A genetic screen for nitrate regulatory mutants captures the nitrate transporter gene NRT1.1.
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.
Wang
Rongchen
R
Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0116, USA.
Xing
Xiujuan
X
Wang
Yong
Y
Tran
Amy
A
Crawford
Nigel M
NM
D000483:Alleles; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D009154:Mutation; D009566:Nitrates / Q000378:metabolism*; D009584:Nitrogen / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*
D027321:Anion Transport Proteins; C101492:NRT1.1 protein, Arabidopsis; D009566:Nitrates; D010940:Plant Proteins; D009584:Nitrogen