Cell Type-Specific Differentiation Between Indica and Japonica Rice Root Tip Responses to Different Environments Based on Single-Cell RNA Sequencing

Abstract

Background: As Oryza sativa ssp. indica and Oryza sativa ssp. japonica are the two major subspecies of Asian cultivated rice, the adaptative evolution of these varieties in divergent environments is an important topic in both theoretical and practical studies. However, the cell type-specific differentiation between indica and japonica rice varieties in response to divergent habitat environments, which facilitates an understanding of the genetic basis underlying differentiation and environmental adaptation between rice subspecies at the cellular level, is little known. Methods: We analyzed a published single-cell RNA sequencing dataset to explore the differentially expressed genes between indica and japonica rice varieties in each cell type. To estimate the relationship between cell type-specific differentiation and environmental adaptation, we focused on genes in the WRKY, NAC, and BZIP transcription factor families, which are closely related to abiotic stress responses. In addition, we integrated five bulk RNA sequencing datasets obtained under conditions of abiotic stress, including cold, drought and salinity, in this study. Furthermore, we analyzed quiescent center cells in rice root tips based on orthologous markers in Arabidopsis. Results: We found differentially expressed genes between indica and japonica rice varieties with cell type-specific patterns, which were enriched in the pathways related to root development and stress reposes. Some of these genes were members of the WRKY, NAC, and BZIP transcription factor families and were differentially expressed under cold, drought or salinity stress. In addition, LOC_Os01g16810, LOC_Os01g18670, LOC_Os04g52960, and LOC_Os08g09350 may be potential markers of quiescent center cells in rice root tips. Conclusion: These results identified cell type-specific differentially expressed genes between indica-japonica rice varieties that were related to various environmental stresses and provided putative markers of quiescent center cells. This study provides new clues for understanding the development and physiology of plants during the process of adaptative divergence, in addition to identifying potential target genes for the improvement of stress tolerance in rice breeding applications.

Keywords: Oryza sativa; adaptative evolution; indica-japonica differentiation; quiescent center cells; single-cell RNA sequencing; transcription factor family.

FIGURE 1

(A) UMAP plots showing the cell types from Oryza sativa ssp. indica variety 9311 and Oryza sativa ssp. japonica variety Nipponbare. (B) Cells from Oryza sativa ssp. indica variety 9311 and Oryza sativa ssp. japonica variety Nipponbare after removal of the batch effect. (C) Bar plot showing the number of differentially expressed genes (DEGs) between indica and japonica rice varieties in the eight cell types. Red bar, DEGs that were upregulated in indica variety 9311; blue bar, DEGs that were upregulated in japonica variety Nipponbare. (D) Shared DEGs in different cell types. Red bar, DEGs that were upregulated in indica variety 9311; blue bar, DEGs that were upregulated in japonica variety Nipponbare.

FIGURE 2

Differentially expressed genes (DEGs) between indica and japonica rice varieties in the WRKY (A), BZIP (B), and NAC (C) transcription factor families. (D) Violin plots showing the expression of differentially expressed genes of WRKY, BZIP, and NAC transcription factors between indica and japonica rice varieties in only one specific cell type. Red, indica rice variety 9311l; blue, japonica rice variety Nipponbare. The white point in each violin plot indicates the median of gene expression, and the error bars indicate variations of gene expression. ** indicates a P < 0.01; *** indicates a P < 0.001.

FIGURE 3

(A) Venn plots showing the differentially expressed genes (DEGs) shared during differentiation of indica-japonica, the comparison of cold-sensitive rice under control and cold-stress conditions and the comparison of cold-sensitive and cold-tolerant rice under cold-stress conditions in terms of the cell type in the cortex, endodermis, epidermis, epidermis near the root hair, metaxylem, root cap, root hair, and stele. Red in the pie chart indicates the percentage of DEGs that were upregulated in the indica variety 9311; blue in the pie chart indicates the percentage of DEGs that were upregulated in the japonica variety Nipponbare. (B,C) GOplot showing the indica-japonica DEGs that overlapped with two sets of cold-induced DEGs (belonging to the WRKY, BZIP, and NAC transcription factor families) in each cell type.

FIGURE 4

(A) The GOplot shows the indica-japonica DEGs that overlapped with drought-induced DEGs (belonging to the WRKY, BZIP, and NAC transcription factor families) in each cell type. The red gradient in (B) indicates the percentage of cell types showing differential gene expression between indica and japonica rice varieties.

FIGURE 5

(A) The heatmap in the left panel shows the indica-japonica DEGs that overlapped with salinity-induced DEGs (belonging to the WRKY, BZIP, and NAC transcription factor families) in each cell type. The red gradient indicates the log FC of DEGs that were upregulated in the indica variety 9311; the blue gradient indicates the log FC of DEGs that were upregulated in the japonica variety Nipponbare. Yellow indicates DEGs in the corresponding dataset. (B) the protein-protein interaction analysis of indica-japonica DEGs that overlapped with salinity-induced DEGs (belonging to the WRKY, BZIP, and NAC transcription factor families).

FIGURE 6

(A) UMAP plot of endodermal cells in rice roots showing the expression of 26 putative QC markers, subclusters and distribution of indica and japonica samples. (B) Heatmap showing the dynamic expression of 26 putative QC markers during the developmental trajectory based on single-cell pseudotime in endodermal cells. (C) Dynamic expression of 4 putative QC markers during the developmental trajectory based on single-cell pseudotime in endodermal cells.