Calcium regulates primary nitrate response associated gene transcription in a time- and dose-dependent manner.

Nitrate (NO) is the primary source of nitrogen preferred by most arable crops, including wheat. The pioneering experiment on primary nitrate response (PNR) was carried out three decades ago. Since then, much research has been carried out to understand the NO signaling. Nitrate is sensed by the dual affinity NO transceptor NPF6.3, which further relays the information to a master regulator NIN-like protein 7 (NLP7) through calcium-dependent protein kinases (CPK10, CPK30, CPK32), highlighting the importance of calcium ion (Ca) as one of the important secondary messengers in relaying the NO signaling in Arabidopsis. In a previous study, we found that Ca regulates nitrogen starvation response in wheat. In this study, 10 days old NO-starved wheat seedlings were exposed to various treatments. Our study on time course changes in expression of PNR sentinel genes; NPF6.1, NPF6.2, NRT2.1, NRT2.3, NR, and NIR in wheat manifest the highest level of expression at 30 min after NO exposure. The use of Ca chelator EGTA confirmed the involvement of Ca in the regulation of transcription of NPFs and NRTs as well the NO uptake. We also observed the NO dose-dependent and tissue-specific regulation of nitrate reductase activity involving Ca as a mediator. The participation of Ca in the PNR and NO signaling in wheat is confirmed by pharmacological analysis, physiological evidences, and protoplast-based Ca localization.