Tracing the dynamics of nitrate responses in xylem elements of hybrid aspen wood

Nitrate, beyond being an important plant nutrient, serves as a signaling molecule. During secondary growth, long-term nitrate application stimulates cambial activity and xylem cell expansion and suppresses xylem maturation, but a short-term response to nitrate has remained unclear in these processes. In this study, we explored short-term responses of nitrate on cambial growth 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 selected to examine the cellular specificity of the response. Using microfluidics coupled with single cell RNA-sequencing (scRNA-seq) in protoplasts isolated from the surface of the woody tissue, we found that most xylem cells could sense and respond to nitrate, and that the cell types having the strongest response were the rays and the fusiform precursors. 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 elements and 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.