The Molecular Mechanism Of Transgenic Tobacco With Altered 14-3-3 Protein And Plasma Membrane H~+-ATPase Expression In Response To Drought Stress

Plasma membrane H+-ATPase is the most abundant protein content on the plant cell membrane, it is one of the main enzymes regulating stomatal opening, involved in plant response to a variety of biological and abiotic stress. The interaction between 14-3-3 and the phosphorylation of plasma membrane H+-ATPase regulate its activity. Plasma membrane H+-ATPase phosphorylation level is influenced by many environmental factors, under drought stress, guard cells of plant through the signal transduction of ABA increases the accumulation of active oxygen (H2O2), H2O2 can inhibit phosphorylation of fava beans to defend the cytoplasm membrane protein H+-ATPase and with the combination of the 14-3-3 protein thus inhibiting plasma membrane H+-ATPase activity, reduce the number of H+pump out, promote stomatal closure, reduce the plant transpiration rate and water loss rate. Tobacco is one of the widely used model plant of botany field research. To take advantage of transgenic tobacco validation aluminum stress induce resistance type aluminum black soy protein (RB) in the 14-3-3 protein and the plasma membrane H+-ATPase of expression and interactions in the role of regulation ability of plant resistance to aluminum, in the Guo Chuanlong master thesis of this laboratory, excessive expression of RB 14-3-3a (SGF14a) and no C end since the inhibitory domain of plasma membrane H+-ATPase {Δ GHA2), at the same time using RNAi technology inhibit 14-3-3 protein and plasma membrane H+-ATPase gene of tobacco expression in Wild type tobacco, it obtain four kinds of transgenic tobacco. In this study, wild type (WT) and four kinds of tobacco 14-3-3 protein and plasma membrane H+-ATPase expression level change of the transgenic tobacco treated with PEG6000 simulating drought stress, molecular mechanism of 14-3-3 proteins and plasma membrane H+-ATPase response to drought stress in tobacco leaves, mainly achieve the following results:Under hydroponic conditions, respectively 2%,5%,10% PEG treatment WT tobacco 0,2,5,12 h, the result shows that with the increase of concentration of PEG treatment and processing time increased, the transpiration rate and stomatal conductivity of WT tobacco gradually reduce, plant water loss rate increased. H2O2 concentration, soluble sugar and proline content of leaves also increase with the increase of the of PEG treatment. With the increase of the concentration of PEG, soluble protein content show a trend of first increased and then decreased, reach the highest in 2%PEG treatment. Antioxidant enzymes SOD activity increase with the increased concentration of processing, and POD, CAT activity reach the highest at 2% PEG treatment, it has a downward trend in 5%,10% PEG treatment. H2O2 fluorescent probes analysis confirm that chloroplast of accumulation H2O2 number in guard cells increase significantly in tobacco, under 2% PEG stress. Expression spectrum analysis results show that 2% PEG induce most of (7) the 14-3-3 genes and 2 germ plasm membrane H+-ATPase (pma2, pma3) gene expression in WT tobacco leaf. With the increase of the processing time, antioxidant enzymes APX expression increasing, downregulation expression of POD. Co-immunoprecipitation analysis results show that Phosphorylation level of plasma membrane H+-ATPase and its interaction with the 14-3-3 protein level decrease with increasing of 2% PEG treatment time, plasma membrane H+-ATPase activity and hydrogen pump activity have a decreasing trend. These results demonstrate that the WT tobacco is a kind of plant that is sensitive to PEG drought stress, by increasing H2O2 accumulation in leaves to reduce the level of phosphorylation of plasma membrane H+-ATPase and its interaction with 14-3-3 proteins to reduce the plasma membrane H+-ATPase activity and stomatal conductance and transpiration water loss, this is an important mechanism of tobacco to adapt to drought stress.Under a fluorescence microscope, observing excessive SGF14a and the lower epidermis of GFP fusion protein transgenic tobacco leaf. It confirms some SGF14a after expression is positioned on the protect cell membranes. Through the analysis of the immune coprecipitation, we select SGF14a overexpression line S23 which has the highest level interaction with phosphorylation of plasma membrane H+-ATPase as experimental material. Spectrum analysis results show that under the absence of PEG and 2% PEG drought stress, the level of gene transcription of nine Nt14-3-3, APX and CAT activity in S23 higher than WT tobacco, which may be induced by the excessive expression of SGF14a raising Nt14-3-3 gene expression. Whether or not PEG drought stress, APX, CAT,and POD activity of S23 leaf are higher than WT, so that the content of H2O2 in S23 leaves is lower than that of the WT. H2O2 fluorescent probe analysis shows that the chloroplast number of accumulation of H2O2in S23 guard cells is lower than WT. In the 2%PEG drought stress, Stomatal aperture, water loss rate, photosynthetic rate, stomatal aperture, transpiration rate, activity of H+-ATPase plasma membrane and hydrogen pump activity are all greater than WT. These results suggest excessive expression SGF14a rises antioxidant enzyme expression levels in transgenic tobacco leaf. Under PEG drought stress, after 12h, the H2O2 in the guard cells reduce, activity of PM H+-ATPase and hydrogen pump activity rise, Stomatal aperture, water loss rate, photosynthetic rate, stomatal aperture and transpiration rate increase, the drought tolerance of transgenic tobacco reduce.Through the analysis of the immune coprecipitation, we select the 14-3-3 inhibit expression strain RE10 which has the lowest level interaction with phosphorylation of plasma membrane H+-ATPase as experimental material of PEG treatment. Spectrum analysis results show that the level of gene transcription of nine Nt14-3-3, APX and CAT activity in RE 10 are lower than WT tobacco, which explain the 14-3-3 expression reduces the antioxidant enzyme inhibition gene expression. Under 2% PEG drought stress, the activity of APX, CAT, POD activity in RE10 leaves are all lower than WT, the content of H2O2 is higher than WT, H2O2 fluorescent probe analysis confirms that the chloroplast number of accumulation of H2O2 in RE 10 guard cells is higher than WT, so the stomatal aperture is less than WT. Under drought stress, water loss rate, photosynthetic rate, stomatal aperture, transpiration rate of RE 10 plants are all lower than WT, which is induced by the RE10 leaf plasma membrane H+-ATPase activity and hydrogen pump activity significantly lower than that WT. These results suggest inhibit Ntl 4-3-3 expression in transgenic tobacco reduces antioxidant enzyme expression levels in transgenic tobacco leaf, under PEG drought stress, after 12h, H2O2 accumulation in the leaves increase, Plasma membrane H+-ATPase phosphorylation level and its interactions with the 14-3-3 protein decrease, Plasma membrane H+-ATPase activity decrease,eventually lead to the transgenic tobacco stomatal aperture decrease, transpiration reduce and drought tolerance enhance.Through activity analysis, we select ΔGHA2 overexpressing G-5 plants whose plasma membrane H+-ATPase activity and hydrogen pump activity are the highest as experimental material of PEG treatment. The results show that the plasma membrane H+-ATPase and hydrogen pump activity in G-5 leaves under drought stress were higher than WT, H2O2 fluorescent probe analysis shows that the chloroplast number of accumulation of H2O2 in guard cells is also lower than WT, its stomatal aperture is 1.5 times that of WT. Under drought stress, after 12h, water loss rate, photosynthetic rate, stomatal aperture, transpiration rate of G-5 are all significantly higher than that of the WT. These results indicate that the excessive expression a constitutively active plasma membrane H+-ATPase increase its stomatal aperture. Under drought stress, the photosynthesis transgenic tobacco enhance.But its the drought tolerance reduce.Through the analysis of the Co-immunoprecipitation, we select the plasma membrane H+-ATPase inhibit expression strain RP1 which has the lowest level interaction with 14-3-3 protein as experimental material of PEG treatment, after 12h. Under 2%PEG drought stress, after 12h, plasma membrane H+-ATPase activity and hydrogen pump activity of RP1 leaves are lower than WT. The chloroplast number of accumulation of H2O2 in RP1 guard cells is Significantly higher than that of WT. Its stomatal aperture is Significantly less than WT. In addition, during drought stress, APX, CAT, POD activities of RP1 leaves is lower than that of WT. Therefore, the H2O2 content in its leaves is higher than WT. Under drought stress, after 12h, water loss rate, photosynthetic rate, stomatal aperture, transpiration rate of RP1 leaves are all lower than WT. These results demonstrate that inhibit plasma membrane H+-ATPase expression make the plasma membrane H+-ATPase activity reduce and stomatal aperture decrease, So it also reduce transpiration of transgenic tobacco under drought stress. Thus the ability of drought tolerance in tobacco is enhanced.