The Role Of Flavonols In The Inhibition Of ABA-induced Stomatal Closure By Ethylene And The Regulatory Mechanism Of Its Synthesis

Stomata composed of a pair of guard cells controls exchange of carbon dioxide and water loss between plants and atmosphere,which is essential for water metabolism,photosynthesis and respiration in plants.The aperture of the stomata pores is regulated by a variety of internal and external factors,such as plant hormones,light,moisture,carbon dioxide and pathogenic bacteria.Flavonols,as important reactive oxygen species scavengers in plants,have been shown to involve in inhibition of ABA-induced stomatal closure by ethylene.However,the detailed role of flavonols and its synthetic regulation mechanism in the process are still unclear.Here,using Arabidopsis thaliana leaves as experimental materials,with pharmacological,genetic and biochemical methods,a systematic study has been done.The main conclusions obtained are as follows:1? Treatment with 10 ?M ABA significantly induced stomatal closure in wild-type(Col-0,Ler-0) leaves,and exogenous flavonol such as quercetin and kaempferol significantly inhibited the effects of ABA-induced stomatal closure.Compared with the wild type,the degree of ABA-induced stomatal closure was significantly enhanced in the tt4-4 mutant,which lacked flavonol synthesis,and the degree of ABA-induced stomatal closure was significantly reduced in the tt3-1 of overproducing-flavonol mutant,suggesting that flavonol inhibited ABA-induced stomatal closure.Treatment with 10 ?M ACC alone significantly inhibited ABA-induced stomatal closure in the wild type.The effect of ABA-induced stomatal closure was also significantly attenuated in the ethylene overproduction mutant eto1,suggesting that both exogenous and endogenous ethylene inhibited ABA-induced stomatal closure.The inhibiting effect of ABA-induced stomatal closure by ACC was weakened in the tt4-4 mutant and enhanced in the tt3-1 mutant,implying that the inhibition of ABA-induced stomatal closure by ethylene could be related to flavonol levels in guard cells.Measurement of endogenous flavonol contents showed that the combined treatment of ACC and ABA significantly improved the levels of flavonols in the guard cells of wild type and tt3-1,and had no significant effect on tt4-4.Correspondingly,the results of endogenous H2O2 contents showed that ABA treatment significantly promoted the level of hydrogen peroxide(H2O2) in guard cells of wild type,tt3-l and tt4-4.Among them,the degree of H2O2 increase in the guard cells of tt3-1 is weaker than that of wild type,and stronger in the tt4-4 mutant.The combined treatment with ACC and ABA significantly reduced the levels of H2O2 in the guard cells of wild type and tt3-1,and had no significant effect on tt4-4.These results showed that flavonols,as the scavengers of H2O2,positively regulated inhibition of ABA-induced stomatal closure by ethylene via reducing H2O2 contents.2?To further study the role of the ethylene signal pathway in the synthesis of flavonols,we used the ethylene receptor mutant etrl-3 and the EIN3 mutant ein3-1 of an important element of ethylene signal transduction.ABA significantly induced the stomatal closure of etr1-3,but ACC didn't inhibit its ABA-induced closure effect.Compared with the wild type,the combined treatment with ACC and ABA didn't induce the increase of flavonol levels in the guard cells of etrl-3,and its ABA-induced H2O2 increase was not inhibited by ACC.ABA also significantly induced the stomatal closure of ein3-1,ACC didn't inhibit its ABA-induced closure effect,the combined treatment with ACC and ABA didn't induce the increase of flavonol levels in the guard cells of ein3-1 and inhibited its ABA-induced H2O2 increase.The above results genetically proved that the ethylene receptor ETR1 and the EIN3 of important element of the ethylene signal pathway are necessary for ethylene to induce flavonol accumulation in guard cells to reduce H2O2 levels and inhibit ABA-induced stomatal