TILLING-by-Sequencing⁺ Reveals the Role of Novel Fatty Acid Desaturases (GmFAD2-2s) in Increasing Soybean Seed Oleic Acid Content

• Main Authors: Naoufal Lakhssassi, Valéria Stefania Lopes-Caitar, Dounya Knizia, Mallory A. Cullen, Oussama Badad, Abdelhalim El Baze, Zhou Zhou, Mohamed G. Embaby, Jonas Meksem, Aicha Lakhssassi, Pengyin Chen, Amer AbuGhazaleh, Tri D. Vuong, Henry T. Nguyen, Tarek Hewezi, Khalid Meksem
• Format: Article
• Language: English
• Published: MDPI AG 2021-05-01
• Series: Cells
• Subjects: high oleic acid; alternative fatty acid pathway; <i>GmFAD2-2</i> subfamily; TILLING-by-Sequencing⁺; subfunctionalization; neofunctionalization
• Online Access: https://www.mdpi.com/2073-4409/10/5/1245

Soybean is the second largest source of oil worldwide. Developing soybean varieties with high levels of oleic acid is a primary goal of the soybean breeders and industry. Edible oils containing high level of oleic acid and low level of linoleic acid are considered with higher oxidative stability and can be used as a natural antioxidant in food stability. All developed high oleic acid soybeans carry two alleles; <i>GmFAD2-1A</i> and <i>GmFAD2-1B</i>. However, when planted in cold soil, a possible reduction in seed germination was reported when high seed oleic acid derived from <i>GmFAD2-1</i> alleles were used. Besides the soybean fatty acid desaturase (<i>GmFAD2-1</i>) subfamily, the <i>GmFAD2-2</i> subfamily is composed of five members, including <i>GmFAD2-2A</i>, <i>GmFAD2-2B</i>, <i>GmFAD2-2C</i>, <i>GmFAD2-2D</i>, and <i>GmFAD2-2E</i>. Segmental duplication of <i>GmFAD2-1A</i>/<i>GmFAD2-1B</i>, <i>GmFAD2-2A/GmFAD2-2C</i>, <i>GmFAD2-2A/GmFAD2-2D</i>, and <i>GmFAD2-2D/GmFAD2-2C</i> have occurred about 10.65, 27.04, 100.81, and 106.55 Mya, respectively. Using TILLING-by-Sequencing+ technology, we successfully identified 12, 8, 10, 9, and 19 EMS mutants at the <i>GmFAD2-2A</i>, <i>GmFAD2-2B</i>, <i>GmFAD2-2C</i>, <i>GmFAD2-2D</i>, and <i>GmFAD2-2E</i> genes, respectively. Functional analyses of newly identified mutants revealed unprecedented role of the five <i>GmFAD2-2A</i>, <i>GmFAD2-2B</i>, <i>GmFAD2-2C</i>, <i>GmFAD2-2D</i>, and <i>GmFAD2-2E</i> members in controlling the seed oleic acid content. Most importantly, unlike <i>GmFAD2-1</i> members, subcellular localization revealed that members of the <i>GmFAD2-2</i> subfamily showed a cytoplasmic localization, which may suggest the presence of an alternative fatty acid desaturase pathway in soybean for converting oleic acid content without substantially altering the traditional plastidial/ER fatty acid production.