Cloning And Functional Charactization Of Panicle Apical Abortion 1019 And Panicle Apical Abortion 74 In Rice

Panicle apical abortion?PAA?causes severe yield losses in rice production and it's related to environmental factors,but details in its development and its molecular basis remain elusive.Herein,two PAA mutants,paa1019 and paa74,was identified among the progeny of the rice elite indica maintainer line Yixiang 1B?YXB?using ethyl methyl sulfonate mutagenesis.The abortion rate of spikelets is different between paa1019 and paa74.Genetic mapping combined with Mutmap analysis revealed that LOC_Os03g20380and LOC_Os02g45160 harbored respectively a single bp substitution that altered transcript sequences.We verified their biological function in rice.The main results are summarized as below:1.We characterized the PAA mutant phenotypes and confirmed that the abortion phenotype of paa1019 and paa74 was resulted from accumulated cell death.DAB staining revealed that H₂O₂ was deposited in paa1019 and paa74 panicles;2.We characterized the abortion spikelets distribution in time and location.Spatiotemporal analysis of spikelet degeneration in both paa1019 and paa74 indicated apical dominance,potentially caused by blocking hormone transportation,especially indole-3-acetic acid/auxin;3.Cross-sections of spikelets along the middle region of the hull,which showed that the cell wall and cytoplasmic membrane of glumes and stamens were deformed during both booting and heading stages,and the anther and stigma were more withered,ovaries were fragile,and the filament and style offered minimal support.I₂-KI staining suggested these were not viable.The structural damage situation in paa74 is slighter than that in paa1019;4.Genetic analysis indicated that the PAA phenotype of paa1019 and paa74 is regulated by a single recessive nuclear gene.By genetic mapping combined with Mutmap analysis,for paa1019,LOC_Os03g20380 encodes calcineurin B-like protein-interacting protein kinase 31?OsCIPK31?and harbored a single bp substitution?C to T?that altered transcript length.For paa74,LOC_Os02g45160 encodes aluminium-activatedmalate transporter 7?OsALMT7?and harbored a single bp substitution?G to A?,which led to a Arginine transform to a lysine.Complementation of paa1019 by transferring the open reading frame of LOC_Os03g20380 from YXB reversed the mutant phenotype,and conversely,gene editing by knockdown of OsCIPK31 and OsALMT7 in YXB generated the PAA phenotype;5.OsCIPK31,a member of the OsCIPK family,to further elucidate the multifaceted functions of OsCIPK31 in rice,we evaluated the performance of paa1019 under cold,salt and drought conditions during the seedling stage.The results demonstrated that OsCIPK31positively regulates cold,salt and pathogen stress tolerances in rice and negatively regulates drought stress tolerance.For OsALMT7,we briefly investigated Al³⁺resistance in WT and paa74 plants and there's no obvious phenotype difference between WT and paa74plants;6.OsCIPK31 was expressed in all tissues examined.The highest expression was found in young roots,and expression was lowest in mature roots.Expression was relatively high in leaves,stems,stamens and panicle necks,and lowest in developing panicles.However,expression levels were increased during panicle development.Subcellular location analysis in the rice protoplast indicated that OsCIPK31 was expressed in the cytoplasm.OsALMT7 was expressed in all tissues examined,the highest expression was found in young and mature roots,corresponding with the transporter function.RNA in situ hybridization was performed and the results demonstrated the preferential expression of OsALMT7 in the stamens of spikelets and the hull of vascular tissues in stems.7.RNA-seq and WGCNA analysis identified OsCIPK31 might be involved in regulating hormone signal transduction and the MAPK signaling pathway and OsCIPK31might be involved in regulating DNA duplication and cell division process.Yeast two hybridization was performed and the results demonstrated there's no interaction between OsCIPK31 and OsALMT7.Meanwhile,the PAA phenotype of double mutant paa1019/74is not aggravated,maybe the same pathway in regulating spikelets development is existed between OsCIPK31 and OsALMT7.