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

Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate ( ) or ammonium nitrate (NHNO), 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 with a 1,785-bp coding sequence exhibited a function in mediating transport associated with tobacco growth on nutrition. Heterologous expression of in the uptake-defective yeast enabled a growth recovery of the mutant on 0.5 mM , suggesting a possible molecular function of 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 NtNRT1.1B. Furthermore, promoter activity assays using a GFP marker clearly indicated that 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 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 N- root influx assays, which showed 15%-20% higher deposition in -overexpressors as well as a high affinity of NtNRT1.1B for at a of around 30-45 µM. Together with the detection of promoter activity in the root stele and shoot-stem vascular tissues, and higher in both xylem exudate and the apoplastic washing fluid of -transgenic lines, 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.