Constitutive Activation of Transcription Factor OsbZIP46 · Constitutive Activation of...

Constitutive Activation of Transcription Factor OsbZIP46Improves Drought Tolerance in Rice1[C][W][OA]

Ning Tang, Hua Zhang, Xianghua Li, Jinghua Xiao, and Lizhong Xiong*

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research(Wuhan), Huazhong Agricultural University, Wuhan 430070, China

OsbZIP46 is one member of the third subfamily of bZIP transcription factors in rice (Oryza sativa). It has high sequencesimilarity to ABA-responsive element binding factor (ABF/AREB) transcription factors ABI5 and OsbZIP23, two transcrip-tional activators positively regulating stress tolerance in Arabidopsis (Arabidopsis thaliana) and rice, respectively. Expression ofOsbZIP46 was strongly induced by drought, heat, hydrogen peroxide, and abscisic acid (ABA) treatment; however, it was notinduced by salt and cold stresses. Overexpression of the native OsbZIP46 gene increased ABA sensitivity but had no positiveeffect 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 todrought and osmotic stresses. Gene chip analysis of the two overexpressors (native OsbZIP46 and the constitutive active formOsbZIP46CA1) 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 suggestthat OsbZIP46 is a positive regulator of ABA signaling and drought stress tolerance of rice depending on its activation. Thestress-related genes activated by OsbZIP46CA1 are largely different from those activated by the other rice ABF/AREBhomologs (such as OsbZIP23), further implying the value of OsbZIP46CA1 in genetic engineering of drought tolerance.

An understanding of how plants respond to variousadverse environmental stresses is a prerequisite fordiscovering promising genes; therefore, such knowl-edge could provide useful insights for generatingcrop plants with improved stress tolerance. A complexnetwork of stress signaling and regulation of geneexpression exists in plants responding and adapting tothe stresses. The stress signals are perceived throughdiverse known and unknown sensors and transducedby various signaling components, including manysecond messengers, phytohormones, signal trans-ducers (such as protein kinases and phosphatases),and transcriptional factors, resulting in the activation

of a large number of stress-related genes and thesynthesis of diverse functional proteins in plants thatfinally lead to various physiologic and metabolic re-sponses to adapt to the stresses (Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006; Hirayama andShinozaki, 2010; Matsukura et al., 2010; Lata andPrasad, 2011).

The phytohormone abscisic acid (ABA) controlsvarious processes of plant growth, including seedgermination and development and abiotic stress tol-erance (particularly drought tolerance). ABA has beenthe most extensively studied stress-related hormone inplants, although the roles of other phytohormones,such as cytokinins, brassinosteroids, and auxins, instress-related processes are emerging (Cutler et al.,2010; Hubbard et al., 2010; Peleg and Blumwald, 2011).Both ABA-dependent and ABA-independent pro-cesses are involved in stress responses (Shinozakiet al., 2003; Yamaguchi-Shinozaki and Shinozaki,2006). The understanding of the molecular basis ofABA responses in plants has been improved dramat-ically mainly due to the recent exciting breakthroughsin unveiling the core signaling of ABA. The majorevents are the identification of the PYR/PYL/RCARreceptors of ABA and the establishment of the detailsof one of the ABA signaling pathways in Arabidopsis(Arabidopsis thaliana). According to the core signalingmodel, the binding of ABA to the receptors PYR/PYL/RCAR inhibits the type 2C protein phosphatases,resulting in the activation of SNF1-related type 2protein kinases (SnRK2s), which can target some ion

1 This work was supported by the National Program for BasicResearch of China (grant no. 2012CB114305), the National Programon High Technology Development (grant no. 2012AA10A303), theNational Natural Science Foundation of China (grant nos. 30725021and 30921091), and the Project from the Ministry of Agriculture ofChina for Transgenic Research (grant nos. 2009ZX08001–021B and2011ZX001–003).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Lizhong Xiong ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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channels and ABA-dependent gene expression byphosphorylating the bZIP transcription factors (Geigeret 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; Raghavendraet al., 2010). Although the major components of ABAsignaling remain unknown in other plants, analyses ofhomologous ABA signaling genes and complementationtests suggest that a similar pathway of ABA core signal-ing as in Arabidopsis also exists in economically impor-tant 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 regulatoryroles in activating ABA-dependent stress-responsivegene expression. Among the transcription factors,members of the bZIP family, which contain a basicregion and a Leu zipper domain, have been identifiedfor their function in the ABA-dependent pathway byrecognizing ABA-responsive elements (ABREs) con-taining an ACGT core motif. As a big transcriptionfactor family, quite a few bZIP transcription factorshave been well characterized functionally with diverseroles in many aspects, including pathogen defense,light signaling, seed maturation, flower development,and, especially, abiotic stress responses and hormonesignal transduction (Uno et al., 2000; Jakoby et al.,2002; Nijhawan et al., 2008). Among the studies of thebZIP family, a noteworthy aspect is that the function ofthe third subfamily (also known as the AREB/ABF/ABI5 subfamily) has been well elucidated because ofthe prominent roles of this subfamily in the ABAsignaling pathway. ABI5 is a genetically identifiedABA signaling component that plays an essential rolein seed germination and ABA-triggered developmen-tal arrest processes after germination in Arabidopsis.In contrast, ABFs or AREBs function mostly in thevegetative 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/ABI5members are major targets of SnRK2 proteinkinases in ABA core signaling (Fujii and Zhu, 2009;Fujita et al., 2009). Noticeably, the members AREB1,AREB2, and ABF3 are master transcription factors thatcooperatively regulate ABA signaling involved inmultiple stress responses and require ABA for fullactivation (Yoshida et al., 2010). AREB1/ABF2 hasbeen further documented for its posttranslationalmodification, 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 mod-ule regulating ABA-mediated gene expression in re-sponse 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 transcriptionfactor genes (Nijhawan et al., 2008). Several membershave been studied for their functions potentially re-lated 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 ofABFs in Arabidopsis, mainly three members of thethird subfamily in rice, TRAB1,OsABI5, andOsbZIP23,have been studied for their roles in ABA-mediatedstress responses (Hobo et al., 1999; Xiang et al., 2008;Zou et al., 2008). TRAB1, which is identified by yeasttwo-hybrid screening for proteins that interact withthe seed development-related transcription factorVP1, can be activated by ABA-dependent phospho-rylation in its Ser-102 residue (Hobo et al., 1999;Kagaya et al., 2002; Kobayashi et al., 2005), but itsbiologic function remains to be clarified. OsABI5,named according to its homology to ABI5 in Arabi-dopsis, was suggested to be involved in ABA signaltransduction and stress responses (Zou et al., 2007,2008). OsbZIP23 was characterized as a key player ofthe bZIP family for conferring ABA sensitivity, salinity,and drought tolerance of rice (Xiang et al., 2008).Several other members were also mentioned for theirinvolvement in ABA and stress (Zou et al., 2007; Lu etal., 2009; Amir Hossain et al., 2010). Among the third(or AREB/ABF/ABI5) bZIP subfamily of rice, there aredozens of stress-responsive ABF homologs includingthe reported members (Xiang et al., 2008). The func-tional redundancy or specificity for a specific memberof this family in the stress responses remains to beaddressed.

