined the relative contribution of CK signaling components in t-Z-induced stomatal closure. Interestingly, stomata
of ahk3-3, arr2-4, and arr10-5 mutants did not close in response to 1 µM t-Z, while those of the ahk2-2tk, cre1-12,
arr1-4, arr11-3, arr12-1, arr14-1, and arr18-2 mutants exhibited WT levels of stomatal closure in response to CK
(Figures 1B and 1C). Similar results were obtained with the arr2-5 and arr2-6 mutant alleles (Supplemental Figures 1B,
which likely represent null mutants as no full-length transcript can be detected by RT-qPCR (Supplemental Figure 1C).
Thus, the histidine kinase AHK3 and the type B response regulators ARR2 and ARR10 are required for CK-mediated
stomatal closure. Consistent with their function in stomatal movements, AHK3, ARR2, and ARR10 are expressed in
guard cells (Supplemental Figure 1D).
AHK3 and ARR2 activate stomatal immunity
As AHK3 and ARR2 are known to participate in apoplastic defense (Choi et al., 2010) and their expression is induced
in guard cells after flg22 treatment (Figure 2A), we evaluated their role in PAMP-triggered stomatal closure and
stomatal defense against bacteria. Epidermal peels were incubated with the PAMP flg22, which promotes stomatal
closure in WT Col-0 plants (Melotto et al., 2006). Interestingly, ahk3-3 and arr2 mutants were defective in flg22mediated stomatal closure (Figure 2B; Supplemental Figure 2A). We then analyzed stomatal movements in ahk3-3 and
arr2-4 mutants after inoculation with the coronatine-deficient Pst DC3000 (Pst COR-) strain, which lacks the ability to
reopen stomata during infection (Melotto et al., 2006). At 2 h post-inoculation (hpi) with Pst COR- bacteria, we
observed bacteria-induced stomatal closure in WT plants, but not in ahk3-3 and arr2-4 mutants (Figure 2C). Because a
defect in stomatal defense often results in enhanced susceptibility to bacteria, plants were spray-inoculated with Pst
COR- bacteria and bacterial growth was evaluated 1 and 3 days later. As expected, ahk3-3 and arr2-4 mutants were
more susceptible than the Col-0 control to surface-inoculated Pst COR- bacteria, with a 4- and 7-fold increase in
bacterial population at 1 and 3 days post-inoculation respectively (Figure 2D, left panel). Since the enhanced
susceptibility of ahk3-3 and arr2-4 mutants to Pst COR- after spray-inoculation could also be caused by a deficiency in
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post-invasive defenses, plants were also inoculated with Pst COR- by syringe-infiltration. Only the ahk3-3 mutant
showed higher susceptibility to Pst COR- after infiltration-inoculation (Figure 2D, right panel), with a modest increase
in bacterial growth (4 fold) at 3 days post-inoculation. Taken together, these results establish a mechanistic connection
between stomatal defense and the CK signaling pathway in guard cells.
Cytokinins compromise COR-mediated stomatal reopening
We observed that activation of the CK signaling pathway in plants overexpressing ARR2 (ARR2 OE) induces
constitutive stomatal closure (Figure 2E). Likewise, without any treatments, IPT1 and IPT3 overexpression lines, which
have elevated CK concentrations (Choi et al., 2010), exhibited partially closed stomata, indicating that endogenous
accumulation of CKs enhances stomatal closure or inhibits stomatal opening (Supplemental Figure 2B). Therefore, we
hypothesized that CK- or ARR2-mediated stomatal closure may enhance stomatal immunity against the Pst DC3000
bacterium, which produces COR to counteract PAMP-triggered stomatal closure and reopen stomata (Melotto et al.,
2006). Treatments of Col-0 WT epidermal peels with 1 µM t-Z impaired Pst DC3000-mediated stomatal reopening
observed at 4 h in control treated plants (Figure 2F). Consistently, Pst DC3000-induced stomatal reopening at 4 h was
also largely compromised in IPT1 and IPT3 OE lines (Supplemental Figure 2B), as well as in ARR2 OE plants (Figure
2E). Since CK-treated plants or plants overexpressing IPT1, IPT3, and ARR2 were defective in Pst DC3000-triggered
stomatal reopening, we expected that these plants are more resistant to Pst DC3000 bacteria. Indeed, in our surface
inoculation assays, the multiplication of Pst DC3000 bacteria at 3 days post-inoculation in CK-treated plants or plants
overexpressing ARR2, IPT1, and IPT3 was greatly reduced as compared with WT plants (Supplemental Figure 2).
Together, these data suggest that CKs promote resistance to bacteria by inhibiting Pst DC3000-mediated stomatal
reopening.
These results imply that the perception of PAMPs carried by bacteria combined with the activation of the CK
pathway impedes COR-mediated inhibition of PAMP-induced stomatal closure. To test this hypothesis, epidermal peels
of WT Col-0 and ARR2 OE plants were treated with COR alone, flg22 alone, or flg22 together with COR, in
combination with t-Z or control solution (Supplemental Figures 2F and 2G). As observed previously (Melotto et al.,
2006), COR reopened stomata of flg22-treated WT controls. Similarly, treatment with COR alone was able to revert tZ- and ARR2-mediated stomatal closure, suggesting that COR interferes with CK signaling in guard cells. Importantly,
when stomatal closure was induced by both flg22 and t-Z or ARR2 overexpression, COR at the concentration used was
not able to reopen stomata. Together, these data suggest that the activation of the CK-signaling pathway via ARR2
potentiates PAMP-signaling and interferes with COR-mediated suppression of PAMP-induced stomatal closure.
As a next step to study the relationship between CK metabolism and plant response to bacterial infection, we
examined the CK content in WT Col-0 leaves at 1 h and 3 h after inoculation with Pst DC3000 bacteria (Figure 2G;
Supplemental Table 1). At 1 h post-inoculation, the levels of c-Z- and iP-type CKs, particularly the bio-active CK bases
and the CK riboside precursors, were higher in Pst-inoculated leaves compared to the Mock control. By contrast, no
meaningful changes in CK levels were observed between Mock- and Pst-inoculated leaves at a later time point (3 hpi).
These data further support a function of CKs in activating the early events of pathogen recognition.
Cytokinin induces ROS production in guard cells
ROS, including superoxide (O2-) and hydrogen peroxide (H2O2), are produced in guard cells after PAMP perception and
have been proposed to function as early secondary messengers in the promotion of stomatal closure by PAMPs, as well
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as by ABA and SA hormones (Lee et al., 1999; Pei et al., 2000; Khokon et al., 2011). Therefore, we tested whether
H2O2 is also an important secondary messenger in CK-mediated stomatal immunity. We monitored H2O2 production in
epidermal strips treated with 1 µM t-Z or 5 µM flg22 using the fluorescent dye 2',7'-dichlorofluorescein diacetate
(H2DCF-DA). An increase in fluorescence was observed in guard cells after t-Z treatment (Figure 3A), indicating that
CK is able to induce ROS production. Notably, t-Z-mediated ROS production was compromised in the ahk3-3 and arr2
mutants and these mutants were also defective in flg22-mediated ROS production (Figure 3A). These results indicate
that AHK3 and ARR2 mediate t-Z- and flg22-induced ROS accumulation in guard cells. We also observed higher levels
of ROS in guard cells of IPT3 and ARR2 overexpression lines as compared to Col-0 WT (Figure 3B and Supplemental
Figure 3A), which could explain their constitutive closed stomata phenotype. Importantly, IPT3 and ARR2
overexpression lines showed enhanced ROS production after flg22 treatment, suggesting an over-activation of stomatal
immunity. Interestingly, ROS accumulation in response to flg22 was reduced after pretreatment with COR (Figure 3B).
This result indicates that COR may act upstream of ROS production in PAMP-mediated stomatal closure. By contrast,
COR application did not reduce ARR2-mediated ROS accumulation. As the H2DCF probe becomes irreversibly
fluorescent upon reaction with ROS (Choi et al., 2012), the enhanced ROS accumulation in the ARR2 OE line could
have occurred before COR treatment.
