This Article Abstract Full Text (PDF) All Versions of this Article: 142/4/1559    most recent pp.106.086223v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (12) Citing Articles via Google Scholar Google Scholar Articles by Kariola, T. Articles by Palva, E. T. Search for Related Content PubMed PubMed Citation Articles by Kariola, T. Articles by Palva, E. T. Agricola Articles by Kariola, T. Articles by Palva, E. T.

ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

EARLY RESPONSIVE TO DEHYDRATION 15, a Negative Regulator of Abscisic Acid Responses in Arabidopsis¹

Viikki Biocenter, Department of Biological and Environmental Sciences, Division of Genetics, University of Helsinki, FIN–00014, Helsinki, Finland

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) is rapidly induced in response to various abiotic and biotic stress stimuli in Arabidopsis (Arabidopsis thaliana). Modulation of ERD15 levels by overexpression or RNAi silencing altered the responsiveness of the transgenic plants to the phytohormone abscisic acid (ABA). Overexpression of ERD15 reduced the ABA sensitivity of Arabidopsis manifested in decreased drought tolerance and in impaired ability of the plants to increase their freezing tolerance in response to this hormone. In contrast, RNAi silencing of ERD15 resulted in plants that were hypersensitive to ABA and showed improved tolerance to both drought and freezing, as well as impaired seed germination in the presence of ABA. The modulation of ERD15 levels not only affected abiotic stress tolerance but also disease resistance: ERD15 overexpression plants showed improved resistance to the bacterial necrotroph Erwinia carotovora subsp. carotovora accompanied with enhanced induction of marker genes for systemic acquired resistance. We propose that ERD15 is a novel mediator of stress-related ABA signaling in Arabidopsis.

Although several components of ABA signaling have been identified, there is still lack of knowledge of how ABA is perceived and the signal transduced partly due to the complexity and redundancy of such signal networks. Mutants affecting ABA responsiveness have defined components of the ABA-signaling pathway, and one class of important players seems to be the type 2C protein phosphatases ABI1 and ABI2 (Leung et al., 1997; Gosti et al., 1999) as well as PP2CA (Tähtiharju and Palva 2001; Yoshida et al., 2005) that appear to act as negative regulators of ABA responses (Merlot et al., 2001; Tähtiharju and Palva, 2001). The dominant-negative alleles of ABI1 and ABI2, abi1-1 and abi2-1, confer ABA insensitivity during vegetative growth as well as in seed germination and cause defects in plant responses to drought stress (Leung et al., 1997). Other known regulators of ABA responsiveness include PKS3, a Ser/Thr protein kinase, a global negative regulator of ABA responses that has been shown to interact with ABI2 (Guo et al., 2002). The farnesyl transferase ERA1 (Cutler et al., 1996) and inositol phosphatase FRY1 (Xiong et al., 2001b) are also negative regulators of ABA responses.

ABA responsiveness of many of the abiotic stress-inducible genes is conferred by the conserved cis-regulatory ABRE sequence (ABA-responsive element), the binding site for the basic-domain Leu zipper-class transcription factors, AREBs (ABRE-binding proteins), or ABFs (ABRE-binding factors; for review, see Yamaguchi-Shinozaki and Shinozaki, 2005). ABA-dependent phosphorylation is required to activate these transcription factors and consequently the expression of ABRE-containing genes (Yamaguchi-Shinozaki and Shinozaki, 2005, 2006).

Recent studies have suggested that part of the regulation of ABA responses takes place posttranscriptionally (Kuhn and Schroeder, 2003). The Arabidopsis (Arabidopsis thaliana) mutants supersensitive to ABA and drought1 (sad1) and hyponastic leaves1 (hyl1) plants show altered response to ABA: Both are hypersensitive to this phytohormone in inhibition of seed germination and show reduced stomatal closure in response to stress. SAD1 is homologous to eukaryotic RNA-binding proteins, while HYL1 encodes a nuclear-localized protein that specifically binds double-stranded RNA (Lu and Fedoroff, 2000; Xiong et al., 2001a). Also, a recent study shows that the mRNA-destabilizing activity of a poly(A)-specific endonuclease, poly(A)-specific ribonuclease (AtPARN), is crucial for proper ABA, salicylic acid (SA), and abiotic stress responses (Nishimura et al., 2005).

Besides its central role in controlling responses to abiotic stress stimuli, recent studies suggest that ABA also influences biotic stress responses and may interfere with signaling that is regulated by the more "traditional" hormones of pathogen defense: SA, jasmonic acid (JA), and ethylene (ET; for review, see Mauch-Mani and Mauch, 2005). Exogenous ABA has been shown to suppress basal as well as JA- and ET-activated transcription of defense genes, whereas ABA-deficient mutants showed a corresponding increase (Anderson et al., 2004). ABA treatment prior to infection increased the susceptibility of Arabidopsis to avirulent Pseudomonas syringae pv tomato, suggesting that ABA interferes with SA-dependent defense responses (Mohr and Cahill, 2003). In both studies, ABA-deficient mutant plants were less susceptible to the pathogen, indicating that decreased ABA levels appear to improve either JA- or SA-dependent defenses (Mohr and Cahill, 2003; Anderson et al., 2004). On the other hand, the -amino-butyric acid-primed accumulation of callose and following resistance to the necrotrophic pathogens Alternaria brassicicola and Plectosphaerella cucumerina has been shown to be dependent on ABA (Ton and Mauch-Mani, 2004; Mauch-Mani and Mauch, 2005).

Here, we report that EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), a small, acidic protein with no known function, is one of the key negative regulators of ABA responses in plants. ERD15 was originally described as a rapidly drought-responsive gene in Arabidopsis (Kiyosue et al., 1994). In this study, we show that alteration of ERD15 expression modulates ABA responsiveness in Arabidopsis. We present evidence showing that the ABA sensitivity of ERD15 overexpression plants is reduced, while RNAi silencing of ERD15 results in hypersensitivity to ABA observed both in seed germination and as enhanced drought and freezing tolerance. We also show that ERD15 is induced by pathogen attack and that overexpression of this gene enhances SA-dependent pathogen defense and plant resistance to Erwinia carotovora. Our results indicate that ERD15 mediates cross talk between abiotic and biotic stress responses.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

The Arabidopsis ERD15 Gene Is Rapidly Induced by Both Biotic and Abiotic Factors

To identify early signaling components of plant defense, we isolated Arabidopsis genes rapidly induced in response to E. carotovora elicitors using suppressive subtractive hybridization (Brader et al., 2001). One of these genes was ERD15, previously characterized as EARLY RESPONSIVE TO DEHYDRATION 15 (Kiyosue et al., 1994). Subsequent characterization of ERD15 expression pattern showed that, in addition to pathogen elicitors and dehydration, the gene was also rapidly induced after E. carotovora infection, as well as by SA, ABA, and wounding (Fig. 1A ). Interestingly, ERD15 was not responsive to methyl jasmonate (MeJA; Fig. 1A), even though E. carotovora is a pathogen that can trigger both SA- and JA-dependent defense signaling in Arabidopsis (Kariola et al., 2003; Li et al., 2004; Kariola et al., 2005). This broad responsiveness of ERD15 to different types of environmental cues could suggest that it is a component of both biotic and abiotic stress responses in Arabidopsis.

