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First published online June 7, 2007; 10.1104/pp.107.099895 Plant Physiology 145:814-830 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
STRESS RESPONSE SUPPRESSOR1 and STRESS RESPONSE SUPPRESSOR2, Two DEAD-Box RNA Helicases That Attenuate Arabidopsis Responses to Multiple Abiotic Stresses1,[OA]Albert Katz Department of Dryland Biotechnologies, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion 84990, Israel
Two genes encoding Arabidopsis (Arabidopsis thaliana) DEAD-box RNA helicases were identified in a functional genomics screen as being down-regulated by multiple abiotic stresses. Mutations in either gene caused increased tolerance to salt, osmotic, and heat stresses, suggesting that the helicases suppress responses to abiotic stress. The genes were therefore designated STRESS RESPONSE SUPPRESSOR1 (STRS1; At1g31970) and STRS2 (At5g08620). In the strs mutants, salt, osmotic, and cold stresses induced enhanced expression of genes encoding the transcriptional activators DREB1A/CBF3 and DREB2A and a downstream DREB target gene, RD29A. Under heat stress, the strs mutants exhibited enhanced expression of the heat shock transcription factor genes, HSF4 and HSF7, and the downstream gene HEAT SHOCK PROTEIN101. Germination of mutant seed was hyposensitive to the phytohormone abscisic acid (ABA), but mutants showed up-regulated expression of genes encoding ABA-dependent stress-responsive transcriptional activators and their downstream targets. In wild-type plants, STRS1 and STRS2 expression was rapidly down-regulated by salt, osmotic, and heat stress, but not cold stress. STRS expression was also reduced by ABA, but salt stress led to reduced STRS expression in both wild-type and ABA-deficient mutant plants. Taken together, our results suggest that STRS1 and STRS2 attenuate the expression of stress-responsive transcriptional activators and function in ABA-dependent and ABA-independent abiotic stress signaling networks.
As a sessile organism, a plant's ability to adapt to abiotic stresses such as heat, cold, drought, and high salinity is crucial for its survival. Plant responses to abiotic stresses involve a complex variety of tolerance mechanisms (Bray et al., 2000  ; Tester and Davenport, 2003; Wang et al., 2003) that are activated and integrated by the expression of thousands of genes (Chen et al., 2002; Kreps et al., 2002; Seki et al., 2002). These genes encode proteins involved in numerous biological processes as well as a large number of proteins of unknown function. Furthermore, the expression of many genes with regulatory functions, such as transcription factors, RNA-binding proteins, calcium-binding proteins, kinases, phosphatases, etc., is altered by stress. These genes are probably involved not only in regulating downstream stress responses but also in stress perception and signaling (for review, see Xiong et al., 2002b; Zhu, 2002; Shinozaki et al., 2003; Bartels and Sunkar, 2005; Yamaguchi-Shinozaki and Shinozaki, 2006).
In recent years, much progress has been made in identifying and characterizing components of stress signaling networks in Arabidopsis (Arabidopsis thaliana). Promoter analyses of cold- and dehydration-responsive genes such as RD29A have revealed two cis-acting elements mediating stress-induced expression, the DRE/CRT and ABRE elements (Yamaguchi-Shinozaki and Shinozaki, 1994
Control of ABA-regulated gene expression is mediated via the ABRE cis-acting element PyACGTGGC, which is bound by bZIP transcription factors known as ABRE-binding (AREB) proteins or AREB factors (ABFs; Choi et al., 2000
Posttranscriptional and posttranslational control of stress gene expression is increasingly being recognized as playing a major role in regulating plant stress responses. For example, in unstressed Arabidopsis lines overexpressing the Na+/H+ antiporter SOS1, levels of SOS1 transcript are similar to wild-type levels. Only under salt stress does SOS1 transcript accumulate to high levels in the overexpressors, suggesting that SOS1 mRNA is unstable in unstressed conditions (Shi et al., 2003
The upstream stress-responsive transcription factors such as ICE1, the DREB/CRT family, the AREB/ABFs, ATMYC2, and AtMYB2, as well as proteins mediating posttranscriptional regulation of gene expression, ultimately control the expression of many downstream stress-responsive genes involved in the response to multiple abiotic stresses (e.g. Maruyama et al., 2004
Identification of the strs Mutants A functional genomics-based screen was performed to identify genes that may function as upstream regulators of multiple abiotic stress responses (P. Kant, M. Gordon, S. Kant, G. Zolla, O. Davydov, Y.M. Heimer, V. Chalifa-Caspi, R. Shaked, and S. Barak, unpublished data). In brief, a microarray analysis of early Arabidopsis heat stress-responsive genes was performed, and the resulting data were combined in a "stress gene" database with data from published microarray analyses examining Arabidopsis responses to a variety of abiotic stresses. The database was queried for a set of regulatory genes whose expression was affected early by multiple abiotic stresses, and Arabidopsis T-DNA insertion mutants defective in each gene were screened for altered sensitivity to abiotic stresses. A preliminary screen of mutants homozygous for the T-DNA insertion identified two mutants exhibiting increased tolerance to salt stress that contained a T- DNA insertion in genes encoding different DEAD-box RNA helicases (Fig. 1, A and B ). Analysis of microarray data in our database indicated that these genes are down-regulated by salt, osmotic, and heat stress, suggesting that the encoded proteins may function to suppress Arabidopsis stress responses. We therefore designated the proteins as STRS1 and STRS2.
