Drought tolerance in Arabidopsis is controlled by the OCP3 disease resistance regulator

Authors


*For correspondence (fax 34 96387 7859; e-mail vera@ibmcp.upv.es).

Summary

Water scarcity and corresponding abiotic drought stress is one of the most important factors limiting plant performance and yield. In addition, plant productivity is severely compromised worldwide by infection with microbial pathogens. Two of the most prominent pathways responsible for drought tolerance and disease resistance to fungal pathogens in Arabidopsis are those controlled by the phytohormones abscisic acid (ABA) and the oxylipin methyl jasmonate (MeJA), respectively. Here, we report on the functional characterization of OCP3, a transcriptional regulator from the homeodomain (HD) family. The Arabidopsis loss-of-function ocp3 mutant exhibits both drought resistance and enhanced disease resistance to necrotrophic fungal pathogens. Double-mutant analysis revealed that these two resistance phenotypes have different genetic requirements. Whereas drought tolerance in ocp3 is ABA-dependent but MeJA-independent, the opposite holds true for the enhanced disease resistance characteristics. These observations lead us to propose a regulatory role of OCP3 in the adaptive responses to these two stresses, functioning as a modulator of independent and specific aspects of the ABA- and MeJA-mediated signal transduction pathways.

Introduction

Plants have evolved complex mechanisms to rapidly sense and adapt to changing environmental conditions that are essential for survival under both biotic and abiotic stresses. Such responses can be achieved by distinct metabolic and physiological adjustments, mediated by different phytohormones often specific to a certain type of stress. Two of the most limiting factors to plant survival are microbial pathogen infections and drought, which are mainly controlled by methyl jasmonate (MeJA) and abscisic acid (ABA), respectively. Increased tolerances to these two stresses represent major challenges for agriculture worldwide. However, little data exists to determine if adaptive responses to these two environmental pressures are linked.

MeJA mediates various defence responses to necrotrophic fungal pathogens, such as Botrytis cinerea and Plectosphaerella cucumerina (Thomma et al., 1998; Liechti and Farmer, 2002; Turner et al., 2002). Increased susceptibility to necrotrophic pathogens is correlated with defects in MeJA accumulation and signalling, and the accompanying loss or delay in the expression of some marker genes, such as PDF1.2, which encodes an antifungal peptide (Penninckx et al., 1998; Thomma et al., 1999; Kachroo et al., 2001). Furthermore, the application of MeJA reduces disease severity caused by some necrotrophs in Arabidopsis and tomato (Thomma et al., 1999; Diaz et al., 2002). However, the genetic control of disease resistance to necrotrophic pathogens, and in particular to B. cinerea, still remains poorly defined.

The phytohormone ABA has a wide range of essential functions in plant adaptation to different environmental pressures, including water deficit. This is achieved through the regulation of complex mechanisms that include transcriptional and post-transcriptional cell reprogramming, and stomatal aperture modulation (Schroeder et al., 2001; Kuhn and Schroeder, 2003). Extensive studies have elucidated part of the regulatory mechanisms of drought-responsive gene expression downstream of ABA-specific positive signalling, mediated by protein kinases, protein phosphatases 2C and transcription factors (Li et al., 2000; Saez et al., 2004; Furihata et al., 2006; Seki et al., 2007).

Despite the awareness that plants must cope with and adapt to situations in which they are simultaneously exposed to several stresses in their natural environment, some stresses such as drought and fungal diseases have remained as mostly separate research fields. There is a growing body of evidence to suggest the existence of a significant overlap between the signalling networks controlling these two plant adaptive processes. Besides its central role in controlling responses to drought, ABA also influences biotic stress responses, and may interfere with signals regulated by disease resistance-associated hormones such as salicylic acid (SA), MeJA or ethylene (ET) (Mohr and Cahill, 2003; Anderson et al., 2004; Flors et al., 2005; Mauch-Mani and Mauch, 2005; Fujita et al., 2006; Adie et al., 2007; Robert-Seilaniantz et al., 2007; de Torres-Zabala et al., 2007; Asselbergh et al., 2008). On the basis of experiments with exogenous application of ABA, inhibition of ABA biosynthesis and/or the use of ABA-deficient mutants, ABA has mostly been considered to act as a negative regulator of disease resistance (revised in Mauch-Mani and Mauch, 2005; Robert-Seilaniantz et al., 2007). However, there are also reports of a positive correlation between ABA and disease resistance. For example, the accumulation of callose primed by β-amino-butyric acid, or following resistance to some necrotrophic fungi, has been shown to be ABA-dependent (Ton and Mauch-Mani, 2004; Mauch-Mani and Mauch, 2005). More recently, Hernández-Blanco et al. (2007) also reported that mutations in three types of cellulose synthases (Cesa4/IRX5, Cesa7/IRX3 and Cesa8/IRX1) conferred enhanced resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. Genetic and transcriptomic analysis indicated that the irx-mediated resistance was accompanied by the upregulation of a number of ABA-responsive genes, thus establishing a link between resistance and ABA. Likewise, it was previously shown that mutant alleles of the CesA8/IRX1 gene conferred tolerance to drought stress in Arabidopsis (Chen et al., 2005). These observations thus suggest that resistance to pathogens and tolerance to drought are somehow interlinked, at least at the level of cellulose synthesis.

