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First published online October 17, 2008; 10.1104/pp.108.128199 Plant Physiology 148:1938-1952 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Characterization of OsbZIP23 as a Key Player of the Basic Leucine Zipper Transcription Factor Family for Conferring Abscisic Acid Sensitivity and Salinity and Drought Tolerance in Rice1,[W],[OA]National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
OsbZIP23 is a member of the basic leucine zipper (bZIP) transcription factor family in rice (Oryza sativa). Expression of OsbZIP23 is strongly induced by a wide spectrum of stresses, including drought, salt, abscisic acid (ABA), and polyethylene glycol treatments, while other stress-responsive genes of this family are slightly induced only by one or two of the stresses. Transactivation assay in yeast demonstrated that OsbZIP23 functions as a transcriptional activator, and the sequences at the N terminus (amino acids 1–59) and a region close to the C terminus (amino acids 210–240) are required for the transactivation activity. Transient expression of OsbZIP23-green fluorescent protein in onion (Allium cepa) cells revealed a nuclear localization of the protein. Transgenic rice overexpressing OsbZIP23 showed significantly improved tolerance to drought and high-salinity stresses and sensitivity to ABA. On the other hand, a null mutant of this gene showed significantly decreased sensitivity to a high concentration of ABA and decreased tolerance to high-salinity and drought stress, and this phenotype can be complemented by transforming the OsbZIP23 back into the mutant. GeneChip and real-time polymerase chain reaction analyses revealed that hundreds of genes were up- or down-regulated in the rice plants overexpressing OsbZIP23. More than half of these genes have been annotated or evidenced for their diverse functions in stress response or tolerance. In addition, more than 30 genes that are possible OsbZIP23-specific target genes were identified based on the comparison of the expression profiles in the overexpressor and the mutant of OsbZIP23. Collectively, these results indicate that OsbZIP23 functions as a transcriptional regulator that can regulate the expression of a wide spectrum of stress-related genes in response to abiotic stresses through an ABA-dependent regulation pathway. We propose that OsbZIP23 is a major player of the bZIP family in rice for conferring ABA-dependent drought and salinity tolerance and has high potential usefulness in genetic improvement of stress tolerance.
Plants can initiate a number of molecular, cellular, and physiological changes in response and adaptation to the ever-changing environment during their growth and development. The phytohormone abscisic acid (ABA) controls various aspects of plant growth throughout development (Finkelstein et al., 2002  ; Himmelbach et al., 2003). It prevents embryos from precocious germination, modulates seedling growth under normal conditions, and regulates seed maturation (Busk and Pages, 1998; Ladyzhenskaya, 2001; Cheng et al., 2002; Chen et al., 2006). ABA also plays important roles in the accumulation of storage proteins and other reserve materials and in the acquisition of desiccation tolerance. During vegetative growth, one of the major roles of ABA is to mediate adaptive responses to various environmental stresses. It has a critical role in the plant adaptation or acclimation to abiotic stresses, such as high salinity, drought, low temperature, and mechanical wounding. The pathways leading to stress adaptation can be divided into two major categories: ABA-dependent and ABA-independent pathways. Underlying the ABA-mediated stress responses is the transcriptional regulation of stress-responsive gene expression dependent on ABA (Shinozaki and Yamaguchi-Shinozaki, 2000; Ladyzhenskaya, 2001; Fedoroff, 2002; Finkelstein et al., 2002; Jakoby et al., 2002; Xiong et al., 2002; Zhu, 2002; Himmelbach et al., 2003).
