| Drought caused plant water stress, the adverse effects for the plant growth, photosynthesis, stomatal movement, respiration, metabolism, and then influence the growth and development of plants. Effect of soil drought stress on plants as the first manifestation was water loss and related physiological and biochemical characteristics of plant stomata, to reduce the plant transpiration and water loss is an important mechanism of plant responses to drought stress through regulation of stomatal conductance and opening. The plasma membrane H+-ATPase is the key enzyme regulating stomatal opening degree, changes in plasma membrane H+-ATPase activity under water stress is one of the important indicators of water stress adaptation of plant.14-3-3proteins is a highly conserved regulatory protein found in eukaryotic cells, it can modulate the activity of plasma membrane H+-ATPase by interactions on its phosphorylation C terminal. The plasma membrane H+-ATPase phosphorylation is regulated by many environmental factors, with studies on the isolated epidermal strips of Vicia demonstrated the H2O2can inhibit plasma membrane H+-ATPase phosphorylated and binding of14-3-3protein to inhibit plasma membrane H+-ATPase activity, reduced H+pump and inhibition of guard cell plasma membrane K+channel, promote stomatal closure. C4plant drought tolerance better than C3plants, maize is a typical C4plant, different maize hybrids under water stress, stomatal resistance increased significantly, to reduce the loss of body water, increased stomatal resistance of different varieties on different rate, drought resistance greatly increased by the strong, but poor drought tolerance increased smaller, this study used PEG simulated drought treatment in two Yunnan maize cultivar (a yellow maize (YM), another white maize (WM)), through analysis the leaves of physiological and biochemical characteristics and14-3-3protein interaction with plasma membrane H+-ATPase and the activity of plasma membrane H+-ATPase of two kinds of maize under drought stress, to discussion14-3-3protein and H+-ATPase in drought stress response mechanism in Maize, the main results are as following: 1In the hydroponic conditions using polyethylene glycol (PEG) simulated drought stress on YM and WM,analysis the relationship between changes of leaf physiological characteristics and14-3-3protein interaction with plasma membrane H+-ATPase and plasma membrane H+-ATPase activity. Results show that in the2%,5%and10%PEG treatment conditions, with the increase of treatment concentration and time, dehydration rate increase, transpiration rate decreased, stomatal conductance decreased in two kinds of corn seedling; in all the same processing conditions, WM plant water loss rate was less than YM, and leaf transpiration rate and stomatal conductance rate decreased significantly greater than YM, shows that drought tolerance of was stronger than YM. In the condition of the same concentration of PEG treatment, the soluble WM protein, soluble sugar content, free proline content leaves of WM was higher than that of YM, that discussion osmotic regulation ability under drought stress in WM was stronger than that of YM; in the antioxidant enzyme system, with the increase of PEG treatment concentration, two kinds of maize leaf CAT activity decreased, but the activity of CAT in WM under2%and5%PEG stress were significantly higher than that of YM, POD activity leaves in WM under the three kinds of concentration of PEG was higher than that of YM, which makes the H2O2content in the leaves was significantly lower than that of YM. With the increase of PEG concentration, the inhibition of phosphorylation level of H+-ATPase and interact14-3-3proteins in WM leaf membrane is stronger than YM, WM leaf membrane H+-ATPase activity and hydrogen pump activity lower than YM leaves, leaf stomatal conductance is less than YM, so that the transpiration rate and stomatal conductance in leaves of WM were lower than those of YM, the drought tolerance is stronger than that of YM. RT-PCR analysis of10%PEG stress in two kinds of maize in5different14-3-3subtypes and3kinds of plasma membrane H+-ATPase gene (HA) changes in transcription, results indicate that the5kinds of14-3-3genes and3kinds of HA genes transcription level in WM leaves are lower than that of YM, the5kinds of14-3-3genes and3kinds of HA genes transcription level are induced in YM and WM under PEG stress, but the transcriptional level in the leaf of WM are lower than YM at all time, perhaps this is an important molecular mechanism that the plasma membrane H+-ATPase activity in WM leaf lower than YM.2Many research results indicated that proline (Pro), gibberellin (GA) and betaine can increase the water absorption capacity and drought tolerance of plants. In this study, were added Pro, GA and betaine treatment to two kinds of corn under10%PEG simulation drought stress to inspect the effects on physiological characteristics, leaf14-3-3protein and plasma membrane H+-ATPase interaction and plasma membrane H+-ATPase activity. The result shows that compared with the single PEG treatment, addition of Pro, GA and betaine under PEG stress not only reduces two maize plants the rate of water loss, but also to reduce the two kinds of maize stomatal conductance and transpiration rate, confirmed that they reduce PEG stress and leaf transpiration.Pro, GA and betaine added under PEG stress make two kinds of corn leaf plasma membrane H+-ATPase activity and H+-pump activity was significantly decreased, leaf stomatal conductance decreased significantly, but the combination of plasma membrane H+-ATPase phosphorylation and14-3-3protein in two kinds of maize leaf had different effect:compared with the single PEG treatment, Pro was enhanced by the phosphorylation level of leaf membrane H+-ATPase and its binding to14-3-3protein of two kinds of corn; GA was attenuated the