An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis

In Arabidopsis the stomatal defense response, a feature of the innate immunity in plants, involves oxylipin-mediated mechanisms that are independent of the phytohormone abscisic acid.


Introduction
Due to their sessile nature and the lack of an adaptive immune system, plants have evolved processes to synthesize a vast array of secondary metabolites devoted to their protection against pathogens. Some of these compounds, known as oxylipins, originate from the incorporation of one or several oxygen atoms in the carbon chain of polyunsaturated fatty acids (PUFAs), mainly linoleic acid (18:2), linolenic acid (18:3), and roughanic acid (16:3). This first step leading to the formation of oxygenated fatty acids can be initiated either by reactive oxygen species (ROS) or enzymes such as lipoxygenases (LOXs), a-dioxygenases, or cytochromes P450. The primary hydroperoxy or hydroxy fatty acids can subsequently be converted into a plethora of metabolites, including aldehydes and oxo-acids, epoxydes, epoxy alcohols, hydroxides, divinyl ethers, and cyclized compounds such as jasmonates or phytoprostanes [1][2][3][4], which are involved in different aspects of plant physiology such as development, fertility, senescence, programmed cell death, and defense [3,[5][6][7][8][9][10].
The 9-and 13-specific LOXs that introduce two atoms of oxygen on carbon 9 or 13 of octadecanoic fatty acids, respectively, contribute to the synthesis of a great number of oxylipins. LOX genes are up-regulated and LOX-derived oxylipins are synthesized upon stress and pathogen challenges, suggesting that LOXs play a role in stress adaptation and pathogen defense [11,12]. The 13-LOX product JA has received much attention as it has been demonstrated to be involved in almost all above-mentioned physiological processes [10,[13][14][15]. The JA signaling pathway is now well documented involving the conjugate jasmonate-Isoleucine (JA-Ile) as the active form of jasmonic acid (JA), the F-box protein Coronatine Insensitive 1 (COI1) as receptor, and Jasmonate Zim Domain (JAZ) transcription factors as repressors whose degradation through the 26S proteasome pathway leads to the transcription of JA-responsive genes [16]. The 9-LOX-derived products have also been identified as signaling molecules regulating plant defense, cell death, as well as lateral root development and singlet oxygen-mediated stress [7,[17][18][19][20][21]. In contrast to JA, the mechanisms whereby 9-LOX products exert their biological functions are still unknown. Due to their enzymatic activity LOX can produce reactive electrophile species oxylipin (RES oxylipin) from fatty acid hydroperoxides. Recently, the mechanisms underlying RES oxylipin signaling has been uncovered. RES oxylipins possess reactive a,b-unsaturated carbonyl or epoxide groups and can add to thiol-or amine-containing compounds, such as glutathione (GSH), proteins, or nucleic acids [22][23][24][25][26][27]. The chemical reactivity of RES oxylipins toward the antioxidant GSH can modify the cellular redox state. RES oxylipins can also target transcription factors and the TGA transcription factors (TGA2, TGA5, and TGA6) were demonstrated to regulate the RES oxylipin-mediated gene transcription [26].
The development of transcriptomic studies and the possibility to access analyses of cell-type-specific gene expressions have enabled us to refine our understanding on the role of genes in plant-specific organs or cells. In this respect, the analyses of celltype-specific leaf transcriptome [28] allowed much progress in the knowledge of guard cell signaling pathways that contribute to stomatal movements in response to various environmental stresses [29][30][31].
Recently, a role of guard cells as an active plant defense mechanism against pathogens has been highlighted by the finding that stomata also function in innate immunity against bacterial invasion [32][33][34]. Soon after pathogen recognition, the two guard cells that flank the stomata actively restrict the opening size preventing entry of microbes and host tissue colonization. This response is referred to as the stomatal defense [35], and it has been suggested that the abscisic acid (ABA) was a key regulator of the pathogen-mediated stomatal closure [32].
The analysis of the cell-type-specific leaf transcriptome of Arabidopsis [28] allowed us to identify the guard cell specifically expressed LOX encoding genes LOX1 and LOX6. By multiple approaches we demonstrate that, in contrast to LOX6, LOX1 plays a major role in the control of stomatal defense and plant innate immunity. We provide evidence that the MAPKs MPK3 and MPK6 and salicylate participate in a LOX1-specific stomatal pathway to respond to pathogens and PAMPs. Moreover, we show that the ABA pathway including the protein kinases OST1, MPK9, and MPK12 only contribute to a small extent to the stomatal response to pathogens. Overall, our data reveal the functioning of an oxylipin-and an ABA-dependent pathway that converge at the level of the anion channel SLAC1 to regulate stomatal closure.

The 9-Specific LOX Encoding Gene LOX1 Is Expressed in Guard Cells
Cell-type-specific transcriptome analysis of Arabidopsis leaves [28] revealed striking differences in the expression of LOX genes ( Figure S1A). Genes encoding 13-LOXs, LOX2, LOX3, and LOX4 [36] were expressed in mesophyll cells, while LOX6 expression was restricted to guard cells. Conversely, expression of the 9-LOX encoding gene LOX1 was detected in guard cells but not in mesophyll cells. Microarray analyses were confirmed by RT-PCR for LOX1, expressed in guard cells and LOX2, LOX3, and LOX4 whose expression is restricted to mesophyll cells ( Figure S1B).

