Structure And Development Of Biphasic Starch In Maize And Rice Endosperm With Inhibition Of Starch Branching Enzyme

The endosperm of cereals,a main storage tissue of seed,is rich in starch.Starch branching enzyme(SBE)is one of the key enzymes involved in starch biosynthesis,and mainly responsible for the production of branch-chains of amylopectin.The inhibition of SBE activity in cereal endosperm can improve resistant starch content,but results in heteromorphous starch and biphasic starch.The structure and formation of the heteromorphous starch have been investigated widely in recent years,but those of biphasic starch are not clear.In this study,maize and rice with inhibition of SBE were investigated to reveal the structure and development of biphasic starch,especially the differences in structures between interior and exterior of biphasic starch granules and the development causes for regional distribution of biphasic starch in endosperm.This study would contribute to the quality cultivation of high resistant starch cereal crops,and provide important information for their utilizations.The main results were as follows:1.Establishment of analysis methods for starch morphological structure and structural properties.In order to further reveal the morphology and structural properties of starch,we established some new methods for in situ observing the growth rings of starch in cereal endosperm,preparing the sample for starch analysis of attenuated total reflectance-Fourier transform infrared spectroscopy(FTIR),and quantitatively measuring the spectral parameters of small-angle X-ray scattering(SAXS).The results showed that the in situ observation method of starch growth ring could be widely used in cereal endosperm.The simple device and sample preparation method for FTIR analysis of starch could be widely applied to analyze the ordered structure of external region of starch granules.The graphical analysis method of starch SAXS spectrum could quantitatively measure the parameters of lamellar structure of starch.These methods were simple,good repeatable,and highly credible,and provided a technical support to analyze the structural properties of starch in this study.2.Morphology and structural properties of starch from endosperm of maize sbe2b mutant.In this study,the morphology and structural properties of endosperm starch were analyzed in normal maize cultivar Mo 17 and sbe2b mutant AF11.The deletion of the ninth exon of the SBEIIb gene in AF11 led to that the AF11 endosperm had significantly lower starch content and higher soluble sugar content than Mo 17 endosperm.Mo 17 had spherical or polygonal starch granules with A-type crystallinity,but AF11 had aggregate and elongated starch granules with B-type crystallinity.AF11 starch had higher amylose content,proportion of long branch-chains of amylopectin,and ordered degree at the external region of granules but lower relative crystallinity,lamellar peak intensity,and lamellar distance than Mo17 starch.These results indicated that the mutation of SBEIIb gene significantly changed the morphology and structural properties of starch,and provided some information for studying structure and development of biphasic starch.3.Structural properties of biphasic starch in maize sbe2b mutant In this study,the structural properties of biphasic starch were investigated in maize sbe2b mutant AF11 using morphological observation and physicochemical determination methods.The exterior of biphasic starch had loose comb-like structure without growing rings and birefringence and was stained red with iodine solution,but the interior had dense crystalline structure with growing rings and birefringence and was stained blue with iodine solution.The biphasic starch was mainly distributed in the inner region of crown endosperm in AF11.Compared with the external endosperm tissue(OE),the internal endosperm tissue(IE)of AF11 had low amylose content and proportion of amylopectin short branch-chains and high proportion of amylopectin long branch-chains.These results indicated that the biphasic starch had low amylose content and high proportion of amylopectin long branch-chains,and its exterior was mainly composed of fewer amylose molecules and more long branch-chains of amylopectin.4.Development causes of the formation and regional distribution of biphasic starch in maize sbe2b mutant.In this study,the normal maize Mo 17 and sbe2b mutant AF11 were used to analyze the causes of formation and regional distribution of biphasic starch by transcriptomics analysis,gene expression level detection and enzyme activity assay.The IE tissue experienced more severe hypoxic stress than the OE tissue in both Mo 17 and AF11,and all genes involved in glycosis were up-regulation in IE tissue.Compared with OE,SBEIIb was down-regulated in IE of Mo17,but it was no significant difference in IE of AF11;DEP1 had no significant difference in IE of Mo 17,but it significantly up-regulated in IE of AF11;SBEIIa had no significant difference in IE of Mo 17,but it significantly down-regulated expression at 15 DAP and significantly up-regulated at 20 DAP in IE of AF11.In addition,the GBSSI activity of starch had no difference between IE and OE of Mo 17,but it was significantly lower in the IE of AF11(biphasic starch)than in the OE of AF11.The above results indicated that the progressive enhancement of SBEIIb functional defects dominated the formation of biphasic starch.The increase in DPE1 expression and the decrease in GBSSI activity reduced the amylose content in periphery region of biphasic starch.Up-regulation of glycolysis in inner endosperm reduced the carbon influx used for starch synthesis,decreased the rate of starch synthesis,and prolonged the time required for starch synthesis,leading to that the IE experienced multiple SBEIIb defect stages from weak to strong and resulted in regional distribution of biphasic starch in endosperm.5.The structure and development of biphasic starch in rice endosperm with inhibition of SBE.In this study,the structure and development of the biphasic starch were analyzed in a japonica glutinous rice cultivar Guang-ling-xiang-nuo(GLXN)and its transgenic rice line GLXN-SBE?/?b with inhibition of SBE?/?b.In GLXN-SBE?/?b-endosperm,the expression levels and activities of SBEI,SBEIIa and SBEIIb were significantly decreased,and the inhibition effect of SBE gene was aggravated with the endosperm development.The GLXN-SBE?/?b-endosperm had typical biphasic starch granules with a comb-like profile that aggravated with the endosperm development.During endosperm development,GLXN starch showed A-type crystallinity,and had similar branch-chain distribution and branching degree of amylopectin.However,the starch gradually changed from A-type to C-type,and the proportion of long branch-chains of amylopectinincreased and the proportion of short branch-chains and branching degree decreased during endosperm development in GLXN-SBE?/?b-.These results indicated that long branch-chains of amylopectin were the main component of the comb-like profiles of biphasic starch granules,and the gradual reduction of SBEs might be responsible for the formation of peripheral profiles of biphasic starch.