Single-cell transcriptomic and cell‑type‑specific regulatory networks in Polima temperature-sensitive cytoplasmic male sterility of Brassica napus L

Abstract

Background: Thermosensitive male sterility (TMS) is a heritable agronomic trait influenced by the interaction between genotype and environment. The anthers of plants are composed of various specialized cells, each of which plays different roles in plant reproduction. In rapeseed (Brassica napus L.), Polima (pol) temperature-sensitive cytoplasmic male sterility (TCMS) is widely used in two-line breeding because its fertility can be partially restored at certain temperatures. The pol-TCMS line exhibits abnormal anther development and pollen abortion at high (restrictive) temperatures (HT, 25 °C) compared to at low (permissive) temperatures (LT, 16 °C). However, the response of different anther cell types to HT and the dynamic regulation of genes under such conditions remain largely unknown.

Results: We present the first single-cell transcriptomic atlas of Brassica napus early developing flower bud tissues in response to HT. We identified 8 cell types and 17 transcriptionally distinct cell clusters via known marker genes under LT and HT treatment conditions. Under HT conditions, changes in the gene expression patterns of different cell clusters were observed, with the number of down-regulated genes in various cell types exceeding that of up-regulated genes. Pseudotime trajectory analysis revealed that HT strongly affected the development of early stamen/anther tissue cells. In combination with the snRNA-seq, WGCNA, and bulk RNA-seq results, we found that many transcription factors play crucial roles in the response to HT, especially heat response family genes.

Conclusions: Our study revealed the transcriptional regulatory network of floral bud tissue in the pol-TCMS line under HT/LT conditions and increased our understanding of high-temperature-induced anther developmental abnormalities, which may help researchers utilize TCMS in the two-line breeding of Brassica plants.

Keywords: Brassica napus; Pol TCMS; Anther; Flower bud; SnRNA-seq; Stamen.

Fig. 1

Identification of cell types in B. napus early flower buds via snRNA-seq. (A) Fertility of B. napus early flower buds at different temperatures. (B) Schematic diagram of snRNA-seq of B. napus early flower buds. (C) Biological replication results of the snRNA-seq data under different temperature treatments. (D) Comprehensive uniform manifold approximation and projection (UMAP) visualization of 17 cell clusters in B. napus early flower buds under LT and HT treatment conditions. Each dot represents a single cell. The color indicates the corresponding cell cluster. (E) Expression patterns of representative cluster-specific marker genes in 17 clusters. The dot diameter indicates the proportion of cells in the cluster that express a given gene. LT: low (permissive) temperature; HT: high (restrictive) temperature

Fig. 2

Identification of B. napus early flower bud expression under different temperature treatments on the basis of snRNA-seq. (A) Comprehensive uniform manifold approximation and projection (UMAP) visualization of 17 cell clusters in B. napus flower buds under different temperature treatments. Each dot represents a single cell. The color indicates the corresponding cell cluster. (B) The number of cells in 17 cell clusters under different temperature treatments. (C) Number of differentially expressed genes (DEGs) in different cell types under HT treatment. (D) Venn diagram analysis of up-regulated differentially expressed genes (DEGs). (E) Venn diagram analysis of down-regulated differentially expressed genes (DEGs). (F) The above represents up-regulated genes in only one cell type and the corresponding enriched pathways. The lower row represents up-regulated genes in more than two cell types and the corresponding enriched pathways. The colored bar represents the z score of − log₁₀ (p value). (G) The above data represent down-regulated genes in only one cell type and the corresponding enriched pathways. The lower row represents down-regulated genes in more than two cell types and the corresponding enriched pathways. The colored bar represents the z score of − log₁₀ (p value)

Fig. 3

Developmental trajectory of different anther cell types in early flower bud tissue. (A) Anatomical diagram of the development of different cell types in plant anthers. (B) Expression patterns of representative early stamen/anther tissue cell cluster-specific marker genes in cluster 16. The dot diameter indicates the proportion of cells in the cluster that express a given gene. (C) Developmental trajectory of epidermal cells and early stamen/anther tissue cells under different temperature treatments. (D) Branching heatmap showing the expression of regulatory genes along pseudotemporal differentiation trajectories of epidermal cells (cluster 1) and early stamen/anther tissue cells (cluster 16). Representative branch-dependent genes and their enriched GO pathways are shown on the right side of the branch heatmap. The color bar indicates the relative expression level

Fig. 4

Identification of key genes via WGCNA and bulk RNA-seq analysis. (A) Number of differentially expressed genes (DEGs) identified via bulk RNA-seq. (B) Gene expression patterns in different cell clusters under different temperature treatments based on WGCNA. (C) Venn diagram showing integrated DEGs in bulk RNA-seq, DEGs in the tapetum cell clusters from snRNA-seq, and genes under the black modules from WGCNA. (D) GO enrichment pathways of up-regulated genes identified via bulk RNA-seq. (E) GO enrichment pathways of down-regulated genes identified via bulk RNA-seq

Fig. 5

Co-expression network construction and TF identification. (A) Expression patterns of transcription factors (TFs) in black modules for different cell types under different temperature treatments. The dot diameter indicates the proportion of cells in the cluster that express a given gene. (B) GO enrichment pathways of down-regulated TFs under HT in the black module. (C) GO enrichment pathways of genes in the black module. (D) Co-expression network construction showing potential hub genes