Lox1
Mutants Are More Susceptible to the Bacterial Pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 In order to determine whether LOX1 contributes to stomatal defense, two lox1 knockout lines (lox1-1 and lox1-2) along with a complemented lox1-2 (lox1-2 35S:LOX1) line and Col-0 as the wildtype (WT) control line were assessed for their resistance to virulent Pst DC3000 upon spray inoculations ( Figure 1A). The two lox1 mutant lines displayed more than 10-fold enhanced growth of Pst DC3000 as compared to the complemented and WT lines, suggesting that LOX1 participates in the control of bacterial leaf colonization.

Lox1 Mutants Are Compromised in Pathogen and PAMP-Induced Stomatal Closure
To test whether LOX1 exerts its effect on plant immunity at the guard cell level, we compared the behavior of stomata of lox1 mutants with WT plants in response to Pst. In contrast to WT and lox1-2 35S:LOX1, lox1 mutants were significantly compromised in their ability to close stomata in response to virulent Pst DC3000 and avirulent Pst DC3000 AvrRpm1 strains ( Figure 1B). Similarly, the flg22-induced stomatal closure was strongly impaired in both lox1 mutants ( Figure 1B). On the contrary, the lox6-1 mutant line (Methods S1), was not compromised in its ability to close stomata in response to flg22 ( Figure S2).
Considering that ABA has been identified as a positive regulator of pathogen and PAMP-induced stomatal closure [33], we investigated also whether lox1 mutants showed altered sensitivity to ABA. We observed that both lox1 mutant lines were as sensitive to exogenously applied ABA as WT and lox1-2 35S:LOX1 ( Figure 1C). Together, these results suggest that LOX1 activity

Author Summary
Stomata are microscopic pores that are present in the epidermis of the aerial parts of higher plants, such as the leaves. These pores, which are flanked by a pair of cells called guard cells, regulate transpiration and the exchange of gas between leaves and the atmosphere. It is well documented that the phytohormone abscisic acid (ABA) is a key regulator that controls the osmotic pressure in guard cells, allowing pore size to be adjusted in response to environmental conditions. Recently, stomata have also been shown to play an important role in the innate immune response. Indeed, upon contact with microbes, plants actively close stomata to prevent the entry of microbes and the consequent colonization of host tissue. This response is known as the stomatal defense response. However, the molecular mechanisms that regulate this defense response are not well understood. Using a variety of approaches, we show in this study that LOX1, a gene that encodes lipoxygenase (LOX) in guard cells, plays a major role in stomatal defense in the model plant Arabidopsis thaliana. Mutations in LOX1 impair stomatal closure and make plants more susceptible to the bacterium Pseudomonas syringae pv. tomato. We also show that several LOX-derived metabolites, the oxylipins, are potent inducers of stomatal closure. Finally, we provide evidence to show that ABA plays only a minor role in stomatal defense response, specifically by modulating this response.
mediates pathogen-and flg22-induced stomatal closure independently of ABA.
PUFAs Induce Stomatal Closure in Col-0 Plants But Fail to Activate This Process in lox1 Mutants Since PUFAs are known LOX substrates, we first tested linoleic acid (18:2) for its ability to induce stomatal closure. We observed that linoleic acid induced significant stomatal closure at 10 nM (Figure 2A). At 100 nM both linoleic (18:2) and linolenic acid (18:3) induced stomatal closure in WT and lox1-2 35S:LOX1 plants but not in both lox1 mutants ( Figure 2B). In contrast, oleic acid (18:1), which is not a substrate of LOXs, was unable to trigger stomatal closure in any plant line ( Figure 2B). These results strongly suggest that products of LOX activity are able to promote stomatal closure.
Fatty Acid Hydroperoxides (FAHs) Activate Stomatal Closure at Lower Doses Than H 2 O 2 or tert-Butyl Hydroperoxide (tBuOOH) LOXs directly generate FAHs by incorporating molecular oxygen into the backbone of PUFAs ( Figure S3). We therefore tested the ability of 9-and 13-HPODE to trigger stomatal closure and compared their activity to H 2 O 2 , which was previously shown to mediate ABA-induced stomatal closure [37] and to tBuOOH as a model organic hydroperoxide. We found that 9-and 13-HPODE promoted stomatal closure at considerably lower concentrations than H 2 O 2 or tBuOOH ( Figure 2C), demonstrating that LOX-derived FAHs are potent inducers of stomatal closure.
Oxidative processes arising from increased cellular hydroperoxide concentrations can be effectively prevented by the addition of free radical scavengers including thiol-containing compounds. Among them, N-acetyl cysteine (NAC) is one of the less potent free radical scavengers [38]. Alternatively, thiols can act as nucleophiles to conjugate with reactive electrophiles through the Michael addition [25,27,39]. In order to get further insights into the mechanisms at the origin of the biological activities of hydroperoxides, epidermal peels were pre-incubated with 1 mM NAC before treatment with 10 mM 9-or 13-HPODE, 40 mM H 2 O 2 , or tBuOOH ( Figure 2D). The inability of NAC to prevent H 2 O 2 -or tBuOOH-induced stomatal closure suggested that NAC did not function as an antioxidant. On the other hand, its specific inhibitory effect on FAHs rather suggested that it could act as a nucleophilic compound.

