Effects Of Environmental Stresses On Enzyme Activities And Gene Expression Of Ascorbic Acid Metabolism In Apple

Fruits are subjected to several environmental stresses including drought, high temperature, and high light that adversely affect growth, metabolism, and yield in the Northwest of China. Ascorbic acid (AsA) is one of the most abundant low molecular weight antioxidants present in plant tissues, and also an important function composition of fruits and others food. In association with other components of the antioxidant system, it can protect plants against oxidative damage. It is well-known that plants can adjust AsA contents by modulating AsA regeneration and biosynthesis. L-galactose pathway is the main AsA biosynthesis pathway in plants, L-galactono dehydrogenase (GalDH) and L-galactono-1, 4-lactone dehydrogenase (GalLDH) is the last two key enzymes for ascorbic acid biosynthesis in this way. Ascorbate-Glutathione cycle is the main AsA regeneration pathway. In this cycle, ascorbate peroxidase (APX) utilizes AsA as electron donor for reduction of H₂O₂, monodehydroascorbate was reduced by monodehydroascorbate reductase (MDHAR) and dehydroascorbate was reduced by dehydroascorbate reductase (DHAR). Oxidized glutathione (GSSG) produced in this cycle is reduced to GSH by glutathione reductase (GR), so they can accomplish the process of reactive oxygen species (ROS, including H₂O₂, HO·, O2·- and 1O2) elimination and AsA regeneration in plant. In order to investigate the response of ascorbate metabolism to environmentmental stresses, and supply theory basis on effects of AsA-GSH cycle on antioxidative stress, ascorbate content, changes of redox state, activities and gene expression of enzymes related to ascorbate metabolism on these parameters were studied in leaves of apple (Malus domestica Borkh. cv Gala) under drought stress, high temperature or high light, and in peels of apple (Malus domestica Borkh. cv Gala) during the growth and development or exposed to sunlight.The results were as follows:1. In order to characterize the response of anti-oxidative system to high temperature, two-year-old potted apple plants were exposed to 28°C as control or high temperature at 40 °C 0, 2, 4, 6, 8 h, respectively. The relative membrane permeability, MDA and H₂O₂ concentrations in apple leaves increased at the high temperature treatment. The contents of total ascorbate, AsA, total glutathione and GSH were the highest at 2 h in the high temperature treatment, followed by a continuous decline with further increases in treatment duration. AsA/DHA and GSH/GSSG declined with the high temperature treatment duration. Gene expression of GalDH and GalLDH reached the highest at 2 h of high temperature treatment. Activity of protective enzymes reached the highest point at 4 h of high temperature treatment duration, and then decreased. Gene expression of APX, DHAR and GR showed the same changes as the enzyme activities. The results suggest that the ascorbate–glutathione cycle is up-regulated in response to high temperature, but its regulation ability declines obviously after reaching the maximum with further increases in treatment duration.2. The relative membrane permeability, MDA and H₂O₂ concentrations in two-year-old potted apple plants increased with the continuance of drought stress and began to decrease after re-watering. The contents of total ascorbate, reduced ascorbic acid (AsA), total glutathione and glutathione (GSH), gene expression of APX, DHAR, GR, GalDH and GalLDH showed the similar changes as the enzyme activities, respectively. were led to an obvious up-regulation in apple leaves while the soil water content is more than 40%⁴5%, followed by an obvious decline with further the increasing of drought level, especially redox state of AsA and GSH. However, levels of them were gotten reversion after re-watering. Moreover, drought stress induced to an significant increase of enzyme activity of not only APX used to scavenge H₂O₂,but also MDHAR, DHAR and GR used to regenerate AsA and GR, especially when the soil water content is above 40%⁴5%. Activities of them were decreased and began to reverse after re-watering. After re-watering, the ration of AsA/DHA and GSH/GSSG showed the slight increase, but still lower than control. These results suggest that the apple leaves would improve the cyclic capability of ascorbate–glutathione system to resist drought stress.3. Levels of components, relative membrane permeability, MDA and H₂O₂, APX, MDHAR, DHAR, GR, AsA, DHA, GSH and GSSG, of AsA-GSH cycle, total ascorbate and total glutathione and the gene expression of APX, DHAR, GR, GalDH and GalLDH in leaves of potted apple plants increased with the high light treatment duration. However, the levels of components in AsA-GSH cycle and the expression of GalDH and GalLDH were all decreased obviously after they reached maximum at 4 h high light treatment duration (except total glutathione). The ratios of AsA/DHA and GSH/GSSG decreased with the duration of high light treatment. The activities and gene expression of enzymes above were down-regulated after 6 h high light treatment duration. The results suggest that the apple leaves would improve the cyclic capability of ascorbate–glutathione system to resist damage of long time high light to plant, and play the important role in the production of ROS mediated by alternate oxygen reduction path and subsequent oxidative damage in stressed plants.4. Ascorbate metabolism, including ascorbate pool, activities of APX, DHAR, MDHAR and GR, gene expression of APX, DHAR, GR, GalDH and GalLDH of bagged apple peels lower than that of non-bagged apple peel during the whole growth and development. These results suggest that the level of AsA-GSH cycle components would decreased in the long time no light environment that resulted by bagged. There were the highest concentrations of ascorbate pool in young fruit peel, and the lowest in mature fruit peel. At the same time, the activities of DHAR, MDHAR and GR come to the lowest in the mature fruit peels along with the decreasing of APX activity.5. The increase activities of APX, MDHAR, DHAR and GR and gene expression of APX, DHAR and GR after removed bags of apple fruit after three month bagged. The concentrations of ascorbic acid and glutathione increased after removed bags, and with the decreasing of AsA/DHA and GSH/GSSG. at the same time. These suggested exposed to sun light from no light directly could increase ability of scavenging reactive oxygen free radical, reduced membrane lipid peroxidation, and the ascorbate-glutathione cycle in the original bagged apple are up-regulated after removed bags in response to non-bagged fruit to minimize photo-oxidative damage and contributes to its acclimation to full sun .In a whole, the ability of AsA regeneration and biosynthesis increased under adversity stress (e.g. drought stress, high temperature, high light and bagged). These changes could avoid AsA oxidation excessively, maintain AsA contents and state of redox and increase capacity of resisting oxidative stress accordingly.