| Drought is one of the major abiotic stress factors to limit plant growth and crop yield, and a variety of mechanism of response to drought stress has formed during the plants long-term evolutionary process. According to regulating the physiological and biochemical characteristics of the stoma guard cells to change the stoma conductance and the opening, and then regulate the transpiration of plants to reduce moisture loss is an important mechanism of plant response to drought stress. Plasma membrane H+-ATPase in higher plant enzymes have dominated role in life activity, regulate many important physiological processes. Plasma membrane H+-ATPase is the key enzyme of regulation of stoma opening, the H+pump action can cause stoma opening, therefore plasma membrane H+-ATPase activity-change is one of the important mechanisms of plant in response to water stress under water stress.14-3-3proteins are a class of highly conserved regulatory protein found in eukaryotic cells, and according to bind the the C-terminal of the phosphorylation of the plasma membrane H+-ATPase increase its activity, and whereas the level of phosphorylation of the plasma membrane H+-ATP enzyme is regulated by the many environmental factors. Soybean is typical C3plants, drought stress is one of the Main reasons for the restricted soybean production, this paper treated with PEG simulated drought stress in two soybean varieties (a kind of soybean (YS) with yellow skin, and another soybeans with black skin (BS)), to study the molecular mechanism of14-3-3protein and plasma membrane H+-ATPase of leaves in response PEG simulated drought stress, and we find the follow result mainly:1. Analysis of the physiological characteristics and the expression of14-3-3protein and plasma membrane H+-ATPase enzyme and interaction and activity correlation of the leaves of YS and BS under hydroponic culture with polyethylene glycol (PEG) simulated drought stress. The result found that with the increase of the concentration and time, the plant water loss rate increased and the leaf transpiration rate and stoma conductivity decreased of two kinds of soybean plants. In all the same processing conditions, YS plant water loss rate is smaller than the BS, and leaf transpiration rate and stoma conductivity decreased more than BS, this result shown that YS drought resistance is better than BS. The soluble protein, soluble sugar content of YS are higher than BS under the same processing, and BS leaf free proline content is higher than that of YS; In antioxidant enzyme system, YS leaf CAT activity decline faster than BS, this result lead to the H2O2content is higher than BS. Analysis of16different subtypes SGF14gene and plasma membrane H+-ATPase (GHA2) transcription level changes of two kinds of soybean by RT-PCT, the result show that the transcription of YS and BS leaves GHA2have induced by PEG stress; the transcription level of background of most SGF14subtype genes of YS leaves is higher than the BS; only5kinds of14-3-3gene(SGF14e、SGF14d、SGF14i、SGF14n、 SGF14p) of YS leaves have a significant role in the induction of transcription under PEG treatment, and have a significant role in the induction of transcription on the BS leaves SGF14. The inhibition of the phosphorylation level of plasma membrane H+-ATPase and the interaction with14-3-3protein are higher the BS, YS leaf plasma membrane H+-ATPase and H+-pump activity also is lower than BS leaf, YS leaf stoma opening is smaller than the BS, leaf transpiration rate and stomatal conductivity is lower than BS, states that this is an important mechanism of drought resistance of YS are better than BS.2. A lot of research results show that the proline (Pro), gibberellin (GA) and betaine can increase the water absorption capacity and the drought resistance of plants. This study explore the relation of leaf plasma membrane H+-ATPase activity of two kinds of soybean and the effect of Pro and GA and betaine to reduce the action in response to drought stress under PEG treatment with Pro and GA and betaine, respectively. And compare with PEG treatment, the result show that the plant water loss-rate of YS and BS are reduced under PEG treatment with Pro and GA and betaine, respectively, confirm to add Pro and GA and betaine has relieved two kinds of soybean drought stress under PEG treatment. However transpiration rate and stomatal conductivity of two kinds of soybean are increased under PEG treatment with Pro, GA and betaine, demonstrate that helps relieve the stress of PEG is not by reducing leaf transpiration