Thiol Reagents Are Powerful Activators of Stomatal Closure
RES oxylipins containing a,b-unsaturated carbonyl groups can result from the metabolization of FAHs by LOX activities ( Figure S3) [40,41]. RES oxylipins are able to add to thiolcontaining compounds such as cysteines of proteins or glutathione according to the Michael addition (for review, see [27]) and are produced during plant microbe interactions [22,26,42]. We investigated whether the two RES oxylipins, 9-KODE and 13-KODE, show a dose-dependent effect on stomata, finding that both compounds induce stomatal closure already at nanomolar concentrations ( Figure 3A).
To test whether the presence of thiol reactive structures was required for the biological activity of 9-and 13-KODE, the ketone group was reduced into nonreactive alcohol. The alcohols, 9-and 13-HODE, were both unable to promote stomatal closure ( Figure 3B). Moreover, the effect of different Michael acceptors along with halogenated organic compounds and isothiocyanate was tested. All these molecules are known as thiol reagents and were potent inducers of stomatal closure ( Figures 3C, D and S4). Additionally, the thiol-containing compound NAC that inhibited the action of FAHs also blocked the activity of 13-KODE and the other thiol reagents PCMB and sulforaphane, suggesting that reactivity toward thiols was critical for biological activity of these compounds. Conversely, NAC did not prevent ABA-induced stomatal closure ( Figure 3D), suggesting that RES oxylipins and ABA do not share a common signaling pathway.
NAC and 13-HODE Inhibit Bacterial-or flg22-but Not ABA-Induced Stomatal Closure The biologically inactive alcohols, 9-and 13-HODE, displaying chemical structures close to the corresponding ketones prompted us to verify whether these alcohols could counteract the biological activity of 9-and13-KODE and different thiol reagents. As shown in Figure 4A, pre-incubation with 1 nM 13-HODE abolished the activity of the three thiol reagents (13-KODE, PCMB, and sulforaphane). Similar results were obtained with 9-HODE (unpublished data). However, the inhibitory effect of 13-HODE was overcome by using higher concentrations (10 or 100 nM) of 13-KODE ( Figure 4B). On the other hand, stoichiometric concentrations of 13-HODE did not alter the ABA-induced stomatal closure ( Figure 4A). Finally, the effects of NAC and 13-HODE were investigated on epidermal peels either inoculated with Pst DC3000 or treated with flg22 ( Figure 4C). In plants containing a functional LOX1, the two compounds abolished stomatal closure induced by both Pst and flg22. Together, these data further support the existence of a biotic stress-mediated stomatal response independent of ABA.

The Bacterial Phytotoxin Coronatine (COR) and Methyl Jasmonate (MJ) Inhibit RES Oxylipin-Induced Stomatal Closure Through Different Mechanisms
COR secreted by virulent strains of Pseudomonas disables stomatal defense in a COI1-dependent manner and notably inhibits PAMP-and ABA-mediated stomatal closure, subsequently triggering stomatal reopening required for pathogen entry and host colonization [32]. To assess whether COR affects the activity of RES-oxylipin, we pre-treated epidermal peels with the toxin prior to treatment with RES oxylipins. As shown in Figure 5A, COR at 4 mM blocked the effects of both 1 nM 13-and 9-KODE. The COR chemical structure resembles that of JA-Ile, the biologically active form of JA, and enables this phytotoxin to interact with the JA-Ile receptor COI1 to hijack the JA-Ile hormone functions. In an attempt to better understand the biological activity of these chemically related compounds, we examined the action of methyl jasmonate (MJ) on guard cells. In our hands, MJ was unable to promote stomatal closure in a range of concentrations between 1 nM and 100 mM ( Figure S4). However, competition experiments of MJ with RES oxylipins (9/13-KODE and 12-OPDA) indicated that pretreatment of epidermal peels with 1 nM MJ reversed the activity of RES oxylipins applied at the same concentration ( Figure 5B). At a 100fold higher concentration of RES oxylipins, this inhibition was no longer observed, suggesting that MJ mediates the inhibition of RES oxylipins activity through a similar mechanism as the alcohols 9/13-HODE ( Figure 4). However, in contrast to COR, MJ did not counteract the action of ABA ( Figure 5C). In addition, our data establish that although COR and MJ are both able to inhibit the RES oxylipin-mediated stomatal closure, in contrast to COR, the MJ-mediated activity is COI1-independent ( Figure 5D).

Salicylic Acid (SA) Is Required for the RES Oxylipin-but Not For ABA-Induced Stomatal Closure
Bacterial-and PAMP-mediated stomatal closures have been demonstrated to be SA-dependent [32] and SA induces stomatal closure in Arabidopsis [35]. In an attempt to clarify the role of SA in the RES oxylipin-and ABA-signaling cascades, we conducted experiments with the transgenic SA-deficient NahG line, along with the two SA biosynthesis mutant lines, sid1-1 and sid2-1 [43,44]. Results shown in Figure 5E demonstrate that these three lines normally responded to ABA, whereas they were all impaired in their ability to respond to the RES oxylipins 9/13-KODE. In addition, our data also indicate that lox1 mutant lines were as sensitive to SA (100 mM) as WT and lox1-2 35S:LOX1 ( Figure 5F), demonstrating that exogenously applied SA can complement for LOX1 deficiency. Together these results support that SA is required to convey the RES oxylipin-but not the ABA-mediated signal leading to stomatal closure.

