Deep tissue profiling of Populus stem at single nucleus level reveals uncharacterized cell types and cell-specific gene regulatory networks
The vascular cambium is responsible for the production of secondary xylem, which comprises the most abundant form of biomass on earth, wood. Despite its massive global importance, the regulatory networks underlying the production of secondary xylem remain partially ambiguous. Countless studies have used whole-tissue (bulk) RNA sequencing (RNA-seq) to characterize transcriptome changes during wood development in Populus and other tree genera. These studies were limited by their resolution and regulatory inferences. In contrast, novel single-cell RNA sequencing (scRNA-seq) technologies provide opportunities to gain insight into the transcriptional program that controls cellular lineages as they differentiate to form the various cell types in wood. In this study, scRNA-seq was carried out utilizing a pool of 11,673 nuclei derived from two replicates of stem samples of Populus trichocarpa to create a single-cell transcriptome atlas with an unprecedented resolution for a tree species. Cell-type specific marker genes were utilized to annotate 20 cell clusters representing the known cell types within the sampled tissue. Reporter gene assays were performed to confirm the cluster-specific cell-type identity of vessel elements, fibers, ray parenchyma, cambial cells, and sub-cell type vessel-associated cells. The developmental trajectory of cambial cells and their xylem-specific derivates was implemented to identify putative regulators that control distinct developmental lineages involved in vascular development and xylogenesis. Finally, an integration with a protoplast derived, single-cell, whole stem dataset was conducted to compare the proficiency of each methodology to capture the transcriptome. Overall design: We have applied single cell sequencing approach utlizing  a novel protocol to isolate nuclei from complex plant tissues (https://doi.org/10.1371/journal.pone.0251149) and 10X Genomics scRNAseq to study stem tissue of Populus trichocarpa. A growth stage in which the stem is undergoing secondary growth was chosen to enable to the high resolution profiling of each cell type as they produce the most abundant biomass material on earth, wood.