Skip to main content

Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus

A Correspondence to this article was published on 03 April 2024

The Original Article was published on 09 January 2023

The Original Article was published on 22 November 2021


Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.

Main text

The application of single-cell RNA sequencing (scRNA-seq) opens a new era for scientists to explore dynamic cell trajectories. One of the biggest challenges of scRNA-seq analyses is the cell type annotation. The first step of scRNA-seq involves cell isolation from the tissue/organ of interest to obtain their transcriptomic profiles. Such isolation usually leads to the loss of morphological information, which complicates subsequent cell type annotation. The use of marker genes is the most common strategy for cell type annotation in scRNA-seq analyses [1]. In plants, numerous marker genes identified by high-throughput spatiotemporal manners in Arabidopsis were used for the cell type annotation [2, 3]. This high-throughput profiling remains largely unexplored in almost all other non-Arabidopsis plant species, which restricts the availability of reliable marker genes.

A common strategy for cell type annotation in non-Arabidopsis species is the use of the orthologs of the marker genes from Arabidopsis, which heavily relies on the functional conservation between Arabidopsis and other species. However, many studies have reported the discrepancy on tissue development and orthologous gene expression patterns between Arabidopsis and other species [4, 5]. Take xylem development for example, Arabidopsis lacks one xylem cell type (ray parenchyma) and the development of another xylem cell type (libriform fiber) is incomplete with respect to woody eudicots [6]. The expression patterns of many genes involved in xylem development are also different between Arabidopsis and other woody species, such as Populus [4,5,6]. This functional and developmental diversity underscores the risks of using Arabidopsis marker genes for cell type annotation in woody eudicots.

During xylem development, stem cells (initials) proliferate into proximal cambium to produce differentiating xylem with two architectural systems, axial system (growing upward and downward) and radial system (growing inward and outward) [7]. Each system possesses its own stem cells as fusiform initials and ray initials (Fig. 1A–C). These initial cells and their proliferating descendant cells are called vascular cambium (Fig. 1B–C). Differentiating xylem is composed of three cell types as vessel elements, libriform fibers (both developed from fusiform initials), and ray parenchyma cells (from ray initials) (Fig. 1D). Four recent studies reported the developmental lineages of differentiating xylem in poplar through scRNA-seq analyses [6, 8,9,10] using the differentiating xylem protoplasts isolated by the same pipeline [11]. The first step of such protoplasting pipeline is stem debarking. Anatomical analyses showed an obvious separation of vascular cambium and differentiating xylem after debarking, and the bark contains phloem and vascular cambium (Fig. 1E). The subsequent protoplast isolation then allows the collection of differentiating xylem protoplasts (Fig. 1E) [6, 8,9,10,11]. Such anatomical results after debarking are very consistent among different species grown in various regions, such as North America, East Asia, and Australia [6, 9, 11, 12]. Among the four studies, Tung et al. [6], Chen et al. [10], and Li et al. [9] used anatomical analyses to ensure the debarking effect as the separation of cambium and differentiating xylem (Fig. 1F). In Chen et al. [10], the cambium was located on the bark side based on the anatomical results, but vast majority of the cells in the “cambium region” clusters resided on the debarked stem using the cell type annotation results, showing the challenges in cell type annotation on the basis of the scRNA-seq data. The anatomical analyses of debarking effect were not reported in Xie et al. [8] (Fig. 1F). Upon the use of the same protoplasting pipeline, the scRNA-seq results between Xie et al. [8] and Tung et al. [6] showed extremely high overlapping rates (99.9%, Additional file 1), which demonstrates the highly similar debarking effects between these two studies. The cell type annotation of Xie et al. [8] on debarked stem included cambium and phloem and conflicted to the suggested debarking effect (Fig. 1F, G). These results highlight the importance of careful anatomical inspection of the samples, in which the separation of tissues by peeling provides critical information on the cell types that can be present in the scRNA-seq data.

