Nitrate reductase is regulated by CIRCADIAN CLOCK-ASSOCIATED1 in Arabidopsis thaliana

Background and aims Nitrate reductase (NR) is highly conserved among plants and has an important role in plant nitrate assimilation pathway. Previous studies show that NR is regulated by circadian clock. However, the molecular mechanism remained elusive. In this study, we tried to find the central circadian clock component which is involved in transcriptional regulation of NR genes, NIA1 and NIA2 in Arabidopsis thaliana . Methods Electrophoresis mobility shift assay (EMSA) was used to test whether recombinant CCA1 protein could directly bind to a fragment of NIA1 and NIA2 promoter and whether the binding requires CCA1 binding site (CBS). Real-time quantitative RT-PCR was used to examine the mRNA expression of NIA1 and NIA2 in CCA1 loss of function mutant ( cca1–1 ) plants contrast with wild type. Results CBSs were found in both upstream regions of NIA1 and NIA2 . EMSA assay confirmed that CCA1 associates with the separate upstream regions of NIA1 or NIA2 in vitro. NIA1 and NIA2 expression analysis in cca1–1 plants showed that NIA1 is activated by CCA1 while NIA2 is repressed by CCA1. Conclusions In summary, our current data provide some molecular evidences that circadian regulation of NIA1 and NIA2 involves action of CCA1 protein binding to the promoters of these genes and regulating their transcription.