Gene Cloning And Functional Analysis Of Rice Floury Endosperm Mutants Flo8 And Fse

Starch accounts for the major storage material in the seeds of cereal crops and thereby the accumulation of starch plays an important role in the formation of crop yield and quality.The process of starch synthesis is complicated and highly regulated.Although many enzymes and regulators have been identified,the knowledge of starch synthesis related genes and their function in seed development have not been well elucidated yet.The abnormal endosperm mutants of rice are ideal materials to study starch synthesis.Therefore,it has important theoretical significance and application value to identify new regulators in the process of starch synthesis.Two stable genetic floury endosperm mutants were obtained from a mutant pool induced by N-methyl-N-nitrosourea(MNU)(in the japonica variety Dianjingyoul background),named fse and flo8.Therefore,this study was divided into two parts:(1)In the flo8 mutant,compared with wild type,the starch content was decreased and the normal physicochemical features of starch were altered.Map-based cloning and transgenic complementary revealed that the mutated gene was previously reported gene Ugp1,while flo8 mutant possessed different mutated site and mutated effects of Ugp1.(2)In the fse mutant,compared with wild type,the starch content was decreased and the normal physicochemical features of starch were altered.Map-based cloning and transgenic complementary showed that FSE encodes a protein containing DDHD domain,which has the activity of phospholipase hydrolysis.The mutation of FSE gene resulted in changing the composition and content of phospholipids in the endosperm cell membrane of rice,which could induce cell membrane stress,induce the endoplasmic reticulum stress,trigger programmed cell death,and ultimately affect the synthesis of starch.The main results are as follows:Part Ⅰ:Gene cloning and functional analysis of flo8 mutant.In the flo8 mutant,compared with wide type,the starch content was decreased and the normal physicochemical features of starch were altered.Map-based cloning and subsequent DNA sequencing analysis revealed a single nucleotide substitution at the 5’-splice junction of the 19th intron of the UDP-glucose pyrophosphorylase 1(Ugp1)gene,resulting in alternative spliced transcripts.Complementation of flo8 mutant restored normal seed appearance by expressing full length coding sequence of Ugpl.The fertility of the mature pollen in the flo8 mutant was similar to that of the wild type as revealed by I2-KI staining.qRT-PCR analysis revealed that Ugp1 was ubiquitously expressed.Mutation affected the expression of most of the genes associated with starch biosynthesis.Meanwhile,western blot and enzyme activity analysis showed the comparability of protein levels and enzyme activities of most tested starch biosynthesis related genes.Our results demonstrate that the mutation of Ugp1 in flo8 mutant is more sensitive to the endosperm development than the pollen development.Part Ⅱ:Gene cloning and functional analysis of fse mutant.1.Compared with wild type,on one hand,the fse mutant showed inhibition of root growth and enhanced root gravitropic response at seedling stage.On the other hand,the fse mutant was slightly shorter than the wild-type in mature plants,and displayed a remarkably slower grain filling rate during seeds development and seed length,seed depth and 1,000-grain weight reduced.In the fse mutant,seed lipid content was higher,whereas crude protein,starch contents and amylose content were significantly lower than that in the wild ype.The pasting property,swelling power of starch,as well as the structure of amylopectin in fse mutant were also remarkable changed.2.The fse mature seed showed floury shrunken endosperm,and scanning electron microscopy images of transverse sections indicated that this endosperm was filled with loosely packed,small,and spherical starch granules with large air spaces.Furthermore,semi-thin sections and iodine staining observation showed the central region of the fse mutant endosperm contained smaller,scattered starch granules,stained weakly with iodine.These results showed the FSE plays an important role in the formation of compound starch granules in rice seed.3.To genetically map the mutation,a F2 population derived from a cross between fse and an indica variety N22.Furthermore,we used 155 floury shrunken endosperm segregants for linkage analysis and fine mapping,ultimately,limited the mutation to the region within 190 kb on the short arm of chromosome 8 between marker WH-2 and WH-4.Candidate gene predicted and sequencing showed a single nucleotide substitution of gene LOC_Os08g01920,leading to a single amino acid changed.Further western bot showed the level of the protein in the fse mutant was down-regulated.Transgenic complementary showed LOC_Os08g01920 was FSE.FSE is a single cope gene in rice genome,and encodes a phosphatidic acid preferring phospholipase A1-Like Protein,containing a DDHD domain.4.Real-time PCR analysis revealed that FSE gene was expressed ubiquitously and exhibited abundantly in developing seeds and root,and Western blot analysis showed FSE protein accumulated as seed development.We can speculate that the mutant phenotype determined by the highly expression in root and developing seed.Cell component separation and western blot showed FSE is a membrane located protein.5.In vitro enzyme activity showed the phospholipase activity of FSE,furthermore,liquid chromatography tandem mass spectrometry(LC-MS/MS)analysis showed phospholipid content and composition also changed in the mutant compared with wild type.Real-time PCR analysis showed ER stress responsive genes were slightly up-regulated while PCD marker genes were significantly up-regulated.In addition,Real-time PCR and Western blot analysis showed starch synthesis related genes were significantly down-regulated in the fse mutant during the early development of endosperm,and sucrose synthase and AGPase activity was only approximately 78%and 44%of that in wild type.Thus,we speculated that FSE mutation resulting in changing the phospholipids composition and content of the endosperm cell membrane,inducing endoplasmic reticulum stress,triggering PCD,and ultimately affecting the synthesis of starch.