The Protein Kinase OST1 Does Not Participate in the Flagellin-Mediated Signaling Cascade
Many steps involved in the ABA-dependent signaling pathway are now well documented [45,46]. More recently, ABA has also been described as a key player in pathogen-and PAMP-induced stomatal responses [33]. It has been demonstrated that the Arabidopsis mutants aba3-1 and ost1-2, which are defective in the hormone biosynthesis and signaling, respectively, were both no longer able to close stomata in response to either Pst or flg22. In order to clarify the function of ABA signaling in pathogen-induced stomatal closure, we first compared the effects of ABA, flg22, and 13-KODE on stomata of the mutant ost1-2. As previously reported, the ost1-2 mutant does not close stomata in response to ABA even at very high concentrations ( Figure 6A). However, ost1-2 plants remained responsive to both flg22 and 13-KODE, albeit at higher doses than WT plants. The stomatal behavior of the aba2-1 mutant, which is defective in ABA production, was also assessed and showed a similar response profile as the ost1-2 mutant ( Figure S5).
In order to get further insights into the signaling steps triggered by the three compounds, we have assessed the activation of OST1 on Arabidopsis cell suspensions. In-gel kinase assays ( Figure 6B) of immunoprecipitated OST1 showed that in accordance with previous work [47][48][49], ABA clearly activated OST1 kinase. In contrast, flg22 and both RES oxylipins 9-and 13-KODE were unable to activate OST1. This is consistent with the hypothesis that different signaling pathways can lead to stomatal closure, but that the optimization of the guard cell response to biotic stress requires ABA-dependent mechanisms. Apart from OST1, a number of other protein kinases have been identified to function in ABA signal transduction, including mitogenactivated protein kinases (MAPKs), which play a prominent role in both biotic and abiotic stress signaling [50,51]. MPK9 and MPK12 were shown to play an important role in ABA-induced closure of stomata [29]. To clarify the role of these MAPKs in ABA and flg22 signaling, we tested mpk9-1/12-1 double mutant plants, which were reported to be impaired in ROS-mediated ABA signaling [29]. Double mutant plants displayed a clear defect in ABA-induced stomatal closure, but were as responsive to flg22 as wild-type plants ( Figure 6C).
To further investigate the role of protein kinases in response to ABA and biotic stress, we carried out kinase assays on cells treated with ABA, flg22, and 9-or 13-KODE. In-gel kinase assays showed flg22-mediated activation of three protein kinases ( Figure 6D) that were previously identified as MPK3, MPK4, and MPK6 [52]. Immunoblotting using the anti-phospho-p44/42 MAPK antibody confirmed the presence of these activated MAPKs in seedlings exposed to flg22 ( Figure 6E). However, none of these MAPKs were activated in response to ABA (Figures 6E), the kinase activities stimulated in ABA-treated cells corresponding to OST1 and other SnRK2s [48]. In order to verify the role of these kinases in the ABA-and PAMP-mediated signaling cascades, the behavior of mpk3 and mpk6 knockout mutants was investigated. As shown in Figure 6F, stomata of mpk3 and mpk6 responded normally to ABA, 18:2 and 9-KODE, whereas their response to flg22 was strongly impaired demonstrating that different sets of MAPKs are required to convey signals generated by the action of ABA and biotic stress and suggesting that RES oxylipin production and action occur downstream of MPK3/6.

Distinct Pathways Mediate ABA and Bacterial Early Gene Expression
To further assess whether early pathogen-mediated signaling cascade depended on ABA, plantlets were submitted either to ABA or to Pst DC3000 or to Pst followed by ABA sprays and sampled over a time scale corresponding to the pathogen-induced stomatal closure. As shown in Figure S6, transcript levels of the three ABA-specific genes, RD29b, ABI1, and ABI2, (Methods S1), significantly increased as soon as 30 min after application of the hormone, whereas transcript levels of these genes did not increase in plants treated with Pst DC3000. In addition, pretreatment with Pst did not modify the response of plants to ABA. Together, these results suggest that during the early phase of the plant-pathogen interaction when stomatal closure takes place, ABA is not produced.