In this paper, we report the functional analysis ofOsbZIP46, another member of the third subfamily thathas high sequence identity to OsbZIP23. This gene wasalso named as ABI5-like1 (ABL1) based on the de-creased ABA sensitivity of a knockout mutant of thisgene (Yang et al., 2011). Overexpression of intactOsbZIP46 showed no significant effect on droughtresistance. We identified a constitutive active form ofOsbZIP46, OsbZIP46CA1, by mutagenesis, and over-expression of OsbZIP46CA1 could activate the expres-sion of the downstream genes and increase droughtresistance. We also found that the posttranslationalmodification of OsbZIP46 may be required for itsfunction.

RESULTS

Identification of OsbZIP46 as a Stress-ResponsivebZIP Factor

We identified the full-length cDNA of OsbZIP46,designated according to Nijhawan et al. (2008), in acDNA library of Minghui 63 (Chu et al., 2003; Zhanget al., 2005; Supplemental Fig. S1). To speculate on thefunction of OsbZIP46, we checked the expressionprofile of OsbZIP46 under different abiotic stressesand phytohormone treatments by real-time quantita-tive reverse transcription (RT)-PCR. We found that theexpression level of OsbZIP46was induced by drought,ABA, and indole-3-acetic acid (IAA; Fig. 1, A and B),

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consistent with a previous report (Yang et al., 2011). Inaddition, we found that the expression of OsbZIP46was affected by temperature stress (induced by heatbut repressed by cold), oxidative stress (induced byhydrogen peroxide [H2O2]), and cytokinin treatment(repressed by kinetin; Fig. 1, A and B) but only slightlyby salt stress. The promoter sequence of OsbZIP46contains many putative stress response-related cis-elements, such as ABRE element (nine hits), MYBrecognition site (13 hits), MYC recognition site (10hits), and one hit of DRE/CRT element (Fig. 1C). Inaddition, transient expression assays in rice protoplastsuggested that the OsbZIP46-GFP fusion protein waslocated in the nucleus; the nuclear localization wasconfirmed by its colocalization with the cyan fluores-cent protein (CFP)-fused nuclear protein GHD7 (Sup-plemental Fig. S2), indicating that OsbZIP46 is anuclear protein, consistent with the result obtainedin onion (Allium cepa) cells (Yang et al., 2011).

Increased ABA Sensitivity of Transgenic Plants with

Overexpression of OsbZIP46

According to the features of OsbZIP46 describedabove, we hypothesized that OsbZIP46 may have apositive role in ABA signaling in rice. To confirm this,we generated the transgenic rice overexpression Osb-ZIP46. We first checked the ABA sensitivity of trans-genic plants at the germination stage. Seeds of threeindependent overexpression lines, two negative lines,and the wild-type Zhonghua11 were germinated inone-half-strength Murashige and Skoog (1/2MS) me-dium containing ABA with a gradient of concentra-tions (0, 1, 3, and 6 mM). The germination rate of theoverexpression lines was identical to the negative linesand the wild-type Zhonghua11 at 0 and 1 mM ABA, butit was significantly lower than that of the negativelines and the wild type at 3 and 6 mM ABA (Fig. 2, Aand B), suggesting that the ABA sensitivity (in terms ofseed germination) of OsbZIP46 overexpression plants

was increased. We also investigated the ABA sensitiv-ity of transgenic plants at the postgermination stage.The lengths of shoot and root of overexpression seed-lings grown for 2 weeks in 1/2MS medium containing3 mM ABA were significantly shorter compared withthose of the negative lines and the wild type, but nodifference was observed for seedlings grown in themedium without ABA (Fig. 2, C–E). These resultssuggested that overexpression of OsbZIP46 can in-crease ABA sensitivity at postgermination stages. To-gether with the decreased ABA sensitivity of themutant of this gene (Yang et al., 2011), we proposethat OsbZIP46 is a positive regulator of ABA signalingin rice.

