Molecular identification and physiological functional analysis of NtNRT1.1B that mediated nitrate long-distance transport and improved plant growth when overexpressed in tobacco.

Abstract

Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate ( ) or ammonium nitrate (NH4NO3), and possesses both high- and low-affinity uptake systems for , little is known about the molecular component(s) responsible for acquisition and translocation in this crop. Here we provide experimental data showing that NtNRT1.1B with a 1,785-bp coding sequence exhibited a function in mediating transport associated with tobacco growth on nutrition. Heterologous expression of NtNRT1.1B in the uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5mM , suggesting a possible molecular function of NtNRT1.1B in the import of into cells. Transient expression of NtNRT1.1B::green fluorescent protein (GFP) in tobacco leaf cells revealed that NtNRT1.1B targeted mainly the plasma membrane, indicating the possibility of permeation across cell membranes via NtNRT1.1B. Furthermore, promoter activity assays using a GFP marker clearly indicated that NtNRT1.1B transcription in roots may be down-regulated by N starvation and induced by N resupply, including , after 3 days' N depletion. Significantly, constitutive overexpression of NtNRT1.1B could remarkably enhance tobacco growth by showing a higher accumulation of biomass and total N, , and even in plants supplied with ; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term 15N- root influx assays, which showed 15%-20% higher deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for at a K m of around 30-45µM. Together with the detection of NtNRT1.1B promoter activity in the root stele and shoot-stem vascular tissues, and higher in both xylem exudate and the apoplastic washing fluid of NtNRT1.1B-transgenic lines, NtNRT1.1B could be considered as a valuable molecular breeding target aiming at improving crop N-use efficiency by manipulating the absorption and long-distance distribution/transport of nitrate, thus adding a new functional homolog as a nitrate permease to the plant NRT1 family.