Comparative WGBS identifies genes that influence non-ripe phenotype in tomato epimutant <Emphasis Type="Italic">Colourless non-ripening</Emphasis>

Whole-genome bisulfite sequencing (WGBS) allows single-base resolution and genome-wide profiling of DNA methylation in plants and animals. This technology provides a powerful tool to identify genes that are potentially controlled by dynamic changes of DNA methylation and demethylation. However, naturally occurring epimutants are rare and genes under epigenetic regulation as well as their biological relevances are often difficult to define. In tomato, fruit development and ripening are a complex process that involves epigenetic control. We have taken the advantage of the tomato epimutant Colourless non-ripening (Cnr) and performed comparative mining of the WGBS datasets for the Cnr and SlCMT3-silenced Cnr fruits. We compared DNA methylation profiles for the promoter sequences of approximately 5,000 bp immediately upstream of the coding region of a list of 20 genes. Differentially methylated regions were found for some of these genes. Virus-induced gene silencing (VIGS) of differentially methylated gene SlDET1 or SlPDS resulted in unusual brown pigmentation in Cnr fruits. These results suggest that comparative WGBS coupled with VIGS can be used to identify genes that may contribute to the colourless unripe phenotype of fruit in the Cnr epimutant.     

Effect of the Colorless non-ripening mutation on cell wall biochemistry and gene expression during tomato fruit development and ripening

The Colorless non-ripening (Cnr) mutation in tomato (Solanum lycopersicum) results in mature fruits with colorless pericarp tissue showing an excessive loss of cell adhesion (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). This pleiotropic mutation is an important tool for investigating the biochemical and molecular basis of cell separation during ripening. This study reports on the changes in enzyme activity associated with cell wall disassembly in Cnr and the effect of the mutation on the program of ripening-related gene expression. Real-time PCR and biochemical analysis demonstrated that the expression and activity of a range of cell wall-degrading enzymes was altered in Cnr during both development and ripening. These enzymes included polygalacturonase, pectinesterase (PE), galactanase, and xyloglucan endotransglycosylase. In the case of PE, the protein product of the ripening-related isoform PE2 was not detected in the mutant. In contrast with wild type, Cnr fruits were rich in basic chitinase and peroxidase activity. A microarray and differential screen were used to profile the pattern of gene expression in wild-type and Cnr fruits. They revealed a picture of the gene expression in the mutant that was largely consistent with the real-time PCR and biochemical experiments. Additionally, these experiments demonstrated that the Cnr mutation had a profound effect on many aspects of ripening-related gene expression. This included a severe reduction in the expression of ripening-related genes in mature fruits and indications of premature expression of some of these genes in immature fruits. The program of gene expression in Cnr resembles to some degree that found in dehiscence or abscission zones. We speculate that there is a link between events controlling cell separation in tomato, a fleshy fruit, and those involved in the formation of dehiscence zones in dry fruits.     

Detection of differentially methylated regions from whole-genome bisulfite sequencing data without replicates

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Recent developments in whole genome bisulfite sequencing (WGBS) technology have enabled genome-wide measurements of DNA methylation at single base pair resolution. Many experiments have been conducted to compare DNA methylation profiles under different biological contexts, with the goal of identifying differentially methylated regions (DMRs). Due to the high cost of WGBS experiments, many studies are still conducted without biological replicates. Methods and tools available for analyzing such data are very limited.We develop a statistical method, DSS-single, for detecting DMRs from WGBS data without replicates. We characterize the count data using a rigorous model that accounts for the spatial correlation of methylation levels, sequence depth and biological variation. We demonstrate that using information from neighboring CG sites, biological variation can be estimated accurately even without replicates. DMR detection is then carried out via a Wald test procedure. Simulations demonstrate that DSS-single has greater sensitivity and accuracy than existing methods, and an analysis of H1 versus IMR90 cell lines suggests that it also yields the most biologically meaningful results. DSS-single is implemented in the Bioconductor package DSS.     

DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development

We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.     

Effect of the Cnr mutation on carotenoid formation during tomato fruit ripening

The characteristic pigmentation of ripe tomato fruit is due to the deposition of carotenoid pigments. In tomato, numerous colour mutants exist. The Cnr tomato mutant has a colourless, non-ripening phenotype. In this work, carotenoid formation in the Cnr mutant has been studied at the biochemical level. The carotenoid composition of Ailsa Craig (AC) and Cnr leaves was qualitatively and quantitatively similar. However, Cnr fruits had low levels of total carotenoids and lacked detectable levels of phytoene and lycopene. The presence of normal tocopherols and ubiquinone-9 levels in the ripe Cnr fruits suggested that other biosynthetically related isoprenoids were unaffected by the alterations to carotenoid biosynthesis. In vitro assays confirmed the virtual absence of phytoene synthesis in the ripe Cnr fruit. Extracts from ripe fruit of the Cnr mutant also revealed a reduced ability to synthesise the carotenoid precursor geranylgeranyl diphosphate (GGPP). These results suggest that besides affecting the first committed step in carotenoid biosynthesis (phytoene synthase) the Cnr mutation also affects the formation of the isoprenoid precursor (GGPP).     

