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Plant Physiol. Apr 2012; 158(4): 1755–1768.
Published online Feb 1, 2012. doi:  10.1104/pp.111.190389
PMCID: PMC3320183

Constitutive Activation of Transcription Factor OsbZIP46 Improves Drought Tolerance in Rice1,[C][W][OA]

Abstract

OsbZIP46 is one member of the third subfamily of bZIP transcription factors in rice (Oryza sativa). It has high sequence similarity to ABA-responsive element binding factor (ABF/AREB) transcription factors ABI5 and OsbZIP23, two transcriptional activators positively regulating stress tolerance in Arabidopsis (Arabidopsis thaliana) and rice, respectively. Expression of OsbZIP46 was strongly induced by drought, heat, hydrogen peroxide, and abscisic acid (ABA) treatment; however, it was not induced by salt and cold stresses. Overexpression of the native OsbZIP46 gene increased ABA sensitivity but had no positive effect on drought resistance. The activation domain of OsbZIP46 was defined by a series of deletions, and a region (domain D) was identified as having a negative effect on the activation. We produced a constitutive active form of OsbZIP46 (OsbZIP46CA1) with a deletion of domain D. Overexpression of OsbZIP46CA1 in rice significantly increased tolerance to drought and osmotic stresses. Gene chip analysis of the two overexpressors (native OsbZIP46 and the constitutive active form OsbZIP46CA1) revealed that a large number of stress-related genes, many of them predicted to be downstream genes of ABF/AREBs, were activated in the OsbZIP46CA1 overexpressor but not (even down-regulated) in the OsbZIP46 overexpressor. OsbZIP46 can interact with homologs of SnRK2 protein kinases that phosphorylate ABFs in Arabidopsis. These results suggest that OsbZIP46 is a positive regulator of ABA signaling and drought stress tolerance of rice depending on its activation. The stress-related genes activated by OsbZIP46CA1 are largely different from those activated by the other rice ABF/AREB homologs (such as OsbZIP23), further implying the value of OsbZIP46CA1 in genetic engineering of drought tolerance.

An understanding of how plants respond to various adverse environmental stresses is a prerequisite for discovering promising genes; therefore, such knowledge could provide useful insights for generating crop plants with improved stress tolerance. A complex network of stress signaling and regulation of gene expression exists in plants responding and adapting to the stresses. The stress signals are perceived through diverse known and unknown sensors and transduced by various signaling components, including many second messengers, phytohormones, signal transducers (such as protein kinases and phosphatases), and transcriptional factors, resulting in the activation of a large number of stress-related genes and the synthesis of diverse functional proteins in plants that finally lead to various physiologic and metabolic responses to adapt to the stresses (Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006; Hirayama and Shinozaki, 2010; Matsukura et al., 2010; Lata and Prasad, 2011).

The phytohormone abscisic acid (ABA) controls various processes of plant growth, including seed germination and development and abiotic stress tolerance (particularly drought tolerance). ABA has been the most extensively studied stress-related hormone in plants, although the roles of other phytohormones, such as cytokinins, brassinosteroids, and auxins, in stress-related processes are emerging (Cutler et al., 2010; Hubbard et al., 2010; Peleg and Blumwald, 2011). Both ABA-dependent and ABA-independent processes are involved in stress responses (Shinozaki et al., 2003; Yamaguchi-Shinozaki and Shinozaki, 2006). The understanding of the molecular basis of ABA responses in plants has been improved dramatically mainly due to the recent exciting breakthroughs in unveiling the core signaling of ABA. The major events are the identification of the PYR/PYL/RCAR receptors of ABA and the establishment of the details of one of the ABA signaling pathways in Arabidopsis (Arabidopsis thaliana). According to the core signaling model, the binding of ABA to the receptors PYR/PYL/RCAR inhibits the type 2C protein phosphatases, resulting in the activation of SNF1-related type 2 protein kinases (SnRK2s), which can target some ion channels and ABA-dependent gene expression by phosphorylating the bZIP transcription factors (Geiger et al., 2009, 2010; Ma et al., 2009; Park et al., 2009; Umezawa et al., 2009, 2010; Cutler et al., 2010; Hubbard et al., 2010; Nishimura et al., 2010; Raghavendra et al., 2010). Although the major components of ABA signaling remain unknown in other plants, analyses of homologous ABA signaling genes and complementation tests suggest that a similar pathway of ABA core signaling as in Arabidopsis also exists in economically important crops such as rice (Oryza sativa; Kobayashi et al., 2004; Fujii and Zhu, 2009; Fujita et al., 2009; Fujii et al., 2011).

In the ABA signaling-mediated stress responses, many transcription factors have crucial regulatory roles in activating ABA-dependent stress-responsive gene expression. Among the transcription factors, members of the bZIP family, which contain a basic region and a Leu zipper domain, have been identified for their function in the ABA-dependent pathway by recognizing ABA-responsive elements (ABREs) containing an ACGT core motif. As a big transcription factor family, quite a few bZIP transcription factors have been well characterized functionally with diverse roles in many aspects, including pathogen defense, light signaling, seed maturation, flower development, and, especially, abiotic stress responses and hormone signal transduction (Uno et al., 2000; Jakoby et al., 2002; Nijhawan et al., 2008). Among the studies of the bZIP family, a noteworthy aspect is that the function of the third subfamily (also known as the AREB/ABF/ABI5 subfamily) has been well elucidated because of the prominent roles of this subfamily in the ABA signaling pathway. ABI5 is a genetically identified ABA signaling component that plays an essential role in seed germination and ABA-triggered developmental arrest processes after germination in Arabidopsis. In contrast, ABFs or AREBs function mostly in the vegetative stage (Choi et al., 2000; Uno et al., 2000; Kang et al., 2002; Kim et al., 2004; Fujita et al., 2011). However, recent reports confirmed that the AREB/ABF/ABI5 members are major targets of SnRK2 protein kinases in ABA core signaling (Fujii and Zhu, 2009; Fujita et al., 2009). Noticeably, the members AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABA signaling involved in multiple stress responses and require ABA for full activation (Yoshida et al., 2010). AREB1/ABF2 has been further documented for its posttranslational modification, which is related to its activity regulation (Fujita et al., 2005; Furihata et al., 2006). Thus far, SnRK2-AREB/ABF has been proven as a major module regulating ABA-mediated gene expression in response to abiotic stresses in Arabidopsis (Kim et al., 2004; Fujita et al., 2005; Furihata et al., 2006; Fujii et al., 2009; Yoshida et al., 2010).