Performance of the Transgenic OsbZIP46 OverexpressionPlants under Abiotic Stress

It has been generally accepted that a positive regu-lator of ABA signaling may also contribute to thetolerance of plants to drought stress. Therefore, weexamined the performance of OsbZIP46 overexpres-sion plants under drought stress with four indepen-dent overexpression lines compared with the wildtype. To our surprise, the overexpression lines showedslightly decreased drought tolerance at the seedlingand reproductive stages. At the seedling stage, thesurvival rate of overexpression lines was lower thanthat of the wild type after drought stress (Fig. 3, A andB). At the reproductive stage, the overexpression lines,drought stressed at the panicle development stage,showed lower relative spikelet fertility compared withthe wild type (Fig. 3, C and D).We repeated this testingseveral times and obtained the same results. We alsotested the overexpression lines for salt stress tolerancebut found no significant difference between the over-expression lines and the wild type (data not shown).These results suggest that overexpression of the nativeOsbZIP46 gene may have a negative effect on droughtstress tolerance.

Figure 1. Expression analysis of OsbZIP46 understress and hormone treatments. A, Expressionlevel of OsbZIP46 under stresses includingdrought (time course 0, 3, and 5 d, recovery 12h), salt (0, 3, 6, and 12 h), cold (0, 3, 6, and 12 h),heat (0, 2, 6, and 12 h), and H2O2 (0, 3, 6, and12 h). B, Expression level of OsbZIP46 underphytohormone treatments including jasmonicacid (JA; 0, 1, 3, and 6 h) and ABA, IAA, GA,kinetin (KT), and brassinosteroid (BR; 0, 3, 6, and12 h). Error bars indicate SE based on threereplicates. C, Distribution of major stress relatedcis-elements in the promoter region ofOsbZIP46.

Activated OsbZIP46 Confers Drought Tolerance

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We checked the expression levels of RAB21, a com-monly used marker gene activated by AREB in ABAand drought responses, in the OsbZIP46 overexpres-sion plants. The expression level of RAB21 in theOsbZIP46 overexpression plants showed no differencecompared with that in the wild type under normalgrowth conditions; however, it was significantly in-creased after exogenous application of ABA (Fig. 3E).This result is in contrast to the result obtained in theOsbZIP23 overexpression rice plant, in which RAB21showed constitutive elevated expression (Xiang et al.,2008). Actually, a large number of drought-responsivegenes were not up-regulated in the OsbZIP46 over-expression plant, as discussed below. Therefore, wesuspected that the native OsbZIP46 protein may not beable to activate the expression of downstream genesand that its activity may be activated through the ABAsignaling pathways.

Activation of the Transcriptional Activity of OsbZIP46

The negative effect of OsbZIP46 overexpression ondrought stress tolerance prompted us to check thetranscriptional activity of OsbZIP46, because it was

predicted to be a typical bZIP transcription factor.First, we tested the transactivation activity of Osb-ZIP46 in yeast. Unlike its close homolog OsbZIP23(Xiang et al., 2008), the full-length OsbZIP46 proteinfused with the GAL4-binding domain had no trans-activation activity in yeast. Partial fragments of Osb-ZIP46 with a series of deletions were then tested. Theresults showed that the mutated forms with a deletionof 204 or 264 amino acids from the C terminus couldactivate the expression of reporter genes, suggestingthat the transactivation domain of OsbZIP46 is locatedin the N-terminal part of the protein. We noticed thatthe activity was detected only when domain D wasabsent, suggesting that domain D may have a prom-inent role in the regulation of the transactivationactivity of OsbZIP46. To confirm this, we generated amutated form of OsbZIP46, which contains 120 aminoacids from the N terminus (containing domains A, B,and C) as a transactivation domain and 105 aminoacids 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. Wefurther checked the activity of the mutated form andfound that it has constitutive transactivation activity in

Figure 2. Increased ABA sensitivity of OsbZIP46 overexpression plants at germination and seedling stages. A, Germinationperformance of OsbZIP46 overexpressor (OE15) and wild-type (WT) seeds on 1/2MS medium containing 0, 1, 3, or 6 mmol L21

ABA at 6 d after initiation. B, Calculation of the germination rates ofOsbZIP46 overexpressors (OE2, OE13, and OE15), negativetransgenic lines (CK9 and CK15), and wild-type seeds. Error bars indicate SE based on three replicates. C, Performance of twoindependent OsbZIP46 overexpression transgenic lines (OE2 and OE15) and two negative transgenic lines (CK2 and CK15) inMS medium containing 3 mM ABA. D and E, Shoot and root lengths of the lines grown on normal (D) or ABA-containing (E)medium. The shoot and root lengths were counted after growth for 14 d (n $ 9 each). Error bars indicate SD.

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yeast, and this mutated form was designated as Osb-ZIP46CA1 (forOsbZIP46 constitutive active form1; Fig. 4).

Performance of Transgenic Rice with Overexpression ofOsbZIP46CA1 under ABA Treatment or Abiotic Stress

Because the domain D-missing mutated form Osb-ZIP46CA1 showed constitutive transactivation activ-ity in yeast, we wondered if it has functions differentfrom the native protein in rice. Therefore, transgenicrice with overexpression of OsbZIP46CA1 was gener-ated and tested for stress tolerance.We first checked the sensitivity to ABA. As ex-

pected, the OsbZIP46CA1 overexpression (CA1-OEhereafter) 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 linesshowed significantly increased drought resistance atboth the seedling and reproductive stages, in contrastto the results of the overexpressor of full-length

OsbZIP46 presented above. At the seedling stage, thesurvival rate of CA1-OE lines was significantly highercompared with the wild-type control after droughtstress (Fig. 5, B and C). At the reproductive stage in thefield, the results indicated that overexpression ofOsbZIP46 can also improve drought resistance (Fig.5, D and E). The water loss rate of the CA1-OE plantswas significantly lower than that in the wild typeand the negative lines (Fig. 5F), which supported theimproved drought resistance phenotype. In addition,the CA1-OE lines were tested for tolerance to osmoticstress in comparison with OsbZIP46 overexpressionplants and the wild-type Zhonghua11. We evaluatedthe osmotic tolerance by using the relative shootlength of plants as a criterion, because the CA1-OElines grew a little lower than the wild type. OsbZIP46-CA1 overexpression had a positive effect on theosmotic stress tolerance, whereas OsbZIP46 overex-pression had a slightly negative effect on osmotic tol-erance (Supplemental Fig. S6).

