The Enzymatic Basis Of Formation Of Heterogeneous Starches In Rice Endosperm With Inhibition Of Starch Branching Enzyme Expression

Many studies show that inhibition of starch branching enzyme(SBE)expression can increase the contents of amylose and resistant starch in cereal endosperm,which has health benefit for human.However,morphologically different(heterogeneous)starches formed in the endosperm with increasing amylose content.The heterogeneous starches have significantly different starch components,crystalline structure and physicochemical properties,and are regionally distributed in the endosperm.However,how the heterogeneous starches formed,and why they exist exclusively in some regions of the endosperm all remain unknown.In this study,the high-amylose resistant starch rice line,TRS,derived from rice variety Te-qing(TQ)with inhibition of SBE?/?b expression was used as plant material.The spatiotemporal accumulation and starch component change of heterogeneous starches including polygonal,aggregate,elongated and hollow granules were investigated,the transcription,expression and activity of starch synthesis key enzymes were analyzed,and the regional distribution of SBEs in endosperm was observed during TRS kernel development.The objective of this study was to reveal the enzymatic basis of formation of heterogeneous starches in rice endosperm with inhibition of SBE expression.This study could provide important reference for the cultivation of high-amylose resistant starch cereal crops,and enrich the knowledge of starch formation and development.The main results were shown as following.1.Spatiotemporal accumulation of heterogeneous starches in developing endosperm.The section observation of whole kernel showed that TRS endosperm contained polygonal,aggregate,elongated,and hollow starches.The polygonal starch was the subgranules of compound starch,formed at 4 day after flowering(DAF),and was mainly distributed in the central endosperm.The aggregate and elongated starches formed at 7 DAF,and were mainly distributed in the middle endosperm.The hollow starch formed at 10 DAF,and was mainly distributed in the outer subaleurone endosperm.2.Starch components of heterogeneous starch.The quantitative determination of amylose,amylopectin and starch contents in developing seed showed that the contents of amylopectin and total starch were significantly lower in TRS seed than in TQ seed,but the amylose content was significantly higher in TRS seed than in TQ seed after 7 DAF.The extent of amylopectin decrease was higher than the extent of amylose increase in TRS seed.Therefore,the decrease of kernel weight and the increase of amylose in isolated starch were mainly due to the inhibition of amylopectin synthesis in TRS seed.The increase of amylose synthesis in TRS kernel further improved the amylose content of isolated starch.The molecular weight distribution of isolated starch and amylopectin showed that the true amylose content of TRS polygonal,aggregate,elongated,and hollow starch was 26.1%,38.3%,62.4%,and 74.9%,respectively.From TRS polygonal,aggregate,elongated to hollow starch,the branching degree of amylopectin gradually decreased,but their extra-long chains of amylopectin were lower than that of TQ starch.The short branch-chains of amylopectin gradually decreased,and the long branch-chains and average branch-chain length of amylopectin gradually increased from TRS polygonal,aggregate,elongated to hollow starch.3.Transcription,expression and activity of starch synthesis key enzymes.Compared with TQ,the transcription,expression and activity of SBE?,SBE?a and SBE?b were significantly inhibited,the activity of soluble starch synthase ? decreased,the expression of starch phosphorylase increased,and the other starch synthesis key enzymes had no significant effect in TRS developing endosperm.For heterogeneous starch,the expression levels of SBE?,SBE?a,SBE?b and SSS?gradually decreased,but that of starch phosphorylase gradually increased from TRS polygonal,aggregate,elongated to hollow starch.There were no differences in the transcription,expression and activity of granule-bound starch synthase ? between TRS and TQ and among TRS four heterogeneous starches.4.Regional distribution of SBE in endosperm.Immunofluorescence analysis conducted in developing endosperm at 10 DAF showed that the SBE?,SBE?a and SBE?b were homogeneously distributed in TQ endosperm but had a region distribution in TRS endosperm.The SBE expression gradually decreased from the interior to the outer of TRS endosperm.The above results indicated that the inhibition of SBE?,SBE?a and SBE?b expression decreased the synthesis of amylopectin in TRS endosperm.The decreased amylopectin in starch might remove steric hindrance and provide extra space for abundant amylose accumulation.The dosages of SBE expression affected the contents of amylopectin and amylose in starch and the structure of amylopectin,leading to the change of starch morphology.The expression levels of SBEs gradually decreased from the inner to the outer of TRS developing endosperm,which resulted in the formation and accumulation of polygonal,aggregate,elongated and hollow starch distributed regionally from the inner to the outer of TRS endosperm.