Rice plants have 89 putative bZIP transcription factor genes (Nijhawan et al., 2008). Several members have been studied for their functions potentially related to stress responses, such as LIP19 (Shimizu et al., 2005), OsBZ8 (Nakagawa et al., 1996; Mukherjee et al., 2006), and RF2a and RF2b (Dai et al., 2004, 2008). Noticeably, in contrast to the comprehensive studies of ABFs in Arabidopsis, mainly three members of the third subfamily in rice, TRAB1, OsABI5, and OsbZIP23, have been studied for their roles in ABA-mediated stress responses (Hobo et al., 1999; Xiang et al., 2008; Zou et al., 2008). TRAB1, which is identified by yeast two-hybrid screening for proteins that interact with the seed development-related transcription factor VP1, can be activated by ABA-dependent phosphorylation in its Ser-102 residue (Hobo et al., 1999; Kagaya et al., 2002; Kobayashi et al., 2005), but its biologic function remains to be clarified. OsABI5, named according to its homology to ABI5 in Arabidopsis, was suggested to be involved in ABA signal transduction and stress responses (Zou et al., 2007, 2008). OsbZIP23 was characterized as a key player of the bZIP family for conferring ABA sensitivity, salinity, and drought tolerance of rice (Xiang et al., 2008). Several other members were also mentioned for their involvement in ABA and stress (Zou et al., 2007; Lu et al., 2009; Amir Hossain et al., 2010). Among the third (or AREB/ABF/ABI5) bZIP subfamily of rice, there are dozens of stress-responsive ABF homologs including the reported members (Xiang et al., 2008). The functional redundancy or specificity for a specific member of this family in the stress responses remains to be addressed.

In this paper, we report the functional analysis of OsbZIP46, another member of the third subfamily that has high sequence identity to OsbZIP23. This gene was also named as ABI5-like1 (ABL1) based on the decreased ABA sensitivity of a knockout mutant of this gene (Yang et al., 2011). Overexpression of intact OsbZIP46 showed no significant effect on drought resistance. We identified a constitutive active form of OsbZIP46, OsbZIP46CA1, by mutagenesis, and overexpression of OsbZIP46CA1 could activate the expression of the downstream genes and increase drought resistance. We also found that the posttranslational modification of OsbZIP46 may be required for its function.

RESULTS

Identification of OsbZIP46 as a Stress-Responsive bZIP Factor

We identified the full-length cDNA of OsbZIP46, designated according to Nijhawan et al. (2008), in a cDNA library of Minghui 63 (Chu et al., 2003; Zhang et al., 2005; Supplemental Fig. S1). To speculate on the function of OsbZIP46, we checked the expression profile of OsbZIP46 under different abiotic stresses and phytohormone treatments by real-time quantitative reverse transcription (RT)-PCR. We found that the expression level of OsbZIP46 was induced by drought, ABA, and indole-3-acetic acid (IAA; Fig. 1, A and B), consistent with a previous report (Yang et al., 2011). In addition, we found that the expression of OsbZIP46 was affected by temperature stress (induced by heat but repressed by cold), oxidative stress (induced by hydrogen peroxide [H2O2]), and cytokinin treatment (repressed by kinetin; Fig. 1, A and B) but only slightly by salt stress. The promoter sequence of OsbZIP46 contains many putative stress response-related cis-elements, such as ABRE element (nine hits), MYB recognition site (13 hits), MYC recognition site (10 hits), and one hit of DRE/CRT element (Fig. 1C). In addition, transient expression assays in rice protoplast suggested that the OsbZIP46-GFP fusion protein was located in the nucleus; the nuclear localization was confirmed by its colocalization with the cyan fluorescent protein (CFP)-fused nuclear protein GHD7 (Supplemental Fig. S2), indicating that OsbZIP46 is a nuclear protein, consistent with the result obtained in onion (Allium cepa) cells (Yang et al., 2011).

Figure 1.
Expression analysis of OsbZIP46 under stress and hormone treatments. A, Expression level of OsbZIP46 under stresses including drought (time course 0, 3, and 5 d, recovery 12 h), salt (0, 3, 6, and 12 h), cold (0, 3, 6, and 12 h), heat (0, 2, 6, and 12 ...

Increased ABA Sensitivity of Transgenic Plants with Overexpression of OsbZIP46

According to the features of OsbZIP46 described above, we hypothesized that OsbZIP46 may have a positive role in ABA signaling in rice. To confirm this, we generated the transgenic rice overexpression OsbZIP46. We first checked the ABA sensitivity of transgenic plants at the germination stage. Seeds of three independent overexpression lines, two negative lines, and the wild-type Zhonghua11 were germinated in one-half-strength Murashige and Skoog (1/2MS) medium containing ABA with a gradient of concentrations (0, 1, 3, and 6 μm). The germination rate of the overexpression lines was identical to the negative lines and the wild-type Zhonghua11 at 0 and 1 μm ABA, but it was significantly lower than that of the negative lines and the wild type at 3 and 6 μm ABA (Fig. 2, A and B), suggesting that the ABA sensitivity (in terms of seed germination) of OsbZIP46 overexpression plants was increased. We also investigated the ABA sensitivity of transgenic plants at the postgermination stage. The lengths of shoot and root of overexpression seedlings grown for 2 weeks in 1/2MS medium containing 3 μm ABA were significantly shorter compared with those of the negative lines and the wild type, but no difference was observed for seedlings grown in the medium without ABA (Fig. 2, C–E). These results suggested that overexpression of OsbZIP46 can increase ABA sensitivity at postgermination stages. Together with the decreased ABA sensitivity of the mutant of this gene (Yang et al., 2011), we propose that OsbZIP46 is a positive regulator of ABA signaling in rice.