Figure 3. Drought tolerance testing of OsbZIP46 overexpression transgenic rice. A, Phenotype of seedlings of OsbZIP46overexpression plants under drought stress. Seedlings from four overexpression lines (OE2, OE9, OE13, and OE15) were grownin barrels eachwith wild-type (CK) plants as a control. B, Survival rates ofOsbZIP46 overexpression plants and the wild type afterdrought stress. Error bars indicate SE based on three replicates. C, Phenotype of OsbZIP46 overexpression plants and wild-typeplants in drought stress at the flowering stage. Three overexpression lines (OE2, OE9, and OE15) were grown in PVC pipes eachwith a wild-type plant as a control. D, Relative spikelet fertility of the plants after drought stress at the flowering stage. Error barsindicate SD (n = 10). E, Expression level of RAB21 inOsbZIP46 overexpression and wild-type plants under normal conditions ortreatment with exogenous ABA.





Distinct Expression Profiles in the OsbZIP46CA1 andOsbZIP46 Transgenic Lines

The different effects of OsbZIP46 and OsbZIP46CA1overexpression on drought resistance might be attrib-uted to different downstream genes activated becauseOsbZIP46CA1 showed constitutive transactivation ac-tivity. To confirm this hypothesis and elucidate themolecular function of this gene, we compared expres-sion profile changes in the OsbZIP46-OE and Osb-ZIP46CA1-OE plants (three independent lines werechecked for each overexpressor) by using AffymetrixGeneChip. Compared with the wild type, transcrip-tomes of both overexpressors had significant changes.With a threshold of 2-fold change, a total of 391 and469 genes were up- and down-regulated, respectively,in the OsbZIP46CA1-OE plants, whereas in the Osb-ZIP46-OE plants, 119 and 390 genes were up- anddown-regulated, respectively (Fig. 6, A and B). Amongthose genes, only 51 and 100 genes were up- anddown-regulated, respectively, in both overexpressors.Interestingly, 13 genes showed opposite expressionchange patterns between the two overexpressors (Fig.6C). These results indicate that significantly moregenes were affected by OsbZIP46CA1 overexpressionthan by native OsbZIP46 overexpression, and most ofthe genes affected in the two overexpressors are dif-ferent. Quantitative RT-PCR was performed to checkthe differently regulated genes in the two overexpres-sors. Among the nine genes checked, all showed thesame expression patterns as in the gene chip results(Fig. 6E; Supplemental Fig. S7).

Gene Ontology (GO) analysis of the differentlyregulated genes in the two overexpressors revealed

that genes in several GO terms under the term “Bio-logical processes” were significantly overrepresented.The GO termwith the highest proportion of differentlyregulated genes is “Response to stimulus” (biotic,abiotic, and endogenous stimuli, etc.), followed byGO terms such as “Signal transduction,” “Proteinmodification,” “Transcription,” “Metabolism,” and“Biosynthesis” (Supplemental Table S1). “Transcrip-tion factor activity” and “Nucleus” have the highestproportion of differently regulated genes under “Mo-lecular function” and “Cellular component,” respec-tively (Supplemental Table S1).

Because the two overexpressors showed distincttranscriptome changes and most of the differentlyregulated genes were attributed to the “Response tostimulus” category, we further classified all these genesbased on their change patterns in the overexpressorsand responsiveness to drought stress. Based on thepublished microarray results (Jain et al., 2007), morethan half of these genes were regulated by droughtstress. Among the 1,204 expression-affected genes, 455and 202 were up- and down-regulated, respectively, bydrought (Supplemental Tables S2–S5). Interestingly,most of the genes up-regulated and down-regulatedin both overexpressors were down- and up-regulated,respectively, by drought stress (Fig. 6D). The genesdifferentially regulated in the two overexpressors canbe classified into five groups. Most of the genes ingroup I (up-regulated in CA1-OE but not changed inOsbZIP46-OE) and group III (up-regulated in CA1-OEbut with opposite changes in OsbZIP46-OE) were in-duced by drought stress, whereas most of the genes ingroup II (down-regulated in CA1-OE but not changedin OsbZIP46-OE) were suppressed by drought stress(Fig. 6D). Notably, many of the genes up-regulated onlyin CA1-OE have been annotated or confirmed for stressresponse or adaptation-related functions, includingdehydrin or late embryogenesis abundant (LEA) pro-teins (five genes), stress-related transcription factors (27genes), kinases (seven genes), phosphatases (eightgenes), amino acid metabolism or transportation pro-teins (three genes), aquaporin (two genes), and lipidtransfer proteins (five genes; Supplemental Table S2).This implies that the increased drought tolerance ofCA1-OE may result from the up-regulation of thesegenes. Interestingly, the differentially expressed genesonly in OsbZIP46-OE (groups IV and V) showed dis-tinct trends of responsiveness to drought; most of thegenes up-regulated in OsbZIP46-OE showed drought-suppressed expression patterns, whereas most ofthe down-regulated genes in OsbZIP46-OE showeddrought-induced expression (Fig. 6D). Especiallyamong these down-regulated genes in OsbZIP46-OE,there are many genes annotated or confirmed for stressresponse or adaptation, including many transcriptionfactors (40 genes), kinases (35 genes), and some otherstress-related functional proteins (LEA protein, lipid-transfer protein; Supplemental Table S5). This resultmay partially explain the reason that overexpressionof OsbZIP46 resulted in decreased drought tolerance.