but by increasing the water absorbing capacity of soybean. Immune co-precipitation (COIP) analysis results show that the phosphorylation level of plasma membrane H+-ATPase and its combination with the14-3-3protein do not change in two kinds of soybean leaf under the stress of PEG with Pro, GA and betaine, and plasma membrane H+-ATPase and H+-pump activity have no difference compared with single PEG treatment, stomata opening increase with a significant, and this results show 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 soybean.3. AMP and VA are plasma membrane H+-ATPase inhibitor, AMP can inhibit phosphorylation of plasma membrane H+-ATPase, and VA can compete the phosphorylation site of plasma membrane H+-ATPase, 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 examine plasma membrane H+-ATPase and14-3-3protein in the regulation of stoma physiological characteristics of two soybean leaves under PEG osmotic stress, BS is treated by5%PEG with AMP and VA, the result show that compared with single PEG treatment the existence of AMP and VA to make BS plant water loss rate reduced12%and14%, respectively, at the same time, leaf transpiration rate and stoma conductivity have dropped significantly. The result of COIP show that the plasma membrane H+-ATPase phosphorylation and levels of its interaction with the14-3-3protein of BS are reduced, enzyme activity and H+pump activity decline, and stoma opening is decreased, and this result illustrate AMP and VA inhibit plasma membrane H+-ATPase and interaction of PM H+-ATPase with the14-3-3proteins to reduce transpiration rate, stoma opening of leaves under the drought stress of PEG. And YS is treated by5%PEG with IAA and MgCl2, the result show that compared with single PEG treatment the existence of IAA and MgCl2to make YS plant water loss rate reduced23%and17.2%, respectively, leaf transpiration rate and stoma conductivity also increase. The result of COIP show that the plasma membrane H+-ATPase phosphorylation and levels of its interaction with the14-3-3protein of YS increased, enzyme activity and H+pump activity increased, and stoma opening is enlarged, and this result illustrate IAA and MgCl2can increase plasma membrane H+-ATPase and14-3-3proteins interact to increase and leaf transpiration, stomatal opening is increasing PEG the effect of drought stress. These results demonstrate that under drought stress in PEG plasma membrane H+-ATPase and the14-3-3protein of two kinds of soybean leaf stomatal physiology characteristic has an important regulatory role. In drought stress exogenous application of AMP and VA can increase soybean drought tolerance ability.4. Recent studies suggest that H2O2can inhibit phosphorylation of plasma membrane H+-ATPase and its combination with the14-3-3proteins, thereby reducing the H+-ATPase activity and reduce the in vitro expression of stoma opening of broad bean. Ascorbic acid (ASA) is a kind of commonly used H2O2scavenger, H2O2generated depends on abscisic acid (ABA), sodium tungstate is ABA synthesis inhibito, the presence of sodium tungstate inhibit to production of H2O2. leaf H2O2content of YS is higher than the BS under PEG treatment, in order to understand under PEG Stress H2O2is involved in regulation of the plasma membrane H+-ATPase activity of BS and YS leaves or not, add to ASA and sodium tungstate with5%PEG drought stress treatment YS, the result show that YS plant water loss-rate increased17.4%and15.7%respectively, leaf transpiration rate increased19.2%and9.1%respectively, and the stomatal conductivity increased17.3%and8.7%respectively, the H2O2content of YS decreased. The result of COIP show that the plasma membrane H+-ATPase phosphorylation and levels of its interaction with the14-3-3protein of YS increased under adding ASA and sodium tungstate, enzyme activity and H+pump activity increased, and stoma opening is enlarged. BS is treated by5%PEG with H2O2, the plant water loss-rate of BS decreased26.3%, leaf transpiration rate and stoma conductivity decreased8.1%and11.2%, respectively, The result of COIP show that the plasma membrane H+-ATPase phosphorylation and levels of its interaction with the14-3-3protein of YS decreased under adding H2O2, enzyme activity and H+pump activity decreased, and stoma opening decreased. These results confirm that under PEG Stress the H2O2to participate BS and YS blade of plasma membrane H+-ATPase activity regulation under drought stress exogenously applied H2O2improve soybean drought tolerance. |
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