Studies On The Relationship Between NADP-ME And Stresses In Rice (Oryza Sativa L)

NADP-malic enzyme (NADP-ME; EC 1.1.1.40) catalyzes the oxidative decarboxylation of malate: L-malate + NADP~+→pyruvate + CO2 + NADPH + H+ in the presence of a bivalent metal ion. NADP-ME, one of key enzymes in organism, acts in a wide range of metabolic pathways in both plants, animals bacterial. In our recent research, we focused in Carbonate stress (NaHCO3, Na2CO3) for plant in the alkali soil (containing NaHCO3, Na2CO3) in the northeast of China. Using molecular biological methods, NADP-ME2 gene was cloned from rice plant under carbonate stress (GeneBank Accession Nos. AB053295). Here, the relationship between NADP-ME and stress was studied in rice plant. Recently, four NADP-ME isozyme genes have been reported. In order to further know the relation between isozyme and stress, we compared the expressing characterization and distinction of four NADP-ME isozymes of rice plant under stress, and the specific activities of NADP-ME were determined in the leaves and roots of rice plant under stress. To clarify the diversity of NADP-ME isozymes in rice, we produced two active GST-fused NADP-ME proteins in Escherichia coli. Obtained two recombinant proteins were used to in vitro investigate their enzymatic properties. The tolerance of yeast expressing NADP-ME protein was analyzed preliminarily. Those results were as followed:1. Four rice NADP-MEs share a high degree of similarity in advance of seventy percent one another. NADP-ME1 protein with the high certainties has the chloroplast transit peptide in its N-terminal amino acid sequence, and using PSORT prediction, it is located in the chloroplasts, NADP-ME2 in the plasma membrane, NADP-ME3 in the endoplasmic reticulum, NADP-ME4 in the cytosol. Using green fluorescence protein (GFP) as molecular labeling, the localization of NADP-ME2 was studied in transformed Arabidopsis plant, and the NADP-ME2-GFP recombinant protein was expressed mainly in the plasma membrane. These results suggest that four rice NADP-ME isozymes are located in different subcellular compartment, and play different roles.2. Gene expression of rice NADP-MEs were analyzed by northern blot. First, rice NADP-ME2 gene expression is stronger in rice leaf and stem, and relatively weak in rice root under normal condition. NADP-ME2 gene expression is not in response to light. So NADP-ME2 may be non-photosynthetic isoform of NADP-ME in rice. However, NADP-ME1 gene (C3 plant plastidic NADP-ME) is regulated by light. This result hasn't been reported. Under NaCl, NaHCO3, Na2CO3, PEG stresses, NADP-ME2 gene expression increased greatly. It indicated that NADP-ME2 was reponse to stress. Four rice NADP-ME gene expressions were analyzed by Semi-quantitative PCR, and the results suggested that each of rice NADP-ME genes has it own response to environmental stresses.3. Four kinds of NADP-ME isoforms were detected through Native-PAGE and activity staining of NADP-ME. This result is not same as the reported one (one NADP-ME band was detectedin C3 plant). NADP-ME activities increased greatly under NaCl, NaHCO3, Na2CO3, PEG stresses. This also suggested that rice NADP-ME is in response to environmental stress at the protein expessing levels.4. Two recombinant rice NADP-ME proteins, NADP-ME2 and NADP-ME3, were compared their enzymatic properties. Two rice NADP-ME genes were successfully expressed in BL21 cells, and active recombinant proteins, NADP-ME2 and NADP-ME3 were obtained by isolation and purification methods. Comparing NADP-ME2 and NADP-ME3, their expression levels are different, NADP-ME2 very lower, and NADP-ME3 high. The affinity of NADP-ME2 for malate was higher than that of NADP-ME3, while the affinity of NADP-ME3 for NADP was higher than that of NADP-ME2. It indicated that NADP-ME2 and NADP-ME3 have the difference in the actions on substrates of malate and NADP. Additionally, maximal velocities of NADP-ME2 and NADP-ME3 for malate were more than maximal velocities for NADP. From above results, it suggested that NADP-ME isozymes were different in rice or other plants.5. The tolerance of yeasts expressing NADP-ME2 or NADP-ME3 was analyzed to know relation NADP-ME and stress from the transciption and expression levels. As yesat is eukaryote organism, it has the similar metabolizing route. NADP-ME2 and NADP-ME3 were constructed yeast express vector, and induced for expressing protein. Analysis of the tolerance, transformant yeast expressing NADP-ME2 protein inceased a little, while compared with the control, transformant NADP-ME3 yeast had no change. The result indicated that the tolerance of yeast highly expressing NADP-ME increased. We further speculate that the tolerance of plant expressing NADP-ME can be enhanced.