LOX1 Activity Controls Stomatal Closure During the Early Stages of Plant-Pst Interactions
In the present work, we show that a defect in LOX1 compromises the ability of plants to close stomata in response to both virulent and avirulent strains of Pst or the PAMP flg22 ( Figure 1). Impairment in ABA responsiveness, which could have explained the lox1 mutant phenotype, was ruled out since mutant and WT lines were similarly able to close stomata upon ABA treatment (Figure 1). On the other hand, involvement of oxylipins arising from LOX1 activity in controlling stomatal access of bacteria into plants was clearly established (Figure 1). Induction of stomatal closure by LOX substrates (PUFAs) or LOX reaction products (FAHs and KOD(T)E) was demonstrated (Figures 2, 3,  and S4). On the other hand, Arabidopsis mutants lacking LOX1 were no longer able to respond to PUFAs (Figure 2), while they still remained responsive to FAHs (unpublished data) and 9/13-KODE ( Figure S7A), indicating that absence of LOX1 activity can be rescued not only by FAHs but also by their metabolites. In line with this result, it is noteworthy that pretreatment of the lox1-2 mutant line with the 9-specific LOX compound 9-KODE (Methods S1), partially restores resistance of LOX1-deficient lines against Pst DC3000 ( Figure S7B), confirming that LOX1 controls stomatal defense.
Dose-response curves of linoleic acid and the two FAHs (9-and 13-HPODE) for stomatal closure showed that these compounds close stomata at concentrations several orders of magnitude lower than ABA (Figures 2 and S8). Our data also demonstrate that the efficiency of oxylipins to close stomata is not position-specific, since 9-and 13-HPODE had the same biological activity and that trienic fatty acids were as active as dienic ones (Figure 2). To better understand the mechanisms responsible for the biological activity of FAHs, their efficiency was compared to that of H 2 O 2 . FAHs were remarkably more active than H 2 O 2 and tBuOOH ( Figure 2). To explain this discrepancy, we postulate that oxidative stress potentially generated by the peroxides might target different signaling components. Unlike H 2 O 2 or tBuOOH, the more hydrophobic FAHs might access and oxidize critical residues in hydrophobic sites of key signaling proteins.
Thiol-containing compounds can protect cells against oxidative injury by scavenging reactive oxygen species (ROS) or by conjugating to reactive electrophile species (RES) often generated from FAHs [27]. NAC, used in the present work for its rather weak antioxidant property [38], did not inhibit the H 2 O 2 -or tBuOOH-induced responses, but it strongly compromised FAH responses ( Figure 2). Hence, it is likely that inhibition of FAH activity by NAC was rather due to its nucleophilic properties. Downstream of FAHs, those oxylipins containing a,b-unsaturated carbonyl structures are powerful RES able to add to thiolcontaining substances [25][26][27]39]. Consequently, the activity of FAHs on stomata can rather be explained by their metabolization into RES-oxylipins ( Figure S3). In good agreement with this hypothesis, the two RES oxylipins 9-and 13-KODE displayed equivalent and very strong activity on guard cells (Figure 3). Five additional RES oxylipins including trienic fatty acid ketones (9and 13-KOTE), two cyclopentenone derivatives (12-OPDA and ProstaglandinA2), and the aldehyde 4-HNE that can be generated by decomposition of FAHs all displayed strong biological activity ( Figure S4).
Among these oxylipins, the JA precursor 12-OPDA displayed a strong activity on stomata, whereas MJ was inactive in a range of concentrations between 1 nM and 100 mM ( Figure S4). The ability of MJ to induce stomatal closure is still controversial ( [53][54][55] and this work), but very recent results show that MJ is active only when ABA endogenous levels reach a critical threshold [56]. In the ABA-deficient line aba2-2 or in plants treated by fluridon, an inhibitor of ABA biosynthesis, MJ was no longer able to promote stomatal closure, indicating that MJ activity on guard cells is intimately dependent on the physiological status of plants. Hence, culture conditions used to produce the biological material can strongly influence this status and modify the ability of plant to respond to MJ.
The a,b-unsaturated carbonyl structure in oxylipins, as soft electrophiles, preferentially add to soft nucleophiles, which are cysteine residues in GSH or proteins [27]. Reducing the RES oxylipins, 9-, and 13-KODE into their corresponding alcohols 9and 13-HODE, the latter being not electrophilic, has allowed us to assess the involvement of this putative mechanism. Given that both alcohols were inactive (Figure 3), reactivity of 9-and 13-KODE toward thiols could explain their biological activity. This hypothesis was confirmed by the fact that a large set of known thiol-reagents, including different natural and artificial Michael acceptors, several halogenated organic compounds, and one isothiocyanate, all promoted stomatal closure at nanomolar concentrations ( Figures 3C, D and S4). Altogether, these results strongly suggest the involvement of a thiol-containing target in the RES oxylipin-mediated cascade.
At stoichiometric concentrations, both the fatty acid alcohols 9/ 13-HODE and MJ were found to block the biological activity of RES oxylipins but not that of ABA (Figures 4 and 5). On the other hand, at concentrations above 1 mM, the Pseudomonas phytotoxin COR disabled both RES oxylipin and ABA activities ([32] and Figure 5). These results suggest that the COR-mediated inhibition of RES oxylipin activity is COI1-dependent, whereas fatty acid alcohols and MJ inhibit this activity by means of another mechanism. This hypothesis is further supported by results shown in Figure 5D, which indicate that, on the mutant coi1-17, COR no longer exerts inhibition of the ketone effects, whereas MJ remains active. Neither NAC, 13-HODE, nor MJ inhibited the activity of ABA (Figures 4 and 5), suggesting that RES oxylipins do not share a common signaling mechanism with ABA. Considering that stomatal closure induced by inoculation with Pst or treatment with flg22 were inhibited by NAC or 13-HODE (Figure 4), it can be concluded that the early stomatal response to pathogens is not under the control of ABA but depends on LOX1-derived RES oxylipins. According to all these data, it is tempting to speculate that thiol-reactive compounds such as RES-oxylipins generated in response to biotic stress would react on guard-cell-specific target(s) bearing a critical cysteine residue buried in a hydrophobic cavity. This covalent binding (alkylation) would then trigger stomatal closure. Competition with thiol-containing compounds such as NAC or the presence of FAH or RES oxylipin metabolites such as 9/13-HODE and jasmonates that could fit into the target cavity without reacting with the critical cysteine residues would prevent the target alkylation and thereby inhibit the biological process. The equal activity of 9-and 13-oxylipins on stomata suggests that these compounds are perceived by the same guard cell target(s). The 9-and 13-LOXs could both contribute to production of metabolites regulating stomatal movements, however in mutants lacking LOX1 activity, (i) PUFAs were not able to rescue stomatal function and (ii) bacteria were unable to trigger stomatal closure. Hence, LOX1 exhibits a specific function for which no other LOXs can be substitutes. Consistent with this assumption, it is clearly established that mutation in LOX6, the 13-LOX encoding gene expressed in guard cells, did not impair stomatal responses to flg22, linoleic acid, or 9-KODE ( Figure S2).
Our examination of the SA function in the stomatal response indicated that the synthesis of SA is required to convey the RES oxylipin (9/13-KODE) signal but not that of ABA and SA acts downstream of these RES oxylipins ( Figure 5). Hence it can be concluded that SA participates in the RES oxylipin-mediated signaling cascade in accordance with previous results indicating that PAMP-or Pst-regulated stomatal closure was SA-dependent [32] but SA is not an intermediate of the ABA-dependent pathway ( Figure 5). Again, these results further support the conclusion that ABA does not act as a regulator of the biotic stress-dependent signal in guard cells.

