Comparing essentiality of SOS1-mediated Na+ exclusion in salinity tolerance between cultivated and wild rice species

Soil salinity is a major constraint that affects plant growth and development. Rice is a staple food for more than half of the human population but is extremely sensitive to salinity. Among the several known mechanisms, the ability of the plant to exclude cytosolic Na+ is strongly correlated with salinity stress tolerance in different plant species. This exclusion is mediated by the plasma membrane (PM) Na+/H+ antiporter encoded by Salt Overly Sensitive (SOS1) gene and driven by a PM H+ -ATPase generated proton gradient. However, it is not clear to what extent this mechanism is operational in wild and cultivated rice species, given the unique rice root anatomy and the existence of the bypass flow for Na+ . As wild rice species provide a rich source of genetic diversity for possible introgression of abiotic stress tolerance, we investigated physiological and molecular basis of salinity stress tolerance in Oryza species by using two contrasting pairs of cultivated (Oryza sativa) and wild rice species (Oryza alta and Oryza punctata). Accordingly, dose- and age-dependent Na+ and H+ fluxes were measured using a non-invasive ion selective vibrating microelectrode (the MIFE technique) to measure potential activity of SOS1-encoded Na+/H+ antiporter genes. Consistent with GUS staining data reported in the literature, rice accessions had (~4–6-fold) greater net Na+ efflux in the root elongation zone (EZ) compared to the mature root zone (MZ). Pharmacological experiments showed that Na+ efflux in root EZ is suppressed by more than 90% by amiloride, indicating the possible involvement of Na+/H+ exchanger activity in root EZ. Within each group (cultivated vs. wild) the magnitude of amiloride-sensitive Na+ efflux was higher in tolerant genotypes; however, the activity of Na+/H+ exchanger was 2–3-fold higher in the cultivated rice compared with their wild counterparts. Gene expression levels of SOS1, SOS2 and SOS3 were upregulated under 24 h salinity treatment in all the tested genotypes, with the highest level of SOS1 transcript detected in salt-tolerant wild rice genotype O. alta (~5–6-fold increased transcript level) followed by another wild rice, O. punctata. There was no significant difference in SOS1 expression observed for cultivated rice (IR1-tolerant and IR29-sensitive) under both 0 and 24 h salinity exposure. Our findings suggest that salt-tolerant cultivated rice relies on the cytosolic Na+ exclusion mechanism to deal with salt stress to a greater extent than wild rice, but its operation seems to be regulated at a post-translational rather than transcriptional level

Evolutionary significance of NHX family and NHX1 in salinity stress adaptation in the genus Oryza

Rice (Oryza sativa), a staple crop for a substantial part of the world’s population, is highly sensitive to soil salinity; however, some wild Oryza relatives can survive in highly saline environments. Sodium/hydrogen antiporter (NHX) family members contribute to Na+ homeostasis in plants and play a major role in conferring salinity tolerance. In this study, we analyzed the evolution of NHX family members using phylogeny, conserved domains, tertiary structures, expression patterns, and physiology of cultivated and wild Oryza species to decipher the role of NHXs in salt tolerance in Oryza. Phylogenetic analysis showed that the NHX family can be classified into three subfamilies directly related to their subcellular localization: endomembrane, plasma membrane, and tonoplast (vacuolar subfamily, vNHX1). Phylogenetic and structural analysis showed that vNHX1s have evolved from streptophyte algae (e.g., Klebsormidium nitens) and are abundant and highly conserved in all major land plant lineages, including Oryza. Moreover, we showed that tissue tolerance is a crucial trait conferring tolerance to salinity in wild rice species. Higher Na+ accumulation and reduced Na+ effluxes in leaf mesophyll were observed in the salt-tolerant wild rice species O. alta, O. latifolia, and O. coarctata. Among the key genes affecting tissue tolerance, expression of NHX1 and SOS1/NHX7 exhibited significant correlation with salt tolerance among the rice species and cultivars. This study provides insights into the evolutionary origin of plant NHXs and their role in tissue tolerance of Oryza species and facilitates the inclusion of this trait during the development of salinity-tolerant rice cultivars

Proto Kranz-like leaf traits and cellular ionic regulation are associated with salinity tolerance in a halophytic wild rice

