Poster: Salinity

Abs # 171: Arabidopsis activation tagged mutants tolerant to NaCl

Presenter:	Price, Jillian M, 9807707p@student.gla.ac.uk
Authors	Price, Jillian M (A)   Thomson, Catriona  (A)   Farcanasu, Illeana  (A)   Dominy, Peter  (A)
Affiliations:	(A): University of Glasgow

We are using Arabidopsis activation tagged lines (Weigel) in large-scale gain-of-function screens to identify single sequences that allow seedlings to survive better under NaCl stress. 23 000 lines (segregated into 230 pools) were screened on MSMO media with reduced K⁺ (200mM) and Ca²⁺ (500mM) concentrations, supplemented with 0.75% sucrose and 80mM NaCl. It is well established that the toxic effects of Na⁺ can be ameliorated by high external K⁺ and Ca²⁺ concentrations. Under these conditions we found this concentration of NaCl to severely inhibit wildtype seedling growth. Therefore, any mutants that were more efficient at K⁺ uptake, Na⁺ efflux, preventing Na⁺ uptake, or show an increased sensitivity to the Ca²⁺ response, should flourish and be easily identified in our screen. Primary screening isolated 482 putative salt tolerant mutants from 106 pools. To date, secondary screening under identical conditions has isolated 52 (23 pools) mutants displaying a strong phenotype, 78 (26 pools) a weak phenotype and 136 (33 pools) false positives. TAIL-PCR is being used to locate the position of the activation tag within the mutant genome. By use of 3 specific nested primers (which hybridize to the tagging vector) and a variety of arbitrary degenerate primers (which hybridize to the plant DNA), TAIL-PCR can effectively isolate the segment of plant DNA adjacent to the activation tag border. The number of insertions per line, and the mode of mutagenesis (i.e. knockout or activation), are being determined by genomic southerns and Q-PCR. In addition we are using ICP-OES to investigate ion distribution within the mutants. The poster will present our progress with this work.