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.