“Understanding the role of SlDML2 expression on ripening initiation in tomato fruit”

Project Summary:

More than one third of fresh fruit produced in the US each year is lost due to spoilage. As the ripening process predisposes fruit to spoilage, the manipulation of ripening regulation is an attractive strategy to reduce losses and promote food security. There are three main facets of ripening regulation: transcriptional control, ethylene signaling and DNA demethylation. The least is known about DNA demethylation, although it has been shown that global DNA demethylation occurs during ripening. Also, chemically-mediated DNA hypomethylation can trigger ripening in immature fruit, suggesting that DNA methylation levels influence the timing of ripening initiation. In tomato, the gene DEMETER-LIKE DNA DEMETHYLASE 2 (SlDML2) is necessary for proper ripening, as fruit fail to ripen when its expression is inhibited. My summer project was to study the role of SlDML2 in ripening initiation using lines that overexpress SlDML2. I characterized the phenotype of these lines along with crosses made between the SlDML2 overexpression plants and several non-ripening mutants that are impaired in DNA demethylation to determine if SlDML2 overexpression could fully or partially restore ripening. While no obvious ripening phenotypes were observed, bisulfite PCR showed that methylation levels are reduced in the strongest overexpression lines indicating that, while the transgene worked, it may not have sufficiently demethylated promoter targets to be effective. I also created a CRISPR/Cas9 construct that will be used to generate an SlDML2 knockout mutant. In conclusion, our results suggest that DNA methylation levels respond to SlDML2 overexpression, although more in depth characterization of these lines is required to assess whether or not there is any ripening change in these lines. If so, this work could be used as a model strategy to control fruit ripening in additional species, thus enhancing food distribution and production systems.

My Experience:

My experience in the BTI-Cornell PGRP REU program has been incredible! I have learned so many new research skills, from plasmid isolation to creating CRISPR/Cas9 constructs, and I have gained experience with plant genetics and sequence data analysis. My mentor, Dan Evanich, was incredible in that he was very eager to ensure that I understood the details of my research, but also was willing to step back and let me carry out procedures on my own. This experience has improved my independence as a researcher and my ability to think about the broader implications of research results and has challenged me to become more familiar with various computational biology skills. My interactions with the various faculty and staff at BTI and Cornell have helped reconfirm that I want to work with plant science research and have given me insight into what I need to do to prepare for graduate school.