The Involvement Of Ethylene In 5-aminolevulinic Acid-induced Stomatal Opening In Leafs And Its Mechanisms

Stomatal movement and its mechanisms have long been a hot topic in the field of plant science.5-Aminolevulinic acid(ALA)is a new plant growth regulator.It can not only promote stomatal opening under darkness,but also inhibit stomatal closing induced by ABA or H2O2.Various signaling substances,including microtubules,PP2A protein phosphatase,flavonols,H2O2,NO,Ca2+,and cytoplasmic pH,have been shown to be involved in the regulation of stomatal movement by ALA.Ethylene is one of the most important hormones in plants,which is also involved in regulation of stomatal movement.However,to date,the relationship of ALA and ethylene in the regulation of stomatal movement is still not clear.Here,using ethylene synthesis inhibitors and receptor competitive inhibitors,we investigated the role of in ALA-ABA/dark-regulated stomatal movement,and revealed the relationship between ethylene and the other signal moleculars in the pathway of ALA-regulated stomatal movement.Our results further explored the mechanisms behind ALA-regulated stomatal movement,providing theoretical basis for its application in agricultural production.The main research results were as follows:1.Using the leaves of Fuji apple(Malus × domenstica CV.Fuji)as materials,we found that ALA treatment promoted the ethylene release in apple leaves.QRT-PCR analysis of leaf abaxial epidermis showed that ALA up-regulated the expression of ACS1 which encodes ACC synthase,a precursor of ethylene synthesis,ACC oxidase gene ACO1,ethylene receptor ETR1 and ethylene transcription factor EIN3.On the other hand,both ethylene synthesis inhibitor aminoethoxyvinylglycine(AVG)and ethylene receptor competitive inhibitor 1-methylcyclopropene(1-MCP)can induce stomatal closure,aggravate ABA/dark-induced stomatal closure,and weaken the inhibitory effect of ALA on ABA or dark-induced stomatal closure.In addition,AVG can promote the increase of H2O2 content in guard cells and inhibit the accumulation of flavonols.The above results suggest that ALA may induce the accumulation of flavonols in guard cells of apple leaves by promoting the biosynthesis and signal transduction of ethylene in guard cells,eliminate the H2O2 accumulation induced by ABA or dark,inhibit the stomatal closure induced by ABA or dark,and consequently open the gas exchange channel required by photosynthesis in apple leaves.2.In order to verify and further study the hypotheses obtained from apples,we studied the role of ethylene in by ALA-ABA/dark-regulated stomatal movement using the seedling cotyledon of YHeml(the ALA overproducing YHeml transgenic Arabidopsis),eto1-2(an negative regulator mutant of ACS),two ethylene insensitive mutants etr1-1 and ein2-5 in Arabidopsis thaliana.Under light conditions,ALA treatment inhibited ABA-induced stomatal closure in wild type seedlings,but it cannot effectively inhibit ABA-induced stomatal closure in ethylene insensitive mutants etr1-1 and ein2-5.The degree of ABA-induced stomatal closure in YHeml was significantly lower than that in wild-type Arabidopsis,while the stomatal aperture of YHeml plants treated with AVG and ABA was close to that of wild-type.The stomatal aperture of etr1-1 and ein2-5 treated with ALA in the dark was significantly less than that of the wild type,and the stomatal aperture of YHeml treated with AVG was less than that of the control group.All the above results indicate that ethylene is involved in the inhibit processor regulation of endogenous and exogenous ALA on ABA/Dark-induced stomatal closure.Endogenous and exogenous ALA inhibited ABA or dark-induced stomatal closure by promoting ethylene biosynthesis and signal transduction.3.In order to further explore the role of ethylene in the stomatal movement regulated by ALA,we used the cotyledons of YHeml transgenic Arabidopsis,ethylene overexpression mutant eto1-2,and flavonol-deficient mutant tt4,as materials to study the relationship between ALA,ethylene and flavonol in the inhibiting process of ABA/Dark-induced stomatal closure.The results showed that:(1)the fluorescence intensities of guard cell flavonol in YHeml and eto1-2 loaded with DPBA fluorescent probe were higher than that of the wild type,while the fluorescence intensity of the flavonol deletion mutant tt4 was significantly lower than that of the wild type.This result indicate that both ALA and ethylene overproduce could induce the accumulation of guard cell flavonols.(2)Ethylene inhibitor AVG treatment weakened the scavenging effect of ALA on H2O2 in guard cells and affected the inhibitory effect of ALA on ABA/Dark-induced ROS burst in guard cells.This result indicate that ethylene is involved in the regulation of ALA on the ROS levels in guard cells.(3)The effect of ALA-induced flavonol accumulation in guard cells of wild type Arabidopsis was inhibited by AVG regardless of light or dark conditions.This result indicate that ethylene is involved in the induction process of ALA-induced flavonol accumulation in guard cells.(4)ALA inhibited the accumulation of ROS in guard cells induced by ABA and dark,but this inhibitory effect was supressed in the flavonol-deficient mutant tt4.This result indicate that ALA inhibits ABA or dark-induced H2O2 accumulation by promoting the accumulation of flavonol in guard cells.The above results demonstrate that ALA promotes ethylene production and signal transduction,and then increases flavonol accumulation in guard cells,decreases ABA or dark-induced H2O2 accumulation,and finally inhibits stomatal closure.In conclusion,using apple and model plant Arabidopsis thaliana as materials,we proved that ALA could induce flavonol accumulation in guard cells by promoting ethylene biosynthesis and signal transduction,thereby inhibit the production of H2O2 in guard cells induced by ABA and dark,and finally inhibit the stomatal closure induced by ABA/dark.