Effect Of High Temperature On Starch Synthesis And Protein Accumulation In Rice Endosperm At Filling Stage

High temperature during reproductive period is one of the crucial factors influencing rice yield and grain quality; Base on the former research work, two early non-waxy indica rice cultivars (J935and J353) differing in cooking and eating quality were chosen to investigate the effects of high temperature on the fine structure of amylopectin, protein body formation and starchy granule distribution by a field experiment of different sowing-date and the controlled temperature treatments in chambers, respectively. In current study, the cDNA microarrays, RT-PCR, protein expression analysis were used to elucidate the underlying metabolisms involved in starchy biosynthesis and storage protein accumulation, as well as their relations to rice grain quality when subjected to high temperature. The main results were showed as follows:1. The high temperature stress at filling stage could deteriorate cooking and eating quality, which was closely related to rice starch granule size distribution as well as starch fine structure. High temperature increased rice starch granule average diameter, caused the percentage of large starch granule increasing, but small and medium-sized starch granule presented with an opposite way. The Phenomenon of starch granule size, affected by high temperature, was one of the explanations for rice starch gelatinization properties involved in high temperature at filling stage. In addition, great changes were also found in rice starch fine structure (chain length) when treated with two temperatures. Among different amylopectin components, long B chain (B3chain, DP≥37) increased significantly with B1chain (DP13-24) components decreasing under high temperature treatment. B3and B1chain presented a similar trend between the two variety, while B2chain (DP25-36) and A chain (DP5-12) differed by variety under high temperature stress. The proportion of B2chain increased in J935, a high quality variety, but decreased in J353. A chain was just opposite to B2chain. For different components in grain developing stage, the short A chain presented a higher proportion at initial stage relative to the other components, but gradually decreased as filling stage. On the contrary with A chain, B1, B2chain and B3chain showed lower proportion at initial stage and higher at later stage.2. The high temperature stress at filling stage may inhibit the relative expression amount to main isoform genes (SUT1and SUT2) for Sucrose Transporter (SUT), implying sucrose transport pathway might be inhibited by high temperature. In addition, the main isoform genes (SuSy2and SuSy3) for sucrose synthase (SuSy) were down-regulation, which indicated sucrose unloading in rice endosperm impaired by high temperature treatment, however, the genes (CIN2and CIN7) related to plasmid-invertase in rice endosperm induced by high temperature. It was showed that high temperature broke the balance between sucrose synthase and plasmid-invertase, and Limited sucrose unloading efficiency. For sugar signal transduction procedure, the gene referred to fructokinase and hexokinase played important role at sugar signal transduction in rice endosperm, the "shift" phenomenon of fructokinase (FrK Ⅰand FrK Ⅱ) and hexokinase (HxK Ⅰand HxK Ⅱ) in the fructose phosphorylation might be an adaptation mechanism to high temperature stress in rice. High temperature can inhibit some signal transduction pathways.3. There were numerous genes involved in starch synthesis, as well as starch fine structure, in rice endosperm exposed to high temperature at filling stage. Among different gene isoform, some genes for starch synthase (SSS Ⅰ, SSS Ⅲa and SSS Ⅳa) was shown up-regulation when exposed to high temperature, but SSS Ⅱa, SBE Ⅰ, SB Ⅵ, which for starch synthase and starch branching synthase presented down-regulation. For starch debranching synthase genes, ISA1showed up-regulation, while PUL presented down-regulation under high temperature stress. Different expression patterns of genes for the three enzymes co-governed amylecptin, and determined its fine structure at high temperture. SSS Ⅰ,SSSⅡa was the two gene isoforms, which was the most important in starch fine structure. Concerned as GBSS Ⅰ, which mainly acted on amylase content, presented up-regulation in J353and down-regulation in J935. The change of GBSS Ⅰ expression pattern was coincide with amylose content when exposed to different temperature treatments at filling stage.4. Transition starch degradation, what always occur in rice grain aleurone, played an important role in supplying carbon source and energy to starch synthesis in rice endosperm. Effect of high temperature on starch degradation metabolism (including phosphorylated degradation pathway and non-phosphorylated degradation pathway) in rice endosperm at filling stage presented similar features:"upstream genes"(like GWD, AMY3and SEX4) showed up-regulation pattern responsible to high temperature stress, which resulted in a rapid accumulation of intermediate product from starch degradation. However,"downstream genes"(like ISA3, PUL and MEX1) mostly inhibited by high temperature, which impaired successive degradation of intermediate product. The expression patterns of "upstream genes" and "downstream genes" under high temperature results in excess accumulation of intermediate product in rice aleurone, which might restrict carbon and energy supply, reduce total starch content in rice endosperm.5. High temperature stress increased the accumulation of all classes of storage proteins and amino acid at early filling stage but decreased the accumulation of prolamin at later stage. High temperature not only accelerated the formation process of protein body (PB Ⅰ and PBⅡ), but also changed relative proportion of storage protein subunit in rice endosperm. For prolamin, high temperature resulted in slightly decreasing of13kDa prolamin subunits with a slight extent, and significantly increasing17-19kDa subunits. For Glutelin, high temperature cause higher content for α-subunit and β-subunit than those to lower temperature. The accumulation of storage protein in rice endosperm was not only regulated by gene related to protein subunit or precursor, but also controlled by the gene involving in protein modification and stress-defense. Compared with the former, the latter was much more sensitive to high temperature. For example, luminal-binding protein precursor gene (BiP), which contributed to prolamin fold and aggregation, as well as protein body Ⅰ (PB Ⅰ) formation, was up-regulation under high temperature. In addition, the genes for protein modification like Protein disulfide isomerase (PDI) and rice basic leucine Zipper factor/rice prolamin box binding factor(RISBZ1/RPBF) were up-regulation when exposed to high temperature.