Spatiotemporal Transcriptomic Atlas of Developing Embryos and Vegetative Tissues in Flax

Abstract

Flax (Linum usitatissimum L.) is an important multipurpose crop widely grown for oil and fiber. Despite recent advances in genomics, detailed gene activities during the important reproductive phase of its development are not well defined. In this study, we employed high-throughput RNA-sequencing methods to generate in-depth transcriptome profiles of flax tissues with emphasis on the reproductive phases of five key stages of embryogenesis (globular embryo, heart embryo, torpedo embryo, cotyledon embryo, and mature embryo), mature seed, and vegetative tissues viz. ovary, anther, and root. These datasets were used to establish the co-expression networks covering 36 gene modules based on the expression patterns for each gene through weighted gene co-expression network analysis (WGCNA). Functional interrogation with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of dominantly expressed genetic modules in tissues revealed pathways involved in the development of different tissues. Moreover, the essential genes in embryo development and synthesis of storage reserves were identified based on their dynamic expression patterns. Together, this comprehensive dataset for developing embryos, mature seeds and vegetative tissues provides new insights into molecular mechanisms of seed development with potential for flax crop improvement.

Keywords: development; embryogenesis; flax; spatiotemporal landscape; transcriptome.

Figure 1

Relationship of the transcriptomes in different tissues and stages during embryonic development in flax. (A) Principal component analysis (PCA) using expressed genes in 9 tissues/stages in flax. X-axis, PC1; Y-axis, PC2. The proportion of variance for each principal component is indicated in brackets of axis titles. Three separated groups are labeled with different colors and in different ovals. GE, globular embryo; HE, heart embryo; TE, torpedo embryo; CE; cotyledon embryo; ME, mature embryo. (B) Heatmap of sample distance from different tissues/stages with two replicates (numbered 1 and 2). The grayscale spectrum represents sample distance calculated by R dist function ranging from 0 (black) to 400 (white), indicating high to low correlations, respectively. The hierarchical tree generated using expressed genes in each sample is shown on the left of the heatmap.

Figure 2

The 36 co-expressed gene modules identified through WGCNA. Module eigengene (ME) values were calculated for each tissue/stage. Line plots were generated based on ME values for all 36 modules. X-axis, nine tissues and stages. T1, anther; T2, GE; T3, HE; T4, TE; T5, CE; T6, ME; T7, ovary; T8, seed; T9, root. Y-axis, ME values. Five modules containing anther-specific (ME2), root-specific (ME7), and embryo-specific genes (ME3, 5, 13) were colored with yellow, blue, and green backgrounds, respectively.

Figure 3

Dominant expressed genes in different tissues. (A) Heatmap of expression dynamics of gene members in five clusters (C1 to C5) representing tissue-specific expressed genes is shown. Genes in each cluster are associated with a color on the left of the heatmap. Different tissues and clusters are shown in different colors, as defined in the right inset. Z-score was applied for each row. For specificity, C1, anther; C2, embryo; C3, ovary; C4, seed; C5, root. (B) GO enrichment (biological process, BP) for each dominant gene cluster. A visual representation of GO enrichment for the genes in five tissue-specific clusters with enriched GO terms in the biological process category (on the y-axis) was plotted against each cluster (x-axis). The GO terms with a significant (p < 0.05) are shown and p-values are associated with colors shown in the right inset, from white (high) to blue (low).

Figure 4

Cluster analysis of embryo-development-essential genes in flax. Heatmaps of clustered embryo-development-essential genes with similar expression patterns were generated. Five clusters were identified, shown with different colors as defined in the right inset. Z-score transformations of expression were performed for each gene across all tissues and stages of embryonic development. Expression levels were indicated by color scheme, from red (high) to blue (low) in corresponding tissues/stages, as defined in the inset. Genes in each cluster are associated with a color on the left of each heatmap3.6. Transcriptomic Regulation of Storage Reserve Genes in Flax.

Figure 5

Storage-related lipid genes and carbohydrate gene-expression patterns during embryonic developmental process in flax. Heatmaps of clustered storage-related lipid and carbohydrate genes with similar expression patterns were generated during embryogenesis. Three clusters for each tissue were identified, shown with different colors as defined in the right inset. Z-score transformations of expression were performed for each gene across all developmental stages. Expression levels were indicated by color scheme, from red (high) to blue (low) in corresponding stages, as defined in the inset. Genes in each species or each cluster are associated with a color on the left of each heatmap. Three clusters represent three categories highly expressed in different adjacent stages.

Figure 6

Expression of fatty-acid metabolic genes during embryonic developmental process in flax. Heatmap of the expression level of fatty-acid metabolic genes in five embryo developmental stages. Expression levels calculated using Log₂ transformed normalized counts are indicated by color gradient, from red (high) to grey (low) in the five stages of embryogenesis.