An NLP-binding site in the 3' flanking region of the nitrate reductase gene confers nitrate-inducible expression in Arabidopsis thaliana (L.) Heynh.

As a signaling molecule, nitrate induces the expression of multiple genes involved in nitrate assimilation in plants. We previously showed that the expression of the Arabidopsis thaliana (L.) Heynh. nitrite reductase gene (NIR1) was activated by nitrate signaling through an interaction between NODULE INCEPETION-like protein (NLP) transcription factors (NLP1-9) and a nitrate-responsive cis-element (NRE) in the NIR1 promoter. By in vitro binding assays, we have also demonstrated the binding of NLP6 to an NRE-like sequence, designated NIA1-NRE1, in the 3' flanking region of an A. thaliana nitrate reductase gene, NIA1. In our current study, we characterize the role of NIA1-NRE1 in NIA1 expression. Deletion analysis of the 3' flanking region of NIA1 revealed that a 345-bp sequence containing NIA1-NRE1 has a dominant role in nitrate-inducible NIA1 expression. We also found that the 35S minimal promoter fused to the 345-bp sequence, or to NIA1-NRE1, can confer nitrate-inducible expression. Furthermore, the NIA1-NRE1-fused 35S minimal promoter is transactivated by NLP6 in vivo. These results suggest that NIA1-NRE1 alone is sufficient to direct nitrate-responsive gene expression and that nitrate-inducible NIA1 expression is regulated through the interaction between NLPs and NIA1-NRE1. Additionally, we show that the distance between the NRE and the gene under the control of the NRE is not critical for this process.