Identity and functional characterisation of the transporter supporting the Na⁺ -dependent high-affinity NO₃ ^- uptake...

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.