Effects Of Long-term Ozone Fumigation On Ascorbic Acid-Glutathione Cycle And Expression Of Related Antioxidant Genes In Ginkgo Biloba L.

Tropospheric ozone?O₃?is a kind of strong oxidative pollutant that harmful to living organisms.The rising concentration of O₃ leads to increasingly serious O₃ pol-lution,which has a bad impact on the sustainable development of urban forests.Ginkgo biloba L.is one of the main tree species for urban greening in northern China.In order to explore the effect of long-term O₃ exposure on growth,physiology and the expres-sion of related antioxidant genes,Ginkgo biloba was fumigated with 80 nmol·mol^-1 and120 nmol·mol^-1 O₃ in 2015,2016 and 2017.The test materials included non-fumigated seedlings?Y0?,O₃ fumigated seedlings?Y1?for the first year,O₃ fumigated seedlings?Y2?for the second year,and O₃ fumigated seedlings?Y3?for the third year.The ma-terials were uniformly planted in 2015.The growth of Ginkgo biloba was observed by measuring photosynthesis,plant height,branch length,basal diameter,leaf area,bud-ding rate,infection rate and mortality rate in the following year.The response of anti-oxidant system and related antioxidant genes to O₃ in Ginkgo biloba leaves was inves-tigated by measuring the levels of reactive oxygen species?ROS?,malondialdehyde?MDA?,non-enzymatic antioxidant substances,antioxidant enzyme activities and the expression levels of related antioxidant genes.It provided a reference for the study of the physiological ecology of urban forest trees under long-term continuous growing season O₃ exposure.The results showed that:?1?O₃ treatment caused net photosynthetic rate?P_n?,stomatal conductance?G_s?,intercellular CO₂ concentration?C_i?and photosynthetic pigment content decreased sig-nificantly.The concentration and time of O₃ treatment had no significant effect on plant height,branch length and basal diameter of different treatment groups,but caused a significant decrease of 39.88%in leaf area of Y3.In the observation of the growth of Ginkgo biloba in the following year,it was found that the budding rate of Y2 and Y3treated with O₃ decreased by 25%and 14.28%respectively in the following year,and the infection rate and mortality rate in the following year increased.?2?The activities of superoxide dismutase?SOD?and ascorbate peroxidase?APX?in Y1 leaves were increased after O₃ treatment,and the dynamic balance of ROS was established and maintained in a short period of time without lipid peroxidation.How-ever,O₃ treatment caused ROS accumulation in Y2 and Y3 leaves.Diaminobenzidine?DAB?staining observation showed that with the increase of O₃ fumigation concentra-tion and time,the accumulation area of hydrogen peroxide?H₂O₂?in Ginkgo biloba seedlings leaves expanded.Increased ROS inhibited the activities of SOD and catalase?CAT?,and stimulated the activities of APX,monodehydroascorbate reductase?MDHAR?,dehydroascorbate reductase?DHAR?and glutathione reductase?GR?.O₃treatment reduced ascorbic acid?AsA?content in Y1 leaves,while Y2 and Y3 leaves were significantly increased.Y2 and Y3 alleviated oxidative stress by regulating the transformation pathway in ascorbic acid-glutathione cycle?AsA-GSH?and/or de novo synthesis of AsA.Although Y2 and Y3 actively regulate the antioxidant system to re-lieve the increase of ROS,they still cause the increase of MDA content,and H₂O₂ con-tent was significantly positively correlated with MDA content.?3?O₃ treatment stimulated the expression of antioxidant enzyme related genes in Ginkgo biloba leaves.SODs,CAT,APXs,glutathione sulfhydryl transferase?GST?and Class?peroxidase?PODs?genes were up-regulated to varying degrees.The change of transcription level of SODs gene family was related to the spatial position of isozyme encoded by gene.The expression of APXs and PODs gene family members were sim-ilar under different concentrations of O₃,and the family members jointly regulated the changes of enzyme activity.In summary:Exposure to O₃ caused a decrease in P_n,but had no significant effect on plant height,branch length,and base diameter.Ginkgo biloba could quickly estab-lish a new balance of redox state under the first year of exposure,while exposure to O₃in continuous growth season made plants more likely to produce and accumulate ROS.But the plants activated the defense system,stimulated the activity of antioxidant en-zymes,regulated the AsA-GSH cycle,increased the content of antioxidants and regu-lated gene expression to maintain the balance of redox states in the cells,and detoxified ROS to ensure the growth of individuals.Therefore,Ginkgo biloba has the ability to adapt to the environment with long-term increase of O₃ concentration and related to the exposure time and concentration,so it may adapt to the environment with increasing O₃ concentration in the future.