RNA methylomes reveal the m6A-mediated regulation of DNA demethylase gene SlDML2 that is required for tomato fruit ripening
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ABSTRACT: For exploring whether mRNA m6A modification participates in the regulation of tomato fruit ripening, we performed m6A-seq in three tomato fruit samples, including wild-type (WT) at 39 days post-anthesis (DPA) and 42 DPA, and Cnr mutant at 42 DPA, with three biological replicates. mRNA methylome analysis reveals that m6A methylation is a prevalent modification in mRNA of tomato fruit and the m6A sites are predominantly enriched in the stop codon and 3’ untranslated region, where m6A deposition has been proved to negatively correlate with gene expression. Hundreds of ripening-induced and ripening-repressed genes, including the SlDML2, were found to harbour changed m6A levels during fruit ripening or in the Cnr mutant, implicating the involvement of m6A modification in the regulation of fruit ripening.
Project description:Background: Fruit color is an important quality trait for nutrition value in tomato (Solanum lycopersicum) and has attracted huge attention for a long time. In order to dissect the yellow-fruit color of a novel tomato mutant n3122, we compared the dynamic transcriptome of the fruit pericarps from the mutant n3122 and its wild type red-fruited tomato cultivar M82. Results: The transcriptomes of fruits from M82 35 DPA (Days Post Anthesis), M82 47 DPA, M82 54 DPA, n3122 35 DPA, n3122 47 DPA, n3122 54 DPA and n3122 60 DPA were sequenced using an Illumina Hiseq 2000 sequencing platform. A total of 5568 differentially expressed genes (DEGs) were commonly identified in the four pairwise comparisons of M82_35 DPA vs n3122_35 DPA, M82_47 DPA vs n3122_47 DPA, M82_54 DPA vs n3122_54 DPA and M82_47 DPA vs n3122_60 DPA. Further Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that carotenoids biosynthesis, ethylene biosynthesis and signaling transduction, and transcription factors associated fruit ripening were different between M82 and n3122 which might be the underlying mechanisms for the yellow-fruit color of tomato. Conclusions: This research provided a global data set of dynamic transcriptomic changes during fruit development and ripening for the wild type red-fruited tomato cultivar M82 and its yellow-fruited mutant n3122, and offered a base for elucidating the molecular mechanisms underlying tomato red/yellow fruit color mutation.
Project description:For exploring whether mRNA m6A modification participates in the regulation of strawberry fruit ripening, we performed m6A-seq in woodland strawberry fruit at three different development stages, including the S6 stage (almost 15 days post-anthsis (DPA)), the RS1 stage (21 DPA), and the RS3 stage (27 DPA), with three biological replicates. mRNA methylome analysis reveals that m6A methylation prevalently distributes in the strawberry transcriptome and highly enrichs in the coding sequence, stop codon and 3’ untranslated region.
Project description:The study of climacteric fruit ripening in tomato has been facilitated by the spontaneous ripening mutants Colorless non-ripening (Cnr), non-ripening (nor), and ripening inhibitor (rin). These mutants effect the genes encoding ripening transcription factors (TFs) SPL-CNR, NAC-NOR, and MADS-RIN causing pleiotropic defects to the ripening program. Here, we demonstrate that some ripening processes occur in the mutant fruit but at later stages of development compared to the wild type. The rin and nor mutant fruit exhibit similar quality traits to wildtype at later stages of ripening and senescence and delayed expression of ripening-associated genes. In addition, we propose that the Cnr mutant has a broader range of effects to fruit development than just fruit ripening. Cnr fruit show distinct differences from wild type in ripening phenotypic traits and gene expression profiles prior to the initiation of ripening. We provide new evidence that some mutants can produce more ethylene than basal levels and demonstrate ABA accumulation is also affected by the mutations. Studies have examined the relationship between the CNR, RIN, and NOR TFs based on protein-protein interactions and transcriptional regulation during fruit ripening. We describe the genetic interactions affecting specific fruit traits by using homozygous double mutants. Cnr predominantly influences the phenotype of the Cnr/nor and Cnr/rin double mutants but additional defects beyond either single mutation is evident in the transcriptome of the Cnr/nor double mutant. Our reevaluation of the Cnr, nor, and rin mutants provides new insights the utilization of the mutants in breeding and studying fruit development.
Project description:Gene-to-gene coexpression analysis is a powerful approach to infer function of uncharacterized genes. To perform non-targeted coexpression analysis of tomato genes, we collected a developmental gene expression dataset using various tissues of tomato plant. Expression data are collected from 24 different tissue types including root, hypocotyl, cotyledon, leaf at different stages, and fruit tissues at 4 different ripening stages from 4 different Solanum lycopersicum cultivars. Fruits were separated to the flesh and the peel. These two tissue types indeed showed remarkably different gene expression profiles. We also collected data from 4 different ripening stages (mature green, yellow, orange, and red) to detail the changes during ripening. By using this gene expression dataset, we calculated pair-wise Pearsonâs correlation coefficients, and performed network-based coexpression analysis. The analysis generated a number of coexpression modules, some of which showed an enrichment of genes associated with specific functional categories. This result will be useful in inferring functions of uncharacterized tomato genes, and in prioritizing genes for further experimental analysis. We used Affymetrix GeneChip Tomato genome Arrays to detail the global gene expression change using 24 different tomato tissue types (67 hybridizations). We collected gene expression data from 24 different tomato tissue types using 67 hybridizations. Root, hypocotyl, cotyledon, and leaf were sampled from 3-week-old or 5-weekâold plant of Solanum lycopersicum cultivar Micro-Tom. Fruit tissues were sampled from S. lycopersicum cultivars Micro-Tom, Anthocyanin fruit (Aft, LA1996), Line27859, and Momotaro 8 (Takii, Japan). From Micro-Tom fruit, the peel and the flesh were separately sampled from 4 different ripening stages: mature green (MG, approximately 30 day after anthesis), yellow (Y, approximately 35 days after anthesis), orange (O, approximately 38-40 days after anthesis), and red (R, approximately 45-48 days after anthesis). From fruits of Aft and Line27859, the peel and the flesh were sampled at mature green (MG, approximately 40 days after anthesis) and red (R, approximately 50-55 days after anthesis) stages. From Momotaro 8, the peel and the flesh were sampled at red (R, 50- approximately 50-55 days after anthesis) stages. For each tissue type, 2-4 biological replicates were made in RNA preparation.
