Xylem cells rapidly sense and respond to nitrate to promote expansion

Nitrate, beyond being an important plant nutrient, serves as a signaling molecule. During vascular development, nitrate stimulates xylem cell expansion and suppresses xylem maturation, but the underlying signaling has remained unclear. In this study, we explored nitrate signaling during vascular development by monitoring transcriptomic changes in developing xylem tissues in a time series after administering potassium nitrate directly to the xylem sap in hybrid aspen ( Populus tremula x P. tremuloides ) trees. Gene expression analysis revealed a dynamic response, with gene expression being transiently activated at two hours post-application for genes related to xylem expansion and at four to eight hours for genes related to xylem maturation. The two-hour timepoint was therefore selected to examine nitrate mediated cell expansion in expanding xylem elements isolated from the surface of the woody tissue. Using microfluidics coupled with single cell RNA-sequencing (scRNA-seq), we found that majority of the xylem cells could sense and respond to nitrate, and that the cells having the strongest response included both xylem fibers and vessel elements at the earliest stages of cell expansion. Among the candidate transcription factors mediating the nitrate-driven xylem cell expansion we discovered the Populus CYTOKININ RESPONSE FACTOR 4 (CRF4 ). Overexpression of the P. tremula CRF4 in hybrid aspen mimicked the stimulatory effect of nitrate on xylem cell expansion, supporting the function of CRF4 in nitrate-mediated xylem cell expansion. Taken together, we identified a coordinated, system-wide response to nitrate whereby nitrate is rapidly and transiently sensed in most of the xylem elementsand provide evidence for nitrate being taken up from the xylem sap into the ray parenchyma and regulate early xylem development through transcriptional circuits unique to xylem cells.