Apoplastic peroxidases, but not NADPH oxidases, are involved in CK-mediated stomatal closure
Two types of enzymes, the apoplastic peroxidases (PRXs) and the plasma membrane NADPH oxidases, have been
proposed to generate a burst of ROS upon pathogen attack or elicitation with PAMPs (Torres, 2010; O'Brien et al.,
2012a). ROS produced by the NADPH oxidases RBOHD and RBOHF are known to be respectively implicated in
PAMP- and ABA-mediated stomatal closure, whereas SA- and chitosan-mediated stomatal closure involves ROS
generated by yet unknown PRXs (Kwak et al., 2003; Khokon et al., 2010b; Khokon et al., 2011; Mersmann et al.,
2010). To investigate the role of ROS and the relative contribution of NADPH oxidases and PRXs in CK-mediated
stomatal closure, we treated Col-0 epidermal peels with t-Z alone or with t-Z together with ascorbate (ASC), which is
known to reduce ROS levels in plants (Lee et al., 1999); diphenylene iodium chloride (DPI), an inhibitor of NADPH
oxidases (Pei et al., 2000); or sodium azide and salicylhydroxamic acid (SHAM), two inhibitors of PRXs (O'Brien et
al., 2012b; Khokon et al., 2010a). We found that t-Z-induced stomatal closure was inhibited by ASC, SHAM, and
sodium azide, but not by DPI (Figure 3C). Likewise, ASC, SHAM, sodium azide but not DPI induced stomatal opening
in the lines overexpressing ARR2 or IPT3 (Figure 3D; Supplemental Figure 3B), suggesting that PRXs, but not NADPH
oxidases, are responsible for CK-mediated stomatal closure.
To further analyze the role of NADPH oxidases in CK-mediated ROS production and stomatal aperture, we
used the rbohD and rbohF mutants. Interestingly, these mutants were not impaired in t-Z-induced stomatal closure
(Figure 3E). Even if we noticed a lower basal level of ROS in rbohD and rbohF guard cells relative to the Col-0 WT,
the rbohD and rbohF mutants consistently exhibited a WT increase in ROS production after t-Z treatment (Figure 3F).
Moreover, the basal expression level of RBOHF was not affected and RBOHD expression was downregulated in ARR2
OE guard cells (Supplemental Figure 3C). Collectively, these results suggest that CK-mediated stomatal closure is
independent of the NADPH oxidases RBOHD and F and rather involves apoplastic peroxidases.
A subset of PRX genes is commonly up-regulated by PAMPs and in ARR2 overexpression line
Apoplastic peroxidases belong to a large multigenic family of 73 members (Valério et al., 2004). To identify the PRX(s)
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implicated in CK-mediated stomatal immunity, we first analyzed public microarray databases (AtGenExpress and
ArrayExpress) to select PRX genes that are both expressed in guard cells and induced by PAMPs or bacterial infection.
We found that PRX4, PRX5, PRX32/33/34, PRX37, PRX50/51, PRX62, and PRX71 fulfill these selection criteria, while
the PAMPs/bacteria-induced PRX38, PRX69, and PRX70 genes are weakly expressed in guard cells (Supplemental
Figure 4).
The expression pattern of PRX genes in whole leaves and in guard cell protoplasts after flg22 treatment as well
as in ARR2 and IPT3 overexpression lines was further analyzed by RT-qPCR (Figure 4; Supplemental Tables 2 and 3).
Except for PRX22, PRX23, and PRX32, which exhibited reduced expression or did not have altered expression after 2 h
of flg22 treatment, the expression of all genes selected was induced by flg22 as compared to control treatments in both
leaves and guard cells. Even if PRX5 and PRX38 expression was highly induced by flg22, their basal expression levels
were either very low or undetectable. Interestingly, the basal expression level of PRX33, 34, 37, 70, and 71 was
moderately up-regulated in leaves of the ARR2 overexpression line relative to the Col-0 WT. Particularly, PRX33,
which showed the highest expression level in the ARR2 OE line, with a 6- and 2-fold induction in leaves and guard cells
respectively (Figure 4), was also 2-fold up-regulated in the IPT3 OE line (Supplemental Table 3). Interestingly, PRX4
expression was lower in ARR2 OE guard cells than in the Col-0 WT. These results suggest that the up- or downregulation of some PRX genes in the ARR2 or IPT3 OE lines might be responsible for their constitutive stomatal
closure.
PRX33 and PRX34 function in CK-mediated stomatal immunity
To analyze the function of flg22-induced PRX genes in stomatal immunity, we isolated available Arabidopsis lines
homozygous for T-DNA insertions (See Methods and Supplemental Figure 5A). Apart from prx4-1, prx4-2, prx33-2,
prx34-1, and prx37-1, which are hypo- or hypermorphic mutants depending on the T-DNA insertions, we could not
detect any full-length transcript by RT-PCR for the remaining mutants, suggesting loss of function mutations
(Supplemental Figure 5B). PRX4 expression was 2-fold down-regulated in the prx4-1 mutant and 7-fold up-regulated in
the prx4-2 mutant (Supplemental Figure 5C). In contrast to previous observations (Daudi et al., 2012; Lyons et al.,
2015), the expression of PRX33 and PRX34 was not affected in the prx33-2 and prx34-1 mutants respectively
(Supplemental Figure 5B). Surprisingly, in the prx34-1 mutant, the downstream tandem-duplicated PRX33 gene was
20-fold overexpressed. The prx37-1 mutant exhibited a 9-fold reduction of PRX37 expression.
We then screened these mutants for their responsiveness to PAMP- and CK-triggered stomatal closure. We
found that the prx4-2 (but not prx4-1), prx33-3, prx34-2, and prx71-1 mutants were not responsive to CK-mediated
stomatal closure (Figure 5A). Similarly, these mutants were fully defective in flg22-mediated stomatal closure, except
for prx71-1, which was partially impaired in this response (Figure 5B). As PRX4 was overexpressed in the
hypermorphic prx4-2 mutant, PRX4 could play a negative role in PAMP- and CK-induced stomatal closure. By
contrast, PRX33, PRX34, and PRX71 may activate PAMP- and CK-mediated stomatal closure. The prx4-2 mutant and
to a lesser extend the prx71-1 mutant were also defective in flg22-triggered ROS production (Figure 5C). By contrast,
the prx34-1 mutant, which exhibits high PRX33 expression, showed constitutive stomatal closure and enhanced ROS
accumulation in guard cells (Figures 5A and 5C). Importantly, the prx33-3 and prx34-2 mutants were impaired in flg22and t-Z-induced ROS production, as well as in bacteria-mediated stomatal closure (Figures 5D and 5E). As expected,
the prx33-3 and prx34-2 mutants were highly susceptible to spray-inoculated Pst COR- bacteria with 5- and 7-fold more
bacteria as compared to WT at 1 and 3 days post-inoculation respectively (Figure 5F, left panel). By contrast, both
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mutants demonstrated WT susceptibility levels after infiltration-inoculation (Figure 5F, right panel), indicating that
PRX33 and PRX34 play an important role in CK-mediated promotion of stomatal defense responses.
ARR2 directly activates the expression of PRX33 and PRX34
Since PRX4, PRX33, PRX34, and PRX71 contribute to flg22- and t-Z-mediated stomatal responses, we next tested
whether ARR2 interacts with the promoters of these PRX genes by using the HA-tagged ARR2 overexpressing line for
chromatin immunoprecipitation (ChIP) experiments (Kim et al., 2006; Choi et al., 2010). The ARR2 DNA-binding
domain specifically interacts in vitro with the AGATT sequence (Sakai et al., 2000). Several DNA regions were
analyzed to cover all potential ARR2-binding sites in the PRX4, PRX33, PRX34, and PRX71 promoters (Figure 6A;
Supplemental Table 4). In addition, the intergenic region, PRX33 coding region, and the EF1 promoter were used as
negative controls. The promoter of the CK-inducible ARR5 gene was used as a positive control. As shown in Figure 6B,
DNA segments containing potential ARR-binding motifs were enriched in the PRX34 promoter. For the PRX4, PRX33
and PRX71 promoters, enrichment was preferentially found in potential binding sites close to the transcriptional start
site. These results indicate that ARR2 binds to the PRX4, PRX33, PRX34 and PRX71 promoters.