View larger version (56K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Induction of the ERD15 gene and characterization of transgenic lines. A, Wild-type Arabidopsis was treated with ABA, SA, MeJA, E. carotovora (Ecc), wounding (W), and with deionized water as control treatment (C). Local samples were collected 0, 0.5, 1, and 1.5 h after the treatment. The samples were analyzed in RNA gel-blot hybridization with gene-specific RNA probe for ERD15. Equal loading of RNA samples was checked using a probe for the constitutively expressed -tubulin (TubA) gene. B, The transgenic Arabidopsis lines generated carrying ERD15 RNAi and overexpression (oex) constructs. Samples for evaluating the efficacy of RNAi silencing of ERD15 as well as the vector control sample (C) were collected 1.5 h after treating the plants with pathogen elicitor preparation. Evaluation of the efficiency of overexpression was done from nontreated samples. All samples were analyzed by RNA gel-blot hybridization with gene-specific RNA probe for ERD15. As a control for equal loading, ethidium bromide (EtBr) staining of RNA is shown at the bottom. C, Phenotypes of Arabidopsis Col-0 vector control, ERD15 oex (lines 21 and 52), and ERD15 RNAi-silenced (lines 8 and 10) plants. Level of ERD15 protein after 4 h of drought stress is shown for each line. D, Induction of ERD15 in wild-type, ERD15 oex, and ERD15 RNAi-silenced plants after biotic (E. carotovora = Ecc) and abiotic (drought) stress. Local leaf samples were collected from ERD15 oex, ERD15 RNAi-silenced, and vector control plants 0, 3, 8, 24, and 48 h after inoculating the plants with Ecc and 0, 0.5, 1, 2, 3, and 6 h after exposing the plants to drought stress. All samples were analyzed by RNA gel-blot hybridization with gene-specific RNA probe for ERD15. As a control for equal loading, EtBr staining of RNA is shown at the bottom.

To explore the possible role of ERD15 in plant defense and stress tolerance, we generated Arabidopsis Columbia (Col-0) lines harboring overexpression or RNAi constructs of ERD15. The effect of the transgenes on ERD15 transcript accumulation was assessed by gel-blot hybridization using a gene-specific RNA probe for this gene. Two overexpression lines with increased and two RNAi lines with clearly decreased expression of ERD15 were employed for further studies (Fig. 1B). ERD15 overexpression but not silencing resulted in some morphological differences from the wild type with more narrow leaves (Fig. 1C). The phenotype of the transgenic lines was further confirmed by determining ERD15 protein levels in the plants after drought exposure. The difference in protein amounts was evident: ERD15 overexpressor lines accumulated more ERD15 protein when compared with the control, whereas in RNAi-silenced lines hardly any protein could be detected (Fig. 1C). The ERD15 expression in the transgenic lines was also characterized following exposure to either biotic (E. carotovora) or abiotic (drought) stress. The rapid but transient induction of ERD15 in response to both types of stimuli was clearly evident in the vector control, whereas the overexpression plants showed a constitutive high level accumulation of the ERD15 transcript. In contrast, in the RNAi-silenced plants, ERD15 expression was almost completely abolished even when induced by either biotic or abiotic stress (Fig. 1D).

Overexpression of ERD15 Sensitizes Plants to Drought

Drought stress rapidly induces ERD15 as shown above (Fig. 1D; Kiyosue et al., 1994). This suggested that the corresponding protein could be involved in abiotic stress adaptation and prompted us to test whether the drought tolerance of the ERD15 transgenic plants was altered. To assess the drought tolerance phenotype of the transgenic plants, we transferred ERD15 overexpression, ERD15 RNAi, and control plants to lower humidity conditions and left them without watering. After 2 weeks of drought stress, the phenotypic difference between the plants was striking and surprising: The majority (72%) of ERD15 overexpression plants were dead, whereas a significant fraction of vector control plants were still alive (Fig. 2, A and B ). Moreover, only 14% of the plants with RNAi-silenced ERD15 were dead, and the survivors appeared healthier than the controls (Fig. 2, A and B). The altered drought tolerance seen after modulation of ERD15 levels strongly indicates that this gene has a role in abiotic stress adaptation in Arabidopsis.

View larger version (53K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Modulation of ERD15 expression affects drought and freezing tolerance of the transgenic plants. A, Drought tolerance of ERD15 RNAi, ERD15 overexpression (oex), wild-type, and vector control plants was tested by keeping them under 50% humidity for 2 weeks without watering. B, Percentage of dead plants after 2 weeks is shown. The values represent the average of three replicates ±SD. Different letters indicate significant differences (P < 0.05) calculated with one-way ANOVA followed by Tukey's HSD test. C and D, The freezing tolerance of vector control, ERD15 oex, and ERD15 RNAi plants was tested in a freezing survival experiment. The plants were photographed before freezing survival (C) and 7 d after the temperature was returned to normal (D). Percentage of dead plants after 7 d is shown. The values represent the average of three replicates ±SD. Different letters indicate significant differences (P < 0.05) calculated with one-way ANOVA followed by Tukey's HSD test.

The altered drought tolerance of the transgenic ERD15 plants and the inducibility of the gene with different abiotic stress stimuli (Fig. 1A) suggested that the transgenic plants might show altered tolerance to related abiotic stresses, such as freezing. To test this possibility, the plants were exposed to freezing temperatures and the survival was assessed. The difference in tolerance between the transgenic lines was evident immediately after the temperature was returned back to 22°C. Most of the ERD15 overexpressors as well as vector control plants appeared to have lost their turgor, whereas ERD15 RNAi plants looked healthy and turgid (data not shown). When the survival was assessed 7 d after exposure to the freeze-thaw cycle, the difference between the lines was clear: Most of the ERD15 RNAi plants had survived without any or with only minor damage, and only a small fraction (11%) of the plants were killed (Fig. 2, C and D). In contrast, the majority of the ERD15 overexpression plants had suffered severe frost damage, and most (84%) of these plants were killed. They appeared even more freezing sensitive than the vector control plants, of which 53% were dead (Fig. 2, C and D).

Freezing tolerance of many temperate plant species, including Arabidopsis, is increased by exposure to low, nonfreezing temperatures, a phenomenon called cold acclimation (Guy, 1990). We characterized whether the modulation of ERD15 levels, besides altering the basal freezing tolerance, also had an impact on the capability of the plants to cold acclimate. Interestingly, all the plants, including ERD15 overexpression plants that showed decreased frost survival without cold acclimation, were capable of (+4°C; 2 d) low-temperature acclimation (data not shown). Taken together, these data argue that, while the modulation of ERD15 expression does not interfere with the ability of the plants to cold acclimate, in nonacclimated plants high-level expression of ERD15 is detrimental to the basal freezing tolerance of Arabidopsis.