Figure 4. Transactivation assay of OsbZIP46. A, Expression construct ofOsbZIP46 in yeast. B, Transactivation assay of truncated OsbZIP46.Fusion proteins of the GAL4 DNA-binding domain and differentportions of OsbZIP46 were checked for their transactivation activityin yeast strain Mav203. FL indicates the full-length OsbZIP46; dC1 todC6 and CA1 indicate the mutated forms of OsbZIP46. The results of acolony-lift filter assay are shown at right; + and2 indicate positive andnegative, respectively, for transactivation activity. [See online article forcolor version of this figure.]

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Because so many genes were differentially regulatedby OsbZIP46 and OsbZIP46CA1 overexpression, wefurther analyzed the promoter sequences of thesegenes. The results suggested that 54% of these geneshave enriched cis-elements featuring ABRE that arepotential binding sites of ABF or AREB proteins ho-mologous to OsbZIP46 (Supplemental Table S6). In-terestingly, the genes up-regulated in CA1-OE but notchanged or with opposite change patterns in theOsbZIP46 overexpressor (groups I and III) and genesdown-regulated in OsbZIP46-OE (group IV) hadhigher ratios of the enriched cis-elements (62% and63%, respectively) than the other two groups (42%and 45%).Three genes, Os11g26790 (also designated as RAB21,

encoding a LEA protein), Os03g19290 (a putativemitochondrial import inner membrane translocasesubunit), and Os05g38290 (a protein phosphatase2C), that were up-regulated only in OsbZIP46CA1-OE but not in OsbZIP46-OE, were selected to test ifOsbZIP46 can directly act on these differentially reg-ulated genes. The pGAD-OsbZIP46 plasmid (contain-ing the putative DNA-binding domain of OsbZIP46

fused to the GAL4 activation domain) and the reporterconstruct pHIS-cis (containing the putative ABRE-containing promoters of the three genes) were cotrans-formed into yeast strain Y187. The reporter gene wasactivated for all three cotransformations (Supplemen-tal Fig. S8), indicating that OsbZIP46 can bind thepromoters of these genes.

OsbZIP46 May Be Activated by Phosphorylation

Significant differences in affecting gene expressionbetween the constitutive activated and native formsof OsbZIP46 indicated that the activity of nativeOsbZIP46 might be regulated by posttranslationalmodifications. In Arabidopsis, stress/ABA-activatedprotein kinases (SAPKs) or SnRK2 have been sug-gested for phosphorylating ABFs or AREBs (Furihataet al., 2006; Fujii and Zhu, 2009; Fujita et al., 2009;Yoshida et al., 2010). Therefore, we checked nineputative SAPK family members from rice with Osb-ZIP46 for yeast two-hybrid analysis. The results sug-gested that OsbZIP46 could interact with OsSAPK2, -6,

Figure 5. ABA sensitivity and drought tolerance testing of OsbZIP46CA1-OE rice. A, Phenotypes of native OsbZIP46overexpression lines (FL-OE9 and FL-OE15), OsbZIP46CA1-OE (CA1-OE5 and CA1-OE7), and the wild type (WT) undermedium containing 3 mM ABA. B, Phenotypes of seedlings of OsbZIP46CA1-OE (CA1-OE1 and CA1-OE7) and the wild typeunder drought stress. Seedlings before (top) and after (bottom) drought stress treatment are shown. C, Survival rate ofOsbZIP46CA1-OE and wild-type plants after drought stress. Error bars indicate SE based on three replicates. D, Phenotype ofOsbZIP46CA1-OE plants under drought stress at the flowering stage. E, Relative yield ofOsbZIP46CA1-OE (CA1-OE1, CA1-OE5,and CA1-OE7) and wild-type plants after drought stress at the flowering stage. Error bars indicate SD (n = 10). F, Water loss rates inleaves cut from the OsbZIP46CA1 overexpressor (CA1-OE1 and CA1-OE3), a negative transgenic line control (NCK), and wild-type plants. Error bars indicate SE based on three replicates.





and -9 (Fig. 7A). The interaction between OsbZIP46and OsSAPKs was further confirmed by bimolecularfluorescence complementation (BiFC; Fig. 7B). An invitro phosphorylation assay demonstrated that theOsbZIP46 protein could be phosphorylated by SAPK2and SAPK6 (Fig. 7C).

Sequence analysis suggested that five putative phos-phorylation target sites are present in OsbZIP46 (Fig.7D). We inferred that the transcriptional activity ofOsbZIP46 might also be related to these putativephosphorylation target sites. To confirm this, we gen-erated single or multiple amino acid substitutions

(Ser/Thr to Asp) of OsbZIP46 (Asp provides a nega-tive charge and can mimic the phosphorylated status)and checked their transactivation activity. The single-substitution mutant PA1 (for phosphorylation activeform 1; with substitution of Thr-129 to Asp) showedslightly increased transactivation activity. But the mul-tiple (three to five) amino acid substitutions couldsignificantly enhance the transactivation activity ofOsbZIP46 (Fig. 7, D and E). Taken together, theseresults suggest that the native OsbZIP46 may beactivated by posttranslational phosphorylation modi-fication.

