Functional characterization and intracellular localization of a Lotus tenuis NHX antiporter

Lotus tenuis is a perennial forage legume widely scattered in the flooding Pampa region of Argentina. Even when it is considered a salt-tolerant species, the molecular basis underlying this trait still remains unknown. Intracellular compartmentation of Na+ into vacuoles by NHX antiporters is an important and well known strategy to reduce ion toxicity during salt stress. In this study, the subcellular localization of Lotus tenuis NHX1 was determined by the transient expression of a LtNHX1-GFP fusion protein in Nicotiana benthamiana agroinfiltrated leaves, confirming that it is a tonoplast-located NHX antiporter. To study LtNHX1 contribution to salinity tolerance, a transgenic Lotus tenuis line was obtained by Agrobacterium-mediated transformation. Overexpression of the transgene was confirmed by PCR and RT-PCR analyses. Compared to wild-type, transgenic plants displayed less sensitivity and grew better under high salt stress, with higher survival rates, shoot length, and dry weight. These results suggest that the activity of the tonoplast-located LtNHX1 antiporter contributes to Lotus tenuis salt tolerance. Key message The constitutive expression of a Na+/H+ antiporter gene from the salt-tolerant forage species Lotus tenuis enhanced salinity tolerance. The vacuolar localization of LtNHX1 protein was confirmed.