Ozone Induces Flavonol Of Ginkgo Biloba Cells Dependently On Nitrate Reductase-mediated Nitric Oxide Signaling

Ozone is a part of the atmosphere,which persists in the stratosphere and troposphere.Ozone in the stratosphere protect all the living things on the earth,however,ozone in the troposphere is pollutant.As the development of modern industry,the concentration of tropospheric ozone has increased markedly and is continuously rising due to all the living,particularly to the plants.It has been reported that ozone exposure causes a reduction in photosynthesis and growth, as well as premature senescence of plants, which in turn causes decreased growth and yields, as well as decreased pathogen tolerance and other possible ecological alterations.Accumulation of secondary metabolites such as flavonoids is one of the early responses of plants to ozone exposure.Nitric oxide (NO) has recently emerged as an important signaling molecule in plants. It has been well documented that NO is widely involved in plant adaptive responses to biotic and abiotic stresses. Accumulating evidence indicates that this free radical, which was first identified as a second messenger in animals, plays important roles in stress-induced secondary metabolite accumulation in plants.Although the accumulation of flavonoid is one of the most effective Protection responses of plants to ozone,the underlying mechanisms of ozone-induced and signal transduction are very poorly understood.In this study,we utilized the Ginkgo biloha cells to study the function of ozone inenhancing flavonoids accumulation in Ginkgo biloba cells.The main results are summarized as follows:1.The plant cell line for the study was induced from the young stems of Ginkgo biloba with the MS medium supplemented with0.5mg/L NAA,1.0mg/L6-BA,0.1mg/L2,4-D,30mg/L sucrose, and8mg/L agar.2.The plant cell line is induced with the medium is adjusted to pH5.9and incubated on an orbital shaker incubator in the dark at100r/min and25℃.3.The Ginkgo biloba cell for the study is induced for six days,which is fresh, losse extent appropriate, uniformly dispersed, high-yield.4.To examine the effect of ozone on secondary metabolite accumulation, we assayed flavonol production of Ginkgo biloba cells exposed with ozone.flavonol production of the cells treated with ozone is significantly increased between15h and30h, being about3.2-fold higher over the control30h after treatment5.The effects of different concentrations of ozone on flavonol production of Ginkgo cells have exposure of the cells to low doses(50nl L-1) of ozonehas no significant effects on flavonol contents of the cells, while flavonol contents of the cells treated with high doses of ozone (>100nl L-1) are significantly increased as compared with those of the control, which suggests that the ozone-induced flavonol production in the cells is dose-dependent.6.Accumulation of NO is one of the early responses of plants to ozone multiple. The results of present work show that NO levels of the cells treated with ozone are immediately increased after ozone treatment, showing that ozone exposure may induce rapid NO generation in the cells. The highest NO level is obtained at about15h after ozone treatment,which is about16times that of the control cells.7.To further investigate whether NO is involved in ozone-induced flavonol production, we examined the effect of NO specific scavenger(cPTIO)on ozone-induced flavonol production of the cells. The results show that pretreatment of the cells with cPTIO inhibits the ozone-induced flavonol production of Ginkgo biloba cells, while cPTIO has no effects on flavonol production of the cells.8.To examine the effect of ozone on NR activity, we assayed NR activity of Ginkgo biloba cells exposed with ozone. NR activity of the cells treated with ozone is significantly increased, being about3.9-fold higher over the control15h after treatment.9.To investigate the biosynthetic route possibly involved in NO generation of Ginkgo biloba cells induced by ozone, we determined nitrate reductase (NR) activity of the cells exposed to ozone. NR activities of the cells exposed with ozone are higher than those of the control cells, showing that ozone exposure may stimulate NR activity of the cells.10.To investigate the biosynthetic route possibly involved in NO generation of Ginkgo biloba cells induced by ozone, we examined the effect of NR inhibitors on ozone-induced NO generation. Treatment of the cells with the widely used NR inhibitors sodium tungstate and Gin not only suppresses the ozone-induced NR activity but also abolishes the ozone-triggered NO generation, which suggests that NR might be involved in ozone-induced NO generation of the cells.11.In order to further assay the role of NR in ozone-induced flavonol accumulation, we examined the effect of NR inhibitors on ozone-induced flavonol production of the cells. Treatment of the cells with sodium tungstate and Gln suppresses the ozone-induced flavonol production. Moreover, the suppression of the inhibitors on ozone-induced flavonol production is restored by exogenous application of NO via its donor sodium nitroprusside, although treatment of the cells with10μM SNP alone has no effect on flavonol production of the cellsIn summary, the results suggests that NR is involved in ozone-induced NO generation of Ginkgo biloba cells,while NO is involved in ozone-induced flavonol production.The results will play an important role in increasing ozone tolerance of the plants.