closure.3?To study the mechanism of ethylene-induced flavonol synthesis in guard cells,we investigated the roles of transcription factors MYB11,MYB12,and MYB111 in flavonol synthesis in guard cells.The results showed that in myb12-If, myb111 and myb11myb12myb111 mutants,the degree of ABA-induced stomata closure was stronger than that of wild type,while the effect of ACC inhibition of ABA-induced stomatal closure was significantly weaker than that of wild type.Compared with wild type,the increase of endogenous flavonol levels in the guard cells after combined treatment with ACC and ABA was significantly reduced in the mutants of myb12-If,myb111,especially myb11myb12myb111,and the endogenous H2O2 levels showed opposite changes.myb11 exhibited a similar response to the wild type for stomata aperture,flavonol and H2O2 levels in guard cells.These results indicated that the transcription factors MYB12 and MYB111 are involved in the regulation of ethylene-induced flavonol synthesis in guard cells to inhibit ABA-induced stomatal closure,while MYB11 may have little effect in the process.4?Compared with the wild type,in the HY5 deficient mutant hy5 in light signal pathway,ABA-induced stomatal closure was greater,the effect of inhibition on ABA-induced stomatal closure by ACC significantly decreased,the endogenous flavonol accumulation of guard cells treated with compound treatment with ABA and ACC was significantly reduced,and endogenous H2O2 levels significantly increased.The hyh mutant had a similar effect to hy5,which was weaker.And the effect was the strongest in hy5hyh.In HY5-OX,the effect of ABA-induced stomatal closure was minimal,and the effects of ABA-induced stomatal closure was not inhibited by ACC,endogenous flavonol levels in guard cells were higher in each treatment,and H2O2 levels were lower.These results indicated that the transcription factors HY5 and HYH regulated the synthesis of flavonols in guard cells,and participate in the inhibition of ABA-induced stomatal closure by ethylene,of which HY5 plays a major role.5?To investigate the effects of miR858a on flavonol synthesis in guard cells,we measured the stomatal aperture,endogenous flavonol levels and H2O2 levels in the guard cells of over-expressing lines MIR858a-OX#5,MIR858a-OX#2 and the attenuated lines STTM858#1,STTM858#2.Compared with the wild type,in STTM858#1 and STTM858#2,the stomatal responses to ABA were stronger,and the inhibiting effects of ABA-induced stomatal closure by ethylene significantly were weaker,endogenous flavonol accumulation in the guard cells of treatment with ACC and ABA was weakened,and endogenous H2O2 levels increased.Compared with the wild type,in MIR858a-OX#5 and MIR858a-OX#2,the stomatal responses to ABA were weaker,the inhibiting effects of ABA-induced stomatal closure by ethylene were significantly stronger,endogenous flavonol accumulation in the guard cells of treatment with ACC and ABA was enhanced,and endogenous H2O2 levels decreased.These results indicated that miR858a positively regulated the flavonol synthesis to reduce H2O2 levels of guard cells in the inhibition of ABA-induced stomatal closure by ethylene.6?In order to investigate the relationship between HY5 and miR858a,we obtained HY5-OX × STTM858#2,hy5 × MIR858a-OX#2 hybrid lines.The results showed that the response of HY5-OX × STTM858#2 to ABA and ACC treatments was similarly to STTM858#2 in terms of stomatal aperture,flavonol and H2O2 levels in the guard cells,and the response of hy5 × MIR858a-OX#2 was similar to MIR858a-OX#2,suggesting that miR858a plays a role downstream of HY5 in the inhibition of ABA-induced stomatal closure by ethylene by promoting flavonol synthesis.In summary,flavonols are involved in the inhibition of ABA-induced stomatal closure by ethylene via reducing H2O2 levels.In this process,the transcription factors MYB12,MYB111,HY5 and miR858a,as positive regulators,are involved in the regulation of flavonol synthesis of the guard cells,and miR85 8a plays a role downstream of HY5.