Single‐cell RNA sequencing profiles of stem‐differentiating xylem in poplar

Wood formation is a complex developmental process, exhibiting a continuous and iterative growth habit because of the existence of stem cells in the vascular cambium (Smetana et al., 2019). The process is controlled by hierarchical gene regulatory networks that regulate specific pathways. Stem-differentiating xylem (SDX), a wood-forming tissue that is part of the inner vascular cambium, differentiates into different types of cells. However, we currently lack distinct genetic markers defining sub-stages or cell types of the SDX differentiation processes. An understanding of the networks and key genes in specific cell types is important for improved feedstock sustainability. To decipher gene markers and the networks in specific cell types of wood formation tissue, protoplasts were prepared for scRNA-seq, which are obtained from the surface of a freshly debarked stem segment of Populus trichocarpa (Figure 1a–e; Lin et al., 2014). Data were pre-filtered at the cell and gene level, resulting in a pool of 12 466 cells with a median of 1566 genes per cell. The uniform manifold approximation and projection (UMAP) algorithm yielded largely overlapping distributions of cells from each of the biological replicates, indicating a high degree of reproducibility (Figure 1f). These unsupervised analyses grouped SDX cells into 12 cell clusters (Figure 1g). The analysis of cell cycle genes revealed that the clustering was not dominated by cell cycle status (Figure 1h). Looking through the clustering profiles, we found that the specialized cells exhibited relatively distinct transcriptome profiles (Figure 1g). Notably, our gene expression and signature analyses provide fresh insights into cluster-specific properties. We then used the following three strategies to annotate cell types in the SDX. First, we used marker genes identified from several well-established high-resolution transcriptome analyses of poplar. Second, we performed RNA in situ hybridization assays (RISH) for eight cluster-specific genes. Third, marker genes were selected to perform transgenic experiment to assign some cell clusters. The single-cell transcriptome atlas enabled us to identify most of the major cell types and several markers in the cluster cloud (Figure 1i,j). The expression pattern of these genes in RISH not only confirmed the above annotations but also enabled us to assign unknown cell clusters (Figure 1k). POPTR_007G044500 (cluster 1) and POPTR_009G101900 (cluster 4) displayed a higher RNA abundance in vessel cells and approximately no signals in other cells. Obvious RNA signals were detected for POPTR_005G150300 (cluster 2) in ray cells, indicating the cluster 2 belongs to ray cells. RNA signals for POPTR_002G104600 (cluster 7) were mainly observed in the cells that belong to phloem cells. Cluster 0 is composed of cambium cells and expressed marker genes such as PtCRF4 (POPTR-015G023200), PtLBD1 (POPTR-008G043900) and PtAIL5 (POPTR-018G091600); clusters 5, 8, 11 are composed of fibre cells and contained marker genes such as PtWPP2 (POPTR-014G021900) and PtNEV (POPTR-001G406300); clusters 1, 3 and 4 are composed of vessel cells and contained marker genes PtCSLC12 (POPTR-005G146900), PtNAC82 (POPTR-007G099400) and PtWAT1 (POPTR-002G029100). PtWAT1 (POPTR-002G029100) and PtCesA8 (POPTR-004G059600) were identified to be upregulated and expressed in vessel and fibre cells in our clustering analysis. To test the applicability of their promoter for cluster-specific expression, we generated transgenic P. tomentosa expressing β-glucuronidase (GUS) under the control of the PtWAT1 and PtCesA8 promoter (PtWAT1pro::GUS, PtCesA8pro::GUS) and examined GUS staining patterns in various tissues (Figure 1l,m). Generally, GUS staining of the PtWAT1pro::GUS and PtCesA8pro::GUS lines displayed vessel and fibre specificity, respectively (Figure 1n,o). This is consistent with their transcriptional signals of cell clusters (Figure 1p,q). We also identified several novel marker genes that were highly and specifically enriched in their respective clusters. To order cells along a reconstructed “trajectory” of SDX cell differentiation, Monocle 2 and STREAM algorithms were employed to disentangle and visualize complex branching trajectories. The vascular cambium is composed of two cell types including fusiform initials and ray initials, and cambium cells divide periclinally inwards to xylem mother cells and outwards to phloem mother cells. The SDX tissue differentiation is essential to understand the wood formation process. It is interesting to note that when considering all clusters of SDX together (phloem cells and unknown clusters were not included), the result revealed that cluster 0 (cambium cells) topologically bifurcated into two trajectories (Figure 1r). A similar output was observed by employing the STREAM software (Figure 1s). The vascular cambium has typically two morphologically distinct types of initials: the axillary elongated fusiform initials that will lead to the formation of the axial system (including vessel and fibre cells) and the smaller isodiametrical ray initials giving rise to the radially orientated parenchymatous rays, supporting that, one trajectory demonstrated gradual transitions from cells of cambium, to fibre and vessel cells and the other from cambium, to fibre and vessel cells and ray cells. To study the evolutionary effects of polyploidy on the transcriptional network underlying wood formation, we reanalysed the functional genomic and transcriptome data for a large number of duplicated gene pairs formed by ancient polyploidy events in poplar (Sundell et al., 2017). Of the 2932 WGD pairs in our marker gene set, we identified 1142 paralogs that were expressed in the same cell type/cluster, suggesting the regulatory divergence contributes to the process of cambial growth and wood formation. TFs were more central in the network than other genes and may serve essential functional roles in cambial growth and wood formation. Notably, compared with the WGD-derived TFs, other WGD paralogous pairs showed more narrow transcription profiles with expression restricted to the same cell type/cluster (Figure 1t; P = 1.450297e-10; Fisher’s exact test). This may reflect the existence of strong genetic control over the TFs, as they perform central roles in regulatory networks. Altogether, the profiling of distinct cell-type-specific transcription in multicellular organisms is essential for elucidating unique developmental regulators and functional genes that give cells their distinctive forms and functions. The data presented here show that profiling of SDX using high-throughput scRNA-seq of thousands of cells offers an unparalleled view of the high heterogeneity and provide key regulators in the wood formation process. This work was supported by the Project of the National Natural Science Foundation of China (Nos. 31872671, 32022057 and 31972954) and the 111 Project (No. B20050). The authors declare no competing financial interest. D.Z. designed the experiments; J.X., M.L. and Y.J. performed the research; J.X. wrote the paper; X.L. revised the manuscript and D.Z. obtained funding and is responsible for this article. All authors read and approved the final manuscript.