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Proc Natl Acad Sci U S A. Jan 25, 2011; 108(4): 1717–1722.
Published online Jan 10, 2011. doi:  10.1073/pnas.1018367108
PMCID: PMC3029766
Plant Biology

Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo

Abstract

Osmotic stress associated with drought or salinity is a major factor that limits plant productivity. Protein kinases in the SNF1-related protein kinase 2 (SnRK2) family are activated by osmotic stress, suggesting that the kinases are involved in osmotic stress signaling. However, due to functional redundancy, their contribution to osmotic stress responses remained unclear. In this report, we constructed an Arabidopsis line carrying mutations in all 10 members of the SnRK2 family. The decuple mutant snrk2.1/2/3/4/5/6/7/8/9/10 grew poorly under hyperosmotic stress conditions but was similar to the wild type in culture media in the absence of osmotic stress. The mutant was also defective in gene regulation and the accumulation of abscisic acid (ABA), proline, and inositol 1,4,5-trisphosphate under osmotic stress. In addition, analysis of mutants defective in the ABA-activated SnRK2s (snrk2.2/3/6) and mutants defective in the rest of the SnRK2s (snrk2.1/4/5/7/8/9/10) revealed that SnRK2s are a merging point of ABA-dependent and -independent pathways for osmotic stress responses. These results demonstrate critical functions of the SnRK2s in mediating osmotic stress signaling and tolerance.

Keywords: abiotic stress, water deficit, osmosensing, signal transduction, phosphorylation

Drought and soil salinity are major environmental factors limiting plant agricultural productivity. Both drought and salinity cause osmotic stress to plants. Osmotic stress activates signaling pathways that lead to rapid changes in gene expression and metabolism, some of which presumably help plants to cope with the stress. One important response to osmotic stress is the accumulation of the stress hormone abscisic acid (ABA). ABA is required for plant resistance to drought or salt stress (16). In the cytoplasm and nucleus, ABA binds to the PYR/PYL/RCAR family of START domain-containing soluble receptors (7, 8). The binding causes the receptors to bind and inhibit the activities of the type A protein phosphatase 2Cs (PP2Cs). Consequently, the PP2Cs lose their ability to interact with and inhibit the subfamily of SNF1-related protein kinase 2s (SnRK2s) such as SnRK2.2, SnRK2.3, and SnRK2.6 (also known as OST1 or SRK2E; refs. 911). The activated SnRK2s can then phosphorylate downstream proteins such as ABA-responsive element (ABRE)-binding transcription factors (ABFs; refs. 1214) and Slow Anion Channel 1 (SLAC1; refs. 15 and 16), NADPH oxidase (17), and a potassium channel (18) to activate ABA-responsive gene expression and cause stomatal closing. In contrast to the recent, rapid progress in our understanding of the ABA signaling pathway, little is known about the signaling pathway that leads from osmotic stress sensing to the activation of ABA accumulation. ABA accumulation under osmotic stress results from increased biosynthesis and reduced catabolism (19). The biosynthesis genes are induced or up-regulated by osmotic stress (20, 21).

The SnRK2 protein kinase family consists of 10 members (SnRK2.1 to -2.10) in Arabidopsis and 10 (SAPK1 to -10) in Oryza sativa (22). Several SnRK2s have been reported to be major kinases in ABA signaling, which regulates various developmental and physiological processes including seed maturation, dormancy, seedling development, and stomatal behavior. The first report of SnRK2 involvement in ABA signaling was for the wheat SnRK2-type protein kinase PKABA1, which acts downstream of ABA during germination and is induced by ABA at the transcript level (23). The Vicia faba SnRK2-type protein kinase AAPK is activated by ABA in guard cells and regulates stomatal closure (24). In Arabidopsis, the snrk2.6 (ost1) mutant cannot exhibit ABA-induced stomatal closure (25, 26), and the snrk2.2/2.3 double mutant is insensitive to ABA in terms of seed germination and seedling growth (27). In the snrk2.2/3/6 triple mutant all examined ABA responses are blocked (2830). Thus, SnRK2.2, -2.3, and -2.6 act redundantly in ABA regulation of guard cells, seed germination and seedling growth.