MeJA has been reported to affect plant transpiration through induction of stomatal closure, similar to ABA. Consistent with this, MeJA treatment not only induces disease resistance against necrotrophic fungi, but also enhances drought tolerance (Raghavendra and Reddy, 1987; Lee et al., 1996; Creelman and Mullet, 1997; Gehring et al., 1997; Wang, 1999). Genetic studies have revealed an interaction between MeJA- and ABA-mediated guard cell signalling, where the production of reactive oxygen species (ROS), ion channel activation and cytoplasmic alkalization are common intermediates in these pathways (Suhita et al., 2003, 2004; Munemasa et al., 2007). These observations indicate that ABA- and MeJA-mediated processes might be linked via crosstalk between the two pathways, leading to drought and pathogen resistance.

OCP3 is a member of the homeobox transcription factor family. In Arabidopsis, OCP3 functions as a negative regulator in the disease resistance response to necrotrophic fungal pathogens such as P. cucumerina and B. cinerea (Coego et al., 2005). ocp3 mutant plants show a remarkable enhancement of resistance towards these necrotrophs, whereas they display normal susceptibility to biotrophic pathogens, including Pseudomonas syringae and Hyaloperonospora parasitica (Coego et al., 2005). Genetic evidence indicates that ocp3-associated enhanced disease resistance requires COI1, an F-box protein. COI1 is a component of E3 ubiquitin ligase, which is involved in the 26S proteosome-mediated protein degradation pathway (Xie et al., 1998; Xu et al., 2002) and functions as an integrator of the cellular responses triggered by MeJA (Chini et al., 2007; Thines et al., 2007).

In this report, we provide evidence that OCP3 also plays a pivotal role in the signal pathway controlling drought tolerance through the modulation of ABA-mediated stomatal closure. This function is ABA-dependent but MeJA-independent. Also, interestingly, ocp3-associated disease resistance to necrotrophs is ABA-independent. Hence, OCP3 can be thought of as a node of integration between ABA- and MeJA-mediated adaptive responses, and therefore OCP3 represents an interesting target to manipulate drought and necrotrophic pathogen resistance in crops.

Results

ABA treatment and dehydration repress OCP3 expression in Arabidopsis

To investigate a possible link between ABA signalling and OCP3, we first studied the effect of ABA on OCP3 transcript levels. Quantitative RT-PCR (qRT-PCR) showed that OCP3 transcript accumulation was repressed rapidly after exogenous application of 100 μm ABA or dehydration stress. At 1 h after ABA application most of the negative effect on OCP3 gene expression had already taken place (Figure 1a). Furthermore, following dehydration Arabidopsis seedlings also showed a decline in the level of OCP3 mRNA that was readily observable after 6 h (Figure 1b). In this case, the observed decline in OCP3 mRNA level was preceded by a transitory increase that peaked at 0.5 h, and started declining 1 h after triggering dehydration. The results indicate that OCP3 gene expression is negatively regulated by ABA.

Figure 1.

 Effect of ABA on OCP3 gene expression and plant growth.
Quantitative RT-PCRs representing the relative mRNA accumulation of OCP3 normalized to that of APT under ABA (a) and dehydration (b) treatments. Error bars represent standard deviations from three independent experiments. Different letters indicate statistically significant differences between genotypes (< 0.005, using anova).
(c) Seedlings (10-days old) grown in MS-Agar plates in the presence (right) or absence (left) of 1 μm ABA.

ocp3 plants show enhanced sensitivity to plant growth inhibition by ABA

To elucidate a possible role of OCP3 in response to ABA, we investigated the efficiency of radicle emergence and early growth of the ocp3 loss-of-function mutant in the presence of ABA. Figure 1c shows a comparative analysis of the growth of ocp3 and wild-type plants in the presence or absence of 1 μm ABA. Interestingly, although the germination rate and the radicle emergence of ocp3 plants was unaffected, the growth of ocp3 plants was arrested compared with that of the wild type. This indicates an enhanced susceptibility of ocp3 plants to ABA.

ocp3 plants show enhanced drought resistance

As a critical aspect of the response of plants to water deficit is reduction in stomatal pore aperture, mediated by ABA, we reasoned that the observed enhanced susceptibility of ocp3 plants to ABA might correlate with an altered response to water scarcity. Indeed, in our assays aimed at detecting plant responses to prolonged periods of drought, ocp3 plants revealed an improvement in their resistance to water deficit (Figure 2a,b). After 12 days without watering, ocp3 plants remained nearly intact, without manifesting major macroscopic symptoms of drought-related stress. Under the same experimental conditions, wild-type plants completely collapsed, and did not recover when watering was resumed. More accurate measurements of water loss over time (see Figure 2c) revealed that after 15 days of water withdrawal, ocp3 plants still retained 50% of their initial fresh weight, whereas wild-type plants had lost 50% of their fresh weight already after 8 days of water withdrawal. The observed drought tolerance was not dependent on any developmental stage of the plant, and occurred in both seedlings and mature plants (Figure 2a,b). The ocp3-associated ABA hypersensitivity and drought tolerance is consistent with OCP3 being a negative regulator of the ABA-mediated plant adaptive responses to water deficit.

Figure 2.

 Drought tolerance of ocp3 mutant plants in different developmental stages, and time-course analysis of transpiration in Col-0 and ocp3 plants.
To simulate drought, water was withheld for 12 (b) and 20 (a) consecutive days from 5-week-old and 3-week-old plants, respectively. Photographs show representative Col-0 (left) and ocp3 (right) plants. Five independent experiments were performed using 20 plants per genotype, with similar results.
(c) Progressive drought stress of 5-week-old Col-0 (full line) and ocp3 (dashed line) plants was initiated on day 0. Water loss measurements were performed by weighing 10 individual pots per genotype at different time points (at the same hour in the day). Data shown are means of water loss percentage ± SD. Three independent experiments were performed with similar results.

