Functional Analysis Of PTST2 Regulating Starch Accumulation In Rice Pollen And Endosperm

Rice endosperm is the main storage tissue of seeds.Starch is abundant in rice endosperm and becomes the essential part of endosperm.The biosynthesis of starch affects the appearance and weight of seed and the properties of starch,which are closely related to the quality and yield of rice.Therefore,it is of great application value to deeply reveal the regulation network of starch synthesis in rice endosperm.Floury endosperm is a type of opaque phenotype of kernel with the abnormal synthesis and accumulation of starch or protein,and has become an ideal material for studying the development of endosperm and the regulation network of starch synthesis in rice.In this study,the floury endosperm m39 mutant was screened from a 60Co-irradiation mutant pool of japonica rice variety Kitaake.The starch granule morphology,endosperm composition and starch physicochemical properties were analyzed,and the mutant gene PROTEIN TARGETING TO STARCH 2 were identified and cloned using map-based cloning and transgene complementation technology.The mechanism of regulating endosperm development and starch metabolization of PTST2 was revealed in developing endosperm of rice.The main results are as follows:1.Phenotype identification of m39 mutant.The plant height after heading and the spikelet seed setting rate of the m39 were obviously reduced.The mature brown kernels exhibited an opaque and floury endosperm,and had significantly low length,width,thickness and weight of kernel.Starch granules were spherical with large gaps and loosely packed in endosperm cells.Compared with Kitaake,the m39 had markedly lower total starch content in its endosperm and apparent amylose content and relative crystallinity in isolated starch.However,the long-branch chain content of amylopectin in m39 was significantly lower than in Kitaake.The starch from m39 had significantly lower gelatinization temperature and enthalpy than Kitaake starch.In addition,the grain filling of m39 was slower than that of Kitaake.The semithin sections observation showed that small,hollow and scattered heterogeneous compound starch formed and accumulated in developing endosperm.The starch granules in the mature pollen grains were abnormally filled,leading to the abortion of pollen grains.2.Mapping and verification of mutant gene.Genetic analysis of the floury endosperm showed that the mutant character was controlled by recessive mononuclear gene.The mutant gene was located between InDel primers 3-7 and 3-10 on the long arm of the third chromosome by map-based cloning technology.There were 49.9 kb between the two primers,and six open reading frames were predicted in this section.The coding regions of six open reading frames in Kitaake and mutant were divided into different fragments.These fragments were amplified and sequenced.Only the guanine(G)at the end of the eighth intron of the LOC_Os03g48170 was replaced by adenine(A).The single base substitution led to that the eighth intron was not spliced,producing a abnormal transcript in m39.The LOC_Os03g48170 full-length genomic sequence of Kitaake was transformed into m39 callus through Agrobacterium tumefaciens,and the pollen fertility and floury kernel of the positive transgenic lines were restored.The analysis of the LOC_Os03g48170 sequence indicated that the LOC_Os03g48170 encoded the PROTEIN TARGETING TO STARCH 2 protein with a plastid localization signal peptide at the N-terminus and a CBM48 domain at the C-terminus.3.Tissue expression and encoded protein subcellular localization of PTST2.PTST2 was expressed in all tissues of rice.The leaf and leaf sheath had the highest level of PTST2 expression,and the developing endosperm maintained a high expression level.The transient expression of tobacco protoplasts showed that PTST2 was located in the chloroplast,but the transgenic rice exhibited the location of PTST2 in the pollen grain and amyloplast stroma.4.PTST2 could bind starch and affected the expression of starch synthesis related genes after mutation.Starch binding experiments in vitro indicated that the CBM48 domain could bind starch,and the mutation CBM48 domain couldn't bind starch,leading to lower expression of starch synthesis related genes AGPL4,AGPS2,GBSSI,SSI,SSIIa,BEI,BEIIb,ISA1,ISA2 and PUL in developing endosperm.The abundance of ISA1 in total protein of m39 endosperm was significantly lower than that of Kitaake.Therefore,the PTST2 can regulate starch synthesis and accumulation in rice pollen and endosperm.Heterogeneous compound starch granules appeared in endosperm,and the long-branch chain content of amylopectin was significantly decreased after PTST2 mutation.The starch granules in the pollen grains were abnormally accumulated,leading to the decrease of spikelet fertility.The research results would expand our knowledge of starch synthesis and its regulation mechanism,and also provide genetic materials for rice quality breeding.