AUTHOR=Jarvis Brice A. , Romsdahl Trevor B. , McGinn Michaela G. , Nazarenus Tara J. , Cahoon Edgar B. , Chapman Kent D. , Sedbrook John C. 

TITLE=CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.)

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.652319

DOI=10.3389/fpls.2021.652319

ISSN=1664-462X

ABSTRACT=<p>Pennycress (<italic>Thlaspi arvense</italic> L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the <italic>FATTY ACID DESATURASE2</italic> (<italic>FAD2</italic>) and <italic>REDUCED OLEATE DESATURATION1</italic> (<italic>ROD1</italic>) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a <italic>FATTY ACID ELONGATION1</italic> (<italic>fae1</italic>) knockout mutation, <italic>fad2 fae1</italic> and <italic>rod1 fae1</italic> double mutants produced ∼90% and ∼60% oleic acid in seed oil, respectively, with PUFAs in <italic>fad2 fae1</italic> as well as <italic>fad2</italic> single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, <italic>rod1 fae1</italic> plants grew like wild type whereas <italic>fad2</italic> single and <italic>fad2 fae1</italic> double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of <italic>ROD1</italic> and <italic>FAE1</italic> may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in <italic>FAD2</italic> will likely require at least partial function to avoid fitness trade-offs.</p>