Overexpression of ERD15 Impairs Development of Freezing Tolerance

The marked effect on drought and freezing tolerance caused by altered ERD15 expression suggested that ABA, a central hormone in drought signaling, might be involved. In addition to low temperature, ABA can also induce the development of freezing tolerance in various higher plants, including potato (Solanum tuberosum; Chen and Gusta, 1983) and Arabidopsis (Lång et al., 1989; Mäntylä et al., 1995). To elucidate the effect of ERD15 on ABA-induced freezing tolerance, we compared the tolerance of axenically grown ERD15 overexpression, ERD15 RNAi, and control plants induced by 60 µM exogenous ABA. The freezing tolerance of the plants was determined 1 and 3 d after ABA treatment by measuring electrolyte leakage (EL₅₀) after exposure to freezing temperatures (Fig. 3A ). Nonacclimated ERD15 overexpression plants appeared more susceptible to freezing than control plants. Although ERD15 transgenic lines were still responsive to exogenous ABA, the freezing tolerance achieved in ERD15-overexpressing plants was significantly lower than in control or ERD15-silenced lines (Fig. 3A).

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Modulation of ERD15 expression alters development of freezing tolerance and seed germination in the presence of ABA. A, Freezing tolerance (EL50) was measured by an electrolyte leakage assay from axenically grown wild-type Col-0, vector control, ERD15 RNAi-silenced, and ERD15 overexpression (oex) plants before (NA = nonacclimated) and 1 and 3 d after treatment with 60 µM ABA. The values represent the average of three replicates ±SD calculated by Probit analysis. At –10°C, ERD15 oex plants had a significantly higher ion leakage than wild-type, vector control, and RNAi plants calculated with one-way ANOVA followed by Tukey's HSD test (P < 0.05). B, Seeds of ERD15 oex, ERD15 RNAi, and vector control were germinated on MS + 2 µM ABA plates. Germination is shown after five (top row) and 10 (bottom row) d. For comparison, the germination of abi1-1 is shown after 10 d. C, Percentage (±SE) of green cotyledons 10 d after germination with 2 µM ABA is shown for two ERD15 oex and two ERD15 RNAi-silenced lines and wild-type and vector control plants. In all cases, similar results were obtained from three independent experiments.

To explore the spectrum of ABA-controlled processes that were affected by modulating ERD15 expression, we elucidated whether the response was specific to stress tolerance in the vegetative parts of the plant or whether it applied also to ABA-regulated processes at other stages of development. Inhibition of seed germination is one of the processes controlled by ABA, and a number of Arabidopsis mutants affecting seed germination due to altered sensitivity to this hormone, such as ABA-insensitive abi1-1 and abi2-1 as well as ABA-hypersensitive abh1 (abscisic acid hypersensitive1), have been characterized.

Germination of the seeds of ERD15 transgenic and control plants was similar in the absence of ABA (data not shown). However, when ABA was added, seeds from ERD15 RNAi-silenced plants germinated poorly and only 10% were able to produce green cotyledons (Fig. 3, B and C). In contrast, seeds of ERD15 overexpression plants exhibited clearly improved seed germination during the first week when compared to control plants, and most of them (approximately 60%) produced green cotyledons 10 d after germination (Fig. 3, B and C). This could be an indication of altered ABA sensitivity; RNAi silencing of ERD15 sensitizes the seeds to exogenous ABA, whereas the overexpression of this gene seems to reduce sensitivity to exogenous ABA in germination.

ERD15 Modulates ABA-Induced Gene Expression

ABA regulates the expression of numerous plant genes involved in plant responses to abiotic environmental stresses, especially those involved in drought response (Yamaguchi-Shinozaki and Shinozaki, 2005). To correlate the ABA-related abiotic stress phenotypes of the transgenic ERD15 plants with corresponding gene expression, we exposed ERD15 overexpression, ERD15 RNAi-silenced, and vector control plants to drought stress, and followed the accumulation of transcripts of two ABA-responsive genes, RAB18 (Lång and Palva, 1992) and LTI78 (Nordin et al., 1991, 1993). In ERD15 overexpression plants, the drought-induced expression of RAB18 was reduced when compared with vector control and ERD15 RNAi-silenced plants. Similar reduction of LTI78 expression was observed in ERD15 overexpression plants (Fig. 4A ). To further explore if the altered inducibility of these genes was due to impaired ABA sensing, the transgenic plants were exposed to ABA and we checked ABA-induced transcript accumulation of RAB18 and LTI78. Similar to drought treatment, expression of these marker genes was reduced in plants overexpressing ERD15. These expression data support the notion that ERD15 interferes with ABA signaling in Arabidopsis and indicate that modulation of ERD15 levels has an impact on ABA responsiveness of the plants (Fig. 4B).

View larger version (45K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Overexpression of ERD15 decreases expression of drought-inducible genes but increases stress-induced ABA accumulation. A and B, Local leaf samples were collected from ERD15 overexpression (oex), ERD15 RNAi-silenced, and vector control plants 0, 0.5, 1, 2, 3, and 6 h after exposing the plants to drought stress (A) and treating the plants with 100 µM ABA (B). Total RNA was extracted and analyzed by RNA gel-blot hybridization with gene-specific probe for RAB18 and LTI78. Equal loading of RNA samples was checked using a probe for the constitutively expressed -tubulin (TubA) gene. C. Accumulation of ABA was determined in ERD15 oex, ERD15 RNAi-silenced, wild-type (Col-0), and vector control plants 0, 0.5, 1, 2, and 3 h after exposing the plants to drought stress. FW, Fresh weight. In both cases, similar results were obtained from two independent experiments.

Drought Induction of ERD15 Is Abolished in abi1-1 and abi2-1 Plants

Our results suggest that ERD15 is involved in ABA signaling and could be a negative regulator of several ABA-controlled processes. Interestingly, ERD15 itself is induced by ABA as well as by drought (Fig. 1, A and D; Kiyosue et al., 1994). To explore the interaction of ERD15 with other regulators of ABA responses, we determined the expression of ERD15 in ABA-insensitive mutants. To this aim, we drought stressed wild-type Landsberg erecta (LE) and ABA-insensitive abi1-1 and abi2-1 mutant plants and characterized ERD15 transcript accumulation. In addition, we employed a double loss-of-function mutant of ABI1 and ABI2, abi1-1R5 abi2-1R1, which has hardly any detectable activity of these two phosphatases (Merlot et al., 2001; Fig. 5 ). In both wild-type plants and in the loss-of-function double mutant abi1-1R5 abi2-1R1, ERD15 was rapidly induced in response to drought. In contrast, in abi1-1 and abi2-1 mutant plants, this gene was already up-regulated in the untreated controls, and no induction but rather a decrease in ERD15 expression was evident after drought exposure (Fig. 5). The observed ERD15 expression in the ABA-insensitive abi1-1 and abi2-1 mutants further strengthen the notion that this gene is involved in ABA signaling in Arabidopsis.

