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

Nitrate (NO₃^-) 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 NO₃^- signaling. Nitrate is sensed by the dual affinity NO₃^- 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 (Ca²⁺) as one of the important secondary messengers in relaying the NO₃^- signaling in Arabidopsis. In a previous study, we found that Ca²⁺ regulates nitrogen starvation response in wheat. In this study, 10 days old NO₃^--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 NO₃^- exposure. The use of Ca²⁺ chelator EGTA confirmed the involvement of Ca²⁺ in the regulation of transcription of NPFs and NRTs as well the NO₃^- uptake. We also observed the NO₃^- dose-dependent and tissue-specific regulation of nitrate reductase activity involving Ca²⁺ as a mediator. The participation of Ca²⁺ in the PNR and NO₃^- signaling in wheat is confirmed by pharmacological analysis, physiological evidences, and protoplast-based Ca²⁺ localization.