STRS1 (At1g31970) is predicted to encode a protein of 537 amino acid residues with an estimated molecular mass of 59.5 kD, while STRS2 (At5g08620) is predicted to be a protein of 563 amino acids with an estimated molecular mass of 62.5 kD. Database searches revealed that both proteins possess all nine conserved motifs that are characteristic of the DEAD-box protein family as well as an upstream conserved Phe (Fig. 1A; de la Cruz et al., 1999 ; Rocak and Linder, 2004). It has been estimated that the Arabidopsis genome encodes over 50 DEAD-box RNA helicases (Aubourg et al., 1999; Boudet et al., 2001), 32 of which were classified by Aubourg et al. (1999) as AtRH1 to AtRH32. Genomic and cDNA sequence analysis indicated that STRS1 is identical to AtRH5, while STRS2 is identical to AtRH25. The N- and C-terminal extensions of DEAD-box RNA helicases are of variable length, and it is thought that they confer substrate specificity (Aubourg et al., 1999; de la Cruz et al., 1999). Counting upstream from the conserved Phe and downstream from motif VI for N termini and C termini, respectively, STRS1 and STRS2 have N-terminal regions of 117 and 81 amino acids and C-terminal regions of 76 and 130 amino acids, respectively. Alignment of STRS1 and STRS2 protein sequences (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) showed that the two proteins exhibit 32% sequence identity and 49% sequence similarity. However, all the sequence identity/similarity resides in the core helicase region of the protein (between the N- and C-terminal regions defined above) containing the conserved motifs, whereas there is no significant sequence similarity between the N-terminal and C-terminal regions.
To explore whether the STRS genes are involved in regulating a variety of abiotic stresses, the stress-responsive phenotypes of two independent T-DNA insertion lines for each gene were analyzed. The two strs1 mutant lines designated strs1 and strs1a contain a T-DNA insertion in exon 6 and exon 9, respectively (Fig. 1B). Real-time PCR analysis of STRS1 gene expression in unstressed wild-type and mutant plants using primers complementary to DNA downstream of the insertion showed that STRS1 transcript was undetectable in both mutant lines (Fig. 1C). However, when primers complementary to DNA upstream of the T-DNA insertion were employed, STRS1 transcripts were detected in strs1 and strs1a plants, albeit at 25% to 30% of wild-type STRS1 transcript levels. This suggests that truncated STRS1 transcripts are produced in both strs1 mutant lines but that they are less stable than wild-type transcripts. Furthermore, while it is unknown whether the truncated transcripts are translated, any truncated protein that may be produced in the strs1 mutants is unlikely to be functional due to the absence of essential protein motifs such as motif V and motif VI (Fig. 1A; de la Cruz et al., 1999 When grown under unstressed conditions, both seedling and adult strs mutants showed no morphological or developmental differences compared to wild type except for a very weak early flowering phenotype (data not shown). Percentage of seed germination of all four mutant lines on Murashige and Skoog (MS) plates in the absence of stress was virtually identical to wild type. However, germination of the strs mutants on MS plates supplemented with NaCl showed substantial tolerance to salt stress (Fig. 2, A and B ). At only 2 d after stratification, the strs lines already showed 2- to 3-fold greater percentage of germination than wild-type seeds. By 5 d after stratification, strs seeds exhibited 95% to 99% germination, whereas wild type showed approximately 60% germination. In fact, under salt stress conditions, the final percentage of germination of wild-type seeds never reached more than 70%. In addition, all the strs lines grew faster than wild type under salt stress. Quantification of fresh weight (FW) at 7 d after germination demonstrated that strs1 and strs1a seedlings exhibited 100% greater FW than wild type, while strs2 and strs2a seedlings showed 37% and 49% greater FW, respectively, than wild type (Fig. 2C).
The strs mutants also showed tolerance to osmotic stress, albeit to a lesser extent than their tolerance to salt stress. When seedlings were germinated and grown on MS plates supplemented with mannitol, all four mutant lines showed between 11% and 33% greater germination than wild-type seedlings by 4 to 5 d after stratification (Fig. 2, D and E). Furthermore, strs1 and strs1a mutants exhibited over 30% greater FW than wild type, while strs2 and strs2a showed approximately 20% greater FW (Fig. 2F).