During the response and adaptation to diverse abiotic stresses, many stress-related genes are induced and the levels of a variety of stress resistance-related functional proteins are accumulated. Numerous genes have been reported to be up-regulated under stress conditions in vegetative tissues (Seki et al., 2002
In Arabidopsis (Arabidopsis thaliana), there are many bZIP genes identified (Jakoby et al., 2002
Rice (Oryza sativa) has become a model plant for monocotyledon species. About 100 putative bZIP sequences (88 and 109 gene models in the sequenced indica and japonica genomes, respectively) were predicted in the rice genome (Guo et al., 2005
A preliminary analysis of the gene expression profiles of the rice genome under various stress conditions revealed that most of the genes in the third (or ABI5) bZIP subfamily were more or less induced by ABA, drought, or high salinity (Y. Xiang, N. Tang, H. Du, H. Ye, and L. Xiong, unpublished GeneChip data). We noticed that a member named OsbZIP23, with the systematic name adopted from the sequence analysis of the bZIP family in rice (Nijhawan et al., 2008
Isolation and Sequence Analysis of OsbZIP23
In our GeneChip analysis of drought-resistant upland rice IRAT109 (japonica) under abiotic stresses (Y. Xiang, N. Tang, H. Du, H. Ye, and L. Xiong, unpublished data), we noticed one gene, designated OsbZIP23 according to Nijhawan et al. (2008)
Expression Profile of OsbZIP23 under Different Stresses To speculate on the physiological and functional relevance of the OsbZIP23 gene, we further checked its expression profile under different abiotic stresses and various chemical treatments. In RNA gel-blot analysis, the transcript level of OsbZIP23 was rapidly and strongly induced by drought, high-salinity, polyethylene glycol (PEG), and ABA treatment but remained unchanged after low-temperature treatment (Fig. 2A ), which agrees with the microarray data very well (Supplemental Table S1). The transcript level of this gene was also checked in a few chemical treatments, including jasmonic acid (JA), salicylic acid (SA), and 2,4-dichlorophenoxyacetic acid (2,4-D), by real-time PCR analysis. The results showed that the expression level of this gene was not obviously affected by any of these treatments (Fig. 2B). We also investigated whether the expression of this gene has any tissue specificity. The results suggested it was relatively higher in seedling and flag leaves than in other tissues checked (Fig. 2C). The strong induction of this gene by abiotic stresses prompted us check its promoter sequence (1,500 bp upstream the transcription start site) by searching the prompter sequence against the PLACE database (http://www.dna.affrc.go.jp/PLACE/). The promoter sequence indeed contains many putative stress response-related cis-elements, such as ABRE (nine hits for the core sequence of ABRE), DRE (three hits), MYC recognition site (eight hits), MYB recognition site (seven hits), and GCC box (four hits; Fig. 2D). The numbers of these response-related cis-elements are apparently higher than those of the other stress-responsive genes in this family, such as TRAB1 (five ABRE, no DRE, seven MYC recognition sites, and five MYB recognition sites) and OsABI5 (no ABRE, no DRE, six MYC recognition sites, and five MYB recognition sites).
OsbZIP23 Is Located in the Nucleus and Has Transactivation Activity in Yeast To confirm that OsbZIP23 is a putative transcription factor, the OsbZIP23-GFP fusion construct was used for in vivo protein targeting in onion epidermal cells by bombarding transformation. The results showed that GFP signal was detected only in the nucleus of the OsbZIP23-GFP-transformed cell (Fig. 3A ), while the control (transformation of GFP construct) showed ubiquitous distribution of GFP signal in the cell, suggesting that OsbZIP23 is a nucleus-localized protein. A putative nuclear localization signal was predicted in the N terminus of the protein by WoLF PSORT (http://wolfpsort.seq.cbrc.jp/). A segment of 60 amino acids containing the nuclear localization signal was fused to GFP, and the fused protein was also located in the nucleus of transformed onion epidermal cells (data not shown).
To check if OsbZIP23 has transcriptional activity, the open reading frame of OsbZIP23 was fused to the GAL4 DNA-binding domain (GAL4-DB) in the vector pDEST32 and the construct was transformed into yeast. Colony-lift filter assay showed that the reporter gene LacZ was expressed only in the yeast cell transformed with the GAL4-DB-OsbZIP23 construct (Fig. 3B), indicating that OsbZIP23 had transcriptional activity in yeast. Transactivation activity assay of a series of shortened OsbZIP23 from both the N and C termini revealed that the amino acid sequences at the N terminus (amino acids 1–59) and a region close to the C terminus (amino acids 210–240) are required for the transactivation activity of the fusion protein in yeast (Fig. 3C). However, deletion from the C terminus up to amino acid 240 did not abolish the transactivation activity of the fusion protein in yeast (Fig. 3C). Nevertheless, no difference in the transactivation activity was detected for the OsbZIP23 sequences from indica and japonica rice, even though there is a polymorphism (amino acids 14–16) in the N-terminal region.
