Discussion
The ability of salinity tolerant plants to restrict the transporter and accumulations of Na+ in leaf tissues and thereby avoid the effects of toxic Na+ ion stress is among the most remarkable salt tolerance traits (L. Li et al., 2001). Plants have evolved physiological and biochemical mechanisms to adapt to salinity stress and many genes are involved in mediating the root-to-shoot translocation of Na+. In the present study, thesitl1 showed significantly improved tolerance to salinity stress due to reduced Na+ uptake in roots and xylem sap (Figure 5). This reduced Na+ concentrations might explain, in part, that relatively lower Na+ amount was translocated to aerial shoots of the sitl1 , which results in decreased H2O2 accumulation and a salinity insensitive phenotype (Figure 4, 5). In order to gather clues into the mechanistic basis for the observed salinity tolerance of thesitl1 , we explored changes in relative mRNA abundances of marker genes involved in antioxidant enzyme genes, and Na+and K+ transporter genes. The reduced Na+ accumulation in both root and leaf tissues of thesitl1 also resulted in decreased mRNA abundances of most antioxidant enzyme genes under salinity stress condition (Figure 6). However, several genes showed significantly different expression levels between the sitl1 and WT under normal condition. For example, OsCAT1 showed decreased and increased mRNA abundances in roots and leaves, respectively. Furthermore, both Na+transporters, OsHKT1;5 and OsSOS1, showed increased mRNA abundance in root under normal condition. These results indicate that Mg+ deficiency in the sitl1 might result in transcriptional reprograming, which led to changes mRNA abundance of these marker genes.
Salinity stress is known to induce changes in various essential ion elements uptakes such as N, P, K, Ca, Mg, and Mn in plants. In rice, several studies have reported that high level of Na+concentration in soils resulted in decreased ability to uptake Mg2+ due to the suppressive effect of Na+ (Akter & Oue, 2018; Hakim et al., 2014; Munns & Tester, 2008). Similar trends are evident when the sitl1 and WT plants exposed to the salinity stress. As shown in Figure 5, content of K, Mg, and Ca in the roots and leaves were reduced under salinity stress condition. These results indicate that some transporters or channels of these cations might reduce their transport activity or co-transporters such as Na+/Mg2+ or Na+/Ca2+ symporters associated with this trend. To the best of our understanding, Na+ and Mg2+ symporters have not been reported in organisms. However, there is an evidence that some transporter can transport both monovalent and divalent ions. For example, both TaHKT2;1 and OsHKT2;4 exhibited strong K+ permeability in X. laevis oocytes ; however, TaHKT2;1 showed a small Mg2+permeability and the OsHKT2;4 showed Mg2+ and Ca2+ permeability in the absence of competing K+ ions, respectively (Munns & Tester, 2008). In addition, Na+/Mg2+ transporter and Na+/Ca2+ exchanger (AtNCL) have been reported to play an important role in Mg2+ and Ca2+ homeostasis in human and plants, respectively (Horie et al., 2011; Wang et al., 2012). Another possibility is that extracellular Mg2+ level could directly alter Na+ influx through non-selective cation channel (Sontia & Touyz, 2007). Similarly, Mg2+ transporter, OsMGT1, which might enhance the transporter activity of OsHKT1;5 suggests decreased Na+ accumulation to the shoots (Davenport & Tester, 2000). In the present study, we identified OsMTP1 is possible causal mutation in the sitl1 via WGS and RNA-seq analyses (Figure 7). The sitl1 showed reduction of both Na+ and Mg2+ ions in roots, leave and xylem sap (Figure 5). This significant reduction in Mg2+ amount in tissues resulted in reduced root growth and chlorophyll content in leaves under normal growth condition (Figure 1-3). However, the Mg2+ deficient symptoms of thesitil1 were restored to the level like that of WT when this mutant was grown under nutrient solution containing 500 µM of Mg2+ (Figure S4). In addition, the sitl1 showed salinity insensitivity under both nutrient solution and DW containing Na+ (50 mM). Taken together, these results suggest that OsMTP1 is related to transport both ions, Na+ and Mg2+ in plants, resulting in salinity insensitivity and Mg2+ deficiency in the sitl1 .