ABA- and dehydration-induced stress marker genes are unaltered in the ocp3 mutant

The enhanced resistance of ocp3 plants to drought, along with the inhibitory effect of ABA on OCP3 gene expression in wild-type plants, prompted us to evaluate whether the expression of ABA-responsive genes in ocp3 mutant plants were affected, compared with the wild type. For this goal, we monitored the expression pattern of some stress marker genes in response to exogenous ABA application and dehydration treatment. This analysis included the widely-used RD29A, RD29B, RD22, RAB18 and KIN1 ABA-responsive genes; the NCED3 gene, encoding a key enzyme in the ABA biosynthesis (Iuchi et al., 2001), and also the P5CS1 and ProDH genes, encoding key enzymes in the biosynthesis and catabolism of the osmoprotectant proline (Pro), respectively (Delauney and Verma, 1993; Kiyosue et al., 1996). As shown in Figure 3a,b, transcripts for these genes were poorly detectable in wild-type and ocp3 mutant plants under non-stressed conditions (time 0 h). Consistent with findings from previous studies (Kurkela and Franck, 1990; Lang and Palva, 1992; Yamaguchi-Shinozaki and Shinozaki, 1993; Yoshiba et al., 1995; Iuchi et al., 2001), ABA and dehydration treatment induced changes in the expression of these marker genes in the wild type. These changes were of a similar magnitude in wild-type and ocp3 plants. This indicates that the drought tolerance exhibited by ocp3 plants was not conferred by an elevated or faster induction of the expression of these marker genes. This reconciles the observation that neither ABA nor Pro accumulation is enhanced in ocp3 plants, both in non-stressed conditions (Figure 4c) and upon drought stress (Figure 4a,b). Instead, ocp3 plants show a delay in the accumulation of these two molecules following the imposed drought stress. These latter results may indicate that OCP3, either directly or indirectly, could regulate ABA biosynthesis upon drought treatment, which is one of the initial events in drought signalling.

Figure 3.

 The loss of function of OCP3 has no major effect on ABA-responsive gene expression and water loss in detached leaves. Time-course expression analysis by RT-PCR of RD29A, RD29B, RD22, RAB18, KIN1, P5CS1, ProDH and NCED3 in Col-0 and ocp3, in response to ABA (a) and dehydration (DRY) (b) treatments. eIF1α was used as a housekeeping gene.
(c) Water loss from detached leaves as a function of time in Col-0 (•), ocp3 (○), Laer (bsl00084) and abi1-1 (bsl00066) plants. Values are means of the percentage of leaf water loss ± SD (n = 15); FW, fresh weight. Three independent experiments were performed with similar results.

Figure 4.

 Determination of proline (Pro) and ABA levels under drought stress in Col-0 and ocp3 plants.
Determination of free Pro levels (a) and ABA (b) under drought stress (•) and control treatment (bsl00066) in Col-0 (continuous line) and ocp3 (dashed line) plants.
(c) Determination of ABA levels under non-stressed conditions in Col-0 and ocp3 plants.
Values represent means ± SD from two independent sets of plants (n = 4) for each time point. Three independent experiments were performed with similar results.

The dehydration response is not affected in ocp3 plants

To further characterize the function of the OCP3 gene under drought stress in vivo, ocp3 plants were analysed for their dehydration response. The abi1-1 mutant, which is less sensitive to ABA and thus unable to control water loss by closing its stomata in response to drought or dehydration stress (Leung et al., 1994, 1997; Allen et al., 1999; Merlot et al., 2001; Hoth et al., 2002), was used as a control. As observed in Figure 3c, ocp3 and wild-type plants displayed similar rates of fresh weight loss over time. In the same conditions, the water content of abi1-1 leaves declined abruptly compared with the wild type. After 3 h, the water loss of abi1-1 leaves was 80%, whereas for the ocp3 and wild-type (Col-0 and Ler) plants it was approximately 25%.

ocp3 plants are more efficient than the wild type in closing stomata

As shown in Figures 2 and 3c, ocp3 plants show an enhanced drought tolerance, but seem to be unaffected in response to dehydration. The dehydration experiments, quantified as leaf water loss (Figure 3c), measure avoidance of low water potential (following Levitt’s terminology; Levitt, 1972), and are typically not applicable to the investigation of drought tolerance mechanisms (Verslues et al., 2006). In fact, mutants deficient in ABA exhibit altered leaf water loss, but not all mutants (although many) with altered ABA sensitivity exhibit this phenotype (Verslues et al., 2006). These indications fit with the apparently contradictory observations from the ABA sensitivity and dehydration response of ocp3 mutant plants.

The rate of plant water loss during drought is largely determined by stomatal aperture/closure, and this mechanism is controlled mainly by ABA. Note that there are no statistical differences between Col-0 and ocp3 plants when comparing stomatal index (the ratio of the number of stomata in a given area divided by the total number of stomata and other epidermal cells in the same area), stomatal frequency (number of stomata per unit area) and the percentage of mature stomata (determined as the number of stomata in which the overlying cuticle had ruptured to reveal an externally-visible pore, divided by the total number of stomata present) (data not shown). To further address the mechanism underlying the drought tolerance in the ocp3 mutant, we therefore decided to measure the stomatal movement dynamics by direct microscopic observation in leaf epidermal strips. As shown in Figure 5, ocp3 plants showed a higher sensitivity to the application of ABA for both ABA-induced stomatal closure (Figure 5a) and ABA-inhibition of light-induced stomatal apertures (Figure 5b). Thus, ocp3 plants exhibited increased sensitivity in the regulation of the ABA stomata closing pathway. This greater efficiency, or speed, in the physiological processes governing the closure of stomata under imposed drought stress, may be the explanation for the observed longer survival of ocp3 plants to prolonged periods of drought.