Figure 2.
Increased ABA sensitivity of OsbZIP46 overexpression plants at germination and seedling stages. A, Germination performance of OsbZIP46 overexpressor (OE15) and wild-type (WT) seeds on 1/2MS medium containing 0, 1, 3, or 6 μmol L−1 ABA ...

Performance of the Transgenic OsbZIP46 Overexpression Plants under Abiotic Stress

It has been generally accepted that a positive regulator of ABA signaling may also contribute to the tolerance of plants to drought stress. Therefore, we examined the performance of OsbZIP46 overexpression plants under drought stress with four independent overexpression lines compared with the wild type. To our surprise, the overexpression lines showed slightly decreased drought tolerance at the seedling and reproductive stages. At the seedling stage, the survival rate of overexpression lines was lower than that of the wild type after drought stress (Fig. 3, A and B). At the reproductive stage, the overexpression lines, drought stressed at the panicle development stage, showed lower relative spikelet fertility compared with the wild type (Fig. 3, C and D). We repeated this testing several times and obtained the same results. We also tested the overexpression lines for salt stress tolerance but found no significant difference between the overexpression lines and the wild type (data not shown). These results suggest that overexpression of the native OsbZIP46 gene may have a negative effect on drought stress tolerance.

Figure 3.
Drought tolerance testing of OsbZIP46 overexpression transgenic rice. A, Phenotype of seedlings of OsbZIP46 overexpression plants under drought stress. Seedlings from four overexpression lines (OE2, OE9, OE13, and OE15) were grown in barrels each with ...

We checked the expression levels of RAB21, a commonly used marker gene activated by AREB in ABA and drought responses, in the OsbZIP46 overexpression plants. The expression level of RAB21 in the OsbZIP46 overexpression plants showed no difference compared with that in the wild type under normal growth conditions; however, it was significantly increased after exogenous application of ABA (Fig. 3E). This result is in contrast to the result obtained in the OsbZIP23 overexpression rice plant, in which RAB21 showed constitutive elevated expression (Xiang et al., 2008). Actually, a large number of drought-responsive genes were not up-regulated in the OsbZIP46 overexpression plant, as discussed below. Therefore, we suspected that the native OsbZIP46 protein may not be able to activate the expression of downstream genes and that its activity may be activated through the ABA signaling pathways.

Activation of the Transcriptional Activity of OsbZIP46

The negative effect of OsbZIP46 overexpression on drought stress tolerance prompted us to check the transcriptional activity of OsbZIP46, because it was predicted to be a typical bZIP transcription factor. First, we tested the transactivation activity of OsbZIP46 in yeast. Unlike its close homolog OsbZIP23 (Xiang et al., 2008), the full-length OsbZIP46 protein fused with the GAL4-binding domain had no transactivation activity in yeast. Partial fragments of OsbZIP46 with a series of deletions were then tested. The results showed that the mutated forms with a deletion of 204 or 264 amino acids from the C terminus could activate the expression of reporter genes, suggesting that the transactivation domain of OsbZIP46 is located in the N-terminal part of the protein. We noticed that the activity was detected only when domain D was absent, suggesting that domain D may have a prominent role in the regulation of the transactivation activity of OsbZIP46. To confirm this, we generated a mutated form of OsbZIP46, which contains 120 amino acids from the N terminus (containing domains A, B, and C) as a transactivation domain and 105 amino acids from the C terminus (containing the bZIP DNA-binding domain and domain E) while the middle part (99 amino acids containing domain D) is absent. We further checked the activity of the mutated form and found that it has constitutive transactivation activity in yeast, and this mutated form was designated as OsbZIP46CA1 (for OsbZIP46 constitutive active form 1; Fig. 4).

Figure 4.
Transactivation assay of OsbZIP46. A, Expression construct of OsbZIP46 in yeast. B, Transactivation assay of truncated OsbZIP46. Fusion proteins of the GAL4 DNA-binding domain and different portions of OsbZIP46 were checked for their transactivation activity ...

Performance of Transgenic Rice with Overexpression of OsbZIP46CA1 under ABA Treatment or Abiotic Stress

Because the domain D-missing mutated form OsbZIP46CA1 showed constitutive transactivation activity in yeast, we wondered if it has functions different from the native protein in rice. Therefore, transgenic rice with overexpression of OsbZIP46CA1 was generated and tested for stress tolerance.

We first checked the sensitivity to ABA. As expected, the OsbZIP46CA1 overexpression (CA1-OE hereafter) plants showed increased ABA sensitivity, just like the intact OsbZIP46 overexpressors (Fig. 5A). We were more interested in the performance of CA1-OE plants under drought stress. The CA1-OE lines showed significantly increased drought resistance at both the seedling and reproductive stages, in contrast to the results of the overexpressor of full-length OsbZIP46 presented above. At the seedling stage, the survival rate of CA1-OE lines was significantly higher compared with the wild-type control after drought stress (Fig. 5, B and C). At the reproductive stage in the field, the results indicated that overexpression of OsbZIP46 can also improve drought resistance (Fig. 5, D and E). The water loss rate of the CA1-OE plants was significantly lower than that in the wild type and the negative lines (Fig. 5F), which supported the improved drought resistance phenotype. In addition, the CA1-OE lines were tested for tolerance to osmotic stress in comparison with OsbZIP46 overexpression plants and the wild-type Zhonghua11. We evaluated the osmotic tolerance by using the relative shoot length of plants as a criterion, because the CA1-OE lines grew a little lower than the wild type. OsbZIP46CA1 overexpression had a positive effect on the osmotic stress tolerance, whereas OsbZIP46 overexpression had a slightly negative effect on osmotic tolerance (Supplemental Fig. S6).