To determine whether PRX33 or PRX34 acts downstream of ARR2 in PAMP- and CK-mediated stomatal
closure, we generated the ARR2 OE/prx33-3 and ARR2 OE/prx34-2 lines by crossing. Introduction of the prx33-3
mutation in the ARR2 OE line restored stomatal aperture and ROS accumulation to WT levels in control conditions
(Figures 6C and 6D). By contrast, the ARR2 OE/prx34-2 line still exhibited partially closed stomata and high ROS
levels, similar to the ARR2 OE plants. Unlike the prx34-2 mutant, ARR2 OE/prx34-2 plants exhibited stomatal closure
in response to flg22 and t-Z (Figures 6C and 6D), albeit to a lesser extend for t-Z treatment compared to the ARR2 OE
line. As compared to the WT Col-0, similar over-accumulation of ROS was observed in ARR2 OE and ARR2 OE/prx342 lines after flg22 treatment. In t-Z-treated conditions, ARR2 OE plants showed a modest increase in ROS level and the
ARR2 OE/prx34-2 line accumulated WT ROS levels. Importantly, ARR2 OE/prx33-3 plants were fully defective in
flg22- and t-Z-induced stomatal closure and ROS production. It should be noted that the PRX33 expression level
remains high in the ARR2 OE/prx34-2 line, similar to the ARR2 OE line (Supplemental Figure 6). By contrast, the upregulation of PRX34 observed in the ARR2 OE line was reverted to the WT level in the ARR2 OE/prx33-3 line. Thus,
even though we cannot exclude a role for PRX34 downstream of ARR2, these results suggest that PRX33 plays a
prominent role in ARR2-, PAMP- and CK-mediated ROS production and stomatal closure.
CK-mediated stomatal closure is mechanistically linked to the SA pathway, but not the ABA pathway
Previous studies have shown that both ABA and SA signaling pathways positively regulate stomatal immunity
(Melotto et al., 2006; Zeng and He 2010; Montillet et al., 2013). Moreover, stomatal closure induced by SA, but not by
ABA, was impaired by PRX inhibitors (Khokon et al., 2010a; Khokon et al., 2011). Interestingly, ahk3-3, arr2-4,
prx33-3, and prx34-2 mutants were impaired in SA-induced stomatal closure and ROS production, but were not
affected in these responses after ABA treatment (Figure 7). Therefore, the signaling cascade involving AHK3, ARR2,
PRX33, and PRX34 may preferentially regulate the CK and SA signaling pathways in an ABA-independent manner.
The SA-signaling regulator NPR1 is required for SA-induced stomatal closure and both NPR1 and SID2 are
known to be involved in stomatal immunity, but are not required for ABA-mediated stomatal closure (Melloto et al.,
2006; Zeng and He, 2010; Montillet et al., 2013). npr1-1 and sid2-2 mutants, which as expected were impaired in flg22triggered ROS production and stomatal closure, did not close stomata or accumulate H2O2 after t-Z treatment (Figure
7
8A; Supplemental Figure 7A). In ARR2 and IPT3 OE lines SA-mediated ROS production was more pronounced as
compared to the WT Col-0 (Supplemental Figure 7B). It was previously shown that ARR2 positively regulates the SA
signaling pathway downstream of NPR1 by direct interaction with the transcription factor TGA3 (Choi et al., 2010).
Surprisingly, t-Z- and flg22-mediated stomatal closure was not affected in the tga3-3 mutant (Supplemental Figure 7C).
Moreover, the tga3-3/ARR2 OE line showed constitutive stomatal closure as in ARR2 OE plants (Supplemental Figure
7D), suggesting that TGA3 is not required for stomatal immunity and ARR2-mediated stomatal closure. Nevertheless,
these results indicate that SA signaling via NPR1 is necessary for CK-mediated ROS production and stomatal closure,
and that CK may activate SA-signaling in guard cells.
To further clarify hormonal crosstalk in PAMP-mediated stomatal closure, we analyzed stomatal movements
after t-Z treatment in the ost1-3 mutant defective in ABA-mediated ROS production and stomatal closure (Mustilli et
al., 2002) and in the ABA-deficient mutant aba1-3. Importantly, ost1-3 and aba1-3 mutants, which were defective in
flg22-triggered stomatal closure (Melotto et al., 2006; Guzel Deger et al., 2015), showed WT stomatal closure after t-Z
treatment (Figures 8B and 8C). Moreover, the t-Z-mediated increase in ROS production was not affected in the ost1-3
mutant (Supplemental Figure 7E). These results suggest that the canonical ABA signaling pathway is not required for
CK-mediated stomatal closure.
DISCUSSION
Cytokinins have been reported to play a positive or negative role in plant-microbe interactions depending on pathogen
life style (Choi et al., 2011). In this study, we uncover a novel and crucial function of CKs in promoting stomatal
defenses and plant resistance to surface-inoculated Pst DC3000 bacteria. In concert with the SA pathway, CK signaling
regulates PRX-mediated ROS production in guard cells for the control of stomatal movements upon PAMP perception.
Thus, our results reveal the multifaceted roles of CKs in plant immunity, not only in post-invasive defense responses in
the apoplastic space (Choi et al., 2010; Großkinsky et al., 2011; Argueso et al., 2012; Hann et al., 2013), but also in the
activation of pre-invasive defense responses at the stomata level.
A plant growth promoting hormone induces stomatal closure
Cytokinins are well-known growth hormones involved in shoot formation, cell division, nutrient mobilization, and leaf
longevity (Hwang et al., 2012). Although it has been reported since the early 1970s that CKs tend to promote stomatal
opening, the stomatal response to exogenous application of CK depends on plant species and on the concentration and
type of CK used (Acharya and Assmann, 2009). Here, we show that in Arabidopsis, exogenous application of the
biologically active t-Z induces stomatal closure in a dose-dependent manner. A significant stomatal closure was
observed from 10 nM with an optimal effect at 1 µM. These concentrations are physiologically relevant as AHK3 has a
high affinity for t-Z with a KD of 4.3 nM (Lomin et al., 2015). Moreover, we found that, within the CK-receptors, only
AHK3 participates in t-Z-mediated stomatal responses. Consistently, AHK3, but not AHK2 and AHK4, activates the
CK-sensitive pARR5:GUS reporter gene in guard cells (Stolz et al., 2010), and CK-induced ARR2 phosphorylation is
specifically mediated by AHK3 (Kim et al., 2006). We also observed that CK-mediated stomatal closure was correlated
with an increase in ROS accumulation in guard cells and the ARR2 overexpression line consistently exhibited
constitutive stomatal closure due to high ROS levels. Accordingly, it was shown recently that an increase in endogenous
CK levels by induction of IPT gene expression in Nicotiana tabacum (tobacco) induces ROS accumulation and a
8
decrease in stomatal conductance (Novák et al., 2013). The observation that a growth promoting hormone induces
stomatal closure is somehow counterintuitive. Although ARR2 overexpression results in smaller stomatal opening, it
may be sufficient for CO2 influx, photosynthesis, and growth. Indeed, the ARR2 overexpressing line consistently
exhibited larger rosette leaves and increased biomass (Supplemental Figure 8), suggesting that constitutive stomatal
closure is not limiting for plant growth, but could prime stomatal immunity.
ARR2-mediated transcriptional activation of PRXs regulates ROS homeostasis in guard cells
Two recent studies highlighted the importance of transcriptional repression of PRX genes in Arabidopsis for the
regulation of ROS homeostasis in root or leaf development (Tsukagoshi et al., 2010; Lu et al., 2014). In rice, the
drought and salt tolerance (DST) transcription factor negatively regulates stomatal closure by activating the expression
of genes encoding ROS-scavenging enzymes including peroxidases (Huang et al., 2009). However, DST homologs
were identified in other cereals but not in Arabidopsis. Here, we found that the transcription factor ARR2 can regulate
the expression of a subset of PAMP-induced PRX genes in leaves, particularly PRX4, PRX33, PRX34, and PRX71,
which are direct ARR2-targets. Interestingly, ARR2 activates PRX33 expression but represses PRX4 expression in
guard cells. Moreover, the cell-wall peroxidases PRX4, PRX33, PRX34, and PRX71, but not the NADPH oxidases
RBOHD and RBOHF, play an important role in CK-mediated stomatal closure. Thus, the CK-signaling component
ARR2 is a novel transcriptional regulator of PRX genes for the control of ROS homeostasis in guard cells.
