Analysis On The Mechanism Underlying The Dual Modulation Of Flavonols On Stomatal Movement

Common wheat(Triticum aestivum L.),one of the world's most important crops,is seriously suffered from drought,so it is urgent to cultivate drought-resistant wheat cultivars.Our lab previously bred wheat introgression cultivar SR3 with superior salt and drought tolerance using the asymmetric somatic hybridization approach.In comparison with its parent wheat cultivar JN177,SR3 genome possesses genome-scale genetic and epigenetic variation,and the variation accouts for the genetic basis of SR3's resistance to salt and drought.Flavonols are important secondary metabolites,and they well accumulate in the guard cells of higher plants.In our previous work,we identified a flavonol synthase gene TaFLS1 from SR3,who has lower methylation level and therefore higher transcript abundance in SR3 than in JN177.Using Arabidopsis TaFLS1 overexpressors and AtFLS1 mutant,flavonols were found to have dual effects on stomatal movement:they promoted stomatal opening under the watered condition,while promoted its closure under drought and therefore lowering water loss rate to enhance drought tolerance.To answer these questions,in this work,we constructed the TaFLS1 overexpression and RNAi lines of wheat,and carried out the following research:1.Flavonols dually modulated stomatal movement and enhanced drought resistance in wheatIn comparison with wildtype,TaFLS1 overexpression lines had higher flavonol contents in the leaves and guard cells of wheat,but its RNAi lines had lower contents.The water-withholding assay indicated that TaFLS1 overexpression enhanced drought tolerance,but its RNAi reduced the ability.TaFLSl overexpression lowered the water loss rate of the detached leaves of wheat,but its RNAi obviously improved the rate.Under the control condition,the proportion of closed stomata was reduced by TaFLS1 overexpression,but increased by its RNAi.After exposure to ABA,the proportion of closed stomata was improved in all lines,among which TaFLS1 overexpression lines had the highest proportion of closed stomata,while the RNAi lines the lowest.Exogenous application of quercetin mimicked the effect of in vivo flavonols,where the application of quercetin alone lowered the proportion of closed stomata,while the application of ABA and quercetin together induced more proportion of closed stomata than the application of ABA alone did.The results indicated that flavonols also performed dual modulation on stomatal movement in wheat,and when suffered from drought they promoted stomatal closure and therefore enhanced drought tolerance via reducing water loss rate.2.The modulation of flavonols on stomatal movement is relative to their regulation on H2O2 in guard cellsH2O2 played central roles in the regulation of stomatal movement.Under the control conditions,when compared to the wildtype,TaFLS1 overexpression lines had lower contents of H2O2 in guard cells,while the RNAi lines had higher contents.When exposed to ABA,the contents of H2O2 in guard cells was elevated in all lines,among which TaFLS1 overexpression lines accumulated the strongest extent of elevation so that their contents were the highest,while the RNAi lines had the least extent and produced lowest contents of H2O2 in guard cells.In comparison with the wildtype,the overexpression lines had lower NADPH oxidase(NOX)activities while the RNAi lines had higher activities in comparison under the normal conditions,while the difference was opposite under drought.In Arabidopsis,after treated with ROS scavengers or when NOX encoding genes were deficient,the application of ABA and flavonols together caused comparable stomatal aperture as the application of ABA alone,and TaFLS1 overexpresion also had no effect on stomatal aperture.The results showed that flavonols dually controlled H2O2 level in guard cells by altering NOX activity to achieve the dual modulating stomatal movement.3.The modulating of flavonols on stomatal movement depended on its controlling NO level in guard cellsNO is a key modulator in the machinery of stomatal movement.Under the normal conditions,both the application of exogenous quercetin and TaFLSl overexpression reduced the activities of nitrate reductase(NR)and the expression of NR encoding gene AtN1Al and TaNR in Arabidopsis and wheat,while under the treatment of ABA,they improved NR activities and the expression of AtNIAl and TaNR.Under the control conditions,in comparison