Study On The Regulation Of Stomatal Opening By Peroxisome Through Acetic Acids

Stomata is a functional micropore and enclosed by a pair of guard cells.The guard cells can continuously perceive the environmental changes of the leaves and then controlling the opening and closing of the pores by their own relaxation or contraction.Stomatal movement regulates plant transpiration and photosynthesis,balances water loss and carbon dioxide fixation.Therefore,stomatal movement is closely related to crop growth,development,resistance,yield and the quality of agricultural products.The decisive factor in regulating stomatal movement is the osmotic potential of guard cells,and organic acids also play an important role in it.With the discovery of the function of malic acid in the regulation of stomatal movement,other evidences on the regulation of stomatal movement by organic acids are gradually revealed.Plants produce acetic acid under drought stress,while the peroxidase-targeting gene At BZU1 can use acetic acid to produce acetyl-coa,and finally produce malic acid(malate2-),balance intracellular K+,regulate cell turgor,and thus regulate stomatal movement.Based on previous studies,this experiment explored the role of organic acids in regulating stomatal movement.It was found that malic acid,oxaloacetic acid and glyoxylic acid could promote stomatal opening under normal conditions,but pyruvic acid,fumaric acid,sodium isocitrate,citric acid,succinic acid and ?-oxolutarate,including acetic acid,could not promote stomatal opening.Based on the uniqueness of glyoxylate metabolism,we hypothesized that peroxisomes may be involved in the regulation of stomatal movement.After the mitochondrial activity was inhibited by the mitochondria-specific inhibitor rotenone,we explored the function of the peroxisome.The results showed that acetic acid promoted the opening of stomata under the conditions of rotenone treatment.We speculate that the peroxisome uses acetic acid to produce malic acid.The experimental results of the mutant bzu1 proved our speculation.The use of the abcb14 mutant further demonstrated that malic acid,which promotes stomatal opening under the conditions in which the inhibitor and acetic acid are used together,is synthesized by the guard cells themselves.Analysis of the expression levels of the enzyme genes involved in the krebs cycle and the glyoxylate cycle in the treated leaves revealed that the mitochondrial activity was decreased and the peroxisome activity was enhanced in the treated leaves.These results suggest that the increase of stomatal opening is due to the formation of malic acid from acetic acid by peroxisome under the condition of co-treatment of inhibitor and acetic acid.Further experiments showed that succinic acid inhibited stomatal opening when the concentration of malic acid in guard cells was too high,and succinic acid and malic acid could coordinately regulate stomatal opening.It has been reported that pyruvate,which is converted from acetic acid,negatively regulates ABA-induced stomatal closure.Therefore,we further explored the role of organic acids in regulating stomatal opening under ABA treatment.The results showed that ABA could induce stomatal closure,while acetic acid and pyruvate could inhibit stomatal closure induced by ABA,but not in bzu1.The expression level of enzymes involved in the tricarboxylic acid cycle and glyoxylic acid cycle in treated leaves was analyzed.It was found that peroxisome's activity was increased.Metabonomics analysis showed that the acetic acid,malic acid and glyoxylic acid had obvious accumulation.According to the rising trend of malic acid content,the increase of their concentration was caused by acetic acid.These results all indicate an increase in the activity of peroxisomes,particularly the activity of the metabolic pathway of malic acid from acetic acid.Thus,we have found a new acetate-malic acid shunt mediated by At BZU1/At ACN1(acetate non-utilizing 1,ACN1)gene in peroxisome,which can regulate the swelling of guard cells to improve drought resistance of Arabidopsis thaliana plants.This not only found a metabolic pathway that can regulate stomatal movement in peroxisome,but also provided strong evidence for the traditional stomatal movement regulation mechanism "malic acid formation theory",and has important application value in improving the resistance of Arabidopsis thaliana to drought stress.