Mechanism Of OsPPR939 In Rice Pollen Development And Plant Growth

Rice is one of the most important food crops in the world,and more than half of the world's population is mainly fed on rice,hence rice plays an important role in ensuring world food security.Rice pollen fertility plays a decisive role in the formation of final yield to a large extent.At present,some genes related to rice pollen development have been cloned and functionally analyzed,but many of these genes are still remins unclear about their regulatory role in the pollen development.Therefore,a comprehensive study on the function of these unknown genes related to rice pollen development will help to further elucidate the molecular mechanism of their regulation on pollen development.In this study,the function of OsPPR939,a rice pollen fertility related gene,was studied in detail by means of reverse genetics and molecular biology.The main results and conclusions of this study are as follows:?1?OsPPR939 encodes a P-subfamily Pentatricopeptide repeat?PPR?protein,which consists of 939 amino acids.This protein contains an N-terminal mitochondrial localization signal peptide and 22 P-type PPR motifs,and no other domains at the C-terminus.?2?Transient expression assay in rice protoplasts and stable expression assay in Arabidopsis revealed that OsPPR939N^1-280 is localized in mitochondria.?3?OsPPR939 was expressed in a constitutive manner in rice,with the highest expression in calli,and a relatively higher expression level in spikelet at stage 6.RNA in situ hybridization of wild-type anthers showed that OsPPR939 was highly expressed in tapetum and male meiocytes.?4?Based on the CRISPR/Cas9-generated mutation,it was found that mutations at different positions of OsPPR939 gene caused complete or partial loss of OsPPR939function,resulting in different degrees of growth retardation and pollen sterility in osppr939 mutants.In osppr939-4 and osppr939-5 mutants,the abundance of mitochondrial nad5 transcript was significantly reduced,and further screening of the splicing of 23 group II introns in the mitochondria of rice revealed that only the splicing efficiency of the nad5 introns 1,2,and 3 decreased.?5?Analyzing the assembly and activity of mitochondrial electron transport chain complex I showed that the abundance of complex I in osppr939-4 mutant was reduced,and the NADH dehydrogenase activity of complex I was seriously decreased.?6?The qRT-PCR and RT-PCR results revealed that the transcription levels of genes involved in alternative respiratory pathways were increased in osppr939 mutant.?7?Transgenic complemention of rice osppr939-4 and Arabidopsis tang2mutants could restore the plant phenotype and nad5 transcript abundance to the wild-type level,which confirmed that the defective phenotype of osppr939-4 was indeed caused by the mutation of OsPPR939.?8?The yeast two-hybrid assay and bimolecule fluorescence complementary experiments proved that OsPPR939 interacts with h subunit of eukaryoutic translation initiation factor 3 OseIF3h.?9?The unaffected transcription of OsPPR939 and reduced abundance of nad5in oseif3h mutant suggested that the translation of OsPPR939 was under regulation of OseIF protein.In summary,this study revealed that OsPPR939 which may be translationally regulated by OseIF3h plays a crucial role in maintaining mitochondrial function,regulating plant growth and pollen fertility by participating in the splicing of mitochondrial nad5 introns 1,2,and 3.