PRXs function in the plant defense response by consuming H2O2 through the peroxidase cycle for cell wall
cross-linking or lignification to block pathogen ingress or by producing ROS through the oxidative cycle during the
PAMP-triggered oxidative burst (Bolwell et al., 2002; O'Brien et al., 2012a). PRXs can directly generate H2O2 from O2
in the presence of a still unknown reductant and upon extracellular alkalinization, which typically occurs upon PAMP
perception. It is known that SA-induced ROS production and subsequent stomatal closure are inhibited by the PRXinhibitor SHAM and are independent of NADPH oxidases, particularly RBOHD/F (Khokon et al., 2011). Similar
observations were made using the fungal PAMP chitosan or yeast elicitor extracts (Khokon et al. 2010a; Khokon et al.,
2010b). However, the identity of these PRXs involved in PAMP- and SA-mediated stomatal closure has remained
puzzling. Here, we provide genetic evidence that at least PRX33 and PRX34 positively regulate SA- and PAMPmediated stomatal closure through the production of ROS. By contrast, PRX4 may inhibit flg22-mediated stomatal
closure probably through the consumption of H2O2 to avoid detrimental effects of ROS excess.
Relative contribution of PRXs and NADPH oxidases to pre-invasive immunity
During post-invasive immunity, PAMP-triggered oxidative burst is dependent on both the plasma membrane NADPH
oxidase RBOHD and cell wall peroxidases PRX33 and PRX34 (O'Brien et al., 2012a). However, PRX33/34 may have a
more important role than RBOHD in downstream defense responses such as callose deposition and expression of PTI
marker genes, suggesting that ROS produced by RBOHD and PRXs are not functionally equivalent in apoplastic
defense responses (Daudi et al., 2012). By contrast, at the guard cell level, previous work clearly indicated that
RBOHD, as part of the FLS2/BIK1 complex, is required for PAMP- and bacteria-induced stomatal closure (Kadota et
al., 2014; Li et al., 2014). Here, we show that at least PRX4, PRX33, PRX34, and PRX71 also function in pre-invasive
immunity. Surprisingly, the disruption of one gene is not compensated by other functional PRX genes, especially for the
tandem duplicated PRX33 and PRX34 genes, which share a high level of sequence similarity (94% at the protein level).
However, although the basal expression level of PRX34 is higher than that of PRX33, our genetic analysis suggests that
9
ARR2-mediated activation of PRX33 expression may compensate for the loss of PRX34 function in the ARR2
OE/prx34-2 line. In addition, the constitutive stomatal closure observed in the ARR2 OE line may also be caused by the
ARR2-mediated transcriptional repression of PRX4, which negatively regulates ROS accumulation in guard cells.
It was previously observed that mutations in either PRX33 or PRX34 down-regulate the expression of both
genes and that RBOHD expression is dependent on the expression of PRX33 and PRX34 (Daudi et al., 2012; O'Brien et
al., 2012b). However, in our genetic backgrounds, the expression of PRX33 in the prx34-2 mutant and PRX34 in the
prx33-3 mutant is not affected (Supplemental Figure 6). Similarly, RBOHD and RBOHF expression is not regulated by
PRX33 or PRX34 (Supplemental Figure 9). Therefore, the observation that plants defective in PRX33 or PRX34 but
with functional RBOHD are impaired in PAMP-mediated stomatal responses, suggests that plasma membrane NADPH
oxidases and cell wall PRXs act cooperatively to produce ROS during PAMP-triggered stomatal closure, perhaps in the
same signaling cascade. Interestingly, a recent study indicates a concerted action of the NADPH oxidase RBOHF and
PRXs that co-localize at the Casparian strips for localized lignin deposition (Lee et al., 2013). A similar mechanism
may occur for ROS production in stomatal defense.
CK together with SA activates stomatal immunity
A previous study demonstrated that CK activates the expression of the SA-biosynthesis gene SID2/ICS1 and induces an
increase in SA level in plants (Choi et al., 2010). Recently, it was shown that repression of ICS1 expression and SA
accumulation by NAC transcription factors is important for COR-mediated stomatal reopening (Zheng et al., 2012; Du
et al., 2014). Interestingly, COR-mediated stomatal reopening was compromised by CKs or in the ARR2 OE line.
Moreover, the overexpression of IPT3 or ARR2 enhanced flg22- and SA-triggered ROS production, indicating that CKs
may over-activate stomatal immunity through ARR2. Importantly, SID2 and NPR1 are required for CK-mediated
stomatal closure and conversely AHK3 and ARR2 regulate the SA pathway in guard cells. As observed before in
mesophyll cells (Choi et al., 2010), it is plausible that CK functions together with SA in guard cells and converges on
ARR2, which acts as a hub that connects the two signaling pathways. We propose that a signaling cascade involving
AHK3, ARR2, PRX33, and PRX34 activates stomatal immunity through the SA pathway in an ABA-independent
manner (Figure 8D). Still, our results indicate that ABA signaling positively regulates stomatal immunity as observed
before (Melotto et al., 2006; Zeng and He, 2010; Du et al., 2014; Guzel Deger et al., 2015). Thus, our study highlights
the existence of two separate branches involving the ABA and the CK/SA pathways, which both contribute to stomatal
defense.
Our results raise the intriguing question on the function of CKs in PAMP- or bacteria-triggered immunity as an
endogenous or exogenous signal. A reduction in endogenous CKs (Naseem et al., 2012) and a decrease in the
expression of IPT3 and CK-induced type-A ARR genes has been reported in the late stages, 1 to 3 days after Pst
DC3000 inoculation (Thilmony et al., 2006). Because type-A ARRs are negative regulators of CK signaling (Hwang et
al., 2012), a reduction of their expression during Pst infection may also de-repress CK signaling. By contrast, the Pst
DC3000 effector HopQ1 was shown to induce an increase in CK levels and an up-regulation of type-A ARRs (Hann et
al., 2013). Our data indicate that CK content increases transiently in leaves soon after Pst DC3000 infection (1 h), likely
due to PAMP perception. By contrast, the expression of CK-responsive ARR5 and ARR6 genes in guard cells was not
affected by flg22 treatment (Figure 4). Alternatively, the CK signaling components AHK3 and ARR2 could be
activated by biotic stresses in a CK-independent manner, which is supported by the observation that flg22 induces
AHK3 and ARR2 expression in guard cells. Although P. syringae has not been reported to produce CKs, many
10
pathogenic bacteria such as Agrobactrerium tumefaciens, and other Pseudomonas strains produce CKs or induce CK
biosynthesis to increase sink activity of the colonized tissues and reallocate nutrients toward the pathogen (Akiyoshi et
al., 1987; Choi et al., 2011). We speculate that during evolution, plants developed a specific signaling pathway in guard
cells to promote pre-invasive immunity against CK-secreting pathogens.
METHODS
All experiments reported here were repeated at least three times with similar results. Statistically significant groups
were determined by Students’ t-tests, or ANOVAs followed by Tukey’s honestly significant difference (HSD) posthoc
test using R software.
Plant materials and growth conditions
Arabidopsis mutant lines in the Col-0 background, except aba1-3 in the Ler background, were obtained from the
Arabidopsis Biological Resource Center at Ohio State University and the European Arabidopsis Stock Centre and are
listed in Supplemental Table 5. All T-DNA insertion mutants were confirmed by genotyping prior to usage using PCR
primers (Supplemental Table 6). T-DNA insertion sites for the prx mutants were determined by sequencing. The IPT1,
IPT3, and ARR2 overexpression lines were previously described (Kim et al., 2006; Choi et al., 2010). F3 plants of the
double transgenic lines ARR2 OE/tga3-3, ARR2 OE/prx33-3, and ARR2 OE/prx34-2 were generated by crossing
homozygous parental lines. The progeny were selected on appropriate antibiotics, and genotyping was performed by
PCR. Four- to five-week-old plants grown on soil in a growth chamber at 21-23°C, 40-60% humidity, and illuminated
with fluorescent tubes at a light intensity of 100 µmol m–2 s–1 light intensity under short-day (10 h light /14 h dark)
conditions were used for all the experiments.
Bacterial infection assay
Bacterial strains Pst DC3000 and Pst DC3000 COR- (DB4G3) (Brook et al., 2004) were cultivated overnight at 28°C in
King’s B medium supplemented with appropriate antibiotics. Bacteria were collected by centrifugation at 3000 g for 5
min at room temperature, and washed twice in 10 mM MgCl2. Plants were surface-inoculated by spraying with a
bacterial solution of 108 cfu/ml in 10 mM MgCl2 containing 0.02% Silwet L-77, and plants were covered to maintain
high humidity until disease symptoms developed. Alternatively, rosette leaves were syringe-infiltrated with a bacterial
solution of 106 cfu/ml in 10 mM MgCl2. After three days, bacterial growth in the apoplast of three leaves per plant and
four plants per genotype was determined as previously described (Katagiri et al., 2002).
