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

Abstract

Pennycress (Thlaspi arvense 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 FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) 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 FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ~90% and ~60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 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 whichmore » 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, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 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 ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.« less

Authors:
 [1];  [2];  [1];  [3];  [3];  [2];  [1]
  1. Illinois State University (ISU), Normal, IL (United States). School of Biological Sciences
  2. Univ. of North Texas, Denton, TX (United States). Dept. of Biological Sciences. BioDiscovery Inst.
  3. Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation. Dept. of Biochemistry
Publication Date:
Research Org.:
Univ. of North Texas, Denton, TX (United States); Illinois State University (ISU), Normal, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1853580
Grant/Contract Number:  
SC0020325; SC0021286
Resource Type:
Accepted Manuscript
Journal Name:
Frontiers in Plant Science
Additional Journal Information:
Journal Volume: 12; Journal ID: ISSN 1664-462X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; plant sciences; CRISPR; MALDI-MSI; oilseed; oleic acid; pennycress; polyunsaturated fatty acid; Thlaspi arvense; triacylglycerol

Citation Formats

Jarvis, Brice A., Romsdahl, Trevor B., McGinn, Michaela G., Nazarenus, Tara J., Cahoon, Edgar B., Chapman, Kent D., and Sedbrook, John C. CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.). United States: N. p., 2021. Web. doi:10.3389/fpls.2021.652319.
Jarvis, Brice A., Romsdahl, Trevor B., McGinn, Michaela G., Nazarenus, Tara J., Cahoon, Edgar B., Chapman, Kent D., & Sedbrook, John C. CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.). United States. https://doi.org/10.3389/fpls.2021.652319
Jarvis, Brice A., Romsdahl, Trevor B., McGinn, Michaela G., Nazarenus, Tara J., Cahoon, Edgar B., Chapman, Kent D., and Sedbrook, John C. Thu . "CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.)". United States. https://doi.org/10.3389/fpls.2021.652319. https://www.osti.gov/servlets/purl/1853580.
@article{osti_1853580,
title = {CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.)},
author = {Jarvis, Brice A. and Romsdahl, Trevor B. and McGinn, Michaela G. and Nazarenus, Tara J. and Cahoon, Edgar B. and Chapman, Kent D. and Sedbrook, John C.},
abstractNote = {Pennycress (Thlaspi arvense 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 FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) 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 FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ~90% and ~60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 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, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 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 ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.},
doi = {10.3389/fpls.2021.652319},
journal = {Frontiers in Plant Science},
number = ,
volume = 12,
place = {United States},
year = {Thu Apr 22 00:00:00 EDT 2021},
month = {Thu Apr 22 00:00:00 EDT 2021}
}

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