Investigation Into Mechanism Of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Activase (RCA) Large Isoform In Heat Acclimation Of Photosynthesis In Rice

With growing global warming, the importance of the research on the heat resistance of plants is further important. As one of the main inhibition points of heat stress, photosynthesis is also the major restriction factor of crop production limited by high temperature. Although photosynthesis of plant has a certain capacity to acclimate to heat, its specific mechanism is still unclear. For several decades, lots of studies have shown that the inhibition of photosynthesis by heat was due to Rubisco activase (RCA). However, various mechanisms of this have not been widely accepted. In order to in-depth study of the heat effect of RCA, RCA heat resistance and RCA thermal adaption improvement, researches were performed on the interaction of RCAL with RCAs, and RCAL with other proteins. Some results are shown as follows:ⅰ) The photosynthetic and fluorescence parameters were distinguished between RCA over-expression and RNAi materials. And it was found that RCAs over-expression lines improved carbon assimilation by the improvement of Rubisco activity. However, the RCAL lines only enhanced photosynthetic electron chain by other function of RCAL. It was proved because of RCAL’s protein binding ability by the subsequent experiments.ⅱ) The stress responsive elements were analyzed through the study of RCA gene promoter, and the primary, secondary and tertiary structure of the protein. A lot of cis-elements of multiple stress related components in the RCA promoter were found. Their secondary structure, binding sites and tertiary structure were different. The transcription and translation levels of the two RCA isoforms were also different in the process of different abiotic stress adaptation. Although both of transcription and translation level in rice leaves rose obviously under various stresses, RCAL increased significantly and the stress treatments significantly increased the quantity of RCAL in chloroplast thylakoid. In addition, co-immuno-precipitation (Co-IP) proved that RCAL in the complex of Rubisco-RCA increased sharply under various stresses. It can be concluded that RCAL plays a more important role in response to abiotic stresses in rice, which may be related with the protein binding ability of RCAL.ⅲ) It was proved that RCAL interacted with CPN60β1 under moderate heat treatment with various evidence, such as co-IP, MS-MS, and BiFC (bimolecular fluorescence complementation). Moreover, the rice heat resistance ability by over-expressing of CPN60β1 were improved. Meanwhile, that RCA combined with the thylakoid membrane under room temperature was confirmed, with the fact that RCA has no trans-membrane domain by bio-information prediction servers. And lots of proteins in thylakoid which could interact with RCA were found by co-IP with RCA antibodies. One of them was identified as the 26kD protein by MS-MS, which was inferred to be a chlorophyll a/b binding protein. With the test of Western-blotting with Arabidopsis chlorophyll a/b binding protein antibody and BiFC between RCAL and chlorophyll a/b binding protein in rice protoplast, we confirmed that the 26kD protein was chlorophyll a/b binding protein.In a word, the protein binding ability of RCA, especially that of RCAL may be the key point in the improvement of the heat resistance in rice or other plants.