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

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 (K_M = 11.1 µM at NO₃⁻ concentrations below 50 µM). ZosmaNRT2 transports NO₃⁻ in a H⁺-independent but Na⁺-dependent manner (K_M = 1 mM Na⁺), with low NO₃⁻ affinity (K_M = 30 µM). When ZosmaNRT2 and ZosmaNAR2 are co-expressed, a Na⁺-dependent high-affinity NO₃⁻ transport occurs (K_M = 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.