Bacterial-Induced Stomatal Closure Is ABA-Independent
A major breakthrough in the understanding of the plant innate immune response against bacterial pathogens was the finding that stomatal closure was a key part of this process preventing bacterial entry in leaves [33]. This work also suggested that PAMP-induced stomatal closure required ABA biosynthesis and components of the ABA signal transduction pathway such as the protein kinase OST1 and nitric oxide biosynthesis.
Our data argue in favor of the existence of an ABA-independent guard cell signaling cascade controlling stomatal closure during the early steps of plant-microbe interaction. First, the ability of lox1 mutant lines to respond to ABA strongly suggested that LOX1 did not act downstream of the hormone (Figure 1). Second, in contrast to ABA, neither flg22 nor the RES oxylipins 9-and 13-KODE could activate OST1 kinase in Arabidopsis cells (Figure 6), suggesting that different early signaling pathways are employed for ABA and flg22 or RES oxylipins. Consistent with these results, the mutant ost1-2, which is insensitive to ABA, still responded to flg22 or 13-KODE (Figure 6), albeit responses were less intense than in WT. Similarly, the stomatal responses of the aba2-1 mutant, which is impaired in ABA synthesis, showed a marked decrease in sensitivity not only to ABA but also to flg22 and 13-KODE ( Figure  S5). Overall, our results strongly suggest that although ABA is not the major regulator of bacterial and PAMP-induced stomatal closure, it contributes to modulate stomatal responses to biotic stress. In a recent genetic screen, Zeng et al. [35] identified Arabidopsis mutants named scord (susceptible to coronatinedeficient Pst DC3000) that were compromised in their ability to close stomata in response to bacteria. Interestingly, a majority of these mutants showed normal closure in response to ABA, confirming our results that ABA does not directly control bacterial-induced stomatal closure. Stomatal closure requires the presence in guard cell membranes of effector proteins that control osmotic pressure. Their transcription and/or posttranslational regulation are known to mainly depend on ABA in an OST1mediated pathway [57]. To explain that mutants affected in ABA synthesis or regulation remained responsive to biotic stress, it must be assumed that still unidentified components also regulate this ultimate step. However, in these mutants, the stomatal closure machinery is likely less efficient, explaining the lower sensitivity of such mutants to biotic stress. Interestingly, the activation of one of these effectors, the slow-type anion channel SLAC1 [58], is required in both ABA-and biotic stress-mediated responses ( Figure S9), suggesting that these pathways converge at the level of the ion channels controlling the osmotic pressure of the guard cell necessary to regulate stomatal aperture.
Numerous MAPK cascades have been reported to convey diverse environmental signals [50,51]. Both MAP kinases MPK9 and MPK12 were identified as critical steps of the guard cell response to ABA, and the mpk9-1/12-1 double mutant is compromised in ABA-induced stomatal closure [29]. However, this mutant responded normally to flg22 (Figure 6), further supporting that distinct ABA-and flg22-mediated pathways exist in guard cells. The two well-characterized MAPKs, MPK3 and MPK6, that are key response elements in both abiotic and biotic stress signaling have also been implicated in ABA signal transduction. MPK3 antisense plants were shown to be partially impaired in ABA-promoted inhibition of stomatal opening, while they responded normally to the hormone in stomatal closure [59]. However, these plants were compromised in their ability to close stomata in response to Xanthomonas campestris inoculation [60]. MPK6 was also reported to mediate ABA-and sugar-regulated seed germination [61] but also responses to other stresses such as pathogen infection, cold and salt stress, and wounding [62][63][64][65]. Our work demonstrated that flg22 but not ABA activated MPK3, MPK4, and MPK6 early after treatment ( Figure 6). A role of MPK3 and MPK6 in flg22-induced stomatal closure was subsequently confirmed by testing mpk3 and mpk6 knockout lines. The fact that both mutants were compromised in the flg22mediated stomatal closure ( Figure 6) suggests that the two MAP kinases do not play a redundant role in this process and agrees with recent findings that these two kinases both play an important but distinct role in plant immunity and signaling [66,67]. Overall, our results demonstrate that MPK3 and MPK6 are involved in the flagellin, but not in the ABA guard cell signaling cascade. The fact that linoleic acid (18:2) and 9-KODE were as active on WT as on mpk3 or mpk6 mutants suggests that LOX1-mediated synthesis of oxylipins is downstream of MPK3/6, although it cannot be ruled out that these two steps are independent. The involvement of these MAPK activities as early specific steps of the flg22-dependent signaling cascade provides another argument supporting the existence of an ABA-independent mechanism leading to stomatal closure.
Finally, gene expression studies of plantlets that were treated with ABA or Pst DC3000 showed that RD29b, ABI1, and ABI2 transcript levels rapidly increased only in response to ABA but not after Pst inoculation ( Figure S6). Additionally, given that plants first treated with Pst and then sprayed with ABA displayed identical responses as those observed in plants treated by ABA only, it can be concluded that ABA is not produced during the very early stage of the interaction with bacteria.
Overall, our data suggest that early signals produced upon challenging of plants with ABA and pathogens are different. As depicted in the proposed model (Figure 7), whereas the response to ABA is transduced principally by OST1 and MPK9/12, microbial signals are conveyed by MPK3/6, LOX1-induced RES oxylipin derivatives, and SA. Our work also provides evidence that ABA is essential to potentiate guard cells to enable them to properly respond to biotic stimuli, and further studies are necessary to characterize these steps and their role in the stomatal responses to other signals. Finally, the present results shed new light on the function of oxylipins in the plant immune response.