In the present study, we found a single nucleotide insertion in plasma membrane-localized OsMTP1 containing CorA-like ZntB type cation transporter domain in the sitl1 (Figure 8). Further analyses of gDNA and cDNA sequences confirmed that the mOsMTP1 may not be functional due to the appearance of a premature STOP codon between 1044 and 1045 inmOsMTP1 transcript. In rice, nine members of OsMRS2/MGT homologs of bacterial CorA-type Mg2+ transporter were isolated and examined their Mg2+ transport activity using a yeast complementation assay (Chen et al., 2017). The ability of Mg2+ transport activity was reported for four (OsMRS2-1, OsMRS2-3, OsMRS2-6 and OsMRS2-9) out of nine members have shown Mg2+ transport activity in yeast CM66. Also, OsMRS2-2 (OsMGT1) was identified to have Mg2+transport ability in X. laevis oocytes and plants (Chen et al., 2017; Saito et al., 2013). Among them, OsMRS2-6 showed most effective cell growth rate in the complementation of the yeast CM66 in liquid medium containing 0.1 mM Mg2+ indicating it to be a high-affinity Mg2+ transporter. In the present study, the yeast complementation assay of the OsMTP1 showed effective cell growth rate which suggested that OsMTP1 functions as Mg2+ transporter (Figure 9a). A phylogenetic tree analysis showed that the OsMTP1 protein was clearly distinct from Arabidopsis and rice MRS2 family; however, the OsMRS2-6 is the most closely related member of the OsMTP1 which shares the ability of Mg2+ transport (Figure S10). The ionomic analysis of the sitl1 showed the reduced Mg2+ content but also reduced Na+ which led us to test the ability of Na+ transport. Heterogeneous expression of OsMTP1 showed increased salinity sensitivity based on yeast cell growth however this salinity sensitivity was affected by Mg2+concentration (Figure 9c-h). As shown in Figure 9, increased concentration of Mg2+ in complete media markedly reduced Na+ senility in yeasts. In addition, thesitl1 showed salinity stress tolerance under deionized water containing NaCl supplemented with no Mg2+ condition which indicated that the OsMTP1 is capable to transport Na+ ions but the Na+ transport activity might be affected by Mg2+ concentration.
The characteristic GMN motif located at the end of the first TM domain is conserved in most members of AtMRS2 and OsMRS2, whereas OsMTP1 has altered GIN tripeptide motif (Figure S13). The conserved GMN motif is suggested to be essential to Mg2+ transport ability, thus if the glycine residue of the GMN motif is substituted by alanine, the Mg2+ transport activity of yeast MRS2 transporter was abolished (Chen, Yamaji, Motoyama, Nagamura, & Ma, 2012; Knoop et al., 2005). Indeed, this conserved GMN motif was shared by five members of OsMRS2-1, OsMRS2-2, OsMRS2-3, OsMRS2-6 and OsMRS2-9 which confirmed their Mg2+ transport activity in yeast and rice. However, further evidence for the importance of functional diversity was reported in CorA family. For instance, TmCorA in T. maritina with the GMN motif has been reported to play a role in cobalt transport with no Mg2+ transport activity (Kolisek et al., 2003) and also OsMRS2-7 with GMN motif failed to show the Mg2+transport activity in the yeast complementation assay (Xia et al., 2011). The OsMTP1 and its orthologs in Gramineae contains well-conserved GIN motif in CorA-like ZntB cation transport domain which was reported to associate with Zn2+ and Cd2+selectivity in S. typhimurium . Subcellular localization, yeast complementation, and inomic analyses suggest that a plasma membrane localized OsMTP1 harboring CorA-like ZntB cation transfer domain might have important roles in regulating both Na+ and Mg2+ homeostasis in rice. However, further studies are required to decipher its exact role.