Screening of landraces from coastal and saline inland regions identified a number of accessions showing modest to significant salinity tolerance that are distinct from traditionally used donors such as Pokkali and Nona Bokra. These accessions are from diverse backgrounds, including nearly all cultivar-groups of O. sativa and also O. glaberrima. To our knowledge this is the first report of significant salinity tolerance from O. glaberrima, and also from the aromatic cultivar-group of O. sativa. Salinity tolerance in rice thus appears to be widespread both geographically and phylogenetically, or, put in another way, tolerance is not well associated with either geographic or cultivar-group origin. Together with the fact that these are landraces and not expected to show relationships apart from gene flow inherent in the species’ history, this suggests that many of these have probably gained tolerance independently and that multiple mechanisms may thus exist.
On the other hand, tolerance is quite well correlated with leaf Na+ concentration across almost all accessions of both O. sativa and O. glaberrima. Despite the diverse origins and relationships of the accessions, tolerance could in almost all cases be explained largely with reference to lower Na+ concentrations in the photosynthetically active leaves. This further suggests that processes controlling this are the predominant mechanisms of tolerance in O. sativa and O. glaberrima, and that tissue tolerance mechanisms (vacuolar sequestration, ROS scavenging, osmotic adjustment, certain hormonal responses) play secondary roles. Na+ exclusion from roots, sequestration of Na+ in roots, stems and basal portions of the leaf (sheath), partitioning of Na+ from leaf to leaf and dilution of Na+ content in a large biomass are mechanisms proposed to influence leaf Na+ concentration. Na+ sequestration is one such mechanism known to operate in a number of species from both the dicots and monocots (e.g., wild and cultivated barleys: [51, 52]; durum and bread wheat [53, 54]). However, the relationship is not universal. For example, it has not been observed in studies on maize and sorghum ([55–57], although see ) and it is an important  but not a universal determinant in wheat . In some cases this may be due to a lack of genotypic variability . The fact that total leaf and shoot Na+ content (not just concentration) also shows a very strong correlation indicates that Na+ sequestration from the leaf blade is a very important contributor to maintaining low tissue Na+ concentrations.
Dilution of Na+ concentrations through a large biomass is also a well-accepted mechanism for maintaining low tissue Na+ concentrations, and Yeo et al.  concluded that Na+ accumulation (content) showed only a poor correlation with performance in rice, being significantly confounded with plant height; tall varieties showed better tolerance and lower Na+ concentrations due simply to dilution of Na+ in the larger volume of tissue produced. The data presented provide an apparent contradiction to the latter, but this may be due to the screening conditions: the latter study conducted screening at relatively low salinity (60 mM) for short periods (10 days). Salt concentration of 150 mM NaCl was used for the physiological characterisation presented here, which is higher than that used in most previous screening studies; the higher salt concentration causes a much greater influx of Na+, which may overwhelm other mechanisms, notably the effect of plant vigour . Under these conditions, growth effectively ceases in all varieties after the application of the salinity treatment. A few of the most highly tolerant varieties will resume growth after some time, but at a greatly reduced rate; over the lifetime of an experiment, even the most highly tolerant variety will produce only about half a new leaf. This growth arrest actually appears to be an adaptive feature, and lines that try to keep growing show a different type of growth arrest – the youngest leaves expand, but soon yellow and die, presumably due to excessive Na+ accumulation. Thus, screening at higher salinity levels may help to reduce the contribution of biomass to tolerance, and so “simplify” the response in this respect.
It is interesting to note that the correlation of SES scores with plant vigour is highest for leaf sheath biomass (r2 = 0.54), followed by total harvested tissue and root biomass (r2 = 0.47 and 0.46, respectively), but much lower for leaf biomass (r2 = 0.14, 0.20 and 0.36 for leaf 4, 5 and 6, respectively). The leaf sheaths and roots are the main tissues known to act as reservoirs for Na+ sequestration, such as that mediated by OsHKT1;5. Thus, the contribution of biomass may be partly to dilute the Na+ taken up, but also to provide a reservoir for sequestration in non-photosynthetic portions.