Statistics
All experiments were performed in triplicates, and statistical differences of means were analyzed with the Student's t test with the following symbols for the classes of probability: * p,0.05, ** p,0.01, and *** p,0.001. To compare more than two means, the ANOVA test was performed and letters have been used to mark statistically identical groups of means. The number of degrees of freedom (dfs), the value of the F statistic and the p value are given in the legend of figures for each group of means.

Transgene Construction and Complementation of the lox1-2 Mutant Line
The Escherichia coli strain TOP10 (Invitrogen) was used for cloning and propagation of the different vectors. Agrobacterium tumefaciens C58 strain transformation was performed by electroporation and was used in all plant transformation experiments. The full-length cDNA clone of LOX1 gene (accession number BT010358) provided by the Nottingham Arabidopsis Stock Center (http://arabidopsis.info) was used for this study. PCR amplification was performed with the cDNA containing vector as template and the primer couple: forward, 59-GGGGACAAGTTTGTA-CAAAAAGCAGGCTTCGAAAACCTGTATTTTCAGG-GAATGTTCGGAGAACTTAGGGATCTG-39 and reverse, 59-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAGA-TAGAGACGCTATTTGGAAT-39. The amplicon was subcloned into the GATEWAY donor vector pDONRZeo (Invitrogen) using the BP clonase II (Invitrogen). The full-length cDNA was subcloned in the destination vector pMDC32 kindly provided by Mark Curtis (Institute of Plant Biology and Zürich-Basel Plant Science Centre, University of Zürich, Switzerland) using the LR clonase (Invitrogen). The homozygous T-DNA line lox1-2 was transformed with empty vector pMDC32 or pMDC32:LOX1 using flower dipping method. T1 plants were screened on germination medium supplemented with Hygromycine (30 mg/L). The T-DNA presence was monitored by PCR analysis using the primer couple: forward, 59-GCGCGATTGCTGATCCCC-39 and reverse, 59-GCCCTCGGACGAGTGCTG-39. The expression of LOX1 in the transgenic lines was measured by semi-quantitative RT-PCR using the primer couples: (LOX1) forward, 59-AGAC-TATCCTTACGCAGTGGA-39 and reverse 59-TGCCGGTGA-CTCCGCCTTC-39, and (EF1) forward, 59-TACCTCCCAGG-CTGATTGTG-39 and reverse, 59-TCTGACCAGGGTGGTT-CATG-39. Seven lox1-2:pMDC32:LOX1 independent lines were retrieved after the screening, T2. Progeny analysis was performed and lines with a single T-DNA locus were selected for further experiments.