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. The basal expression level of ERD15 is increased in abi1-1 and abi2-1 mutants. Wild-type (LE), abi1-1, abi2-1, and abi1-1R5 abi2-1R1 plants were exposed to drought stress, and local leaf samples were collected 0, 0.25, 0.5, 1, 2, and 3 h after this. Total RNA was extracted and analyzed by RNA gel-blot hybridization with gene-specific probe for ERD15. As a control for equal loading, the samples were probed with the constitutively expressed -tubulin (TubA) gene. In both cases, similar results were obtained from three independent experiments.

The inducibility of ERD15 by a pathogen and pathogen-derived elicitors (Fig. 1), as well as the recent studies suggesting involvement of ABA in the pathogen response of plants (Mohr and Cahill, 2003; Anderson et al., 2004; Mauch-Mani and Mauch, 2005), prompted us to investigate the role of ERD15 in resistance to pathogens. To assess the possible contribution of ERD15 to plant defense, the transgenic lines as well as control plants were locally inoculated with E. carotovora, and symptom development as well as bacterial growth were followed. ERD15 overexpression plants displayed enhanced resistance to this pathogen: The majority of the inoculated leaves showed no or minor symptom development after 24 h (Fig. 6A ), and a clear reduction was seen in the pathogen growth (Fig. 6B). In contrast, in both vector control and ERD15 RNAi plants, the disease symptoms spread rapidly and the inoculated leaves were almost completely macerated after 24 h (Fig. 6A). Also, the bacterial growth was clearly improved when compared to ERD15 overexpression plants (Fig. 6B). These results demonstrate that overexpression of ERD15 promotes plant resistance against E. carotovora.

View larger version (50K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. ERD15 overexpression (oex) plants are resistant to E. carotovora infection. A, Two to three leaves of Arabidopsis ERD15 oex, RNAi-silenced, and vector control lines were inoculated by infiltration with E. carotovora. Infiltrated leaves are indicated with arrows. ERD15 oex, RNAi-silenced, and vector control plants 24 h after bacterial inoculation are shown. Percentage of infected leaves 24 h after inoculation is shown. Different letters indicate significant differences (P < 0.05) calculated with one-way ANOVA followed by Tukey's HSD test. B, Growth of E. carotovora in planta 0 and 24 h after the inoculation. Colony forming units of four to six plants were determined from the time points in three independent experiments. The values represent the average of four replicates ±SE.

To explore the cause for the dramatic improvement in plant resistance to E. carotovora in ERD15 overexpression lines, we elucidated the role of different defense pathways in this resistance. Enhanced resistance to E. carotovora in Arabidopsis can be generated either by induction of JA/ET-mediated (Vidal et al., 1998; Norman-Setterblad et al., 2000; Kariola et al., 2003) or SA-mediated defenses (Palva et al., 1994; Kariola et al., 2003; Li et al., 2004).

To distinguish between these possibilities, we explored the effect of ERD15 levels on expression of defense pathway-specific marker genes following induction of defense responses. To avoid possible problems due to differences in the progress of infection, we used SA and MeJA in addition to pathogen inoculation of the plants. First, we monitored expression of PDF1.2, a JA/ET-responsive gene (Penninckx et al., 1996), and found that the induction was both delayed and decreased in ERD15 overexpression plants in response to both MeJA and E. carotovora when compared with ERD15 RNAi and vector control plants (Fig. 7A ).

View larger version (74K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. The expression of SAR marker genes is enhanced in ERD15 overexpression (oex) plants. Local leaf samples were collected from ERD15 oex, ERD15 RNAi-silenced, and vector control plants 0, 3, 8, 24, and 48 h after inoculation with E. carotovora and treatments with SA and MeJA. Total RNA was extracted and analyzed by RNA gel-blot hybridization with gene-specific probe for PDF1.2 after treatment with MeJA and after inoculation with E. carotovora (Ecc; A) and with PR2 after treatment with SA and inoculation with Ecc (B). Equal loading of RNA samples was checked using a probe for the constitutively expressed -tubulin (TubA) gene. In both cases, similar results were obtained from three independent experiments.

Insensitivity to ABA Enhances Resistance to E. carotovora in Arabidopsis

The altered sensitivity to ABA and pronounced differences in resistance to E. carotovora in ERD15 overexpression and RNAi-silenced plants prompted us to elucidate the contribution of ABA to the resistance of Arabidopsis against this pathogen. To assess this, wild-type LE plants, along with the ABA-insensitive mutants abi1-1 and abi2-1, were inoculated with E. carotovora and symptom development was followed. Already 24 h after inoculation with the pathogen, the difference in resistance between the plant lines was obvious (Fig. 8A ). In LE plants the maceration had proceeded considerably, whereas most abi1-1 and abi2-1 plants showed clearly reduced symptom development (Fig. 8A). The decreased maceration in abi1-1 and abi2-1 plants was accompanied with a distinct reduction in the pathogen growth (Fig. 8B).

View larger version (71K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Insensitivity to ABA enhances SA-dependent defense gene expression and improves resistance to E. carotovora. A, Wild-type (LE), abi1-1, and abi2-1 mutants were inoculated by infiltration with E. carotovora and shown 24 h after infiltration. Infiltrated leaves are indicated with arrows. B, Growth of E. carotovora in planta 0 and 24 h after the inoculation. Colony forming units of four to six plants were determined from the time points in two independent experiments. The values represent the average of four replicates ±SE. C, Local leaf samples were collected from wild-type (LE), abi1-1, and abi2-1 mutants 0, 4 (LE and abi1-1), 8, 24, and 48 h after treatment with SA. Total RNA was extracted and analyzed by RNA gel-blot hybridization with gene-specific probe for PR1. Equal loading of RNA samples was checked using a probe for the constitutively expressed -tubulin (TubA) gene. In both cases, similar results were obtained from two independent experiments.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
ERD15 is rapidly but transiently induced in response to various stress factors and stress-related hormones, such as dehydration (Kiyosue et al., 1994), ABA, wounding, SA, the plant pathogen E. carotovora (Fig. 1), salt, and low temperature (data not shown) in Arabidopsis. Rapid response to such a wide variety of abiotic and biotic factors suggests a significant role for this gene in mediating plant stress responses. However, the actual function of ERD15 has been an enigma since it was first characterized (Kiyosue et al., 1994). In this study, we provide evidence that ERD15 controls ABA-mediated stress responses in Arabidopsis and propose that ERD15 is a novel, negative regulator of ABA signal transduction related to these processes.

