“Structural diversity in cardenolides: Gaining insights into the biosynthetic pathway”
Thanks to its phytochemical defense diversity, the Erysimum genus is better equipped to avoid herbivory than other plants in the Brassicaceae family. Erysimum produces two classes of toxic compounds, glucosinolates and cardenolides, a type of cardiac glycosides. Cardenolides act as inhibitors of the Na+, K+- ATPase ion channel, an essential and conserved membrane protein in animal cells that helps maintain the electrochemical gradient involved in many cell and organ functions. In human medicine, cardenolides are used as a treatment for heart arrhythmias and congestive heart failure. Despite their medical and ecological importance, the cardenolide biosynthetic pathway remains unknown. To address this knowledge gap, the Jander lab has established Erysimum cheiranthoides (wormseed wallflower) as a model system for studying cardenolide biosynthesis. Along with this, the lab applied mutagenesis and discovered plants with altered phenotypes, including the absence of derived cardenolides such as cannogenol and strophanthidin, presumably because of a mutation in a cytochrome P450 that hydroxylates digitoxigenin. This mutation is useful for studying the evolution of the biosynthetic pathway, as the Erysimum cytochrome P450 is homologous to a known Arabidopsis thaliana cytochrome P450 that hydroxylates steroid-like compounds. Transient overexpression of the P450 enzyme via Agrobacterium tumefaciens infiltration, implemented in E. cheiranthoides mutants that are deficient in derived cardenolides, successfully complemented the mutant phenotype. The cytochrome p450 was also transiently expressed in Nicotiana benthamiana plants via A. tumefaciens infiltration and addition of the substrate digitoxigenin. Yeast transformation was used to express the p450 in Saccharomyces cerevisiae, with addition of digitoxigenin to determine whether the derived compounds are produced.
This summer research internship has been one of the best experiences of my life, and I know it will mark an important milestone in my professional career. I remember that at the beginning of the internship I had expectations of getting an insight into academic life and the commitments that come associated with working in research, as well as acquiring new skills that would serve me in a possible future career as a research scientist myself. But today, reflecting and admiring the experience from the other side, I am certain that I got many other things out of this summer. The challenges of moving to an English-speaking country for two months, in the role of a short-term researcher, have been considerable. I have had opportunities to prove to myself that I have a strong sense of responsibility and resilience, and I have gained a lot of confidence in myself and my skills. I highly value the experience of working in a high-level research laboratory, and doing science has made me realize that it has to be collaborative. It was invaluable working with my mentor, Gordon Younkin, who was there to guide and support me but let me have the driver’s seat in developing my own project, to which I could contribute my knowledge.