Functional complementation and heterologous overexpression assay in yeast
A complementation assay was performed using the yeast mutant CM66, which lacks two magnesium transporters (ALR1 and ALR2), and CM52 (wild-type) derived from FY833 (Saito et al., 2013). The mutant CM66 yeast cells cannot grow on Mg2+ concentrations of <4mM in culture medium (Winston, Dollard, & Ricupero-Hovasse, 1995). Heterogeneous expression of the OsMTP1 gene in CM66 showed this yeast stain was able to complement growth on both solid and liquid media supplanted with 0.1 and 1 mM Mg2+, whereas expression of the mOsMTP1 gene in CM66 did not alter yeast cell growth at lower levels of Mg2+ compared to the CM66 and EV (Figure 9a,b). This result indicates that heterogeneous expression of OsMTP1 is able to direct Mg2+ uptake into yeast.
The observed salinity insensitive phenotype and the reduced Mg2+ and Na+ concentrations in plant tissues and in xylem sap in the sitl1 led us to hypothesize that the OsMTP1 could have ability to uptake not only Mg2+but also Na+ ion as a function of cotransporter activity (Figure 4-5). We next explored whether heterogeneous expression of the OsMTP1 in WT yeast can reduce cell growth on standard media containing NaCl due to increasing Na+ uptake by OsMTP1 (Figure 9c-h). Indeed, heterologous expression of OsMTP1 showed reduced cell growth rate of yeast due to higher sensitivity to NaCl (0.5 and 1 M) compared to control lines (Figure 9c-f). The OsMTP1 transformants showed no remarkable difference in their growth when medium containing 1 M NaCl along with Mg2+ at a low 0.1 mM concentration (Figure 9g). However, this reduced cell growth of the OsMTP1 transformant could be rescued in the presence of 1 mM Mg2+ (Figure 9h). These results demonstrate that the OsMTP1 protein might play important roles to uptake both Mg2+ and Na+ ions, and the higher concentrations of Mg2+ can compete with the rate of Na+ influx at the plasma membrane.