Phenotypic Analysis and Gene Cloning of Rice Floury Endosperm Mutant fse4

【Objective】Starch is the main energy reserve of rice endosperm. The biosynthesis of starch is complex, requiring a large number of synthetic enzymes and regulators. Screening rice endosperm defective mutants and cloning the underlying genes will lay theoretical basis for starch biosynthesis and its regulation. 【Method】 A stable genetic floury and shrunken endosperm mutant termed as fse4 (floury and shrunken4) were obtained from the mutant library of Ningjing 3 (WT), which was induced by N-methyl-N-nitrosourea (MNU). An F2 mapping population was generated by crossing the fse4 mutant with Dular (an indica rice variety) and the gene was finally isolated. The floury seeds segregated from the fse4 heterozygous plants were used to observe the morphological features, and the physicochemical properties of the brown rice flour were analyzed. The endosperm structure was observed with a scanning electron microscopy by the semi-thin section technology. The expression of starch synthesis related genes during grain filling was determined by qRT-PCR; Immunoblotting was used to detect the accumulation of proteins related to starch synthesis. The amino acids contents of each mature endosperm were determined with the fully automatic amino acid analyzer.【Result】The 1000-grain weight and grain size were significantly reduced in fse4. Compared with WT, the contents of total starch, amylose and total protein were signi?cantly lower in fse4, while the lipid content was signi?cantly higher. The starch viscosity, breakdown viscosity and setback viscosity of the fse4 mutant were lower than WT. The endosperm of the mutant had many single dispersed starch granules with large spaces between each other. Using 1568 recessive individuals, FSE4 was narrowed down to a 252 kb region. Sequencing revealed a single base substitution in the first exon of the delta 1-pyrroline-5-carboxylate synthetase (P5CS), resulting in a conserved amino acid variation. Most of the genes related to starch synthesis were downregulated in fse4 and the protein accumulation related to starch synthase were reduced. The contents of various amino acids in fse4 rice flour were increased or decreased, the total free amino acids contents in fse4 seeds was 2.6 times higher than those in WT. Exogenous proline was applied during the germination of fse4 seeds, and the embryonic lethal phenotype was partially recovered.【Conclusion】FSE4 encode the key rate-limiting enzyme P5CS of proline synthesis, which plays an important role in the biosynthesis and metabolism of amino acids in endosperm and affects the accumulation of starch.