Identity and functional characterisation of the transporter supporting the Na + -dependent high-affinity NO 3 - 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 NO 3 - . In 2000, we reported the first physiological evidence for the Na + -dependent high-affinity NO 3 - uptake in this plant. Now, to determine the molecular identity of this process, we searched for NO 3 - 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 NO 3 - -deficiency, while ZosmaNRT2 and ZosmaNPF6.3 expressions were low and unaffected by NO 3 - . NO 3 - transport capacity, kinetic properties and H + or Na + -dependence were examined by heterologous expression in the Hansenula polymorpha high-affinity NO 3 - transporter gene disrupted strain (∆ynt1). ZosmaNPF6.3 functions as a H + -dependent NO 3 - transporter, without functionality at alkaline pH and apparent dual kinetics (K M  = 11.1 µM at NO 3 - concentrations below 50 µM). ZosmaNRT2 transports NO 3 - in a H + -independent but Na + -dependent manner (K M  = 1 mM Na + ), with low NO 3 - affinity (K M  = 30 µM). When ZosmaNRT2 and ZosmaNAR2 are co-expressed, a Na + -dependent high-affinity NO 3 - transport occurs (K M  = 5.7 µM NO 3 - ), mimicking the in vivo value. These results are discussed in the physiological context, providing evidence that ZosmaNRT2 is a Na + -dependent high-affinity NO 3 - 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.