In vitro assays have shown that SnRK2.2, -2.3, and -2.6 are strongly activated by ABA, whereas SnRK2.7 and -2.8 are weakly activated by ABA (13, 31). Interestingly, all of the SnRK2s except SnR2.9 can be activated by osmotic stress in Arabidopsis protoplasts (31). Activation of SnRK2 homologs was also observed in tobacco (32), soybean (33), and rice (34). The osmotic stress activation of the SnRK2s suggests that the kinases play roles in osmotic stress signaling. However, the physiological role of SnRK2s in osmotic stress responses has remained unclear. The extreme-drought stress sensitivity of the snrk2.2/3/6 triple mutant may be explained by defective guard-cell regulation due to ABA insensitivity (28, 29). It is unclear whether these kinases may also have a direct role in osmotic stress signaling. Although root growth of the snrk2.8 mutant on agar plates has increased sensitivity to drought and overexpression of SnRK2.8 or the wheat TaSnRK2.4 improved drought tolerance (35, 36), drought tolerance in soil was similar among the wild type, snrk2.7, snrk2.8, and the snrk2.7/2.8 double mutant (37). The difficulty in defining the physiological roles of the SnRK2s in osmotic stress signaling and tolerance is likely a result of a high degree of functional redundancy among these genes. According to published microarray data (genevestigator: https://www.genevestigator.com/gv/index.jsp), the expression of all ten members of SnRK2 genes is detectable in most tissues. To determine whether redundancy masks defects caused by mutations in some of the SnRK2s, we constructed a decuple mutant carrying loss-of-function mutations in all 10 members of the SnRK2 family in Arabidopsis. This decuple mutant and other combinations of snrk2 mutants allowed us to directly determine a role for these genes in osmotic stress signaling and tolerance.

Results

Construction of the snrk2.1/2/3/4/5/6/7/8/9/10 Decuple Mutant.

To generate the decuple mutant snrk2.1/2/3/4/5/6/7/8/9/10, we crossed snrk2 T-DNA mutants (Fig. S1A) sequentially and produced a mutant homozygous for snrk2.1/3/4/5/6/7/8/9/10 but heterozygous for snrk2.2 (herein after referred to as 2hetero). We also identified the snrk2.1/4/5/7/8/9/10 septuple mutant (herein after referred to as the septuple) and the snrk2.1/3/4/5/6/7/8/9/10 nonuple mutant, in which only SnRK2.2 is of the wild-type allele (herein after referred to as 2W).

To identify a decuple mutant among the progenies of 2hetero, seeds were screened on MS agar plates containing 5 μM ABA because the snrk2.2/3/6 triple mutant is insensitive to ABA in seed germination (28). Germinated seeds were confirmed to be the decuple mutant by genomic PCR, and RT-PCR data confirmed that the decuple mutant did not express any intact SnRK2 mRNAs (Fig. 1). Typically <5% of the seeds germinated on the ABA plate rather than the expected 25% homozygous for snrk2.2; this is likely because snrk2.2/3/6 triple mutant seeds frequently did not mature (28). Thus, many of the decuple mutant seeds also failed to mature, resulting in <25% germination rate. Consistent with this result, siliques of 2hetero plants contained aborted seeds (Fig. S2A).

Fig. 1.
Isolation of the decuple mutant. (A) RT-PCR analysis with SnRK2s and Tubulin primers using total RNA extracted from seedlings of the wild type (Col-0) and the decuple mutant as the template. (B) Photograph of wild type (WT) and snrk2.2/3/6 (236), snrk2.1/4/5/7/8/9/10 ...

Because the snrk2.2/3/6 triple mutant has a wilty phenotype (28), the decuple mutant plants grown in soil were covered with plastic lids to maintain high humidity (96–100% relative humidity). Under these high-humidity conditions, the decuple mutant plants could grow, bolt, and flower, but were much smaller than the snrk2.2/3/6 triple mutants, which were also smaller than the wild type (Fig. 1B). In addition, the decuple mutant failed to produce seeds even though it produced flowers with a pistil and stamens (Fig. S2B).