Figure 5.
ABA sensitivity and drought tolerance testing of OsbZIP46CA1-OE rice. A, Phenotypes of native OsbZIP46 overexpression lines (FL-OE9 and FL-OE15), OsbZIP46CA1-OE (CA1-OE5 and CA1-OE7), and the wild type (WT) under medium containing 3 μm ABA. B, ...

Distinct Expression Profiles in the OsbZIP46CA1 and OsbZIP46 Transgenic Lines

The different effects of OsbZIP46 and OsbZIP46CA1 overexpression on drought resistance might be attributed to different downstream genes activated because OsbZIP46CA1 showed constitutive transactivation activity. To confirm this hypothesis and elucidate the molecular function of this gene, we compared expression profile changes in the OsbZIP46-OE and OsbZIP46CA1-OE plants (three independent lines were checked for each overexpressor) by using Affymetrix GeneChip. Compared with the wild type, transcriptomes of both overexpressors had significant changes. With a threshold of 2-fold change, a total of 391 and 469 genes were up- and down-regulated, respectively, in the OsbZIP46CA1-OE plants, whereas in the OsbZIP46-OE plants, 119 and 390 genes were up- and down-regulated, respectively (Fig. 6, A and B). Among those genes, only 51 and 100 genes were up- and down-regulated, respectively, in both overexpressors. Interestingly, 13 genes showed opposite expression change patterns between the two overexpressors (Fig. 6C). These results indicate that significantly more genes were affected by OsbZIP46CA1 overexpression than by native OsbZIP46 overexpression, and most of the genes affected in the two overexpressors are different. Quantitative RT-PCR was performed to check the differently regulated genes in the two overexpressors. Among the nine genes checked, all showed the same expression patterns as in the gene chip results (Fig. 6E; Supplemental Fig. S7).

Figure 6.
Gene chip analysis of OsbZIP46 overexpressors. A and B, Scatterplots of the expression profiles of whole-genome genes in FL-OE (A) and CA1-OE (B) compared with the wild type (WT). The x and y axes indicate the chip hybridization signal in the overexpressor ...

Gene Ontology (GO) analysis of the differently regulated genes in the two overexpressors revealed that genes in several GO terms under the term “Biological processes” were significantly overrepresented. The GO term with the highest proportion of differently regulated genes is “Response to stimulus” (biotic, abiotic, and endogenous stimuli, etc.), followed by GO terms such as “Signal transduction,” “Protein modification,” “Transcription,” “Metabolism,” and “Biosynthesis” (Supplemental Table S1). “Transcription factor activity” and “Nucleus” have the highest proportion of differently regulated genes under “Molecular function” and “Cellular component,” respectively (Supplemental Table S1).

Because the two overexpressors showed distinct transcriptome changes and most of the differently regulated genes were attributed to the “Response to stimulus” category, we further classified all these genes based on their change patterns in the overexpressors and responsiveness to drought stress. Based on the published microarray results (Jain et al., 2007), more than half of these genes were regulated by drought stress. Among the 1,204 expression-affected genes, 455 and 202 were up- and down-regulated, respectively, by drought (Supplemental Tables S2S5). Interestingly, most of the genes up-regulated and down-regulated in both overexpressors were down- and up-regulated, respectively, by drought stress (Fig. 6D). The genes differentially regulated in the two overexpressors can be classified into five groups. Most of the genes in group I (up-regulated in CA1-OE but not changed in OsbZIP46-OE) and group III (up-regulated in CA1-OE but with opposite changes in OsbZIP46-OE) were induced by drought stress, whereas most of the genes in group II (down-regulated in CA1-OE but not changed in OsbZIP46-OE) were suppressed by drought stress (Fig. 6D). Notably, many of the genes up-regulated only in CA1-OE have been annotated or confirmed for stress response or adaptation-related functions, including dehydrin or late embryogenesis abundant (LEA) proteins (five genes), stress-related transcription factors (27 genes), kinases (seven genes), phosphatases (eight genes), amino acid metabolism or transportation proteins (three genes), aquaporin (two genes), and lipid transfer proteins (five genes; Supplemental Table S2). This implies that the increased drought tolerance of CA1-OE may result from the up-regulation of these genes. Interestingly, the differentially expressed genes only in OsbZIP46-OE (groups IV and V) showed distinct trends of responsiveness to drought; most of the genes up-regulated in OsbZIP46-OE showed drought-suppressed expression patterns, whereas most of the down-regulated genes in OsbZIP46-OE showed drought-induced expression (Fig. 6D). Especially among these down-regulated genes in OsbZIP46-OE, there are many genes annotated or confirmed for stress response or adaptation, including many transcription factors (40 genes), kinases (35 genes), and some other stress-related functional proteins (LEA protein, lipid-transfer protein; Supplemental Table S5). This result may partially explain the reason that overexpression of OsbZIP46 resulted in decreased drought tolerance.

Because so many genes were differentially regulated by OsbZIP46 and OsbZIP46CA1 overexpression, we further analyzed the promoter sequences of these genes. The results suggested that 54% of these genes have enriched cis-elements featuring ABRE that are potential binding sites of ABF or AREB proteins homologous to OsbZIP46 (Supplemental Table S6). Interestingly, the genes up-regulated in CA1-OE but not changed or with opposite change patterns in the OsbZIP46 overexpressor (groups I and III) and genes down-regulated in OsbZIP46-OE (group IV) had higher ratios of the enriched cis-elements (62% and 63%, respectively) than the other two groups (42% and 45%).