The strs mutants were next tested for altered basal and acquired thermotolerance. For basal thermotolerance, seeds were sown on MS plates, stratified for 4 d at 4°C, and then exposed to 1 to 4 h of 45°C (Hong and Vierling, 2000
Acquired thermotolerance results from prior exposure to a pretreatment such as a sublethal high temperature (Lindquist, 1986 ). To assess whether the strs mutants possessed enhanced acquired thermotolerance, a quantitative hypocotyl assay was performed (Hong and Vierling, 2000). In brief, seedlings were grown on vertical MS plates in the dark for 2 to 3 d before application of heat stress treatments. The increase in hypocotyl length was recorded 3 d after the heat stress. Figure 3D shows that a heat stress of 45°C for 2 h killed both wild-type and mutant seedlings, as evidenced by lack of hypocotyl elongation. However, a pretreatment of 38°C followed by 45°C for 2 h or 3 h allowed seedlings to survive. The hot1-3 mutant was most severely affected by 2 h of heat stress, exhibiting an 80% reduction in hypocotyl elongation compared to the control (no heat treatment). Hypocotyl elongation of wild-type seedlings was reduced by 42%, whereas strs1 and strs2 exhibited a drop of only 4% and 13%, respectively. A heat stress of 3 h killed the hot1-3 mutant, while wild-type and strs seedlings showed a further reduction in hypocotyl elongation with the mutants displaying approximately one-half the reduction observed in wild type. A similar effect was observed for the strs1a and strs2a mutants. Taken together, these results suggest that the strs mutants exhibit enhanced basal and acquired thermotolerance. Plants were also tested for freezing tolerance, but no difference could be observed between wild type and mutants (data not shown).
To gain insight into the molecular basis of the stress-tolerant strs mutant phenotypes, we next investigated the expression of the well-characterized stress-responsive marker gene, RD29A (Yamaguchi-Shinozaki and Shinozaki, 1994
In unstressed plants, no differences in RD29A expression were observed between wild-type and mutant plants (Fig. 4, A–C), indicating that loss of STRS function alone is not sufficient for enhanced RD29A expression. We therefore surmised that derepressed expression of upstream transcription factors in the strs mutants might account for the enhanced RD29A expression. Consequently, we analyzed the expression of two members of the DREB transcription factor family that mediate stress-responsive RD29A expression via DRE elements in the RD29A promoter (Stockinger et al., 1997 ; Liu et al., 1998). In wild-type plants, DREB1A/CBF3 expression was induced by salt, drought, and cold stress with peak expression at 6 h after onset of salt stress and 3 h after onset of drought or cold stress (Fig. 4, D–F). Under salt and drought stress, expression dropped sharply by 6 h after stress and thereafter slowly declined, whereas under cold stress a more gradual decrease in DREB1A/CBF3 expression was observed. Furthermore, peak expression of DREB1A/CBF3 was an order of magnitude higher under cold stress than in salt and drought stress, reflecting the primary role of DREB1A/CBF3 in cold-responsive gene expression (Liu et al., 1998; Shinwari et al., 1998). In the strs mutants, expression kinetics of DREB1A/CBF3 was comparable to wild type, but fold-induction of DREB1A/CBF3 expression was higher in the mutant lines compared to wild type. The DREB2 proteins play a major role in drought and salt stress signaling networks (Liu et al., 1998; Nakashima et al., 2000). DREB2A expression was induced in wild-type plants by salt and drought stress and by cold stress, although cold-induced expression levels were two orders of magnitude lower than drought-induced expression (Fig. 4, G–I). Peak expression of DREB2A occurred at identical time points to RD29A expression. Once again, enhanced stress-induced expression of DREB2A was observed in the strs mutants with expression displaying similar kinetics to that observed in wild-type plants. No difference was observed in either DREB1A/CBF3 or DREB2A expression between unstressed wild-type and strs mutant plants. This suggests that the STRS proteins are not acting to repress stress-responsive gene expression in unstressed plants. Rather, the STRS proteins attenuate gene expression once it has been induced by stress.
We next tested whether the STRS genes also regulate stress-responsive genes that are not controlled by the DREB signaling subnetwork by analyzing the salt- and drought-induced expression of two non-DRE element genes, RD19 and RD22 (Yamaguchi-Shinozaki et al., 1992 To ensure that the effects of the strs mutations were specific, we analyzed expression of the housekeeping gene ACTIN2 (ACT2). Figure 4, P to R, demonstrates that ACT2 expression was unaffected either by stress treatments or by absence of STRS1 or STRS2, thereby suggesting a specific role for the STRS proteins in Arabidopsis abiotic stress responses.
The molecular basis for the increased tolerance to heat stress of the strs mutants was examined by analyzing expression of the gene encoding HSP101. This protein has been shown to be essential for both basal and acquired thermotolerance (Hong and Vierling, 2000
Heat shock proteins are primarily regulated at the transcriptional level by heat shock transcription factors (HSFs; Wu, 1995 ). In Arabidopsis, there are 21 putative HSFs (Nover et al., 2001), and of those that have been studied, some are constitutively expressed, while the expression of others is induced by heat stress (Lee et al., 1995; Prandl et al., 1998; Miller and Mittler, 2006). HSF4 and HSF7 are two genes whose expression is induced by heat stress (Prandl et al., 1998; AtGenExpress database, http://www.arabidopsis.org/info/expression/ATGenExpress.jsp), and we examined whether expression of HSF4 and HSF7 is enhanced in the strs mutants. Figure 5, B and C, shows that in wild type and strs mutants, the expression of both HSF4 and HSF7 was induced by heat stress, reaching peak expression at 30 min after onset of stress. Moreover, their expression was enhanced in the strs mutants predominantly at, and after, peak expression. As a control, the expression of TUBULIN5 (TUB5) in response to the heat treatment was examined. ACT2 expression was not used as a control, because its expression is affected by heat stress (data not shown). TUB5 expression was unaffected by heat stress in wild type and strs mutant seedlings (Fig. 5D), demonstrating that HSP101, HSF4, and HSF7 expression was specifically affected in the strs mutants. Thus, although there is, as yet, no evidence that HSF4 and/or HSF7 directly regulate HSP101 expression, our results suggest that the enhanced HSP101 expression observed in the strs mutants is due to derepression of upstream HSFs. Taken together, our results suggest that the STRS proteins act as general attenuators of Arabidopsis stress responses and that increased salt-, drought-, cold-, and heat-induced gene expression in the strs mutants is due, at least in part, to derepressed, stress-mediated expression of upstream transcriptional activators.
