Nitrate Signaling by the Regulatory GeneNIT2inChlamydomonas

Abstract

Positive signaling by nitrate in its assimilation pathway has been studied in Chlamydomonas reinhardtii. Among >34,000 lines generated by plasmid insertion, 10 mutants were unable to activate nitrate reductase (NIA1) gene expression and had a Nit À (no growth in nitrate) phenotype. Each of these 10 lines was mutated in the nitrate assimilation-specific regulatory gene NIT2. The complete NIT2 cDNA sequence was obtained, and its deduced amino acid sequence revealed GAF, Gln-rich, Leu zipper, and RWP-RK domains typical of transcription factors and transcriptional coactivators associated with signaling pathways. The predicted Nit2 protein sequence is structurally related to the Nin (for nodule inception) proteins from plants but not to NirA/Nit4/ Yna proteins from fungi and yeast. NIT2 expression is negatively regulated by ammonium and is optimal in N-free medium with no need for the presence of nitrate. However, intracellular nitrate is required to allow Nit2 to activate the NIA1 promoter activity. Nit2 protein was expressed in Escherichia coli and shown to bind to specific sequences at the NIA1 gene promoter. Our data indicate that NIT2 is a central regulatory gene required for nitrate signaling on the Chlamydomonas NIA1 gene promoter and that intracellular nitrate is needed for NIT2 function and to modulate NIA1 transcript levels.

Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism

“…Transcript abundance for the gene encoding NIT2, a transcription factor regulating nitrate metabolism, was increased 6-fold, while that for NAB1, a transcription factor regulating light-harvesting proteins, was decreased 16-fold, consistent with previously described physiological changes in response to N deprivation (Mussgnug et al, 2005;Camargo et al, 2007). The transcript level for the GSP1 mt + gamete-specific transcription factor was decreased 3-fold at the 48-h sample point.…”

Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants

“…We did not find enrichment of the NRE sequence among the NLP7-bound fragments. Moreover, Chlamydomonas NIT2, a RWP-RK protein related to NLP7, also has been shown to bind sequences unrelated to NRE elements 26 , indicating that the RWP-RK proteins are not likely candidates for NRE-binding proteins.…”

A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas

“…This protein, containing GAF and RWP-RK domains, shows conservation with plant nodule inception-like proteins that might function similarly (Camargo et al, 2007;Castaigns et al, 2009). By contrast, the mechanism of negative regulation of NIA1 expression is poorly understood.…”

“…In Chlamydomonas, NIT2 is essential for nitrate assimilation by mediating transcriptional activation of NIA1, the gene encoding NR, in response to nitrate (Schnell and Lefebvre, 1993;Ferná ndez et al, 1998;Camargo et al, 2007). This protein, containing GAF and RWP-RK domains, shows conservation with plant nodule inception-like proteins that might function similarly (Camargo et al, 2007;Castaigns et al, 2009).…”