Three genes, Os11g26790 (also designated as RAB21, encoding a LEA protein), Os03g19290 (a putative mitochondrial import inner membrane translocase subunit), and Os05g38290 (a protein phosphatase 2C), that were up-regulated only in OsbZIP46CA1-OE but not in OsbZIP46-OE, were selected to test if OsbZIP46 can directly act on these differentially regulated genes. The pGAD-OsbZIP46 plasmid (containing the putative DNA-binding domain of OsbZIP46 fused to the GAL4 activation domain) and the reporter construct pHIS-cis (containing the putative ABRE-containing promoters of the three genes) were cotransformed into yeast strain Y187. The reporter gene was activated for all three cotransformations (Supplemental Fig. S8), indicating that OsbZIP46 can bind the promoters of these genes.

OsbZIP46 May Be Activated by Phosphorylation

Significant differences in affecting gene expression between the constitutive activated and native forms of OsbZIP46 indicated that the activity of native OsbZIP46 might be regulated by posttranslational modifications. In Arabidopsis, stress/ABA-activated protein kinases (SAPKs) or SnRK2 have been suggested for phosphorylating ABFs or AREBs (Furihata et al., 2006; Fujii and Zhu, 2009; Fujita et al., 2009; Yoshida et al., 2010). Therefore, we checked nine putative SAPK family members from rice with OsbZIP46 for yeast two-hybrid analysis. The results suggested that OsbZIP46 could interact with OsSAPK2, -6, and -9 (Fig. 7A). The interaction between OsbZIP46 and OsSAPKs was further confirmed by bimolecular fluorescence complementation (BiFC; Fig. 7B). An in vitro phosphorylation assay demonstrated that the OsbZIP46 protein could be phosphorylated by SAPK2 and SAPK6 (Fig. 7C).

Figure 7.
Interaction between OsbZIP46 and SAPKs may be activated by phosphorylation. A, Yeast two-hybrid assays of OsbZIP46 and SAPK members. Control A and control C indicate the positive and negative controls, respectively. 3-AT, 3-Amino-1,2,4-triazole; SC-LTH, ...

Sequence analysis suggested that five putative phosphorylation target sites are present in OsbZIP46 (Fig. 7D). We inferred that the transcriptional activity of OsbZIP46 might also be related to these putative phosphorylation target sites. To confirm this, we generated single or multiple amino acid substitutions (Ser/Thr to Asp) of OsbZIP46 (Asp provides a negative charge and can mimic the phosphorylated status) and checked their transactivation activity. The single-substitution mutant PA1 (for phosphorylation active form 1; with substitution of Thr-129 to Asp) showed slightly increased transactivation activity. But the multiple (three to five) amino acid substitutions could significantly enhance the transactivation activity of OsbZIP46 (Fig. 7, D and E). Taken together, these results suggest that the native OsbZIP46 may be activated by posttranslational phosphorylation modification.

DISCUSSION

Constitutive Activation Is a Useful Tool for Functional Analysis of Transcription Regulators

Many reports suggest that overexpression of some stress-inducible transcription factors can increase the abiotic stress tolerance of plants (Ciftci-Yilmaz and Mittler, 2008; Dubos et al., 2010; Matsukura et al., 2010; Lata and Prasad, 2011), but some special members need additional modifications to exhibit their full functions. For example, overexpression of DREB2A in transgenic plants could not improve stress tolerance, but overexpression of DREB2ACA, carrying a deletion of the central negative regulatory domain (NRD), activated the expression of many stress-inducible genes and improved tolerance to drought and heat stress in transgenic Arabidopsis (Sakuma et al., 2006a, 2006b). Similarly, AREB1, a homologous gene of OsbZIP46, has been reported to fully function after posttranslational modification (Fujita et al., 2005). However, such modification of transcription factors for stress tolerance was seldom reported in crops.

Although OsbZIP46/ABL1 has recently been reported as a positive regulator of ABA signaling by mutant analysis of this gene (Yang et al., 2011), its actual role in regulating stress tolerance remains unknown. Normally, overexpression of positive regulators of ABA signaling from the bZIP family, such as OsbZIP23 (Xiang et al., 2008) and OsbZIP72 (Lu et al., 2009), can result in increased drought tolerance. However, OsbZIP46 overexpression rice did not display the expected increase but a decrease of drought tolerance. We found that the intact OsbZIP46 was insufficient to induce the expression of the target gene unless the exogenous ABA was applied. In yeast, full-length OsbZIP46 had no transactivation activity. Considering the fact that OsbZIP46 is a homolog of TRAB1, which was previously reported with ABA-dependent phosphorylation activity (Kagaya et al., 2002), these preliminary results suggest that the function of OsbZIP46 may also need posttranslational level modifications. Therefore, we managed to produce a constitutively active form of OsbZIP46 in yeast and tested its function for stress tolerance in rice.

As expected, OsbZIP46CA1 overexpression significantly increased the drought tolerance of rice at both the seedling and reproductive stages, which is in contrast to the drought-sensitive phenotype of OsbZIP46 overexpression plants. Microarray analysis of the two overexpressors provided strong evidence for the differences in drought tolerance. In short, many stress tolerance-related genes were activated only in the OsbZIP46CA1 overexpression lines but not in the OsbZIP46 overexpression lines, supporting the hypothesis that OsbZIP46CA1 also possesses constitutive transcriptional activity in rice and thus activates the expression of stress-related target genes, leading to increased drought tolerance. Therefore, the generation of constitutive activated forms for posttranslational modification-required regulatory proteins (at least for some transcription factors) seems to be a promising way to probe their functions, which may be especially true when functional redundancy exists for the gene being studied or overexpression of the native form of the gene results in unexpected outcomes, as is the case for OsbZIP46 discussed below.