Figure 5.

 Influence of ocp3 mutation on ABA-mediated stomatal closure. Stomatal apertures were measured on epidermal peels. Values are means ± SEM (> 60). *Significant difference from the wild type (P < 0.05), using the Student’s t-test. These blind experiments were repeated at least five times.
(a) Effect of ocp3 mutation on ABA inhibition of light-induced stomatal opening. Stomata were pre-opened under light for 2.5 h, and then incubated in the indicated concentrations of ABA for 2.5 h under light.
(b) Stomata were pre-closed in darkness for 2.5 h, and then incubated in the indicated concentrations of ABA for 2.5 h under light.
(c) Micrographs representing the dynamics of ABA-mediated stomatal closure in Col-0 and ocp3 plants.

Thus, ocp3 plants show an enhanced drought tolerance that correlates with an enhanced sensitivity to ABA in terms of both growth inhibition and stomatal closure. In contrast, this mutant is unaffected in other plant responses mediated by ABA, such as leaf water loss or expression of ABA-responsive marker genes (e.g. RD29A, RD29B, RD22, KIN1, RAB18, NCED3, ProDH or P5CS1). All of these results suggest that OCP3 may be acting specifically in one of the multiple output branching pathways for ABA.

Loss of ABA biosynthesis and perception suppresses all major aspects of drought resistance in ocp3 plants

To further evaluate the biological function of OCP3 in an ABA-mediated response to drought stress, we generated a double ocp3 abi1-1 Arabidopsis mutant line. As abi1-1 plants are more susceptible to drought than wild-type plants, to simulate drought and yet observe a difference between abi1-1, ocp3 and the wild type, water was withheld for only 9 days instead of the regular 12 days applied in previous experiments. Because the abi1-1 mutant is in the Ler background and the ocp3 mutant is in the Col-0 background, parental Ler and Col-0 were both used as controls in these experiments. Furthermore, the erecta (er) mutation has been involved in the generation and organization of anatomical structures of the stomatal complex (Shpak et al., 2005), and also in the regulation of transpiration efficiency (Masle et al., 2006). In order to avoid interference with the er mutation, in our studies we segregated the er mutation in our double mutant lines by marker-assisted selection (see Experimental procedures). As shown in Figure 6a, in ocp3 abi1-1 plants, all features characteristic of the ocp3 mutation related to drought resistance were abolished. ocp3 abi1-1 plants no longer demonstrated the characteristic drought resistance attributable to ocp3. This was accompanied by the loss of the observed enhanced sensitivity of ocp3 seedlings to ABA, and also by the reduced expression of ABA-regulated marker genes, such as RD29A, RD29B and to a lesser extent RD22 (Figure 6b,c). The requirement of an intact ABI1 for OCP3 to function as a negative regulator of drought tolerance was further corroborated by the effect of the abi1-1R1 mutation, characterized as a reduction-of-function allele of abi1-1 (Gosti et al., 1999). The analysis of ocp3 abi1-1R1 double mutant plants under drought stress revealed that the abi1-1R1 mutation also suppressed the drought resistance phenotype of ocp3 plants (Figure 6a). Applying the same rationale, the lack of ABA biosynthesis as conferred by an aba2-1 mutation (Leon-Kloosterziel et al., 1996) blocked the ocp3-associated drought tolerance in ocp3 aba2-1 plants (Figure 6a). Moreover, a further defect in ABA signal transduction, such as that conferred by the abi5-1 mutation (Finkelstein and Lynch, 2000), was also found to block the ocp3-associated drought tolerance phenotype when assayed in an ocp3 abi5-1 double mutant background (Figure 6a). All these observations favour the interpretation that OCP3 is a negative regulator in an ABA-mediated drought-response pathway.

Figure 6.

 Effect of disruption on ABA synthesis or perception in ocp3 drought tolerance, plant growth in presence of ABA, and ABA-induced marker gene expression.
(a) Double-mutant drought tolerance analyses were performed as described in Figure 2b, but by withholding water for only 9 days instead of the regular 12 days.
(b) Seedlings (10-days old) grown in MS-Agar plates in the presence (right) or absence (left) of 1 μm ABA.
(c) Time-course expression analysis by RT-PCR of RD29A, RD29B and RD22 in response to ABA. eIF1α was used as a housekeeping gene.

The abi1-1 mutation does not suppress the enhanced disease resistance to necrotrophic fungal pathogens of ocp3 plants

OCP3 was originally discovered by virtue of its role as a negative regulator of disease resistance to necrotrophic fungi (Coego et al., 2005). This phenotype was suppressed in the ocp3 coi1-1 double mutant (Figure 7a,b and Coego et al., 2005), indicating a requirement for an intact MeJA response in ocp3-associated disease resistance. However, disruptions of the SA or ET pathways did not affect the ocp3 resistance phenotype (Coego et al., 2005).

Figure 7.

