Molecular identification of tobacco NtAMT1.3 that mediated ammonium root-influx with high affinity and improved plant growth on ammonium when overexpressed in Arabidopsis and tobacco

- A functional ammonium transporter NtAMT1.3 was isolated from tobacco.
- Expression of NtAMT1.3 complemented the growth on NH4+ of both yeast and Arabidopsis mutant defective in NH4+-uptake.
- NtAMT1.3 targets to the plasma membrane and mediated NH4+ root-influx with high affinity at Km of ca. 51 μM.
- Transcriptional expression of NtAMT1.3 occurs in both roots and leaves and is upregulated by N-starvation and NH4+.
- Overexpression of NtAMT1.3 improved the growth on low NH4+ of tobacco plants.

Although biological functions of ammonium (NH4+) transporters (AMTs) have been intensively studied in many plant species, little is known about molecular bases responsible for NH4+ movement in tobacco. Here, we reported the identification and functional characterization of a putative NH4+ transporter NtAMT1.3 from tobacco (Nicotiana tabacum). Analysis in silico showed that NtAMT1.3 encoded an integral membrane protein containing 464 amino acid residues and exhibiting 10 predicted transmembrane α-helices. Heterologous functionality study demonstrated that NtAMT1.3 expression facilitated NH4+ entry across plasma membrane of NH4+-uptake defective yeast and Arabidopsis qko mutant, allowing a restored growth of both yeast and Arabidopsis mutant on low NH4+. qPCR assay revealed that NtAMT1.3 was expressed in both roots and leaves and significantly up-regulated by nitrogen starvation and resupply of its putative substrate NH4+ and even nitrate, suggesting that NtAMT1.3 should represent a nitrogen-responsive gene. Critically, constitutive overexpression of NtAMT1.3 in tobacco per se improved obviously the growth of transgenic plants on NH4+ and enhanced leaf nitrogen (15% more) accumulation, consistent with observation of 35% more NH4+ uptake by the roots of transgenic lines in 20 min root-influx test. Together with data showing its plasma membrane localization and saturated transport nature with Km of about 50 μM for NH4+, we suggest that NtAMT1.3 acts an active NH4+ transporter that plays a significant role in NH4+ acquisition and utilization in tobacco.