Map-Based Cloning And Functional Analysis Of A Key Gene FLO11 Involved In Amyloplast Develoment In Rice Endosperm

Starch is the major component of cereal endosperm,occupying 60%-80%of total weight of grain,thus normal starch synthesis is significant for yield and quality for crop.Starch formation is a complex process in which a variety key starch enzymes and regulatory factors are involved.Finaly,starch is stored in a specific plastid,amyloplast,referd as starch granule.Defective starch synthesis and abnormal amyloplast development usually lead to defective endosperm,such as floury endosperm and shrunken endosperm.The function of these key starch synthesis enzymes was clearly studied by studying floury endosperm mutants and abnormal starch granule mutants.Howerver,the molecular mechanism of starch synthesis and amyloplast development is unchlear.Endosperm occupies the majority of rice grain and is the main place of storage starch,thus rice endosperm is the best material for the study of starch metabolism and amyloplast development.Identification more defective endosperm mutants is significance for isolating new genes to elucidate the molecular mechanism of starch synthesis and amyloplast development.In this study,we identified a floury endosperm mutant flo11.We characterized its phenotype in detail and discussed the molecular mechanism of floury phenotype formation in flo11.The main results of this study as follows:1.A floury endosperm mutant was isolated from screening irradiated indica rice variety N22.The architecture of the mutant exhibited no visible differences with wild-type plants during whole vegetative growth phase.While the grain-filling rate was slower at late stages and 1000-grain weight was also mildly reduced in the mutant.At mature stage,grain was opaque in the mutant,thus the mutant was named as flo11(floury endosperm 11)Scanning electron microscopic(SEM)analysis revealed that the center and periphery regions of flo11 endosperm were loosely packed with spherical polyhedral starch granules.The contents of total starch,amylose,and lipid the amylopectin structures were as well as physicochemical property similar between wild type and the flo11 mutant.2.Semi-thin sections revealed that wild type endosperm contained obvious compound SGs,consisting of several dozen homogeneous polyhedral and sharp edged starch granules.While in the opaque parts of flo11,the size and appearance of amyloplasts varied significantly.We observed amounts of single starch grains in flo11 mutant.In addition,two types of abnormal amyloplasts were readily observed.One type was larger and irregularly-shaped with bumpy membrane boundaries compared with the nearly round or oval amyloplasts with smooth membrane boundaries in wild type and numerous small starch granules assembled to form such compound starch grain structure with bumpy surface;the other type was stained weakly with iodine and their internal starch granule structures were almost nondetectable.Besides,there are larger normal developmental amyplasts in flo11 mutant.Transmission electron microscopy(TEM)images revealed that starch granules were smaller and amyloplast development was delayed in floll mutant.3.To identify the flo11 locus,we constructed an F2 population from a cross between the flo11 mutant and japonica cultivar Nipponbare.We mapped the flo11 locus to a 360 kb region between marker P37 and P18 interval on the short arm of chromosome 12.Twenty three open reading frames(ORFs)in this region were predicted by NCBI(https://www.ncbi.nlm.nih.gov/)and RiceXPro(http://ricexpro.dna.affrc.go.jp/).Sequence analysis revealed that only LOC_Os12g14070 had one base pair(bp)deletion in the second exon in the flo11 mutant,leading to a frame shift and presumably forming a truncated loss-of-function polypeptide with N-terminal 229 amino acids.Complementation test showed LOC_Os12g14070 was the target gene.LOC_Os12g14070 encodes a member of the Hsp70 family.4.Real-time PCR analysis revealed FLO11 was expressed in roots,leaves,leaf sheaths,stems,spikelets and developing seeds of various developmental stages,whlie FLO11 was abundantly expressed in developing seeds.Western blot and GUS activity analysis was consistent with its expression pattern.All above results indicated that the expression of OsHsp70cp-2 peaks at developing seeds.5.Protein sequence alignment analysis revealed that FLO11 is highly homologous to the plastid-localized Hsp70 of other species.Subcellular localization result demonstrated that FLO 11 is a plastid-localized protein in rice.FLO 11 is highly homologous with OsHsp70cp-1,which was studied,thus FLO11was named OsHsp70cp-2.The endosperm of rice transgenic rice was observed.OsHsp70cp-2-GFP was localized in endosperm amyloplast,indicating OsHsp70cp-2 is important for amyloplast development.6.BiFC and CoIP experiments in vivo showed that OsHsp70cp-2 was associate with inner membrane translocons Tic110 and Tic40,suggesting that OsHsp70cp-2 may be involved in protein transport in amyloplasts.