BIOSYNTHESIS AND FUNCTION OF CARDIAC GLYCOSIDES IN THE CRUCIFER GENUS <i>ERYSIMUM</i>

<i>Erysimum</i> is a diverse genus within the Brassicaceae consisting of several hundred species distributed across the temperate northern hemisphere. Like most members of the mustard family, <i>Erysimum</i> produces evolutionarily ancestral glucosinolates as a defense against herbivores. However, its recent and rapid radiation has been partially attributed to its 'escape from herbivory' via the evolution of a different group toxic compounds called cardiac glycosides or cardenolides. Cardiac glycosides have been used to treat heart conditions for hundreds of years and are on the World Health Organization's list of essential medicines. However, the biosynthetic pathway remains unknown. In Chapter 1 of this dissertation, I provide background information and describe the development of genetic resources for the study of cardiac glycoside biosynthesis in <i>Erysimum cheiranthoides</i>, including an improved genome assembly and a protocol for floral dip stable transformation. In subsequent chapters, I use metabolomic and transcriptomic datasets to identify candidate genes for cardiac glycoside biosynthesis and test candidate gene function using CRISPR/Cas9-mediated gene editing, in vitro assays with purified recombinant proteins, and pathway reassembly in heterologous systems. In total, I identified and characterized seven enzymes that are involved in cardiac glycoside biosynthesis in <i>E. cheiranthoides</i>. In Chapter 2, I describe EcCYP87A126, a cytochrome P450 that initiates cardiac glycoside biosynthesis via sterol side chain cleavage. Chapter 3 explores Ec3βHSD (a hydroxysteroid dehydrogenase), Ec3KSI (a ketosteroid isomerase), EcP5βR2 (a progesterone 5β-reductase), and EcDET2 (a steroid 5α-reductase), which are involved in oxidation and reduction of the steroid core and help to explain variation in cardiac glycoside structure that is observed across the <i>Erysimum</i> genus. Finally, I discuss two 2-oxoglutarate dependent dioxygenases that are required for cardiac glycoside biosynthesis in Chapter 4. Through the identification of these enzymes, I begin to untangle the evolutionary history of cardiac glycoside biosynthesis in the genus, and I use cardiac glycoside-deficient mutant lines to better understand their role in protection against insect herbivores in an already well-defended plant lineage. These results represent a step forward in our understanding of cardiac glycoside biosynthesis and function, with implications for engineering the pathway in heterologous systems.

Name thesis:degree_name

Ph. D., Plant Biology

Level thesis:degree_level

Doctor of Philosophy

Discipline thesis:degree_discipline

Plant Biology

Grantor

Cornell University

Year dc:date.issued

2024

Author dc:creator

• Younkin, Gordon

Committee members dc:contributor.committeemember

• Schroeder, Frank
• Moghe, Gaurav

• cardenolides
• cardiac glycosides
• Erysimum
• evolution
• herbivory
• metabolism

Statement dc:rights

• Attribution-NonCommercial 4.0 International

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc/4.0/

Language dc:language.iso

en

DOI

https://doi.org/10.7298/42t2-w321

Dc Identifier Other

ProQuest Submission ID: 14212
ProQuest Publication ID: 31238499

OAI identifier oai:identifier

oai:ecommons.cornell.edu:1813/116043