Figure 6. Gene chip analysis of OsbZIP46 overexpressors. A and B, Scatterplots of the expression profiles of whole-genomegenes in FL-OE (A) and CA1-OE (B) compared with the wild type (WT). The x and y axes indicate the chip hybridization signal inthe overexpressor and the wild type, respectively. The red and green dots indicate the probe sets withOE-wild type signal ratios ofgreater than 2 and less than 0.5, respectively. C, Venn diagram for the proportions of genes affected by the overexpression ofOsbZIP46 native form and OsbZIP46CA1. FL-up and FL-down indicate genes up- and down-regulated, respectively, inOsbZIP46-OE; CA1-up and CA1-down indicate genes up- and down-regulated, respectively, in OsbZIP46CA1-OE. D, Drought-responsive patterns of all the differentially regulated genes in the two overexpressors. The asterisk indicates genes with the sameregulated pattern between CA1-OE and FL-OE. Group I genes are up-regulated in CA1-OE; group II genes are down-regulated inCA1-OE; group III genes are up-regulated in CA1-OE but down-regulated in FL-OE; group IV genes are down-regulated in FL-OE;and group V genes are up-regulated in FL-OE. E, The differentially regulated patterns in the two overexpressors revealed by genechip analysis are validated by real-time quantitative RT-PCR.

Tang et al.




DISCUSSION

Constitutive Activation Is a Useful Tool for FunctionalAnalysis of Transcription Regulators

Many reports suggest that overexpression of somestress-inducible transcription factors can increase theabiotic stress tolerance of plants (Ciftci-Yilmaz andMittler, 2008; Dubos et al., 2010; Matsukura et al., 2010;Lata and Prasad, 2011), but some special members needadditional modifications to exhibit their full functions.For example, overexpression of DREB2A in transgenicplants could not improve stress tolerance, but over-expression of DREB2ACA, carrying a deletion of thecentral negative regulatory domain (NRD), activated theexpression of many stress-inducible genes and im-proved tolerance to drought and heat stress in trans-genic Arabidopsis (Sakuma et al., 2006a, 2006b).Similarly, AREB1, a homologous gene of OsbZIP46, hasbeen reported to fully function after posttranslational

modification (Fujita et al., 2005). However, such modi-fication of transcription factors for stress tolerance wasseldom reported in crops.

Although OsbZIP46/ABL1 has recently been repor-ted as a positive regulator of ABA signaling by mutantanalysis of this gene (Yang et al., 2011), its actual role inregulating stress tolerance remains unknown. Nor-mally, overexpression of positive regulators of ABAsignaling from the bZIP family, such as OsbZIP23(Xiang et al., 2008) and OsbZIP72 (Lu et al., 2009), canresult in increased drought tolerance. However, Osb-ZIP46 overexpression rice did not display the expectedincrease but a decrease of drought tolerance. We foundthat the intact OsbZIP46 was insufficient to induce theexpression of the target gene unless the exogenousABA was applied. In yeast, full-length OsbZIP46 hadno transactivation activity. Considering the fact thatOsbZIP46 is a homolog of TRAB1, which was previ-ously reported with ABA-dependent phosphorylation

Figure 7. Interaction between OsbZIP46 and SAPKs may be activated by phosphorylation. A, Yeast two-hybrid assays ofOsbZIP46 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, synthetic complete-Leu-Trp-His medium. B, Confirmation of the interaction of OsbZIP46 and SAPK2/SAPK6/SAPK9 by BiFC. YFP, Yellow fluorescent protein. C, In vitro phosphorylation assay of OsbZIP46 by SAPK kinases.Molecular mass markers are indicated on the right. The arrow indicates the position of the GST-fused intact OsbZIP46 protein(60.7 kD). D, Single or multiple amino acid substitutions (Ser/Thr to Asp) of OsbZIP46 by site-directed mutagenesis. PA,Phosphorylation active, the mutated forms of OsbZIP46; WT, wild type, the native OsbZIP46. The white circles indicate theputative phosphorylation target sites, and the blue circles indicate the mutated sites in the mutated forms of OsbZIP46. E,Transactivation assay of OsbZIP46 mutants in yeast. X-gal assay and His test results are shown. CK2 and CK+ are negative andpositive controls, respectively.





activity (Kagaya et al., 2002), these preliminary resultssuggest that the function of OsbZIP46 may also needposttranslational level modifications. Therefore, wemanaged to produce a constitutively active form ofOsbZIP46 in yeast and tested its function for stresstolerance in rice.

As expected, OsbZIP46CA1 overexpression signifi-cantly increased the drought tolerance of rice at boththe seedling and reproductive stages, which is incontrast to the drought-sensitive phenotype of Osb-ZIP46 overexpression plants. Microarray analysis ofthe two overexpressors provided strong evidence forthe differences in drought tolerance. In short, manystress tolerance-related genes were activated only inthe OsbZIP46CA1 overexpression lines but not in theOsbZIP46 overexpression lines, supporting the hy-pothesis that OsbZIP46CA1 also possesses constitu-tive transcriptional activity in rice and thus activatesthe expression of stress-related target genes, leading toincreased drought tolerance. Therefore, the generationof constitutive activated forms for posttranslationalmodification-required regulatory proteins (at least forsome transcription factors) seems to be a promisingway to probe their functions, which may be especiallytrue when functional redundancy exists for the genebeing studied or overexpression of the native form ofthe gene results in unexpected outcomes, as is the casefor OsbZIP46 discussed below.

