Plant research is ushering in a new era of single-cell transcriptomics. However, a comprehensive single-cell transcriptomic landscape of the whole organism has not been achieved in any plant species.
To generate a comprehensive cell atlas of the model plant Arabidopsis with biological focus on leaf senescence and nutrient allocation, we collected 20 tissues that represent the key developmental stages and transitions throughout the entire life cycle, including two stages of root (6d and 11d after germination), shoot at 6d (cotyledon, SAM and leaf primordia), six stages of the second pair of true leaves (rosette) from expansion to senescence (from 14 to 49d, with 7-day interval), inflorescence stem (apical, middle and basal regions) at 42d, cauline leaf at 42d, three stages of flowers (flower bud to fully opened flowers) at 49d and six successive timepoints of siliques (0 to 5d after anthesis with 1-day interval).
An Arabidopsis single-nucleus atlas decodes leaf senescence and nutrient allocation
1. Summary
Characterizing cell-type composition and functions across life-cycle of the model organism Arabidopsis is crucial for researches in plant biology. Here we generated a comprehensive Arabidopsis single-nucleus transcriptomic atlas using over one million nuclei from tissues encompassing multiple developmental stages. Our analyses identified cell types that have not been characterized in previous single-protoplast studies, and revealed cell-type conservation and specificity across different organs. Through time-resolved sampling, we revealed highly coordinated onset and progression of senescence among the major leaf cell types. We originally formulated two molecular indexes to quantify the ageing state of leaf cells at single-cell resolution. Additionally, facilitated by weighted gene co-expression network analysis (WGCNA), we identified hundreds of novel hub genes that may integratively regulate leaf senescence. Inspired by the functional validation of novel hub genes, we built a systemic scenario of carbon and nitrogen allocation among different cell types from source leaves and sink organs.
Figure 1 A comprehensive cell atlas of Arabidopsis.
A. Schematic illustration of the sampling strategy in this study. A total of 20 tissues (T1-T20) were collected from vegetative growth to reproductive growth, including two seedling stages (6 and 11 DAG) and six different rosette stages (S1-S6) from 14 to 49 DAG. T1-T2: whole root from two stages of seedling (6d and 11d after germination); T3: shoot at 6d (including cotyledon, SAM and leaf primordia); T4: stem (whole stem including apical, middle and basal regions) at 42d; T5: cauline leaf at 42d; T6-T11: six stages of the second pair of true leaves from expansion to senescence (as shown by arrows); T12-T14: three stages of flowers (flower bud to fully opened flowers) at 49d; T15-T20: six successive timepoints of siliques (0 to 5d after anthesis with 1 day interval). Tissues T6-T11 are indicated by arrows in the individuals from S1-S6.
B. Numbers of profiled nuclei and captured genes of each sample. DAG, days post germination. DPA, days post anthesis.
C. UMAP of global clustering of all cells colored by organs.
D. UMAP of single-nucleus atlas of Arabidopsis colored by major cell types.
2. Data description
2.1 Raw data
In total, 20 samples were collected at indicated day post-germination for all tissues including seedling, cotyledon, hypocotyl, root, rosette leaf, stem, lateral leaf, flower, and silique. Then we used an in-house nuclei isolation protocol (see method) and DNBelab C Series Single-Cell Library Prep Set (MGI, 1000021082) for snRNA data generation as previously described (Han et al., 2022). The concentration of DNA library was measured by Qubit (Invitrogen). Libraries were sequenced by DNBSEQ-T7RS.
2.2 Expression matrix
The raw sequencing reads were filtered and demultiplexed by PISA (Version 1.1.0) (https://github.com/shiquan/PISA), and aligned to the TAIR10 reference genome using STAR (version 2.7.4a) (Dobin et al., 2013) with default parameters. Over one million nuclei from 20 samples passed the accepted droplet-based single nuclei filtering criteria with > 200 genes/nucleus. Following the majority of previous studies, we used a more rigorous criteria with > 500 genes/nucleus to obtain high-quality nuclei for downstream analyses. A total of 913,769 nuclei passed our quality control, with 1610 mean genes and 2451 mean UMIs captured in each nucleus. Cells from each of the 20 samples were independently clustered to obtain single-sample maps, then integrated to generate multi-stage organ level maps and a comprehensive all-cell transcriptome atlas.
3. Results
3.1 Root (6 DAG)
UMAP clustering of root (6 DAG) and annotation of cell types. DAG, days after germination.
3.2 Root (11 DAG)
UMAP clustering of root (11 DAG) and annotation of cell types. DAG, days after germination.
3.3 Shoot (6 DAG)
UMAP clustering of shoot (6 DAG) and annotation of cell types.
3.4 Stem
UMAP clustering of stem (42 DAG) and annotation of cell types. DAG, days after germination.
3.5 Cauline
UMAP clustering of cauline (42 DAG) and annotation of cell types. DAG, days after germination.
3.6 Rosette at stage 1 (14 DAG)
UMAP clustering of rosette at stage 1 (14 DAG) and annotation of cell types. DAG, days after germination.
3.7 Rosette at stage 2 (21DAG)
UMAP clustering of rosette at stage 2 (21 DAG) and annotation of cell types. DAG, days after germination.
3.8 Rosette at stage 3 (28DAG)
UMAP clustering of rosette at stage 3 (28 DAG) and annotation of cell types. DAG, days after germination.
3.9 Rosette at stage 4 (35 DAG)
UMAP clustering of rosette at stage 4 (35 DAG) and annotation of cell types. DAG, days after germination.
3.10 Rosette at stage 5 (42 DAG)
UMAP clustering of rosette at stage 5 (42 DAG) and annotation of cell types. DAG, days after germination.
3.11 Rosette at stage 6 (49 DAG)
UMAP clustering of rosette at stage 6 (49 DAG) and annotation of cell types. DAG, days after germination.
3.12 Early flower
UMAP clustering of early flower and annotation of cell types.
3.13 Middle flower
UMAP clustering of middle flower and annotation of cell types.
3.14 Late flower
UMAP clustering of late flower and annotation of cell types.
3.15 Silique (0 DPA)
UMAP clustering of silique (0 DPA) and annotation of cell types. DPA, days post anthesis.
3.16 Silique (1 DPA)
UMAP clustering of silique (1 DPA) and annotation of cell types. DPA, days post anthesis.
3.17 Silique (2 DPA)
UMAP clustering of silique (2 DPA) and annotation of cell types. DPA, days post anthesis.
3.18 Silique (3 DPA)
UMAP clustering of silique (3 DPA) and annotation of cell types. DPA, days post anthesis.
3.19 Silique (4 DPA)
UMAP clustering of silique (4 DPA) and annotation of cell types. DPA, days post anthesis.
3.20 Silique (5 DPA)
UMAP clustering of silique (5 DPA) and annotation of cell types. DPA, days post anthesis.