Since OsbZIP23 was strongly induced by ABA, we tested if OsbZIP23 is involved in ABA sensitivity of rice, which is an important aspect of ABA-dependent regulation. To confirm this, an overexpression construct with the OsbZIP23 gene under the control of the maize (Zea mays) ubiquitin promoter (Fig. 4A ) was transformed into rice Zhonghua 11. The expression level and copy number of T-DNA was checked by northern- and Southern-blot analyses (Supplemental Figs. S2 and S3). ABA sensitivity was examined for two independent homozygous T2 overexpression lines (S20 and S26, containing a single copy of the T-DNA). The results indicated that OsbZIP23-overexpressing lines were hypersensitive to ABA at the germination stage (Fig. 4B). At 6 d after seed imbibition, transgenic seeds had only about 50% of the relative germination rate (ratio of germination rate in the ABA treatment to that in water control) with 1 µM ABA in the medium, while the wild type showed nearly 100% of the relative germination rate under the same conditions. In the treatment with 2 µM ABA, the relative germination rate of the wild type was about 90%, but the relative germination rate of overexpression lines was reduced to about 15%. Under the same dose of ABA, the germinated wild-type plantlets continued to grow, albeit at a slower rate than on ABA-free medium, but transgenic plantlets grew very slowly. Such a hypersensitivity of OsbZIP23-overexpressing lines was also confirmed at the postgermination stage. In this experiment, transgenic and wild-type seeds were geminated on normal Murashige and Skoog (MS) medium, and the seedlings were then transferred to medium containing 3 µM ABA. The shoot and root length were measured at 10 d after being transferred. The results showed that OsbZIP23 overexpressors had significantly (P < 0.05) shorter roots and shoots than the wild type (Fig. 4, C and D), while on normal MS medium all transgenic plants showed no difference from the wild type (Supplemental Fig. S4).
Decreased ABA Sensitivity of the Osbzip23 Mutant The function of OsbZIP23 was further investigated by loss-of-function mutant analysis. A mutant line of OsbZIP23 in the background of Zhonghua 11, with a T-DNA inserted in the second intron (Fig. 5A ), was obtained from the Rice Mutant Database (http://rmd.ncpgr.cn/). After being confirmed for the T-DNA insertion site and the abolishment of OsbZIP23 expression (Supplemental Fig. S5), homozygous mutant plants derived from a single T-DNA insertion were checked for an ABA sensitivity phenotype. In a seed germination experiment with application of ABA in the medium, the mutant had significantly higher germination rates (about 80% and 20% in the medium with 3 µM and 8 µM ABA, respectively) than that in WT' (wild-type plants segregated from a heterozygous Osbzip23 mutant; about 50% and 0% in the medium with 3 µM and 8 µM ABA, respectively; Fig. 5, B and C). In the normal medium, the germination rates of mutant and WT' plants had no significant difference. We also checked the ABA sensitivity of the mutant in the medium containing ABA at the postgermination stage. The mutant also showed a significantly higher relative growth (50% of the shoot length on normal medium) than WT' (30%; Fig. 5D). After a DNA fragment containing genomic sequence of the OsbZIP23 gene and its native promoter was retransformed into the mutant, the germination rate of the retransformed transgenic seeds in the ABA-containing medium was decreased to a level similar to that in WT' (Fig. 5, B–D), indicating that the decreased ABA sensitivity of the mutant can be complemented by the OsbZIP23 gene. These results indicated that OsbZIP23 plays a critical role in mediating ABA sensitivity in rice.
Stress Tolerance of OsbZIP23 Transgenic Plants
The strong induction of OsbZIP23 expression by drought and high-salinity stresses suggested that this gene might be involved in stress resistance. Homozygous plants of the two independent T2 overexpression lines (S20 and S26) and the mutant of this gene were selected for drought resistance testing. At seedling stage, the overexpression lines showed increased and decreased drought resistance, respectively, compared with the wild type (Fig. 6, A and B
). We evaluated the drought stress tolerance at reproductive stage in PVC pipes under the rain-off shelter according to the well-established drought stress method (Yue et al., 2006
Since OsbZIP23 is expressed predominantly in leaves (Fig. 2C), we further checked if water loss rate in leaves was affected in the transgenic plants during the dehydration process. The results showed that the leaves from the overexpression plants had significantly lower (P < 0.05) rates of water loss than wild-type plants (Fig. 6E). But the mutant showed no significant difference of water loss rate compared with the wild type (data not shown). This result prompted us to check the density and aperture of stomata in the overexpression and mutant plants, but no significant differences were observed under normal and drought stress conditions (data not shown). To evaluate salt tolerance of the transgenic rice, four-leaf plants were irrigated with water containing 200 mM NaCl, and leaf death rate was checked at 10 d after treatment. The results showed that the overexpressors had significantly more green leaf area left (approximately 80%) than the wild-type control (approximately 38%) or the mutant (approximately 20%; Fig. 7 ), suggesting that overexpression of OsbZIP23 in rice can also improve salt tolerance.
