The Mechanism Investigation Of Arabidopsis Mitochondrial Pyruvate Carrier 1 In Plant Drought Response

Drought is one of the most serious and common abiotic stresses to limit plant development and crop yield.Plants have evolved diverse ways to adapt to or tolerate the drought stress.Of these different ways,plant stomata play an essential role,since most of the water absorbed by the plant roots from the soil is lost through stomatal transpiration.The plant stomata are composed of a pair of guard cells which surround each stoma.The control of stomatal aperture is achieved by regulating tugor in the pair of guard cells.Therefore,searching the regulative mechanism of stomatal movement and finding the functional genes which take part in drought stress response,are significant to improve the drought resistance and to increase the crop production.Many factors can affect stomatal movement,such as light,CO2,humidity and so on,among factors that affect stomatal movement,and the phytohormone abscisic acid(ABA)is quite important regulator.The ABA-triggered activation of the guard cell anion channels results in the efflux of anions,which in turn reduces the turgor of the guard cells to close stomata.In addition,the accumulation of endogenous ABA in the guard cell can promote the generation of reactive oxygen species(ROS).ROS had been proposed to function as important second messengers in ABA signaling,particularly in ABA-induced activation of ion channels and stomatal closure.Moreover,the production of ROS in response to ABA is the result of activation of plasma membrane NADPH oxidases,and RbohD and RbohF are the major catalytic subunits in this response.Pyruvate carriers are subcellularly located on inner membrane of mitochondria,and transport pyruvate from cytosol into mitochondria for subsequent TCA cycle,and play essential roles in cellular metabolism and signaling.Pyruvate,a product of glycolysis,has a central role in cellular metabolism.The transport of Pyruvate into the mitochondria,facilitated by proteins termed Mitochondrial Pyruvate Carriers(MPCs),is necessary for the functioning of the tricarboxylic acid cycle.There are more and more researches on the function of MPCs,which take part in the regulation of cell metabolism and the response to a lot of stress.In our study,we performed a set of experiments to investigate the function and molecular mechanism of Arabidopsis thaliana Mitochondrial Pyruvate Carrier AtMPC1 by using molecular biology,genetics,electrophysiology,physiology techniques.The A.thaliana gene MPC1 has been isolated and shown to display MPC activity when heterogeneously expressed in yeast.AtMPC1 is deposited in the mitochondria when expressed with GFP as fusion protein in A.thaliana protoplasts and where the mitochondrial pyruvate carriers are supposed to be subcellularly located Besides,we also observed high abundance of AtMPCl in the stomatal guard cells by using its promoter droved GUS staining.The stomata of mpcl mutants were more sensitive to ABA treatment than those of wild type plants in terms of the ABA induced stomatal closure,and this phenotype was consistent with the observation that ABA activated more amplitude slow-type anion currents efflux of mpcl mutant guard cells.In addition,the mpcl mutant plants lost water more slowly and thus to survive more to drought stress.Consistent with these consequences of the absence of AtMPC1 protein,the effect of over-expressing the gene had the opposite phenotypic effect and the C lines behaved similarly to Col-0.So,we can conclude that AtMPCl is a negative regulator of ABA-mediated stomatal closure and the drought response.In addition,the measurements of the tissue content of Pyruvate showed that,the Pyruvate was increased by ABA or drought treatment in both Col-0 and the mpcl mutants,and the Pyruvate content in mpcl mutants was significant higher that in wild type plants.When Col-0 guard cells were exposed to Pyruvate,the effect was to reduce the stomatal aperture alone with Pyruvate concentration-dependent,while treatment with Pyruvate was able to activate the slow-type anion channels.Besides,this process that the increased Pyruvate content induced stomatal closure and activated the slow-type anion channels of guard cells was dependent on the function of RbohD/RbohF NADPH oxidases and reactive oxygen species concentrations in guard cells.It is consistent with the observation that Pyruvate induces ROS production in the guard cell.Therefore,Pyruvate may regulate stomatal closure by inducing an increase in ROS production.The results revealed the functional mechanism of AtMPC1 in the regulation of stomatal movement and the drought response.