Functional Analysis Of The Na~+/H~+Exchanger PeNHX3in Populus Euphratica

Salt stress is one of the most severe environmental factors limiting the productivity of crop plants. Therefore, understanding the mechanisms underlying plant salt tolerance is critical for developing crop production. Populus euphratica is a tree halophyte that exists in saline desert areas in northwest China. Thus, exploring how P. euphratica deals with salt will be helpful for the development of salt tolerant crops.Recent genomics study found that the genes encoding ion transporters were selectively expanded in the P. euphratica genome, suggesting that ion transporters controlling cellular salt movement may play critical roles in this tree halophyte. Na+, K+/H+antiporters are H+-coupled cotransporters that transfer the Na+or K+across a membrane in exchange for protons (H+). Studies have shown that Na+, K+/H+antiporters have decisive roles in Na+/K+transport and salt tolerance in plants.In this study, we studied the ion transport activity, cellular localizations and salt tolerance of PeNHX3using molecular techniques including gene cloning, yeast growth, GFP labeling and genetic transformation. Our goal is to explore the role of PeNHX3in ion transport and slat tolerance in P. euphratica. Our study will pave the way for the understanding of the mechanisms governing salt tolerance in P. euphratica and for the development of salt tolerant crop plants. The major results are listed below:1. PeNHX3gene was cloned from the total RNA of P. euphratica callus. We compared the gene sequence of our cloned PeNHX3with the GenBank sequence (gene no. FJ589741.1) and found that there were two point mutations in our cloned sequence:A282was mutated to C2g2, T555was mutated to C555. However, these point mutations are synonymous, i.e. the codons are changed from ATA into ATC, and GTT into GTC, respectively. These two new codons still encode isoleucine and valine, respectively.2. The transport activity was analyzed by expressing the PeNHX3gene in yeast mutants lacking multiple ion carriers. PeNHX3conferred resistance to high Na+, high K+and Hygromycin B stress. These results suggest that PeNHX3are able to transfer Na+and K+. 3. Using the tobacco transient expression system and GFP fusion protein to analyze the subcellular localization of PeNHX3in the tobacco epidermal cells. The results showed that PeNHX3was mainly localized in the vacuolar membranes of the tobacco epidermal cells, suggesting that moving Na+into vacuoles to maintain a low level of Na+in cytosol and avoid the toxic effect of high Na+is the mechanism of enhancing salt tolerance by PeNHX3in plants.4. In order to analyze the function of PeNHX3in plants, we generated transgenic plants by transforming the PeNHX3gene into nhxl and sosl mutants. We have obtained the T1and T2transgenic plants. We are now screening the homozygous plants and performing growth analysis for the transgenic plants.