with the wildtype,TaFLSl overexpression lines of Arabidopsis and wheat had lower NO contents in guard cells,but TaFLS1 RNAi wheat lines and Arabidopsis AtFLS1 mutant flsl-1 had higher;Under the treatments of ABA,JA or drought,the NO levels in guard cells were improved in all lines,among which TaFLSl overexpressors had the highest contents but the RNAi lines and flsl-1 had the lowest.On the other hand,NO donor SNP drastically induced the expression of TaFLSl in wheat and AtFLSl in Arabidopsis,and improved the contents of flavonols in guard cells of Arabidopsis and wheat.These data demonstrated that flavonols dually modulated NO levels in guard cells via dually controlling NR activities,and in turn NO can regulate the synthesis and content of flavonols,showing that there is a close relationship between NO and flavonols.We further analyzed the role of flavonols in ABA-induced stomatal closure.In comparison with the application of ABA alone,the application of ABA and flavonols together induced higher proportion of closed stomata,and smaller stomatal aperture.When NR activity was inhibited by applying the NR inhibitor,the proportion of closed stomata in wheat and the stomatal aperture in Arabidopsis after application of ABA and flavonols together were comparable to those after application of ABA alone.After exposure to ABA,in comparison with the wildtype,TaFLSl overexpression wheat lines had higher proportion of closed stomata,and its overexpression Arabidopsis lines had smaller stomatal aperture.After treated with the NR inhibitor to inhibit NR activity,TaFLS1 overexpression has no contribution to promote ABA-induced stomata1 closure in wheat and Arabidopsis.Similarly,in NR mutants nial and nia2,TaFLS1 overexpression did not promote ABA-induced stomatal closure.These findings indicated that flavonols dually modulated stomatal movement via controlling NO levels in guard cells,and meanwhile the promotion of flavonols on ABA-induced stomatal closure is dependent on NO.4.The relationship of NO and H2O2 in the modulation of flavonols on stomatal movementThe close cross-talk between H2O2 and NO is embedded in the machinery of stomatal movement.In this work,we found that in comparison with the wildtype,TaFLS1 overexpression reduced NOX activities under the control conditions,but improved them under the treatment of NO donor SNP.Different from that flavonols reduced H2O2 levels in guard cells under the control conditions SNP promoted ABA-induced H2O2 accumulation under ABA treatment in Arabidopsis wildtype.In NR mutants nia1 and nia2,the application of flavonols alone as well as the application of ABA and flavonols together both had no effect on H2O2 level in guard cells.Similarly,in nia1 and nia2,TaFLS1 overexpression did not reduced H2O2 level in guard cells under the control conditions,or promote the level after ABA treatment.The results indicate that the modulation of flavonols on H2O2 level in guard cells depends on NO.On the other hand,in comparison with the wildtype,TaFLS1 overexpression reduced NR activities under the control conditions,but improved them under the treatment of H2O2.Different from that flavonols reduced NO levels in guard cells under the control conditions but promoted ABA-induced NO accumulation under ABA treatment in Arabidopsis wildtype.In NOX mutants rbohd and rbohf,the application of flavonols alone as well as the application of ABA and flavonols together both had no effect on NO level in guard cells.Similarly,in rbohd and rbohf,TaFLS1 overexpression did not reduced NO level in guard cells under the control conditions,or promote the level after ABA treatment.The results indicate that the modulation of flavonols on NO level in guard cells depends on H2O2In line with the previous results,this work finds that the accumulation of flavonols in guard cells dually modulates stomatal movement via the dual control of NO and H2O2 levels in guard cells:flavonols promote stomatal opening under the normal conditions,which is beneficial to the exchange of gas and water and enhance efficiency of photosynthesis and transpiration;they promote stomatal closure under drought,which lowers water loss rate and enhance drought tolerance.These findings firstly bring new modulator(secondary metabolites)into the machinery of stomatal movement.Thus,this work provides evidence to deepen our insights into the mechanism of stomatal movement,and a candidate gene for wheat(crop)drought tolerance molecular breeding.