Molecular Mechanism Of Rice Fertility Regulated By Programmed Cell Death Of Tapetum

Rice is one of the most important food crops and an ideal model plant of monocotyledons for molecular genetic researches. After completed sequencing of rice genomes, the most key step for functional genomics is characterizing every gene in rice using T-DNA or transposable element insertion mutant collection. Here, we isolated a mutant line osapi5-1 in our T-DNA insertion mutant library, showing the male and female sterility, which is co-segregated with the T-DNA insertion site in osapi5. Histological and histological chemistry examination of osapi5-1 mutant showed that delayed degeneration of tapetum and defects in formation of male gametophytes were caused by inhibition of the tapetal PCD process. While, sterile embryo sacs was resulted from degeneration of functional macrospores with unknown reasons after meiosis in the osapi5-1. Successful complementary test and RNAi of OsAPI5 verified the loss-of-function of the OsAPI5 was the reason for male and female sterility of osapi5-1. OsAPI5 encodes a homologue gene of Apoptosis inhibitor 5 (Api5), a member of ancient conserved gene family, which exist widely in protists, plants and animals. OsAPI5 was characterized as a nuclear protein with transcriptional activation activity in yeast. QRT-PCR results showed that the OsAPI5 is a constitutive expression gene which with preferred expression in tapetum. Two DEAD-box ATP-dependent RNA helicases, AIP1 and AIP2 (for API5 Interacting Protein 1 and 2), were identified as its physical partners by yeast two hybrid methods. This interacteion was further confirmed by BiFC (Bimolecular Fluorescence Complementation, BiFC) and Co-IP (Co-Immunoprecipitation). Only three amino acids are different between AIP1 and AIP2 and these differences do not influence the interaction between AIP1/2 and OsAPI5. Expression analyses showed that AIP1 and AIP2 were also ubiquitous expressed genes. The subcellular localization of AIP1 and AIP2 in Arabidopsis mesophyll protoplasts is nuclear and cytoplasm, while the dimerization of AIP1/2 taken placed in nuclear specifically. Suppressed expression of AIP1/2 simultaneously by artificial micro RNA (amiRNA-AIPl/2) resulted in male sterility of the transgenic plants, in which delayed degeneration of tapetum were also observed. AIP1/2 can complement the lethal phenotype of yeast sub2 mutant and then were considered to an ortholog of Saccharomyces cerevisiae (yeast) SUB2p. To find the downstream genes of the OsAPI5 and AIP1/2, we compared the expression profiles between WT and osapi5-1 and a rice cysteine protease gene OsCP1 was down-regulated about 2-fold in osapi5-1. Suppressed expression of OsCP1 caused a significant defect in pollen development, which showed similar phenotypes with those of osapi5 and amiRNA-AIP1/2 transgenic plants. QRT-PCR analyses verified that the expression level of OsCP1 in osapi5-1 and amiR-AIP1/2 transgenic plants anthers was decreased dramatically. Next, we identified a promoter region CP1-P-1 of OsCP1, which interacts specifically with AIP1 in yeast and further confirmed in vitro by EMSA (Electrophoretic Mobility Shift Assay) and ChIP assay in vivo. Taken together, we speculate that OsAPI5-AIP1/2 complex, just like a native "hybrid" transcription factor, regulates the tapetum degenenration of rice anthers by up-regulating the OsCP1 through the binding to the promoter of OsCPl. These results provide some important molecular network of OsAPI5, AIP1/2 and OsCPl for understanding the molecular mechanisms of postmeiotic anther development in rice.