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.

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.

Abscisic acid signaling and biosynthesis are feedback-regulated by transcription factor OsbZIP23 that targets diverse genes involved in drought resilience.

Many stress responsive genes have been reported with an effect on improving stress resistance in model plants under greenhouse conditions. Towards identification of genes for drought resistance breeding, seven well documented genes (CBF3, SOS2, NCED2, NPK1, LOS5, ZAT10, and NHX1) in stress resistance were selected in this study and transformed into rice cultivar Zhonghua 11 under the control of constitutive promoter Actin1 and stress-inducible promoter of a rice HVA22 homolog, and transgenic rice were tested for drought resistance under field conditions. A total of 1598 independent transgenic T₀ plants were generated. The percentages of single copy and expression of the transgenes were 36.7% and 57.6%, respectively. For each gene construct, 30 T₁ families with expression of transgene were selected for drought resistance testing at the reproductive stage in field, and 10 of them were tested in PVC pipes with a defined stress protocol at the same stage. Relative yield and relative spikelet fertility were used as two major criteria to evaluate drought resistance performance because significantly decreased yield was observed in the T₁ generation. Transgenic families of eight constructs (HVA22P:CBF3, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, Actin1:NHX1, and HVA22P:NHX1) showed significantly higher RY than wild-type (WT) under both drought stress field and PVC tube conditions. Transgenic families of 9 constructs (HVA22P:SOS2 and CBF3, LOS5, ZAT10, and NHX1 by both promoters) showed significantly higher relative spikelet fertility than WT in the field or PVC pipes. In the field drought resistance testing of T₂ families derived from the T₁ families with relatively lower yield decrease, transgenic families of seven constructs (HVA22P:CBF3, Actin1:NPK1, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, and HVA22P:ZAT10) showed significantly higher yield per plant than WT, and families of nine constructs (Actin1:CBF3, HVA22P:CBF3, HVA22P:SOS2, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, and Actin1:NHX1) had higher spikelet fertility than WT. In general, LOS5 and ZAT10 showed relatively better effect than the other five genes in improving drought resistance of transgenic rice under field conditions. The results and experience obtained from this study could be a useful reference for drought resistance engineering in rice.