Overexpression or knockout of this gene had no significant effect on changing cold tolerance (data not shown). In addition, there was no significant difference in plant morphology (such as root depth and volume, plant height, and numbers of tillers and spikelets) between the transgenic lines and the wild type under normal growth conditions (data not shown), although spikelet fertility of transgenic plants was slightly lower than in the wild type (Supplemental Table S2), a phenomenon that has been observed very frequently in the early generations of transgenic rice produced by the tissue culture method. In general, overexpressing OsbZIP23 had no detrimental effect on the growth and development of rice.
Toward identification of the target genes regulated by OsbZIP23 and thus elucidation of the molecular mechanism of stress tolerance mediated by this gene, we further checked the genome-wide expression profile changes in the OsbZIP23-overexpressing and knockout plants using the Affymetrix GeneChip. Two independent OsbZIP23-overexpressing lines, the mutant line, and the wild type (Zhonghua 11) were used to check the expression profiles under the same growth conditions at the four-leaf stage.
A total of 795 genes showed greater than 2-fold higher (P < 0.01) expression levels in both the OsbZIP23-overexpressing lines and in the wild type (Supplemental Table S3). Even though a limited number of genes with greater than 2-fold changes are statistically significant, we still chose 2-fold as an experiential threshold because those genes with greater than 2-fold changes can seldom be confirmed by real-time PCR. Noticeably, nearly half of the up-regulated genes have been annotated or evidenced for stress response or adaptation (Supplemental Table S3). Examples are genes encoding stress-related functional proteins, such as dehydrin family proteins (four genes), late embryogenesis abundant (LEA) proteins (five genes), seed storage/lipid transfer proteins (14 genes), amino acid metabolism or transportation proteins (33 genes), cell membrane stability-related proteins (eight genes), and stress-related regulatory factors such as transcription factors (25 genes), protein kinase (seven genes), and phosphatase (10 genes). Real-time PCR was performed to confirm the expression of 16 up-regulated genes with their annotations related to stress tolerance, and the results suggested that all of the chosen genes showed elevated expression levels in the overexpression lines (Fig. 8A
), although the absolute values of the fold changes showed some variation for some genes between microarray and real-time PCR analysis. On the other hand, all 16 genes showed decreases or no significant changes in expression levels in the mutant based on real-time PCR analysis (Fig. 8B). Checking of additional genes (Supplemental Fig. S6) by real-time PCR further consolidated the GeneChip data. We also checked the stress responsiveness of the up-regulated genes in the overexpression lines in our previous microarray analysis of rice under abiotic stresses (Zhou et al., 2007
A total of 1,017 genes showed decreased expression (greater than 2-fold) in the OsbZIP23-overexpressing lines compared with the wild type (Supplemental Table S4), suggesting that overexpression of OsbZIP23 can also suppress the expression of a large number of genes. The predicted functions of these down-regulated genes are extremely diverse, and there was no obvious predominance of certain categories. In contrast to the high proportion of up-regulated genes in the overexpression lines that were induced by stresses (Supplemental Table S3), very limited numbers of the down-regulated genes were responsive (neither induced nor suppressed) to the stresses based on our previous microarray data (Supplemental Table S4). In the mutant, 86 and 141 genes were up- and down-regulated, respectively, compared with the wild type. The stress responsiveness of these genes was also checked in our previous GeneChip data. Among the 86 up-regulated genes, nine, 28, and five genes were induced by drought, ABA, and salt, respectively, while 23, nine, and 14 genes were down-regulated by drought, ABA, and salt stress, respectively (Supplemental Table S5). Among the 141 down-regulated genes, 50, 23 and seven genes were induced by drought, ABA, and high-salinity stress, respectively, and 37, 12, and 20 genes were down-regulated by drought, ABA, and salt stress, respectively (Supplemental Table S6). Some of these expression level-altered genes have also been annotated or evidenced with direct or indirect functions in stress response or adaptation, although most of them are annotated as having unknown functions in the database. By comparing the expression profile changes between the overexpressor and the mutant of OsbZIP23, 37 genes showed reverse direction of expression level changes (Supplemental Table S7). Among them, 24 genes were up-regulated in the overexpressor but down-regulated in the mutant, and 13 genes were down-regulated in the overexpressor but up-regulated in the mutant. These genes might be regulated specifically by OsbZIP23. Taken together, the expression profiling analysis revealed dramatic changes of expression profiles in both the overexpressor and the mutant of OsbZIP23, and a significant portion of the expression level-altered genes are related to stress response and adaptation. The altered ABA sensitivity and stress tolerance of the overexpressor and the mutant may have resulted from cumulative effect of the expression changes of these genes.
