Evaluating the contribution of tonoplast and plasma membrane transporters in salinity tissue tolerance in barley

Salinity is one of the major abiotic stresses affecting the world food supply. Salt affected soil has area coverage of 950 million ha which accounts for 10% of the land surface of the globe. Fifty percent of the irrigated land (230 million ha) is affected by salinity costing the world food production $US27 billion per annum. In Australia alone, 67% of the agriculture land is affected by transient salinity costing the Australian economy $AUD 1330 million per annum. In comparison to other strategies, the use of salt tolerant plants is cost effective and sustainable way of controlling salinity. While numerous attempts have been made to develop salt tolerant varieties over the last few decades, most of the efforts were focused on tackling individual components or genes contributing to overall salt tolerance. As a result, the progress in the field was much slower than expected and we still lack truly salt tolerant varieties in the farmers‚ÄövÑvº field. Salinity tolerance is a multi-faceted physiological trait, and all the beneficial effects of improving a function of one gene/mechanism may be lost or overturned by plethora of other contributing factors. This calls for a need of pyramiding‚ÄövÑvp several key traits in one ideotype. Before this can be implemented into the practice, the essentiality and a relative contribution of various traits should be quantified, for each particular species. The aim of this PhD study was to identifying the major contributing mechanisms to salt tolerance in barley. The major focus was on the following aspects: (1) essentiality of transcriptional vs post translational factors in mediating plant adaptive responses to salinity; (2) quantifying the relative contribution of osmo- and tissue-tolerance mechanisms in barley; (3) identifying components involved in Na+ sequestration in vacuole; and (4) revealing the role of H+-ATPase in vacuolar ion sequestration and overall salinity stress tolerance. In the first part of this PhD study, the relative contribution of ionic, osmotic and oxidative stresses to the overall salinity tolerance in barley was studied, both at the whole plant and cellular level. In addition, the gene expression profile of key genes in ionic and oxidative homeostasis (NHX, RBOH, SOD, AHA and GORK) as a way of comparing the contribution of transcriptional and post translational factors for salinity tolerance was also investigated. The major findings can be summarized to two main points. These are: (i) tissue tolerance is the dominant component in which root K+ retention and lower sensitivity to stress induced hydroxyl radical production are the main ones; (ii) responses at the post-translational level is more important than at the transcriptional level for understanding the mechanisms for salt tolerance. Overall, for better tissue tolerance, sodium sequestration, K+ retention and resistance to oxidative stress are important salt tolerance mechanisms. It is concluded that every crop improvement programs for salinity stress tolerance should take into consideration all this components. In the second part of this PhD study, we showed that unlike a number of reports for saltsensitive salt excluder‚ÄövÑvp species (such as Arabidopsis or rice), expressing AtNHX1 in barley had no beneficial effect on plant performance under saline conditions. AtNHX1 Arabidopsis tonoplast Na+/H+ exchanger was expressed in barley (Hordeum vulgare L., cv. Golden Promise) and the plants grown under saline growth condition. The transgenic plants were compared with null segregant for biomass, water content, gas exchange, and Na+ and K+ content of the leaf. The transgenic barley plants expressing AtNHX1 has not shown significant difference from the null segregant for any of the trait at least under our experimental conditions. The lack of phenotype in barley which adapts salt including‚ÄövÑvp strategy was explained by one or more of the following: (i) low level of activity of vacuolar H+-PPiase and vacuolar H+-ATPase causing poor proton gradient; (ii) the lack of controlling passive leak of sodium via Na+ permeable slow activating and fast activating channels in the vacuole; (iii) insufficient ATP pool to assist the H+ pumping activity; (iv) the AtNHX1 protein may not be folded properly, inactive or mis-targeted. In the last part of this PhD study, improvement in salinity stress tolerance was obtained in barley crops by expressing V-ATPase subunit C gene. Most of the reports until now have not shown improvement at the grain yield level and also have not explained the physiological mechanisms behind. Also, no previous attempt to express the V-ATPase subunit C was done in barley. Accordingly, we expressed AtVHA-C gene in barley and grown under 300mM NaCl. The barley plants expressing the gene were compared with wild type plants for dry biomass, leaf pigment content, stomatal conductance, grain yield, and leaf Na+ and K+ content. The transgenic barley plant expressing AtVHA-C have shown smaller reduction in biomass and grain yield compared to wild type plants. The beneficial effect in the AtVHA-C expressing plant was due to better maintenance of stomatal conductance resulted from the accumulation of Na+ and K+ in the leaf that lead to osmotic adjustment and less reliance on the de novo synthesis of organic osmolytes. To recap, salinity tolerance is a multigenic physiological trait involving various mechanisms that may differ between species. For barley, the dominant mechanism appears to be the tissue tolerance. This includes better K+ retention in the root; reduced tissue sensitivity to oxidative stress; and more efficient sodium sequestration in the leaf. We have shown that for a better sodium sequestration all the components such as proton pumping for generating proton gradient in the tonoplast membrane, control of the back leak of sodium through Na+ permeable SV and FV channels, and ensuring properly folded, active and correctly targeted NHX protein to the tonoplast all should be considered and modified as one set, to achieve a positive outcome and improve crop salinity tolerance. Finally, we have also shown the importance of vacuolar H+-ATPase towards salinity tolerance by the contribution of vacuolar H+-ATPase for the accumulation of Na+ and K+ in the leaf where they contribute towards osmotic adjustment, and hence, conservation of energy otherwise spent for organic osmolyte production.