Proteomic Analysis Of Rice Leaves In Response To Drought And Osmotic Stress

Drought is one of the major abiotic stresses limiting plant growth and production, especially for drought susceptive genotypes. Rice (Oryza sativa L.) is not only one of the most widely cultivated food crops throughout the world, but also the model plant species. The omics study, e.g. proteomics, plays an important role in system analysis of molecular mechanism under drought resistance.The upland rice IRAT109 is a variety with high ability of drought resistance, which was widely used for the gene discovery and genetic improvement of rice drought reisitance. This variety was used as a core material in our experiment. Proteomic tool based on two dimensional electrophoresis and mass spectrometry integrated with morphological and physiological survey were used here to investigate the responses in rice leaves to partial root osmotic stress (PROS) and whole root osmotic stress (WROS).The main results were as follows:1. The WROS stress promoted the the concentration of ABA and proline in rice leaves and caused stress symptoms like leaf rolling and membrane leakage. PROS stimulated ABA and proline content, but did not caused severe leaf damage, shows the stress signals were induced, followed by a serious of stress resistance response, such as osmotic regulation.2. Quantitative image analysis of rice leaves based on 2-DE revealed a total of 67 protein spots with significantly different expression levels in WROS and PROS plants when compared to well watered (WW) plants, including 36 spots only in WROS treatment and 15 spots only in PROS treatment. Additionally, there are 11 of the upregulated and 3 of the downregulated protein spots were observed in both the PROS and WROS treatments. Furthermore, there were 2 protein spots showed opposite responses between PROS and WROS when compared with the WW condition. A total of 58 protein spots were successfully identified by MALDI-TOF/TOF analysis. These proteins were classified to 12 categories based on their functions, e.g.stress defense, and photosynthesis.3. Differentially expressed proteins induced by PROS stress mainly involved in photosynthesis and carbohydrate metabolism, e.g. promoted the degradation of rubiso; enhanced the expression of glyceraldehyde-3-phosphate dehydrogenase and aconitate hydratase, which is a key enzyme of glycolysis and TCA cycle, respectively; and induced the expression of ATPases. Besides these observations, there are two proteins, which show upregulation only in the PROS plants, including anthranilate synthase and nickel-binding protein 2A. The upregulation of anthranilate synthase is probable related to the role of ABA pretreatment, which overcoming the growth inhibition by stress. For the nickel-binding protein 2A, the precise function of this protein in plant stress response is unknown, although the previous research considered it as a candidate gene for drought resistance.4. For the WROS-treated plants, many molecular chaperones and heat-shock proteins, which also function as molecular chaperones, were mostly upregulated. The enhanced expression of these proteins can assist protein refolding to promote the survival and growth of the plant under stress conditions. In addition, the accumulation of redox homeostasis-related proteins (monodehydroascorbate reductase, peroxidase, and glutathione S-transferase), would enhance the ability of plant to scavenge reactive oxygen species (ROS), and reduced the stress damage. Furthermore, the down-regulation of proteins belongs to secondary metabolism (S-adenosylmethionine synthetase and caffeic acid 3-O-methyltransferase), suggesting a decreased synthesis of lignin and other secondary metabolites under stress conditions. This change may serve to decrease any unnecessary consumption of material and energy.In order to obtain a better understanding of the response of upland rice IRAT109 to drought stress, integrated analysis of proteome, transcriptome and metabolome was conducted for the rice leaf samples at seedling stage. The main results were as follows:1. A total of 71 protein spots showed significantly altered expression while 60 of them were successfully identified with 22 down-regulated (Figure.2A) and 38 up-regulated. These proteins were classified into 11 categories according their functions, e.g. protein folding and assembly, carbohydrate metabolism. The functionally classification showed that the down-regulated proteins were mainly associated with translation, and the up-regulated proteins were mainly related to protein folding and assembly.2. The transcriptome analysis showed 4756 differentially expressed genes, of which 2528 were up-regulated and 2228 were down-regulated. Some of these genes act as the regulated genes, such as signal transduction and transcription; some act as functional genes, such as redox homeostasis, carbohydrate metabolism and amino acid metabolism. In addition, there were many categories of genes that were detected as differentially expressed in terms of mRNA levels but not protein levels, such as nucleic acid metabolism, lipid metabolism, and non-protein coding. 3. Pathways analysis using the KEGG pathway database identified 47 significantly changed pathways (p< 0.05) related to the differentially expressed proteins and genes. Five pathways had significant changes in both mRNA and protein levels. And it is interesting to found that most of the significantly changed pathways were related to basic metabolism (e.g., carbon fixation, urea cycle, fatty acid metabolism and amino acid biosynthesis). In addition, several pathways related to secondary metabolism were also found to be significantly changed (e.g., phenylpropanoid biosynthesis, limonene and pinene degradation).4. Metabolic profiling based on GC-MS showed 37 differentially expressed metabolites. These metabolites were classified into 6 categories including:organic acids (38%), sugars (19%), amino acids (16%), fatty acids (14%), sugar alcohols (5%), and others (8%).5. The integrated analysis of differentially expressed mRNAs, proteins, and metabolites provided us a globe understanding of metabolism regulation for rice drought resistance:under drought stress, photosynthesis is limited, leading to the decreased output of energy and carbohydrates. However, resistance to drought stress requires large amounts of energy and substances for some processes, such as ROS scavenging and osmolyte synthesizing. The extra energy and substances consumption conflicts with the lower photosynthesis rate under drought stress conditions. The mobilization of storage substances is needed to solve this problem. Carbohydrates and fatty acids are two of the most important forms of rice leaf reserves. Drought stress enhanced the glycolysis and fatty acids degradation, protmoted the TCA cycle and the synthesis of amino acids, induced a substance transformation and energy flux from carbohydrates and fatty acids to amino acids. These processes, on the one side, will helpful for the accumulation of amino acids, e.g. proline, to enhance the osmotic regulation ability, on the other side, make a good prepare for the protein synthesis. Furtermore, the increased production of ATP from the glycolysis and TCA cycle, provided sufficient energy to confor the drought stress.