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Antiporter Characterization of Arabidopsis mutants lacking multiple vacuolar/endosomal Na+/H+ antiporters.

 

Characterization of Arabidopsis mutants lacking multiple vacuolar/endosomal Na+/H+ antiporters.

Background
Throughout the biological kingdom, from bacteria, algae, fungi and worms to plants and humans, Na+/H+ antiporters play a major role in the pH and Na+ homeostasis of cells. Na+/H+ antiporters are integral membrane proteins residing in the plasma membranes and endomembranes of different cells. Arabidopsis thaliana vacuolar/endosomal Na+/H+ 1 ( AtNHX1) has been previously shown to play important roles in cellular homeostasis, including the sequestration of Na+ and K+ ions into the vacuole, the regulation of vacuolar pH and leaf development. Overexpression of AtNHX1 has also lead to increased salt tolerance in a number of plants. We are now interested in characterizing the function of additional members of the AtNHX family which remain largely unknown. Preliminary results indicate that members of the AtNHX family, have unique and novel roles in plant growth and development.

atnhx chart

 

 

Amino acid sequence similarity predicts that AtNHX antiporters belong to two sub-groups: AtNHX1-4 and AtNHX5-6. While the first group is >75% similar, the second group is <30% similar to the first. Phylogenetic analyses of AtNHX predicts that AtNHX 5 and 6 are putatively localized in endosomes/TGN while AtNHX 1-4 are at the membrane of cell vacuoles.

atnhx chart

Characterization of single and multiple AtNHX knockout lines
To determine roles of all endosomal AtNHX members, we characterized single knockout lines of AtNHX 1 to 6 and generated multiple knockout lines of the different NHXs. Single to quadruple knockouts lines of nhx1/nhx2/nhx3/nhx4 were generated independently from single and double knockouts of nhx5 and nhx6.

T-DNA insertion lines for AtNHX 1 to 6

athnx chart

Apart from generating multiple knockout lines of AtNHX1-6 we also created translational fusions between NHX genomic sequences and the reporter gene GFP, as well as GUS reporter lines. We are currently using such lines to better understand the localization of different NHX members both at tissue and cellular levels.

Preliminary results reveal unique and diverse functions for each member of AtNHX 1 through 6, with individual role(s) for each member in regulation of cell volume, ion homeostasis, cell growth and plant growth and development.