| Rice (Oryza sativa L.) is the world’s staple food crop. Most half the world’s population eats the rice as the main grain. Rice male sterility and the wide application of heterosis played an important role in agricultural production. The molecular mechanism of floral organ development is a hotspot in plant developmental biology researches. The development of rice floral organ directly affects the yield and quality. Therefore, understanding the molecular mechanism of floral organ development is very important for rice breeding. In flowering plants, tapetum plays an important role in the process of pollen development. It supports pollen wall formation and the subsequent pollen development, while in the late stage it is crucial for the development of fertile pollen that the Tapetum degenerates, what is considered to be triggered by programmed cell death (PCD). However, the molecular basis regulating tapetum PCD remains poorly understood.The rice male sterile mutant which named tapetum degeneration retardation (tdr), exhibit aborted degeneration of the middle layer and tapetum, as well as collapse of microspores. The TDR encodes a putative basic/helix-loop-helix (bHLH) protein and it is mainly expressed in the tapetum. The downstream genes regulated by TDR have been isolated by using chromatin immunoprecipitation (ChIP) and ChIP-sequencing, and a bunch of downstream genes are shown to be likely direct targets of TDR. Among these genes, OsADF (LOC_Osl0g03660), encoding a F-box protein was found. F-box protein is an expanding family member of eukaryotic protein characterized by an F-box motif which has specificity of substrate recognition in the ubiquitin-mediated proteolysis. These proteins have been proved to be critical for many physiological processes, such as cell-cycle transition, signal transduction, gene transcription, male sterility, programmed cell death and so on. To better understand the role of OsADF during pollen development, OsADF gene has been isolated from rice and its expression patterns and functions were analyzed. The data provide some important information to understand the molecular mechanism of pollen development. The main results and conclusions are as follows:1. OsADF was proved to be the direct targets of TDR through ChIP-PCR analyses and the electrophoretic mobility shift assay (EMSA). Furthermore, additional evidence is provided that the expression of OsADF is positively regulated by TDR, OsADF could expressed in the tdr mutant when it was driven by the TDR promoter.2. Bioinformatics analysis showed that OsADF belongs to F-box protein family. RT-PCR, promoter activity assay of OsADF and in situ hybridization analyses indicated that OsADF expression is mainly detectable in rice tapetal cells and microspores from stage 9 to stage 12 of anther development. OsADF-YFP fusion protein was located in the cell membrane.3. Plants in which OsADF was over expressed showed reduced pollen viability. Moreover, rice plants in which OsADF was silenced exhibited a similar phenotype as TDR deficient (tdr) plants with abnormal degeneration retardation of the Tapetum as well as collapse of microspores and reduced pollen fertility. To determine the morphological defects of anthers of the OsADF-RNAi lines, transverse sections were examined. These observations suggested that knockdown of OsADF could alters pollen development in rice. Real-time PCR analysis indicated that the expression of target gene OsADF from transgenic plant was lower than that of the Nipponbare.These results suggest that the TDR downstream gene OsADF plays a crucial role in anther development of rice. These data provide an important biological insight in understanding of anther development in rice. |
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