Regulation of xylem ion loading and ionic relations in barley and wheat leaves in the context of salinity stress tolerance

Soil salinization is the accumulation of water-soluble salts in the soil to a level that impacts on agricultural production. Approximately 20% of the world's cultivated land, or 6% of the world total area, is threatened by salinity. Hence, soil salinization is becoming the main challenge of the modern agriculture. Of all the cereals, wheat (Triticum aestivum) is a moderately salt-tolerant crop while barley (Hordeum vulgare) is classified as relatively salt tolerant. Within Triticum genera, durum wheat (Triticum turgidum ssp. durum) is less salt-tolerant than bread wheat (Triticum aestivum). Salinity tolerance is a complex trait, both physiologically and genetically. Both rapid and efficient osmotic adjustment and effective control of Na+ delivery to the shoot are absolutely critical for salinity stress tolerance. Reducing Na+ delivery from root to shoot can be achieved by either minimizing entry of Na+ to the xylem from the root symplast or maximizing Na+ retrieval from the xylem (e.g. by active means) or by reduced rate of transpiration resulted from stomata closure (e.g. by passive means). Specific details of their coordination and the relative contribution of these components towards salinity stress tolerance in wheat and barley have been not fully revealed until now. Hence, the major aim of this PhD project was to investigate the physiological and molecular mechanisms of regulating Na+ transport from root to shoot and its link with plant osmotic adjustment and the overall plant performance under saline conditions. The following specific objectives were addressed: * To quantify the relative contribution of organic and inorganic osmolytes towards osmotic adjustment in barley * To link osmotic adjustment and stomatal characteristics with salinity stress tolerances in contrasting barley accessions * To evaluate predictive values of various physiological indices for salinity stress tolerance in wheat and barley * To investigate the physiological and molecular mechanisms mediating xylem Na+ loading in wheat and barley Abstract ii Working along these lines, a broad range of barley (Hordeum vulgare and Hordeum spontaneum) genotypes contrasting in salinity stress tolerance were used to investigate the causal link between plant stomatal characteristics, tissue ionic relations, and salinity tolerance. In total, 46 genotypes (including two wild barleys) were grown under glasshouse conditions and exposed to a moderate salinity stress (200 mM NaCl) for five weeks. The overall salinity tolerance correlated positively with stomata density, leaf K+ concentration and the relative contribution of inorganic ions towards osmotic adjustment in the shoot. At the same time, no correlation between salinity tolerance and stomatal conductance or leaf Na+ content in shoot was found. Taken together, these results indicate the importance of increasing stomata density as an adaptive tool to optimise efficiency of CO2 assimilation under moderate saline conditions, as well as benefits of the predominant use of inorganic osmolytes for osmotic adjustment, in barley. Another finding of note was that wild barleys showed rather different strategies dealing with salinity, as compared with cultivated varieties. Then, a large number of wheat (Triticum aestivum and Triticum turgidum) cultivars were screened by using a broad range of physiological indices, to evaluate predictive values of various physiological indices for salinity stress tolerance in wheat cultivars. In general, most of the bread wheats showed better Na+ exclusion that was associated with higher relative yield. Leaf K+/Na+ ratio and leaf and xylem K+ contents were the major factors determining salinity stress tolerance in wheat. Other important traits included high xylem K+ content, high stomatal conductance, and low osmolality. Bread wheat and durum wheat showed different tolerance mechanisms, with leaf K+/Na+ content in durum wheat making no significant contributions to salt tolerance, while the important traits were leaf and xylem K+ contents. These results indicate that Na+ sequestration ability is much stronger in durum compared with bread wheat, most likely as a compensation for its lesser efficiency to exclude Na+ from transport to the shoot. Based on the large screening of barley genotypes for their ability to exclude Na+ for its loading into the xylem and delivery to the shoot, four genotypes contrasting in salinity stress tolerance were selected for further studies of molecular and physiological mechanisms