From grass to yeast; functional insights from heterologous expression of LfHKT2;1 in ion regulation

• Heterologous Expression : LfHKT2;1 was heterologously expressed in Saccharomyces cerevisiae mutants (trk1, trk2), restoring growth under hygromycin B stress, indicating its potential role in ion transport. • Ion Uptake Capabilities : The expression of LfHKT2;1 conferred the ability to uptake both sodium (Na+) and potassium (K+), facilitating growth recovery in K+-deficient conditions. • K+ Uptake Activity : Increased concentrations of K+ in the growth medium reduced hypersensitivity to hygromycin in trk1, trk2 mutants expressing LfHKT2;1, demonstrating enhanced K+ uptake activity. • Na+ Hypersensitivity : Hypersensitivity to Na+ was observed in trk1, trk2 mutants expressing LfHKT2;1, suggesting the presence of an additional Na+ uptake system facilitated by the transporter. • Regulation by K+ Availability : LfHKT2;1 expression under galactose-inducible promoter conditions showed higher transcript accumulation at lower K+ concentrations, highlighting its regulation by K+ availability. Saccharomyces cervisceae mutants lacking the major potassium trasnporters trk1 and trk2 show hypersensitivity to aminoglycoside antibiotic hygromycin (hyg). This study demonstrates that expression of the inward K + transporter LfHKT2;1 can suppress this hygromycin sensitivity in the trk1, trk2 double mutant strain. Growth complementation was performed on solid yeast peptone dextrose (YPD) media supplemented with hygromycin B. Both, wild type and trk1 trk2 yeast strains exhibited growth inhibition in the presence of hygromycin. However the potassium uptake-deficient trk1 trk2 strain (control) showed complete growth arrest when exposed to hygromycin, while expression of LfHKT2;1 resulted in growth recovery. Increased sodium concentrations caused cellular toxicity in the trk1 trk2 strain, which was exacerbated by the addition of hygromycin to the media. The hypersensitivity of trk1 trk2 mutant yeast cells expressing LfHKT2;1 to sodium suggested the presence of an additional sodium uptake system on the membrane, which was further confirmed by transient GFP expression assays. These results provide conclusive evidence that heterologous expression of LfHKT2;1 confers both sodium and K + uptake capabilities in hygromycin supplemented YPD media, thereby rescuing the growth of K + transport-deficient S. cerevisiae mutants. This highlights the potential of plant gene expression in yeast as a valuable tool for studying ion transport mechanisms and gene function under stress conditions.