Drought-induced RD22 and AtMYC2 expression is mediated by ABA (Abe et al., 1997
The STRS proteins could attenuate ABA-dependent stress-responsive gene expression either by modulating ABA signaling to its target genes or by acting directly in the ABA signaling pathways. In the latter case, it would be expected that STRS expression would respond to ABA. Figure 6C shows that exposure of wild-type seedlings to ABA led to rapid suppression of STRS expression, indicating that STRS1 and STRS2 can function as components of the ABA-dependent signaling subnetwork. However, the fact that the STRS proteins regulate stress-mediated DREB expression as well (Fig. 4) suggests that STRS1 and STRS2 also function in the ABA-independent stress signaling subnetwork. To test this hypothesis, we examined stress-mediated expression of STRS1 and STRS2 in wild type and the ABA-deficient aba2-1 mutant (Leon-Kloosterziel et al., 1996 ). Figure 6, D and E, shows that salt stress led to a reduction in STRS expression in both wild-type and aba2-1 plants, thus demonstrating that STRS1 and STRS2 can respond to stress signals in the absence of ABA. This finding supports the contention that STRS1 and STRS2 regulate both the ABA-independent and ABA-dependent stress signaling subnetworks.
Because the absence of STRS1 and STRS2 led to enhanced expression of ABA-responsive genes (Figs. 4, L–O, and 6A), we surmised that the strs1 and strs2 mutants might exhibit an ABA-hypersensitive phenotype. We therefore examined the effect of the strs mutations on ABA inhibition of seedling germination. Wild-type and mutant seed (both independent strs1 and strs2 T-DNA insertion lines) were germinated on MS plates containing 0, 0.5, 1, or 2 µM ABA. On control plates, no difference in germination could be observed between wild-type and mutant plants (Fig. 6F). Surprisingly, however, the strs mutant lines exhibited an ABA-insensitive phenotype, whereby ABA progressively inhibited wild-type germination more severely at each concentration than it did the strs mutants. This finding is similar to that found in the hos5 and fry1 mutants that also exhibit up-regulated stress gene expression but display an ABA-insensitive germination phenotype (Xiong et al., 1999
Inspection of our "stress gene" database along with results from testing STRS expression in wild-type and aba2-1 plants (Fig. 6, D and E) suggested that STRS1 and STRS2 expression is down-regulated by salt, osmotic, and heat stress. To confirm this observation and to examine the detailed temporal expression of STRS1 and STRS2, we analyzed STRS gene expression in wild-type plants in response to salt, drought, heat, and cold stresses. Salt and drought stresses led to an over 50% reduction in STRS1 and STRS2 expression by 1 h after the onset of stress (Fig. 7, A and B ). Expression continued to decline to about 20% and 10% of control levels under salt and drought stress, respectively, with STRS expression progressively rising thereafter. However, the later rise in STRS expression levels was considerably less under drought stress than under salt stress. Furthermore, under salt stress, STRS1 expression exhibited greater stress-mediated repression than STRS2. Down-regulation of STRS expression was even more rapid after onset of heat stress (Fig. 7C). Expression levels reached their nadir by 2 h of heat stress and began rising again by 3 h of heat stress.
We also noticed that trough STRS expression coincided with peak expression of RD29A and DREB2A at 6 and 12 h after the onset of salt or drought stress, respectively (compare Fig. 7A with Fig. 4, A and G; and Fig. 7B with Fig. 4, B and H). Furthermore, the fast post-peak recovery of STRS expression under salt and the slow recovery of expression under drought were also reflected in the kinetics of the post-peak decline of RD29A and DREB2A expression. Similarly, trough STRS expression coincided with peak HSP101 expression at 2 h of heat stress (compare Fig. 7C with Fig. 5A). These results suggest a remarkable temporal correlation between trough expression of the STRS genes and the peak expression of downstream stress-responsive genes within a particular stress treatment. In contrast to salt, drought, and heat stress, cold treatment had no effect on the expression of the STRS genes (Fig. 7D). This result is in agreement with the finding that the strs mutants did not exhibit enhanced freezing tolerance (data not shown) and confirmed the notion that STRS1 and STRS2 are not involved in attenuation of cold stress-regulated gene expression. However, the strs mutants did display enhanced expression of the DREB genes and a downstream target gene in response to cold stress (Fig. 4, C, F, and I). This finding suggests that in the absence of STRS1 and STRS2, stress-induced expression of genes normally attenuated by the STRS proteins will show enhanced expression in response to any signal that triggers stress-responsive gene expression. This will occur even if that signal does not normally down-regulate STRS expression. One limitation of using relative real-time PCR quantification is that, unlike northern analysis, a visualization of overall expression levels between various genes is not possible. Therefore, to determine expression levels of STRS1 and STRS2 compared to other stress-responsive genes, we quantified STRS transcript copy number in unstressed plants and compared them with transcript copy numbers of DREB1A/CBF3, RD29A, and HSP101 (Table I ). STRS1 and STRS2 exhibited comparable amounts of unstressed transcript levels to each other, but these were an order of magnitude higher than RD29A and HSP101 and two orders of magnitude higher than DREB1A/CBF3 transcript levels. STRS1 and STRS2 expression was detected in all organs from unstressed plants that were examined (Table II ). However, transcript copy numbers differed according to the organ analyzed. Highest STRS expression was observed in inflorescences and lowest levels in green siliques and roots.