Project description:The increased susceptibility of ripe fruit to fungal pathogens poses a substantial threat to crop production and marketability. Here, we coupled transcriptomic analyses with mutant studies to uncover critical genes and processes governing ripening-associated susceptibility in tomato (Solanum lycopersicum) fruit. Using wild-type unripe and ripe fruit inoculated with three fungal pathogens—Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer—we identified common pathogen response genes reliant on chitinases, WRKY transcription factors, and reactive oxygen species detoxification. Interestingly, susceptible ripe fruit demonstrated a more extensive defense response than resistant unripe fruit, indicating that the magnitude and diversity of defense response does not significantly impact the interaction. To tease apart individual features of ripening that may be responsible for susceptibility, we utilized three tomato non-ripening mutants: Cnr, rin and nor. Fruit from these mutants displayed different patterns of susceptibility to fungal infection. Functional analysis of the genes altered during ripening in the susceptible genotypes revealed losses in the maintenance of cellular redox homeostasis. Moreover, jasmonic acid accumulation and signaling coincided with the activation of defenses in resistant fruit. Lastly, based on high gene expression in susceptible fruit, we identified and tested two candidate susceptibility factors, pectate lyase (PL) and polygalacturonase (PG2a). CRISPR-based knockouts of PL, but not PG2a, resulted in more than 50% decrease in the susceptibility of ripe fruit, demonstrating that PL is a major susceptibility factor. Ultimately, this study demonstrates that targeting specific genes that drive susceptibility is a viable strategy to improve resistance of tomato fruit against fungal pathogens.
Project description:In this study, we show the potential link between m6A deposition and fruit expansion in tomato (Micro-tom). m6A level was increased during fruit expansion. Later, a parallel analysis of the m6A -seq and RNA-seq in expanding fruit were preformed to evaluate global correlation between m6A modification and fruit expansion. Our m6A -seq reveal that thousands of protein-coding genes contains m6A modification with a consensus motif and primarily enriched in the 3’untranslated regions. Gene ontology analysis indicate that the distribution of m6A in expanded fruit is largely associated with plant-specific processes relating to the chloroplast function. Moreover, we further uncover a positive correlation between m6A methylation and the mRNA abundance. Specifically, large number of fruit expansion-related genes, which involved in hormone and endoreplication, were m6A modified and actively expressed compared to non- m6A modified genes, suggesting a potential involvement of m6A modification in fruit expansion. Collectively, our result suggests a dynamic role of m6A modification involving in fruit expansion.
Project description:Transcription profiling of post-anthesis unfertilized pistil development and senescence in Arabidopsis, in a serie including samples from seven different stages according to pistil age in days post-anthesis (dpa). The time course started at anthesis and ended at 12-14 dpa, several days later of the loss of fruit set capacity. Keywords: Developmental time course
Project description:To decipher the mechanisms by which FvALKBH10B regulates fruit ripening, we performed m6A sequencing (m6A-seq) using mRNA from fruits of wild type and Fvalkbh10b mutant. The libraries for sequencing were prepared with 3 replicates, in which the mRNA samples were independently prepared. FvALKBH10B mutation leads to a delay in fruit ripening and causes global m6A hypermethylation of 1859 genes.
Project description:The tomato (Solanum lycopersicum) MADS-box transcription factor RIPENING INHIBITOR (RIN) acts as a master regulator of tomato fruit ripening. We previously identified a direct RIN target gene Solyc07g052960, which encodes a putative GRAS family protein belonging to the SHORT-ROOT (SHR) branch, but its role was unknown. RNA interference (RNAi)-mediated gene silencing reduced Solyc07g052960 expression in transgenic fruits, but the fruits appeared to ripen normally. However, the transgenic fruits at the ripening stage showed a marked decrease of the expression levels of several ripening-induced genes, especially involved in cell wall modification and secondary metabolism. This suggests that Solyc07g052960 participates in the regulation of these processes as one component of the RIN-activated transcriptional cascade regulating fruit ripening in tomato.
Project description:The tomato (Solanum lycopersicum) MADS-box transcription factor RIPENING INHIBITOR (RIN) acts as a master regulator of tomato fruit ripening. We previously identified a direct RIN target gene Solyc07g052960, which encodes a putative GRAS family protein belonging to the SHORT-ROOT (SHR) branch, but its role was unknown. RNA interference (RNAi)-mediated gene silencing reduced Solyc07g052960 expression in transgenic fruits, but the fruits appeared to ripen normally. However, the transgenic fruits at the ripening stage showed a marked decrease of the expression levels of several ripening-induced genes, especially involved in cell wall modification and secondary metabolism. This suggests that Solyc07g052960 participates in the regulation of these processes as one component of the RIN-activated transcriptional cascade regulating fruit ripening in tomato. For a preliminary screening, we monitored global gene expression in tomato fruits from untransformed control (Ailsa Craig [AC] cultivar) and three transgenic lines with Solyc07g052960 knockdown by RNAi (lines T-7, T-22 and T-23) at the ripening (pink coloring) stage using microarray without biological replication.