Reinforcement of CHH methylation through RNA-directed DNA methylation ensures sexual reproduction in rice

Abstract

DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well studied in Arabidopsis (Arabidopsis thaliana), most mutations in RdDM genes cause no remarkable developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 (OsRDR2) in rice (Oryza sativa). Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH (H = A, C, or T) methylation. Moreover, male and female reproductive development was disturbed, which led to sterility in osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation may regulate the expression of multiple key genes involved in stamen development, meiosis, and pollen viability. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of CHH methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity, which includes OsRDR2 activity. Our results, which revealed a global increase in CHH methylation through enhancement of RdDM activity in reproductive organs, suggest a crucial role for OsRDR2 in the sexual reproduction of rice.

Figure 1

Gene silencing of 35S::OsGA2ox1. A, Morphology of 110-d-old TP309, dwarf GAE, semi-dwarf GAE, and GAS plants. Scale bar = 10 cm. B, Genotyping transgenic OsGA2ox1. OsActin served as the control. GAE, OsGA2ox1ectopic expression; GAS, OsGA2ox1silencing. C, The mRNA level of OsGA2ox1 as indicated by semi-quantitative RT-PCR. OsActin served as the control. D, Western blot detection of the protein level of SLR1. E, Chop-PCR assay on the gene body of OsGA2ox1 in the indicated genotypes. F, sRNA northern blot detection of the sRNA levels on the gene body of OsGA2ox1. G, Chop-PCR assay of the 35S promoter in the indicated genotypes.

Figure 2

Map-based cloning of FEM1. A, Morphology of 3-month-old TP309, GAE, GAS, fem1-1, and fem1-2 plants. Scale bar = 10 cm. B, Genotyping transgenic OsGA2ox1. OsActin served as the control. C, The mRNA level of OsGA2ox1 as indicated by semi-quantitative RT-PCR. OsActin served as the control. D, Chop-PCR assay of the gene body of OsGA2ox1. E, Chop-PCR assay of the 35S promoter. F, Chop-PCR assay of endogenous loci. LMR, Losing DNA methylation region. G, Diagram showing the map-based cloning of fem1-1. Mutations of fem1-1 and fem1-2 are indicated. H, Morphology of 3-month-old plants of GAS, fem1-1, and three complementary lines (C1–C3). Scale bar = 10 cm. I, Genotyping of complementary lines. J, The mRNA level of OsGA2ox1 in the indicated genotypes as determined by semi-quantitative RT-PCR.

Figure 3

osrdr2 mutants are sterile. A, Morphology of the WT and various osrdr2 mutants. Scale bar = 10 cm. B, Morphology of the WT, osrdr2–6/osrdr2–7, osrdr2–6, and osrdr2–7 mutants. Scale bar =  10 cm. C, Panicle morphology of the WT, osrdr2–3, osrdr2–4, and osrdr2–5. Scale bar = 5 cm. D, Panicle morphology of the WT, osrdr2–6/osrdr2–7, osrdr2–6, and osrdr2–7. Scale bar = 5 cm. E, Unfilled (top) and filled (bottom) rice grains of the WT, osrdr2–3, osrdr2–4, and osrdr2–5. Scale bar = 1 cm. F, Unfilled (top) and filled (bottom) rice grains of the WT, osrdr2–6/osrdr2–7, osrdr2–6, and osrdr2–7. Scale bar = 1 cm. G, Seed setting rate of various genotypes (Data are means ± sd (Standard Deviation), n = 15) (^**P < 0.01; ns, no significant difference by Student’s t test). H, Seed setting rate from reciprocal crosses between the WT and osrdr2 mutants (Data are means ± sd, n = 10).

Figure 4

OsRDR2 regulates CHH methylation and 24-nt siRNA biosynthesis. A and B, Average CG, CHG, and CHH methylation levels on genes (A) and TEs (B) in the panicles of various genotypes (R1 represents WT-Replicate 1; R2 represents WT-Replicate 2). C, Average CHH methylation levels on TEs of different lengths in panicles of the indicated genotypes. D and E, The number (D) and length (E) of various DMRs in panicles of osrdr2–3. F, Box plots showing DMR lengths. Different letters indicate a significant difference according to Fisher’s least significant difference (LSD) (P < 0.05). Here and all subsequent box plots, the box indicates the upper and lower quartiles, the black lines are medians, and the whiskers corresponding to the minimum and maximum range. G, Genomic distribution of CHH hypo-DMRs in osrdr2–3 panicles. “Gene” indicates a DMR that overlaps with the gene body; “TE” indicates a DMR that overlaps with the TE body; “Gene and TE” indicates a DMR that overlaps with both the gene body and TE body; “Intergenic” indicates a DMR that does not overlap with either the gene body or the TE body. H, Abundance of small RNAs of different length relative to 21-nt in WT and osrdr2 panicles. I, Box plots indicating the methylation level of CG, CHG, and CHH, and 24-nt siRNA abundance on CHH hypo-DMR in osrdr2–3 panicles (^**P < 0.01 by two-tailed z-test). J, Integrated Genome Browser view of CHH methylation levels, 24-nt siRNAs abundance on one representative CHH hypo-DMR in panicles of the indicated genotypes.

