| The arid and semi-arid regions in China cover a very large area, drought stress is one of the major abiotic stress factors to limit plant growth and crop yield, a variety of response mechanism of drought stress of Plant has formed in the process of long-term evolution, the plant regulate the stomatal guard cells physiological and biochemical characteristics to change the stomatal conductance and the degree of opening, thereby regulating plant transpiration reduce the water loss is an important mechanism of plant response to drought stress. Plasma membrane H+-ATPase is the most abundant protein in cell membrane, and it participates the regulation in a variety of biological processes, and it also responds to a variety of environmental stresses, subjected to drought stress, the plant stomatal opening and closing is mainly affected by the regulation of the plasma membrane H+-ATPase, plasma membrane H+-ATPase activity is an important mechanism of plant adaptation to drought stress.14-3-3proteins are widely present in eukaryotes and the high homology, and increase its activity by phosphorylation of the plasma membrane H+-ATPase of the C-terminal binding, the phosphorylation of the plasma membrane H+-ATPase regulated by the many environmental factors. Broad bean is C3plants and more sensitive to drought stress, it’s stomatal guard cells easily observed and leaf epidermis easily prepared and a commonly used to study plant response to drought stress mechanism. In this study two broad bean (a Vicia faba cultivated in the yellow soil referred YV, another cultivation on the red soil referred RV), explored broad bean14-3-3proteins and plasma membrane H+-ATPase response PEG simulated drought stress mechanism. In this paper, we found the following results:1.Using a red soil Vicia faba (RV) cultivar and a yellow soil Vicia faba (YV) cultivar as the materials, changes in physiological characteristics and function of plasma membrane (PM) H+-ATPase were analyzed in the two Vicia faba cultivars treated with polyethylene glycol (PEG) simulation drought stress under hydroponic conditions. The results indicated under the treatment conditions of2%,5%and10%PEG with an increase in PEG concentration and treatment time, an elevation in the water loss rate, a decrease in leaf transpiration rate and stomatal conductance were observed in the two broad bean. Under all the same treatment conditions, the water loss rate in YV was less than that of RV, whereas, the reduce in YV leaf transpiration rate and stomatal conductance was larger than those of RV, indicating that drought tolerant of YV was stronger than that of RV. Under the same treatment conditions, the leaf soluble protein and soluble sugar and free proline content in YV was higher than those in RV. In the antioxidant enzyme system, the increase in SOD and POD activity in YV leaves was significantly faster than that of RV, leading to a higher H2O2content in YV leaves than that in RV leaves. Four different subtypes of14-3-3genes and two different plasma membrane H+-ATPase enzyme (VHA) changeof the transcriptional level by RT-PCR analysis, the results suggest that two broad bean leaves14-3-3genes transcription levels is similar with two kinds of the VHA genetic,5%PEG stress on the the YV and RV leaves14-3-3genes and VHA gene transcription has induced,and in RV the effects is stronger than in YV. As a YV result, under drought stress conditions, the inhibition in the PM H+-ATPase phosphorylation evel and its interaction with14-3-3protein in YV leaves was stronger than those in RV leaves. The PM H^-ATPase and proton pump activity in YV leaves was lower than those in RV leaves. Consequently, the stomata aperture in YV leaves was less than that in RV leaves. Leaf transpiration rate and stomatal conductivity in YV was lower than those in RV leaves. This might be an important mechanism underlying the stronger drought tolerance in YV that that in RV.2.Proline (Pro), gibberellin (GA) and betaine as osmotic regulation substances, can keep cells water. InThis study, Applied to Pro, GA and betaine in the5%PEG to investigat that simulation of the two broad beans, Pro, GA and betaine mitigation drought stress effect and leaves the plasma membrane H+-ATPase activity correlation. This study respectively under5%PEG simulated drought stress with Pro, GA and betaine processing two kinds of beans, Pro, GA and betaine alleviates the two beans drought stress and blade plasma membrane H+-ATPase activity of relevance. Applied to Pro, GA and betaine can decrease the plant water loss-rate, leaves transpiration rate and stoma conductance higher than PEG treatment, confirmed with Pro, GA and betaine has alleviate drought stress PEG two broad bean. Immune co-precipitation (COIP) analysis results show