Salinity detection and control of sodium transport in Arabidopsis thaliana.

Abstract

Soil salinity is a major abiotic stress, reducing crop yields and endangering global food security. With salt affected areas increasing, understanding the molecular mechanisms of salinity stress is of great importance. Plant salinity stress can be categorised into two phases, the initial shoot ion independent osmotic stress and the later ionic stress. Osmotic stress occurs as soon as the plant encounters salt in the soil and results in an immediate reduction in the shoot growth rate. Ionic stress is caused by the accumulation of ions such as Na+ and Cl- in the cytosol of cells in the shoot and results in the inhibition of cellular processes and induces premature leaf senescence. The two Arabidopsis thaliana ecotypes Col-0 and C24 have previously been identified as interesting candidates to study plant salinity tolerance. The Col-0 ecotype is less salt tolerant than the C24 ecotype, based on its reduction in dry weight under stressed conditions. This is despite C24 accumulating significantly more Na⁺ in the shoot than Col-0. Interestingly, C24 also appeared to be less responsive to salt stress, as transcript levels of several key salt responsive genes are not substantially altered in response to salt stress. The AtHKT1;1 gene is one key gene found to be not up-regulated in C24 during salt stress. AtHKT1;1 encodes a protein likely to be involved in the retrieval of Na⁺ from the xylem thereby reducing the amount of Na⁺ translocating to the shoot. In this thesis the C24 and Col-0 HKTs are compared at the protein and transcriptional levels. Electrophysiological analysis in Xenopus oocytes and a functional assay in yeast confirm Na⁺ transport properties of both AtHKT1;1 proteins and, interestingly, indicated AtHKT1;1 from both ecotypes had the ability to transport K⁺. To determine the difference in expression profile between the two ecotypes, a series of AtHKT1;1promoter::GFP and AtHKT1; 1promoter: :AtHKT1;1cDNA constructs were tested in Arabidopsis. Results suggest that both the Col-0 and C24 AtHKT1;1 promoters are able to drive expression of the downstream genes, suggesting that differences in the promoter region are not responsible for the lack of AtHKT1;1 expression in C24. A transposable element identified in the second intron of the C24 AtHKT1;1 genomic sequence may be important in causing the lack of AtHKT1;1 expression in roots. Furthermore, the reduced responsiveness of C24 to salt stress is investigated in relation to how salt is initially perceived by the plant. An assay using aequorin bioluminescence was used to compare the responses in the salt stress inducible Ca²⁺-signatures of Col-0 and C24 seedlings. Excitingly, C24 appears to be missing part of the Ca²⁺ signature observed in the salt responsive plant Col-0, suggesting that C24 may not detect the ion component of salt stress. This potentially provides a suitable screening methodology for the identification of as yet unknown components in the early stages of the salt signalling pathway. An attempt is made to develop a screening assay suitable for performing QTL analysis on an available Col-0  C24 mapping population, based on measuring changes in transcript levels of salt responsive genes.