Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction

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 x 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.