One gene known to contribute significantly to Na+
sequestration in rice and other species is HKT1;5
]. Allele mining of this gene revealed seven major allele groups within O. sativa
, and comparison of leaf Na+
concentrations across a number of diverse landraces allows a tentative hypothesis to be proposed as to the relative strength of the various alleles:
It should be noted that the most highly active allele, found in traditional donors such as Pokkali, Nona Bokra and others, has almost certainly originated within the aromatic cultivar-group, despite these being indica types. Indeed, although the sample size and fold changes are small, it seems that the most highly tolerant lines are those from the indica cultivar-group that also possess this Aromatic allele; these are often more tolerant than lines from the aromatic cultivar-group. It may be that some feature of the indica cultivar-group genetic background is in some way synergistic with the action of the Aromatic allele. Alternatively, it has been noted that many aromatic lines (according to the functional definition) have lower salt tolerance due to their inability to produce gamma aminobutyric acid – the same mutation that confers their aromaticity . Although the aromaticity of most of the lines in this study hasn’t been tested, it may be that the HKT1;5 allele from traditional aromatic lines has evolved higher activity to compensate for this deficiency and, when transferred into other genetic backgrounds, its full effect is seen.
In many cases low tissue Na+ concentrations (and therefore tolerance) can be largely explained by the apparent relative activity of the particular HKT1;5 allele present in a line. This suggests that it is not sufficient to declare a line as a major new donor of tolerance without first determining the HKT1;5 allele present, and this should be a component of future screening efforts. However, several exceptions do exist and the association of low leaf Na+ concentration with the HKT1;5 allele is not as tight as that for SES score. Examples of these exceptions include accessions such as Carolina Gold (from Peru, tropical japonica cultivar-group, Japonica allele of HKT1;5), Gachia (Bangladesh, aromatic cultivar-group, Japonica allele) and several accessions from the Philippines and China (indica cultivar-group, Hasawi allele). These all possess much lower tissue Na+ concentrations and higher tolerance than would be predicted from their HKT1;5 allele. Likewise, tolerant O. glaberrima lines showed very low leaf Na+ concentrations, yet all share an OgHKT1;5 allele with several accessions that are manifestly not tolerant and have quite high leaf Na+ concentrations (data not shown); thus, it seems likely that these are also using some other mechanisms apart from OgHKT1;5 that are, nonetheless, highly effective. Also, varieties from Iran and Turkey would fit in this category. Although these mostly possess the Aromatic allele of OsHKT1;5, they appear to possess an additional mechanism that limits the amount of Na+ entering the root (as opposed to reducing the amount of Na+ translocated to the shoot) and so, unlike varieties such as Pokkali and FL478, they possess both low shoot and low root Na+ concentrations (Figure 8). Thus, in all these cases it seems likely that alternative mechanisms besides Saltol/OsHKT1;5 (for example, reduced transpirational bypass flow, alternative sequestration mechanisms) are contributing to a reduction in shoot Na+ content and concentration.
Thus, while maintaining low tissue Na+ concentrations appears to be the predominant trait conferring tolerance in most rice genotypes, the actual mechanisms conferring low tissue Na+ concentration may be quite diverse. Genetic evidence from multiple QTL studies (e.g. [20, 22]) shows that while HKT1;5 contributes a major QTL for Na+ exclusion, a number of other minor QTLs also exist. The FL478 × Hasawi F2 population presented here also suggests that these mechanisms can be alternately separated and combined genetically using molecular markers. Hasawi is a landrace from Saudi Arabia that shows intermediate tolerance and tissue Na+ concentrations (Wei et al. in preparation, current data). It is in the aus cultivar-group, and is expected to contain QTLs/mechanisms distinct from those found in traditional donors from India and Bangladesh, such as Pokkali, the presumed tolerant donor for FL478 [60, 61]. Examination of an F2 population derived from these parents showed transgressive segregation in both the tolerant and sensitive directions. This strongly suggests that the mechanisms present in the two parental lines are distinct and can be combined to produce plants with even higher tolerance. Thus, although maintenance of low tissue Na+ concentrations appears to be the predominant mechanism of tolerance in O. sativa and probably O. glaberrima, there appear to be many mechanisms by which this can be achieved, and these mechanisms are possibly additive.