Measurements of stomatal aperture
Stomatal experiments were conducted as previously described (Desclos-Theveniau et al., 2012). Leaf peels were
collected from the abaxial side of young fully expanded leaves and floated in stomatal buffer (10 mM MES-KOH, 30
mM KCl, pH 6.15) for 2.5 h under light (100 µmol m–2 s–1) to ensure that most stomata were opened before treatments.
Solutions of chemicals or bacterial suspension at 108 cfu.ml-1 in 10 mM MgCl2 were directly added in the stomatal
buffer. Purified chemicals, except flg22 peptide (AnyGen, Korea), were purchased from Sigma and used at the
following concentrations: 5 µM flg22, 1 µM trans-Zeatin (t-Z), 1 µM ABA, 10 µM SA, 1.6 µM (0.5 ng/µL) coronatine
(COR), 1 mM ascorbic acid (ASC), 2 mM salicylhydroxamic acid (SHAM), 20 µM diphenyleneiodonium chloride
11
(DPI), 1 µM sodium azide. Control solutions were stomatal buffer containing 0.01% ethanol for ABA and SA, 1%
ethanol for SHAM, 0.1% DMSO for DPI, 10 µM NaOH for t-Z, and water for COR, sodium azide, and ASC. After
treatments, epidermal peels were further incubated under light for 2 h and observed under a light microscope (Carl
Zeiss, Axioplan 2). Stomatal apertures of at least 100 stomata in random areas were measured using ImageJ 1.42
software.
Monitoring ROS in guard cells
The fluorescent dye 2',7' dichlorofluorescein diacetate (H2DCFDA, Sigma) was used to measure H2O2 levels in guard
cells. After 2.5 h incubation in stomatal buffer, epidermal peels were incubated with 50 µM H2DCFDA in 0.1% DMSO
for 15 min. Excess H2DCFDA was then removed by washing 3 times for 20 min with stomatal buffer. Then, 5 µM
flg22, 1 µM t-Z, 1 µM ABA, 10 µM SA, or control solutions were added. After 30 min incubation, H 2DCFDA
fluorescence was observed with a fluorescence microscope (Carl Zeiss, Axioplan 2) and fluorescence intensity of at
least 60 stomata was analyzed using ImageJ software.
Quantification of endogenous cytokinins
Young fully expanded rosette leaves were syringe-inoculated with 108 cfu/ml Pst DC3000 bacteria or Mock solution
(10 mM MgCl2) and collected at 1 and 3 h post-inoculation. For each biological replicate, two infiltrated leaves from
three different plants were pooled and freeze-dried. Extraction and quantification of cytokinins was performed as
described previously (Kojima et al., 2009) with an UPLC-ESI-qMS/MS (AQUITY UPLC™ System/Xevo-TQS;
Waters) with an ODS column (AQUITY UPLC BEH C18 , 1.7 µm, 2.1 × 100 mm, Waters).
Analysis of transcriptome databases
The transcriptome datasets related to biotic stress response were downloaded from AtGenExpress (TAIR) and include
time-course experiments on the leaves of five-week-old Arabidopsis plants collected 2, 6, and 24 h after inoculation
with the bacterial strains Pst DC3000, Pst avrRpm1, Pst DC3000 hrcC-, and Pseudomonas syringae pv. phaseolicola
(ME00331; F. Brunner and T. Nürnberger, unpublished), or 1 and 4 h after treatment with HrpZ, NPP1, flg22, and LPS
elicitors (ME00332; F. Brunner and T. Nürnberger, unpublished). The datasets for leaves and guard cells (E-GEOD19520; Pandey et al., 2010) and for guard cells and mesophyll cells in which transcriptional inhibitors (actinomycin D
and cordycepin) were added or not during protoplast isolation (E-MEXP-1443; Yang et al., 2008) were downloaded
from ArrayExpress (EMBL-EBI). For each profile, the intensities of probes were log2-transformed and then normalized
using the GCRMA method (Wu et al., 2004). Fold-changes were calculated as log2-median-differences between the
normalized values in control and biotic stress-treated conditions. The heatmaps were generated by hierarchical
clustering with euclidean distance and complete linkage.
Isolation of guard cell protoplasts
For each condition, about 50 young fully expanded leaves were directly collected or immerged for 2 h in stomatal
buffer containing 0.02% (v/v) Silwet-L77 with or without 1 µM flg22. Guard cell protoplasts were isolated as
previously described (Obulareddy et al., 2013) in the presence of transcriptional inhibitors 0.01% (w/v) cordycepin and
0.0033% (w/v) actinomycin D. For each sample, about 106 guard cell protoplasts were obtained for RNA extraction and
their purity was above 98%.
12
Gene expression analysis
Three fully expanded rosette leaves were either not treated or syringe-infiltrated with 1 µM flg22 or control solution (10
mM MgSO4) and plants were left for 2 h in a growth room before assessment. For leaf samples, total RNA was extracted
using Trizol reagent (Invitrogen) and genomic DNA was removed by digestion with DNase I (DNA-free; Ambion)
according to the manufacturer's protocol. For guard cells, RNA was extracted using the RNeasy Plant Mini Kit with incolumn Dnase 1 digestion (Qiagen). One microgram (leaves) or 200 ng (guard cells) of total RNA were reverse
transcribed using 500 ng of oligo(dT)15 and the ImProm-II Reverse Transcription System following the manufacturer’s
instructions (Promega). cDNA was diluted 2-fold before quantitative PCR or PCR. PCR amplifications were done with
2 µL of the first-strand cDNA as template, 0.025 unit of Ex Taq DNA polymerase (Takara), 250 µM dNTP, and 0.5 µM
of primers in a total volume of 20 µL. The cycling conditions were 95°C for 5 min for one initial step followed by 95°C
for 30 s, 60°C for 30 s, and 72°C for 1 min, for 25 to 45 cycles. The PCR was terminated with one extra step at 72°C for
10 min. Quantitative real-time PCR reactions were performed on a Light Cycler 2 (Roche) using 10 µL SYBR Premix
Ex Taq (Takara), 2 µL of cDNA, and 0.5 µM of primers in a total volume of 20 µL per reaction. The cycling conditions
were composed of an initial 20 s denaturation step at 95°C, followed by 45 cycles of 95°C for 7 s, 60°C for 10 s, 72°C
for 13 s. A melting curve was run from 65°C to 95°C to ensure the specificity of the products. Data were analyzed with
the delta Ct method using Light Cycler 2.0 software. Elongation factor 1 alpha (EF1) and ubiquitin 1 (UBQ1) were used
as reference genes for normalization of gene expression levels. In Figure 4 and Supplemental Tables 2 and 3, Col-0 WT
without any treatment or control treatment were considered as controls (expression level=1). RT-PCR and qRT-PCR
primer sequences are listed in Supplemental Table 7.
Chromatin immunoprecipitation
ChIP was performed essentially as described (Gendrel et al., 2005) with minor modifications. Four rosette leaves from
at least 18 plants (1.5 to 2 g) of 35S:ARR2-HA (Choi et al., 2010) or wild-type (WT) Col-0 (used as a negative control)
were cross-linked under vacuum for 10 min in a solution containing 0.4 M sucrose, 10 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% formaldehyde. Glycine was added to a final
concentration of 125 mM to stop the cross-linking. Leaves were ground in liquid nitrogen, resuspended with 25 mL of
extraction buffer 1 (0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF
and 1X protease inhibitor cocktail [PI, Roche]) and filtered through four layers of Miracloth (Calbiochem). The filtrate
was centrifuged at 3,000 g and 4°C for 20 min. The pellet was resuspended in 1 mL of extraction buffer 2 (0.25 M
sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and 1X
PI) and centrifuged at 12,000 g and 4°C for 10 min. The pellet was resuspended in 300 µL of extraction buffer 3 (1.7 M
sucrose, 10 mM Tris-HCl pH 8.0, 0.15% Triton X-100, 2 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and
1X PI), loaded on top of 1.5 mL of extraction buffer 3, and centrifuged at 16,000 g and 4°C for 1 h. The nuclear pellet
was resuspended in 500 µL nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, and 1X PI) and
sonicated 5 times (15 s each) to yield DNA fragments of 0.5 to 1 kb. After centrifugation at 12,000 g and 4°C for 5 min,
the supernatant was diluted 10-fold with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 167 mM
NaCl, and 1.1% Triton X-100) and pre-cleared with pre-equilibrated salmon sperm DNA/protein G agarose beads
(Millipore) for 1 h at 4°C. Then, 1 mL of the pre-cleared chromatin solution was immunoprecipitated with 5 µL of an
anti-HA antibody (Abcam, catalog number ab9110) overnight at 4°C and extracted by incubating with 60 µL of pre-
13
equilibrated salmon sperm DNA/protein G agarose beads for 2 h at 4°C. The beads were successively washed with 1
mL of washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 20 mM Tris-HCl pH 8.0) containing 150 mM
NaCl, 1 mL of washing buffer containing 500 mM NaCl, and twice with TE buffer (10 mM Tris-HCl pH 8.0, and 1 mM
EDTA). The immunocomplexes were eluted twice from the beads with 250 µL of elution buffer (1% SDS and 0.1 M
NaHCO3) and then reverse cross-linked overnight with a final concentration of 200 mM NaCl at 65°C. After removing
proteins with proteinase K, DNA was purified by phenol-chloroform extraction and ethanol precipitation. Precipitated
DNA was resuspended with 50 µL of TE buffer and 2 µL was used as a template for quantitative real-time PCR
analysis. PCR conditions were the same as for qRT-PCR and the sequences of primers are listed in Supplemental Table
8. A 10 µL aliquot of sonicated chromatin was reverse cross-linked as an input control. Input samples were first used to
normalize the results. Fold difference was then calculated by calculating the ratios between normalized results from WT
Col-0 plants and from ARR2-HA plants. Finally, the fold enrichment was calculated as the ratio between the probes and
EF1 as a negative control.
