Drought And Light Stress On Rice Thylakoid Membrane Proteome

The Effects of drought and high light intensity on leaf photosynthetic rate, stomatal conductance and photosynthetic photochemical activity (chlorophyll fluorescence) of rice (Oryza sativa L. ssp japonica, cv. Nipponbare) were investigated. The changes of thylakoid membrane proteins under drought and high light intensity were also analyzed using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) techniques. The main results are as follows:1. Under the drought condition, leaf water potential was decreased along with the decreas of soil water content (SWC). The inhibitions of leaf photosynthesis rate and stomatal conductance were observed under drought conditions. However, significant decreases of Fv/Fm,ΦPSⅡand qP and increases of qNP were only observed under severe drought stress, implying that non-stomatal factor exerted its effect on photosynthesis under severe drought scondition2. High light intensity had obvious inhibitory influences on leaf photosynthesis rate and chlorophyll fluorescence of rice. However, its effects were less severe than those of drought.3. The method of isolation and purification of chloroplasts and thylakoid membrane from rice leaves were explored in the present study. It was found that differential centrifugation combined with density gradient centrifugation was better than differential centrifugation alone to get the chloroplast with high integrity ratio.4. We also compared the effects of 4 different lysis bufferes (containing different detergents, i.e. 4%CHAPS, 2%CHAPS/2%SB3-10, 4%ASB-14 and 4%TritonX-100) in the solubilization of membrane proteins. The results showed that lysis buffer containing 4%CHAPS was the best one to solube membrane proteins.5. Drought stress had significant effects on rice thylakoid membrane proteome. Analysis on about 350 thylakoid membrane proteins visualized in 2-DE gel using related analysis softwares, we found that the espression of 20 thylakoid membrane proteins significantly changed during drought stress. Among 20 proteins, 3 proteins were disappeared, 3 new proteins were detected, 7 proteins were up-regulated (more than 3-fold) and 7 proteins were down-regulated (more than 3-fold) under drought stress. We further characterized and analyzed these 20 membrane proteins with the MALDI-TOF/MS and online BLAST, and found these 20 proteins were belonged to the subunit or homologs of 11 chloroplast proteins.6. Under high light intensity, only 4 thylakoid membrane proteins were found to be significantly affected. Among them, one protein was disappeared, two new proteins were appeared, one protein down-regulated when exposed to high light intensity. MS characterization showed these four different proteins were similar to counterparts of above drought-responsed thylakoid membrane proteins.