Fig. 1.

scRNA-seq and lcmRNA-seq reveal cell clusters involved in SDX development. A Schematic of the workflow for lcmRNA-seq of libriform fibers, vessel elements, and ray parenchyma cells (shown as black area) and scRNA-seq of SDX protoplasts. For SDX protoplast isolation, the stems of greenhouse-grown P. trichocarpa were processed by bark peeling and cell-wall enzyme digestion. B Ten cell clusters, Ptr1 to Ptr10, were obtained through unsupervised K-means clustering and visualized by UMAP. V, vessel element. FuIP, fusiform intermediate precursor. RO, ray organizer. FuEP, fusiform early precursor. RP, ray precursor. FuO, fusiform organizer. F, libriform fiber. R, ray parenchyma cell. C–N LCM for three cell type collection and their transcriptomic correlations to scRNA-seq results. Real transverse or tangential sections of P. trichocarpa stems for libriform fiber (red area) (C), vessel element (blue area) (G), and ray parenchyma cell (pink area) (K) collection. Scale bars, 200 μm. The blue and pink rectangles at the left bottom in G and K are the magnified vessel elements and ray parenchyma cells. Scale bars, 25 μm. The illustrations for three cell type collection from transverse and tangential perspectives (D, E, H, I, L, M). In E and I, the white box represents a vessel element with empty lumen. In L, the pink box with dashed lines is the area for ray parenchyma cell collection. The other dashed line box indicates the potential contamination caused by collecting the ray parenchyma cells not from the middle of a section. Such dashed line box contains dark-gray area as ray parenchyma cells and light-gray area as the neighbor libriform fibers shown as the red asterisk. In K–M, the white and gray arrows indicate the collected and avoided ray parenchyma cells, respectively. The transcriptomic correlations of libriform fibers (F), vessel elements (J), and ray parenchyma cells (N) to the single-cell transcriptomes, respectively. O–R The transcript abundance of HD-ZIP III (O), ANT (P), VND6 (Q), and scUPlcmUP genes in ray parenchyma cells (R) in scRNA-seq results. S The proposed cell lineages during SDX development shown on a schematic transverse section of P. trichocarpa, including ray, fiber, vessel lineages, and a cell-type undetermined fusiform lineage. The SDX protoplast isolation releases around six layers of SDX cells from the stem surface after debarking, so around six layers are labeled with colors. The phloem part removed with tree bark and the inner part (inner than six layers) are labeled as gray. Fusiform organizer (purple), fusiform early precursor (green), fusiform intermediate precursor (brown), vessel element (blue), libriform fiber (red), ray organizer (orange), ray precursor (yellow), and ray parenchyma cell (pink)