Our results demonstrate that overexpression of ERD15 decreases tolerance of the transgenic plants to stresses that involve ABA signaling: drought and freezing. Accordingly, silencing of ERD15 resulted in improved drought as well as freezing tolerance of the plants. Overexpression of ERD15 was also accompanied by decreased expression of the ABA-responsive genes RAB18 and LTI78 (Fig. 4B). Our results are best explained by altered responsiveness to ABA due to modulation of ERD15 levels. Overexpression of ERD15 results in reduced sensitivity to ABA, while silencing of the gene results in ABA hypersensitivity. The altered responsiveness of ERD15 transgenic plants was also observed in seed germination in the presence of ABA: Silencing of ERD15 resulted in hypersensitivity to this phytohormone, whereas the seeds of overexpression plants demonstrated reduction of sensitivity. Furthermore, overexpression of ERD15 resulted in increased accumulation of ABA, a phenotype observed with other ABA-insensitive mutants (Lång and Palva, 1992; Mäntylä et al., 1995; Verslues and Bray, 2006; Fig. 4C). Interestingly, besides abiotic stress, the modulation of ERD15 expression had an impact on the biotic stress tolerance of Arabidopsis as well: Overexpression of this gene enhanced the induction of SAR response and resistance to the pathogen E. carotovora.

The altered ABA sensitivity of transgenic ERD15 plants can be explained as a result of changed expression of a negative regulator of ABA responses (Fig. 9 ). ABA is the central hormone mediating drought responses and overexpression of ERD15 decreased the drought and freezing tolerance of the plants, a likely consequence of enhanced activity of a negative regulator. Freezing is closely related to drought stress since it involves cellular dehydration (Thomashow, 1999). The fact that neither overexpression nor silencing of ERD15 had an effect on the capability of the plants to improve their freezing tolerance in response to low temperature underlines the ABA-specific role of ERD15. We propose that the enhanced activity of a negative regulator, ERD15, confers the observed reduction in ABA sensitivity. It is possible that the plant tries to compensate this reduced ABA sensitivity by producing more ABA. Lack of feedback can explain the moderately increased basal endogenous as well as the increased stress-induced ABA level in ERD15 overexpression plants in comparison to controls (Fig. 4C). A similar, feedback regulation-related increase in ABA levels has previously been observed in the ABA-insensitive abi1-1 and abi2-1 mutants (Lång and Palva, 1992; Verslues and Bray, 2006).

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Hypothetical model presenting the role of ERD15 in ABA responses. The activation of ABA-signaling pathway by abiotic stress leads to drought and freezing tolerance. ERD15 negatively regulates the transduction of ABA signal, possibly downstream of the protein phosphatases ABI1 and ABI2. The negative effect of ERD15 on ABA signaling enhances SA-dependent defense seen as improved induction of PR genes and leading to enhanced resistance to the pathogen E. carotovora. Simultaneously, the enhanced SAR response down-regulates JA-dependent defense responses.

Not only does ERD15 control abiotic stress tolerance, but it also has a clear impact on biotic stress responses as demonstrated by the improved resistance of ERD15 overexpression plants to the plant pathogen E. carotovora. Consequently, it seems that the insensitivity or slow response to the ABA signal is not necessarily bad for the plant stress responses but could improve disease resistance. We hypothesize that the enhanced resistance of the overexpression plants could be due to the observed reduction in ABA sensitivity (Fig. 9). This is clearly different from previous studies where ABA deficiency, not insensitivity, has been proposed as the basis for the decreased pathogen susceptibility (Mauch-Mani and Mauch, 2005).

Until recently, the main focus of ABA research has been in its role in abiotic stress responses, and, thus, the role of this hormone in plant-pathogen interactions still poses many questions (Mauch-Mani and Mauch, 2005). Mohr and Cahill (2003) demonstrated that when Arabidopsis was either drought stressed or treated with ABA prior to infection with an avirulent strain of P. syringae pv tomato, the outcome was necrosis and chlorosis, symptoms of a susceptible interaction. Related studies with the ABA-deficient tomato mutant sitiens indicate that depletion of ABA enhances the resistance of these mutant plants against the fungal pathogen Botrytis cinerea, and the susceptibility of the sitiens plants can be restored by application of ABA (Audenaert et al., 2002).

Both Audenaert et al. (2002) and Thaler and Bostock (2004) showed that depletion of ABA enhanced SA-dependent defense responses and suggest an antagonistic effect of ABA on SA-mediated defense. SA-dependent defenses have been shown to be effective against E. carotovora (Li et al., 2004; Kariola et al., 2005), and, indeed, the SAR response was enhanced in Arabidopsis plants overexpressing ERD15, evidenced by enhanced induction of SAR markers as well as resistance to E. carotovora. Alterations in the hormone levels do not seem to be causing these differences, since basal contents of SA or JA are not altered in these transgenic lines (data not shown). We propose that the reduced ABA sensitivity of ERD15 overexpression plants has a positive impact on the SA-dependent defense responses (Fig. 9). This is further supported by the improved resistance the ABA-insensitive abi1-1 and abi2-1 mutant plants display to E. carotovora, also accompanied by a stronger SAR response (Fig. 8). Interestingly, recently characterized AtPARN, involved in mRNA degradation, could be a common component for ABA- and SA-signaling pathways: It has a prominent role not only in ABA- but also in SA-mediated stress responses in Arabidopsis (Nishimura et al., 2005).

An antagonism has also been reported between ABA and JA signaling: Anderson et al. (2004) demonstrated that exogenous ABA down-regulated JA- or ET-dependent defense genes. This could in turn improve SA-dependent defenses since the mutual antagonism between SA and JA signaling is well established (Petersen et al., 2000; Kunkel and Brooks, 2002; Li et al., 2004, 2006; Glazebrook, 2005). This antagonism may also explain the observed down-regulation of the JA/ET-dependent PDF1.2 gene by the enhanced SAR response in plants overexpressing ERD15.

How is ERD15 able to modulate ABA responses? Recently, ERD15 was described to have a PAM2 motif that enables the interaction with the C terminus of poly(A)-binding proteins (PABP; Albrecht and Lengauer, 2004; Kozlov et al., 2004), an interaction demonstrated in a yeast two-hybrid assay by Wang and Grumet (2004). PABPs are important in the regulation of translation and mRNA stability since they bind to the poly(A) tails of mRNAs before these are taken to the translational machinery (Belostotsky, 2003; Albrecht and Lengauer, 2004). Several mutations in genes encoding proteins involved in RNA metabolism, such as the mRNA cap-binding ABH1, have been shown to affect ABA sensitivity in Arabidopsis (Hugouvieux et al., 2001; Kuhn and Schroeder, 2003). A recent study by Razem et al. (2006) characterized the RNA-binding protein FCA as a receptor for ABA, which further strengthens the prominent role of posttranscriptional regulation in ABA signal transduction. ERD15 with its PABP-binding ability combined with the effect it has on ABA sensing fits this category well, and future studies including microarray analysis should further clarify the role of ERD15 in plant stress responses. (A microarray analysis of ABA-induced gene expression in ERD15 overexpression plants compared to control plants has been performed and the data can be found in the Web pages of NASC Affymetrix http://affymetrix.arabidopsis.info/, experiment reference no. NASCARRAYS-321.)

