Identity and functional characterisation of the transporter supporting the Na⁺‐dependent high‐affinity NO₃⁻ uptake in Zostera marina L.

Abstract

Zostera marina is a seagrass, a group of angiosperms that evolved from land to live submerged in seawater, an environment of high salinity, alkaline pH and usually very low NO3−. In 2000, we reported the first physiological evidence for the Na+‐dependent high‐affinity NO3− uptake in this plant. Now, to determine the molecular identity of this process, we searched for NO3− transporters common to other vascular plants encoded in Z. marina's genome. We cloned two candidates, ZosmaNPF6.3 and ZosmaNRT2 with its partner protein ZosmaNAR2. ZosmaNAR2 expression levels increase up to 4.5‐fold in Z. marina leaves under NO3−‐deficiency, while ZosmaNRT2 and ZosmaNPF6.3 expressions were low and unaffected by NO3−. NO3− transport capacity, kinetic properties and H+ or Na+‐dependence were examined by heterologous expression in the Hansenula polymorpha high‐affinity NO3− transporter gene disrupted strain (∆ynt1). ZosmaNPF6.3 functions as a H+‐dependent NO3− transporter, without functionality at alkaline pH and apparent dual kinetics (KM = 11.1 µM at NO3− concentrations below 50 µM). ZosmaNRT2 transports NO3− in a H+‐independent but Na+‐dependent manner (KM = 1 mM Na+), with low NO3− affinity (KM = 30 µM). When ZosmaNRT2 and ZosmaNAR2 are co‐expressed, a Na+‐dependent high‐affinity NO3− transport occurs (KM = 5.7 µM NO3−), mimicking the in vivo value. These results are discussed in the physiological context, providing evidence that ZosmaNRT2 is a Na+‐dependent high‐affinity NO3− transporter, the first of its kind to be functionally characterised in a vascular plant, that requires ZosmaNAR2 to achieve the necessary high‐affinity for nitrate uptake from seawater.