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 ( ${\text{NO}}_3^{-}$ ) or ammonium nitrate (NH₄NO₃), and possesses both high- and low-affinity uptake systems for ${\text{NO}}_3^{-}$ , 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 ${\text{NO}}_3^{-}$ transport associated with tobacco growth on ${\text{NO}}_3^{-}$ nutrition. Heterologous expression of NtNRT1.1B in the ${\text{NO}}_3^{-}$ uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5 mM ${\text{NO}}_3^{-}$ , suggesting a possible molecular function of NtNRT1.1B in the import of ${\text{NO}}_3^{-}$ 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 ${\text{NO}}_3^{-}$ 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 ${\text{NO}}_3^{-}$ , 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, ${\text{NO}}_3^{-}$ , and even ${\text{NH}}_4^{+}$ in plants supplied with ${\text{NO}}_3^{-}$ ; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term ¹⁵N- ${\text{NO}}_3^{-}$ root influx assays, which showed 15%-20% higher ${\text{NO}}_3^{-}$ deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for ${\text{NO}}_3^{-}$ at a K _m of around 30-45 µM. Together with the detection of NtNRT1.1B promoter activity in the root stele and shoot-stem vascular tissues, and higher ${\text{NO}}_3^{-}$ 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.

Keywords: long-distance transport; nitrate transporter NtNRT1.1B; nitrogen use efficiency; overexpression in tobacco; promoter activity; yeast complementation.