NRT1.1s and NRT2.1 affect rhizosphere and apoplastic pH during nitrate uptake in Medicago truncatula

Nitrate is the primary source of nitrogen for plants in most environments. Nitrate uptake from the rhizosphere into root cells is mediated by Nitrate Transporter 2 (NRT2) and NRT1.1 proteins via proton symport, with the apoplast serving as an intermediate compartment. Using a simple root immersion system, we investigated the effects of NRT transporters on external media pH (pHmed) and apoplastic pH (pHapo) in M. truncatula nitrate transporter mutants. Monitoring of pHmed showed that loss of NRT2.1 largely prevented nitrate induced alkalization of the media, while loss of NRT1.1A/B reduced media alkalization only during the initial stages of nitrate uptake, suggesting they have fundamentally different physiological roles. Use of the pH sensitive dye HPTS showed that the pHapo of root cells initially decreased during nitrate uptake, and this required NRT1.1A/B. Unexpectedly, the observed changes in pHmed during nitrate uptake in WT and the mutants were not reflected in pHapo. To explain these discrepancies, we propose a model in which the depletion of protons in the media occurring during nitrate symport is offset by the buffering of protons in the cell wall and the activity of the H+-ATPase, allowing a lower pHapo to be maintained. Such a mechanism would enable efficient nitrate uptake across a range of external pH values.