Metabolism Of Glutathione And Ascorbate And The Signal Function Of Ja In Regulating Glutathione And Ascorbate Metabolism In Agropyron Cristatum Under Drought Stress

The native grass Agropyron cristatum is a perennial wild forge with high nutritional value, which widespreadly grows in the semiarid area of loess hilly-gully region on the Loess Plateau of northwestern China. With a strong resistance to the drought stress and capacity of water and soil conservation, A. cristatum is important for the ecological construction in this region. Up to date, there are lacks in the studies of drought-resistant mechanism of A. cristatum. Thus, the drought-resistant mechanism of A. cristatum has not been really revealed. Water stress usually induces the accumulation of reactive oxygen species (ROS), which cause oxidative damage to plants. Plants could protect themselves against oxidative damage by enhancing antioxidant system including antioxidative enzymes and nonenzymatic compounds. Therefore, there is a close relationship between drought resistance and antioxidant capacity. As ascorbate and glutathione are two crucial nonenzymatic compounds involved in defence against oxidative stress, they are important components of antioxidant system. So they are important roles in defence against oxidative stress. The ascorbate and glutathione contents are modulated by the regeneration and biosynthesis of ascorbate and glutathione. APX, GR, DHAR and MDHAR, which are involved in the regeneration of ascorbate and glutathione, are also the main enzymes involved in the antioxidant metabolism in plants. Therefore, ascorbate and glutathione metabolism are important parts of the antioxidant metabolism in plants. However, the response of ascorbate and glutathione metabolism in native grass A. cristatum to water stress and the signal regulation in this process remain unknown. In view of above reasons, we investigate the response of ascorbate and glutathione metabolism in native grass A. cristatum to water stress from three perspectives including physiological, molecular and signal perspectives, in order to reveal the antioxidant metabolism and drought-resistant mechanism of A. cristatum, and provide theoretical basis for its application in the ecological construction in the semiarid area of loess hilly-gully region on the Loess Plateau of northwestern China. The main results of our research are as follows.1. A. cristatum protected itself from oxidative damage under drought stress by increasing the activities of SOD, CAT, APX, GR, DHAR, MDHAR, GPX, and the contents of GSH, AsA and Car. S. bungeana protected itself by increasing the activities of SOD, CAT, POD, APX, GR, DHAR, MDHAR, GPX, and the AsA content. C. genessongorica protected itself by increasing the activities of SOD, CAT, APX, GR, DHAR, MDHAR, GPX, and the contents of AsA and Car. Bothriochloa ischaemum protected itself by increasing the activities of SOD, CAT, POD, APX, GR, DHAR, MDHAR, GPX, and the contents of AsA and Car. The sequence of antioxidant capacity of four grasses was A. cristatum> C. genessongorica> B. ischaemum> S. bungeana according to the results of comprehensive evaluation by subject function method. Therefore, A. cristatum, C. genessongorica and B. ischaemum can be considered as suitable grasses applied to eco-environmental rehabilitation in the semiarid area of loess hilly-gully region. As A. cristatum showed stronger antioxidant capacity, especially for its higher contents of AsA and GSH than other grasses among four grasses under drought stress. Thus, we select A. cristatum as the experimental material for the study of metabolism of glutathione and ascorbate and its signal regulation under drought stress.2. A. cristatum could maintain the levels of AsA and GSH, and their redox status under drought stress, by increasing the activities of GalLDH, y-ECS, APX, GR, DHAR and MDHAR involved in the metabolism of glutathione and ascorbate. By this way, A. cristatum protected itself from oxidative damage induced by drought stress. However, there are differences in the responses of the metabolism of glutathione and ascorbate in the leaves in A. cristatum to different drought stress levels with the prolonging stress days. During first 20 days of stress treatment, there was significant enhancement in the metabolism of glutathione and ascorbate under severe drought stress. After 20 days of stress treatment, the activities of GalLDH, y-ECS, APX, GR, DHAR and MDHAR decreased significantly under severe drought stress, because A. cristatum suffered more severe oxidative damage induced by severe drought stress. However, the activities of GalLDH, y-ECS, APX, GR, DHAR and MDHAR maintained higher during the whole stress time under moderate drought stress. These results suggested that A. cristatum could effectively protect itself from oxidative damage induced by moderate drought stress for long time. However, A. cristatum could effectively protect itself from oxidative damage induced by severe drought stress only for limited time, and its antioxidant capacity decreased significantly with the prolonging stress days.3. We obtained the fragments of key enzymes involved in the metabolism of glutathione and ascorbate and reference gene by using primers designed according to the conserved sequence of the closely related plants, such as wheat, barley and rice, etc.. The GenBank accession numbers of above gene fragments are as follows:GalLDH (GenBank accession number GQ457296),γ-ECS (GenBank accession number GQ457297),APX (GenBankaccession number GQ457300),GR (GenBank accession number GQ457301),DHAR (GenBank accession number GQ457298),MDHAR (GenBank accession number GQ457299),β-actin (GenBank accession number GQ457302)4. Water stress-induced JA accumulation participates in the regulation of ascorbate and glutathione metabolism by increasing the transcript levels and activities of APX, GR, MDHAR, DHAR, GalLDH, the contents of AsA, GSH and total ascorbate, and the ratios of AsA/DHA and GSH/GSSG, which, in turn, enhances the antioxidant ability and protects A. cristatum against oxidative stress induced by water stress. Meanwhile, our results also suggested that the transcriptional control of enzymes involved in the ascorbate and glutathione metabolism is the main way of the regulation of the ascorbate and glutathione metabolism regulated by JA under water stress.5. H2O2 and NO all participated in the signal transduction of JA in regulating the ascorbate and glutathione metabolism. JA-induced H2O2 production mediated NO generation, which in turn, resulted in upregulation of the transcript levels and activities of APX, GR, MDHAR, DHAR, GalLDH andγ-ECS, and the increases in the contents of AsA, GSH, total ascorbate and total glutathione. These results provide new knowledge to the regulation of the antioxidant metabolism by JA in plants.6. The protein kinase MEK1/2 participated in the signal transduction of JA in regulating transcript levels and activities of APX, GR, MDHAR, DHAR and GalLDH, and the contents of AsA, GSH, total ascorbate and total glutathione. The MEK1/2-mediated signal pathway does not induce the production of H2O2 and NO in the signal transduction of JA in regulating the ascorbate and glutathione metabolism. JA-induced H2O2 and NO production activated MEK1/2-mediated signal pathway, which in turn, resulted in upregulation of the transcript levels and activities of APX, GR, MDHAR, DHAR and GalLDH, and the increases in the contents of AsA, GSH, total ascorbate and total glutathione. These results provide new knowledge to the regulation of the antioxidant metabolism by JA in plants.