The bZIP transcription factors play important roles in diverse biologic processes, such as seed mutation, flower development, and stress responses. In Arabidopsis, quite a few members of the third subfamily of the bZIP family have been reported for their roles in regulating ABA-mediated stress responses. These bZIP transcription factors include ABI5 (Carles et al., 2002 ; Lopez-Molina et al., 2002; Bensmihen et al., 2005), ABF1 to ABF4 (Choi et al., 2000; Kang et al., 2002; Kim et al., 2004), and EEL (Bensmihen et al., 2002). In rice, however, only two members in this subfamily, TRAB1 (Hobo et al., 1999; Kagaya et al., 2002) and OsABI5 (Zou et al., 2007, 2008), have been reported, but their biological functions are largely unclear. Here, we report the isolation and characterization of a rice bZIP transcription factor, OsbZIP23, which belongs to the third subfamily of the bZIP family. Our data suggest that OsbZIP23 may be major player in the bZIP family in mediating ABA-dependent pathways for conferring stress tolerance, on the basis of the following arguments.
First, the OsbZIP23 gene has stronger stress-induced expression than other members of the family. OsbZIP23 is strongly induced by drought and high-salinity stresses and by PEG and ABA treatment (Fig. 2A; Supplemental Table S1). Meanwhile, more stress-related cis-acting elements, including ABRE, DRE, MYBRS, MYCRS, and GCC boxes, are present in the 1.5-kb promoter region of OsbZIP23 than in other members of this family (Fig. 2D). ABRE, MYBRS, and MYCRS can be recognized by the AREB/ABF, MYB, and MYC transcription factors, respectively. These cis-elements and the corresponding transcription factors have important roles in ABA signaling and abiotic stress responses (Yamaguchi-Shinozaki and Shinozaki, 2005
Second, overexpression and knockout of the OsbZIP23 gene resulted in significant changes in ABA sensitivity and stress resistance. The OsbZIP23-overexpressing rice plants showed significantly increased sensitivity to exogenous ABA at both germination and postgermination stages. To the contrary, the null mutant of this gene showed significantly decreased sensitivity to ABA at both germination and postgermination stages. Importantly, the phenotype of decreased ABA sensitivity in the mutant can be complemented by the OsbZIP23 gene under the control of its native promoter. The overexpression and null mutants of OsbZIP23 also showed opposite phenotypes in stress tolerance. The seedlings of the OsbZIP23 overexpression lines showed increased drought and salt tolerance, but the mutant plants performed slightly poorer (but still significantly) than the wild type under the stress conditions. This is also true for the drought resistance tested at the heading stage. Considering the facts that there are quite a few closely related members in the third subfamily of bZIP in rice (Fig. 1) and that a few other members in this subfamily are also induced by ABA and/or stresses, we originally suspected that there is a functional redundancy for OsbZIP23. But the mutant of the OsbZIP23 gene showed quite a strong phenotype (especially for ABA sensitivity), suggesting that it is a major (if not dispensable) player for mediating ABA-dependent stress responses. In rice, OsABI5 is so far the only bZIP transcription factor that was characterized for its role in stress tolerance through a transgenic approach. OsABI5-overexpressing plants were sensitive to ABA but also to high-salinity stress and PEG treatment, while the transgenic plants of antisense OsABI5 exhibited increased tolerance to salt and PEG treatment (Zou et al., 2007 Third, overexpression of OsbZIP23 can change the expression levels of a large number of genes. It is not very surprising to detect expression changes of a certain number of genes in an overexpressor of a transcription factor. However, the numbers of the genes