Analysis of fruit-specific SLDML2 overexpression on ripening-related DNA demethylation in tomato fruit
Global agriculture today is facing the daunting problem of feeding a growing population on a decreasing amount of arable land. While increasing yields represents one strategy to address this issue, another is to reduce the spoilage of food that is already being produced. Fleshy fruits like tomato represent an attractive target for this improvement, as they are nutritionally-rich, but prone to spoilage upon ripening. Thus, the development of approaches to control the progression of ripening can be used to reduce food waste. My project focuses on the initiation of ripening in tomato fruit, with the goal of understanding the tight genetic controls underlying this process. Ripening in tomato is regulated by three interacting systems consisting of transcription factors, ethylene production, and DNA demethylation. I specifically focused on the role of DNA demethylation during ripening, as well as the gene DEMETER-LIKE DNA DEMETHYLASE 2 (SlDML2) that controls this process. I conducted my project using transgenic plants where expression of the SlDML2 gene occurs much earlier in fruit development than it would normally, and I characterized these lines using a combination of DNA methylation and expression analyses. I extracted DNA from two different fruit tissues, and used bisulfite conversion and sequencing to evaluate the differences in methylation levels between them. I also performed real-time polymerase chain reactions (qPCR) to measure the expression level of SlDML2 in different fruit tissues. I then evaluated this methylation and expression data while also considering observed fruit phenotypes to infer the role that SlDML2 and subsequent demethylation plays in ripening initiation. Ultimately, furthering this type of knowledge can lead to significant improvements in our ability to control the ripening process in a commercial setting and contribute to a more sustainable and less wasteful global food system.
My experience at the Boyce Thompson Institute this summer has been amazing to say the least! Through the program I have been able to learn countless new laboratory techniques, gain insight in applying to graduate school and NSF funding opportunities, learn about an array of ongoing research in plant science, as well as be surrounded by a fun and encouraging group of people. My research specifically allowed me to delve into my interest in epigenetics and learn about the role it plays in tomato ripening. My mentor, Dan Evanich, was a phenomenal teacher and role model, and helped me understand and become confident in using techniques such as bisulfite conversion, demethylation analysis, qPCR, and DNA extraction. I also learned that research comes with many challenges, such as changing or restarting experiments when they don’t go as well as hoped, or hurriedly moving samples out of a freezer when it unexpectedly starts to thaw. Overarchingly I really enjoyed and learned so much from every component of the experience BTI had to offer, and appreciate how formative of program it has been as I consider my plan and ambitions for a future in scientific research.