Extensive transcriptomic and epigenomic remodelling occurs during Arabidopsis thaliana germination

Abstract

Background: Seed germination involves progression from complete metabolic dormancy to a highly active, growing seedling. Many factors regulate germination and these interact extensively, forming a complex network of inputs that control the seed-to-seedling transition. Our understanding of the direct regulation of gene expression and the dynamic changes in the epigenome and small RNAs during germination is limited. The interactions between genome, transcriptome and epigenome must be revealed in order to identify the regulatory mechanisms that control seed germination.

Results: We present an integrated analysis of high-resolution RNA sequencing, small RNA sequencing and MethylC sequencing over ten developmental time points in Arabidopsis thaliana seeds, finding extensive transcriptomic and epigenomic transformations associated with seed germination. We identify previously unannotated loci from which messenger RNAs are expressed transiently during germination and find widespread alternative splicing and divergent isoform abundance of genes involved in RNA processing and splicing. We generate the first dynamic transcription factor network model of germination, identifying known and novel regulatory factors. Expression of both microRNA and short interfering RNA loci changes significantly during germination, particularly between the seed and the post-germinative seedling. These are associated with changes in gene expression and large-scale demethylation observed towards the end of germination, as the epigenome transitions from an embryo-like to a vegetative seedling state.

Conclusions: This study reveals the complex dynamics and interactions of the transcriptome and epigenome during seed germination, including the extensive remodelling of the seed DNA methylome from an embryo-like to vegetative-like state during the seed-to-seedling transition. Data are available for exploration in a user-friendly browser at https://jbrowse.latrobe.edu.au/germination_epigenome .

Keywords: Alternative splicing; Arabidopsis; DNA methylation; Germination; RNA-seq; Small RNA; Transcription factor.

Fig. 1

Overview of the extensive transcriptomic and epigenetic remodelling that occurs during seed germination. a (i) The time course examined in this study. Transcriptomes and sRNAs were analysed at all time points. H denotes freshly harvested seeds, collected before two weeks of dry, dark ripening. DNA methylation was analysed at 0 h (after ripening), 48 h S, 6 h SL, 24 h SL and 48 h SL. (ii) The number of differentially methylated regions (DMRs), sRNAs and genes that were identified and differentially expressed (DE) over germination are shown (as total numbers for all time points combined/compared). b An example of an unannotated DE locus (XLOC_000109), with nearby differential methylation and overlapping sRNAs, as shown in the AnnoJ genome browser

Fig. 2

Alternative splicing of RNA processing genes occurs during germination. a Heatmap of the log ratios of isoform1/isoform2 for the 620 genes with at least two isoforms and average expression greater than 0.1 transcript per million (tpm). Only the genes that had a maximum isoform1/isoform2 ratio of > 2 and a minimum isoform1/isoform2 ratio of < 0.5 over the time course are shown. b Of the 620 genes displaying isoform variation, 612 were differentially expressed during germination. The proportion of these falling into the three clusters compared to the genome is shown (Cluster 1: increasing over time course, highest expression in seedling, Cluster 2: transiently peaking, Cluster 3: decreasing expression over time course with highest expression in seeds). c GO enrichment analysis showing the top three enriched categories. Examples of genes showing variations in isoform expression including (d) SR45, (e) PIF6 and (f) PhyB. The solid lines represent the average of three replicates

Fig. 3

Analysis of DE unannotated loci during seed germination. a Relative expression levels of the 163 DE unannotated loci identified during germination. An enrichment of genes showing transient expression during germination (Cluster 2) is seen. b Top five enriched GO categories of the 66 genes that had significant hits (E < 0.01) following BLAST analysis. c Expression profiles of the four genes encoding proteins involved in developmental processes (MEE5, MEE28, MEE38 and LOM2) are shown. The identifiers of the unannotated loci that are homologous to these genes are shown in brackets (See Additional file 3: Table S2. for chromosomal co-ordinates). d Example of an unannotated locus expressed transiently during germination

Fig. 4

Modelling the TF network controlling germination. Simplified DREM model annotated with TFs based on DAP-seq binding data. Only the top three TFs (strongest associations) and summary of TF families involved are shown. In order to simplify the model only four time points were used to calculate the log2 fold changes of DEGs relative to 0 h: 12 h S, 48 h S, 12 h S L and 48 h S L. Transcriptionally upregulated TFs are coloured in blue, downregulated TFs are shown in red. The full model is presented in Additional file 2: Figure S3

Fig. 5

Characterisation of mutants in predicted germination regulatory TFs for model validation. a Percent of seeds with extruded radicles at 36 h SL. Each data point corresponds to 50 scored seeds from a single parent plant. Horizontal bars indicate the mean for each genotype. Asterisks denote significant differences compared to WT (logistic regression, p < 10⁻⁵). b Number of misexpressed genes at 24 h SL compared to WT (q < 0.01)

Fig. 6

Differential expression of microRNAs over seed germination. a Of the annotated miRNAs, 165 were differentially expressed during germination and their relative expression levels were hierarchically clustered. b Expression profiles of (i) miRNA159a and (ii) miRNA160a-5p and their targets genes, which have been shown to have a role in regulation during seed germination. c Expression profiles of (i) miRNA781a and (ii) miRNA400 (and their target genes), which are known for a role in other (non-germination) conditions/stages in Arabidopsis. These are two of the 19 genes that showed highest expression in the dry seed. d Expression profiles of (i) miRNA851a and (ii) miRNA858a (and their target genes). Note that targets only predicted for miR858a are indicated with (a) next to the AGI. These are two of the five miRNAs showing a transient increased expression during germination before decreasing in abundance at the end of the time course

Fig. 7

Differential expression of sRNAs during seed germination. a Heatmap of sRNA abundances for loci with differential sRNA accumulation (p adj < 0.01) during the time course. sRNA levels shown are the regularised log2 expression values (normalised by locus). b Overlap between sRNA clusters and genomic features. Non-significant enrichments are transparent. Numbers indicate the number of sRNA loci overlapping the features

Fig. 8

Significant demethylation occurs from seed to seedling. a Heatmaps showing DNA methylation levels (as a percentage) in DMRs in CG, CHG and CHH contexts. b Overlap of DMRs and sRNA loci (by cluster). c Overlap between DMRs and genomic features. Non-significant enrichments are transparent. Numbers indicate the number of overlaps