Based on our results, we suggest that ERD15 is a negative regulator of the early stages of stress-related ABA signaling in Arabidopsis (Fig. 9). It prevents the plants from responding too fast after the onset of abiotic stress, possibly by acting as a capacitor attenuating the ABA response: Only after input of sufficient stimuli is the capacitor overflown and the downstream response triggered. This system would ensure that the plant responds only when it becomes essential to invest assets in stress adaptation. It would be a waste of resources to activate a large-scale response before it is certain that the stress prevails and adaptation is necessary. Heil (2002) introduced a similar concept for biotic stress. Elucidating the mechanistic role of ERD15 in detail and identifying the possible translational partners and the specific transcripts this protein regulates will be of great interest for future studies and give new insights into plant ABA signaling.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotypes Col-0 and LE and mutant plants derived from LE were used in all experiments. Seeds were germinated on Murashige and Skoog (MS) medium (Sigma-Aldrich) plates and seedlings transferred either to soil or to MS in 12-well plates (Cellstar; Greiner Bio-One) after 1 week. Plants were grown in 1:1 peat:vermiculite (Finnpeat B2; Kekkilä Oyj) with a 12-h-light period at 22°C. Four- to 5-week-old plants were used for experiments.

Generation of Transgenic Plants

A 790-bp full-length fragment for ERD15 was cloned from a cDNA library of Arabidopsis plants treated with CF by PCR using the following primer pair: 5'-GACATATTTATCAACTTGATCAACTTGAG-3' and 5'-CGGAATTCAACTCTAGTTCTCATTTCTCTTC-3'. The full-length PCR fragment was digested with XbaI and EcoRI, cloned into a pBluescript II SK vector (Stratagene), and sequenced to verify the sequence. The plasmid harboring the full-length fragment for ERD15-designed pBluescript-ERD15 was digested with XbaI and EcoRI, and then subcloned into the corresponding sites of the binary vector pCP60, which is derived from pBIN19 containing the 35S promoter of Cauliflower mosaic virus, multiple cloning sites, and NOS, resulting in the overexpression construct S-pCP60-ERD15, with the 35S promoter directing expression in the sense orientation of the full-length ERD15.

A 511-bp DNA fragment was obtained using the ERD15 full-length fragment as template with the primer pair 5'-CGGAATTCTCAGCGAGGCTGGTGGATG-3' and 5'-AGGGAGCTCTGAGAATGGCGATGGTATCAGGA-3', digested with EcoRI and SacI, and then cloned into the EcoRI-SacI sites of pBluescript-ERD15. Since this fragment is in antisense orientation, the vector was called pBluescript-ERD15-loop. The XbaI-SacI fragment from the loop construct was cloned into pCP60, resulting in RNAi construct A-pCP60-ERD15. The fidelity of all constructs was confirmed by restriction and sequence analysis. Arabidopsis transformation was performed as described previously (Clough and Bent, 1998). Transgenic progeny lines with single insertion loci were selected on MS plates containing kanamycin and carried to homozygosity. The empty vector pCP60 was used to generate transgenic control plants in a similar manner.

Production of Polyclonal Anti-ERD15 Serum

Polyclonal antibodies against ERD15 were raised by immunizing a rabbit four times subcutaneously at 21-d intervals. Before the immunization, a preimmune blood sample was taken. For the primary immunization, 300 µg of purified ERD15 protein (received from Jack Leo and Adrian Goldman) emulsified with complete Freund's adjuvant (MP Biomedicals) was used. In subsequent boosters, 300 µg of ERD15 and incomplete Freund's adjuvant were used. Serum was collected 1 week after the last immunization. Specificities of the preimmune serum and the anti-ERD15 serum were determined by western blotting (1:100–1:50,000 dilutions) using anti-rabbit IgGs conjugated with alkaline phosphatase (Promega) secondary antibodies.

Protein Extraction and Western-Blot Analyses

Protein extraction was done as described by Lång et al. (1989). Ten micrograms of protein extract was loaded to 12 5% SDS-PAGE gels. SDS-PAGE and western blotting were done according to standard protocols. Anti-ERD15 serum was diluted 1:1,000 and used for immunodetection of ERD15. A goat-anti-rabbit antibody conjugated with alkaline phosphatase was diluted 1:10,000 and used as secondary antibody. Detection was made using NBT/BCIP as substrate. ERD15 was detected from drought-stressed samples.

Pathogen Strains and Plant Stress Treatments

Erwinia carotovora subsp. carotovora strain SCC1 (Rantakari et al., 2001) was propagated in Luria medium (Miller, 1972) at 28°C. An overnight culture was centrifuged for 7 min (6,500g), the pellet resuspended in 1 mL of 0.9% NaCl, and diluted to the appropriate concentration. The plants were infected by infiltrating E. carotovora subsp. carotovora SCC1 culture (approximately 10⁴–10⁵ cfu/plant) with a needleless syringe. The plants were infected at approximately 200 µmol m^–2 s^–1 photon flux density at approximately 80% humidity in a growth chamber with a 12-h-light period.

MeJA was applied to the plants as 100 µM and SA as 5 mM both by pipetting 5- x 5-µL droplets on the leaves. ABA was added by spraying as 100 µM solution (soil-grown plants) and by pipetting to MS media to final concentration of 60 µM (axenically grown plants). Wounding was done by pressing two leaves per plant with forceps. Salt was added by infiltrating 0.9% NaCl solution to two leaves per plant. Plants were exposed to drought stress by cutting off leaves and leaving them to dry on Whatman 3 paper for different periods of time for gene expression and determination of ABA. To see the drought phenotype, the plants were put to growth chamber with 50% humidity and left without watering for 2 weeks.

Assessment of Freezing Tolerance

To determine the degree of freezing tolerance, two methods were used. In freezing survival test, 3-week-old soil-grown plants were placed at –2°C in a phytotron for 1 h, after which freezing of the plants was initiated by spraying the plants with ice cold tap water. The plants were kept at –2°C for additional 4 h. The temperature was then decreased by 2°C per hour until it reached –10°C and kept there for 20 h. The temperature was allowed to return slowly to 22°C during 20 h. The plants were moved to normal growth conditions and assessed visually after 7 d.

In the electrolyte leakage test (Sukumaran and Weiser, 1972), axenically grown plants were harvested without roots and wrapped in moist Miracloth (Calbiochem). Plants were placed in test tubes in a controlled freezing bath. Extracellular freezing was initiated at –1.5°C by touching the samples with a frosted wire. After a 1.5-h equilibrium period, the temperature of the bath was decreased by 2°C per hour. Samples were taken at 1°C or 2°C intervals and thawed on ice overnight. Leaking electrolytes were extracted with deionized water (20 mL) by shaking for 1 h at room temperature and the conductivity was measured. The samples were then frozen in liquid nitrogen, reextracted with the original solution by shaking for 1 h at room temperature, and the conductivity was measured again. Plants showing leakage of 50% (EL₅₀) or more were considered dead and EL₅₀ values were calculated by Probit analysis with SPSS 10 (SPSS).

