Studies On Ascorbic Acid Metabolism During Drought Stress And Senescence In Apple

Ascorbic acid (AsA, vitamin C) 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-1,4-lactone dehydrogenase(GalLDH) is the last key enzyme 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 response of ascorbate metabolism to drought stress and senescence, and supply theory basis on effects of AsA metabolism on antioxidative stress, ascorbate content, changes of redox state, activities of enzymes related to ascorbate metabolism, and effects of exogenous AsA on these parameters were studied in Malus sieversii and Malus hupehensis seedlings, leaves and fruits of apple (Malus domestica Borkh. cv Gala) under drought stress or during senescence.The results were as follows:1. Ascorbate contents and activities of related metabolism enzymes had no significant differences in Malus sieversii and Malus hupehensis seedlings in well-water condition. Under drought stress, the changes trends of contents on AsA, total AsA, GSH and total GSH were consistent in Malus sieversii and Malus hupehensis seedlings, but the increased extent of them in Malus sieversii were more than that of Malus hupehensis. The leaves of Malus sieversii had higher activities of GalLDH,DHAR and GR, and higher AsA/DHA and GSH/GSSG than that of Malus hupehensis under drought stress. These suggested that Malus sieversii have higher abilities of AsA biosynthesis and regenerating than that of Malus hupehensis under drought stress.2. Levels of components, H₂O₂, APX, MDHAR, DHAR, GR, AsA, DHA, GSH and GSSG, of AsA-GSH cycle, total ascorbate and total glutathione and activity of GalLDH in leaves of potted apple (Malus domestica Borkh. cv Gala) seedling increased under low soil water content of 11.25%¹2.50%. However, the levels of components in AsA-GSH cycle and the activity of GalLDH were all decreased obviously after they reached maximum. The activities of enzymes above were down-regulated during rewatering phase, and the contents of AsA, DHA ,GSH, GSSG decreased during rewatering, but AsA/DHA,GSH/GSSG increased slowly although they were lower than that of control plant. These suggested that regulation of AsA contents could increase the capability of resist drought stress by regulating activities of regeneration and synthesis enzymes in apple plants. 5 mmol·L^-1 exogenous AsA reduced injuries resulted from membrane lipid peroxidation and leakage electrolyte during drought stress. The plants of supplying exogenous AsA had higher content of AsA, total AsA, GSH and total GSH, in consistent with higher enzymes activities of MDHAR,DHAR and GR in this period than that of plant under drought stress. These changes increased drought-resistance, kept higher ratio of AsA/DHA and GSH/GSSG after rewatering, and suggested the plant of supplying exogenous AsA could recovery rapidly after rewatering.3. Ascorbate metabolism, including AsA content, metabolism relative enzymes activities and relationships among them, was investigated in leaves of apple (Malus domestica Borkh. cv Gala) of various ages, i.e. young leaves, expanding leaves, mature leaves, aged leaves, in this paper. The results showed that the activity of MDHAR decreased, and the activities of APX and DHAR were all decreased when they reached maximum at the 24th and 36th hour of senescence respectively. GalLDH increased rapidly and the activity reached maximum at the 6th hour and then decreased. AsA/DHA, AsA pool and GSH pool all increased and then decreased all along with the senescence, and GSH/GSSG decreased obviously. The results also showed that total AsA content and AsA content changed accordingly,reaching maximum in mature leaves and minimum in aged leaves, and changes of GalLDH activity were accordant with AsA content. With the decline of APX activities and accumulation of H₂O₂, the activities of MDHAR, DHAR and GR which conduce to regeneration of AsA were lowest at aged leaves,so as to the extent of AsA oxidation was reached maximum in ages leaves. These results suggested that AsA contents were mainly due to ability of synthesis in apple leaves of various ages.4. Contents of total AsA and total GSH, activities of APX, DHAR and GalLDH increased, but activities of MDHAR decreased during senescence prophase of detached leaves of apple (Malus domestica Borkh. cv Gala). These leaves could delay senescence by increasing regeneration and biosynthesis to maintain AsA content. But these parameters decreased along with senescence, and AsA/DHA and GSH/GSSG decreased obviously during senescence.The results also showed that 5mmol·L^-1 exogenous AsA inhibited membrane lipid peroxidation and decreased H₂O₂ content, but had little effect on relative membrane permeability. Exogenous ASA maintained high activities of APX, DHAR and GalLDH, but delayed the activity decline of MDHAR, and decreased oxidative content of GSH, This suggested 5mmol·L^-1 exogenous AsA could increase adaptions to oxidative stress in plant and delay senescence of detached leaves of apple.5. The higher activities of MDHAR and DHAR could redound to the regeneration of AsA, while increased the abilities of elimination of ROS in the apple (Malus domestica Borkh. cv Gala) fruits under room temperature during prophase storage. But AsA, DHA, GSH, GSSG, total AsA, total GSH contents decreased obviously during anaphase storage. The apple fruits of supplying 10 mmol·L^-1 exogenous AsA had lower peak value of respiration rate and ethylene production rate than that of CK during storage, and had lower relative membrane permeability, contents of MDA and H₂O₂. 10 mmol·L^-1 exogenous AsA increased activities of related enymes of AsA-GSH cycle (APX,MDHAR,DHAR and GR) and contents of AsA and GSH correspondingly during prophase storage, and had higher ratio of AsA/DHA. These suggested exogenous AsA could increase ability of scavenging reactive oxygen free radical, reduced membrane lipid peroxidation. But these above-mentioned parameters had no significant difference during anaphase storage.In a whole, the ability of AsA regeneration and biosynthesis increased under adversity stress (e.g., drought stress and senescence etc.). These changes could avoid AsA oxidation excessively, maintain AsA contents and state of redox and increase capacity of resisting oxidative stress accordingly. Applicating of suitable concentration exogenous AsA improved the function of antioxidant system, reduced active oxygen species (ROS) accumulation and decreased membrane permeability membrane lipid peroxidation under stess, enhanced drought resistance and delayed senescence and reduced injuries caused by stress in plants accordingly.