Functional Characterization Of Rice Stress-Related BZIP Transcription Factors

Plants have evolved complicated protective mechanisms to survive the adverse environmental condition. Hereinto, bZIP transcription factors play pivotal roles in the abscisic acid (ABA)-mediated abiotic stress tolerance of plants. Isolation and identification of the stress-related bZIP factors may provide opportunities for rice stress tolerance improvement. In this research, stress-responsive bZIP genes from rice were overexpressed in rice, and stress tolerance and ABA sensitivity of the transgenic plants were tested. Biological function of a few bZIP genes involved in the ABA response and stress tolerance were identified. We further characterized the functions of two OsbZIP transcription factors and a protein BIP4 that interacts with OsbZIP23, a previously reported key regulator of ABA and stress response in rice. The main results are as follows:1, Twenty-two rice bZIP genes that are stress-inducible in cDNA microarray analysis were construted for over-expression in rice Zhonghua 11 (Oryza sativa L. ssp japonica). The over-expression levels of these transgenes were checked by Northern or Real-time quantitative PCR analysis. ABA sensitivity and stress tolerance of transgenic plants at seedling and reproductive stage were tested. Transgenic plants of two gene, named ST10 (also designated as OsbZIP46) and ST14, showed significant changes in drought tolerance and ABA sensitivity. These two genes were selected for functional characterization.2, Expression of OsbZIP46 was strongly induced by drought, heat, hydrogen peroxide, and ABA treatment; however, it was not induced by salt and cold stresses. OsbZIP46 was a nuclear protein. Overexpression of the native OsbZIP46 gene increased ABA sensitivity but had no positive effect on drought resistance. The expression level of RAB21 in the OsbZIP46 overexpression plants showed no difference compared with that in the wild type under normal growth conditions; however, it was significantly increased in the overexpression plants after exogenous application of ABA. The activation domain of OsbZIP46 was defined by a series of deletions. The native OsbZIP46 has no transactivation activity and a region (domain D) was identified as having a negative effect on the activation. We produced a constitutive active form of OsbZIP46 (OsbZIP46CA1), which contains the N terminal transactivation domain and C terminal bZIP DNA binding domain, while the domain D is absent. Overexpression of OsbZIP46CAl in rice significantly increased the ABA sensitivity and the tolerance to drought and osmotic stresses. Gene chip analysis of the two overexpressors (native OsbZIP46 and the constitutive active form OsbZIP46CAl) revealed that a large number of stress-related genes, many of them predicted to be downstream genes of ABF/AREBs, were activated in the OsbZIP46CAl overexpressor but not (even down-regulated) in the OsbZIP46 overexpressor. OsbZIP46 can interact with OsSAPK2, OsSAPK6 and OsSAPK9, the homologs SnRK2 protein kinases that phosphorylate ABFs (bZIPs) in Arabidopsis. In vitro phosphorylation assay demonstrated that the OsbZIP46 protein could be phosphorylated by OsSAPK2 and OsSAPK6. We generated OsbZIP46 phosphorylation active forms (OsbZIP46PA) by single or multiple amino acid substitutions (Ser/Thr to Asp) of OsbZIP46, and these phosphorylation active forms showed significantly enhanced transactivation activity.2, Expression of ST14 was induced by drought, but also by ABA treatment; however, it was not induced by salt and cold stresses. We detected the expression of ST14 in various tissues and found that the expression level in panicle and leaf were higher than others. ST14 was a nuclear protein and functioned as a transcriptional activator in the transactivation assay in yeast. It was demonstrated that the N terminal (amino acids 1-46) was required for its transactivation activity. The transgenic rice overexpressing ST14 was hypersensitive to ABA in post-germination growth compared to wild type, and it also showed significantly improved tolerance to drought stress. The water lose rate of transgenic plants was significantly lower than wild type, which was corresponding to the improved drought tolerance phenotype. Real-time quantitative PCR analysis revealed that many stress related genes including LEA class were up-regulated in the rice plants overexpressing ST14. Additionally, The expression levels of genes encoding ABA 8’-hydroxylases, key enzymes in the oxidative catabolism, were down-regulated and the ABA content was increased in the ST14-overexpressors.3, We identified a novel OsbZIP23-interacting protein BIP4 by yeast two-hybrid screening. BIP4 was localized in the nucleus and interacted with OsbZIP23 in vivo. BIP4 interacted with OsbZIP23 probally through the D domain. Sequence analysis indicated that BIP4 is function-unknown protein with significant homologs existing in other plants. The expression of BIP4 was induced by ABA and drought stress, but the induction was much less and slower than that of OsbZIP23. Overexpression of BIP4 in rice resulted in decreased ABA sensitivity, whereas two allelic bip4 mutants (bip4-1 and bip4-2) showed increased ABA sensitivity and drought tolerance. The expression levels of OsbZIP23-targeting genes, including RAB21 and OsPP2C that are positively regulated by OsbZIP23, were inversely correlated to the expression level of BIP4. The mRNA level of OsbZIP23 was not affected in BIP4-overexpressing and the bip4 mutant plants, but the protein level expression of OsbZIP23 was up-regulated in BIP4-overexpressing and down-regulated in the bip4 mutant plants. We further identified a BIP4-interacting protein FIP1 (BIP-Four-Interacting Protein 1, which is predicted to be a E3 ubiquitin ligase) by yeast two-hybrid screening. The null fipl mutant showed increased ABA sensitivity and drought tolerance. The functional analyses of BIP4 and FIP1 suggest that the degradation of OsbZIP23 depends on ubiquitin/26S proteasome system.