Species of wild rice (Oryza spp.) possess a wide range of stress tolerance traits that can be potentially utilized in breeding climate-resilient cultivated rice cultivars (Oryza sativa) thereby aiding global food security. In this study, we conducted a greenhouse trial to evaluate the salinity tolerance of six wild rice species, one cultivated rice cultivar (IR64) and one landrace (Pokkali) using a range of electrophysiological, imaging, and whole-plant physiological techniques. Three wild species (O. latifolia, O. officinalis and O. coarctata) were found to possess superior salinity stress tolerance. The underlying mechanisms, however, were strikingly different. Na+ accumulation in leaves of O. latifolia, O. officinalis and O. coarctata were significantly higher than the tolerant landrace, Pokkali. Na+ accumulation in mesophyll cells was only observed in O. coarctata, suggesting that O. officinalis and O. latifolia avoid Na+ accumulation in mesophyll by allocating Na+ to other parts of the leaf. The finding also suggests that O. coarctata might be able to employ Na+ as osmolyte without affecting its growth. Further study of Na+ allocation in leaves will be helpful to understand the mechanisms of Na+ accumulation in these species. In addition, O. coarctata showed Proto Kranz-like leaf anatomy (enlarged bundle sheath cells and lower numbers of mesophyll cells), and higher expression of C4-related genes (e.g., NADPME, PPDK) and was a clear outlier with respect to salinity tolerance among the studied wild and cultivated Oryza species. The unique phylogenetic relationship of O. coarctata with C4 grasses suggests the potential of this species for breeding rice with high photosynthetic rate under salinity stress in the future

Sodium sequestration confers salinity tolerance in an ancestral wild rice

Wild rice Oryza rufipogon, a progenitor of cultivated rice Oryza sativa L., possesses superior salinity tolerance and is a potential donor for breeding salinity tolerance traits in rice. However, a mechanistic basis of salinity tolerance in this donor species has not been established. Here, we examined salinity tolerance from the early vegetative stage to maturity in O. rufipogon in comparison with a salt-susceptible (Koshihikari) and a salt-tolerant (Reiziq) variety of O. sativa. We assessed their phylogeny and agronomical traits, photosynthetic performance, ion contents, as well as gene expression in response to salinity stress. Salt-tolerant O. rufipogon exhibited efficient leaf photosynthesis and less damage to leaf tissues during the course of salinity treatment. In addition, O. rufipogon showed a significantly higher tissue Na+ accumulation that is achieved by vacuolar sequestration compared to the salt tolerant O. sativa indica subspecies. These findings are further supported by the upregulation of genes involved with ion transport and sequestration [e.g. high affinity K+ transporter 1;4 (HKT1;4), Na+/H+ exchanger 1 (NHX1) and vacuolar H+-ATPase c (VHA-c)] in salt-tolerant O. rufipogon as well as by the close phylogenetic relationship of key salt-responsive genes in O. rufipogon to these in salt-tolerant wild rice species such as O. coarctata. Thus, the high accumulation of Na+ in the leaves of O. rufipogon acts as a cheap osmoticum to minimise the high energy cost of osmolyte biosynthesis and excessive ROS production. These mechanisms demonstrated that O. rufipogon has important traits that can be used for improving salinity tolerance in cultivated rice

Leaf mesophyll K+ and Cl- fluxes and reactive oxygen species production predict rice salt tolerance at reproductive stage in greenhouse and field conditions

Extensive research on salinity tolerance in rice has been mostly carried out at the seedling stage in single experimental trials. Here, we aimed to understand the roles of ion transport and oxidative responses of leaf mesophyll in salinity tolerance of rice (Oryza sativa L.) at its reproductive stage using comparative investigations in both greenhouse and field trials. Two experimental trials were conducted to assess the salt tolerance of three rice cultivars at their reproductive stage in greenhouse and field. We employed agronomic, physiological, electrophysiological, molecular and cell imaging techniques to compare physiological response of control and salinity stressed rice plants. Salinity had the most severe effect to Koshihikari, followed by Doongara and Reiziq. We found that K+ retention and low recovery Cl− efflux in mesophyll cells confers salt tolerance in rice. Moderate to strong correlations were found between growth parameters and net K+ flux (r2=0.45–0.60), and net Cl− flux (r2=0.47–0.72). Also, dynamic ROS production and regulation of the NADPH oxidase gene, OsRBOHD, in mesophyll cells is crucial for salt tolerance of rice at the reproductive stage. OsRBOHD expression was significantly correlated to recovery ion fluxes (r2=0.45–0.64). This study brings together, for the first time, potential links between cellular ionic stress and oxidative stress components of salinity tolerance in rice at the reproductive stage in both greenhouse and field conditions. Our study will provide guidance to examine crop salinity tolerance at reproductive stages in controlled environments and natural climatic conditions in the future