Flavonols Accumulation Is Involved In Inhibition Of ABA-induced Stomatal Closure By 5-Aminolevulinic Acid

Stomata serve as the gateway for gas exchange.Aperture of stomata,which is closed related to plant photosynthesis,plays an important role in plant biomass and yield.Thus,increasing stomatal aperture,and hence improveing the absorption and fixation of CO2,might be efficient approches to enhance plant capacity.Many researches have proved that5-aminolevulinic acid(5-ALA),an essential precursor of porphyrin synthesis,is a potential plant growth regulator,which can improve plant growth and enhance plant resistance to various abiotic and biotic stresses.Our previous studies demonstrate that 5-ALA promotes stomata opening under dark conditions and inhibits ABA-and H2O2-induced stomata closure by downregulation reaction oxygen species(ROS)in guard cells.However,the physiological mechanism behind 5-ALA regulating ROS in guard cells remains unclear.Flavonoids are important secondary antioxidant metabolites,which involved in scavenging ROS in cells.Since,5-ALA could up-regulate the phenylpropanoid metabolism and improve secondary metabolites accumulation,such as fruit flavonoids and anthocyanins.Using the DPBA,flavonols fluorescent dye,we found that flavonols accumulated in guard cells,and flavonols inhibit ABA-induced stomatal closure via decreasing ROS content in guard cells.In this tiny model,hence,it is of important theoretical significance to explore the effects of 5-ALA on flavonols metabolism,and the relationship between 5-ALA-induced stomatal movement and flavonols accumulation.The main results are as follows:1.5-ALA pretreatment inhibited ABA-induced stomatal closure via decreasing ROS accumulation in apple guard cells.Using flavonol specific fluorescent dye DPBA,we found that DPBA fluorescence specifically appeared in guard cells,while there were no fluorescence signals in pavement cells.We observed that DPBA fluorescence in guard cells increased when isolated epidermis peels were pretreated with 5-ALA for 2 h or 4 h.Meanwhile,pretreatment of quercetin or kaempferol,main components of flavonols,also inhibited stomatal closure induced by ABA,and decreased ROS content in the guard cells of apple leaves.Furthermore,exogenous flavonols could eliminate the effect of H2O2 that induced stomatal closure in apple leaves.Therefore,it can be deduced that 5-ALA-induced stomatal opening may be associated with its up-regulation of flavonol levels in guard cells,which then eliminates ROS levels.Thus,flavonols may be involved in stomatal regulation induced by 5-ALA in apple leaves.To verify this hypothesis,we use the model plant Arabidopsis thaliana,which possess diverse genetic resources,as experimental material for the further research.2.To better understand underlyging mechanisms stomatal regulation by 5-ALA,we studied with WT,YHeml(over-producing 5-ALA transgenic plants)and tt4(CHALCONE SYNTHASE mutation)with cotyledons of Arabidopsis thaliana(Columbia).Pretreatment with 5-ALA and YHeml could inhibit ABA induced stomatal closure,while stomata of tt4 were more sensitive to ABA.Using DCF as a monitor to evaluate the content of ROS,we found that pretreated with 5-ALA and YHeml could decrease ABA-incuced ROS markedly,but this effect was impaired in tt4 mutant.All of these results indicate that flavonols anabolism pathway is involved in 5-ALA-induced stomatal regulation.3.In order to investigate whether and how 5-ALA promote flavonols,accumulation in guard cells,responses of flavonols content in guard cells were studied using WT,YHeml and tt4 with cotyledons of Arabidopsis thaliana.We found that flavonols accumulated specifically in the guard cells,which was similar to what we found in apple leaves.We also found that pretreatment with 5-ALA for 2 h or 4 h could enhance DPBA fluorescent intensity in the cotyledons of WT.At the same time,the DPBA fluorescent intensity of YHeml was 4-5 fold of WT,and there were no DPBA fluorescent signals in tt4.Furthermore,results of qRT-PCR showed that 5-ALA up-regulated expression of genes in the pathway of flavonols synthesis.These results imply that 5-ALA could up-regulate gene expression of flavonols anabolism and improve flavonols accumulation in guard cells.4.Using the quercetin and kaempferol,main components of flavonols,as the exogenous flavonols to test the capability of flavonols regulating redox equilibrium.Exogenous flavonols impaired stomatal closure induced by ABA,decreased the ROS content,and recovered the ability of tt4 to regulate stomatal aperture.Furthermore,pretreated with exogenous flavonols to WT and tt4 also could enlarge stomatal aperture by decreasing H2O2.Together,these results suggest that either the endogenous flavonols induced by 5-ALA or exogenous flaovnols could regulate stomatal movement by decreasing ROS accumulation.To summary,using apple leaves and the model plant Arabidopsis thaliana,we proved that flavonols accumulation induced by 5-ALA is involved in reducing ABA-induced ROS,and then inhibiting ABA-induced stomatal closure.