phosphorylation level of leaf membrane H+-ATPase and its binding to14-3-3protein in WM, but enhanced the phosphorylation level of plasma membrane H+-ATPase and its binding to14-3-3protein in YM leaves; betaine was attenuated the phosphorylation level of membrane H+-ATPase and its binding to14-3-3protein in two kinds of maize leaf. These results indicate that, effects of Pro, GA and betaine on plasma membrane H+-ATPase phosphorylation may not directly bind on the14-3-3protein same point in C tip, but in other domains related to regulation of plasma membrane H+-ATPase activity under PEG simulated drought stress, the modification in these domains indirect regulation of plasma membrane H+-ATPase interaction with14-3-3protein.3Adenosine-5,-monophosphate (AMP) and vanadate (VA) are inhibitors of plasma membrane H+-ATPase, the former can inhibit14-3-3protein combined with phosphorylation of plasma membrane H+-ATPase and decreased membrane H+-ATPase activity; while IAA and MgCl2are the activator of plasma membrane H+-ATPase, can enhance the quality of H+-ATPase phosphorylation and promote the integration of14-3-3protein and phosphorylation of plasma membrane H+-ATPase, increase the activity of plasma membrane H+-ATPase. The results showed that under PEG drought stress, leaf plasma membrane H+-ATPase and14-3-3protein interaction in YM is stronger than that of WM, in order to further investigate the regulation of physiological characteristics of stoma by plasma membrane H+-ATPase and14-3-3protein under PEG simulated drought stress in two kinds of maize leaves, use the10%PEG simulated drought stress and add AMP,VA to treatment YM, the results found to significantly reduce the YM plant water loss rate compared to single PEG treatment, at the same time, leaf transpiration rate and stomatal conductance decreased significantly reduce.COIP analysis showed that AMP significantly decreased the phosphorylation of plasma membrane H+-ATPase and its binding to14-3-3protein, the presence of VA also reduced cell phosphorylation of membrane H+-ATPase and its binding capacity to14-3-3protein, two application were significantly decreased the activity of H+-pump activity and plasma membrane H+-ATPase, stomatal angle was also decreased, indicating that AMP and VA through inhibits interaction of membrane H+-ATPase and14-3-3protein to reduce transpiration, stomatal conductance, alleviate drought stress, application the exogenous of AMP and VA can significantly increased maize drought tolerance under drought stress. Using IAA and MgCl2treatment WM, compared with the single PEG treatment, the presence of IAA and MgCl2make the leaf transpiration rate and stomatal conductance increased significantly, and increased the rate of water loss. COIP analysis showed that the plasma membrane H+-ATPase phosphorylation level was significantly increased, enhanced binding ability with14-3-3protein, increased enzyme activity and H+-pump activity, leaf stomatal conductance increased significantly. These results suggest that plasma membrane H+-ATPase interaction with14-3-3protein increased stomatal opening and transpiration under PEG drought stress, with IAA and MgCl2. These results were confirmed, plasma membrane H+-ATPase and14-3-3protein plays an important role in regulating physiological characteristics of stomatal in two kinds of Maize leaves in PEG simulation drought stress. Potted WM plants treatment with PEG adding VA can significantly alleviate drought stress symptoms in leaves, potted YM plants treatment with PEG adding MgCl2increased drought stress symptoms in leaves of maize.4Recent studies indicate that H2O2can inhibit phosphorylated of plasma membrane H+-ATPase and its binding to14-3-3protein, thereby reducing the activity of H+-ATPase and reduce the stomata opening of vitro epidermis of Vicia. Ascorbic acid (ASA) is a common scavenger of H2O2, H2O2generated by abscisic acid (ABA), sodium tungstate is an inhibitor of ABA synthesis, sodium tungstate has also inhibited produce of H2O2. The contents of H2O2in YM leaves under PEG drought stress was higher than that of WM, in order to understanding H2O2is involved control the leaf membrane H+-ATPase activity under the stress of PEG in two kinds of corn, the addition of ASA and sodium tungstate treatment of YM in10%PEG drought stress, although the results of the transpiration rate and stomatal conductance rate in leaves of YM were increased, H2O2content in leaves significantly reduced, plant water loss rate increased significantly. The COIP analysis results show that the addition of ASA and sodium tungstate make the phosphorylation level of plasma membrane H+-ATPase increased significantly to the YM leaves, the interaction of plasma membrane H+-ATPase and14-3-3protein was significantly enhanced, enzyme activity and H+pump activity was significantly increased, stomatal conductance also enhanced. These results suggest that ASA and sodium tungstate has increase plasma membrane H+-ATPase phosphorylation through decreased the content of H2O2in the leaves, thereby enhancing its interaction with14-3-3protein, increased the stomatal conductance and transpiration of leaves, plant water loss rate increased. Add H2O2treatment WM under10%PEG stress, the WM plant water loss rate decreased significantly, leaf transpiration rate and stomatal conductance decreased. COIP analysis showed that adding H2O2to the WM leaves the phosphorylation level of plasma membrane H+-ATPase decreased significantly, while binding to14-3-3protein decreased, enzyme activity and H+-pump activity was significantly decreased, the stomatal aperture reduced. These results confirm the regulation under the stress of PEG and H2O2to membrane H+-ATPase activity in two kinds of corn leaf, significantly improve drought tolerance in maize under drought stress and exogenous H2O2. |
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