3.3 Identification of differentially expressed proteins related to rice development and quality formation
A total of 748 unique proteins were identified based on their specific expressions when exposed to high temperature. After removing 302 proteins with lower scores and unknown functional classification, 39 proteins related to rice development and quality formation were distinguished (Table 2, Figure 6). In this study, the expression of 16.9 kDa class Ⅰ heat shock protein in rice grains was significantly reduced 3 days after heating, which was down-regulated by 84% compared with CK treatment. However, the expression of heat shock factor binding protein 1 involved in heat shock protein synthesis was significantly increased. Furthermore, the expression levels of HSP70s at early grain-filling stages were only about half of normal treatment (0.47, 0.5, and 0.43 times reduction at 3d, 6d 9d after flowering, respectively). Results showed the most significant change was the 26.7 kDa heat shock protein of the sHSPs family, and its expression was increased significantly at 6, 9, and 12 days after flowering, with 6.78, 3.06, and 5.44 folds compared to the CK treatment. At the 12th day after flowering, the expressions of 18.0kda class II heat shock protein, 24.1kda heat shock protein and heat shock protein 82 were up-regulated to varying degrees (2.26, 2.01, 3.63 folds respectively) when subjected to high temperatures.
Expression of proteins related to rice quality formation were also regulated under field warming conditions. Expressions of glutelin anabolism proteins were regulated at the beginning of grain-filling. For example, the glutelin type-A 3, glutelin type-B 1, glutelin type-B 5-like expression levels of rice grains were significantly up-regulated by 2.79, 2.49, and 3.3 folds, respectively. At the same time, the expression levels of glutelin type-B 1-like and glutelin type-B 2-like proteins were decreased significantly. Furthermore, the expression levels of globulin 19 kDa globulin, globulin 1s allele and basic 7S globulin were increased significantly, which is consistent with the increase of globulin content measured in mature grains. In this study, the significant decrease in prolamin under warming conditions was mainly at the 12th day after flowering and expression analysis of prolamin PPROL 14E and prolamin PPROL 14E-like proteins showed that they were both decreased significantly (0.33, 0.38 folds) at this stage comparted to the normal temperature treatment. As the main component of rice grains, the accumulation of rice grain starch has been proved to be sensitive to elevated temperature. The synthesis of rice grain starch is mainly regulated by a series of protein families, including SSS, SBE, DBE, GBSS. In this study, the GBSS was down-regulated at the 6th day after flowering under increased temperature. While proteins related to amylopectin synthesis obtained no significant changes when compared to the CK. The expression levels of granule-bound starch synthase at 6d, 9d and 12d after flowering were significantly lower than that of the control and the expression levels of soluble starch synthase responsible for the synthesis of amylopectin SS4 and SSS2-3 were decreased under high temperatures.
Results of this study showed that another key growth and development process regulated by the increased temperature during grain-filling stage was the photosynthetic system of rice plant. In this study, the expression levels of the chlorophyll a-b binding protein 1B-21, chlorophyll a-b binding protein P4, and chlorophyll a-b binding protein 7 in chlorophyll ab binding protein were significantly up-regulated at the beginning of grain-filling (2.22, 2.03, 3.3 times), when compared to the CK. On the other hand, the expression of PSB28, which is responsible for water splitting, had a downward trend through the grain-filling period, and reaching a significant level at 12 days after flowering. Furthermore, protein TIC 62 (translocon at the inner envelope membrane of chloroplasts) responsible for the dynamic balance of the proteome was significantly downregulated at 9d after flowering. Photosynthesis is the process by which light energy is converted into chemical energy and stored, and thus it is essential for the accumulation of rice grain assimilation.