Negative Effect of OsbZIP46 Overexpression onDrought Tolerance

We observed that overexpression of both OsbZIP46and OsbZIP46CA1 increased ABA sensitivity. How-ever, the OsbZIP46 overexpressor showed slightlyreduced drought tolerance. Logically, increased ex-pression of a positive regulator of ABA signaling willmost likely have some positive effect, or at least notnegative, on stress tolerance, even if the regulatorneeds ABA- or stress-triggered posttranslational mod-ification for its full activity. We propose that OsbZIP46is an activation-required transactivator, but its activa-tion varies depending on the stresses or ABA treatment.Because OsbZIP46 is one of the positive regulators ofABA signaling (Yang et al., 2011), we propose thatoverexpressed OsbZIP46 may just match up with thehigh dosage requirement for mediating the signalingtriggered by exogenous ABA. While under the droughtstress that triggers ABA signaling more slowly thandoes the exogenous application of ABA, the preaccu-mulated OsbZIP46 in the overexpressor might have anegative effect on mediating stress signaling throughendogenous ABA. This is very likely true, because thenative OsbZIP46 contains a negative domain (domainD) for activation activity in yeast. Microarray analysisrevealed that arrays of drought stress-related geneswere actually down-regulated in the OsbZIP46 over-expressor (Fig. 6D; Supplemental Table S5), and some ofthese genes were not induced or were significantly lessinduced by drought compared with those in the wild

type (data not shown). Nevertheless, the decreaseddrought resistance might also result from other un-known negative effects of the overexpression of Osb-ZIP46.

Possible Mechanisms for the Constitutive Activityof OsbZIP46CA1

Because the deletion of domain D of OsbZIP46resulted in constitutive transactivation activity in yeast(Supplemental Fig. S11), we proposed that domain Dis a negative regulatory domain and has a pivotal rolein the regulation of OsbZIP46 activity. It has beenreported that the conserved Leu in the LxLxL motifis important for repression in some transcription re-pressors, such as the ethylene response factor (ERF)-associated amphiphilic repression (EAR) motif foundin 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 ofdomain D with the conserved EAR motif. Althoughthe LxLxL motif was not found, domain D contains asequence signature of LxxxxLxxxL (Supplemental Fig.S1D). In addition, considering that DREB2A contains aPEST sequence in the NRD that plays a role in thestability of the protein (Sakuma et al., 2006a), and thePEST sequence, existing in some rapid-turnover pro-teins, serves as a signal for proteolytic degradation andregulates the activity of proteins by controlling theiraccumulation (Rechsteiner and Rogers, 1996), wewondered whether domain D of OsbZIP46 has asimilar role as NRD in DREB2A. However, no poten-tial PEST sequence was identified in OsbZIP46 usingthe epestfind server (http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind; data not shown). Itwould be interesting to further test which part ofdomain D is essential for the negative effect on trans-action activity.

It cannot be excluded that the deletion of domain Dmay cause a conformational change that mimics thechanges resulting from posttranslational modification,such as phosphorylation of the native OsbZIP46. Here,our results showed that the transactivation activity ofOsbZIP46 in yeast was related to multiple putativephosphorylation sites and that OsbZIP46 may bephosphorylated by three homologs of SnRK2 kinases,OsSAPK2, OsSAPK6, and OsSAPK9 (Fig. 7). An invitro phosphorylation assay confirmed that OsbZIP46protein can be phosphorylated by OsSAPK2 andOsSAPK6. Because we failed to obtain soluble ex-pressed proteins for OsbZIP46CA1, it remains to bechecked if there is any change in the phosphorylationof OsbZIP46CA1 by the OsSAPKs. It has been welldocumented that the phosphorylation of ABF/AREBby SnRK2 is an essential part of the complete ABAsignaling pathway in Arabidopsis (Fujii and Zhu,2009; Fujita et al., 2009). Moreover, the genes of theSnRK2 family (also designated SAPK, for osmoticstress/ABA-activated protein kinase) in rice were

Tang et al.




identified for their ABA-inducible characteristics(Kobayashi et al., 2004). Especially TRAB1, a homologof OsbZIP46, has been identified as phosphorylated bySAPKs (Kagaya et al., 2002; Kobayashi et al., 2005).RAB21, the putative target gene of OsbZIP46, wasactivated in OsbZIP46 overexpression rice plants onlyafter treatment with exogenous ABA. These resultstogether imply that the transcriptional activity of Osb-ZIP46 may be mainly regulated by ABA-dependentphosphorylation.

Specificity of OsbZIP46 Compared with OtherABF Members

OsbZIP46 is member of the third (or ABF/AREB/ABI5) subfamily of bZIP transcription factors. Al-though most of the ABF members are thought to beinvolved in ABA and abiotic stress responses, accord-ing to studies in Arabidopsis, the results from Osb-ZIP46 suggest that it has some specificity in addition toits common features compared with other ABF mem-bers.At the protein sequence level, most members in the

third subfamily of the bZIP family, including Osb-ZIP46, contain a typical basic region and the Leuzipper domain as well as one of five other conservedmotifs (named domains A, B, C, D, and E, respectively)that are predicted to contain five phosphorylationsites, suggesting that most members of the ABF sub-family maybe involved in stress or ABA signalingthrough phosphorylation by the protein kinase. More-over, the third subfamily bZIP members of rice andArabidopsis could be further classified into fourgroups (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, andOsABI5) and has the highest similarity to ABF1 to -4 ofArabidopsis (Hobo et al., 1999; Xiang et al., 2008; Zouet al., 2008).However, our results showed that the expression of

