Interacting MeZFP29 and MebZIPW regulates MeNRT2.2 from cassava responding to nitrate signaling

Cassava is a significant tropical cash crop. MeZFP29 interacting with MebZIPW improves MeNRT2.2, encoding a high-affinity nitrate transporter, through binding to NREs under low nitrate and shows a nitrate-signaling-triggered regulation. Cassava (Manihot esculenta) is a globally significant tropical root crop and exhibits exceptional adaptability to native soil fertility. MeNRT2.2 encodes a high-affinity nitrate transporter in cassava and heterologous overexpression of MeNRT2.2 in Arabidopsis increases nitrate transportation and utilization under nitrogen scarcity. However, the responsive mechanism of MeNRT2.2 to nitrate remains unclear. In this study, we identified a nitrate-responsive fragment of 450 bp located upstream of the start codon of MeNRT2.2, and two potential regulators, MeZFP29 and MebZIPW, of MeNRT2.2. Two regulators specifically bound to nitrate-responsive cis-element (NRE), i.e., TGCATT and CAGATG, in the 450 bp fragment and together significantly stimulated promoter activity. Furthermore, we confirmed the interaction between two regulators in vivo and in vitro via Y2H, BiFC, Co-IP, and GST-pull-down assays. In addition, the distribution of MeZFP29 and its heterodimers (MeZFP&MebZIPW) are determined by nitrate signaling, i.e., in the cytoplasm and nucleus under nitrogen limitation, and predominantly in nucleus under sufficient nitrate. In contrast, MebZIPW consistently localizes to the cytoplasm and nucleus regardless of nitrate conditions. Moreover, overexpression of MeZFP29 in Arabidopsis enhanced growth and chlorophyll content, particularly, under low nitrate conditions, while MebZIPW did not. These findings not only confirm the regulation of MeZFP29 and MebZIPW on MeNRT2.2, but also illustrate the nitrate signaling-triggered promotion and feedback on MeNRT2.2. Our study provides a novel approach to enhancing nitrogen-use efficiency in cassava by modulating the regulators under moderate nitrogen levels as low as possible.