Accession Numbers
All sequence data from this article can be found in the Arabidopsis Genome Initiative data library and accession
numbers are listed in Supplemental Table 9.
Supplemental Data
Supplemental Figure 1. ARR2 participates in t-Z-mediated stomatal closure.
Supplemental Figure 2. Cytokinin inhibits the COR-dependent reopening of stomata.
Supplemental Figure 3. CK-mediated stomatal closure is dependent on apoplastic peroxidases.
Supplemental Figure 4. The expression of a set of PRX genes is induced by bacteria or PAMPs in leaves.
Supplemental Figure 5. Molecular characterization of prx mutants.
Supplemental Figure 6. PRX33 and PRX34 expression in ARR2 OE/prx33-3 and ARR2 OE/prx34-2 lines.
Supplemental Figure 7. CK functions with SA in guard cells independently of the ABA pathway.
Supplemental Figure 8. ARR2 overexpression enhances plant growth.
Supplemental Figure 9. RBOHD and RBOHF expression is not affected in prx33-3 and prx34-2 mutants.
Supplemental Table 1. Cytokinin concentrations in Col-0 (WT) leaves at 1 hr and 3 hrs after inoculation with Pst
DC3000 bacteria.
Supplemental Table 2. Expression levels of PRX genes in guard cell protoplasts of Col-0 (WT) and ARR2
overexpressing line (ARR2 OE), and in Col-0 after flg22 treatment.
Supplemental Table 3. Expression levels of PRX genes in whole leaves of Col-0 (WT), ARR2 and IPT3 overexpressing
lines (ARR2 OE and IPT3 OE), and in Col-0 after Control or flg22 treatments.
Supplemental Table 4. Positions of putative B-type ARR binding sites (AGATT) in PRX4, PRX33, PRX34 and PRX71
promoters.
Supplemental Table 5. Description of the mutant lines used in the study.
Supplemental Table 6. PCR primers used for genotyping the mutant lines.
Supplemental Table 7. Primers used for RT-PCR and quantitative RT-PCR.
Supplemental Table 8. Primers used for ChIP qPCR.
Supplemental Table 9. Gene sequences used in this article and the corresponding accession numbers.
14
Supplemental Dataset 1. ANOVA tables.
ACKNOWLEDGMENTS
We thank Jean-Luc Montillet for critical reading of the manuscript and helpful discussions. We thank Tatsuo Kakimoto
for ahk2-2tk seeds and Ming-Che Shih for prx4-2 seeds. D.A. was funded by the European Union’s Seventh Framework
Programme for research, technological development and demonstration under grant agreement no GA-2010-267243 –
PLANT FELLOWS. I.H. was supported by the Next-Generation BioGreen 21 Program (PJ01184402), Rural
Development Administration, Republic of Korea and Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2014R1A2A1A10052592).
No conflict of interest is declared.
AUTHOR CONTRIBUTIONS
D.A. conducted the experiments. S.L. contributed to the ChIP experiments and bacterial infection assay. S.L. and D.S.
analyzed the transcriptome databases. J.C. contributed plant materials and assisted with the stomatal assays. Y.T. and
H.S. analyzed the cytokinin levels. D.A., S.L., and I.H. analyzed the data. D.A. and I.H. designed the research and
wrote the manuscript with comments from all authors.
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19
Stomatal aperture (µm)
3
a
b
2.5
b
bc
2
c
1.5
1
0.5
0
0
0.01 0.1
1
10
Stomatal aperture (µm)
B
A
Control
3
t-Z
2.5
2
***
***
***
1.5
1
0.5
0
Col-0 ahk2-2tk ahk3-3 cre1-12
t-Z (µM)
Stomatal aperture (µm)
C
Control
3
t-Z
2.5
2
1.5
***
***
Col-0
arr1-4
***
***
***
***
1
0.5
0
arr2-4 arr10-5 arr11-3 arr12-1 arr14-1 arr18-2
Figure 1. Cytokinin induces stomatal closure through AHK3, ARR2, and ARR10.
(A) Stomatal response of Arabidopsis Col-0 WT epidermal peels to different concentrations of tzeatin (t-Z). Results are shown as the mean of ≥100 stomata measurements ± SE. Different letters
indicate significant differences at P < 0.01 based on a Tukey’s HSD test (See ANOVA table;
Supplemental Dataset 1).
(B) and (C) Stomatal apertures in epidermal peels of Col-0 WT, cytokinin receptor mutants (B) and
type-B response regulator mutants (C) after 2 h of incubation with Control solution (10 µM NaOH)
or 1 µM t-Z. Results are shown as the mean of ≥100 stomata measurements ± SE. Asterisks
indicate significant differences between Control and t-Z treatment based on a two-tailed Student's ttest (***P<0.001).
B
**
2
1.5
1
0.5
flg
22
c
c
2
1.5
b
1
0.5
0
Control
flg22
D
2
arr2-4
a
Col-0
ahk3-3
8
7
b
1.5
1
0.5
0
c c
6
b
ab b
5
b
7
a
6
a
4
3
3
2
1 dpi
3 dpi
ah
k3
-
Pst COR-
Spray
Mock
1h
4h
Control
2.5
2
a
b
1.5
Pst
b b
t-Z
a
2
a
b
b
1
3h
**
1.5
1
*
0.5
0
t-Z
0.5
0
0
Pst
Mock
1h
Pst
Mock
4h
ZR
Pst
0.5
c-
Mock
1
active CKs
ZR
0
1.5
c-
0.5
*
***
t-Z
R
1
2
t-Z
R
1.5
b
iP
b b
2.5
iP
b
1h
Z
b
3
c-
2
F
Stomatal aperture (µm)
a
a
Pst DC3000
*
Z
a
Mock
t-Z
2.5
3.5
Relative CK levels
3
G
ARR2 OE
c-
Col-0
Relative CK levels
Stomatal aperture (µm)
E
a
5
4
2
Mock
Infiltration
arr2-4
C
ol
-0
2.5
Col-0
ahk3-3
a a a
a
log (cfu/cm2)
Stomatal aperture (µm)
C
a
t-Z
R
Ps
cZR
Ps
iP
R
Ps
C
on
tro
l
flg
22
C
on
tro
l
0
a a
2.5
arr2-4
t-Z
R
Ps
cZR
Ps
iP
R
Ps
2.5
3
ahk3-3
iP
R
**
Col-0
ar
r2
-4
ARR2
iP
R
AHK3
Stomatal aperture (µm)
Fold change
3
3
A
CK precursors
Figure 2. The CK signaling components AHK3 and ARR2 participate in stomatal immunity.
(A) RT-qPCR analysis of AHK3 and ARR2 transcript levels in guard cell protoplasts after treatment
with Control solution (MES buffer) or 1 µM flg22 for 2 h. Transcript levels were normalized to
UBQ1. Error bars indicate SE of three independent experiments (n = 3). Asterisks indicate
statistically significant differences between Control and flg22 treatments based on a two-tailed
Student's t-test (**P<0.01).
