The Studies On Function And Characterization Of APX And CAT Isoenzymes From Rice And Their Relationship With Salinity Stresses

Rice is a kind of main staple crop around the world, but the production and quality of rice is severely influenced by adverse environmental stresses causing great losses in productivity. However, under environmental conditions like salinity, the excess generation of reactive oxygen species (ROS) from plant cells leads to the disrupted cellular homeostasis and a series of damage to cells. Recently, how to efficiently scavenge the excess ROS, to make balance between the production and breakdown of ROS in cells, to promote the scavenging enzymes and metabolism of antioxidants, ultimately to reduce the toxicity of ROS plants endured is a key in the field of plant resistant mechanism. In this paper, we aimed the isoenzymes of ascorbate peroxidase (APX) and catalase (CAT) from rice (Oryza saliva L.), which acted as the important ROS scavenging enzymes. The comprehensive studies was done among APX and CAT isoenzymes, including the transcriptional characterization of interest genes of two isoenzymes related to several stresses, especially to carbonate stress; the comparative analysis of two isoenzymes characterization and function in vivo and vitro under stresses; and characterization and the relevant physiological and biochemical metabolism of transgenic mutants as well. The results elucidated the relationship between plants salt tolerance and ROS metabolism, and explored the plants salt tolerant mechanism, also obtained applicable plant salt tolerant materials through plant genetic engineering. The results in this paper as followed:Based on the data base released from Genbank and information of rice geneome projects, the open reading frame (ORF) clones of APXa, APXb encoding rice APX and CAT1, CAT2 encoding rice CAT isoenzymes were isolated by RT-PCR respectively. The identity of deduced amino acid sequences of APXa and APXb, CAT1 and CAT2 was 92%, 76.4% respectively. The possibility of APXa and APXb isoenzymes located in microbody peroxisome was 47.4% and 50.8% respectively, in cytoplasm was 45%; the possibility of CAT1 and CAT2 isoenzymes located in microbody peroxisome was 74.8%, 64% respectively, in cytoplasm was 45%. Northern blot to clarify the characterization of the expression of these genes showed the results that there was a significant increase of APXa. APXb, CAT1 and CAT2 mRNA levels than control by detecting rice seedlings exposed to NaCl, NaHCO₃, Na₂CO₃, PEG and H₂O₂ at different levels, and it exhibited in a different transcript levels between in leaves and in roots. In details, APXa and APXb showed that it is abundant transcripts under 80mM NaCl together in leaves; however, APXb mRNA from roots expressed higher under 30mM NaHCO₃, 15mM Na₂CO₃ than that was under 80mM NaCl with the highest was under 15mM H₂O₂, but APXa was not the same. CAT1 and CAT2 performed in a slight difference, the highest expression of CAT1 in leaves was under 15mM H₂O₂, while CAT2 showed that the relative higher expression among 80mM NaCl, 15mM Na₂CO₃ and 15mM H₂O₂ stress both in leaves and roots.Combined the different mRNA expression of interested genes, the stress-induced changesof APX and CAT isoenzymes in rice were assayed. Using native PAGE, five APX isoforms and three CAT isoforms were observed from rice extracts under stresses, more kinds were found in leaves than in roots. The total activity of APX or CAT were examined, which individual activity response to kinds, intensity, and length of stresses given. The contents of H2O2, ascorbate in plants under stresses showed the relevance to the total activity, while activity of APX and CAT were different between leaves and roots. These results collectively suggested that stresses activate or suppress APX and CAT performance depending on the actual conditions. From the performance of active isoenzymes of APX and CAT, we investigated the relationship between stresses and APX, CAT in taci rice.In order to clarify the diversity and individual function of isozymes both APX and CAT from rice, a method of producing large quantities of these proteins using protein recombined technique is needed, to express and purify and characterize the enzymatic properties of the recombinants of interested genes products in vitro. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb, and of two rice CAT genes including CAT1 and CAT2 as well. The genes were cloned into the pGEX-6p-3 vector to allow expression of APX or CAT as a GST fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg. g"1 cells (dry weights), respectively. Specific activities were 15 and 20 111M ascorbate. min"1. mg"1 protein, respectively. The Km values for ascorbate were 4 and 1 mM, respectively, and those for H2O2 were 0.3 and 0.7mM, respectively. The native APXa and APXb were obtained after the fusion proteins were cleaved by PreScission Protease. Our results suggest that the two rice isoenzymes (APXa and APXb) have different properties. The GST-CAT 1 and GST-CAT2 fusion proteins were induced to express correctly in Escherichia coli respectively.The ORF of rice APXa, APXb, CAT1, CAT2 were constructed into plant expressional vector pBI121 respectively, and introduced into tobacco (N. plum) respectively using a Agrobacterium-mediated system. The regenerated plantlets were obtained through the optimal selection, and then Tl progenies seeds of N. plum were harvested after the normal pollination under careful cultivation. PCR and Southern blot determination of transgenic tobacco plants confirmed that the rice APXa, APXb, CAT1 and CAT2genes were integrated into genome of tobacco respectively, and that the effective expression of foreign genes in vitro.The comprehensive analysis of transgenic tobacco plants were done on mRNA transcript, physiological metabolism and salt tolerance. Northern blot results showed that genes of rice APXa, APXb, CAT1 and CAT2 express effectively in tobacco plants, which the expressional density of APXa and CAT2 were higher than that APXb, CAT1 respectively in leaves. The different expression of mRNA from the foreign genes would alter the transgenic tobacco plants intrinsic activity and relevant physiological metabolism.The different bonds subjected to CAT isoenzymes native PAGE were presented in the leaves of transgenic tobacco plantlets of CATI-17, CAT2-11 respectively, whereas that wasnot observed from the untransformed tobacco plants. The total activity of APX or CAT was elevated in transgenic tobacco plants and different from each other plants. The H2O2 contents in transgenic tobacco plants were lower than that in untransformed tobacco plants, no obvious changes were checked with a longer stress period. Most transgenic tobacco plants showed a lower relative electronic conductivity than the untransformed tobacco plants, and proved that less membrane damage in transgenic plants than that in untransformed tobacco plants. The transgenic tobacco plants with APXa, APXb, CAT1 and CAT2 genes can survive in the medium containing 240mM NaCl or 8mM Na2CO3 respectively with the retarded growth rate of leaf and root, however, the untransformed tobacco plants could not grow even in the medium containing 120mM NaCl or 8mM N...