Project description:In this study, we identified METTL16 as an mRNA m6A methyltransferase that plays a vital role in the floral transition in Arabidopsis. Transcriptome-wide analysis of RNA methylome in the mettl16 mutant revealed that m6A modification enriched near the stop codon and within the 3ʹ untranslated region. Deficiency of METTL16 leads to decreases in m6A levels of approximately 471 transcripts, indicating that it is responsible for the methylation of a small group of mRNAs. The mettl16 mutant displayed an early flowering phenotype, and the level of FLOWERING LOCUS C (FLC) was markedly decreased in the mutant. Importantly, METTL16-mediated m6A methylation affects the splicing of FLC, thereby influencing its transcript level to regulate floral transition. Our study identified METTL16 as a novel m6A methyltransferase and suggests a close molecular link between METTL16-mediated m6A methylation and FLC splicing in flowering time control.
Project description:The autonomous flowering pathway promotes flowering in rapid-flowering accessions of Arabidopsis thaliana by reducing the expression of the key flowering repressor gene FLC. Although previous studies identified several autonomous pathway components, little is known about how these components cooperate in the regulation of flowering time. Here, we identified an autonomous pathway complex (APC) composed of three known autonomous pathway components (FLD, LD, and SDG26) and three previously uncharacterized components (EFL2, EFL4, and APRF1). Loss-of-function mutations of all of these components result in increased FLC expression and delayed flowering. The late-flowering phenotype is independent of photoperiod and can be overcome by vernalization, supporting the inference that the complex regulates flowering time specifically through the autonomous pathway. Our ChIP-seq analyses indicated that the APC components are required for the suppression of the histone modifications (H3Ac, H3K4me3, and H3K36me3) associated with the activation of gene expression and for the promotion of the histone modification (H3K27me3) associated with the repression of gene expression at FLC. We also found that the C-terminal coiled-coil domain of SDG26 binds to DNA in vitro and that the DNA-binding ability mediates the association of SDG26 with FLC chromatin in vivo. These results reveal the molecular basis of the autonomous pathway.
Project description:The autonomous flowering pathway promotes flowering in rapid-flowering accessions of Arabidopsis thaliana by reducing the expression of the key flowering repressor gene FLC. Although previous studies identified several autonomous pathway components, little is known about how these components cooperate in the regulation of flowering time. Here, we identified an autonomous pathway complex (APC) composed of three known autonomous pathway components (FLD, LD, and SDG26) and three previously uncharacterized components (EFL2, EFL4, and APRF1). Loss-of-function mutations of all of these components result in increased FLC expression and delayed flowering. The late-flowering phenotype is independent of photoperiod and can be overcome by vernalization, supporting the inference that the complex regulates flowering time specifically through the autonomous pathway. Our ChIP-seq analyses indicated that the APC components are required for the suppression of the histone modifications (H3Ac, H3K4me3, and H3K36me3) associated with the activation of gene expression and for the promotion of the histone modification (H3K27me3) associated with the repression of gene expression at FLC. We also found that the C-terminal coiled-coil domain of SDG26 binds to DNA in vitro and that the DNA-binding ability mediates the association of SDG26 with FLC chromatin in vivo. These results reveal the molecular basis of the autonomous pathway.
Project description:Arabiposis plants with conbinations of different FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) alleles grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D). Genotypes: Columbia wild type (Col-0): fri FLC Columbia with introgressed FRI from Sf-2: FRI FLC Columbia with introgressed FRI and deleted FLC (flc-3): FRI flc Columbia with deleted FLC (flc-3): fri flc Time points: 0, 2, and 4 days after shift to long days Keywords = flowering Keywords: time-course
Project description:Quantitative variation in expression of the Arabidopsis floral repressor FLC influences whether plants overwinter before flowering or have a rapid cycling habit, enabling multiple generations a year. Genetic analysis has identified activators and repressors of FLC expression, but how they interact to set expression level is poorly understood. Here, we show that antagonistic functions of the FLC activator FRIGIDA (FRI), and the repressor FCA, at a specific stage of embryo development, determines FLC expression and flowering. FRI antagonizes an FCA-induced proximal polyadenylation to increase FLC expression and delay flowering. Sector analysis shows that FRI activity during the early heart stage of embryo development maximally delays flowering. Opposing functions of co-transcriptional regulators during an early embryonic developmental window thus set FLC expression levels and determine flowering time.
