Transcriptome Profiling And Physio-chemical Analysis Reveals Intricate Gene Networks In Grain Filling Caryopses That May Contribute To Chalky Grains In Rice (oryza Sativa L.)

Grain endosperm chalkiness of rice is a varietal physical characteristic that negatively affects not only the appearance and milling properties but also the cooking texture and palatability of cooked rice. Chalky grains have a lower density of starch granules compared to vitreous ones, and are therefore more prone to breakage during milling. In many rice-producing areas, high chalkiness represents one of the most serious problems that decrease grain quality. However, chalky grain is a complicated quantitative trait and the molecular mechanisms underlying its formation are still poorly understood. In this study, we performed a comparative transcriptome analysis of the caryopses of a pair of near-isogenic lines, CSSL50 with high chalkiness and its low chalkiness parental line Asominori. Corroborated with the phenotypic and physico-biochemical observations, our genome-wide transcription analysis supports the notion that rice grain endosperm development is controlled by delicate, but complex genetic networks. Our results revealed that grain endosperm chalkiness of rice formation entails an intricate gene network, including genes involved in carbon metabolism, starch biosynthesis and degradation, metabolism of cellulose, hemicellulose and pectin, signal transduction and most interestingly, we found that ROS signaling and homeostasis are closely related to the formation of rice grain endosperm chalkiness. The important results were as follows:1.The results of analyses at the phenotypic, physiological and biochemical levels showed that the occurrence of endosperm chalkiness in rice might associate with the changes of enzyme activities, the increased of grain-filling rate and the structure of starch granule. Asominori amyloid is of the form of polygon, tightly arranged, few inter-space among the granules, and good transparency; however, the amyloid on the chalky portion of CSSL50 seed granule endosperm is of the form of ellipse, loosely arranged, big interspace among the granules, and poor transparency. Besides, the SuSy, AGPase, SBE, and DBE for high chalkiness CSSL50 is remarkably higher than that for low chalkiness Asominori. What is more, on the 10-15th day after grain filling, the seed grain filling speed for CSSL50 is remarkably higher than that for Asominori. These results show that the high chalkiness of CSSL50 is most likely due to the dynamic change of starch-composed enzyme and high seed grain filling speed, and to the fine structures of starch granules.2.Based on the results of gene expression profiles between Asominori and CSSL50, we found the pathways of signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis might be play a significant role in the formation of chalkiness in rice. the occurrence of endosperm chalkiness in rice might be closely related to these functional and regulatory pathways. For the vast majority of probes, expression appeared unchanged between Asominori and CSSL50. Using the selection criteria of P value<1%, we identified a total of 623 probe sets with a significant change. Of these, expression of 324 was up-regulated and 299 down-regulated. Based on the bioinformatics tools such as Gene ontology and JAFA , and the metabolic and functional features of rice, a total of 623 differentially expressed genes were classified into 18 major categories. 68.8% of these identified genes were implicated in the first six functional groups (signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis), suggesting that the occurrence of endosperm chalkiness in rice might be closely related to these functional and regulatory pathways.3.From three aspects of expression of gene difference, carbon- metabolization-related enzyme activities and substance content, we found that there exists a close relationship between the biosynthesis/hydrolysis of starch and non-starch polysaccharide and the formation of endosperm chalkiness, namely, the enhanced starch biosynthesis and the decreased biosynthesis of non-starch polysaccharide may result in the occurrence of chalkiness in rice endosperm.4.Based on the results in this study on gene chips, RT-PCR, and physiochemical data (eg., H2O2 content), it is found that the antioxidation system in high chalkiness isogen CSSL50 is activized. It is also found that the hydrogen peroxide content of high chalkiness CSSL50 is remarkably higher than that of low chalkiness background parent strain Asominori. Meanwhile, the expression level of antioxidation genes such as SOD, APX, GPX, MDAR,and PrxR in high chalkiness CSSL50 is remarkably higher than that in Asominori, which shows that the antioxidation system in high chalkiness CSSL50 seed granule endosperm is activized and the ability of eliminating extra oxidation free radicals (such as H2O2, etc.) in CSSL50 is higher than that in Asominori. Besides, the expression level of GST, Glx, and Trx genes in high chalkiness CSSL50 is also remarkably higher than that in Asominori and the expression level of LOX gene in CSSL50 is remarkably lower than that in Asominori,which is further testified by the RT-PCR result.Then it is concluded that the function of antioxidation repair system in CSSL50 seed granule endosperm is remarkably higher than that in Asominori, which shows that the emergence process of the rice endosperm chalkiness begins in the environment of activized (increased) antioxidation level, and is fulfilled through changing carbohydrate metabolization.5 . To conclude, exterior stressful conditions are likely to further influence the carbohydrate metabolization of rice by changing the oxidation-reduction balance in the plant, finally leading to the emergence of chalkiness, which agrees with the conclusion of a dissertation by Wan et al (2008)—"The stresses of coldness, drought, salt, heavy metal,etc can lead to the fact that H2O2 content in the rice is increased and oxidation-reduction system in the rice plant is activized, finally affecting the carbohydrate metabolization."As for the high chalkiness CSSL50, the cause for forming its chalkiness is similar to the case in which a general low chalkiness variety can produce high chalkiness in coercive environment.