Decuple Mutant Is Deficient in Osmotic Stress-Activated Protein Kinase Activity.

The activation status of SnRK2s in vivo can be detected by in-gel kinase assays with histone as the substrate. Such assays using Arabidopsis crude extracts have revealed that osmotic stress activates several protein kinases (26, 31, 38). We performed in-gel kinase assays using extracts from plants exposed to osmotic stress in liquid culture. Four bands could be detected in extracts from wild-type plants under untreated control conditions (Fig. 2A). The two bands around 40–42 kDa were probably from activities of SnRK2s in the SnRK2.1/4/5/7/8/9/10 group because they were reduced or disappeared in the septuple and decuple mutants. The two larger bands (around 48 and 60 kDa) were outside the size range of the SnRK2s and did not change in the decuple mutant, and thus were clearly not from SnRK2s. In extracts from wild type treated with 0.8 M mannitol for 20 min, at least three bands around 42 kDa were activated (Fig. 2 A and B). According to previous experiments (27), SnRK2.2 is included in the upper band. In the snrk2.1/4/5/7/8/9/10 septuple mutants, the lower and middle bands almost disappeared, but the upper band remained. In the decuple mutant, the remaining upper band also disappeared (Fig. 2 A and B). These results indicate that the mannitol-activated bands in the in-gel kinase assay are SnRK2s and that SnRK2 activities were eliminated in the decuple mutant.

Fig. 2.
In-gel kinase assay. In-gel kinase assay with proteins extracted from the wild type (Col-0) and snrk2.2/3/6 (236), snrk2.1/4/5/7/8/9/10 septuple (sep), and snrk2.1/2/3/4/5/6/7/8/9/10 decuple (dec) mutants grown in liquid culture with or without a 20-min ...

Decuple Mutant Is Hypersensitive to Osmotic Stress.

We examined the effect of lack of SnRK2s on seedling growth under osmotic stress conditions. Because the decuple mutant did not produce seeds, obtaining plants of the decuple mutant required repeated screening of 2hetero progeny. The decuple mutants identified on 5 μM ABA plates were transferred to 1/2 MS plates without ABA for recovery by further incubation for 5 d. The seedlings were subsequently transferred to control plates or plates containing a low concentration of PEG (−0.4 MPa; ref. 39). Similar sized seedlings of the wild type, snrk2.2/3/6, and the septuple mutant were also transferred to control or PEG plates. Although growth of all of the genotypes was similar on control plates, growth of the decuple mutant, but not growth of the wild type, was severely inhibited on PEG medium (Fig. 3A). In addition, leaves of the decuple mutant seedlings turned yellow under PEG treatment (Fig. 3A). The sensitivity of snrk2.2/3/6 and the septuple mutant to PEG was variable; some seedlings grew well, whereas others grew poorly. Quantitative data showed that PEG greatly reduced the fresh weight and root growth of the decuple mutant but only moderately reduced the fresh weight and root growth of snrk2.2/3/6 and the septuple mutant (Fig. 3B). Compared with the snrk2.2/3/6 triple mutant, the septuple mutant was more affected by osmotic stress and had less fresh weight. The growth of the 2W nonuple mutant was similar to that of the septuple mutant (Fig. 3B). These results indicate that SnRK2s are required for growth under osmotic stress conditions and that there is high redundancy because the presence of SnRK2.2 alone could largely rescue the decuple mutant phenotype.

Fig. 3.
Seedling growth under osmotic stress. (A) Photographs of seedlings 18 d after transfer to the control medium (1/2 MS/1% sucrose) or medium containing PEG (−0.4 MPa). (B) Seedling fresh weight and root length for seedlings treated as in A. For ...

Decuple Mutant Is Impaired in Osmotic Stress-Induced ABA Accumulation.

