A Study On The Function Pathways Of PLDα1Gene In Regulating Plant Drought Resistance

With aggravating water resource crisis, drought has become an important factor of restrictingagricultural and social development in China. Developing water-saving agriculture to improve water useefficiency （WUE） is the only way to achieve sustainable agricultural development. Enhancing WUE anddrought tolerance of crop cultivars by exploring gene recourses on the basis of mechanism studies onhigh WUE and drought tolerance of plants is the main goal of biological water saving. Phospholipase D（PLD）, found widely in diverse plants, is a class of important transmembrane signaling enzymes, andhave been reported to be involed in many biotic and abiotic stress responses.In this research, Agrobacterium tumefaciens-mediated method was adopted to respectivelytransform the overexpression vector and RNAi vector of PLD1gene into Poplar84K（P.alba×P.glandulos）. The result of PCR test and southern blot analysis of transgenic plants showedthat the target gene fragment had been integrated into the chromosome of Poplar84K, and obtain thesurvival super-expressed plants and RNAi plants with PLD1gene.The three genotypes of OE, RNAi, and wild type （WT） were treated with drought stress in thegreenhouse to evaluate the droght resistance phenotypically. The results showed that, compared with WT,drought resistance was increased in OE type transgenic plants, while decreased in RNAi type transgenicplants, indicating that PLD1gene positively regulates drought resistance of palnts.To clarify the function pathways of PLD1gene in regulating plant drought resitance, osmoticadjustment ability, stomatal regulation trait, the signal ion flux, and the membrane lipidomics weremeasured and analyzed after the plants were treated with water stress. The main results are as follows.（1） No significant differences in saturated osmotic potential and osmotic adjustment ability wereshown among the three genotypes after water stress treatment, indicating that PLD1gene is notinvolved in the osmoregulation ways to improve plant drought resistance.（2） Under water stress, compared with WT plants, OE type transgenic plants shew increase in thestomatal conductance, while RNAi type transgenic plants shew decrease in stomatal conductance.Stomatal conductance reduction may lead to a decrease in transpiration rate under water stress, whichcontrol effectively water loss through transpiration. This suggests that PLD1gene regulate droghtresistance of plants through the pathway of stomatal regulation.（3） Ca²⁺, H⁺, and K⁺fluxes in roots of the three genotype after water stress treatment weresimultaneneously measured by using the Non-invasive Micro-test Technique. The results show that theefflux of Ca²⁺and influxes of H⁺in OE type transgenic plants were promoted, in contrast, that in RNAitype transgenic plants were inhibited. This further supports that PLD1gene is involved in droughtsignal transduction pathways. Through this study, one signal transduction pathway in plant may bespeculated as: extracellular water stress signal-transmembrane signal transduction enzymePLD1-Ca²⁺-stomatal regulation-Regulation drought resistance. Based on this, inhibition of PLD1expression blocked the signal transduction pathway to some extent, then the stomatal regulation was inhibited, which resulted in large amount of water loss through transpiration and decrease in droughtresistance of plants.（4） Lipidomic analysis of the cell membrane in two transfer genotypes under water stressconditions found that PLD1enzyme selectively hydrolyze PC in vivo, leading to a lower level of PCand a higher level of PA in OE type transgenic plant than that in RNAi type transgenic plants. Suchvariation in lipid composition can not stabilize cell membrane under stress. However, results of plantgrowth, net photosynthetic rate, stomatal conductance, transpiration rate indicated that overexpression ofPLD1enhanced the drought resistance of plants. PLD enzymes play both lipid degradation role andsignal transduction role in living organism. This result imlies that, under water stress conditions, thesignal transduction role PLD1enzymes plays contributed dominantly.