that under the stress of PEG after add Pro, GA and betaine, the level of phosphorylation of the plasma membrance H’-ATPase increased and its combination with the14-3-3protein phosphorylation level ability increased, plasma membrane H+-ATPase activity decreased when add Pro, GA, after adding betaine was increased, the H+-pump activity compared with single PEG treatment no difference, but increased significantly, stomata opening shows that plasma membrane H+-ATPase and the14-3-3protein interaction is not involved in Pro, GA and betaine alleviates the PEG simulated drought stress of two kinds of beans, because of leaf transpiration decrease amplitude is small, Pro, GA, and betaine alleviates the PEG stress may mainly by increasing the horsebean water absorption ability.3.AMP and VA are plasma membrane H+-ATPase inhibitor, AMP can inhibit plasma membrane H+-ATPase phosphorylation, and VA can compete plasma membrance H+-ATPase phosphorylation site, prevent the protein with phosphorylation plasma membrane H+-ATPase/14-3-3protein, reduce plasma membrane H+-ATPase activity; IAA and MgCl2are the activator of plasma membrane H+-ATPase, can promote the14-3-3protein with phosphorylation of plasma membrane H+-ATPase combination, improve the plasma membrane H+-ATPase activity. In order to further investigation plasma membrane H+-ATPase and14-3-3protein of two broad bean leaves in the regulation of stomatal physiological characteristics under Polyethylene PEG drought stress. Under IAA and MgCl2treatment, YV water loss-rate increased,leaves transpiration rate and stoma conductivity also increased,but H2O2content decreased in leaves,COIP analysis results show that the level of phosphorylation of the plasma membrance H+-ATPase increased, activity increased and leaves stoma opened. IAA and MgCl2by lowering the H2O2content, increase the plasma membrane H+-ATPase and14-3-3proteins interact to increase and leaf transpiration, stomatal opening is increasing the effect of drought stress. By drought stress, applied AMP and VA, the water loss-rate of RV is lower than PEG treatment alone, at the same time, transpiration rate and stoma conductivity decreased, but H2O2content increased in leaves. COIP analysis results show that the level of phosphorylation of the plasma membrance H+-ATPase reduced, activity decreased and leaves stoma closed. MP and VA can restrain by adding H2O2content to restrain plasma membrane H+-ATPase and the14-3-3protein interactions in order to reduce the RV and leaf transpiration, stomatal opening to alleviate the drought stress. These results indicate that plasma membrane H+-ATPase and14-3-3proteins have an important role in the regulation of stomatal physiological characteristics of two Vicia faba under drought stress. Under drought stress exogenous application of AMP and VA can increase the drought tolerance of broad beans.4. H2O2can the phosphorylation of the plasma membrance H+-ATPase, inhibition the activity of plasma membrance H+-ATPase, and regulating stoma. Ascorbic acid (ASA) is a kind of commonly used H2O2scavenger, H2O2generated depends on abscisic acid (ABA), sodium tungstate is ABA synthesis inhibitor,sodium tungstate can inhibit the production of H2O2. Under drought stress, H2O2content of YV is higher than that of the RV,to understand the H2O2whether to regulate the plasma membrane H+-ATPase activity of RV and YV,adding different concentration of ASA and sodium tungstate treatment YV in5%PEG,the results show that YV water loss-rate increased,leaves transpiration rate and stoma conductivity also increased,but H2O2content decreased in leaves,COIP analysis results show that the level of phosphorylation of the plasma membrance H+-ATPase increased, activity increased and leaves stoma opened. ASA and sodium tungstate increase the plasma membrane H+-ATPase and14-3-3proteins interact to increase and leaf transpiration, stomatal opening is increasing the effect of drought stress. By drought stress, adding different concentrations of H2O2treatment the RV, the water loss-rate of RV is lower than PEG treatment alone, at the same time, transpiration rate and stoma conductivity decreased, but H2O2content increased in leaves. COIP analysis results, show that the level of phosphorylation of the plasma membrance H+-ATPase reduced, activity decreased and leaves stoma closed. H2O2restrain plasma membrane H+-ATPase and the14-3-3protein interactions in order to reduce the RV and leaf transpiration, stomatal opening to alleviate the drought stress. These results confirmed that H2O2involved in the regulation of plasma membrane H+-ATPase activity of RV and YV.Under drought stress exogenous application of H2O2can increase the drought tolerance of broad beans. |
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