Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit

DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals. Active DNA demethylation in plants is initiated by a family of 5-mC DNA glycosylases/lyases (i.e., DNA demethylases). Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato. In this study, we generated loss-of-function mutant alleles of a tomato gene, SlDML2, which is a close homolog of the Arabidopsis DNA demethylase gene ROS1. In the fruits of the tomato mutants, increased DNA methylation was found in thousands of genes. These genes included not only hundreds of ripening-induced genes but also many ripening-repressed genes. Our results show that SlDML2 is critical for tomato fruit ripening and suggest that active DNA demethylation is required for both the activation of ripening-induced genes and the inhibition of ripening-repressed genes.

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Global increase in DNA methylation during orange fruit development and ripening

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815441116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1430-1436 ◽

Cited By ~ 37

Author(s):

Huan Huang ◽

Ruie Liu ◽

Qingfeng Niu ◽

Kai Tang ◽

Bo Zhang ◽

...

Keyword(s):

Dna Methylation ◽

Fruit Ripening ◽

Tomato Fruit ◽

Dna Demethylation ◽

Epigenetic Mark ◽

Dna Hypermethylation ◽

Active Dna Demethylation ◽

Orange Fruit ◽

Global Increase ◽

Genomic Regions

DNA methylation is an important epigenetic mark involved in many biological processes. The genome of the climacteric tomato fruit undergoes a global loss of DNA methylation due to active DNA demethylation during the ripening process. It is unclear whether the ripening of other fruits is also associated with global DNA demethylation. We characterized the single-base resolution DNA methylomes of sweet orange fruits. Compared with immature orange fruits, ripe orange fruits gained DNA methylation at over 30,000 genomic regions and lost DNA methylation at about 1,000 genomic regions, suggesting a global increase in DNA methylation during orange fruit ripening. This increase in DNA methylation was correlated with decreased expression of DNA demethylase genes. The application of a DNA methylation inhibitor interfered with ripening, indicating that the DNA hypermethylation is critical for the proper ripening of orange fruits. We found that ripening-associated DNA hypermethylation was associated with the repression of several hundred genes, such as photosynthesis genes, and with the activation of hundreds of genes, including genes involved in abscisic acid responses. Our results suggest important roles of DNA methylation in orange fruit ripening.

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A DEMETER-like DNA demethylase governs tomato fruit ripening

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1503362112 ◽

2015 ◽

Vol 112 (34) ◽

pp. 10804-10809 ◽

Cited By ~ 126

Author(s):

Ruie Liu ◽

Alexandre How-Kit ◽

Linda Stammitti ◽

Emeline Teyssier ◽

Dominique Rolin ◽

...

Keyword(s):

Stress Responses ◽

Fruit Ripening ◽

Developmental Process ◽

Tomato Fruit ◽

Dna Demethylation ◽

Biochemical Processes ◽

Active Dna Demethylation ◽

Expression Of Genes ◽

Tomato Fruit Ripening ◽

Genes Encoding

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

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DNA Demethylation Pathways: Recent Insights

Genetics & Epigenetics ◽

10.4137/geg.s12143 ◽

2013 ◽

Vol 5 ◽

pp. GEG.S12143 ◽

Cited By ~ 4

Author(s):

Cong-jun Li

Keyword(s):

Dna Methylation ◽

Mammalian Cells ◽

De Novo ◽

Dna Demethylation ◽

Epigenetic Mark ◽

Sequencing Technologies ◽

Active Dna Demethylation ◽

Mammalian Genomes ◽

Genome Context

DNA methylation is a major epigenetic regulatory mechanism for gene expression and cell differentiation. Until recently, it was still unclear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether or not active demethylating activity is involved. Even the role of molecules and the mechanisms underlying the processes of active demethylation itself is blurred. Emerging sequencing technologies have led to recent insights into the dynamic distribution of DNA methylation during development and the role of this epigenetic mark within a distinct genome context, such as the promoters, exons, or imprinted control regions. This review summarizes recent insights on the dynamic nature of DNA methylation and demethylation, as well as the mechanisms regulating active DNA demethylation in mammalian cells, which have been fundamental research interests in the field of epigenomics.

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Postharvest handling induces changes in fruit DNA methylation status and is associated with alterations in fruit quality in tomato (Solanum lycopersicum L.)

