| Drought stress affects rice reproduction and results in severe yield loss. The developmental defects and gene regulation network in reproductive tissues under drought stress are largely unknown. In this study, rice plants subjected to drought stress during reproductive stage were examined for floral development and transcriptomic changes. Results showed that male fertility was dramatically affected, with differing pollen viability in flowers of the same panicle due to aberrant anther development. Examination of local starch distribution revealed that starch accumulated abnormally in terms of position and abundance in anthers of water-stressed plants. Microarray analysis using florets of different sizes identified>1600drought-responsive genes, most of which were specifically regulated in only one or two particular sizes of florets, suggesting developmental stage-dependent responses to drought. Genes known to be involved in tapetum and/or microspore development, cell wall formation or expansion, and starch synthesis were found more frequently among the genes affected by drought, while meiosis and MADS-box genes were less frequently affected. In addition, pathways related to gibberellin acid signaling and abscisic acid catabolism were reprogrammed by drought. Our results strongly suggest interactions between reproductive development, phytohormone signaling and carbohydrate metabolism in water-stressed plants.We identified1617candidates involved in the reproductive development under drought stress using microarray analysis. To further validate the genes vital for the floral development under drought stress, we selected dozens of genes, and obtained rice and Arabidopsis mutants with these gene loss or gain-of-function. Subsequently we studied their phenotype under NaCl, ABA, PEG treatment and drought stress. The results showed that Arabidopsis mutant3-ooac was sensitive to NaCl; rice mutant cao exhibited extremely low pollen fertility and Arabidopsis mutant cao was sensitive to ABA; overexpression of OsPP2C in rice and Arabidopsis enhanced plant drought tolerance; another rice mutant msp could produce larger seeds. We decided to focus on OsERF, because it exhibited a distinct drought-resistance phenotype.The OsERF is named as OsERF101according to the phylogenetic analysis of AP2/EREBP family. Promoter-GUS fusion protein staining, In situ hybridization and qRT-PCR analysis showed that, under normal growth conditions, the OsERF101was not expressed in root, young leaves or old leaves while it was highly expressed in old leaves under drought stress, Additionally, expression level of OsERF101was high in flowers during the early stage of microspore development, especially in tapetum and microspore cells, and then decreased. Moreover, expression of OsERF101was induced by PEG and ABA treatment.Overexpression of OsERF101enhanced drought tolerance of transgenic plants at seedling and reproduction stages. Increasing proline content and peroxidase activity in transgenic plants could be one reason of improving drought-tolerance. Overexpression of OsERF101also increased the yield by increasing pollen fertility and seed setting rate. We checked the expression level of drought-responsive and development-regulated genes, found that OsERF101could activate ABA-responsive gene expression. In addition, Oserf101mutants and RNAi plants showed a low fertility under normal growth conditions. We detected three different anther types among Oserf101and RNAi plants:â… ) the normal anthers, â…¡) the anthers without pollen sacs and â…¢) the deflated anther with abnormal or none pollen. These indicated that OsERF101also involved in anther development process.More interestingly, OsERF101was located in the cytoplasm instead of the nucleus under normal growth condition, although it is a member of AP2/EREBP transcription factors. It can be observed in the nucleus only if its amino-terminal signal peptide was cleaved. We hypothesized that OsERF101possibly regulates the anther development under drought stress by multi-targeting way, and more experiments are required to confirm this hypothesis.
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