Mechanisms Of The Reguration Of Microtubule Maintenance And Nadph Oxidase Activity By Phosphalipase D And Phosphatidic Acid In Arabidopsis Thaliana

Drought and salinity are the major environmental threats to agricultural productivity, inducing severe damage such as devastation of cell membrane, DNA degradation, nutrition scarcity, and cell death in agricultural plants. Therefore, it is very important to gain insight on the inner responding system for drought and salinity, particularly the relation and crosstalk of them, and the regulation mechanisms, which are facilitated to increase the tolerance ability of agricultural plants to drought and salinity, and to improve the productivity and quality of agricultural plants.In Arabidopsis genomes, There are 12 PLD genes with different structures, distinguishable biochemical and regulatory properties, which involve in different physiological, biochemical and cell processes. We focus on Arabidopsis to study how to regulate the activity of MAP65-1, maintain the cell microtubule stability, and increase the plant salinity tolerance by Phospholipase D and PA. The homozygote of TUA6-GFP crossed with pldal was obtained, with PLD protein deletion assayed by western blotting. When treated with 50 mM NaCl, the microtubules of cytoledons and hypocotyls in pldal depolymerizaed heavily, inducing death of the epidermal cells. The results showed that PA derived from PLD involved in maintaining the stability of microtubules under 50 mM NaCl treatment. The microtubule-disrupting drug-oryzalin increased seedlings microtubules damage and cell death in pldal hypocotyl cells. The applied PA discharged the phenotypes above. Oryzalin promoted PLD activity in a short time in WT. Those results indicated that the depolymeraization of microtubules induced the death of cell and seedlings, and PLDal and PA played an important role in stablizing the cell micotubules under salt stress.There are 9 MAP65 genes in Arabidopsis genomes have. The recombinant proteins of MAP65-1, MAP65-5, MAP65-6 and MAP65-8 were expressed in E.Coli. PA binding to MAP65-1 protein was confirmed by immunoblotting and immunoprecipitation. The ability of PA binding to MAP65-1 increased during NaCl treatment. PA increased MAP65-1 binding to microtubules in vitro, resulting in more bundle formation and microtubule stabilization. PA increased MAP65-1 binding to microtubules in vitro, resulting in more bundle formation and microtubule stabilization. Overexpression of MAP65-1 enhanced salt tolerance in wild-type mesophyll protoplasts, but not in pldal. However, applied PA increased the salt tolerance in overexpressed MAP65-1 protein in pldal mesophyll protoplasts.PLDal protein did not bind to neither tubulin (αβ) nor MAP65-1, and PA did not bind to tubulin. PLD8 protein was microtubule-binding protein in vivo, but did not bind to MAP65-1. The change of microtubules of pldδwere the same as wide type under 25 mM NaCl treatment. However, more depolymerizaed microtubules were abserved in pldal protoplasts. The map65-1 mutant displayed salt-sensitive phenotype, which was similar to pldal mutant. The results above showed that the putative complex PA-MAP65-1-microtubules in plants played an important role in keeping the pattern and structure of microtubules, and enhanced the resistance ability to the later stage of salinity.Applied ABA and Ca2+ induced microtubules depolymerization in guard cell, and promoted stomatal closure. However, there was little effect on pldal by ABA and Ca2+. PPM and oryzalin induced the microtubules depolymerization in guard cells, but did not influence the status of open stamato. However, applied PA or disrupting-drug and ABA simultaneously deploymerized microtubules and induced stomatal closure. The Ca2+ content in guard cells increase by applying ABA or disrupting-drugs, but responded slowly in pldal. These results indicated that there was a crosstalk during the colsure of stamato, which including cell skeleton, Ca2+ and PLDal/PA in ABA signal transduction.We investigated the effect of PA on NADPH oxidase activity in guard cells. The PA derived from PLD induced by applied ABA binded to and activated NADPH oxidase (Rboh) activity in plasma membrane, which produced ROS to induce stomatal closure. The RbohD and RbohF were both expressed in Arabidopsis guard cells. Overexpressed RbohD protein in rbohD mutant increased the ROS production in guard cells, and PA promoted the further ROS generation in the overexpressed cells. However, when the four PA-binding Arg sites in N terminus of RbohD were mutated to Ala,PA did not induce ROS production. In the non-PA binding RbohD overexpressed seedlings, ABA did not induce ROS generation and stomatal closure in rbohD. The results indicated the putative PA-RbohD complex maybe one of important mechanisms to regulate ROS in ABA signal pathway in plant guard cells.