The Maize ZmbHLH118 Transcription Factor Regulates Vacuolar Nitrate Loading by the NO ₃ ⁻ Transporter ZmCLCa

Nitrate (NO₃ ^-) is a major nutrient promoting plant growth and crop yield. Vacuolar nitrate storage is essential for nitrate acquisition and remobilization within the plant. However, the transcriptional regulation of transporters facilitating nitrate influx into the vacuole remains unclear. Here, we identified a bHLH transcription factor, ZmbHLH118, that negatively regulates the expression of ZmCLCa, thereby modulating nitrate vacuolar loading and uptake in maize (Zea mays). ZmbHLH118 overexpression and ZmCLCa loss-of-function in maize impaired root NO₃ ^- uptake, and further reduced nitrate content and plant growth. Moreover, ZmbHLH118 directly binds to the promoter and inhibits the expression of ZmCLCa, which encodes a tonoplast-localized nitrate transporter. Electrophysiological analysis showed that ZmCLCa mediates NO₃ ^- fluxes across the vacuolar membrane, indicating that ZmCLCa-mediated NO₃ ^- influx is required for vacuolar nitrate storage. Our data identify the ZmbHLH118 as a molecular actor of the transcriptional regulation of ZmCLCa in response to extracellular nitrate. This provides insights into the regulatory mechanisms acting upstream of the CLCa-mediated vacuolar nitrate transport. Finally, field assays showed that the regulation mechanisms of vacuolar nitrate affect maize growth and grain yield, highlighting their value for nitrogen use efficiency improvement in crop plants.