(B) and (C) Stomatal apertures in WT Col-0, ahk3-3 and arr2-4 mutants exposed to Control
solution and 5 µM flg22 (B) or Mock control (10 mM MgCl2) and 108 cfu/ml COR-deficient Pst
DC3000 (Pst COR-) bacteria (C) for 2 hrs. Data are means ± SE (n ≥ 100).
(D) Bacterial growth in WT Col-0, ahk3-3 and arr2-4 mutants assessed at 1 day (1 dpi) and 3 days
(3 dpi) after spray-inoculation with Pst COR- at 108 cfu/ml (left panel), or at 0 and 3 days after
syringe-infiltration with Pst COR- at 106 cfu/ml (right panel). The dashed line indicates the
apoplastic bacterial population (cfu/cm 2) at 0 dpi. Values are the means ± SE (n = 4).
(E) and (F) t-Z or ARR2 overexpression inhibits Pst DC3000-mediated stomatal reopening.
Stomatal apertures in Col-0 and the ARR2 overexpressing (ARR2 OE) line (E), or Col-0 WT
treated with 1 µM t-Z or Control solution (F) exposed to Mock control or 108 cfu/ml Pst DC3000
(Pst) for 1 and 4 h. Data are means ± SE (n ≥ 100). Different letters indicate significant differences
at P < 0.05 (D) and P < 0.001 (B), (C), (E) and (F) based on a Tukey’s HSD test (See ANOVA
tables; Supplemental Dataset 1).
(G) Relative CK levels in Col-0 WT leaves after infiltration with Mock control (10 mM MgCl2) and
108 cfu/ml Pst DC3000 bacteria for 1 h (upper panel) and 3 h (lower panel). CK concentration was
calculated as pmol/g dry weight, and the relative levels of CKs were normalized to a value of 1 in
Mock treated control. Data represent the means and SE values of three independent biological
replicates. Asterisks indicate significant difference between Mock and Pst DC3000 treatments
based on a two-tailed Student's t-test (*P<0.05; **P<0.01; ***P<0.001).
A
B
Control
flg22
t-Z
Control COR
Col-0
arr2-4
ARR2 OE
arr2-4
ahk3-3
b
50
arr2-5
40
c
30
20
10
a
a
a
a
a
a
a
a
a
a
0
Control
flg22
t-Z
C
Stomatal aperture (µm)
Col-0
ROS production
(fluorescence intensity)
Col-0
D
2.5
a
a
a
2
a
b
b
1.5
1
0.5
0
Control Control ASC
DPI
SHAM Azide
3
Stomatal aperture (µm)
ROS production
(fluorescence intensity)
Col-0
2.5
ARR2 OE
100
c
80
60
20
b
b
40
b
a
0
Control
COR
ARR2 OE
a
a a
a
flg22
flg22 +
COR
a a
a
1
0.5
0
Control
ASC
DPI
SHAM
Azide
t-Z
rbohD
Col-0
3
2.5
a
a
rbohF
a
2
b
1.5
b
1
0.5
0
Control
t-Z
b
F
ROS production
(fluorescence intensity)
Stomatal aperture (µm)
E
rbohD
Col-0
35
30
25
20
15
10
5
0
a
b
b
1.5
b
a
a
Col-0
2
flg22 flg22+COR
c
rbohF
c
c
Control
Col-0
rbohD
a
b
b
Control
rbohF
t-Z
t-Z
Figure 3. Cytokinin induces ROS production and stomatal closure through apoplastic peroxidases.
(A) ROS production detected by H 2DCFDA fluorescence in guard cells of Col-0 WT, ahk3-3, arr2-4 and
arr2-5 mutants 30 min after treatment with Control solution, 5 µM flg22 or 1 µM t-zeatin (t-Z).
(B) Coronatine reduces flg22-mediated ROS accumulation. ROS production detected by H 2DCFDA
fluorescence in guard cells of Col-0 WT and ARR2 overexpression (OE) lines 30 min after treatment with
Control solution or 5 µM flg22. Peels were incubated with 1.6 µM coronatine (COR) for 30 min before the
addition of flg22 (flg22+COR). In (A) and (B), a representative stomate is shown (upper panel). Scale bars
represent 5 µm.
(C) Stomatal aperture in Col-0 WT exposed to Control solutions, 1 µM t-Z, and 1 µM t-Z together with 1
mM ascorbate (ASC), 20 µM DPI, 2 mM SHAM, or 1 µM sodium azide for 3 h.
(D) Stomatal aperture in Col-0 WT and ARR2 OE lines exposed to Control solution, 1 mM ASC, 20 µM
DPI, 2 mM SHAM, or 1 µM sodium azide for 3 h.
(E) Stomatal apertures in Col-0 WT, rbohD, and rbohF mutants after 2 h of incubation with Control
solution or 1 µM t-Z. In (C) to (E), values are means ± SE (n ≥ 100).
(F) ROS detected by H2DCFDA fluorescence in guard cells of WT Col-0, rbohD, and rbohF mutants 30
min after treatments with Control solution and 1 µM t-Z. In (A), (B), and (F) values are means ± SE (n ≥ 60
stomata). Different letters indicate significant differences at P < 0.001 (A) to (E) and P < 0.05 (F) based on
a Tukey’s HSD test (See ANOVA tables; Supplemental Dataset 1). A representative stomate is shown
(right panel). Scale bars represent 5 µm.
Leaves
PRX4
PRX5
PRX22
PRX23
flg22
*
**
Col-0 ARR2 OE
Control
PRX4
ND
ND
*
*
PRX32
flg22
**
PRX22
ND
ND
ND
ND
PRX23
ND
ND
ND
ND
*
PRX33
***
PRX34
*
*
PRX34
PRX38
*
**
**
PRX38
***
***
*
PRX50
PRX51
**
**
PRX50
*
PRX51
*
**
PRX62
PRX37
PRX62
PRX70
**
**
PRX71
**
PR1
5
PRX32
**
PRX69
***
PRX5
PRX33
PRX37
Col-0 ARR2 OE
**
ND
ND
**
PRX69
*
PRX70
PRX71
**
ARR5
ARR6
0
-5
***
***
**
**
Figure 4. Expression of PRX genes in leaves and guard cells after PAMP treatment or in the ARR2
overexpression line.
Expression analysis of PAMP-inducible PRX genes in leaves (left panel) and guard cell protoplasts
(right panel) after flg22 or control treatments for 2 h, and in Col-0 WT and ARR2 overexpressing
lines (ARR2 OE). Transcript levels were determined by RT-qPCR and normalized to both UBQ1
and EF1. PR1, ARR5 and ARR6 were used as positive controls. The changes in transcript levels
relative to Col-0 are color-coded. ND, not detected. Asterisks indicate statistically significant
differences between WT and ARR2 OE lines or between Control and flg22 treatments based on a
two-tailed Student’s t-test (*P<0.05; **P<0.01; ***P<0.01). Numerical expression values are shown in
Supplemental Tables 2 and 3.
Fold change (log2)
Control
Guard cells
Control
***
2.5
2
1.5
***
***
Col-0
prx4-1
***
***
***
***
***
***
1
0.5
0
B
Control
prx4-2 prx33-3 prx34-1 prx34-2 prx37-1 prx38-1 prx50-1 prx51-1 prx62-1 prx69-1 prx71-1
flg22
3
***
2.5
2
1.5
***
***
Col-0
prx4-1
***
***
***
1
***
***
***
***
***
0.5
0
a
a
prx33-3
Col-0
3
2.5
a
flg22
a
prx34-2
a
b
1
0.5
0
Mock
Pst COR-
Control
prx33-3
Col-0
7
ac
a
2
1.5
F
a a
flg22
t-Z
Infiltration
prx34-2
7
c c
6
5
a a
a a a
b b
b
a
5
a
4
4
3
3
2
2
1 dpi
a
a
6
3 dpi
Spray
34
-2
ab
c
pr
x
a a
prx34-2
33
-3
b
140
120
100
80
60
40
20
0
pr
x
c
prx33-3
b
Col-0
ROS production
(fluorescence intensity)
d
Control
E
D
C
ol
-0
prx34-1
prx71-1
log (cfu/cm2)
70
60
50
40
30
20
10
0
prx4-2 prx33-3 prx34-1 prx34-2 prx37-1 prx38-1 prx50-1 prx51-1 prx62-1 prx69-1 prx71-1
Col-0
prx4-2
C
ROS production
(fluorescence intensity)
Stomatal aperture (µm)
t-Z
3
Stomatal aperture (µm)
Stomatal aperture (µm)
A
Figure 5. PRX4, PRX33, PRX34, and PRX71 are involved in flg22- and t-Z-mediated stomatal
closure.
