Silencing this gene inhibits tomato fruit ripening . DNA methylation in plants regulates molecular processes such as gene expression and transposon silencing, which in turn modulates biological and developmental processes, and response to biotic and abiotic stress . During tomato fruit ripening, DNA demethylation is active, such that the global DNA methylation level is reduced . Demethylation is especially active in the promoter of ripening genes that contain the RIN binding sites . Post harvest handling strategies may be viewed as types of stress on the tomato fruit , but there are few reports related to post harvest induced changes in the tomato fruit methylome, especially early harvest and low-temperature storage. Based on current knowledge, post harvest chilling leads to hypermethylation of the promoter of ripening genes in red tomato fruit , suppressing their expression, and partially explaining the poor quality of refrigerated tomato. This chilling-induced methylation is reversible when fruit is rewarmed . Furthermore, Zhang et al. followed up and demonstrated that post harvest ethylene is able to stimulate increases in SlDML2 transcripts and DNA demethylation in tomato. Still in tomato, besides DNA methylation, there are also additional epigenetic regulatory mechanisms influencing fruit ripening. Methylation-directed mRNA changes, i.e., epitranscriptomic regulation that is mainly governed by mRNA demethylase, is also widespread in fruit ripening .
Another epigenetic mark- histone modifications after translation, planting blueberries in pots is commonly conserved in fleshy fruit species regulating ethylene biosynthesis . Histone lysine demethylase encoded by SlJMJ6 could regulate this histone methylation, therefore, promote tomato fruit ripening . In conclusion, post harvest practice is needed to control tomato fruit ripening speed, however, it may negatively reduce fruit quality. To maintain fruit quality as well as extend its shelf-life, a deep understanding on fruit ripening mechanism is necessary. The tomato ripening network is complex. There are raising questions regarding DNA methylation on tomato fruit ripening: though, the recent studies shown that post harvest chilling and ethylene have effect on global DNA methylation levels and fruit volatiles content, they didn’t focus on the post harvest practices that are commonly used in fresh tomato industry, i.e., early harvest and low-temperature storage; the correlation between post harvest induced DNA methylation and important fruit quality biomarkers including sugar, acid, firmness, and etc. is still unclear; the global DNA demethylation appears to take place during tomato fruit ripening, and is triggered by demethylase. However, the key players initiating demethylase gene expression, i.e., SlDML2 and SlJMJ6 are unknown. This could be studied by focusing on the transcriptional regulation of demethylase gene; the current knowledge only considers that DNA demethylation in the gene promoter activates ripening genes expression. However, the ripening transcription factors may also regulate DNA methylation by binding SlDML2. The potential evidence is that the ripening mutants, including rin, nor and Cnr, all have decreased SlDML2 expression level in fruit ripening. A further study is needed for testing their regulatory relationship; hormone, a key player in fruit ripening, may play an important role in regulating DNA demethylation during ripening. Their relationship is still undetermined.Tomato is one of the world’s most popular fresh-market vegetables , and is also an important research model for fleshy-fruit development .
Tomato is highly perishable after harvest , and determining adequate post harvest handling and storage conditions is important for extending fruit shelf-life and reducing post harvest losses , which is necessary given the complicated modern supply chain for fresh produce . Early harvest and low-temperature storage can delay fruit ripening and extend shelf-life, but there can often be unintended consequences, such as lower fruit sensory quality, reduced consumer satisfaction, and as a result, fewer repeat purchases . For example, Mature green fruit ripened off-the vine at temperatures lower than ambient will have maximal shelf-life, but they may not have a fully realized sensory profile . These poor sensory attributes compared to ‘on-the-vine’ ripened tomato occur because fruit nutrient supply from the mother plant is prematurely disrupted, and there may be additional losses of flavor- and taste-associated compounds occurring during storage which collectively leads to post harvest waste . Further, exposing tomatoes to temperatures below 10°C can severely disrupt normal ripening leading to post harvest chilling injury . This physiological disorder is cumulative in its effect, as the consequences are normally presented during rewarming, and include unusual Tomato is one of the world’s most popular fresh-market vegetables , and is also an important research model for fleshy-fruit development . Tomato is highly perishable after harvest , and determining adequate post harvest handling and storage conditions is important for extending fruit shelf-life and reducing post harvest losses , which is necessary given the complicated modern supply chain for fresh produce . Early harvest and low-temperature storage can delay fruit ripening and extend shelf-life, but there can often be unintended consequences, such as lower fruit sensory quality, reduced consumer satisfaction, and as a result, fewer repeat purchases . For example, Mature green fruit ripened off-the vine at temperatures lower than ambient will have maximal shelf-life, but they may not have a fully realized sensory profile .