mediating xylem Na+ and K+ loading and linking it with overall plant performance and sequestration of Na+ and K+ in leaf tissues. We report that both leaf and xylem K+/Na+ ratios correlated positively with overall plant salt tolerance after prolonged (3 weeks) exposures to salinity stress. Interestingly, it was Na+ but not K+ content that determined this correlation. At the same time, it was found that accumulation of Na+ in xylem sap in salt-tolerant genotypes (TX9425 and CM72) reached a peak 5 days after salt application and then declined. In contrast, salt-sensitive genotypes were less efficient in controlling xylem Na+ loading and showed a progressive increase in xylem Na+ concentrations. We then used the MIFE (microelectrode ion flux measurement) technique to study some aspects of salt stress signalling and Na loading into the xylem. This was achieved by measuring net fluxes of Ca2+, K+ and Na+ from xylem parenchyma tissue of control- and salt-grown plants in response to a range of known second messengers such as H2O2, ABA, or cGMP. Our results indicate that NADPH oxidase-mediated apoplastic H2O2 production acts upstream of xylem Na+ loading and is causally related to ROS-inducible Ca2+ uptake systems in the xylem parenchyma tissue. ABA was also able, directly or in-directly, to regulate the process of Na+ retrieval from xylem. The above findings were further supported by molecular experiments revealing that salt-tolerant barley genotypes (CPI and CM72) upregulate transcript levels of HvHKT1;5 and HvSOS1 to take up Na+ and transport them to shoot for osmotic adjustment shortly after salt addition. Salinity stress tolerance in durum wheat is strongly associated with plant's ability to control Na+ delivery to the shoot. Two loci, termed Nax1 and Nax2, were recently identified as being critical for this process and were suggested to confer activity of HKT1;4 and HKT1;5 transporters from HKT gene family, respectively. At the functional level these transporters are assumed to actively retrieve Na+ from the xylem thus limiting the rates of Na+ transport from roots to shoots. In this work we show that Nax loci also affect activity and expression levels of SOS1-like Na+/H+ exchanger in both root cortical and stelar tissues. Net Na+ efflux measured from salt-treated stele by non-invasive ion flux measuring MIFE technique declined in the following sequence Tamaroi (parental line) > Nax1 = Nax2 > Nax1:Nax2 lines. This efflux was amiloride (a known inhibitor of Na+/H+ exchanger)-sensitive and was mirrored by net H+ flux changes. SOS1 relative transcript levels were 6 to 10 fold lower in Nax lines compared with Tamaroi. Thus, it appears that Nax loci confer two highly complementary mechanisms, both contributing to reducing xylem Na+ content. One of them is enhanced retrieval of Na+ back into the root stele via HKT, and another one reduced rate of Na+ loading into the xylem via SOS1. It is suggested that such duality may play important adaptive role by providing plant with a greater versatility to respond to changing environment and control Na+ delivery to the shoot. In conclusion, this project has found that different crop species and genotypes adopt different strategies to resist salinity stress. In barley, it was found that the predominant use of inorganic osmolytes contributes to osmotic adjustment in the shoot. While rapid Na+ delivery to the shoot seems to be an effective strategy to ensure normal shoot growth under saline condition, xylem Na+ loading should be tightly controlled and stopped once sufficient amount of Na+ was delivered to the shoot. Salt-sensitive barley cultivars failed to slow down the xylem Na+ loading once osmotic adjustment was achieved, while tolerant genotypes were efficient in controlling this process. Both HvHKT1;5 and HvSOS1 transporters were found to be involved in control of xylem Na+ loading and delivery to the shoot. The above process is also intrinsically linked with NADPH oxidase-mediated apoplastic H2O2 production that acts upstream of xylem Na+ loading and is causally related to ROS-inducible Ca2+ uptake systems in the xylem parenchyma tissue. In wheat, bread and durum wheat can be differentiated by their reliance on Na+ exclusion and Na+ sequestration, respectively. The discovered role of NAX loci as controller of expression level and activity of SOS1-like transporters and existence of two highly complementary mechanisms conferring xylem Na+ loading/retrieval and reported insights into regulation of activity of membrane transporters expressed at xylem parenchyma interface open new prospects of cereal breeding for salinity tolerance.