Project description:Co-transcriptional processing of nascent transcripts is coupled with transcription through the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). The mRNA modification N6-methyladenosine (m6A) occurs co-transcriptionally and impacts pre-mRNA processing. How alternative splicing of pre-mRNA is co-transcriptionally regulated in an m6A-dependent manner is not well understood. Furthermore, splicing regulation through RNAPII pausing has been frequently suggested but the underlying control mechanism remains unclear. Here, we show that the m6A reader protein hnRNPG directly binds to the phosphorylated CTD of RNAPII using Arg-Gly-Gly (RGG) motifs in its low-complexity region. This RGG-phospho-CTD interaction and nascent RNA binding by hnRNPG enables its co-transcriptional association with RNAPII, resulting in transcriptome-wide regulation of alternative splicing and transcript abundance. m6A sites near exon splice sites in nascent mRNA further modulate hnRNPG binding and exon splicing. Exon inclusion is associated with RNAPII pausing downstream of the m6A site. Our results reveal an integrated nuclear mechanism of m6A-mediated gene regulation, in which an m6A reader protein regulates gene expression by using RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA, while the interplay between hnRNPG binding and RNAPII pausing modulates alternative splicing regulation.
Project description:Polyploidy is a widespread phenomenon in flowering plant species. Polyploid plants frequently exhibit considerable transcriptomic alterations after whole-genome duplication (WGD). It is known that the transcriptomic response to tetraploidization is ecotype-dependent in Arabidopsis. Nevertheless, the biological significance and the underlying mechanism are unknown. Here, we showed that 4x Col-0 and 4x Ler presented different flowering times, with a delayed flowering time in 4x Col-0 but not in 4x Ler. We found that the expression of FLOWERING LOCUS C (FLC), the major repressor of flowering, was significantly increased in 4x Col-0 but subtle change in 4x Ler. Moreover, the level of a repressive epigenetic mark, trimethylation of histone H3 at lysine 27 (H3K27me3), was significantly decreased in 4x Col-0 but not in 4x Ler, potentially leading to different transcription levels of FLC and flowering time in 4x Col-0 and 4x Ler. Apart from the FLC locus, hundreds of genes showed differentially H3K27me3 alterations in 4x Col-0 and 4x Ler. Comparably, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) and transcription factors required for H3K27me3 deposition presented differential transcriptional changes between 4x Col and Ler, potentially account for differential H3K27me3 alterations in 4x Col-0 and Ler. Last, we found that the natural 4x Arabidopsis ecotype Wa-1 presented early flowering time, associated with low expression and high H3K27me3 of FLC. Taken together, our results showed a role of H3K27me3 alterations in response to genome duplication in Arabidopsis autopolyploids and that flowering time variation potentially functions in autopolyploid speciation.
Project description:N6-methyladenosine (m6A) is a crucial epigenetic modification in mRNA and the most abundant methylation in eukaryotes. However, research on m6A modification in radish (Raphanus sativus) is not as extensive as in model plants. In this study, we analyzed m6A modification during the vegetative and reproductive growth stages of radish using m6A-seq and RNA-seq to explore its potential role in bolting and flowering. The results showed that m6A peaks significantly increased during the reproductive growth stage compared to the vegetative stage, with more m6A modification sites in the stop codon, 3' UTR, and promoter regions. Overexpression of the RsALKBH10B gene led to a global reduction in m6A modification and resulted in an early bolting and flowering phenotype, with transcription levels of key flowering factors RsSOC1 and RsFT increasing by 10-40 fold. Correlation analysis between differential m6A modification and differentially expressed genes indicated that genes such as RsSUF4, RsAGL13, and RsCDF5 might regulate bolting and flowering. Overexpression of RsSUF4 delayed bolting and flowering and decreased overall m6A modification levels; in positive transgenic plants, higher m6A levels of RsSUF4 were associated with lower transcription levels. These findings suggest that m6A methylation may regulate gene expression related to bolting and flowering in radish by affecting mRNA stability, ultimately leading to bolting and flowering.
Project description:Arabiposis plants with conbinations of different FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) alleles grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D). Genotypes:; Columbia wild type (Col-0): fri FLC; Columbia with introgressed FRI from Sf-2: FRI FLC; Columbia with introgressed FRI and deleted FLC (flc-3): FRI flc; Columbia with deleted FLC (flc-3): fri flc; Time points:; 0, 2, and 4 days after shift to long days
Project description:Plants of three different genotypes (FRI FLC, FRI flc and fri flc) were induced to flowering by shifting from short day conditions to long day conditions. FRI=FRIGIDA, FLC=FLOWERING LOCUS C.