10.1101/2020.10.08.331819 ◽

2020 ◽

Author(s):

Jiaqi Zhou ◽

Bixuan Chen ◽

Karin Albornoz ◽

Diane M Beckles

Keyword(s):

Dna Methylation ◽

Solanum Lycopersicum ◽

Fruit Quality ◽

Fruit Ripening ◽

Tomato Fruit ◽

Transcriptional Analysis ◽

Marker Genes ◽

Tomato Fruit Ripening ◽

Postharvest Handling ◽

Early Harvest

AbstractPostharvest handling of tomato (Solanum lycopersicum L.), specifically low-temperature storage and early harvest are used to extend shelf life, but often reduce fruit quality. Recent work suggests that DNA methylation dynamics influences fruit ripening through the demethylase SlDML2 gene. However, the influence of postharvest handling on DNA methylation in relation to fruit quality is unclear. This work aimed to clarify these issues by analyzing DNA methylation using methyl-sensitive amplification polymorphism (MSAP), semi-quantitative transcriptional analysis of marker genes for fruit quality (RIN; RIPENING INHIBITOR) and DNA methylation (SlDML2; Solanum lycopersicum L. DNA demethylase 2), and, fruit biochemical quality biomarkers. Multivariate analysis of these data supported the view that DNA methylation of fruit was influenced more by postharvest handling than ripening stage, however, fruit quality was influenced mainly by ripening. Fruit chilled postharvest were distinct in their DNA methylation state and quality characteristics, which implied that these three phenomena i.e., chilling, methylation, and quality are highly connected. In addition, different postharvest handling methods modulated SlDML2 transcript levels but had little effect on the level of RIN transcripts in fruit that reached the Turning stage after early harvest, and cold storage. These data collectively helped to advance our interpretation of tomato fruit ripening. In conclusion, our findings revealed that postharvest-induced variation in fruit quality is in relation to DNA methylation. Long-term this work will help better connect physiological changes in tomato fruit to events happening at the molecular level.

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Tet proteins: on track towards DNA demethylation?

BioMolecular Concepts ◽

10.1515/bmc-2012-0022 ◽

2012 ◽

Vol 3 (5) ◽

pp. 395-402 ◽

Cited By ~ 1

Author(s):

Nathalie Véron

Keyword(s):

Dna Methylation ◽

Enzymatic Activity ◽

Dna Demethylation ◽

Epigenetic Mark ◽

Developmental Processes ◽

Tet Proteins ◽

Active Dna Demethylation ◽

Demethylation Pathway ◽

Cellular Integrity ◽

Tet Protein

AbstractDynamic DNA methylation is a prerequisite for many developmental processes and maintenance of cellular integrity. In mammals however, mechanisms of active DNA demethylation have for long been elusive. The discovery of the ten-eleven translocation (Tet) family of enzymes that oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC) provided new means by which DNA methylation could actively be reversed. This review focuses on the possible mechanisms of DNA demethylation via Tet proteins and their metabolites 5hmC, 5fC and 5caC. Additionally, it discusses the roles of the three Tet protein family members Tet1, Tet2 and Tet3 as developmental regulators, probably in part independent of their enzymatic activity. By contrast, recent evidence suggests a function of 5hmC as an epigenetic mark on its own, going beyond the expectation of only acting as an intermediate in an active DNA demethylation pathway.

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A novel protein complex that regulates active DNA demethylation in Arabidopsis

10.1101/2020.02.21.958371 ◽

2020 ◽

Cited By ~ 1

Author(s):

Pan Liu ◽

Wen-Feng Nie ◽

Xiansong Xiong ◽

Yuhua Wang ◽

Yuwei Jiang ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Protein Complex ◽

Excision Repair ◽

Dna Demethylation ◽

Loss Of Function ◽

Dna Hypermethylation ◽

Active Dna Demethylation ◽

Domain Protein ◽

Genomic Regions

SUMMARYActive DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo.Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

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Regulatory link between DNA methylation and active demethylation in Arabidopsis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502279112 ◽

2015 ◽

Vol 112 (11) ◽

pp. 3553-3557 ◽

Cited By ~ 106

Author(s):

Mingguang Lei ◽

Huiming Zhang ◽

Russell Julian ◽

Kai Tang ◽

Shaojun Xie ◽

...

Keyword(s):

Dna Methylation ◽

De Novo ◽

Gene Promoter ◽

Dna Demethylation ◽

Target Sequence ◽

Loss Of Function ◽

Dna Hypermethylation ◽

Active Demethylation ◽

Active Dna Demethylation ◽

Regulatory Link

De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression.

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Chromoplast formation during tomato fruit ripening. No evidence for plastid DNA methylation

Plant Molecular Biology ◽

10.1007/bf00017913 ◽

1991 ◽

Vol 16 (1) ◽

pp. 11-19 ◽

Cited By ~ 32

Author(s):

Maria Rosa Marano ◽

N�stor Carrillo

Keyword(s):

Dna Methylation ◽

Fruit Ripening ◽

Tomato Fruit ◽

Plastid Dna ◽

Tomato Fruit Ripening

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Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation

International Journal of Molecular Sciences ◽

10.3390/ijms22031072 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1072

Author(s):

Woo Lee Choi ◽

Young Geun Mok ◽

Jin Hoe Huh

Keyword(s):

Dna Methylation ◽

Strand Break ◽

Dna Methyltransferases ◽

Dna Demethylation ◽

Dna Glycosylase ◽

Epigenetic Mark ◽

Gene Expressions ◽

Independent Manner ◽

Single Strand Break ◽

Active Dna Demethylation

In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.

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