Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster

Abstract

Using a new Arabidopsis (Arabidopsis thaliana) mutant (Atnrt2.1-nrt2.2) we confirm that concomitant disruption of NRT2.1 and NRT2.2 reduces inducible high-affinity transport system (IHATS) by up to 80%, whereas the constitutive high-affinity transport system (CHATS) was reduced by 30%. Nitrate influx via the low-affinity transport system (LATS) was unaffected. Shoot-to-root ratios were significantly reduced compared to wild-type plants, the major effect being upon shoot growth. In another mutant uniquely disrupted in NRT2.1 (Atnrt2.1), IHATS was reduced by up to 72%, whereas neither the CHATS nor the LATS fluxes were significantly reduced. Disruption of NRT2.1 in Atnrt2.1 caused a consistent and significant reduction of shoot-to-root ratios. IHATS influx and shoot-to-root ratios were restored to wild-type values when Atnrt2.1-nrt2.2 was transformed with a NRT2.1 cDNA isolated from Arabidopsis. Disruption of NRT2.2 in Atnrt2.2 reduced IHATS by 19% and this reduction was statistically significant only at 6 h after resupply of nitrate to nitrogen-deprived plants. Atnrt2.2 showed no significant reduction of CHATS, LATS, or shoot-to-root ratios. These results define NRT2.1 as the major contributor to IHATS. Nevertheless, when maintained on agar containing 0.25 mm KNO(3) as the sole nitrogen source, Atnrt2.1-nrt2.2 consistently exhibited greater stress and growth reduction than Atnrt2.1. Evidence from real-time PCR revealed that NRT2.2 transcript abundance was increased almost 3-fold in Atnrt2.1. These findings suggest that NRT2.2 normally makes only a small contribution to IHATS, but when NRT2.1 is lost, this contribution increases, resulting in a partial compensation.

Figure 1.

Schematic representation of AtNRT2.1 and AtNRT2.2 genes with T-DNA insertions. Gray boxes indicate locations of promoters; black and white boxes represent exons and untranslated regions, respectively. In Atnrt2.1-nrt2.2 mutants (Salk_035429), the T-DNA insertion has deleted all of AtNRT2.2 and part of the AtNRT2.1 gene; in Atnrt2.1 mutants (Salk_141712), the T-DNA insertion is at 236 bp before the putative start codon; in Atnrt2.2 mutants (Salk_043543), the T-DNA insertion is 15 bp upstream of the putative start codon. The diagram is not drawn to scale.

Figure 2.

Real-time PCR expression patterns for AtNRT2.1 and AtNRT2.2 in wild-type (WT), Atnrt2.1-nrt2.2 (A), Atnrt2.1 (B), and Atnrt2.2 (C) plants, respectively. Expression levels of mutants are given relative to wild type (set at 1). Results are the means of two separate experiments using three replicate analyses for each experiment.

Figure 3.

Growth of wild type (WT) and Atnrt2.1-nrt2.2, Atnrt2.1, and Atnrt2.2 mutants on agar containing 2.5 mm KNO₃.

Figure 4.

Growth of wild type (WT) and Atnrt2.1-nrt2.2, Atnrt2.1, and Atnrt2.2 mutants on agar containing 0.25 mm KNO₃.

Figure 5.

Time course of IHATS influx in wild-type and mutant lines following resupply of nitrate to plants nitrogen deprived for 7 d. Plants were grown in 1 mm NH₄NO₃ for 4 weeks and then deprived of nitrogen for 1 week. Plant roots were then exposed to 1 mm KNO₃ for the times shown prior to measuring ¹³NO₃⁻ influx from 100 μm KNO₃. Values shown are means (n = 6) together with ses. •, Wild type; ○, Atnrt2.2; ▴, Atnrt2.1; ▪, Atnrt2.1-nrt2.2.

Figure 6.

Concentration dependence of the HATS influx in wild-type and mutant lines after 6-h resupply of nitrate to plants nitrogen deprived for 7 d. Plants were grown in 1 mm NH₄NO₃ for 4 weeks and then deprived of nitrogen for 1 week. Plant roots were then exposed to 1 mm KNO₃ for 6 h prior to measuring ¹³NO₃⁻ influx at various concentrations of KNO₃. Values shown are means (n = 6) together with ses. •, Wild type; ○, Atnrt2.2; ▴, Atnrt2.1; ▪, Atnrt2.1-nrt2.2.