Ripening Oranges with Epigenetics
Have you ever tried to peel an orange that wasn’t quite ripe yet? The peel sticks to the fruit, coming off in frustratingly small segments. Over the years, scientists have found that many different factors regulate the process of fruit ripening. The most well-known of these are plant hormones like ethylene, auxin, and abscisic acid (ABA) . If you’ve ever placed a piece of unripe fruit in a bag with a banana and seen it ripen ̶ sometimes overnight ̶ you’ve witnessed the work of ethylene. In addition to hormones and transcription factors, which turn specific genes on or off, epigenetic modifications are also important regulators of fruit ripening .
One important type of epigenetic modification is the addition of methyl groups to DNA, called DNA methylation. A change in DNA methylation during fruit ripening was first observed in tomatoes, where global DNA methylation steadily decreased as the fruit ripened. Recent research has shown that DNA methylation might regulate ripening in citrus fruits as well . In a new study published in PNAS, Huang and colleagues set out to characterize DNA methylation with single base-pair resolution in the Newhall navel orange as it ripens.
To assess overall methylation levels across the orange genome, the authors used whole-genome bisulfite sequencing analysis on DNA from oranges 90, 120, 150, 180, and 210 days after blooming. Surprisingly, they found that over the course of these five time points, DNA methylation gradually rose until it reached its highest levels at 210 days. This observation was the opposite of what was found in tomatoes, which decrease DNA methylation during ripening, and suggests that both DNA hyper- or hypo-methylation can contribute to ripening in a fruit-specific manner.
What causes the increase in global DNA methylation in ripening oranges? The researchers reasoned that during ripening, the expression levels of enzymes that either add or remove methyl groups from DNA, called DNA methyltransferase and DNA demethylase, respectively, may change to promote hyper-methylation. To test these possibilities, the authors measured the expression level of seven methyltransferase genes and four demethylase genes during ripening. They found that the expression of methyltransferase genes did not change over the ripening period, but DNA demethylase expression significantly decreased. These results indicated that the global increase in DNA methylation observed during orange ripening is likely not a result of increased methylation, but through a reduction in the removal of methyl groups from DNA.
Next, the researchers determined if the increase in DNA methylation had an effect on overall gene expression. Using RNA sequencing (RNA-seq), Huang and colleagues found that the number of genes with altered expression levels increased as ripening progressed, which corresponded with the increase in DNA methylation. When the expression of genes associated with hypermethylated regions of DNA was analyzed, it was found that 1,113 of those genes decreased expression, while 950 genes increased their expression. These results were surprising because DNA methylation is typically associated with repression of gene expression.
To better understand the role of the genes that are differentially expressed as a result of DNA methylation during ripening, the authors used Gene Ontology (GO) analysis to find any common functions among the genes. They found that many of the genes with decreased expression were involved in processes like photosynthesis and fruit development. The fact that these genes had lower expression levels during ripening made sense because processes like photosynthesis occur before fruit ripening begins. The genes that had increased expression included those that were involved in the ABA response, which is known to be important for ripening of non-climacteric fruits, which include oranges, strawberries, and lemons . These findings show that increased DNA methylation likely contributes to ripening by regulating the expression of genes that are important for fruit development at specific stages.
Finally, the researchers asked if a DNA methylation inhibitor could block orange ripening if applied directly to the fruit. Over the course of ripening, the researchers saw that the orange treated with the DNA methylation inhibitor (5-azacytidine) failed to change from green to orange – a process called degreening and an important indicator of ripening. In terms of gene expression, a gene that normally decreases expression during ripening failed to decrease, and one that typically increases in expression did not increase, emphasizing the importance of DNA hyper-methylation in orange fruit ripening.
Overall, this study shows that a global increase in DNA methylation occurs in Newhall navel oranges as they ripen, in contrast to the ripening process of tomatoes. This study also provides the first detailed characterization of DNA methylation in a citrus fruit during ripening and the associated changes in gene expression. More research needs to be done to uncover the molecular pathways involved in orange ripening and the divergent role of DNA methylation in ripening different fruits. However, the contribution of this study to a better understanding of citrus fruit ripening makes enjoying a perfect, easy-to-peel orange all that much sweeter.
Original paper: Huang H, Liu R, Niu Q, Tang K, Zhang B, Zhang H, Chen K, Zhu JK, Lang Z (2019). Global increase in DNA methylation during orange fruit development and ripening. Proc Natl Acad Sci U S A, 201815441. doi: 10.1073/pnas.1815441116.
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