STRS1 and STRS2 Expression Is under the Control of the Circadian Clock
Transcript profiling has shown that the expression of many stress-responsive genes is under the control of the circadian clock (Harmer et al., 2000
We have identified STRS1 and STRS2 as negative regulators of multiple abiotic stress responses in Arabidopsis. Disruption of the STRS1 and STRS2 genes by T-DNA insertions increases the tolerance of strs mutant seedlings to salt and osmotic stresses and enhances basal and acquired thermotolerance (Figs. 2 and 3). Consistent with their stress-tolerant phenotypes, the strs mutants exhibit enhanced expression of stress-responsive genes and their upstream transcriptional activators (Figs. 4 and 5). However, stress-responsive gene expression is not up-regulated in unstressed strs mutants. These findings, coupled with results showing stress-mediated down-regulation of STRS gene expression in wild-type plants (Fig. 7), suggest that STRS1 and STRS2 are required to attenuate expression of upstream stress signaling components.
The STRS1 and STRS2 genes encode proteins that are members of the large family of approximately 50 Arabidopsis DEAD-box RNA helicases, a larger number than the sequenced genomes of other organisms, including the fly and worm (Boudet et al., 2001
The los4-1 mutant exhibits severely reduced cold induction of DREB/CBF expression and its target genes and is more sensitive to cold stress (Gong et al., 2002 Although our results suggest that STRS1 and STRS2 attenuate transcript accumulation of upstream stress transcription factors, their mode of action is unclear at present. They may directly affect transcription, pre-mRNA processing, mRNA stability, or other aspects of stress transcription factor RNA metabolism. Alternatively, they may function by regulating transcripts of enhancers or repressors of stress transcription factors. It is tempting to speculate, however, that STRS1 and STRS2 are involved in degrading the stress-induced mRNAs because the abundance of the STRS transcripts decreases as the abundance of the stress marker transcripts increases. If the STRS proteins themselves are short lived and their abundance parallels that of their transcripts, then a decline in STRS protein would allow accumulation of the stress-induced transcripts. The virtually identical phenotypes of the strs1 and strs2 mutants plus the close pattern of expression of the two genes in response to the various stresses suggest that STRS1 and STRS2 may function together in a complex. It should also be noted that although the strs mutant phenotypes did not result from a perturbation of general gene expression, the STRS proteins may have additional functions to those in abiotic stress responses. This premise is supported by the observation that the strs mutants have a slightly early flowering phenotype, at least under long-day (16 h light:8 h dark) conditions (data not shown) and that the highest expression of both genes was detected in flowers (Table II).
STRS1 and STRS2 are two of several negative regulators of stress responses that have been identified in recent years. These include HOS1 (Lee et al., 2001
Attenuation of the stress signaling networks by negative regulators is thus clearly important for proper regulation of the response to abiotic stresses. Constitutive activation of the stress response by ectopic expression of DREB1A/CBF3 leads to severe growth retardation in unstressed plants (Kasuga et al., 1999
Our results further demonstrate the distinct regulation of the drought and salt signaling subnetworks and the cold signaling subnetwork. First, while strs mutants exhibit tolerance to salt and osmotic stresses (Fig. 2), they do not appear to be tolerant to freezing stress (data not shown). However, this finding must be further explored because freezing tests were carried out on detached leaves, whereas all other tolerance assays were performed on seedlings. Moreover, enhanced cold-induced gene expression was observed in the strs mutants (Fig. 4, C, F, and I), suggesting that mutant plants might indeed show tolerance to cold stress under certain conditions. Nevertheless, the freezing tolerance results are supported by the discovery that STRS1 and STRS2 expression is unaffected by cold stress, whereas it is down-regulated by salt and drought stresses (Fig. 7). In addition to the phenotypes of various stress repressor mutants such as hos1 and hos5-1 described above, there are several other lines of evidence pointing toward distinct signaling subnetworks for salt and drought stresses on the one hand and cold stress on the other hand. For instance, the DREB1 proteins and ICE1, the upstream regulator of DREB1A/CBF3 expression, are mainly involved in regulating cold-induced gene expression, while DREB2A and DREB2B control salt- and drought-induced gene expression (Liu et al., 1998