Figure 5

Increase of CHH methylation in panicles depends on OsRDR2. A and B, Average CG, CHG, and CHH methylation levels on genes (A) and TEs (B) in panicles and seedlings. C, Average CHH methylation levels on TEs of different length in panicles and seedlings. D, Venn diagram of the overlap between CHH hyper-DMRs of panicles > seedlings and hypo-DMRs of osrdr2–3 < WT in panicles. Box plots indicate the methylation levels of CHH and 24-nt siRNA abundance on common and specific regions in panicles or seedlings of various genotypes (^**P < 0.01 by two-tailed z-test). E, Chop-PCR assay of several panicle CHH hyper-DMRs. Values were calculated relative to the first sample.

Figure 6

Increase of CHH methylation in stamens and pistils depends on OsRDR2. A, Venn diagram showing the overlap between stamen CHH hyper-DMRs (stamen > seedling) and osrdr2–3 CHH hypo-DMRs in stamens. Box plots indicate the methylation levels of CHH and 24-nt siRNAs abundance on the overlapped and specific regions in stamens or seedlings of the indicated genotypes (^**P < 0.01 by two-tailed z-test). B, Venn diagram showing the overlap between pistil CHH hyper-DMRs (pistils > seedlings) and CHH hypo-DMRs of osrdr2–3 in pistils. Box plots indicate the methylation levels of CHH and 24-nt siRNA abundance on the common and specific regions in pistils or seedlings of the indicated genotypes (^**P < 0.01 by two-tailed z-test).

Figure 7

TE expression in stamens and pistils of osrdr2. A and B, Box plots showing the transcriptional levels of expressed TEs (exp), upregulated TEs (up), and downregulated TEs (down) in A, stamens, and B, pistils, of the WT and osrdr2–3. (^**P < 0.01 by two-tailed z-test). C, Ratio of different types of TEs among the indicated TEs. D, Ratio of TEs with different length among the up- or downregulated TEs in osrdr2 stamen and pistil.

Figure 8

Increase of DNA methylation regulates gene expression in stamens. A, Stamen morphology of the WT (Nip) and osrdr2 mutants at Stage 12. Scale bars = 1 mm. B, Scanning electron micrographs of Stage 12 stamens of the WT and osrdr2 mutants. Scale bars = 200 µm. C, Transverse sections of anthers at five stages of development in the indicated genotypes. Msp: microspore, BP: binucleate pollen grain, MP: mature pollen grain, T: tapetum. Scale bar = 20 µm. D, Integrated Genome Browser view of CG, CHG, and CHH methylation levels on OsTDL1A, MIL1, and MSP1 in various organs of the indicated genotypes. E, Relative expression levels of OsTDL1A, MIL1, and MSP1 in stamens of the WT and osrdr2 mutants. Ubq (LOC_Os03g13170) served as the control (^**P  < 0.01; ^*P < 0.05; by Student’s t test). Data are means ± sd, n = 3.

Figure 9

Expression levels of RdDM genes are upregulated in reproductive tissues. A, Heatmap showing the relative expression levels of DNA methylation-related genes in various tissues from the Rice XPro database (https://ricexpro.dna.affrc.go.jp/GGEP/index.php). B, Western blot detection of the protein levels of OsRDR2 in various tissues of two independent OsRDR2::OsRDR2-3xFLAG transgenic rice plants. C, DNA methylation-related genes overlapped with panicle-, stamen-, and pistil-CHH hyper-DMRs. D, Percentages of DNA methylation-related genes overlapped with panicle CHH hyper-DMRs (left), stamen CHH hyper-DMRs (middle), and pistil CHH hyper-DMRs (right) (^**P < 0.01; by Fisher’s two-sided test). E, Integrated Genome Browser view of CHH methylation levels on OsNRPD1b in various organs of the indicated genotypes. F, Heatmap showing the expression levels of DNA methylation-related genes in WT seedlings, WT pistils, and osrdr2–3 pistils.