Negative Effect of OsbZIP46 Overexpression on Drought Tolerance

We observed that overexpression of both OsbZIP46 and OsbZIP46CA1 increased ABA sensitivity. However, the OsbZIP46 overexpressor showed slightly reduced drought tolerance. Logically, increased expression of a positive regulator of ABA signaling will most likely have some positive effect, or at least not negative, on stress tolerance, even if the regulator needs ABA- or stress-triggered posttranslational modification for its full activity. We propose that OsbZIP46 is an activation-required transactivator, but its activation varies depending on the stresses or ABA treatment. Because OsbZIP46 is one of the positive regulators of ABA signaling (Yang et al., 2011), we propose that overexpressed OsbZIP46 may just match up with the high dosage requirement for mediating the signaling triggered by exogenous ABA. While under the drought stress that triggers ABA signaling more slowly than does the exogenous application of ABA, the preaccumulated OsbZIP46 in the overexpressor might have a negative effect on mediating stress signaling through endogenous ABA. This is very likely true, because the native OsbZIP46 contains a negative domain (domain D) for activation activity in yeast. Microarray analysis revealed that arrays of drought stress-related genes were actually down-regulated in the OsbZIP46 overexpressor (Fig. 6D; Supplemental Table S5), and some of these genes were not induced or were significantly less induced by drought compared with those in the wild type (data not shown). Nevertheless, the decreased drought resistance might also result from other unknown negative effects of the overexpression of OsbZIP46.

Possible Mechanisms for the Constitutive Activity of OsbZIP46CA1

Because the deletion of domain D of OsbZIP46 resulted in constitutive transactivation activity in yeast (Supplemental Fig. S11), we proposed that domain D is a negative regulatory domain and has a pivotal role in the regulation of OsbZIP46 activity. It has been reported that the conserved Leu in the LxLxL motif is important for repression in some transcription repressors, such as the ethylene response factor (ERF)-associated amphiphilic repression (EAR) motif found in some ERFs and SUPERMAN (Fujimoto et al., 2000; Ohta et al., 2001; Hiratsu et al., 2002, 2003; Kazan, 2006) and the repression domain found in auxin/IAA genes (Tiwari et al., 2004). We compared the sequence of domain D with the conserved EAR motif. Although the LxLxL motif was not found, domain D contains a sequence signature of LxxxxLxxxL (Supplemental Fig. S1D). In addition, considering that DREB2A contains a PEST sequence in the NRD that plays a role in the stability of the protein (Sakuma et al., 2006a), and the PEST sequence, existing in some rapid-turnover proteins, serves as a signal for proteolytic degradation and regulates the activity of proteins by controlling their accumulation (Rechsteiner and Rogers, 1996), we wondered whether domain D of OsbZIP46 has a similar role as NRD in DREB2A. However, no potential PEST sequence was identified in OsbZIP46 using the epestfind server (http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind; data not shown). It would be interesting to further test which part of domain D is essential for the negative effect on transaction activity.

It cannot be excluded that the deletion of domain D may cause a conformational change that mimics the changes resulting from posttranslational modification, such as phosphorylation of the native OsbZIP46. Here, our results showed that the transactivation activity of OsbZIP46 in yeast was related to multiple putative phosphorylation sites and that OsbZIP46 may be phosphorylated by three homologs of SnRK2 kinases, OsSAPK2, OsSAPK6, and OsSAPK9 (Fig. 7). An in vitro phosphorylation assay confirmed that OsbZIP46 protein can be phosphorylated by OsSAPK2 and OsSAPK6. Because we failed to obtain soluble expressed proteins for OsbZIP46CA1, it remains to be checked if there is any change in the phosphorylation of OsbZIP46CA1 by the OsSAPKs. It has been well documented that the phosphorylation of ABF/AREB by SnRK2 is an essential part of the complete ABA signaling pathway in Arabidopsis (Fujii and Zhu, 2009; Fujita et al., 2009). Moreover, the genes of the SnRK2 family (also designated SAPK, for osmotic stress/ABA-activated protein kinase) in rice were identified for their ABA-inducible characteristics (Kobayashi et al., 2004). Especially TRAB1, a homolog of OsbZIP46, has been identified as phosphorylated by SAPKs (Kagaya et al., 2002; Kobayashi et al., 2005). RAB21, the putative target gene of OsbZIP46, was activated in OsbZIP46 overexpression rice plants only after treatment with exogenous ABA. These results together imply that the transcriptional activity of OsbZIP46 may be mainly regulated by ABA-dependent phosphorylation.

Specificity of OsbZIP46 Compared with Other ABF Members

OsbZIP46 is member of the third (or ABF/AREB/ABI5) subfamily of bZIP transcription factors. Although most of the ABF members are thought to be involved in ABA and abiotic stress responses, according to studies in Arabidopsis, the results from OsbZIP46 suggest that it has some specificity in addition to its common features compared with other ABF members.

At the protein sequence level, most members in the third subfamily of the bZIP family, including OsbZIP46, contain a typical basic region and the Leu zipper domain as well as one of five other conserved motifs (named domains A, B, C, D, and E, respectively) that are predicted to contain five phosphorylation sites, suggesting that most members of the ABF subfamily maybe involved in stress or ABA signaling through phosphorylation by the protein kinase. Moreover, the third subfamily bZIP members of rice and Arabidopsis could be further classified into four groups (I–IV) according to a phylogenetic analysis (Xiang et al., 2008). OsbZIP46 belongs to group II, which includes the previously reported stress or ABA-related rice bZIP members (TRAB1, OsbZIP23, and OsABI5) and has the highest similarity to ABF1 to -4 of Arabidopsis (Hobo et al., 1999; Xiang et al., 2008; Zou et al., 2008).