Fig. 1
figure 1

Xylem anatomy and cell type annotation. AC Schematics of a woody plant (A), the cambium (fusiform and ray initials) on the transverse section (B), and the tangential section (C) of the stem. D Xylem cell morphologies via axial and radial systems. Vessel elements (labeled as vessel) and libriform fibers (labeled as fiber) both derived from fusiform initials. Ray parenchyma cells derived from ray initials. E Debarking and differentiating xylem protoplast isolation. F Anatomical analyses during stem debarking of four previous studies (shown as Tung et al. 2023 [6], Chen et al. 2021 [10], Li et al. 2021 [9], and Xie et al. 2022 [8]). The anatomical data is not available in Xie et al. G Annotated cell types on stem after debarking of four previous studies. H Schematics of laser capture microdissection (LCM) procedure to harvest three cell types for in situ cell transcriptomes, including libriform fiber (red area), vessel element (blue area), and ray parenchyma cell (pink area). I The correlation was obtained by the following two steps: (i) first round correlation analyses between the in situ cell transcriptomes and scRNA-seq transcriptomes. For each cell in scRNA-seq, a correlation coefficient was obtained. (ii) Second round correlation analyses between the results from (i) and the transcript abundance of each marker gene in each cell. The correlation (shown as LCM vs marker genes) were illustrated as the results from second round correlation analyses. F, libriform fibers. V, vessel elements. R, ray parenchyma cells. One, two, and three asterisks represent Student’s t-test p < 0.05, 0.01, and 0.001, respectively. ns, no significant difference. J Inconsistent xylem cell type annotations (shown as black area) (Chen et al. for vessel elements; Li et al. for vessel elements; Xie et al. for libriform fibers, vessel elements, and ray parenchyma cells)

Due to the lack of reliable marker genes in differentiating xylem, Tung et al. [6] generated in situ cell transcriptomes using laser capture microdissection for all three cell types in differentiating xylem (Fig. 1H) to conduct the annotation. Li et al. [9] and Chen et al. [10] used a mixture of marker genes from poplar and Arabidopsis for differentiating xylem cell type annotation. As a result, the marker genes used from both studies (Li et al. [9] for vessel elements; Chen et al. [10] for xylem cells composed of at least vessel elements and libriform fibers) unexpectedly annotated libriform fibers (Fig. 1I). Although Xie et al. [8] used all marker genes from poplar, their marker genes for libriform fibers annotated both libriform fibers and ray parenchyma cells, their vessel element markers annotated all three xylem cell types, and their ray parenchyma marker annotated vessel elements (Fig. 1I). The problems in cell type annotation of these three studies [8,9,10] (Fig. 1J) demonstrate the needs of in situ cell transcriptomes for the cell type annotation.

Because of the cell type annotation differences, four different models were proposed on the differentiating xylem developmental lineages [6, 8,9,10]. Starting from vascular cambium, Tung et al. [6] separated fusiform and ray lineages and further divided fusiform lineages into vessel elements and libriform fibers (Fig. 2), whereas Chen et al. [10] merged the lineages of vessel elements and libriform fibers together (Fig. 2). In the other two studies, Li et al. [9] and Xie et al. [8] both mixed ray parenchyma lineage with libriform fibers or even vessel elements (Fig. 2). Previous anatomical analysis [13, 14] unambiguously showed that axial and radial systems are architecturally independent, thus rendering the proposed models by Li et al. [9] and Xie et al. [8] almost impossible. Taken together, the model proposed by Tung et al. [6] would be the most plausible one, on the basis of the available scientific data, for explaining differentiating xylem development.

Fig. 2
figure 2

Proposed cell lineages for xylem development. Schematics of proposed developmental cell lineages from vascular cambium to differentiating xylem of the four previous studies (shown as Tung et al. 2023 [6], Chen et al. 2021 [10], Li et al. 2021 [9], and Xie et al. 2022 [8]). Blue color represents vascular cambium. Brown and gray colors represent two distinct developmental lineages

This analysis highlights the necessity of using in situ cell transcriptomes for cell type annotation. Few marker genes from either the source species or the homologs of Arabidopsis would be inadequate for cell type annotation. The inconsistency of the models among the four studies on developmental lineages demonstrates the importance of the incorporation of anatomical knowledge. Mathematical algorithm-based reconstruction using scRNA-seq data may not faithfully describe the real-world biological processes. The combination of anatomical data and in situ transcriptomes coupled with scRNA-seq analyses would lead to a more reliable conclusion.

Availability of data and materials

Sequence data was downloaded from the four previous studies [6, 8-10]. For scRNA-seq data analysis, the raw reads from Tung et al. [6] were downloaded from National Center for Biotechnology Information (NCBI) under accession number GSM5453537. The raw reads from Xie et al. [8] were downloaded from National Genomics Data Center under BioProject accession number PRJCA014789. The raw reads from Li et al. [9] were downloaded from NCBI under BioProject accession number PRJNA703312. The raw reads of wood tissues in Chen et al. [10] were downloaded from National Genomics Data Center under BioProject accession number PRJCA005543. For lcmRNA-seq data analysis, the raw reads from three replicates of libriform fibers, vessel elements, and ray parenchyma were downloaded from NCBI under accession number GSE180121.