 Effect of coi1-1 and abi1-1 mutation on ocp3-associated drought tolerance and Botrytis cinerea disease resistance.
(a) Progress of necrosis development in representative single leaves 72 h after inoculation with a suspension droplet of B. cinerea spores.
(b) Lesion diameter measurements 3 days after B. cinerea inoculation. Data points represent average lesion diameter ± SE from a minimum of 50 lesions. Different letters indicate statistically significant differences between genotypes (< 0.005 using anova). Three independent experiments were performed with similar results.
(c) Double-mutant drought tolerance analysis performed as described in Figure 2b.

Given the requirement of an intact ABA signalling pathway for the ocp3-associated drought tolerance, we decided to test whether ABA perception is also necessary for the enhancement of disease resistance to necrotrophic fungi in the ocp3 mutant. To this end, we analysed the response of ocp3 and ocp3 abi1-1 to B. cinerea infection. Interestingly, the disease resistance associated with the ocp3 mutation remained intact in the ocp3 abi1-1 double mutant. The disease caused by the fungus on these plants was markedly decreased compared with the wild type. Conversely, and in accordance to what was previously observed (Coego et al., 2005), ocp3 requires COI1 to manifest the enhanced disease resistance to B. cinerea (Figure 7a,b). These data thus reveal that disease resistance to necrotrophic fungal pathogens in ocp3 mutant plants requires an intact MeJA signalling pathway, but does not require an intact ABA pathway. ABA has been shown to have divergent effects on the response to necrotrophic pathogens, and these seem to depend not only on the plant–pathogen interaction under consideration, but also on the timing of infection and the experimental conditions used (for a summary of these divergent effects see Asselbergh et al., 2008). This may explain the differences found by some authors in the response of the abi1-1 mutant to infection by B. cinerea. In our hands, and under our experimental conditions of using fully-developed plants, the abi1-1 mutant behaves as wild-type plants do towards this pathogen.

The coi1-1 mutation does not suppress the ocp3 enhanced tolerance to drought

To further investigate the possible link between ABA and MeJA, we wondered whether or not the OCP3 function in ABA-mediated drought response was dependent on an intact MeJA signalling pathway. For this purpose, we analysed the drought response of ocp3 coi1-1 double-mutant plants. As shown in Figure 7c, the drought-tolerant phenotype conferred by ocp3 remained unaffected in an ocp3 coi1-1 double mutant. Hence, in contrast to abi1-1, the coi1-1 mutation does not suppress the drought tolerance observed in ocp3 plants. This suggests that the drought tolerance attributable to ocp3 is exhorted through an ABA-dependent pathway that is MeJA-independent.

The plant transcription factor MYC2 has been proposed to be a key regulator in the crosstalk mechanism between ABA and MeJA signalling pathways, and gain-of-function experiments have confirmed the relevance of MYC2 in the activation of these two pathways (Lorenzo et al., 2004). To address whether MYC2 gene expression might be altered in ocp3 plants upon treatment with ABA and MeJA, and thus explain some of the ocp3-associated phenotypes, we measured MYC2 transcript levels by qRT-PCR following these two treatments. Figure 8a,b reveals that although transcript levels for MYC2 remained invariant in ocp3 compared with wild-type plants under resting conditions, the transcripts accumulated to higher levels following treatment with both ABA and MeJA in ocp3 plants. These data reinforce the consideration that OCP3 may be a key player in the interconnection of these two hormone pathways upon signal perception.

Figure 8.

 Effect of the application of abscisic acid (ABA) and methyl jasmonate (MeJA) on MYC2 transcript accumulation in Col-0 and ocp3 plants.
Quantitative RT-PCRs representing relative mRNA accumulation of MYC2 normalized to that of ACT8 under treatments with ABA (a) and MeJA (b) in Col-0 (black bars) and ocp3 (grey bars) plants. Error bars represent standard deviations from three independent experiments. *Significant difference from the wild-type (P < 0.05), using the Student’s t-test.

Discussion

OCP3 is a transcription factor that negatively regulates disease resistance against the necrotrophic fungal pathogens B. cinerea and P. cucumerina, but not against biotrophs such as P. syringae and H. parasitica (Coego et al., 2005). OCP3 transcript accumulation rapidly decreases in response to infection by necrotrophs. Accordingly, the loss-of-function ocp3 mutant shows a remarkable enhanced disease resistance compared with the wild type (Coego et al., 2005). In this study we provide evidence that OCP3 controls an ABA-mediated drought response through the modulation of stomatal aperture. We propose that OCP3 is an early negative regulator of ABA signal transduction related to this process. In addition, we also found that OCP3 regulates necrothophic fungal disease resistance independently of ABA. On the other hand, OCP3 function in drought tolerance is independent of MeJA, which is required for resistance to necrotrophs. Thus, we propose that OCP3 acts at a nexus of signal integration, controlling drought and necrotrophic fungal infections.

OCP3 is a negative regulator in a specific branch of the ABA-mediated drought-response network controlling stomatal movement

ABA is the central hormone mediating drought responses, and increases in endogenous ABA (provoked by dehydration treatment), as well as the exogenous application of ABA, resulted in decreased levels of OCP3 transcript (Figure 1a,b). The loss of function of OCP3 resulted in increased sensitivity to ABA, as demonstrated by comparison of the ABA-mediated plant growth inhibition in ocp3 and wild-type plants (Figure 1c). Accordingly, ocp3 mutant plants showed an increased tolerance to drought (Figure 2). Interestingly, both the dehydration response as well as the expression pattern of some stress-marker genes (including RD29A, RD29B, RD22, RAB18, KIN1, NCED3, ProDH and P5CS1) in response to exogenous ABA application, and dehydration treatment, were not affected in the ocp3 mutant (Figure 3a,b,c). Neither enhanced accumulation of ABA nor enhanced accumulation of Pro, with respect to wild-type plants, was observed in ocp3 plants following drought stress (Figure 4). In contrast, ocp3 plants showed a higher sensitivity to ABA application for both ABA-induced stomatal closure and ABA-inhibition of light-induced stomatal aperture (Figure 5). Thus, ocp3 plants exhibited an increased sensitivity to ABA not only in terms of growth inhibition, but also in the regulation of the stomatal closing pathway.

In addition, double mutant analysis revealed that OCP3 function requires intact ABA synthesis and perception, reinforcing our hypothesis that OCP3 functions in the ABA signalling pathway. ABI1 encodes a protein phosphatase 2C involved in ABA signalling. abi1-1 plants are insensitive to ABA, and are unable to close their stomata in response to both drought stress and ABA application (Gosti et al., 1999). Consistent with an upstream location of OCP3 in the ABA-mediated drought stress response, the abi1-1 mutation blocked the ABA sensitivity in an ocp3 abi1-1 double mutant background (Figure 6b). Also, ocp3 abi1-1 plants lost the drought tolerance associated with ocp3, and this was accompanied by the loss of induction of ABA marker gene expression (Figure 6a,c). An abi1-1 reduction-of-function allele (abi1-1R1) also suppressed the characteristic ocp3 drought resistance, as demonstrated in an ocp3 abi1-1R1 double mutant, thus confirming the requirement of ABI1. Similarly, a disruption in ABA biosynthesis or ABA perception (achieved by combining ocp3 with aba2-1 or abi5-1, respectively), also suppressed the drought tolerance attributed to ocp3 (Figure 6a). All these results strengthen the conclusion that OCP3 is involved in an early ABA signalling response mediating drought tolerance.

After drought stress perception, ABA levels increase rapidly, followed by physiological and transcriptional reprogramming (reviewed in Seki et al., 2007). In order to prevent water loss, plants close the stomatal pores through a complex and specific signalling network in the guard cells. Later, if the stress continues, plants activate a battery of defence systems throughout the plant to avoid cellular damage resulting from the loss of water potential and dehydration (Verslues et al., 2006). Our results suggest that OCP3 could be acting in the first steps of the plant response to drought stress. Accordingly, the OCP3 transcript is rapidly repressed by exogenous application of ABA, and this repression occurs before stomatal closure. In the ocp3 mutant, the ABA-mediated stomatal closure is much more rapid, and the ocp3 plants tolerate a drought imposed stress better. However, OCP3 does not seem to be implicated in the latter response of the plant against dehydration, as indicated by the unaltered expression pattern of some classical ABA and dehydration marker genes in the ocp3 mutant background (Figure 3). In addition, ocp3 plants do not synthesize more Pro and ABA than the wild type, neither under non-stressed conditions nor upon the imposition of a prolonged period of drought (Figure 4). All these results suggest that OCP3 is involved in the regulation of an early specific branch of the ABA-mediated drought-response network.

OCP3 independently regulates drought tolerance and necrotrophic disease resistance through parallel ABA and MeJA signalling pathways

The role of ABA in plant–pathogen interactions is complex, with studies reporting positive or negative interactions depending on the system analysed. Recent studies (revised in Mauch-Mani and Mauch, 2005; Robert-Seilaniantz et al., 2007; Asselbergh et al., 2008) have improved our knowledge in this field, but whether or not the responses to biotic and abiotic stresses are linked, and how plants manage in coexisting stress situations remain almost unexplored questions.

In this work, we have used the ocp3 mutant as a tool to investigate this subject. ocp3 mutant plants show both enhanced ABA-mediated drought tolerance (this study) and MeJA-mediated necrotrophic fungal disease resistance (Coego et al., 2005). Using a double-mutant approach, we asked the question of whether ocp3-associated disease resistance requires ABA signalling, and whether ocp3-associated drought tolerance requires MeJA signalling. Confirming previous findings (Coego et al., 2005), the coi1-1 mutation was able to suppress the disease resistance of ocp3 to B. cinerea, but intriguingly, the drought tolerance remained intact in ocp3 coi1-1 double mutants. On the other hand, the abi1-1 mutation abolished the ocp3 characteristic drought resistance without affecting the disease resistance response to necrotrophs (Figure 7).

In summary, our observations indicate that plants respond to infection by necrotrophic pathogens and drought stress through parallel signalling pathways, where OCP3 seems to play a regulatory role. The ABA and MeJA pathways appear to work independently, but are both regulated by OCP3 (Figure 9). This mode of genetic regulation could give flexibility to the fine tuning of plant responses towards different kinds of environmental stress. In addition, our study helps address why certain environmental cues can predispose a plant to respond to other apparently unrelated insults. Our data demonstrate that it is possible to increase the resistance of a plant towards two of the most important environmental pressures, namely drought and disease caused by necrotrophic fungal pathogens. These two types of environmental stresses have significant economic implications, resulting in decreased yields of crop plants. The potential to assure crop productivity by the sole manipulation of a single gene that functions as an integrator of two environmental signals has far-reaching economic potential. Further studies on a genome-wide scale will allow a deeper understanding of the regulatory networks controlled by OCP3.

Figure 9.

 A genetic model for the common regulation of drought and pathogen stress responses through OCP3.
Abscisic acid (ABA) and methyl jasmonate (MeJA) signalling pathways modulate drought and pathogen responses. Specific branches of these pathways are commonly controlled by OCP3 to negatively regulate drought tolerance and disease resistance to necrotrophic fungal infections.

Experimental procedures

Plant materials and growth

The PCR-based detection of ocp3 and coi1-1 was performed as described (Coego et al., 2005), whereas the primers used for the abi1-1 mutant allele were (5′-GATATCTCCGCCGGAGAT-3′ and 5′-CCATTCCACTGAATCACTTT-3′). Genomic DNA was extracted from young leaves of Arabidopsis plants as described by Edwards et al. (1991). The PCR product was digested with NcoI (New England Biolabs; http://www.neb.com), resulting in 572- and 258-bp fragments in the wild type, and an 830-bp fragment in abi1-1. Homozygous abi5-1 mutant plants were selected by their ABA insensitivity. For the abi1-1R1 mutant allele, the primers used were (5′-GCTAAGGAGAAACCGATGCTC-3′ and 5′-TCACTCGCCAAAATCAGACA-3′). The PCR product from genomic DNA was digested with SacI (New England Biolabs), resulting in 272- and 390-bp fragments in the wild type, and a 662-bp fragment in abi1-1R1.

For homozygous aba2-1 mutant plants, the primers used were (5′-GGATACGTGTGAACTGTGTTTCG-3′ and 5′-GATAGACATGATAAATTGGCGG-3′). The 334-bp PCR product from genomic DNA was digested with AciI (New England Biolabs), resulting in 174-, 105-, 44- and 11-bp fragments in the wild type, and 174-, 116- and 44-bp fragments in aba2-1. In the double-mutant analysis, the same phenotype was observed for, at least, two independent double mutant lines generated.

Seeds of mutants and wild-type Arabidopsis thaliana were kept at 4°C for 3 days, and were sown in jiffy7 peat pellets (Clause Tezier Ibérica; http://www.clausetezier-iberica.com) or on a turf substrate mix. Plants were grown in a growth phytochamber with a light intensity of approximately 150–200 μE m−2 sec−1 at 23°C, under 10-h light/14-h dark cycles and 60% humidity.

ABA, MeJA and dehydration treatments

For ABA and MeJA response assays, surface-sterilized seeds were sown in round Petri dishes containing 2.2 g l−1 MS (Duchefa Biochemie; http://www.duchefa.com), 5 g l−1 sucrose and 6 g l−1 Agargel (Sigma-Aldrich; http://www.sigmaaldrich.com), and were transferred to a phytochamber and grown as described previously for 10 days. Plants were transferred to liquid MS medium supplemented either with sucrose (5 g l−1), and, where indicated, with 100 μm ABA (Sigma-Aldrich), or 100 μm MeJA (Sigma-Aldrich).

For dehydration (DRY) treatments, plants were grown for 10 days on MS-Agar plates, as described for the ABA-response assays. After this time, plants were removed from MS-Agar plates and kept in empty Petri dishes to enable dehydration.

Drought tolerance assays

For drought tolerance assays, plants were grown in different conditions, including diverse developmental stages, photoperiods, substrates and pots. In all cases, comparative differences were found between genotypes. The length of imposed water stress was normally 12 days (unless indicated otherwise). A total of 20 plants per genotype were analysed. Photographs of representative plants were taken at the end of this time period.

Progressive water loss measurements

Water loss occurred primarily through plant transpiration, as evaporation was prevented by covering the soil surface with Parafilm, as described in (Vartanian et al., 1994). The percentage of plant water deficit was calculated as (Wf/Wi) × 100, where Wf is the weight of the pot at each time point, and Wi is the initial weight (day 0).

Leaf water loss assays

Detached leaves from 5-week-old plants were exposed to room temperature (22°C). Leaves were weighed at various time intervals, and the loss of fresh weight (%) was used to indicate water loss.

Proline determination

Free Pro was measured as described by Bates et al. (1973). A 100-mg portion of frozen plant material was homogenized in 1 ml of 3% sulphosalicylic acid, and the residue was removed by centrifugation. A 500-μl volume of the extract was reacted with 500 μl of acid ninhydrin [2.5 g ninhydrin warmed in 100 ml of glacial acetic acid, 6 m phosphoric acid and water (6:3:1 v/v/v) until dissolved] for 30 min at 90°C, and the reaction was then terminated in an ice bath. The reaction mixture was extracted with 1 ml of toluene. An 800-μl aliquot of the chromophore-containing toluene was warmed to room temperature, and its optical density was measured at 520 nm. The quantity of Pro was determined from a standard curve in the range of 20–100 μg.

ABA determination

To determine changes in ABA subjected to drought stress, full plants were harvested and frozen immediately in liquid nitrogen, and then ground to a fine powder. Before extraction, a mixture of internal standards containing 100 ng [2H6]ABA was added. Liofilized tissue (70 mg) was homogenized in 2.5 ml of ultrapure water; after centrifugation (5000 g for 40 min), the supernatant was recovered, acidified and partitioned twice against diethylether, as described in Flors et al. (2008). After evaporation, the solid residue was resuspended in 1 ml of a water/methanol (90:10) solution, and then filtered through a 0.22-lm cellulose acetate filter. A 20-μl aliquot of this solution was then directly injected into an ultra-performance Waters Acquity liquid chromatography (UPLC) system (Waters; http://www.waters.com). The UPLC was interfaced to a triple quadrupole tandem mass spectrometer (TQD; Waters), using an orthogonal Z-spray electrospray interface. LC separation was performed using an Acquity UPLC BEH C18 analytical column (2.1 × 50 mm by 1.7 μm; Waters) at a flow rate of 300 μl min−1. Standard curves were obtained by injecting pure compounds at different concentrations (10, 25, 50, 70, 100 and 150 ng). Quantifications where carried out with Mass Lynx v1.4 (Waters), using the internal standard as the reference for extraction recovery, and the standard curve as the quantifier.

Stomatal aperture measurements

Stomatal aperture was measured according to a previously published procedure, with minor modifications (Zhang et al., 2004). Epidermal peels were stripped from fully expanded leaves of 5-week-old plants, and were floated in a solution of 30 mm KCl and 10 mm 2-(N-morpholine)-ethanesulphonic acid (MES-KOH; pH 6.15) in Petri dishes. After incubation for 2.5 h under cool white light (150–200 μE m−2 sec−1) at 22°C, to induce stomatal opening, appropriate concentrations of ABA were added. For the inhibition of stomatal opening, the epidermal peels were pre-treated in the above solution in the dark for 2.5 h before adding ABA. Stomatal apertures were recorded under a Nikon Eclipse E600 microscope with a Nikon DXm1200F digital camera, and were analysed using Act-1 software (Nikon; http://www.nikon.com). Measurements were performed as described by Ichida et al. (1997) using the free software ImageJ 1.36b (Broken Symmetry Software; http://brokensymmetry.com).

RT-PCR and qRT-PCR

RNA was isolated with Trizol (Invitrogen; http://www.invitrogen.com). For RT-PCR, RevertAid M-MuLV Reverse Transcriptase (Fermentas; http://www.fermentas.com) was used, following the manufacturer’s instructions. The resulting single-stranded cDNA was then used as the template in semi-quantitative PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR). RT-PCRs and qRT-PCRs were carried out with gene-specific primers, designed using Primer Express 2.0 (Applied Biosystems; http://www.appliedbiosystems.com): OCP3 (At5g11270), 5′-AAGCTGGGCGTCGTAAAACTAGTA-3′ and 5′-TGGCGGTTTTTCATCTGGTAGTGT-3′; ABI1 (At4g26080), 5′-GTCGAGATCCATTGGCGATAGA-3′ and 5′-TGCCATCTCACACGCTTCTTC-3′; APT (At1G27450), 5′-CCTTTCCCTTAAGCTCTG-3′ and 5′-TCCCAGAATCGCTAAGATTGCC-3′; RD29A (At5g52310), 5′-TGCACCAGGCGTAACAGGTAA-3′ and 5′-TTGTCCGATGTAAACGTCGTCC-3′; RD29B (At5g52300), 5′-GCGCACCAGTGTATGAATCCTC-3′ and 5′-TGTGGTCAGAAGACACGACAGG-3′; RD22 (At5g25610), 5′-AGGTGGCTAAGAAGAACGCACC-3′ and 5′-TGGCAGTAGAACACCGCGAAT-3′; ELF1α (At5g60390), 5′-GCACAGTCATTGATGCCCCA-3′ and 5′-CCTCAAGAAGAGTTGGTCCCT-3′); RAB18 (At5g66400), 5′-ATGGCGTCTTACCAGAACCGT-3′ and 5′-CCAGATCCGGAGCGGTGAAGC-3′; KIN1 (At5g15960), 5′-GCTGGCAAAGCTGAGGAGAA-3′ and 5′-TTCCCGCCTGTTGTGCTC-3′; NCED3 (At3g14440), 5′-CAACGGAGCTAACCCACTTCA-3′ and 5′-ACCCTATCACGACGACTTCATCT-3′; P5CS1 (At2g39800), 5′-TTTATGGTGCTATAGATCACA-3′ and 5′-GAATGTCCTGATGGGTGTAAAC-3′; ProDH (At3g30775), 5′-GTTGGTGAGAGGGGCTTACA-3′ and 5′-AACGACACCGAAACCAGAAC-3′; ACT8 (At1g49240), 5′-GAGGATAGCATGTGGAAGTGAGAA-3′ and 5′-AGTGGTCGTACAACCGGTATTGT-3′. qRT-PCRs were performed using the SybrGreen PCR Master Mix (Applied Biosystems) in a ABI PRISM 7000 sequence detector. Cycle thresholds (Cts) were obtained using the 7000 system sds software core application v1.2.3 (Applied Biosystems), and the data were transformed with the formula 2(40–Ct). Both RT-PCR and qRT-PCR analyses were performed at least three times using sets of cDNA samples from independent experiments.

Botrytis cinerea bioassays

In B. cinerea infections, 5-week-old plants were inoculated, as described by Coego et al. (2005), with a suspension of fungal spores of 2.5 × 104 spores per mL. The challenged plants were maintained at 100% relative humidity. Disease symptoms were evaluated by determining the lesion diameter of at least 40 lesions, 3 days after inoculation.

Acknowledgements

We thank B. Wulff, R. Solano and J. Dangl for comments on the manuscript, and for helpful discussions. We also thank J. Salinas for providing seeds of the abi1-1, abi5-1 and aba2-1 mutants, and J. Giraudat for providing seeds of the abi1-1R1 mutant. The authors would like to thank the editor and anonymous reviewers for their valuable suggestions. We acknowledge the support of the Spanish Ministry of Education and Science (Grant BFU2006-00803 to PV) for financial support.

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