The strs mutant phenotype showing increased basal and acquired thermotolerance (Fig. 3) and enhanced HSP101, HSF4, and HSF7 expression (Fig. 5) reveals interactions between the heat, salt, and osmotic signaling subnetworks. Analysis of the expression of all 21 Arabidopsis HSF genes has demonstrated that many HSFs are induced by multiple abiotic stresses, again illustrating the links between the signaling subnetworks (Miller and Mittler, 2006
The finding that STRS1 and STRS2 regulate the ABA-dependent stress signaling subnetwork (as well as the ABA-independent subnetwork) and that their expression is down-regulated by ABA (Fig. 6) provides another connection between heat stress responses and responses to other abiotic stresses. It has been found that ABA biosynthesis and signaling mutants are defective in acquired thermotolerance, while addition of exogenous ABA protects Arabidopsis plants from heat-induced oxidative damage (Larkindale and Knight, 2002
In summary, this study has identified two negative regulators of ABA-dependent and ABA-independent upstream abiotic stress transcriptional activators. Furthermore, STRS1 and STRS2 are regulatory nodes linking salt, osmotic, heat, and ABA signaling subnetworks. Because STRS1 and STRS2 are members of the large family of DEAD-box RNA helicases, additional studies may identify other DEAD-box RNA helicases involved in abiotic stress signal transduction. Indeed, we have recently identified several other putative STRS genes in both our functional genomics screen and by inspection of the AtGenExpress database (http://www.arabidopsis.org/info/expression/ATGenExpress.jsp) that may be down-regulated by multiple abiotic stresses. Studies of their T-DNA insertion mutants are apace to determine whether they have similar phenotypes to the strs1 and strs2 mutants. Our findings also illustrate the growing importance of RNA metabolism in the regulation of Arabidopsis abiotic stress signal transduction (e.g. Lu and Fedoroff, 2000 ; Hugouvieux et al., 2001; Xiong et al., 2001a, 2002a; Borsani et al., 2005; Gong et al., 2005; Cao et al., 2006).
Plant Materials and Growth
All Salk T-DNA insertion mutants were obtained from the Arabidopsis Biological Resource Center (ABRC). The Salk ID for each mutant is as follows: strs1, Salk_062509; strs1a, Salk_147039; strs2, Salk_028850; and strs2a, Salk_005131 (Alonso et al., 2003
The F2 generation of homozygous mutants was used for all experiments. The wild-type control Arabidopsis (Arabidopsis thaliana) accession was Columbia. For plate experiments, seeds were surface sterilized by soaking in a solution of 50% bleach for 10 min and then rinsing five times with sterile water. Seeds were sown on nutrient agar plates containing MS salts (Murashige and Skoog, 1962
For salt and osmotic stress assays, 50 to 100 surface-sterilized wild-type and mutant seeds were sown on plates containing MS media with or without NaCl (salt stress) or mannitol (osmotic stress). Four replicate plates were used per treatment, and germination (emergence of radicals) was scored daily for 6 to 7 d until no further germination was observed. FW of seedlings was measured 10 d after stratification. Thermotolerance assays were performed essentially according to Hong and Vierling (2000)
Fifty wild-type seedlings were germinated and grown on MS plates in the growth room for 7 d. After this period, seedlings were transferred to a tabletop growth chamber (LE509, MRC) at 22°C and entrained with a photoperiod of 12 h light (100 µmol photons m–2 s–1)/12 h dark. After 4-d entrainment, the photoperiod was changed to continuous light, and seedlings were harvested every 3 h over the third and fourth circadian cycles (Green and Tobin, 1999
Total RNA was isolated, and cDNA was prepared according to Kant et al. (2006)
We express our appreciation to the ABRC for Arabidopsis T-DNA insertion mutant seeds and to Professors Elizabeth Vierling and Gadi Galili for their kind gift of the hot1-3 and aba2-1 mutants, respectively. We are much obliged to Dr. Matthew Hannah for carrying out the freezing tolerance assays. We are grateful to The Sol Leshin Fund for funding this research and to the Israel Science Foundation for funding the real-time PCR system. Many thanks to all members of the Barak laboratory for their support. We are also indebted to Prof. Yair Heimer, Dr. Gidon Grafi, Dr. Rachel Green, and the reviewers for critical reading of the manuscript. Finally, special thanks to Nick Poore of Bancroft's School for starting the journey. Received March 22, 2007; accepted May 30, 2007; published June 7, 2007.
1 This work was supported by The Sol Leshin Fund and by the Israel Science Foundation (to S.B.). 
2 These authors contributed equally to the article.
3 Present address: Department of Plant Agriculture, Crop Science Building, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
4 Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
5 Present address: Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beersheva 84105, Israel. 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: Simon Barak (simon{at}bgu.ac.il).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.099895 * Corresponding author; e-mail simon{at}bgu.ac.il; fax 972–8–6596752.
Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15: 63–78 Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K (1997) Role of Arabidopsis MYC and MYB homologs in drought and abscisic acid-regulated gene expression. Plant Cell 9: 1859–1868[Abstract] Albrecht V, Weinl S, Blazevic D, D'Angelo C, Batistic O, Kolukisaoglu U, Bock R, Schulz B, Harter K, Kudla J (2003) The calcium sensor CBL1 integrates plant responses to abiotic stresses. Plant J 36: 457–470[CrossRef][Web of Science][Medline] Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657 Aubourg S, Kreis M, Lecharny A (1999) The DEAD box RNA helicase family in Arabidopsis thaliana. Nucleic Acids Res 27: 628–636 Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. CRC Crit Rev Plant Sci 24: 23–58[CrossRef] Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123: 1279–1291[CrossRef][Web of Science][Medline] Boudet N, Aubourg S, Toffano-Nioche C, Kreis M, Lecharny A (2001) Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila. Genome Res 11: 2101–2114 Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to abiotic stresses. In BB Buchanan, W Gruissem, RL Jones, eds, Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD, pp 1158–1203 Cao S, Jiang L, Song S, Jing R, Xu G (2006) AtGRP7 is involved in the regulation of abscisic acid and stress responses in Arabidopsis. Cell Mol Biol Lett 11: 526–535[CrossRef][Web of Science][Medline] Chen W, Provart NJ, Glazebrook J, Katagiri F, Chang HS, Eulgem T, Mauch F, Luan S, Zou G, Whitham SA, et al (2002) Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell 14: 559–574 Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong X, Agarwal M, Zhu JK (2003) ICE1: a regulator of cold-induced transcriptome and freezing. Genes Dev 17: 1043–1054 Choi HI, Hong JH, Ha JO, Kang JY, Kim SY (2000) ABFs, a family of ABA-responsive element binding factors. J Biol Chem 275: 1723–1730 de la Cruz J, Kressler D, Linder P (1999) Unwinding RNA in Saccharomyces cerevisiae: DEAD box proteins and related families. Trends Biochem Sci 24: 192–198[CrossRef][Web of Science][Medline] Dong CH, Agarwal M, Zhang Y, Xie Q, Zhu JK (2006) The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci USA 103: 8281–8286 Fujita M, Fujita Y, Maruyama K, Seki M, Hiratsu K, Ohme-Takagi M, Phan Tran LS, Yamaguchi-Shinozaki K, Kazuo Shinozaki K (2004) A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. Plant J 39: 863–876[CrossRef][Web of Science][Medline] Fujita Y, Fujita M, Satoh R, Maruyama K, Parvez MM, Seki M, Hiratsu K, Ohme-Takagi M, Shinozaki K, Yamaguchi-Shinozaki K (2005) AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis. Plant Cell 17: 3470–3488 Furihata T, Maruyama K, Fujita Y, Umezawa T, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K (2006) ABA-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc Natl Acad Sci USA 103: 1988–1993 Gong Z, Dong CH, Lee H, Zhu J, Xiong L, Gong D, Stevenson B, Zhu JK (2005) A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. Plant Cell 17: 256–267 Gong Z, Lee H, Xiong L, Jagendorf A, Stevenson B, Zhu JK (2002) RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance. Proc Natl Acad Sci USA 99: 11507–11512 Green RM, Tobin EM (1999) Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression. Proc Natl Acad Sci USA 96: 4176–4179 Harmer SL, Hogenesch JB, Straume M, Chang HS, Han B, Zhu T, Wang X, Kreps JA, Kay SA (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290: 2110–2113 Hoagland DF, Arnon DI (1950) The water culture for growing plants without soil. Berkeley California Agriculture Experimental Station Circular 347: 39 Hong SW, Vierling E (2000) Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc Natl Acad Sci USA 97: 4392–4397 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] Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280: 104–106 Kang JY, Choi HI, Im MG, Kim SY (2002) Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell 14: 343–357 Kant S, Kant P, Raveh E, Barak S (2006) Evidence that differential gene expression between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila. Plant Cell Environ 29: 1220–1234[CrossRef][Medline] Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K (1999) Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17: 287–291[CrossRef][Web of Science][Medline] Kreps JA, Wu Y, Chang HS, Zhu T, Wang X, Harper JF (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 130: 2129–2141 Kurihara Y, Takashi Y, Watanabe Y (2006) The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12: 206–212 Larkindale J, Hall JD, Knight MR, Vierling E (2005) Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol 138: 882–897 Larkindale J, Knight MR (2002) Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol 128: 682–695 Lee BH, Henderson DA, Zhu JK (2005) The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. Plant Cell 17: 3155–3175 Lee H, Xiong L, Gong Z, Ishitani M, Stevenson B, Zhu JK (2001) The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning. Genes Dev 15: 912–924 Lee JH, Hubel A, Schoffl F (1995) Derepression of the activity of genetically engineered heat shock factor causes constitutive synthesis of heat shock proteins and increased thermotolerance in transgenic Arabidopsis. Plant J 8: 603–612[CrossRef][Web of Science][Medline] Leon-Kloosterziel KM, Gil MA, Ruijs GJ, Jacobsen SE, Olszewski NE, Schwartz SH, Zeevaart JAD, Koornneef M (1996) Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci. Plant J 10: 655–661[CrossRef][Web of Science][Medline] Linder P (2006) Dead-box proteins: a family affair—active and passive players in RNP-remodeling. Nucleic Acids Res 34: 4168–4180 Lindquist S (1986) The heat shock response. Annu