RNA Gel-Blot Analyses

Isolation of total RNA, labeling of DNA probes with digoxigenin (DIG), and RNA gel-blot analysis was performed as described previously (Kariola et al., 2003), and the membranes were hybridized with PCR-labeled gene-specific DNA or RNA DIG probes. DIG labeling of RNA, hybridization, and detection were done according to the manufacturer's instruction (Roche, Basel). A 790-bp cDNA fragment cloned to pCR2.1 (Invitrogen) was used as a template for an ERD15 (At2g41430)-specific RNA probe synthesized with T7 RNA-polymerase (Promega). DNA probes were amplified by PCR from the cDNA of PR1 (At2g14610; Uknes et al., 1992) and PR2 (At3g57260; Chen et al., 1995). PDF1.2 (At5g44420) and GST1 (At1g02930) probes were obtained from the Arabidopsis Biological Resource Center (GenBank accession nos. T04323 and N37195).

Quantification of Plant Hormones

Drought-stressed leaves (80–150 mg) were frozen and ground in liquid nitrogen, and ABA, SA, and JA were quantified with the vapor-phase extraction method described by Schmelz et al. (2003) using 40 ng of ¹³C₁-SA, 20 ng of dihydrojasmonic acid (Montesano et al., 2005), and 10 ng of D₆-ABA from Icon Isotopes as internal standard for each sample. GC-MS analysis was performed on a Trace-DSQ from Thermo as described previously (Montesano et al., 2005).

	ACKNOWLEDGMENTS

 
We thank Leila Miettinen and Hanne Mikkonen for excellent technical assistance. We also thank Anita Hegedus for the pCP60 plasmid. Jack Leo and Adrian Goldman are acknowledged for producing the ERD15 protein used in the immunization of the rabbits for production of anti-ERD15 serum.

Received July 3, 2006; accepted October 12, 2006; published October 20, 2006.

	FOOTNOTES

 
¹ This work was supported by the Academy of Finland (projects 79776, 202886, and 1213509; Finnish Centre of Excellence Programme 2000–2005 and 2006–2011), the Viikki Graduate School of Biosciences, the Helsinki Graduate School in Biotechnology, and Biocentrum Helsinki.

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.plantphysiol.org) is: E. Tapio Palva (tapio.palva{at}helsinki.fi).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.086223

^* Corresponding author; e-mail tapio.palva{at}helsinki.fi; fax 358–9–19159076.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Albrecht M, Lengauer T (2004) Survey on the PABC recognition motif PAM2. Biochem Biophys Res Commun 316: 129–138[CrossRef][Web of Science][Medline]

Anderson JP, Badruzsaufari E, Schenk PM, Manners JM, Desmond OJ, Ehlert C, Maclean DJ, Ebert PR, Kazan K (2004) Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell 16: 3460–3479[Abstract/Free Full Text]

Audenaert K, De Meyer GB, Höfte MM (2002) Abscisic acid determines basal susceptibility of tomato to B. cinerea and suppresses salicylic acid dependent signaling mechanisms. Plant Physiol 128: 491–501[Abstract/Free Full Text]

Belostotsky DA (2003) Unexpected complexity of poly(A)-binding protein gene families in flowering plants: three conserved lineages that are at least 200 million years old and possible auto- and cross-regulation. Genetics 163: 311–319[Abstract/Free Full Text]

Brader G, Tas É, Palva ET (2001) Jasmonate-dependent induction of indole glucosinolates in Arabidopsis by culture filtrates of the non-specific pathogen Erwinia carotovora. Plant Physiol 126: 849–860[Abstract/Free Full Text]

Chen T, Gusta L (1983) Abscisic acid-induced freezing resistance in cultured plant cells. Plant Physiol 73: 71–75[Abstract/Free Full Text]

Chen Z, Malamy J, Henning J, Conrath U, Sanchez-Casas P, Silva H, Ricigliano J, Klessig DK (1995) Induction, modification, and transduction of the salicylic acid signal in plant defense responses. Proc Natl Acad Sci USA 92: 4134–4137[Abstract/Free Full Text]

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline]

Cutler S, Ghassemian M, Bonetta D, Cooney S, McCourt P (1996) A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science 273: 1239–1241[Abstract]

Finkelstein RR, Gibson SI (2002) ABA and sugar interactions regulating development: cross-talk or voices in a crowd? Curr Opin Plant Biol 5: 26–32[CrossRef][Web of Science][Medline]

Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43: 205–227[CrossRef][Web of Science][Medline]

Gosti F, Beaudoin N, Serizet C, Webb AA, Vartanian N, Giraudat J (1999) ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling. Plant Cell 11: 1897–1910[Abstract/Free Full Text]

Guo Y, Xiong L, Song CP, Gong D, Halfter U, Zhu JK (2002) A calcium sensor and its interacting protein kinase are global regulators of abscisic acid signaling in Arabidopsis. Dev Cell 3: 233–244[CrossRef][Web of Science][Medline]

Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu Rev Plant Physiol Plant Mol Biol 41: 187–223[Web of Science]

Heil M (2002) Ecological costs of induced resistance. Curr Opin Plant Biol 5: 1–6[Medline]

Hugouvieux V, Kwak JM, Schroeder JI (2001) An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis. Cell 106: 477–487[CrossRef][Web of Science][Medline]

Kariola T, Brader G, Li J, Palva ET (2005) Chlorophyllase, a damage control enzyme, affects the balance between defense pathways in plants. Plant Cell 17: 282–294[Abstract/Free Full Text]

Kariola T, Palomäki TA, Brader G, Palva ET (2003) Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defence responses and cell death in Arabidopsis. Mol Plant Microbe Interact 16: 179–187[Web of Science][Medline]

Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K (1994) ERD15, a cDNA for a dehydration-induced gene from Arabidopsis thaliana. Plant Physiol 106: 1707[CrossRef][Web of Science][Medline]

Kozlov G, De Crescenzo G, Lim NS, Kahvejian A, Trempe JF, Elias D, Ekiel I, Sonenberg N, O'Connor-McCourt M, Gehring K (2004) Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase. EMBO J 23: 272–281[CrossRef][Web of Science][Medline]

Kuhn MK, Schroeder J (2003) Impacts of altered RNA metabolism on abscisic acid signaling. Curr Opin Plant Biol 6: 463–469[CrossRef][Web of Science][Medline]

Kunkel BN, Brooks DM (2002) Cross talk between signaling pathways in pathogen defense. Curr Opin Plant Biol 5: 325–331[CrossRef][Web of Science][Medline]

Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol 49: 199–222[CrossRef][Web of Science][Medline]

Leung J, Merlot S, Giraudat J (1997) The Arabidopsis ABSCISIC ACID INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell 9: 759–771[Abstract]

Li J, Brader G, Kariola T, Palva ET (2006) WRKY70 modulates the selection of signaling pathways in plant defense. Plant J 46: 477–491[CrossRef][Web of Science][Medline]

Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16: 319–331[Abstract/Free Full Text]

Lu C, Fedoroff N (2000) A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin and cytokinin. Plant Cell 12: 2351–2366[Abstract/Free Full Text]

Lång V, Heino P, Palva ET (1989) Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 77: 729–734

Lång V, Palva ET (1992) The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh. Plant Mol Biol 5: 951–962

