Characterization of a Two-Component High-Affinity Nitrate Uptake System in Arabidopsis. Physiology and Protein-Protein Interaction

Abstract

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mM nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 3 atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mM nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.

Nitrate transporters and peptide transporters

“…In Arabidopsis, there are two NAR2 genes, NAR2.1 (AtNRT3.1, At5g50200) and NAR2.2 (AtNRT3.2, At4g24720). AtNAR2.1 is known to participate in high-affinity nitrate uptake [63,65]. In the nar2.1 null mutant, cHATS is reduced by up to 89%, while iHATS is reduced by up to 96% [65].…”

Environmental nitrate signals through abscisic acid in the root tip

“…6 In the Arabidopsis root, 6 different transporters have been shown to transport nitrate from the environment into root cells. 6,20 Under conditions of low external nitrate, uptake is mediated primarily by the high-affinity transporter, AtNRT2.1, and to a lesser degree by AtNRT2.2 and AtNRT2.4 [21][22][23] ; under nitrate starvation conditions uptake is mediated mainly by AtNRT2.5, with some contribution by AtNRT2.1. 24,25 At high concentrations of environmental nitrate, the dual-affinity transporter AtNPF6.3 (AtNRT1.1) and the low-affinity transporter AtNPF4.6 (AtNRT1.2; AtAIT1) come into play.…”

Reciprocal regulation between AtNRT2.1 and AtAMT1.1 expression and the kinetics of NH4+ and NO3− influxes

“…This gene makes only a small contribution to the NO 3 − HATS, except that when AtNRT2.1 is lost, its contribution increases and results in a partial compensation (Li et al, 2007). NAR2.1/NRT3.1 has been described as a trans-membrane protein that is involved in the NRT2.1 transporter post-translational regulation, but it has no transporter activity in itself (Okamoto et al, 2006;Orsel et al, 2006). Surprisingly, atnar2.1 null mutants display stronger HATS activity reduction than the double mutant atnrt2.1-atnrt2.2 (Orsel et al, 2006).…”