Genetic identification and genomic organization of factors affecting fruit texture

Fleshy fruits are an essential part of the human diet providing vital vitamins, minerals and other health-promoting compounds. The texture of the ripe fruit has a significant effect on quality and influences consumer acceptance, shelf-life, resistance, and transportability. The development of rational approaches to improve texture and shelf-life depend on understanding the biological basis of fruit ripening. Until recently, work has focused on the isolation of ripening-related genes from a variety of fleshy fruits. However, little is known about the genes that regulate this complex developmental process or whether similar regulatory genes are active in all fruiting species. A major breakthrough would be the identification of generic genes associated with texture and other aspects of ripening in fleshy fruits. In tomato, a small number of single gene mutations exist, such as ripening-inhibitor (rin), non-ripening (nor), Never-ripe (Nr), and Colourless non-ripening (Cnr) which have pleiotropic effects resulting in the reduction or almost complete abolition of ripening. These mutations probably represent lesions in regulatory genes. The cloning of the wild-type alleles of RIN and NOR is reported by Moore et al. in this issue. This review focuses on the texture characteristics of the Cnr mutant. A possible framework for the molecular regulation of fruit texture is discussed and quantitative genetic approaches to determining the generic attributes of fruit texture are explored.     

Virus-induced gene silencing in tomato fruit

Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants. Here we report that either by syringe-infiltrating the tobacco rattle virus (TRV)-vector into the surface, stem or carpopodium of a tomato fruit attached to the plant or by vacuum-infiltrating into a tomato fruit detached from the plant, TRV can efficiently spread and replicate in the tomato fruit. Although VIGS can be performed in tomato fruit by all of the means mentioned above, the most effective method is to inject the TRV-vector into the carpopodium of young fruit attached to the plant about 10 days after pollination. Several reporter genes related to ethylene responses and fruit ripening, including LeCTR1 and LeEILs genes, were also successfully silenced by this method during fruit development. In addition, we found that the silencing of the LeEIN2 gene results in the suppression of tomato fruit ripening. The results of our study indicate that the application of VIGS techniques by the described methods can be successfully applied to tomato fruit and is a valuable tool for studying functions of the relevant genes during fruit developing.     

Global analysis of DNA methylation in hepatocellular carcinoma via a whole-genome bisulfite sequencing approach

Alterations in DNA methylation patterns are considered early events in hepatocellular carcinoma (HCC). However, their mechanism and significance remain to be elucidated. We studied the genome-wide DNA methylation landscape of HCC by applying whole-genome bisulfite sequencing (WGBS) techonlogy. Overall, HCC exhibits a genome-wide hypomethylation pattern. After further annotation, we obtained 590 differentially hypermethylated genes (hyper-DMGs) and 977 differentially hypomethylated genes (hypo-DMGs) from three groups. Hyper-DMGs were mainly involved in ascorbate and alternate metabolism pathways, while hypo-DMGs were mainly involved in focal adhesion. By integrating the DMGs with HCC-related differentially expressed genes (DEGs) and DMGs from the TCGA database, we constructed prognostic model based on thirteen aberrantly methylated DEGs, and verified our prognostic model in GSE14520 dataset. This study compares the patterns of global epigenomic DNA methylation during the development of HCC, focusing on the role of DNA methylation in the early occurrence and development of HCC, providing a direction for future research on its epigenetic mechanism.     

Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature (15 degrees C) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.     

Downregulation of RdDM during strawberry fruit ripening

Background

Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during the ripening of non-climacteric fruits are unknown.

Results

Here, we generate single-base resolution maps of the DNA methylome in immature and ripe strawberry. We observe an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. Application of a DNA methylation inhibitor causes an early ripening phenotype, suggesting that DNA hypomethylation is important for strawberry fruit ripening. The mechanisms underlying DNA hypomethylation during the ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes are not upregulated during the ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation are downregulated during strawberry ripening. Further, ripening-induced DNA hypomethylation is associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that a downregulation of RdDM contributes to DNA hypomethylation during strawberry ripening.

Conclusions

Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climacteric fruit and suggest a novel function of RdDM in regulating an important process in plant development.

     

Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.