However, our results showed that the expression of OsbZIP46 is inducible by drought, heat, H2O2, and ABA treatment and repressed by cold but apparently not affected by salt stress. These results suggest a different expression profile compared with OsbZIP23, in which expression is inducible by drought, salt, and ABA treatment but not cold (Xiang et al., 2008). We compared the expression profile of OsbZIP46 across various tissues and stages with its homology members by searching the expression profiles database CREP (Wang et al., 2010) and found that their expression patterns were also different (Supplemental Fig. S3). The OsbZIP46 overexpression rice showed unexpectedly reduced drought tolerance, which is distinct from the OsbZIP23 overexpression rice, which showed significantly increased drought and salt stress tolerance (Xiang et al., 2008). This may suggest that different members of the third bZIP subfamily may function in stress tolerance with a different spectrum of stresses or different degrees of the same stress. To support this, we compared the transcriptomes of the OsbZIP46 (OsbZIPCA1 as well) and OsbZIP23 overexpression plants and found that the genes affected by overexpression of the two ABF members are largely different (only less than one-quarter of the regulated genes are overlapped; Supplemental Fig. S9; Supplemental Table S7). Furthermore, unlike OsbZIP23 and most other members in this subfamily, the native form OsbZIP46 has no transactivation activity in yeast. This further suggests that the activity of OsbZIP46 in planta may strongly rely on the posttranslational modification. However, considering that the domain D sequence and putative phosphorylation target sites also exist in other ABF members, it remains intriguing to identify which part of domain D or which phosphorylation target sites determine the specificity of stress responses.

CONCLUSION

We characterized a constitutive active form of transcription factor OsbZIP46. Comparison analysis revealed that OsbZIP46 is a positive regulator of ABA signaling and regulates drought stress resistance of rice by modulating many stress-related genes, and its function on activating downstream genes depends on posttranslational modification. This constitutive active form of OsbZIP46 has promising usefulness in transgenic breeding of drought resistance.

MATERIALS AND METHODS

Generation of Transgenic Rice Plants

The full-length cDNA of OsbZIP46 was obtained from a cDNA library of Minghui 63 rice (Oryza sativa indica) and confirmed by sequencing with primers SP6 (5′-ATTTAGGTGACACTATA-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′). The sequence-confirmed clone containing the full-length cDNA of OsbZIP46 was digested by KpnI and BamHI and cloned into the KpnI and BamHI sites of the binary expression vector pCAMBIA1301U (driven by a maize [Zea mays] ubiquitin promoter). The construct was introduced into Zhonghua11 rice (Oryza sativa japonica) by Agrobacterium tumefaciens-mediated transformation. To create the overexpression construct of OsbZIP46CA1, the OsbZIP46CA1 coding region was PCR amplified with KpnI-BamHI (indicated by lowercase letters) linker primers 5′-ATAggtaccATGGAGTTGCCGGCGGATG-3′ and 5′-ATAggatccTCAGCATGGACCAGTCAGTG-3′ based on the template of a truncated fragment of OsbZIP46 with an internal deletion. The sequence-confirmed PCR fragment of OsbZIP46CA1 was also cloned into pCAMBIA1301U and then transformed into Zhonghua11.

Stress Treatment of Plant Materials

To check the expression level of the OsbZIP46 gene under various abiotic stresses or phytohormone treatment, Zhonghua11 rice plants were grown in soil (for drought, cold, and heat stress) or hydroponic culture medium (for others) for about 3 weeks under normal conditions. The seedlings at the four-leaf stage were treated with abiotic stress, including drought stress (removing the water supply), salt stress (200 mm NaCl), heat stress (exposing plants to 42°C), cold stress (seedlings were transferred to a growth chamber at 4°C), and oxidative stress (treated with 1% H2O2 solution), followed by sampling at the designated times. For phytohormone treatment, 0.1 mm ABA, brassinosteroid, IAA, kinetin, GA, or jasmonic acid was sprayed on the leaves and added to the culture medium of the rice plants.

To test the ABA sensitivity or osmotic stress tolerance of transgenic plants at the seedling stage, positive transgenic lines were selected by germinating seeds on 1/2MS medium containing 25 mg L−1 hygromycin; negative transgenic lines and the wild type were also germinated on normal 1/2MS medium. The seedlings (12 plants each, three repeats) were transplanted to normal 1/2MS medium or 1/2MS medium containing 3 μm ABA or 150 mm mannitol and grown for 14 d. For testing the ABA sensitivity of transgenic plants at the germination stage, the positive T3 transgenic families, negative transgenic lines, and the wild type (30 seeds each, three repeats) were germinated on 1/2MS medium containing a gradient concentration of ABA (0, 1, 3, and 6 μm), and the germination rate of the treated seeds was calculated after 13 d.

To test the other abiotic stress tolerances of transgenic plants, the positive transgenic lines were also germinated on hygromycin-containing medium. For testing at the seedling stage, positive transgenic and wild-type plants (30 plants each, three repeats) were grown in a half-and-half manner in barrels filled with sandy soil. At the four-leaf stage, stress testing was conducted, including drought (irrigation was withheld for 1 week followed by recovery for 1 week) and salt (irrigated with 200 mm NaCl solution for 1 week). Drought testing at the reproductive stage was conducted in polyvinyl chloride (PVC) pipes; the details of drought treatment and trait measurement for these tests were basically the same as described previously (Yue et al., 2006; Xiao et al., 2007). One positive transgenic plant and one wild-type plant each were planted per PVC tube (10 plants each). Drought stress was initiated at the panicle development stage by discontinuing watering of plants, and the plants were recovered with irrigation at the flowering and seed maturation stages.

To detect the water loss rate under dehydration conditions, leaves of positive transgenic plants, negative transgenic controls, and wild-type plants were cut, exposed to air at room temperature, and weighed at the designated times.

