Study On The Effects Of H₂S,PLD?1 And ABA In Arabidopsis Under Drought Stress

Drought stress has become one of the major factors that limit crop production in the world.Plant responds to drought stress mainly by changing a series of physiological,biochemical and molecular mechanisms,for example,reducing the growth rate,closing stomata,and increasing the antioxidant system to adaption.Understanding the characteristics of plant responding to drought resistance at physiological and molecular levels,revealing its drought-resistance mechanism will provide a theoretical basis for improving the plant growth in dryland and breeding drought-tolerant varieties.As a gas small molecule,H₂S participates in the regulation of plant growth and development,and responses to abiotic stress.H₂S can promote root formation and seed germination,and enhance the antioxidative resistance in plant.Phospholipase is a kind of hydrolytic enzyme for the degradation of phospholipids.Phospholipase D?PLD?is one of important enzyme belonging to phospholipase's family,which catalyzes the hydrolysis of phosphodiester bonds.PLD and its product phosphatidic acid?PA?are important second messengers involved in maintaining cell membrane stability and regulating various cell signaling pathways.In Arabidopsis,PLD genes are divided into six categories,named as PLD?,PLD?,PLD?,PLD?,PLD?and PLD?,which are involved in different cellular processes coordinately or independently.The phytohormone ABA can be induced by low temperature and drought stress.ABA regulates the process of plant growth as signal molecule and plays an important role in the abiotic stress response.The gas signal molecules H₂S,phytohormone ABA and PLD can regulate the physiological processes such as seed germination,root growth,stomatal movement and stress response in plants.Environmental stress triggered the production of H₂S,ABA and PLD.H₂S and ABA interacted to regulate the stomatal movement under drought stress.H₂S depended on the PLD?1 activity to produce H₂O₂.ABA affected the lipid metabolism.PLD?1 and PA regulated NADPH and ROS during the stomatal closure caused by ABA.Although studies showed there may be connected among H₂S,PLD and ABA,the definite relationship of these three singnal molecules responding to stress is not yet reported.Therefore,using the seedlings of wild-type Arabidopsis thaliana WT,PLD?1deficient mutant plda1,L-cysteine desulfyrase?L-DEs?deficient mutant lcd and the ABA1 deficient mutant aba1 as materials,and 0.3M mannitol treatment as drought stress,the signal relationship and the effects of H₂S,PLD?1 and ABA in response to drought stress were investigated.The results are as follows:1.The H₂S content,PLD and L/D-DEs activities and relative gene expression in wild-type Arabidopsis changed significantly under drought stress.The activity of L-DEs and LCD relative gene expression were significantly higher than D-DEs.It indicated that L-DEs was the main pathway for H₂S production under drought stress.In the germination rate experiment,the pld?1 and lcd mutants are more sensitive to drought stress than WT.For application NaHS,the seed germination and endogenous H₂S production of WT,plda1 and lcd were induced under stress,while the application of PA,the germination rate and H₂S content were increased in WT and plda1 under drought stress,but had no significant effect on the germination rate and H₂S content in lcd.The results of RT-qPCR analysis were similar to it.The results showed that the signaling molecules H₂S and PLD?1 played a vital role under the drought stress response in Arabidopsis and H₂S may be located at the downstream of PLD?1 to participate in the regulation of Arabidopsis seed germination.2.ABA response to drought stress,the ABA content depended partly on the ABA1relative expression,indicating that other genes regulate the ABA content.the application of NaHS promoted the H₂S content in WT,lcd and aba1 under drought stress.The relative expression of LCD and ABA1 genes were up-regulated under drought stress,for the application of ABA,the ABA and H₂S content,the relative expression of LCD and ABA1 were all were up-regulated in WT and aba1 under drought stress,but had no significant effect on H₂S content and LCD relative expression in lcd,indicating that ABA was connected with the expression of LCD.The relative expression of PLD?1 and LCD genes were down-regulated in aba1compared with WT;exogenous ABA activated the PLD activity in WT and aba1under drought stress;the exogenous PA significantly induced the relative expression of PLD?1 in aba1 under drought stress,showed that the signal molecule H₂S,PLD?1,and ABA played a vital role response to the drought stress in Arabidopsis,and H₂S is located at downstream of ABA to participated in the regulation of signaling process.When NaHS was applied,the stomata aperture were increased in WT,pld?1,lcd and aba1 under drought stress,indicating that NaHS promoted stomata closure under drought stress.It indicated that NaHS reduced transpiration to alleviate the water loss caused by drought stress.It also indicated that H₂S is located at the downstream of PLD?1,ABA under drought stress.3.The degree of membrane damage?EL,MDA?,ROS levels,antioxidant enzyme activities?SOD,POD,CAT?were all increased in WT,pld?1 and lcd under drought stress,in addition,the activities and the gene relative expressions of antioxidant enzymes?SOD,POD,CAT?in pld?1 and lcd were higher than that in WT under drought stress,showed that the pld?1 and lcd were more sensitive to drought compared with WT.Exogenous NaHS reduced ROS levels,EL and MDA content in WT,pld?1 and lcd;Exogenous NaHS significantly increased the SOD,POD activities and the SOD,POD relative expression in WT,pld?1 and lcd under drought stress.It is indicated that H₂S protects Arabidopsis from oxidative damage by maintaining membrane integrity under drought stress.