MAP-based Cloning And Functional Analysis Of A Key Gene FLO7 Involved In Regulation Of Amyloplast Development In Rice Endosperm

Starch content in cereal seeds accounts for over 90%of the total weight,so the accumulation of starch plays an important role in the formation of crop yield and quality.The process of starch synthesis is complicated and sophisticated which requires the participation of many enzymes and regulatory factors.At last,starch is converted to starch granules and stored in amyloplast.Studies on some new opaque endosperm mutants showed that these new genes can not be added into the old model which has been established,so we need to construct a bigger or a cross model using those novel mutants.To seek for new genes related to starch synthesis,we performed a large-scale screen in a T-DNA-inserted mutant pool and isolated a floury endosperm mutant line,named flo7.The results of cytological observation indicated that the development of amyloplast in the peripheral endosperm is affected in flo7.Map-based cloning showed that FL07 encodes a 364 amino acids protein of unknown function.Bioinformatics prediction results displayed that FL07 contains a plastid transit peptide at the N terminus and a domain of unknown function 1338(DUF1338)at the C terminus.FLO7-GFP fusion proteins were positioned in amyloplast stroma by subcellular localization.Tissue expression analysis showed that FL07 in rice is a constitutive expression gene and is highest expressed in seed endosperm in which its expression is specific in peripheral endosperm.The stability of mutant protein was declined in vivo and in vitro degradation experiment.The main results are as follows:1.The mature seeds in mutant flo7 were opaque.The outer of the endosperm in the flo7 grains was floury white and scanning electron microscopic examination revealed that the SGs in flo7 outer endosperm were loosely packed but no difference in the inner edosperm.The flo7 achieved a markedly slower grain-filling rate and the mean weight of seeds(per 1000 grains)was only approximately 87%that of the wild-type.In the flo7 mutant,seed amylose,protein and amino acid contents were lower,whereas the lipid content was significantly higher than the wild-type.The structural changes in amylopectin were also significant.2.Compound SGs consisted of many starch granules in amyloplast and growed bigger.The stroma surrounded starch granules in early developing seeds and the space was quickly occupied by developmental starch granules.However,the SGs in flo7 developed slowly and large space of amyloplasts were full of stroma so that the filling rate of amyloplasts became slowly.Semi thin section observation showed that the amyloplasts in the middle filling stages were disintegrated and the starch granules were discretely distributed but not formed the shape of polygon in the flo7 endosperm.These results suggested that FL07 plays a key role for the formation of the normal structure of compound SGs in rice endosperm.3.We first crossed the flo7 mutant with an indica varity Peiai64 to generate a F2 mapping population and the flo7 locus was mapped to a 83 kb genomic region flanked by the markers Z5 and Z24.Sequece analysis revealed a single nueotide substitution in Os10g0463800,which led to a premature stop codon.The FL07 gene encodes an unknown function protein containing 364 amino acids which is a single copy gene and conserved from lower plants to higher plants.The FL07 protein harbors a plastid transit peptide at N-terminal,and a domain of unknown function 1338(DUF1338)at C-terminal.4.FL07 was expressed in root,stem,leaf sheath,leaf,panicle and seed and the highest expression was investigated in endosperm.Western blot showed that the content of FL07 accumulated gradually in different grain filling stages.The expression patterns obtained in GUS staining were approximately the same as that obtained using real-time quantitative PCR.In-situ hybridization identified that the mRNA of FL07 was specifically localized in the endosperm of outer portion.We can conclude that the mutant phenotype is determined by the specificity expression of FL07 in endosperm.5.N-terminal transit peptide ensured FL07 correct localization in chloroplast.In rice transgenic plants,the FL07 protein was localized in amyloplast stroma.Through continuous observation of wild-type protein and mutant protein expressed in tobacco leaves,we found that compared with wild type,the stability of the mutant protein was decreased.The same results were once again proved in the rice protoplast system.Therefore,the missed amino acids in the C-terminal are essential for the stable accumulation of FL07.The 28 genes related to starch synthesis and carbohydrate metabolism were similarly expressed between wild-type and flo7 endosperm.Zymogram analyses showed that there were no significant differences in the activities of DBE isozyme(ISA),SS isoforms(SSI and SSIIIa),PHO isoforms(PHO1 and PHO2)and BE isoforms(BEI,BEIIa and BEIIb)between the wild-type and the flo7 mutant.These data suggest that the process of starch synthesis in flo7 mutant is not affected.