Map-Based Cloning And Functional Analysis Of Flo18 Involved In Rice Endosperm Development

Rice（Oryza sativa L.）is one of the most important food in the world,which has made a great contribution to world food reserves.In recent years,with the efforts of breeding experts and the implementation of high-yield and super rice breeding programs,the yield of China’s rice has been greatly improved,which is the cornerstone of national food security.However,with the improvement of people’s consumption level and quality of life,the demand for high-quality rice has gradually increased,and improving the quality of rice has become one of the important objectives of breeding.Endosperm accumulates large amounts of storage substances,such as starch and protein,and plays a crucial role in seed germination and seedling growth.It is also the main part of rice for eating.Starch is the most important chemical component in endosperm and an important factor in determining rice quality.Despite some advances have been made in the discovery of genes regulating endosperm development and starch synthesis in rice,the rice endosperm development and regulatory network of starch synthesis remains obscure,and it is necessary to further explore and research new regulatory factors.In this study,we isolated and characterized rice floury endosperm mutant named flo18,with deffects on both endosperm development and vegetative growth.We performed map-based cloning of the FLO18 gene and analyzed the molecular biological functions of the gene.The results are as follows:（1）flo18 is a mutant with abnormal endosperm developmentThe flo18 mutant was obtained from an MNU（N-methyl-N-nitrosourea）-treated population of japonica rice cv.Ningjing 3.Compared with the wild type,the mature kernels of flo18 was opaque,1000-grain weight and grain thickness decreased significantly.The amylose,starch,protein contents were reduced in dry weight of mature flo18 seeds compared to that of wild type,only lipid contents were elevated.In addition,the seed germination and seedling growth of flo18 were retarded,and could not grow into normal plants,resulting in death at seedling stage.（2）cytological observation of developing endosperm in flo18The semi-thin section observation of endosperm at early development stage showed that the shape and size of starch grains in the endosperm of flo18 mutant were changed,and the aleurone layer cells were abnormal,and more starch remained in the pericarp.At the same time,the expression level of sugar transporter genes in endosperm was significantly decreased.These results suggest that the extracellular pathway of sucrose transfer maternal tissue to filial endosperm was disturbed in flo18 endosperm,and starch synthesis was affected.In summary,the mutation of FLO18 affected the development of starch granules and aleurone layer in endosperm,and the transportation of nutrients from maternal tissue into endosperm.（3）FLO18 encodes a P-type PPR proteinWe constructed an F₂ population by crossing flo18^+/-heterozygous plants with an indica cv N22.The FLO18 locus was finally mapped to 57 kb on the long arm of chromosome 7.Sequencing analysis revealed that Os07g0688100 contained a single nucleotide substitution of thymidine（T）to adenosine（A）in the coding region,which led to a premature translation stop.Transgenic complementation experiments proved that Os07g0688100 is the mutated gene FLO18.According to prediction on the website,Os07g0688100 encodes a PPR protein of 665 amino acids,and it contained 15 PPR motifs and belonged to the P-type PPR protein group.Subcellular localization experiments indicated that FLO18 was localized in mitochondria.FLO18 was ubiquitously expressed in all tissues,with relatively higher expression in leaves and developing endosperm.（4）flo18 mutation affected the assembly and activity of Complex I in mitochondria.To clarify the origin of a potential respiratory perturbation,several respiratory chain complexes were analyzed by Blue Native（BN）-PAGE and in-gel NADH oxidase activity staining,and it revealed that flo18 mutation affects the assembly and activity of complex I in mitochondria.A structural defect in mitochondria in flo18 mutant endosperm was observed by transmission electron microscopy（TEM）.We measured the ATP content of developing endosperm at 6 DAF and found that ATP content was significantly decreased in the flo18mutant.These results suggested that the function and structure of mitochondria in flo18 was disrupted.Moreover,the defects of complex I in flo18 activated the alternative respiratory pathway,The expression of AOX genes in endosperm was induced at both transcriptional and protein levels significantly.（5）flo18 mutation affected 5’processing of nad5 mRNANo difference was observed between mitochondrial transcripts of wild type and the flo18mutant in an analysis of 36 genes using RT-PCR.Most editing sites in mitochondria were also investigated and there was no divergence between wild type and flo18 mutant.The 5’and 3’termini of the mitochondrial transcripts were analyzed by Circular RT-PCR（c RT-PCR）and differences in the nad5 termini were observed.Northern blot verify that flo18accumulated high levels of nad5 precursor mRNA which was not completely processed.Subsequent clones sequencing of c RT-PCR products revealed that loss of FLO18 function caused an absence of short nad5 5’termini.These findings indicated an essential role of FLO18 in 5’processing of the nad5 5’UTR.In summary,this study has identified a key gene FLO18 that regulates rice endosperm development and starch synthesis.As a mitochondria-targeted PPR protein,FLO18 may play an important role in mitochondrial function and endosperm development by involved in the processing of the 5’termini of the mitochondrial coding gene nad5 mRNA.The experimental results provide a new idea for studying the regulation of PPR protein on rice endosperm development,and also provide a theoretical basis for rice quality improvement.