(A) and (B) Stomatal apertures in Col-0 WT and prx mutants exposed to Control solution, 5 µM flg22
(A), or 1 µM t-Z (B) for 2 h. Values are means ± SE (n ≥ 100). Asterisks indicate significant
differences between Control and flg22 or t-Z treatments based on a t test (***P<0.001).
(C) and (D) ROS production detected by H2DCFDA fluorescence in guard cells of Col-0 WT, prx4-2,
prx34-1, and prx71-1 mutants (C) and prx33-3 and prx34-2 mutants (D) 30 min after treatments with
Control solution, 5 µM flg22, or 1 µM t-Z. Values are means ± SE (n ≥ 60).
(E) Stomatal apertures in Col-0 WT, prx33-3, and prx34-2 mutants exposed to Mock control or 10 8
cfu/ml COR-deficient Pst DC3000 (Pst COR-) bacteria for 2 h. Values are means ± SE (n ≥ 100). In
(C) to (E), different letters denote significant differences at P < 0.001 (Tukey’s HSD test).
(F) Bacterial growth in Col-0 WT, prx33-3, and prx34-2 mutants assessed at 1 day (1 dpi) and 3 days
(3 dpi) after spray-inoculation with Pst COR- at 108 cfu/ml (left panel), or at 0 and 3 days after syringeinfiltration with Pst COR- at 106 cfu/ml (right panel). The dashed line indicates the apoplastic bacterial
population (cfu/cm2) at 0 dpi. Values are the means ± SE (n = 4). Different letters indicate significant
differences at P < 0.05 based on a Tukey’s HSD test (See ANOVA tables; Supplemental Dataset 1).
A
a
b
-2000
c
-1000
a
ATG
b
-2000
a
c
-1000
-2000
PRX4
d
e
ATG
-4000
PRX33
b
-1000
f
g
-2000
ATG
PRX71
h
-1000
ATG
PRX34
Stomatal aperture (µm)
ProARR5
C
2.5
2
a
b
c
ProPRX4
Col-0
ARR2 OE
a
a
b
a
b
1.5
b
b
c
a
b
b
b
0.5
0
flg22
t-Z
g
a
h
b
ProPRX71
D
ab
b
f
ProPRX34
ARR2 OE/prx33-3
ARR2 OE/prx34-2
b
e
ProPRX33
1
Control
d
ROS production
(fluorescence intensity)
8
7
6
5
4
3
2
1
0
ProEF1
Fold enrichment
(ARR2-HA / Col-0)
B
Col-0
ARR2 OE
18
16
14
12
10
8
6
4
2
0
c
ARR2 OE/prx33-3
ARR2 OE/prx34-2
c
b
b
b
a
a
Control
b
b
b
a
flg22
a
t-Z
Figure 6. ARR2 directly regulates PRX4, PRX33, PRX34, and PRX71 expression.
(A) Schematic representation of PRX4, PRX33, PRX34, and PRX71 genes with dots indicating
putative ARR-binding motifs (AGATT), black and grey boxes the exons and UTR, respectively.
Positions of PCR primers for ChIP are indicated by letters (a to h).
(B) Chromatin immunoprecipitation in Col-0 and ARR2-HA OE plants followed by qPCR of the
PRX4, PRX33, PRX34, and PRX71 promoters. ARR5 and EF1 promoters were respectively used
as positive and negative controls. qPCR results were normalized against the input samples and
fold enrichment was calculated by calculating the ratios between normalized results from ARR2HA OE and Col-0 (WT) plants. Error bars represent SE of three independent experiments.
(C) and (D) Stomatal apertures (C) and ROS production detected by H2DCFDA fluorescence in
guard cells (D) of the WT Col-0 and ARR2 OE, ARR2 OE/prx33-3, and ARR2 OE/prx34-2 lines
exposed to Control solution, 5 µM flg22, or 1 µM t-Z. Values are means ± SE, n ≥ 100 in (C) and n
≥ 60 in (D). Different letters indicate significant differences at P < 0.001 based on a Tukey’s HSD
test (See ANOVA tables; Supplemental Dataset 1).
3
b
2
c c c
1.5
1
0.5
0
C
ROS production
(fluorescence intensity)
a a
a a a
2.5
B
arr2-4
ahk3-3
Control
SA
Col-0
ahk3-3
ABA
25
b
20
arr2-4
b
b
5
a
a
a
b
a
a
0
Control
SA
3
2.5
ABA
a
a
prx33-3
bc
1.5
prx34-2
a a
a
2
c c c
1
0.5
0
Control
D
15
10
Col-0
Stomatal aperture (µm)
Col-0
ROS production
(fluorescence intensity)
Stomatal aperture (µm)
A
Col-0
30
SA
ABA
prx33-3
prx34-2
b
b b
25
b
20
15
10
a
a a
a
a
5
0
Control
SA
ABA
Figure 7. CK signaling components are required for stomatal responses induced by SA but not by
ABA.
(A) and (B) Stomatal apertures in Col-0 WT, ahk3-3, and arr2-4 mutants (A), and prx33-3 and prx342 mutants (B) after 2 h of incubation with Control solution, 10 µM SA or 1 µM ABA. Values are means
± SE (n ≥ 100).
(C) and (D) ROS production detected by H2DCFDA fluorescence in guard cells of Col-0 WT, ahk3-3,
and arr2-4 mutants (C), and prx33-3 and prx34-2 mutants (D) 30 min after treatment with Control
solution, 1 µM ABA, or 10 µM SA. Values are means ± SE (n ≥ 60). Different letters indicate
significant differences at P < 0.001 based on a Tukey’s HSD test (See ANOVA tables; Supplemental
Dataset 1).
Stomatal aperture (µm)
3
2.5
a a a
2
ab
sid2-2
ab
b
c
1.5
b
d
1
0.5
0
Control
C
Stomatal aperture (µm)
npr1-1
Col-0
Ler
4
3.5
3
2.5
2
1.5
1
0.5
0
t-Z
flg22
Col-0
3
2.5
ost1-3
a a
2
a
b b
1.5
c
1
0.5
0
Control
t-Z
flg22
D
aba1-3
a a
Stomatal aperture (µm)
B
A
SID2
ABA
flg22
SA
OST1
FLS2
NPR1
CK
flg22
AHK3
FLS2
a
b b
c
?
RBOHF
Control
t-Z
flg22
PRX71
RBOHD PRX34
PRX33
ARR2
PRX4
?
H2O2
Stomatal closure
Figure 8. CK-mediated stomatal closure is mechanistically linked to the SA pathway.
(A) to (C) Stomatal apertures in Col-0 WT, npr1-1, and sid2-2 plants (A), Col-0 WT and ost1-3 (B),
and Ler WT and aba1-3 (C) after 2 h of incubation with Control solution, 1 µM t-Z, or 5 µM flg22.
Values are means ± SE (n ≥ 100). Different letters indicate significant differences at P < 0.001 based
on a Tukey’s HSD test (See ANOVA tables; Supplemental Dataset 1).
(D) Hypothetical model for the regulation of stomatal immunity by cytokinins. This model is based on
the information provided in this study and references cited in the discussion. In guard cells,
perception of the PAMP flg22 by FLS2 is mechanistically linked to ABA and SA signaling pathways,
respectively illustrated by a blue and a brown shading. PRX33, PRX34, and PRX71 activatee
(arrows), while PRX4 represses (T-bars) PAMP-triggered ROS production. Independently of ABA and
through a signaling cascade involving AHK3, ARR2, PRX33, and PRX34, CK (green shading)
together with SA induces ROS production and stomatal closure to promote pre-invasive defense
responses. Dashed lines and question marks indicate indirect and uncertain connections,
respectively.
Regulation of reactive oxygen species homeostasis by cytokinins modulates stomatal immunity in
Arabidopsis
Dominique Arnaud, Seungchul Lee, Yumiko Takebayashi, Daeseok Choi, Jaemyung Choi, Hitoshi
Sakakibara and Ildoo Hwang
Plant Cell; originally published online March 2, 2017;
DOI 10.1105/tpc.16.00583
This information is current as of June 17, 2017
Supplemental Data
/content/suppl/2017/03/02/tpc.16.00583.DC1.html
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