These poor sensory attributes compared to ‘on-the-vine’ ripened tomato occur because fruit nutrient supply from the mother plant is prematurely disrupted, and there may be additional losses of flavor- and taste-associated compounds occurring during storage which collectively leads to post harvest waste . Further, exposing tomatoes to temperatures below 10°C can severely disrupt normal ripening leading to post harvest chilling injury . This physiological disorder is cumulative in its effect, as the consequences are normally presented during rewarming, and include unusual softening, poor flavor and taste, and, visual defects such as uneven ripening, pitting and decay . Tomato quality attributes are determined by many genetic, physiological and biochemical factors that occur as the fruit ripens. This leads to the characteristic and desirable changes in color, texture, flavor and taste. Measurements of fruit firmness can act as a proxy for texture and juiciness , color is an indicator of ripening stage and visual quality , and the ratio of sugar-to-acid contributes to an appealing tomato taste and is used as a marker of this attribute . The biochemical changes that lead to these and other events are controlled by programmed developmental pathways that are disrupted by off-the-vine ripening and low-temperature storage . Tomato fruit ripening, and therefore quality is mediated in part, by upstream changes in DNA methylation . The promoter region of many ripening genes remains methylated during early fruit development until the onset of ripening . In tomato, DNA demethylation is critical for fruit ripening and quality, while in contrast, post harvest chilling reverses this process and promotes methylation of many ripening genes, and it is associated with reduced quality . Two genes have important roles in these observations: SlDML2 and RIN which both increase in expression during tomato fruit development. SlDML2 encodes a DNA demethylase, that activates hundreds of ripening-related genes in tomato fruit by removing the methyl-group from their promoter region . One important target of SlDML2 is RIN . RIN is a central fruit ripening transcription factor that is also, powerfully regulated by the DNA methylation levels of its promoter regions . RIN and RIN-induced genes and transcription factors are suppressed through hypermethylation, in pre-ripened fruit or fruit exposed to chilling . The dilemma raised here is that consumers prefer full-flavored tomato fruits , but post harvest practices designed to extend shelf-life often reduce fruit quality. The former observations provide a cornerstone for understanding how post harvest techniques result in the loss of flavor in tomato fruit. Here, we researched if changes in fruit DNA methylation status are induced by different post harvest practices i.e. early harvest at Mature green, and low-temperature storage, and, raspberries in pots if there is a relationship between changes in DNA methylation and fruit quality parameters. An unbiased overview of changes in ripening-associated genome methylation was determined using Methyl-sensitive amplification polymorphism . This is a simple method that indicates changes in a limited set of methylation sites across genomes . In MSAP, two restriction enzymes HpaII and MspI that recognize the same CCGG sequence, but with differential sensitivity to methylation at the inner or outer cytosine are used. DNA methylation status is then determined first, by analyzing the number and sizes of the generated fragments after enzyme digestion, and second, by comparing fragments from tissues at different developmental stages, environmental treatment etc. to indicate how these conditions influence global methylation status .
To sum up, our aim was to use MSAP to clarify how industry practices influence fruit global DNA methylation levels. This is a first step to extend shelf-life while helping improve fruit quality. Long-term, reducing post harvest losses and increasing market consumption could be possible if the relationships of fruit quality and DNA methylation are understood.All harvested fruits were soaked in 0.25% sodium hypochlorite and rinsed with nanopore water, wrapped with paper towels until dry before storage or further analysis. To explore potential changes at the molecular level, and in tomato fruit quality due to post harvest practices, specifically, early harvest and low-temperature storage, we harvested fruit at Mature green , and stored them at different temperatures until they reached the Turning stage . Turning is the ripening stage just before Red ripe for ‘MicroTom’, which is similar to the Pink stage for conventional tomatoes . To investigate post harvest chilling injury, we stored Mature green fruit at 5°C for 14 days in order to induce this disorder . Tomato fruit are unable to ripen at this temperature, so after chilling they were allowed to recover at 20°C until they reached Turning.The DNA methylation data generated by MSAP were grouped into one of three classes based on treatment-induced changes i.e., de novo methylation, demethylation, or no change in methylation status. These data were depicted in two ways; first, showing details of the relative abundance of individual sites that led to the above classification , and second, providing an overview of the data i.e. the percentage of each methylation class induced by the treatment .We found distinctive global DNA methylation levels in fruit at the same ripening stage i.e. Turning, due to post harvest handling. This conclusion was drawn based on the MSAP data , which indicated that there were different de novo methylation and demethylation events occurring across assorted Turning fruit, and only a limited number of the examined sites did not change. ‘FHT’ fruit were ripened under optimal condition. Early-harvested fruit that were ripened at 20°C was the most similar in methylation patterns to ‘FHT’, while the most contrastable DNA methylation fragmentation state was observed between ‘FHT’ and ‘5T’. Comparing the methylation levels in ‘FHM’ vs. ‘FHT’, we found that 26.13% of the genomic sites tested represented DNA demethylation events, which is greater than the percentage representing de novo methylation events . This indicates that the overall cytosine methylation was reduced during ‘on-the-vine’ ripening. To focus on the chilling effect on Mature green fruit, chilled ‘5M’ were compared to those that were freshly harvested ‘FHM’ . The data showed that 39.64% of the bands underwent de novo methylation, 35.14% of the bands were unchanged, and 25.23% of bands were demethylated in ‘5M’ relative to ‘FHM’. Thus, the data from 111 generated DNA fragments show that DNA methylation status was influenced by chilling.After transferring previously chilled fruit to room temperature, fruit were able to resume ripening. Under this recovery treatment, more genomic sites were demethylated, compared to those that underwent de novo methylation . The high percentage of demethylation events during rewarming is similar to the trend seen during normal ripening . This suggested that the normal demethylation events that occured during ripening recovered when the fruit were rewarmed, which allowed the fruit to reach Turning after two weeks chilling . To summarize all observations generated by the MSAP analysis, post harvest handling induced changes in fruit global DNA methylation status, and the rank of their methylation status based on similarity to “on-the-vine” ripened fruit at Turning was ‘20T’ > ‘12.5T’ > ‘5T’; demethylation occurred as the fruit ripened; chilling at the onset of fruit ripening inhibited the demethylation trend in normal fruit ripening; and DNA demethylation occurred in the following rewarming process, but some of these demethylated sites were different to the fruit under normal ripening.DNA methylation has been reported to play a critical role in regulating fruit ripening. In the current work, we focused on potential changes in DNA methylation dynamics due to post harvest handling.