with expression level changes in the OsbZIP23 overexpressor and mutant are indeed unexpected. Nearly 800 genes showed greater than 2-fold up-regulation in the overexpression lines. Actually, more than 2,000 genes showed up- or down-regulation in the overexpressors at a threshold of 1.5-fold, although most of the genes with 1.5- to 2-fold (some of them are significant at P < 0.05) changes cannot be confirmed by real-time PCR (data not shown). On the other hand, knockout of this gene also resulted in expression changes of hundred of genes. Importantly, many of the up- or down-regulated genes in the OsbZIP23 overexpressor and mutant are predicted or shown to be involved in stress tolerance. Since OsbZIP23 has transactivation activity in yeast and many genes in rice can be affected in their expression levels in the OsbZIP23 overexpressor and mutant, we speculate that OsbZIP23 functions as a major transcriptional activator among the bZIP family to regulate genes involved in ABA sensitivity and stress tolerance in rice.
The results of leaf water-loss experiments indicated that OsbZIP23 overexpressors have stronger water-holding capability than wild-type plants, but the results of scanning electron microscopy suggested that stomata may not be involved in the drought resistance in this case, since neither density nor aperture of stomata showed differences between the overexpressor and the wild type. Nevertheless, the GeneChip profiling analysis provided informative clues for dissecting the molecular mechanism of the improved stress tolerance of transgenic rice plants overexpressing OsbZIP23. Among the up-regulated genes in OsbZIP23-overexpressing plant lines, a large number of genes have been reported or predicted to be involved in stress tolerance. We noticed that more than 10 genes encoding dehydrin or LEA proteins were significantly up-regulated in the overexpressors. Dehydrin family proteins have been reported for their roles in the plant-protective reactions against dehydration (Nylander et al., 2001 Although more than 1,000 genes showed decreased expression in the overexpression lines, only a small portion of them might be related to abiotic stress according to their annotations. It cannot be excluded that some of these down-regulated genes may also contribute to the improved stress tolerance or other growth or developmental aspects, since a majority of these genes are unknown in their functions. The expression profiling analysis of the mutant has also provided some informative clues on the stress tolerance conferred by the OsbZIP23 gene. For example, most of those genes up-regulated in the OsbZIP23 overexpression plants were down-regulated or not changed (because of partial functional redundancy) from their expression levels in the mutant. Many of the genes down-regulated in the mutant are also stress responsive and/or annotated with putative functions in stress response or tolerance. Even though a full scenario of the molecular mechanism of the stress resistance conferred by OsbZIP23 remains to be completely revealed, the expression changes of all the well-annotated or evidenced stress-related genes in the overexpression or mutant line suggest that enhanced protection ability at the cellular level (such as osmotic adjustment, stabilization of functional proteins, and cell membrane) may be a major contribution to the stress tolerance conferred by OsbZIP23. In conclusion, this study showed that OsbZIP23 belongs to the third subfamily of bZIP transcription factors and positively regulates ABA sensitivity and stress resistance in rice. Alteration of OsbZIP23 expression can change the expression levels of more than 1,000 genes, and many of these genes are involved in stress responses or tolerance. Our results also suggest that the OsbZIP23 gene has high potential usefulness in improving the stress resistance of crops.
