A genetic screen for nitrate regulatory mutants captures the nitrate transporter gene NRT1.1

Abstract

Nitrate regulatory mutants (nrg) of Arabidopsis (Arabidopsis thaliana) were sought using a genetic screen that employed a nitrate-inducible promoter fused to the yellow fluorescent protein marker gene YFP. A mutation was identified that impaired nitrate induction, and it was localized to the nitrate regulatory gene NLP7, demonstrating the validity of this screen. A second, independent mutation (nrg1) mapped to a region containing the NRT1.1 (CHL1) nitrate transporter gene on chromosome 1. Sequence analysis of NRT1.1 in the mutant revealed a nonsense mutation that truncated the NRT1.1 protein at amino acid 301. The nrg1 mutation disrupted nitrate regulation of several endogenous genes as induction of three nitrate-responsive genes (NIA1, NiR, and NRT2.1) was dramatically reduced in roots of the mutant after 2-h treatment using nitrate concentrations from 0.25 to 20 mm. Another nrt1.1 mutant (deletion mutant chl1-5) showed a similar phenotype. The loss of nitrate induction in the two nrt1.1 mutants (nrg1 and chl1-5) was not explained by reduced nitrate uptake and was reversed by nitrogen deprivation. Microarray analysis showed that nitrate induction of 111 genes was reduced and of three genes increased 2-fold or more in the nrg1 mutant. Genes involved in nitrate assimilation, energy metabolism, and pentose-phosphate pathway were most affected. These results strongly support the model that NRT1.1 acts as a nitrate regulator or sensor in Arabidopsis.

Figure 1.

Nitrate induction of NRP-YFP in wild-type and Mut21 roots. Transgenic seedlings (containing the NRP-YFP construct) grown with ammonium but no nitrate for 4 d were treated with either 20 mm KNO₃ or 20 mm KCl in the presence of 2.5 mm ammonium succinate for 16 h. Fluorescent (A and C) and visible light (B and D) images were captured with a fluorescent microscope to visualize YFP expression.

Figure 2.

Mapping of nrg1 (Mut21). Shows schematic diagrams of the Arabidopsis chromosome 1 showing where nrg1 mapped. Exons are shown in large black boxes. Amino acid and nucleotide changes found in Mut21 are also shown. WT, Wild type.

Figure 3.

Nitrate induction of endogenous genes. Wild-type (WT) and two nrt1.1 mutant seedlings (nrg1 and chl1-5) were grown on 2.5 mm ammonium succinate for 5 d on agarose plates then treated with either 20 mm KNO₃ or 20 mm KCl in the presence of 2.5 mm ammonium succinate for 2 h. Root mRNA levels were determined by qPCR. Error bars represent sd of biological replicates (n = 3).

Figure 4.

Nitrate induction of endogenous genes after 24 h of nitrogen deprivation. Plants were grown and treated as described in the legend to Figure 3, except at day 4 plants were transferred to nitrogen-free medium for 24 h then treated with 20 mm nitrate or chloride for 2 h with no added ammonium succinate. Root mRNA levels were determined by qPCR. Error bars represent sd (n = 3). WT, Wild type.

Figure 5.

Titration of the nitrate induction response. Seedlings were grown 5 d on agarose plates with 2.5 mm ammonium succinate (same as Fig. 3), then treated with various concentrations of KNO₃ or KCl for 2 h in the presence of 2.5 mm ammonium succinate before roots were collected for RNA preparation. NiR mRNA levels were determined by qPCR. Error bars represent sd (n = 3). WT, Wild type.

Figure 6.

Nitrate accumulation. Seedlings grown 5 d with 2.5 mm ammonium succinate were treated with various concentrations of KNO₃ (same as for Fig. 5) in the presence of 2.5 mm ammonium succinate for 2 h. Whole seedlings were then collected for nitrate assays as described in “Materials and Methods.” Error bars represent sd (n = 3). WT, Wild type.