Poster: Membrane Transport
Abs #
1211: Cloning and characterization of wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump
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Presenter: |
Brini, Faical , brinifr@yahoo.fr |
Authors | Brini, Faical (B) Masmoudi, Khaled (B) Gaxiola, Roberto A. (A) Berkowitz, Gerald A. (A) | | Affiliations: |
(A): Agricultural Biotechnology Lab, Dept. Plant Science
(B): Center of Biotechnology at Sfax (CBS)
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Salinity (and water deficits) limit wheat crop growth; osmotic adjustment capacity (i.e. solute accumulation) impacts wheat cultivar response to these abiotic stresses. Prior work (Gaxiola et al., Proc. Natl. Acad. Sci. USA 96:1480 & 98:11444; Apse et al., Science 285:1256) indicates that overexpression of the Arabidopsis vacuolar Na/H antiporter (NHX1) and pyrophosphatase H-pump (AVP1) provide enhanced cellular-level tolerance to salinity (and water deficit in the case of AVP1) by increasing the capability to accumulate cations (Na and/or K) in the vacuole. We cloned the wheat orthologs of NHX1 and AVP1 using PCR, primers corresponding to conserved regions of the respective coding sequences, and a wheat cDNA library as template. Wheat NHX1 and AVP1 are highly homologous to barley, then rice, and less similar to Arabidopsis orthologs. Function of wheat NHX1 as a cation/proton antiporter was demonstrated using yeast mutants. Wheat NHX1 was cloned into the pRS8 plasmid, allowing for constitutive expression driven by the PMA1(plasma membrane H pump) promotor. A yeast mutant lacking yeast NHX1, and the double mutant lacking NHX1 and ENA1(encoding the plasma membrane Na efflux ATPase) were transformed with pRS8-wheat NHX1. The nhx1 and the nhx1,ena1 mutants show enhanced sensitivity to Na and the toxic cation hygromycin B. Expression of wheat NHX1 in both of these yeast mutants complemented growth in the presence of hygromycin, presumably due to ability of the wheat antiporter to facilitate sequestration of this toxic cation (i.e. as occurs with Na) in the vacuole. Functional characterization of the wheat AVP1 ortholog using appropriate yeast mutants is underway. Supported by USDA-RSED awards FG-TN102 & 5831483022.
