Genome-wide redistribution of 24-nt siRNAs in rice gametes

Abstract

Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.

Figure 1.

Small RNA composition of ovary, egg cell, sperm cell, and seedling shoot. (A) Principle component analysis (PCA) plot of tissues by miRNA expression pattern. (B) miRNA159 family expression. y-axis values are relative to the total number of miRNA reads in each tissue. Color code is the same as in A. (C) Small RNA compositions. y-axis values are relative to the total number of reads that mapped to the genome. (D) siRNA abundance by length and category. “siRNAs” refers to all 20- to 25-nt small RNAs that mapped to the genome but were not included in any category in C. “Uniquely mapped” refers to the subset that map with a MAPQ value of at least 20. “Gypsy” is the subset that overlaps with an annotated Gypsy element, and “TIR” is the subset that overlaps with an annotated DNA transposon of the Tc1/Mariner, PIF/Harbinger, Mutator, or hAT superfamilies. “CentO” is the subset that overlaps CentO tandem repeats. y-axis values are the number of siRNAs of each length normalized by the total number of siRNAs. In all panels, error bars are 95% confidence intervals based on biological replicates of each tissue.

Figure 2.

siRNA distributions in ovary, egg cell, sperm cell, and seedling shoot. (A) Whole-genome 24-nt siRNA heat maps. Top track: gene density; second to fifth tracks: 24-nt siRNAs from seedling shoot, sperm cell, egg cell, and ovary. (B) Metagene coverage plot for 24-nt siRNAs. Coverage is measured over 100-bp intervals and normalized per 1000 total siRNAs. Vertical grid lines indicate 500-bp intervals. (TSS) Transcription start site, (poly[A]) polyadenylation site. (C) Venn diagram showing number and overlap of 24-nt siRNA loci. The genome was divided into 100-bp intervals and categorized as 24-nt siRNA loci based on coverage of 24-nt siRNAs from seedling shoot, sperm cell, and egg cell. (D) Number of 24-nt siRNA loci and abundance and enrichments of siRNAs from each tissue. Abundance (% 24-nt siRNAs in loci) is the number of 24-nt siRNAs that overlapped with the loci relative to the total number of 24-nt siRNAs from the tissue. Enrichment is the abundance normalized by the number of 24-nt loci. In locus types, “spec.” is short for “specific,” the set of loci only identified from a single tissue. (E) siRNA abundance at 24-nt siRNA loci. “Intersect.” refers to intersection loci, those identified as 24-nt siRNA in all three tissues. y-axis values are percent of siRNAs of each length relative to the total number of siRNAs that mapped to the genome. Error bars are 95% confidence intervals based on biological replicates of each tissue.

Figure 3.

DNA methylation across tissues. (A) DNA methylation metagene plots for post-bisulfite adapter tagging (PBAT) libraries. Plots indicate average DNA methylation values over 100-bp intervals. Vertical grid lines indicate 500-bp intervals. DNA methylation is measured as the proportion of methylated cytosines relative to total cytosines in each sequence context. (TSS) Transcription start site, (poly[A]) polyadenylation site. Published data from Park et al. (2016) and Kim et al. (2019). (B) DNA methylation metagene plot for conventional WGBS libraries; x- and y-axes as in A. (C) DNA methylation of 24-nt siRNA loci for PBAT libraries. Center line is median; box spans interquartile range; whiskers span 2.5th to 97.5th percentiles. (D) DNA methylation of 24-nt siRNA loci for conventional WGBS libraries, as in C; color code as in C. (E) Abundance of hypomethylated CHH differential methylated regions (hypo-CHH DMRs) in drm2 mutant relative to wild-type mature embryo. Abundance is measured as the percent of hypomethylated regions relative to total number of regions with sufficient read coverage for statistical significance. Dotted line indicates genome-wide average percent hypo-CHH DMRs. Color code as in C.

Figure 4.

siRNA and DNA methylation in ddm1 mutant leaf. (A) DNA methylation metagene plots. Plots indicate average DNA methylation values over 100-bp intervals in ddm1 (osddm1a osddm1b double mutant), drm2, and wild-type leaf. Vertical grid lines indicate 500-bp intervals. DNA methylation is measured as the proportion of methylated cytosines relative to total cytosines in each sequence context. (TSS) Transcription start site, (poly[A]) polyadenylation site. Data source: Tan et al. (2016). (B) DNA methylation of 24-nt siRNA loci. Center line is median; box spans interquartile range; whiskers span 2.5th to 97.5th percentile. (C) siRNA abundance at 24-nt siRNA loci in ddm1 and wild-type leaf. “Intersect.” refers to intersection loci, those identified as 24-nt siRNA in all three tissues. y-axis values are percent of siRNAs of each length relative to the total number of siRNAs that mapped to the genome. Error bars are 95% confidence intervals. Data source: Tan et al. (2018).

Figure 5.

Schematic representation of 24-nt siRNAs and CHH methylation in different tissues. Gray segments represent euchromatic DNA and colored segments represent heterochromatic DNA, the same region in each tissue. Tissue-specific 24-nt siRNAs are abundant in either sperm cell, egg cell, or seedling leaf (tissue-specific loci), while shared 24-nt siRNAs are abundant in all three (intersection loci).