Map-based Cloning And Functional Analysis Of FLO10 Involved In Rice Endosperm Development

In cereal crops,endosperm is the main energy storage organ,storing a large amount of starch,which is the main source of human food.The increase in the number of people and the ever-increasing need for a better life of the people have made the demand for rice in terms of quantity and quality increasingly high.The development of endosperm and the nutrients accumulation in endosperm directly affect the yield and quality of rice.Previous studies have identified many key enzymes for starch synthesis and some regulatory factors.However,the development and grain filling process of endosperm is still poorly understood,and new regulatory factors need to be explored.In this study,we isolated a rice endosperm defective mutant,floury endosperm10(flo10).The molecular function of FLO10 were studied and the role of FLO10 in endosperm development and grain filling process were discussed.The main results as follows:(1)The flo10 mutant was isolated from an N-methyl-N-nitrosourea(MNU)-treated mutant pool of the indica cv N22.Compared with wild type,flo10 exhibited defective grain development evidenced by smaller and opaque grains at maturity.During endosperm development,the grain filling rate and starch accumulation offlo10 were significantly lower than those of wild type.The length,width and thickness of flo10 mature grains decreased significantly,and the same as 1000-grain weight.The total starch and amylose content of flo10 decreased,while the protein and lipid content increased.Also,the flo10 mutant exhibited obviously slower growth during the vegetative and reproductive stages.In conclusion,the mutation inflo10 causes defective starch synthesis in endosperm and retarded plant growth.(2)Semithin sections observation at the early stage of endosperm development showed that flo10 mutant endosperm cells had smaller SGs,smaller aleurone cells with sparse contents.In addition,more starch grains were retained in the pericarp of the flo10 mutant,while less starch was accumulated in the starchy endosperm.The expression of sugar transporter-encoding genes,including OsSUTs and OsSWEETs,were significantly affected during early endosperm development in flo10.These results imply that flo10 developed an abnormal aleurone layer,which might obstruct the nutrition transfer from maternal tissues into the endosperm.(3)To identify the FLO10 gene,we constructed an F2 mapping population by crossing flo 10 with japonica cv Nipponbare.The,flo10 locus was narrowed down to a 57.9 kb genomic region containing 15 putative ORFs.Sequencing analysis revealed a single cytosine(C)deletion in the coding region of the LOC＿Os03g07220 gene,causing a premature translation stop at the N terminus.Transgenic complementation and gene knockout experiments proved that LOC＿Os0307200 is FL010.FLO10 was widely expressed in all tissues examined,with relatively higher expression in leaves,leaf sheaths and developing endosperm.FLO10 encodes a protein comprising 1269 amino acids,which harbors 26 typical PPR repeats and belongs to the P subfamily.Subcellular localization experiments showed that FLO 10 protein was localized in mitochondria but not in chloroplasts.(4)Of the 33 mitochondrial genes analyzed,only the mature nadl transcript was obviously reduced in flo10.The splicing efficiency analysis of group II introns coding by mitochondrial genome showed that the splicing efficiency of nadl intron 1 dramatically reduced in.flo10.Northern blot experiments showed that flo 10 accumulated high levels of both dissociated nadl exon 1 and exons 2-5 precursors,compared with the mature nadl mRNA in wild-type and complemented lines.Circular RT-PCR combined with clone sequencing further confirms the increase of nadl exon 1-intronla and exons 2-5 precursors.and reveals that the 5’UTR processing of nadl exon 1 precursors is also affected in flo10.(5)nad1 encodes a core subunit of respiratory chain complex I.Blue native PAGE analyses showed that the abundant and activity of complex I in flo10 decreased significantly.The ATP content in flo10 endosperm was only about 33%of that in wild type and complementary lines.The defect of complex I in flo10 activates the alternative respiratory pathway,and decreased the cristae and inner membrane area of mitochondria.In conclusion,the splicing defect of nadl intron 1 in flo10 leads to the decrease of assembly and activity of respiratory chain complex I,leading to the decrease of ATP content,the induction of alternative respiratory pathway and structural change of mitochondria in rice endosperm.In conclusion,FL010 encodes a mitochondria-localized P-type PPR protein.FLO 10 participates in the splicing of mitochondrial nadl intron 1,affecting the ATP production of mitochondria in endosperm,which may further affect the process of nutrient uptake from maternal tissues to filial endosperm during seed development.This study provides an important basis for studying the regulation of P-type PPR protein on rice endosperm development,and lay a theoretical foundation for improving rice yield and quality.