Rev Biochem 55: 1151–1191[CrossRef][Web of Science][Medline] Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10: 1391–1406 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2– Lorsch JL (2002) RNA chaperones exist and DEAD box proteins get a life. Cell 109: 797–800[CrossRef][Web of Science][Medline] 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: 2351–2365 Lu PI, Chen NZ, An R, Su Z, Qi BS, Ren F, Chen J, Wang XC (2007) A novel drought-inducible gene, ATAF1, encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis. Plant Mol Biol 63: 289–305[CrossRef][Web of Science][Medline] Maruyama K, Sakuma Y, Kasuga M, Ito Y, Seki M, Goda H, Shimada Y, Yoshida H, Shinozaki K, Yamaguchi-Shinozaki K (2004) Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcription factor using two microarray systems. Plant J 38: 982–983[CrossRef][Web of Science][Medline] Miller G, Mittler R (2006) Could heat shock transcription factors function as hydrogen peroxide sensors in plants? Ann Bot (Lond) 98: 279–288 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473–497[CrossRef] Nakashima K, Shinwari ZK, Sakuma Y, Seki M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2000) Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression. Plant Mol Biol 42: 657–665[CrossRef][Web of Science][Medline] Nover L, Bharti K, Doring P, Mishra SK, Ganguli A, Scharf KD (2001) Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need? Cell Stress Chaperones 6: 177–189[Web of Science][Medline] Park W, Li J, Song R, Messing J, Chen X (2002) CARPEL FACTORY, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 12: 1484–1495[CrossRef][Web of Science][Medline] Prandl R, Hinderhofer K, Eggers-Schumacher G, Schoffl F (1998) HSF3, a new heat shock factor from Arabidopsis thaliana, derepresses the heat shock response and confers thermotolerance when overexpressed in transgenic plants. Mol Gen Genet 258: 269–278[CrossRef][Web of Science][Medline] Queitsch C, Hong SW, Vierling E, Lindquist S (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12: 479–492 Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16: 1616–1626 Rocak S, Linder P (2004) DEAD-box proteins: the driving force behind RNA metabolism. Nat Rev Mol Cell Biol 5: 232–241[CrossRef][Web of Science][Medline] Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell 18: 1292–1309 Schwer B (2001) A new twist on RNA helicases: DExH/D box proteins as RNPases. Nat Struct Biol 8: 113–116[CrossRef][Web of Science][Medline] Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, et al (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high salinity stresses using a full-length cDNA microarray. Plant J 31: 279–292[CrossRef][Web of Science][Medline] Shi H, Lee BH, Wu SJ, Zhu JK (2003) Overexpression of a plasma membrane Na+/H+ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21: 81–85[CrossRef][Web of Science][Medline] Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6: 410–417[CrossRef][Web of Science][Medline] Shinwari ZK, Nakashima K, Miura S, Kasuga M, Seki M, Yamaguchi-Shinozaki K, Shinozaki K (1998) An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression. Biochem Biophys Res Commun 250: 161–170[CrossRef][Web of Science][Medline] Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94: 1035–1040 Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16: 2001–2019 Suzuki N, Rizhsky L, Liang H, Shuman J, Shulaev V, Mittler R (2005) Enhanced tolerance to environmental stress in transgenic plants expressing the transcriptional coactivator Multiprotein Bridging Factor 1c. Plant Physiol 139: 1313–1322 Tanner NK, Linder P (2001) DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol Cell 8: 251–262[CrossRef][Web of Science][Medline] Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Ann Bot (Lond) 91: 1–25 Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K (2000) Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proc Natl Acad Sci USA 97: 11632–11637 Wang W, Vinocur B, Altman A (2003) Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta 218: 1–14[CrossRef][Web of Science][Medline] Wu C (1995) Heat stress transcription factors. Annu Rev Cell Dev Biol 11: 441–469[CrossRef][Web of Science][Medline] Xiong L, Gong Z, Rock CD, 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 1: 771–781[CrossRef][Web of Science][Medline] Xiong L, Ishitani M, Lee H, Zhu JK (1999) HOS5: a negative regulator of osmotic stress-induced gene expression in Arabidopsis thaliana. Plant J 19: 569–578[CrossRef][Web of Science][Medline] Xiong L, Lee H, Ishitani M, Tanaka Y, Stevenson B, Koiwa H, Bressan RA, Hasegawa PM, Zhu JK (2002a) Repression of stress-responsive genes by FIERY2, a novel transcriptional regulator in Arabidopsis. Proc Natl Acad Sci USA 99: 10899–10904 Xiong L, Lee HH, 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 Xiong L, Schumaker KS, Xhu JK (2002b) Cell signaling during cold, drought and salt stress. Plant Cell (Suppl) 14: S165–S183 Yamaguchi-Shinozaki K, Koizumi M, Urao S, Shinozaki K (1992) Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol 33: 217–224 Yamaguchi-Shinozaki K, Shinozaki K (1994) A nove1 cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress. Plant Cell 6: 251–264[Abstract] 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] Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247–273[CrossRef][Medline] This article has been cited by other articles:
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0.05). C, FW of wild type and two independent alleles of strs1 and strs2 10 d after stratification on MS plates with and without 125 mM NaCl. Data are mean ± SD (n = 4). Bars with different letters indicate significant difference at P 
