Graduation Term
Summer 2025
Degree Name
Master of Science (MS)
Department
School of Biological Sciences
Committee Chair
John C. Sedbrook
Committee Co-Chair
Thomas M. Hammond
Committee Member
Viktor Kirik
Abstract
Advances in genome editing are accelerating the domestication and value enhancement of winter annual crops like pennycress and rye, which could serve as excellent sources of animal feed, making them more profitable for farmers along with contributing to sustainable agriculture and bioenergy production. Pennycress (Thlaspi arvense L.) is a Brassica species with a natural growth cycle from fall to spring. Domesticated pennycress varieties have been developed for the U.S. Midwest Corn Belt and other temperate growing regions to produce seed oil and meal for biofuels and animal feed uses. As a viable feedstock for renewable diesel and sustainable aviation fuel (SAF), domesticated pennycress seed oil offers a promising pathway toward “climate smart” greener energy solutions. To make pennycress profitable, efforts are underway to improve the quality of pennycress seed meal by lowering levels of sinapic acid. Studies in rapeseed have shown that sinapate esters with sinapoylcholine contribute to the bitter taste, astringency, and dark color of seed products (Husken et al., 2005, Molecular Breeding). During seed oil processing, sinapate esters get oxidized and form complexes with proteins, thus lowering the digestibility of the meal. We utilized CRISPR-Cas9 genome editing technology to introduce loss-of-function mutations in two key genes of the phenylpropanoid biosynthetic pathway, namely FERULIC ACID 5-HYDROXYLASE (F5H) and REDUCED EPIDERMAL FLUORESCENCE 1 (REF1). We found that pennycress f5h and ref1 single knockout mutants produced seeds with substantially reduced amounts of sinapate esters and exhibited growth indistinguishable from wild-type controls. Moreover, senesced stems of pennycress f5h mutants had drastic reductions in S lignin content similar to Arabidopsis f5h mutants. Interestingly, pennycress does not accumulate the secondary metabolite sinapoyl malate in leaves like Arabidopsis but instead flavonoid compounds which were also reduced in pennycress ref1 and f5h mutants. We also investigated the role that sinapoylcholine plays in modulating ABA homeostasis during seed germination in pennycress. Taken together, our studies show that mutations in f5h and ref1 in pennycress substantially reduced sinapate esters in the seeds which should improve meal quality. The plants grow similar to wild type, making these mutations worth considering for pennycress domestication. Parallel efforts in rye (Secale cereale L.) aim to enhance its value as animal feed and cover crop through CRISPR targeting of lignin biosynthesis genes (CWG1, CWG2, CWG3). While rye offers excellent biomass potential, its genetic complexity and recalcitrant tissue culture pose challenges for genome editing. We designed high-specificity guide RNAs and validated CRISPR constructs for CWG1, CWG2 and CWG3. Successful lignin modification in rye through genome editing has the potential to enhance saccharification efficiency, thereby improving cell wall digestibility for ruminant feed applications, while preserving its agronomic advantages as a resilient winter cover crop.
Access Type
Thesis-Open Access
Recommended Citation
Lima, Arjuman, "Altering the Phenylpropanoid Pathway to Decrease Sinapine Content in Pennycress Seeds and Lignin Content in Winter Cereal Rye Biomass" (2025). Theses and Dissertations. 2181.
https://ir.library.illinoisstate.edu/etd/2181
DOI
https://doi.org/10.30707/ETD.1763755358.815272