Mäntylä E, Lång V, Palva ET (1995) Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana. Plant Physiol 107: 141–148[Abstract]

Mauch-Mani B, Mauch F (2005) The role of abscisic acid in plant-pathogen interactions. Curr Opin Plant Biol 8: 1–6[Medline]

Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J (2001) The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. Plant J 25: 295–303[CrossRef][Web of Science][Medline]

Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Mohr PG, Cahill DM (2003) Abscisic acid influences the susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv. tomato and Peronospora parasitica. Funct Plant Biol 30: 461–469[CrossRef]

Montesano M, Brader G, Ponce De Leon I, Palva ET (2005) Multiple defence signals induced by Erwinia carotovora ssp. carotovora elicitors in potato. Mol Plant Pathol 6: 541–549[CrossRef]

Nawrath C, Metraux JP (1999) Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell 11: 1393–1404[Abstract/Free Full Text]

Nishimura N, Kitahata N, Seki M, Narusaka Y, Narusaka M, Kuromori T, Asami T, Shinozaki K, Hirayama T (2005) Analysis of ABA Hypersensitive Germination2 revealed the pivotal functions of PARN in stress response in Arabidopsis. Plant J 44: 972–984[CrossRef][Web of Science][Medline]

Nordin K, Heino P, Palva ET (1991) Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh. Plant Mol Biol 16: 1061–1071[CrossRef][Web of Science][Medline]

Nordin K, Vahala T, Palva ET (1993) Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh. Plant Mol Biol 21: 641–653[CrossRef][Web of Science][Medline]

Norman-Setterblad C, Vidal S, Palva ET (2000) Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora. Mol Plant Microbe Interact 13: 430–438[Web of Science][Medline]

Palva TK, Hurtig M, Saindrenan P, Palva ET (1994) Salicylic acid induced resistance to Erwinia carotovora subsp. carotovora in tobacco. Mol Plant Microbe Interact 3: 356–363

Penninckx IAMA, Eggermont K, Terras FRG, Thomma BPHJ, De Samblanx GW, Buchala A, Métraux JP, Manners JM, Broekart WF (1996) Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8: 2309–2323[Abstract]

Petersen M, Brodersen P, Naested H, Andreasson E, Lindhart U, Johansen B, Nielsen HB, Lacy M, Austin MJ, Parker JE, et al (2000) Arabidopsis map kinase 4 negatively regulates systemic acquired resistance. Cell 115: 679–689

Razem FA, El-Kereamy A, Abrams RS, Hill DA (2006) The RNA-binding protein FCA is an abscisic acid receptor. Nature 439: 290–294[CrossRef][Medline]

Rantakari A, Virtaharju O, Vähämiko S, Taira S, Palva ET, Saarilahti HT, Romantschuck M (2001) Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster. Mol Plant Microbe Interact 14: 962–968[Web of Science][Medline]

Schmelz EA, Engelberth J, Alborn HT, O'Donnell P, Sammons M, Toshima H, Tumlinson JH III (2003) Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants. Proc Natl Acad Sci USA 100: 10552–10557[Abstract/Free Full Text]

Schroeder JI, Allen GJ, Hugouvieux V, Kwak JM, Waner D (2001) Guard cell signal transduction. Annu Rev Plant Physiol Plant Mol Biol 52: 627–658[CrossRef][Web of Science][Medline]

Sukumaran NP, Weiser CJ (1972) An excised leaflet test for evaluating potato frost tolerance. Hort Sci 7: 467–468

Tähtiharju S, Palva ET (2001) Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana. Plant J 26: 461–470[CrossRef][Web of Science][Medline]

Thaler SJ, Bostock RM (2004) Interactions between abscisic-acid-mediated responses and plant resistance to pathogens and insects. Ecology 85: 48–58[CrossRef][Web of Science]

Thomashow M (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50: 571–599[CrossRef][Web of Science][Medline]

Ton J, Mauch-Mani B (2004) -Amino-butyric acid-induced resistance against necrotrophic pathogens is based on ABA-dependent priming for callose. Plant J 38: 119–130[CrossRef][Web of Science][Medline]

Uknes S, Mauch-Mani B, Moyer M, Potter S, Williams S, Dincher S, Chandler D, Slusarenko A, Ward E, Ryals J (1992) Acquired resistance in Arabidopsis. Plant Cell 4: 645–656[Abstract/Free Full Text]

Verslues PE, Bray EA (2006) Role of abscisic acid (ABA) and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation. J Exp Bot 57: 201–212[Abstract/Free Full Text]

Vidal S, Eriksson ARB, Montesano M, Denecke J, Palva ET (1998) Cell wall-degrading enzymes from Erwinia carotovora cooperate in the salicylic acid-independent induction of a plant defense response. Mol Plant Microbe Interact 11: 23–32

Wang X, Grumet R (2004) Identification and characterization of proteins that interact with the carboxy terminus of poly(A)-binding protein and inhibit translation in vitro. Plant Mol Biol 54: 85–98[CrossRef][Web of Science][Medline]

Xiong L, Gong Z, Rock C, Subramanian S, Guo Y, Xu W, Galbraith D, Zhu JK (2001a) Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis. Dev Cell 6: 771–781

Xiong L, Lee BH, Ishitani M, Lee H, Zhang C, Zhu JK (2001b) FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev 15: 1971–1984[Abstract/Free Full Text]

Yamaguchi-Shinozaki K, Shinozaki K (2005) Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci 10: 88–94[CrossRef][Web of Science][Medline]

Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57: 781–803[CrossRef][Medline]

Yoshida T, Nishimura N, Kitahata N, Kumori T, Ito T, Asami T, Shinozaki K, Hirayama T (2005) ABA-Hypersensitive Germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs. Plant Physiol 140: 115–126[Medline]

Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247–273[CrossRef][Medline]

Related articles in Plant Physiol.:

On the Inside

Peter V. Minorsky
Plant Physiol. 2006 142: 1341-1342. [Full Text]  

This article has been cited by other articles:

				C. Sirichandra, A. Wasilewska, F. Vlad, C. Valon, and J. Leung The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action J. Exp. Bot., April 1, 2009; 60(5): 1439 - 1463. [Abstract] [Full Text] [PDF]

				A. Welling and E. T. Palva Involvement of CBF Transcription Factors in Winter Hardiness in Birch Plant Physiology, July 1, 2008; 147(3): 1199 - 1211. [Abstract] [Full Text] [PDF]

				A. Wasilewska, F. Vlad, C. Sirichandra, Y. Redko, F. Jammes, C. Valon, N. F. d. Frey, and J. Leung An Update on Abscisic Acid Signaling in Plants and More ... Mol Plant, March 1, 2008; 1(2): 198 - 217. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 142/4/1559    most recent pp.106.086223v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (12) Citing Articles via Google Scholar Google Scholar Articles by Kariola, T. Articles by Palva, E. T. Search for Related Content PubMed PubMed Citation Articles by Kariola, T. Articles by Palva, E. T. Agricola Articles by Kariola, T. Articles by Palva, E. T.