Nitrate transiently rewires transcription in developing xylem of Populus wood to promote cell expansion via CRF4.

Nitrate functions both as a nutrient and a signaling molecule, but how nitrate is perceived and translated into developmental outputs within woody tissues is unknown. Here, we show that developing xylem in hybrid aspen (Populus tremula × P. tremuloides) mounts a rapid, cell-type-specific transcriptional response to nitrate that promotes xylem cell expansion. To isolate local nitrate signaling in wood-forming tissues, we delivered nitrate directly into the xylem sap and profiled transcriptomic responses from 2 to 48 h after treatment. Bulk RNA sequencing revealed a transient response peaking within 2-4 h. Single-cell RNA sequencing at the peak response resolved nitrate signaling across developing xylem cell types and showed that nitrate induced cell-type-specific programs linked to nitrogen transport, cell wall remodeling, and developmental progression, while repressing lignin-biosynthetic genes. These data indicate that nitrate acts transiently in wood-forming tissues to fine-tune early xylem development. Among nitrate-responsive transcription factors, CYTOKININ RESPONSE FACTOR 4 (CRF4) emerged as a candidate regulator of this response. CRF4 overexpression in hybrid aspen stimulated xylem cell expansion, whereas simultaneous knock-out of CRF4 and its paralog, CRF3, significantly attenuated the nitrate-induced expansion. Together, our findings identify a local nitrate signaling module in developing wood and establish CRF4 as a component linking nitrate sensing to xylem cell expansion.