Quantification of Gene Expression

Total RNAs of the rice leaves were extracted using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The DNase-treated RNA was reverse transcribed using SuperScript reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Real-time quantitative PCR was performed on an optical 96-well plate with an ABI PRISM 7500 real-time PCR system (Applied Biosystems) using SYBR Premix Ex Taq (TaKaRa). The PCR thermal cycles were as follows: 95°C for 10 s, followed by 45 cycles at 95°C for 5 s and 60°C for 34 s. The rice Actin1 gene was used as the endogenous control, and relative expression levels were determined as described previously (Livak and Schmittgen, 2001). For RNA gel blotting, 15 μg of total RNA from each sample was separated on a 1.2% agarose gel containing 1% formaldehyde and then transferred onto a nylon membrane. Hybridization and washing conditions were based on standard protocols (Sambrook et al., 1989).

Subcellular Localization

To determine the subcellular localization of OsbZIP46, OsbZIP46 was cloned into pM999-33 vector to produce an OsbZIP46-GFP fusion construct driven by a cauliflower mosaic virus 35S promoter (35S::OsbZIP46:GFP). Rice protoplasts prepared from etiolated shoots were cotransformed with 35S::OsbZIP46:GFP and 35S::Ghd7:CFP as a nuclear marker by polyethylene glycol treatment. The florescence signal was observed with a confocal microscope (Leica) at 24 h after transformation.

Transactivation and Two-Hybrid and One-Hybrid Assays in Yeast

The single or multiple amino acid substitutions (Ser/Thr to Asp) of OsbZIP46 were produced with the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene). The procedure was performed according to the instruction manual. For the transactivation assay, the intact, point-mutated, or truncated fragments of OsbZIP46 were fused in frame with the yeast GAL4 DNA-binding domain in the vector pDEST32 (Invitrogen). Fusion proteins were expressed in Mav203 yeast cells (MATα; leu2–3,112; trp1–901; his3Δ200; ade2–101; gal4Δ; gal80Δ; SPAL10::URA3; GAL1::lacZ; HIS3UASGAL1::2SYL@3SIH; can1R; cyh2R; Invitrogen). The colony-lift filter assay (X-gal assay) was performed according to the manufacturer’s manual (Invitrogen).

The yeast two-hybrid assay was performed using the ProQuest Two-Hybrid System (Invitrogen). The coding regions of OsbZIP46 and SAPKs were cloned into the vector pDEST32 and pDEST22 using Gateway technology (Invitrogen) to generate bait and prey vectors, respectively. The two vectors were cotransformed into the yeast strain Mav203, and the transformants were identified according to the manufacturer’s instructions.

For the one-hybrid assay, a fusion protein of OsbZIP46 and the GAL4 activation domain was produced in the vector pGADT7-Rec2 (Clontech). Then, the fusion protein was cotransformed with the reporter vector (pHIS2-cis) into Y187 yeast cells (MATα; ura3–52; his3–200; ade2–101; trp1–901; leu2–3, 112; gal4Δ; gal80Δ; met–; URA3::GAL1UAS-GAL1TATA-LacZ; MEL1; Clontech). The DNA-protein interactions were determined by the growth of the transformants on the nutrient-deficient medium. The detailed procedure conducted was derived from the manufacturer’s manual (Clontech).

BiFC

Rice SAPK genes were cloned into the pVYCE vector and fused to the C-terminal 156 to 239 amino acids of yellow fluorescent protein, and OsbZIP46 was cloned into pVYNE vector and fused to the N-terminal 1 to 155 amino acids of yellow fluorescent protein. Combinations of BiFC constructs were expressed transiently in rice leaf protoplasts via polyethylene glycol transformation. The fluorescence was detected by confocal microscopy (Leica).

Protein Phosphorylation Assay

Glutathione S-transferase (GST)-fused OsbZIP46 (or OsbZIP46CA1) and OsSAPK genes were constructed into vector pGEX-6P (GE Healthcare) and expressed in Escherichia coli (BL21). GST fusion protein production was induced by isopropyl-β-d-thiogalactoside (0.1 mm) overnight at 18°C. Bacterial lysates were applied to glutathione Sepharose (GE Healthcare), and GST fusion proteins were eluted with 10 mm glutathione by following the manufacturer’s instructions. For the kinase assay, SAPK and OsbZIP46 proteins were incubated in kinase assay buffer (50 mm Tris-HCl, 10 mm MgCl2, 10 mm MnCl2, 1 mm dithiothreitol, 0.2 mm ATP, and 1 μCi of [γ-32P]ATP). The reaction was incubated at room temperature for 30 min and terminated by the addition of 5 μL of sample buffer and heating at 100°C for 5 min. After separation on a 12% SDS-PAGE gel, the gel was exposed to Kodak x-ray film for detecting the protein phosphorylation.

Microarray Analysis

Three independent OsbZIP46 overexpression lines (30 positive transgenic plants each), three independent OsbZIP46CA1-OE lines (30 positive transgenic plants each), and the wild type (two independent biological replicates; 30 plants each) were sampled for microarray experiments. The process of microarray analysis was conducted according to the standard protocol of the Affymetrix GeneChip service (CapitalBio). The differentially expressed genes (up- or down-regulated) between the overexpression transgenic plants and the control (the wild type) were selected with a significance threshold of P < 0.01 and analyzed with the Excel add-in and MAS 3.0 molecule annotation system (http://bioinfo.capitalbio.com/mas3/; Supplemental Tables S1S7). The results were then confirmed by real-time quantitative RT-PCR.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AK103188.

Supplemental Data

The following materials are available in the online version of this article.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Jian Xu and Lei Wang (Huazhong Agricultural University) for providing plasmid pM999-33 and 35S::Ghd7:CFP, respectively.

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