Single cell sequencing of plants – getting to the root

09.02.2023

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Plant organs are composed of specialized cells, ensuring division of labor between various organs or their parts, and the integrity of the whole organism. Unique transcriptional programs are the basis for the diversity of cell types and thanks to single cell sequencing, it is possible to capture them, to study heterogeneity among and within cell populations and to get insights into the cells’ microenvironment. It is possible to gain in-depth understanding of different cell types, cell states and to discover rare or transient cell populations.

In contrast to the well-established single cell sequencing of mammalian cells, single cell sequencing of plant tissues possesses additional challenges. Main differences are the presence of a cell wall, which hinders tissue separation and acquisition of individual cells, and the larger cell size (1). Protoplast isolation has become the method of choice and has been applied successfully to a number of plant species and tissues (summarized e.g., by 1, 2).

The GEXSCOPE^® Single Cell RNA Library Kit can be used with protoplast suspension to generate a sequencing ready NGS library that is compatible with Illumina sequencing instruments. Singleron’s patented SCOPE-chip^® technology enables the partitioning of single cells by gravity. No pressurized system is necessary, making this technology particularly suitable for fragile cells, such as protoplast.

Plant single cell sequencing with Singleron’s SCOPE-chip technology

Arabidopsis root is an ideal model system for investigating cell type-specific gene expression and cell differentiation in plants (3) and is the most profiled plant tissue at single cell resolution (1). Multiple publications utilized single cell solutions to characterize major cell types, rare cell populations, trajectories of cell differentiation or even transcriptional states in mutant lines (e.g., 4, 5).

Arabidopsis root was used to demonstrate the compatibility of Singleron’s technology with plant samples. Roots of eight-day-old Arabidopsis seedlings were used for protoplast isolation (6). After fluorescence-activated sorting (FACS), GEXSCOPE Single Cell RNA Library Kit Cell V2 was used for single cell NGS library preparation. Pre-processing and mapping were performed with CeleSCOPE^® v1.10.0, using TAIR 10 genome as a reference. Further processing was performed using R package Seurat v4.3.0 (7). 8,878 high-quality cells were identified, which were characterized by median of 1,761 genes per cell and median mitochondrial proportion of 0.33%. After clustering, 19 distinct cell populations were identified. Seven clusters showed a signature of stele cells and included clearly defined populations of phloem and xylem cells. Additionally, a distinct population of root cap cells – characterized by expression of ANAC033 transcription factor – was detected. Populations of trichoblasts and atrichoblasts were identified based on accumulation of GLABRA 2 (GL2) and EXTENSIN 17 (EXT17) transcripts, respectively (Figure 1).

Figure 1: UMAP with cell labels and feature plots of selected marker genes. Major cell types were captured with RNA sequencing of 8,878 single cells. UMAP with cell-type labels (left) and expression of selected cell-type markers (right). Color scale represents log-normalized, corrected UMI counts. Transcription factor ANAC033 is specific to root cap cells, GLABRA 2 (GL2) is a marker gene of atrichoblasts and EXTENSIN 17 (EXT17) is a marker gene of trichoblasts.

Trajectory analysis was performed using Monocle3 (8). The developmental branch from meristematic cells to trichoblasts is highlighted here. Transcript levels of AT5G62330 vanish in meristematic cells, which is followed by transcript accumulation in trichoblasts. Transcript accumulation of METHYL ESTERASE 15 (MES15) is restricted to a narrow time window and to the trichoblast population only.

Figure 2: Trajectory analysis and trichoblast development. Trajectory analysis rooted in the super-cluster of meristematic cells identified three main branches of development, one of them specific to trichoblasts (left). Visualization of pseudotime-dependent gene expression predicts that METHYL ESTERASE 15 (MES15) expression is restricted to short duration and trichoblasts only.

For more information check our AppNote “Single cell sequencing of plants”.

References

1. Bawa, G; et al., (2022). Single-Cell RNA Sequencing for Plant Research: Insights and Possible Benefits. International Journal of Molecular Sciences, 23(9). https://doi.org/10.3390/ijms23094497

2. Shaw, R; et al., (2021). Single-Cell Transcriptome Analysis in Plants: Advances and Challenges. Molecular Plant, 14(1), 115–126. https://doi.org/10.1016/j.molp.2020.10.012

3. Shahan, R; et al., (2022). A single-cell Arabidopsis root atlas reveals developmental trajectories in wild-type and cell identity mutants. Developmental Cell, 57(4), 543-560.e9. https://doi.org/10.1016/j.devcel.2022.01.008

4. Denyer, T; et al., (2019). Spatiotemporal Developmental Trajectories in the Arabidopsis Root Revealed Using High-Throughput Single-Cell RNA Sequencing. Developmental Cell, 48(6), 840-852.e5. https://doi.org/10.1016/j.devcel.2019.02.022

5. Ryu, KH; et al., (2019). Single-cell RNA sequencing resolves molecular relationships among individual plant cells. Plant Physiology, 179(4), 1444–1456. https://doi.org/10.1104/pp.18.01482

6. Bargmann, BOR; et al., (2010). Fluorescence Activated Cell Sorting of Plant Protoplasts. Vis. Exp. (36), e1673, doi:10.3791/1673

7. Hao, Y; et al., (2021). Integrated analysis of multimodal single-cell data. Cell, 184(13), 3573-3587.e29. https://doi.org/10.1016/j.cell.2021.04.048

8. Trapnell, C; et al., (2014). The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Biotechnol. 32, 381-386. https://doi.org/10.1038/nbt.2859

Resources
CeleScope – https://github.com/singleron-RD/CeleScope [accessed 2023-01-28]
The Arabidopsis Information Resource (TAIR) –www.arabidopsis.org [accessed 2023-01-28]

Acknowledgement

We would like to thank Dr. Magdalena Marek (Max-Planck-Genome-Centre Cologne) for preparing the root protoplast suspension and Prof. Dr. Stanislav Kopriva (Institute for Plant Sciences, University of Cologne) for providing plant material for sequencing of Arabidopsis rosettes.

A post by Singleron team