We analyzed ABA and H2O2 contents in the mutant plants under osmotic stress. After a 12-h exposure to media containing PEG (−1.2 MPa), ABA increased by ~10- to 17-fold in the wild type, snrk2.2/3/6 triple mutant, and septuple mutants. However, the ABA content increased by only about twofold in the decuple mutant in response to the same PEG treatment (Fig. 4A). These results suggest that the SnRK2s are critical for ABA accumulation under osmotic stress.

Fig. 4.
ABA and H2O2 contents of the mutants under osmotic stress. ABA (A) and H2O2 (B) contents were measured in the wild type (WT) and in the snrk2.2/3/6 triple (t), the septuple (s), and the decuple (d) mutants 12 h after transfer to control medium (1/2 MS/1% ...

Some mutants that are sensitive to osmotic or salt stress accumulate more reactive oxygen species (ROS; ref. 40). ROS may cause cell damage, even though ROS at low levels are also used as signaling molecules. We examined the possibility that the growth defect of the decuple mutant under osmotic stress resulted from ROS accumulation as indicated by H2O2 concentration. After a 12-h exposure to medium containing PEG (−1.2 MPa), H2O2 contents increased in all mutants and in the wild type. The H2O2 concentration was similar in the decuple mutant and the wild type (Fig. 4B). These results suggest that the phenotype of the decuple mutant under osmotic stress is not a result of ROS over accumulation.

Decuple Mutant Is Defective in Osmotic Stress Induction of Gene Expression.

Osmotic stress induces the expression of many genes in Arabidopsis (41). To examine the effect of snrk2 mutants on osmotic stress-induced gene expression, we performed quantitative RT-PCR (qPCR) of several known osmotic-stress-induced genes (41). In the wild type, expression of RD29A, NCED3, RD26, and PKS5 was induced in seedlings grown in liquid culture after 3 h of treatment with 0.8 M mannitol. Induction of these genes was substantially reduced in the decuple mutant and in the snrk2.2/3/6 triple mutant. Effect of the snrk2.1/4/5/7/8/9/10 septuple mutant varied between genes, with PKS5 showing some reduction but other genes not showing any significant reduction (Fig. 5A). These results indicate that SnRK2s are important for osmotic-stress-induced gene expression, with SnRK2/3/6 regulating all four genes tested, whereas SnRK1/4/5/7/8/9/10 have more specific effects on some stress-regulated genes.

Fig. 5.
Expression of genes induced by osmotic stress. (A) Expression of osmotic-stress–upregulated genes RD29A, NCED3, RD26, and PKS5 assayed by quantitative RT-PCR in Col-0 (WT) and the snrk2.2/3/6 triple (t), snrk2.1/4/5/7/8/9/10 septuple (s), and ...

To analyze genome-wide gene expression changes in the mutants, we performed microarray analysis using total RNA extracted from liquid cultured plants of wild type, snrk2.2/3/6 triple mutant, the septuple mutant, and the decuple mutant after 3-h treatment with 0.8 M mannitol. For the wild type and the decuple mutant, RNA was also extracted from seedlings that were not treated with mannitol. First, we examined the expression of SnRK2s. The transcript level of SnRK2.7, which was mutated at the C-terminal end (Fig. S1 B and C), in the decuple mutant was one-third of that in the wild type (Table S1). Reduction of transcripts for other SnRK2s confirmed the mutations in the decuple mutant (Table S1). Second, we performed genome-wide comparison of gene expression in wild type and the decuple mutant after mannitol treatment.

To gain a better understanding of impact of mutations in SnRK2s in the osmotic stress response, we focused on the 464 genes induced by more than fourfold in the wild type after mannitol treatment and compared their expression between the wild type and mutants. We calculated expression in the mutants as a percentage of expression in the wild type for each gene and made histograms to show the distribution. The expression of many induced genes after the treatment was less in snrk2.2/3/6 than in the wild type, whereas nearly all of these genes had unchanged expression (close to 100%) in the septuple mutant compared with wild type (Fig. 5B). In contrast, the decuple mutant was even more affected in gene expression than snrk2.2/3/6, with a vast majority of induced genes being expressed at lower level in the decuple mutant than in wild type or snrk2.2/3/6. (Fig. 5B). The median of the percentage expression relative to the wild type was 70–80% for snrk2/3/6, 100–110% for the septuple mutant, and 40–50% for the decuple mutant. Without mannitol treatment, the expression of these 464 induced genes was similar in the decuple mutant and the wild type (relative expression was close to 100%; Fig. 5C), indicating that the mutations did not affect the basal level of expression of the selected genes but did affect their response to osmotic stress. As a control, we quantified the expression of 464 randomly selected genes for which expression was not changed by osmotic stress in the wild type; for these noninduced genes, the expression was similar in all mutants and the wild type (relative expression was close to 100%; Fig. S3). These results indicate that the decuple snrk2 mutant has a strong global effect on gene induction in response to osmotic stress, an effect that cannot be detected even in the septuple mutant.

Decuple Mutations Affect Proline Accumulation and IP3 Synthesis.

We also analyzed other osmotic stress responses in the snrk2 mutants. We measured proline accumulation because proline is one of the compatible osmolytes accumulated by cells under osmotic stress (42). Proline accumulation was reduced in the snrk2.2/3/6 triple mutant and in the decuple mutant, but was greater than wild type in the septuple mutant after incubation for 12 h in liquid culture containing 0.8 M mannitol (Fig. 6A). In the microarray data, the expression of P5CS1, which is a rate-limiting factor of proline biosynthesis (43), in the triple, septuple, and decuple mutants is 65%, 112%, and 54%, respectively, of that in the wild type. This trend of proline accumulation was also observed when plants were treated with ABA (Fig. 6B). In addition to the enhancement in the septuple mutant, ABA-induced proline accumulation was slightly increased in the snrk2.1/4/5/10 quadruple mutant and greatly increased in the snrk2.1/4/5/9/10 quintuple mutant relative to the wild type (Fig. 6C). These results suggest that mannitol-induced proline accumulation reflects ABA-induced proline accumulation. Thus, SnRK2.2/3/6 is important for osmotic-stress-induced proline accumulation, whereas SnRK2.1/4/5/7/8/9/10 and especially SnRK2.1/4/5/9/10 may act as negative regulators of ABA-induced proline accumulation. The difference between the snrk2.1/4/5/10 quadruple and the snrk2.1/4/5/9/10 quintuple mutants suggests that SnRK2.9 has a negative role in ABA-induction of proline accumulation.

Fig. 6.
Osmotic stress or ABA-induced accumulation of proline and IP3. (A) Proline content measured in liquid cultured seedlings of the wild type (WT) and the snrk2.2/3/6 triple (t), the septuple (s), and the decuple (d) mutants before (control) and after a 12-h ...

We also analyzed an ABA-independent response. Inositol 1,4,5-trisphosphate (IP3) accumulation is induced by osmotic stress but not by ABA (44), although ABA induces IP3 accumulation in a longer (≥30 min) time course (45, 46). After a 20-min exposure to 0.8 M mannitol, IP3 accumulated in liquid culture of the wild type and snrk2.2/3/6 but did not accumulate in the decuple mutant and was reduced in the septuple mutant (Fig. 6D). IP3 accumulation was not observed under 50 μM ABA treatment for 20 min (Fig. S4). These results indicate that SnRK2s are also required for this ABA-independent response to osmotic stress.

Discussion

Although osmotic stress is an important problem in crop production, the osmotic stress signaling pathway has been largely unknown. SnRK2s have been proposed as key components of the pathway because they are activated by osmotic stress. Results of our experiments using the decuple mutant, which were impaired in all members of the SnRK2 family, clearly show the importance of SnRK2s in the osmotic stress pathway in vivo. In the decuple mutant, all full-length mRNA of SnRK2s are disrupted (Fig. 1), resulting in elimination of the kinase activity of SnRK2s induced by osmotic stress (Fig. 2). The decuple mutants show a clear growth defect under osmotic stress, but grow as well as the wild type under nonstressed conditions (Fig. 3), indicating the essential and specific role of SnRK2s in the response of plants to osmotic stress. Disruption of normal osmotic stress response was also observed for gene expression (Fig. 5) and accumulation of ABA (Fig. 4A), proline (Fig. 6A), and IP3 (Fig. 6D), all of which are important responses for stress adaptation. The decuple mutant grows poorly in soil but grows as well as the wild type on culture plates, suggesting that our soil conditions were not optimal for the decuple mutant. The roles of SnRK2.8 and SnRK2.6 in metabolism (47, 48) may also have been a factor. The observed sterility of the decuple mutant may have been caused by the high humidity, which was required for the survival of the decuple mutant plants but may have interfered with development.

SnRK2s show high redundancy. Even when nine of the genes are knocked out, the phenotype is not severe as long as SnRK2.2 is intact (Figs. 1B and and3).3). SnRK2.2 as well as SnRK2.3 and -2.6 help mediate the ABA-dependent pathway (28), and osmotic stress signaling has been considered to be divided between ABA-dependent and -independent pathways. Thus, having at least one of the ABA-activated SnRK2s is sufficient for growth and development in soil and for coping with osmotic stress in culture media. On the other hand, snrk2.2/3/6, which is insensitive to ABA, grew normally when subjected to osmotic stress on culture plates (Fig. 3), indicating that these ABA-activated SnRK2s are not essential for short-term osmotic stress tolerance. IP3 accumulation, which is independent of ABA (Fig. S4B and ref. 44), differed between the decuple and septuple mutants (Fig. 6D), indicating that SnRK2.2/3/6 functions not only in the ABA-dependent pathway, but also in the ABA-independent pathway. This is consistent with the observation that SnRK2.2/3/6 is activated not only by ABA, but also by osmotic stress (Fig. 2, ref. 38). This would suggest that ABA-dependent and -independent pathways converge at the SnRK2s or their substrates.

Even in the decuple mutant, osmotic-stress-induced responses (in terms of gene expression and accumulation of ABA and proline) are greatly reduced but are not abolished, indicating that some osmotic stress signaling remains. One possibility is that the snrk2.7 mutation may not be a null and the truncated SnRK2.7 (Fig. S1 B and C) may retain sufficient activity to trigger the observed residual responses, even though this activity is below the detection level in the in-gel kinase assay. Another possibility is that there are SnRK2-independent osmotic-stress-response pathways. Some CDPKs and SnRK3s are involved in osmotic- and salt-stress pathways (3, 49, 50). It is possible that they share some substrates with SnRK2s (51). This would result in some residual osmotic stress response in the decuple mutant, although this residual response is insufficient to rescue the impaired growth of the decuple mutant.

The results obtained with the snrk2.1/4/5/7/8/9/10 septuple mutant are less straightforward to explain. Plants lacking SnRK2.1/4/5/9/10, which is not activated by ABA, have increased sensitivity to ABA in terms of ABA-induced proline accumulation (Fig. 6C). This is consistent with the finding that overexpression of a rice SnRK2.1/4/5/9/10 homolog (SAPK6) made tobacco ABA insensitive (52). The results suggest that these kinases are negative regulators of the ABA pathway. Microarray data (Fig. 5B) and mannitol-induced proline accumulation data (Fig. 6A) seem to reflect ABA responses more than ABA-independent responses. Although snrk2.2/3/6 is more affected than the septuple mutant in terms of osmotic stress-induced gene expression, the septuple mutant is more affected than snrk2.2/3/6 in terms of the sensitivity of growth to osmotic stress on PEG plates (Fig. 3) where the impaired stomatal control of snrk2.2/3/6 is less of a factor than in soil-grown plants. The reduced growth of the septuple mutant could be caused by the impairment of certain key ABA-independent osmotic responses. Although the decuple mutant and the snrk2.2/3/6 triple mutant showed similar defects in osmotic stress induction of NCED3 expression (Fig. 5A), ABA deficiency was observed in the decuple mutant, but not the triple mutant (Fig. 4A). The results suggest that the osmotic-stress-induced ABA accumulation was not limited by the NCED3 gene expression level but rather by other factor(s) controlled by the SnRK2.1/4/5/7/8/9/10s. The SnRK2.1/4/5/7/8/9/10s could be important for the posttranslational activation of NCED3 and other ABA biosynthesis enzymes.

