Mechanisms Of Melatonin In Regulating Stomatal Immunity In Nicotiana Tabacum And Panax Notoginseng

Plants have developed a variety of response mechanisms to resist adverse during long-term evolution.Reactive oxygen species(ROS)as second messengers,play important roles in plant response to biological and abiotic stresses.The stomata formed by guard cells are the main gate for gas and water exchange,and also an important way for foliar pathogens invasion plant leaves.Plants respond to the invasion of pathogenic bacteria by stimulating the production of ROS in guard cells to promote the closure of stomata.This mechanism of actively limiting the invasion of pathogenic bacteria is called stomatal immunity.in plants,Melatonin and flavonols are important compounds,and play an important role in the regulation of plant growth and stomatal opening,however,it is unclear whether melatonin and flavonol are involved in plant stomata immunity and also their regulatory mechanisms.In this study,we take the characteristic economic crop tobacco and Panax notoginseng in Yunnan province,as main research objects,to analyze the regulatory mechanism of melatonin and flavonol signaling in plant stomatal immunity,and provide a new theory for the development of green sustainable agriculture to improve plant disease resistance.The main results were shown as below:1.The interaction regulation mechanism of melatonin,flavonol and ROS in stomatal immunity was analyzed in wild-type tobacco.The experimental results showed that the pathogenic bacteria Pst DC3000 induced the expression of tobacco melatonin synthesis(NtSNAT)and receptor(NtPMTR1)genes,and exogenous addition of melatonin can significantly reduce the Number of colonies in tobacco leaves,indicating that the melatonin is involved in the infection of tobacco-responsive to pathogen Pst DC3000.Further research found that treatment with melatonin and flagellin(flg22)induced tobacco stomata closure and the production of ROS in guard cells,while the fluorescence intensity of flavonol compounds in guard cells decreased significantly.In contrast,the addition of kaempferol,inhibited melatonin and flg22-induced stomatal closure and the ROS levels in guard cells,that indicates,melatonin and flg22 signal may promote the production of ROS and induce stomata closure by inhibiting the synthesis of flavonol.Therefore,this study subsequently analyzed the changes in the expression levels of melatonin on flavonol synthesis(NtFLS1,NtCHI)and disease resistance genes as well.The results showed that the expression levels of NtFLS1 and NtCHI began to decrease after 2 hours of exogenous melatonin(MT)and pathogen co-treatment(MT+Pst DC3000),then reached the lowest level after 8 hours of treatment;and after 24 hours of treatment they were significantly increased or restored to control levels,It shows that melatonin and flg22 may be used as a signal to inhibit the synthesis of flavonol compounds in a short time and improve ROS production and also stomatal immunity.2.At the genetic level,it was verified that flavonols are involved in melatonin and flg22-induced stomatal immunity.The results showed,the number of colonies in the leaves of NtCHI and NtFLS2 overexpressing plants increased by 6 and 2 times after three days of Pst DC3000 infection,compared with wild-type tobacco,while the number of colonies in RNAi plants decreased significantly by 2.3 and 1.8 times.Compared withMT,the number of colonies in leaves of NtCHI and NtFLS2 overexpressing plants decreased by 1.6 and 6.5 times,also decreased significantly by 15 and 11 times,in leaves of RNAi plants.The results of stomata opening indicated that melatonin and flg22 induced NtCHI and NtFLS2 overexpression plants the size of the stomata is larger than RNAi plants,which may be related to the high levels of flavonols in NtCHI and NtFLS2 overexpression plants.3.In transgenic tobacco which overexpressing the melatonin synthesis gene GmSNAT,we further verified the mechanism of melatonin and flavonol signaling to regulate stomatal immunity.The results showed the number of colonies in the leaves of GmSNATOE2 and GmSNATOE5 plants was 23%and 14%of the wild-type wild after Pst DC3000 treatment,and the fluorescence intensity of flavonol compounds in GmSNATOE2 and GmSNATOE5 was significantly lower than wild type,but the ROS content was significantly increased;However,the exogenous kaempferol obtained the opposite result,indicating that overexpression of GmSNAT reduced the flavonol content in tobacco stoma.When melatonin and kaempferol(MT+Kae)or flagellin and kaempferol(flg22+Kae)co-treatment,the content of flavonols and ROS in guard cells returned to comparable levels.Stomatal sensitivity analysis found,melatonin-treated GmSNATOE2 and GmSNATOE5 plants,the size of stomatal is lower than the control group,while melatonin and kaempferol co-treated(MT+Kae),it can be restored to the level of the control,when flagellin and kaempferol co-treated(flg22+Kae),it cannot be completely restored to blank group level,compared with flagellin treated(flg22).4.Under natural conditions,we tested the disease resistance,ROS content in guard cells of Panax notoginseng in Yunnan province,which is used as medicine.after 2 hours treatment with melatonin and flagellin,stomata significantly closed,and the number of colonies in Panax notoginseng leaves decreased significantly on the third day,after pretreatment with 10μM melatonin.Observation of reactive oxygen species in guard cells,we can see,exogenous melatonin can significantly increase the reactive oxygen species which is required for stomatal close.Use the same concentration of melatonin to spray Panax notoginseng in field,it was found that exogenous melatonin can significantly reduce(about 18%)the disease susceptibility,but there is no difference between spraying once or twice.These results show that exogenous melatonin can significantly reduce the susceptibility rate of Panax notoginseng and improve plant resistance to pathogenic bacteria.