Molecular Analysis of Salt Tolerance Proteins

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Soil salinity is a major factor in reducing plant growth and productivity. Most crop plants are highly sensitive to salt stress that tends to cause a water deficit due to osmotic stress, and that causes several biochemical problems due to toxic excess intracellular sodium. The degree of salt tolerance of some plant species varies greatly. Sodium/Hydrogen (Na+/H+) antiporters are membrane proteins that remove salt from inside the cells of plants and yeast. They remove salt, (sodium) in exchange for a proton that is on the outside of the cell. Overexpression of Na+/H+ antiporters has been shown to confer improved salt tolerance in plants allowing them to grow in saltier soils. The purpose of our project is to understand how Na+/H+ antiporters work, that is, to better understand the way that they remove salt out of cells and allow plants to grow in soil with salt present. To do this we are using a model system of yeast to study how Na+/H+ antiporters function. Yeast have a Na+/H+ antiporter that is very similar to plant Na+/H+ antiporters. We have made a yeast strain that has its own Na+/H+ antiporter knocked out. It doesn’t grow in medium that has external salt. Plant and yeast Na+/H+ antiporter proteins are similar and can replace each other and function normally. We can return a plant Na+/H+ antiporter into the yeast or an altered form of the protein. Then we can test if it is functioning by checking if the yeast grow in medium containing salt. We have made molecular models of the proteins, based on computer programs that analyze the proteins sequence. To confirm which part of the protein is important we are making mutations in the plant and yeast proteins, that are based on the molecular models. We then return the mutant protein back into the knockout yeast and test if salt tolerance is returned. This allows us to determine which parts of the protein are important. Another part of the project is to try to get a picture of how the protein looks determining its shape, or topology in the cell membrane. To do this, we made an altered Na+/H+ antiporter protein that has all its cysteine amino acid residues removed, but is still functional. We will reintroduce single cysteine amino acids in specific places in the protein and express it in our knockout yeast. We will then use specific chemical compounds that react with cysteine amino acids. We can do this in such as way as to determine if the amino acid is on the outside of the cell, on the inside of the cell or within the membrane of the cell. We will do this repeatedly and in specific spots on the protein based on our computer models. We will then be able to build a verified picture of the protein. Another part of the project is to make a hyperactive salt tolerance protein. We will randomly mutagenize several Na+/H+ antiporter proteins. Then we will reintroduce them into the yeast and grow the yeast in high salt containing medium. We can then analyze the mutations that allowed super salt tolerance. Collating this information in different Na+/H+ antiporters may allow us to design super salt tolerance proteins that might be useful to reintroduce back into plants, making them more able to grow in salt containing soil. Overall, the project will help us understand how the protein works, allowing us to better design proteins that can be used to improve salt tolerance in plants.