Chemicals and Synthesis of 9-and 13-HODE
Oxylipins and other chemicals used in this study are given in Table S2.  In red are the steps whose activity has been demonstrated in this work by genetic approaches. Downstream of biotic stimuli (Pst or flg22), both MPK3 and MPK6 are required to activate production of LOX1dependent oxylipins such as FAHs (9-HPOD(T)E) and/or reactive ketones (9-KOD(T)E). Our pharmacological data strongly suggest that the latter compounds modulate the activity of putative thiol-containing targets responsible for biotic signal transduction. Downstream of this step, SA is required to convey the biotic stress signal. In a COI1-dependent manner, COR inhibits the accumulation of SA through the activity of three NAC transcription factors and orchestrates the stomatal reopening [74]. On the other hand, the ABA-dependent pathway that is triggered upon abiotic stress requires the protein kinases OST1 and MPK9 or MPK12. As one of the final effectors required for the regulation of osmotic pressure in guard cells, activation of the slow-type anion channel SLAC1 is necessary for stomatal closure in response to both biotic stress and ABA. Not depicted in this model are the data provided by this work and supporting the involvement of ABA in the guard cell potentiation required for optimizing responses to biotic stress. doi:10.1371/journal.pbio.1001513.g007 INRA Toulouse, France). The alcohols, 9-HODE, (10E,12Z)-9hydroxy-10,12-octadecadienoic acid and 13-HODE, (9Z,11E)-13hydroxy-9,11-octadecadienoic acid were produced by sodium borohydride reduction of the corresponding ketones according to the following procedure: 10 mL of 10 mM 9-or 13-KODE ethanolic solution was added to 20 mL of 5% (w/v) NaBH 4 in 0.2 N NaOH and incubated 5 min at room temperature. After addition of 100 mL of 0.5 M potassium acetate pH 4.0, products were extracted 3 times with 200 mL of the solvent mixture hexane/diethyl ether, 70/30 (v/v). The organic phase was then evaporated to dryness under a nitrogen stream, and the residue was dissolved in absolute ethanol. Purity of compounds was controlled by HPLC chromatography according to Montillet et al. [70]. The chromatographic profiles were recorded at 234 and 280 nm for alcohol and ketone detections, respectively. None of the alcohol preparations were contaminated with a detectable amount of ketone.

Pseudomonas syringae Growth Assay
Plants were grown on soil in a controlled environment chamber under an 8 h light regime (150-200 mE?m 22 ?s 21 ) at 22uC and 65% relative humidity. Pst DC3000 was grown for 24 h at 28uC on NYGA solid medium supplemented with 50 mg/mL Rifampicin. For in planta bacterial growth assays, 4-wk-old plants were spray-inoculated with bacterial suspensions at 5?10 7 cfu/mL in 10 mM MgCl 2 with 0.03% (v/v) Silwet L-77 (Lehle Seeds). In planta bacterial titers were determined at day 3 by shaking leaf discs in 10 mM MgCl 2 with 0.01% (v/v) Silwet L-77 at 28uC for 1 h as previously described [71]. At least five plants per genotype were used for each sampling.

Measurements of Stomatal Aperture
After 3 d of stratification of seeds at 4uC, wild-type, mutant, and transgenic lines of A. thaliana were grown on soil in a plant growth chamber with an 8 h light period (250 mE?m 22 ?s 21 ) at 23uC and a 16 h dark period at 19uC, and relative humidity of 75%. The abaxial side of leaves of 4-to 5-wk-old plants was stuck on cover slips and peeled. Samples were then placed in Petri dishes containing 10 mM MES/Tris pH 6.0, 30 mM KCl, and 1 mM CaCl 2 (working buffer). After 30 min in darkness, epidermal peels (except dark controls) were transferred for 2 h under light (300 mE?m 22 ?s 21 ) at 22uC in order to assure that most stomata were open before treatments. For promotion of closure assays, stock solutions of compounds (in water or ethanol as indicated in the figures) were directly diluted in the working buffer in contact with epidermal peels. Stock solutions prepared in ethanol were diluted so that the final solvent concentration did not exceed 0.1% (v/v). Pst DC3000 and Pst DC3000 AvrRpm1 were grown for 18 h in a rotating incubator at 30uC and 200 rpm in liquid LB medium supplemented with 50 mg/mL of Rifampicin and 50 mg/mL of Rifampicin plus 10 mg/mL of tetracycline, respectively. For treatments, bacterial suspensions were centrifuged and resuspended in the working buffer at a final concentration of 10 8 cfu/mL. After treatments, samples were further incubated under light for 2.5 h (or 1 h for treatments with bacteria) before stomatal aperture measurements according to Merlot et al. [72]. To avoid bias results, blind experiments were performed so that experimenters in charge of stomatal measurements were not informed of the evaluated genotypes or treatments. Values reported are the means of at least three independent experiments, for which 60 aperture widths were measured each time. Error bars represent standard deviations of the means.

Immunoblotting
Arabidopsis thaliana Col-0 seedlings were grown in liquid MS medium (SIGMA) for 14 d in a growth chamber (24uC, 16 h photoperiod). Seedlings were treated with water (mock) or 1 mM flg22 for 10 min or with 0.1% ethanol (mock) or 100 mM ABA for 10, 20, 30, or 60 min. For protein extraction, ground samples were resuspended in SDS-PAGE loading buffer, boiled, and centrifuged at 8,000 g for 2 min. Supernatants, containing total protein extracts, were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% BSA in TBST, membranes were probed with 1/5,000 anti-phospho-p44/ 42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (Cell Signaling) followed with 1/10,000 horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (SIGMA). HRP activity was detected using the ECL Plus reagent kit (GE Healthcare) and the GeneGnome imaging system (Syngene). Coomassie blue staining of blots was carried out for protein visualization.