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.

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

“…Because Pol IV- and RDR2-generated siRNAs are the hallmarks of RdDM [ 4 , 15 ], we assessed CHH methylation on siRNA clusters. On both bill-siRNA and pau-siRNA clusters, the CHH methylation levels were significantly higher in inflorescences than in seedlings ( Figure 3 B), which is consistent with our previous finding that CHH methylation is globally higher in reproductive organs than in seedlings [ 11 ]. The CHH methylation on bill-siRNA and pau-siRNA clusters is significantly lower in osrdr2 inflorescences than in wild-type (WT; Nipponbare) inflorescences ( Figure 3 B), demonstrating that OsRDR2 -dependent bill-siRNAs as well as OsRDR2 -dependent pau-siRNAs are required for DNA methylation in inflorescences.…”

“…In a previous study, we found that the whole-genome methylation level (WGML) in the CHH context was higher in inflorescences than in seedlings mainly because of the up-regulation of RdDM machinery [ 11 ]. However, 24 nt siRNA abundance on the hyper-methylated differentially methylated regions (hyper-DMRs) of inflorescence relative to seedlings was significantly lower in inflorescences than in seedlings ( Figure S1A ).…”

“…About 90.2% of inflorescence siRNA clusters were also identified in seedlings, and only 1971 clusters were inflorescence-specific ( Figure S1B ). On all seedling siRNA clusters (siRNA clusters that were seedling-specific or common to seedlings and inflorescences), the siRNA abundance was higher in seedlings than in inflorescences, but the CHH methylation was lower in seedlings than in inflorescences ([ 11 ]; Figure S1B ). By contrast, the 24 nt siRNA abundance and CHH methylation were both higher in inflorescences than in seedlings on the inflorescence-specific siRNA clusters ( Figure S1B ).…”

“…Capsella pol iv affected male gamete development [ 10 ]. In fem1 ( osrdr2 ) mutants of rice, the development of both male and female reproductive organs was defective [ 11 ]. Together, these results indicate that 24 nt siRNAs are important for reproductive development of rice and other plants.…”

A null allele of the pol IV second subunit impacts stature and reproductive development in Oryza sativa

“…ago4ab and rdr2 RNAi lines also showed reduced height and panicle size, and the ago4ab line phenocopied dcl3 in the flag leaf angle (Wei et al., 2014). Mutation in rice RDR2 showed defects in pollen grain development (L. Wang et al., 2022), while drm2 mutants showed a wide range of developmental phenotypes including complete sterility (Moritoh et al., 2012).…”