The Rise and Fall of Billionaire siRNAs during Reproductive Development in Rice

Abstract

RNA polymerase IV-dependent siRNAs, usually 24 nt in length, function in the RNA-directed DNA methylation that is responsible for de novo methylation in plants. We analyzed 24 nt siRNAs in inflorescences and found that among the 20,200 24 nt siRNA clusters, the top 0.81% highly expressed clusters accounted for more than 68% of the 24 nt siRNA reads in inflorescences. We named the highly expressed siRNAs as billionaire siRNAs (bill-siRNAs) and the less-expressed siRNAs as pauper siRNAs (pau-siRNAs). The bill-siRNAs in inflorescences are mainly derived from the ovary. Female gametes produced more bill-siRNAs than male gametes. In embryos and seedlings developed from fertilized egg cells, the bill-siRNAs from gametes disappeared. The endosperm, which develops from the fertilized central cell, also contained no bill-siRNAs from gametes but did contain newly and highly expressed siRNAs produced in different regions. In contrast, bill-siRNAs from the ovaries were maintained in the seed coat. The biosynthesis of bill-siRNAs in various tissues and cells is dependent on OsRDR2 (RNA-dependent RNA polymerase 2) and Pol IV (DNA-dependent RNA polymerase IV). Similar to the pau-siRNAs, the first base of bill-siRNAs is enriched at adenine, and bill-siRNAs can direct DNA methylation in various tissues.

Keywords: reproductive development; rice; siRNA.

Figure 1

Rice inflorescences produce bill-siRNAs. (A) Comparison of 24 nt siRNA distributions on the rice genome between inflorescences and seedlings in Nipponbare. Y-axis indicates siRNA abundance with RPTM within 500 kb windows. (B) Cumulative expression plot of 24 nt siRNAs in inflorescences and seedlings in Nipponbare. The abundance of each cluster is indicated by RPTM based on two replicates; their cumulative expression was ranked and plotted. siRNA clusters were analyzed if their abundance ≥ 12 RPTM in combined replicates (n = 20,200 in inflorescence, n = 57,424 in seedling). (C) Screen shots of Integrative Genomics Viewer (IGV) showed the abundance of bill-siRNAs in inflorescences and seedlings.

Figure 2

Characteristics of bill-siRNAs in inflorescences. (A) Percentage of four bases on the first nucleotide acid of bill-siRNAs and pau-siRNAs. (B) Lengths of bill-siRNA clusters and pau-siRNA clusters in Nipponbare and TP309. ** indicates p < 0.01 by the Wilcoxon sum test. (C) Relative abundance of bill-siRNA clusters and pau-siRNA clusters in Nipponbare and TP309. ** indicates p < 0.01 by the Wilcoxon sum test. (D) Comparison of histone modification of H3K9me2, H3K27me3, H3K4me1, H3K27ac, and H3K4me3, and Pol II occupancy on bill-siRNA clusters and on pau-siRNA clusters. A total of 20,000 randomly selected 350 bp regions were analyzed and served as control. ** indicates p < 0.01, and ns indicates not significant by the Wilcoxon sum test. (E) Comparison of expression level of bill-siRNA cluster- and pau-siRNA cluster-adjacent genes in Nipponbare and TP309. Three hundred randomly selected genes served as controls. ** indicates p < 0.01, and * indicates p < 0.05 by the Wilcoxon sum test.

Figure 3

Accumulation of siRNAs and CHH methylation on bill-siRNA clusters depend on OsRDR2. (A) Boxplots showing the 24 nt siRNA abundance in seedlings and inflorescences of WT and osrdr2 mutant. ** indicates p < 0.01 by the Wilcoxon sum test. (B) Boxplots showing the methylation levels of CG, CHG, and CHH in seedlings and inflorescences of the WT and osrdr2 mutant on bill-siRNA clusters and pau-siRNA clusters in inflorescences. ** indicates p < 0.01 by the Wilcoxon sum test. (C) Screen shots of IGV indicating 24 nt siRNA abundance and CHH methylation levels in seedlings and inflorescences of the WT and osrdr2 mutant on two representative bill-siRNA clusters. (D) Chop-PCR assay suggesting the CHH methylation level in seedlings and inflorescences of the WT and osrdr2 mutant.

Figure 4

Bill-siRNAs in inflorescences are mainly derived from the ovary. (A) Cumulative expression plot of 24 nt siRNA clusters in seedlings, inflorescences, ovaries, bracts, branches, and stamens in Nipponbare. (B) Violin plots showing 24 nt siRNA abundance in different tissues on various genotypes on various bill-siRNA clusters. Quantile lines indicate the 1/4 and 3/4 percentiles. Black dots represent the median. ** indicates p < 0.01 by the Wilcoxon sum test. (C) Venn diagram indicating the overlap between bill-siRNAs in inflorescences and bill-siRNA clusters in the other four tissues. (D) Boxplot showing 24 nt siRNA abundance on the combined bill-siRNA clusters (inflorescences, ovaries, bracts, branches, and stamens) in various tissues. ** indicates p < 0.01 by the Wilcoxon sum test. (E) Venn diagram indicating the overlaps between bill-siRNA clusters in four tissues. (F) sRNA northern blotting indicating the sRNA levels of different tissues on one representative bill-siRNA cluster.

Figure 5

Bill-siRNAs of gametes are absent in embryos. (A) Cumulative expression plot of 24 nt siRNA clusters in egg cells, sperm, and embryos. (B) Overlap between bill-siRNA clusters in egg cells and embryos. (C) Overlap between bill-siRNA clusters in sperm and embryos. (D) Heatmaps indicating the siRNA abundance on bill-siRNA clusters in egg cells and sperm.

Figure 6

Endosperm produces new bill-siRNAs. (A) Cumulative expression plot of 24 nt siRNA clusters in the ovary without the egg cell, sperm, and endosperm. (B) Overlap between bill-siRNA clusters of the ovary without the egg cell and endosperm. (C) Overlap between bill-siRNA clusters of sperm and endosperm. (D) Heatmaps showing the siRNA abundance on bill-siRNA clusters in ovary, sperm, and endosperm.

Figure 7

Biosynthesis of bill-siRNAs in the endosperm is dependent on OsRDR2 and Pol IV. (A) Overlap between bill-siRNAs in the endosperm of Nipponbare and TP309. (B) Boxplots showing the methylation levels in CG, CHG, and CHH on bill-siRNA and pau-siRNA clusters of the endosperm. (C) Comparison of H3K27me3 enrichment on bill-siRNA clusters and pau-siRNA clusters in the endosperm. ** indicates p < 0.01 by the Wilcoxon sum test. (D) Boxplots showing siRNA abundance on bill-siRNA clusters of TP309 endosperm. ** indicates p < 0.01 by the Wilcoxon sum test.

Figure 8

Bill-siRNAs derived from the ovary are retained in the seed coat. (A) Cumulative expression plot of 24 nt siRNA clusters in seedlings and seed coats in TP309 background. (B) Overlap for bill-siRNA clusters in the seed coat and in the ovary without the egg cell (upper), and in the ovary with egg cell (lower). (C) Boxplots showing siRNA abundance in the indicated tissues on TP309 seed coat bill-siRNA clusters. ** indicates p < 0.01 by the Wilcoxon sum test.