  1. Seyfferth C, Renema J, Wendrich JR, Eekhout T, Seurinck R, Vandamme N, Blob B, Saeys Y, Helariutta Y, Birnbaum KD, De Rybel B, et al. Advances and opportunities in single-cell transcriptomics for plant research. Annu Rev Plant Biol. 2021;72:847–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN, et al. A gene expression map of the Arabidopsis root. Science. 2003;302:1956–60.

    Article  CAS  PubMed  Google Scholar 

  3. Denyer T, Ma X, Klesen S, Scacchi E, Nieselt K, Timmermans MCP, et al. Spatiotemporal developmental trajectories in the Arabidopsis root revealed using high-throughput single-cell RNA sequencing. Dev Cell. 2019;48:840–52.

    Article  CAS  PubMed  Google Scholar 

  4. Lu SF, Sun YH, Shi R, Clark C, Li LG, Chiang VL, et al. Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell. 2005;17:2186–203.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Garcia-Lorenzo M, Sjodin A, Jansson S, Funk C. Protease gene families in Populus and Arabidopsis. BMC Plant Biol. 2006;6:30.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Tung CC, Kuo SC, Yang CL, Yu JH, Huang CE, Liou PC, Sun YH, Shuai P, Su JC, Ku C, Lin YJ, et al. Single-cell transcriptomics unveils xylem cell development and evolution. Genome Biol. 2023;24:3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fischer U, Kucukoglu M, Helariutta Y, Bhalerao RP. The dynamics of cambial stem cell activity. Annu Rev Plant Biol. 2019;70:293–319.

    Article  CAS  PubMed  Google Scholar 

  8. Xie JB, Li M, Zeng JY, Li X, Zhang DQ, et al. Single-cell RNA sequencing profiles of stem-differentiating xylem in poplar. Plant Biotechnol J. 2022;20:417–9.

    Article  CAS  PubMed  Google Scholar 

  9. Li H, Dai X, Huang X, Xu M, Wang Q, Yan X, et al. Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus. J Integr Plant Biol. 2021;63:1906–21.

    Article  CAS  PubMed  Google Scholar 

  10. Chen Y, Tong S, Jiang Y, Ai F, Feng Y, Zhang J, et al. Transcriptional landscape of highly lignified poplar stems at single-cell resolution. Genome Biol. 2021;22:319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lin YC, Li W, Chen H, Li Q, Sun YH, Shi R, et al. A simple improved-throughput xylem protoplast system for studying wood formation. Nat Protoc. 2014;9:2194–205.

    Article  CAS  PubMed  Google Scholar 

  12. Bossinger G, Spokevicius AV. Sector analysis reveals patterns of cambium differentiation in poplar stems. J Exp Bot. 2018;69:4339–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Evert RF. Esau's Plant Anatomy: Meristems, Cells, and Tissues of the Plant Body: Their Structure, Function, and Development. Hoboken: John Wiley & Sons, Inc; 2006.

  14. Larson PR. The vascular cambium: development and structure. Berlin: Springer Nature; 1994.

Download references

Review history

The review history is available as Additional file 2.

Peer review information

Wenjing She was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.


Y.J.L. was supported by Young Scholar Fellowship Columbus Program and other programs from Ministry of Science and Technology of Taiwan (MOST) (109–2636-B-002–003, 110–2628-B-002–026, 111–2628-B-002–020 and 111–2311-B-002–021) and National Science and Technology Council (NSTC) (112–2311-B-002–023-MY3). Y.L.C was supported by MOST Young Scholar Fellowship Einstein Program (109–2636-B-006–009, 110–2636-B-006–008, 111–2636-B-006–015 and 112–2636-B-006–006) and Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at National Cheng Kung University.

Author information

Authors and Affiliations



Y.J.L. and Y.L.C. wrote the manuscript. J.A.H. performed data analyses. J.A.H., S.C.K., C.T.K., C.C.T., J.H.Y., T.H.C., C.K., D.Z., J.X., T.M., and Q.L. revised the manuscript. S.C.K. established the analyzing pipeline.

Authors’ information

Twitter handles: @cyl0828 (Ying-Lan Chen); @joweihsieh (Jo-Wei Allison Hsieh); @chuanku_ (Chuan Ku); @YCJimmyLin (Ying-Chung Jimmy Lin).

Corresponding authors

Correspondence to Ying-Lan Chen or Ying-Chung Jimmy Lin.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Supplementary figure S1.

Additional file 2.

 Review history.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, YL., Hsieh, JW.A., Kuo, SC. et al. Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus. Genome Biol 25, 85 (2024).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: