Genetic Transformation Of Petpkl And Pegpx And The Relevance To Plant Salt Tolerance

Salt stress is one of the main environmental factors which restrain plant growth and reduce agricultural productions. Improving salt-resistance and outputs of crops under salt stress have received much attention for many years. Populus euphratica Olive (P. euphratica) is a salt resistant tree species widely distributed in the desert areas of north-west China. It usually forms a forest ecosystem in severe salinized desert areas with some halophytic shrubs. Although salt-resistant mechanism of P. euphratica both in physiological and molecular aspects has been studied intensively, the molecular mechanism underlying K+homeostasis and ROS homeostasis still needs to be elucidated. In this study, PeTPK1gene and PeGPX gene were cloned from P. euphratica, which were up-regulated under salt stress by means of microarray analysis, and transformed into BY-2cells and tobacco plantlet respectively. We examined their contributions to K+and ROS howmeostasis control in salinized plants and cells.The main experimental results are as follows:1. Vacuolar K+ion channels of the two-pore K+(TPK) family play an important role in maintaining K+homeostasis. In a search for K+channels involved in KT homeostasis and salt resistance, we cloned a putative K+channel gene from Populus euphratica. Sequence analysis showed that PeTPK1possesses the universal K-channel-specific pore signature, TXGYGD, suggesting its role in transmembrane K+transport. Over-expression of PeTPK1with CaMV35S promoter improved salt tolerance of tobacco BY-2cells, but did not enhance the cell tolerance to hyperosmotic stress caused by mannitol (200-600mM). Fresh and dry weights of the transgenic cells were higher than that of wild-type cells after3-weeks of NaCl stress (100and150mM). Compared to wild-type cells,PeTPK1-transgenic cells showed higher cell viability and lower membrane permeability during the period of a short-term salinity (100mM NaCl,24h). Under salt stress, the K+content in PeTPK1-transgenic cells is higher than that of wild-type cells; consistenly, the wild-type cells have much higher Na+than that of PeTPK1-transgenic cells. SIET data reveal that shocked callus cells and protoplasts of the PeTPK1-transgenic line exhibited a transient K+efflux, which was significantly higher than the wild-type cells. As EYFP-PeTPK1fusion protein located exclusively in the vacuolar membrane, we speculate that the salt-induced K+translocation is likely mediated through P. euphratica two-pore K+channel TPK1, which functions as an outward channel favoring K+efflux from the vacuole, thus enables tobacco BY-2cells to regulate cytosolic K+homeostasis under salt stress.2. The full length GPX cDNA encoding a hydrophobic protein of231amino acids was cloned from P. euphratica, designated as PeGPX. PeGPX was over-expressed in tobacco plants and improved the salt tolerance of tobacco plants. After15d of salt treatment, transgenic plantlets survived in the salt medium, and grew new roots in the medium. In contrast, the leaf of wild-type plantlet shrinked upon the salt treatment, and these plantlets could not grow new roots under the same salt medium. The data of photosynthetic shows that net photosynthetic rate of transgenic tobacco plants is less affected than that of in wild-type plants. Noteworthy, transgenic plants had much higher GPX enzyme activity than that of wild-type tobacco under salt stress. In conclusion, our results reveal that PeGPX gene could improve the capacity of salt tolerance in tobacco plants, but the functions of PeGPX in salt stress signaling of P.euphratica still needs many studies.In conclusion, our data confirmed that over-expression of PeTPK1improved salt tolerance of tobacco cells in terms of cell growth, viability, membrane permeability and K-efflux. The results can explain our finding that salinized P. euphratica redistributed K+within cell compartments for K+homeostasis in cytoplasm. PeGPX over-expression increase the capacity of salt tolerance in tobacco plants, implying that PeGPX gene plays an important role in ROS homeostasis control under salt stress. Therefore, our results suggest that PeTPK1and PeGPX benefit salt tolerance in P. euphratica.