Because osmotic-stress-induced kinase activity of SnRK2.9 was not detected in a protoplast assay (31), it was not clear whether SnRK2.9 plays a role in osmotic stress responses in vivo. In our results, the snrk2.1/4/5/9/10 quintuple mutant accumulated more proline in response to ABA than the snrk2.1/4/5/10 quadruple mutant (Fig. 6C). When these results are considered along with the results that ABA-induced proline accumulation of the snrk2.9 single mutant did not significantly differ from that of the wild type (110 ± 8%, n = 5), they suggest that SnRK2.9 is also involved in negatively regulating the ABA pathway, but this role may be redundant with that of other SnRK2s.

Although SnRK2s have been known as osmotic stress-activated kinases, the physiological roles of the kinases in vivo have been unclear. The defects in osmotic stress responses of the snrk2.1/2/3/4/5/6/7/8/9/10 decuple mutant revealed critical functions of these kinases in vivo.

Materials and Methods

T-DNA Insertion Lines.

The snrk2.2, -2.3, and -2.6 mutants were described in ref. 28. The seeds of snrk2.1, -2.4, -2.5, -2.7, -2.8, -2.9, and -2.10 T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Center (53). Homozygous plants were identified by PCR screening using primers designed by the I-Sect website (http://signal.salk.edu/cgi-bin/tdnaexpress; primer sequences are given in Table S2).

In-Gel Kinase Assays.

In-gel kinase assays were described in ref. 27. Histone was purchased from Sigma (St. Louis, MO).

Physiological Assays.

Plants in soil and seedlings in agar plates were routinely grown under 16 h of light (~75 μmol m−2 s−1) and 8 h of dark at 23 °C.

For quantification of ABA, H2O2, proline, and IP3, tissues were ground in liquid nitrogen. ABA was extracted with 80% methanol, 500 mg/L citric acid, and 10 mg/L butylated hydroxytoluene. After centrifugation to remove debris, the supernatant was dried and reconstituted with Tris-buffered saline (25 mM Tris/100 mM NaCl/1 mM MgCl2, pH 7.5). ABA was measured with a Phytodetek ABA measurement kit (Agdia, Inc., Elkhart, IN) following the manufacturer's instructions. H2O2 was extracted with 20 mM PBS and measured by using an Amplex Red H2O2 kit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions and ref. 40. Proline was assayed by using the ninhydrin-based colorimetric assay (54). IP3 was assayed with an inositol-1,4,5-trisphosphate [H-3] RRA Kit (Perkin-Elmer, Waltham, MA) following the manufacturer's instructions.

RT-PCR and qPCR Analysis of Gene Expression.

Total RNA was purified from seedlings by using TRIzol (Invitrogen) following the manufacturer's instructions. Reverse transcription reactions were performed using 2 μg of total RNA and SuperScriptII reverse transcriptase (Invitrogen). PCR was then performed for 35 cycles. Real-time qPCR analysis was performed with the iQ5 real-time PCR detection system and SYBR Green (Bio-Rad, Hercules, CA). PCR (15 μL) was performed with 1 μL of RT reaction. Relative amount of target mRNA was determined based on ΔΔCt method using Actin2 as an internal control. Three technical replicates were performed for each experiment. Primers used for both RT-PCR and qPCR are given in Table S2 or in ref. 27.

Microarray Analysis.

Biologically duplicated total RNA was purified from seedlings by using the RNeasy plant kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Hybridization to Arabidopsis genome ATH1 array (Affymetrix, Santa Clara, CA) was performed by the Core Instrumental Facility of the Institute for the Integrative Genome Biology, the University of California, Riverside. Data were analyzed with affylmGUI (55).

Supplementary Material

Supporting Information:

Acknowledgments

We thank the Arabidopsis Biological Resource Center for providing the T-DNA insertion mutants. This work was supported by National Institutes of Health Grant R01GM059138 (to J.-K.Z.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1018367108/-/DCSupplemental.

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