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Physiological basis of differential salinity stress tolerance between cultivated and wild rice species
Soil salinity is a major environmental issue constraining successful crop production that affects more than 20% of irrigated land worldwide. Salinity stress imposes deleterious impacts on crop growth resulting in substantial yield losses. Rice (Oryza sativa L.) is arguably one of the most important staple food crops but is also known as the most salt sensitive amongst the cereals. Salinity stress adversely affects rice growth by reducing water availability, damaging photosynthetic machinery, imposing oxidative stress, and causing Na+ ion toxicity. Salinity stress tolerance in plants is a complex multigenic trait, both genetically and physiologically. Salt tolerance has been characterized in wild rice relatives, but they lost during the process of domestication. In this project, 6 rice cultivars (Oryza sativa L.) and 4 wild rice genotypes (Oryza alta, Oryza barthii, Oryza australiensis, and Oryza punctata) contrasting in their salt tolerance were utilized to investigate the physiological and molecular basis of salinity tolerance in Oryza species, for potential re-introgression of key traits into cultivated O. sativa elite germplasm. All ten rice genotypes were grown in glasshouse and treated with moderate (50 mM NaCl) or acute (100 mM NaCl) salinity stress for three weeks. Salinity stress significantly (P < 0.05) reduced physiological and growth indices. However, the impact of salinity-induced growth reduction differed substantially among genotypes. Interestingly, tolerant genotypes (cultivated and wild rice species) showed better control over gas exchange properties, exhibited higher tissue tolerance, and retained higher K+ concentrations despite higher Na+ accumulation in leaves. Moreover, wild rice genotypes showed better control over Na+ xylem loading and its delivery to shoots with an efficient vacuolar Na+ sequestration for achieving osmotic adjustment. Tolerant wild rice genotypes showed relatively lower and steady xylem sap Na+ concentration over the period of three weeks. Contrary to this, sensitive genotypes managed to avoid initially Na+ loading but failed to accomplish this in the longer term (after three weeks), hence showed higher sap Na+ concentration after three weeks of salt treatment. Conclusively, our results suggest that wild rice genotypes possess an efficient control over xylem Na+ ion loading, relies on tissue tolerance mechanism and allows a rapid osmotic adjustment by using Na+ ion as cheap osmoticum for osmoregulation. As a result, our results showed that salinity tolerance occurs in this sequence: Pokkali > O. alta > O. barthii > IR1 > Nipponbare > H-86 > O. australiensis > Pusa Basmati > O. punctata > IR29. Potassium (K+) is a vital nutrient for maintaining proper functioning and activation of several important enzymes. Maintaining high cytosolic K+/N+ ratios in the cytoplasm is an important determinant of salinity tolerance in different crops. However, it remains elusive whether this trait is also crucial for wild rice species in conferring salinity stress tolerance. To examine the potential role of root K+ retention in salinity tolerance, contrasting pairs of cultivated rice: IR1 (tolerant), IR29 (sensitive) and wild rice species: O. alta (tolerant), O. punctata (sensitive) were established for a better understanding of mechanistic basis of salinity stress tolerance and examined the operation of key different ion transporters/channels mediating ion homeostasis among rice genotypes. Net ion flux (K+, H+ and Ca2+) measurements were evaluated in response to NaCl and ROS (H2O2 and OHˉ) using non-invasive Microelectrode Ion Flux Measuring (MIFE) technique. Our results showed that salinity-induced K+ efflux measured from root elongation zone (EZ) of wild rice group was significantly higher (~2- 3-fold) compared to the root mature zone (MZ). Pharmacological experiments revealed that TEA and GdCl3 markedly suppressed salt-induced K+ efflux (>80% inhibition) measured from EZ of a rice cultivar IR1 suggesting the possible involvement of GORK and NSCC channels in the salinity-induced K+ efflux from the root epidermal cells. Moreover, the causal relationship between H+ and K+ fluxes showed significant difference between the cultivated and wild rice groups regarding NaCl-induced K+ loss and H+-ATPas activity. Cultivated rice group showed relatively lower K+ efflux but greater H+ efflux suggesting that cultivated rice tend to actvate H+-ATPase pumping for Na+ exclusion. Wild rice, however, showed much lower H+-ATPase activity and higher K+ loss. Pharmacological experiments demonstrated that H+ efflux was inhibited by orthovanadate (>90% inhibition) suggesting that H+-ATPase is involved as a major salt-induced H+ efflux in the salt tolerant cultivar IR1. Wild rice species showed significantly higher ROS-induced K+ efflux (2-3-fold) in root EZ than MZ. ROS-induced K+ efflux was greatly inhibited by TEA and GdCl3, a general K+ channel blocker and NSCC blocker, respectively (> 90% inhibition) confirmed the involvement of GORK and NSCC in salt tolerant rice cultivar IR1. While root K+ retention is considered as an important determinant of salinity tolerance for many crops, this was not the case for wild rice. Wild rice showed higher K+ efflux in response to both NaCl and ROS compared to cultivated rice. The reason may be the signalling role of K+, which acts as a "metabolic switch" by inhibiting energy consuming anabolic reactions and allowing energy to be saved for adaptations and repairs, which may provide the advantage to wild rice in salinity-limited conditions.
Another purpose of this study was aimed to evaluate the potential role of cytosolic Na+ exclusion in roots as a mechanistic basis for salinity stress tolerance in different rice species. The plant ability to exclude cytosolic Na+ is strongly correlated with salinity stress tolerance. Hence, dose and age-dependent Na+ and H+ fluxes were measured using electrophysiological protocol known as “recovery protocols” for the potential activity of SOS1-like genes. SOS1-like genes have been shown to mediate Na+ efflux for the cytosolic exclusion of excessive salt from differentially active root epidermal cells. However, their role in salinity tolerance among different rice genotypes remains to be shown. Electrophysiological experiments indicated that salt tolerant rice genotypes had (~4-6-fold) greater net Na+ efflux in root EZ compared to the sensitive genotypes. The activity of plasma membrane (PM) Na+/H+ exchanger was more evident in root epidermis of the tolerant rice genotype IR1. However, H+ flux measured in root MZ was nonsignificant. Our pharmacological experiments confirmed that amiloride suppressed Na+ efflux in root EZ of cultivar IR1 by more than 90% (significant at P < 0.05) indicating the possible involvement of PM Na+/H+ exchanger activity in root elongation zone. Our data suggest cultivated rice utilized cytosolic Na+ exclusion mechanism to greater extent than wild rice in conferring salinity stress tolerance. The genus Oryza includes both cultivated and wild rice relatives which represents a rich and diverse source of genetic variability for salinity tolerance. Wild rice relatives such as O. alta, O. barthii, O. australiensis, and O. punctata showed much genotypic variability of key physiological mechanisms such as Na+ sequestration in vacuoles, greater control over Na+ xylem loading, better control over stomatal conductance and higher tissue tolerance. Despite elite cultivars being focussed only for introgressing Na+ exclusion mechanism, future research for salinity tolerance should be targeted on above mentioned mechanisms which are present in wild rice relatives.
- PhD Thesis