OsbZIP46 is inducible by drought, heat, H2O2, andABA treatment and repressed by cold but apparentlynot affected by salt stress. These results suggest adifferent expression profile compared with OsbZIP23,in which expression is inducible by drought, salt, andABA treatment but not cold (Xiang et al., 2008). Wecompared the expression profile of OsbZIP46 acrossvarious tissues and stages with its homology membersby searching the expression profiles database CREP(Wang et al., 2010) and found that their expressionpatterns were also different (Supplemental Fig. S3).The OsbZIP46 overexpression rice showed unexpect-edly reduced drought tolerance, which is distinct fromthe OsbZIP23 overexpression rice, which showed sig-nificantly increased drought and salt stress tolerance(Xiang et al., 2008). This may suggest that differentmembers of the third bZIP subfamily may function instress tolerance with a different spectrum of stresses ordifferent degrees of the same stress. To support this,

we compared the transcriptomes of the OsbZIP46(OsbZIPCA1 as well) and OsbZIP23 overexpressionplants and found that the genes affected by over-expression of the two ABF members are largely dif-ferent (only less than one-quarter of the regulatedgenes are overlapped; Supplemental Fig. S9; Supple-mental Table S7). Furthermore, unlike OsbZIP23 andmost other members in this subfamily, the native formOsbZIP46 has no transactivation activity in yeast. Thisfurther suggests that the activity of OsbZIP46 in plantamay strongly rely on the posttranslational modifica-tion. However, considering that the domain D se-quence and putative phosphorylation target sites alsoexist in other ABF members, it remains intriguing toidentify which part of domain D or which phospho-rylation target sites determine the specificity of stressresponses.

CONCLUSION

We characterized a constitutive active form of tran-scription factor OsbZIP46. Comparison analysis re-vealed that OsbZIP46 is a positive regulator of ABAsignaling and regulates drought stress resistance ofrice by modulating many stress-related genes, and itsfunction on activating downstream genes depends onposttranslational modification. This constitutive activeform of OsbZIP46 has promising usefulness in trans-genic 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#-TAATACGACT-CACTATAGGG-3#). The sequence-confirmed clone containing the full-lengthcDNA 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#-ATAggtaccATGGAGTTGCCGGCG-GATG-3# and 5#-ATAggatccTCAGCATGGACCAGTCAGTG-3# based on thetemplate 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), andoxidative 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 L21 hygromycin; negative trans-

genic 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 mM 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 mM), 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 mMNaCl 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 (Invitro-

gen) 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�Cfor 5 s and 60�C for 34 s. The rice Actin1 gene was used as the endogenouscontrol, and relative expression levels were determined as described previously

(Livak and Schmittgen, 2001). For RNA gel blotting, 15 mg 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 Assaysin Yeast

The single or multiple amino acid substitutions (Ser/Thr to Asp) of

OsbZIP46 were produced with the QuikChange Multi Site-Directed Muta-

genesis 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 (MATa; leu2–3,112; trp1–901; his3D200;

ade2–101; gal4D; gal80D; SPAL10::URA3; GAL1::lacZ; HIS3UASGAL1::HIS3@LYS2;

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 (MATa; ura3–52; his3–200; ade2–101; trp1–901; leu2–3,

112; gal4D; gal80D; met–; URA3::GAL1UAS-GAL1TATA-LacZ; MEL1; Clontech).

The DNA-protein interactions were determined by the growth of the trans-

formants 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, andOsbZIP46

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 trans-

formation. 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-b-D-thiogalactoside (0.1 mM) overnight at 18�C. Bacte-rial lysates were applied to glutathione Sepharose (GE Healthcare), and GST

fusion proteins were eluted with 10 mM glutathione by following the man-

ufacturer’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 mCi of [g-32P]ATP). The reaction

was incubated at room temperature for 30 min and terminated by the addition

of 5 mL of sample buffer and heating at 100�C for 5 min. After separation on a12% 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 trans-

genic 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 S1–S7).

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.

Supplemental Figure S1. Sequence analysis of OsbZIP46.

Supplemental Figure S2. Subcellular localization analysis of OsbZIP46.

Supplemental Figure S3. Expression profile of OsbZIP46 and its homol-

ogous genes in various tissues and at different stages.

Supplemental Figure S4. Overexpression of OsbZIP46.

Supplemental Figure S5. Overexpression of OsbZIP46CA1.

Supplemental Figure S6.Osmotic stress tolerance testing ofOsbZIP46 and

OsbZIP46CA1 transgenic rice.

Supplemental Figure S7. Real-time quantitative RT-PCR for validating

results from the gene chip.

Tang et al.




Supplemental Figure S8. Identification of putative target genes of Osb-

ZIP46.

Supplemental Figure S9.Comparison of genes regulated byOsbZIP23 and

OsbZIP46 overexpression.

Supplemental Figure S10. Increased ABA sensitivity of OsbZIP46-OE rice

at germination stage.

Supplemental Figure S11. Transactivation assay of truncated OsbZIP46

without the D domain.

Supplemental Table S1. GO analysis of the differential expressed genes.

Supplemental Table S2. Up-regulated genes in transgenic rice plants

overexpressing OsbZIP46CA1.

Supplemental Table S3. Down-regulated genes in transgenic rice plants

overexpressing OsbZIP46CA1.

Supplemental Table S4. Up-regulated genes in transgenic rice plants

overexpressing native OsbZIP46.

Supplemental Table S5. Down-regulated genes in transgenic rice plants

overexpressing native OsbZIP46.

Supplemental Table S6. Genes that are differentially regulated by native

OsbZIP46 and OsbZIP46CA1 overexpression and containing enriched

cis-element ABRE.

Supplemental Table S7. Genes similarly regulated in both OsbZIP23 and

OsbZIP46 overexpression rice.

ACKNOWLEDGMENTS

We thank Jian Xu and Lei Wang (Huazhong Agricultural University) for

providing plasmid pM999-33 and 35S::Ghd7:CFP, respectively.

Received October 31, 2011; accepted January 31, 2012; published February 1,

2012.

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