Generation of Transgenic Plants and Knockout Mutant
The full-length cDNAs of OsbZIP23 were amplified from upland rice (Oryza sativa IRAT109) by RT-PCR with the following primers: 5'-TAAGGTACCATCCCACCTCTCCTCAGGTT-3' and 5'-TAAGGATCCGGCAGCTTCACCATCCTACT-3' (the underlined sequences are for restriction sites KpnI and BamHI, respectively). The sequence-confirmed PCR fragment was digested by KpnI and BamHI and ligated into the binary expression vector pCAMBIA1301U, which was also digested with KpnI and BamHI, thus allowing the genes to be driven by a maize (Zea mays) ubiquitin promoter. The construct was introduced into japonica cv Zhonghua 11 by Agrobacterium-mediated transformation (Hiei et al., 1994
Mutant seeds of OsbZIP23 were obtained from the Rice Mutant Database (http://rmd.ncpgr.cn/; Zhang et al., 2006
The planting and drought treatment in the PVC pipes (1 m in length and 0.2 m in diameter) was essentially the same as described previously (Yue et al., 2006
To detect the transcript levels of genes, IRAT109 rice plants were grown in the greenhouse with a 14-h-light/10-h-dark cycle. Two-week-old seedlings were treated with chemical or abiotic stress. Chemical treatments were conducted by spraying leaves with 0.1 mM ABA followed by sampling at 0, 3, 6, 12, and 24 h, or spraying leaves with 0.1 mM JA, 0.1 mM SA, or 0.1 mM 2,4-D followed by sampling at 0, 1, 2, 6, and 12 h, or irrigating the plants with 20% PEG 6000 followed by sampling at 0, 1, 5, and 12 h. Abiotic stress treatments for gene expression level analysis were essentially the same as in our previous report (Xiang et al., 2007
Total RNA was isolated from rice leaves using TRIzol reagent (Invitrogen). For real-time PCR analysis, first-strand cDNAs were synthesized from DNaseI-treated total RNA using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed on an optical 96-well plate with an ABI PRISM 7500 real-time PCR system (Applied Biosystems). Each reaction contained 10 µL of 2x SYBR Green Master Mix Reagent (Applied Biosystems), 1.0 µL of cDNA samples, and 200 nM gene-specific primer in a final volume of 20 µL. The thermal cycle used was as follows: 95°C for 3 min, then 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The rice Actin1 gene (accession no. X16280) was used as an internal control with primers 5'-TGGCATCTCTCAGCACATTCC-3 and 5'-TGCACAATGGATGGGTCAGA-3'. The relative expression levels were determined as described previously (Livak and Schmittgen, 2001
The subcellular localization vector was constructed by replacing the GUS fragment of pCAMBIA1391Xb (CAMBIA) with a ubiquitin promoter-GFP cassette. The coding region of OsbZIP23 cDNA was amplified from IRAT109 rice using the following primer pair: 5'-TAAGGTACCATCCCACCTCTCCTCAGGTT-3' (the underlined part is for the KpnI site) and 5'-TAAAAGCTTGATCAGAGACGGGAACCTGA-3' (the underlined part is for the BamHI site). The amplified fragment was inserted upstream of and in frame with GFP. The procedure of bombarding onion (Allium cepa) epidermal cells was conducted as described by Dai et al. (2007)
RNA samples of transgenic plants of overexpressors, the T-DNA insertion mutant, and the wild type were extracted using TRIzol (Invitrogen) as described by the manufacturer. Three independent samples (replicates) were used for each line. Probe labeling and chip hybridization were carried out through the Affymetrix custom service (CapitalBio) by following the standard protocol (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Normalization was performed according to the standard Affymetrix protocol to allow the comparison of the samples for each set of experiments. The Excel add-in for significance analysis of microarrays was used to identify differentially expressed genes between the control and transgenic plants. Partial up- or down-regulated genes from GeneChip analysis were confirmed by real-time PCR analysis using the rice Actin1 gene as an internal control for the calibration of relative expression. Stress responsiveness of the genes that were up- or down-regulated in the overexpression or mutant plant were checked in our previous expression profiling analysis of rice under various stress conditions (Zhou et al., 2007 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AK072062.
The following materials are available in the online version of this article.
Received August 18, 2008; accepted October 15, 2008; published October 17, 2008.
1 This work was supported by the National Special Key Project of China on Functional Genomics of Major Plants and Animals, the National Program on the Development of Basic Research, the National Natural Science Foundation of China, the Ministry of Education of China (grant no. NO 707045), the European Union FP6 INCO-MPC2 project (grant no. INCOCT–2005–015468), and the Rockefeller Foundation.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Lizhong Xiong (lizhongx{at}mail.hzau.edu.cn).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.128199 * Corresponding author; e-mail lizhongx{at}mail.hzau.edu.cn.
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