Lesquerella FAD3-1 gene is responsible for the biosynthesis of trienoic acid and dienoic hydroxy fatty acids in seed oil

Authors: LEE, KYEONG-RYEOL - Rural Development Administration - Korea; KIM, EUN-HA - Rural Development Administration - Korea; JEON, INHWA - Rural Development Administration - Korea; LEE, YONGJI - Sejong University; Chen, Grace; KIM, HYUN UK - Sejong University

Submitted to: Industrial Crops and Products
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2019
Publication Date: 4/8/2019
Citation: Lee, K., Kim, E., Jeon, I., Lee, Y., Chen, G.Q., Kim, H. 2019. Lesquerella FAD3-1 gene is responsible for the biosynthesis of trienoic acid and dienoic hydroxy fatty acids in seed oil. Industrial Crops and Products. 134:257–264. https://doi.org/10.1016/j.indcrop.2019.04.008.
DOI: https://doi.org/10.1016/j.indcrop.2019.04.008

Interpretive Summary: Lesquerella (Physaria fendleri) contains unusual hydroxy fatty acid (HFA), lesquerolic acid (C20:1-OH), at 55–60% in seed oil which has industrial value. Lesquerella also contains about 10-15% a-linolenic acid (C18:3) and 1% desipolic acid (C18:2-OH). To investigate the biosynthetic enzyme which is responsible for C18:3 and C18:2-OH synthesis in lesquerella, two fatty acid desaturase 3 (FAD3) genes were cloned and functional characterized using Arabidopsis FAD3-deficient mutant and Arabidopsis CL37 line which contains 17% hydroxy fatty acid in seed oil. Our study revealed that lesquerella FAD3-1 (PfFAD3-1) is a functional enzyme which can synthesize C18:3 and C18:2-OH. However, PfFAD3-2 is a non-functional enzyme although its protein is almost intact. PfFAD3-2’s function may be disrupted by point mutation in the region where is crucial for the desaturase activity. Besides, PfFAD3-1 prefers using C18:2 rather than C18:1-OH as substrate. This study enhances our understanding of molecular mechanisms underlying FAD3 activity.

Technical Abstract: Lesquerella (Physaria fendleri) contains a major unusual hydroxy fatty acid, lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid, C20:1-OH), at 55–60% in seed oil which has industrial value. The remaining seed oil comprises mainly common fatty acids including a-linolenic acid (octadec-cis-9,12,15-enoic acid, C18:3) at 10.7–15.8%. C18:3 is produced from linoleic acid (octadec-cis-9,12-enoic acid, C18:2) by fatty acid desaturase 3. Previous seed transcriptome analysis uncovers two fatty acid desaturase 3 (FAD3) transcripts, PfFAD3-1 and PfFAD3-2. To determine the activity of PfFAD3-1 and PfFAD3-2, PfFAD3-1 and PfFAD3-2 were introduced into an Arabidopsis FAD3-deficient mutant (fad3-2) which has reduced C18:3 from 20.0% in wild-type to 1.6% in fad3-2. Among 20 T2 transgenic lines expression PfFAD3-1, C18:3 increased variably from 2.5–29.9% demonstrating that PfFAD3-1 acted as a functional FAD3. Among 32 T2 transgenic lines expressing PfFAD3-2, C18:3 content ranged from 1.0–3.6%, showing that PfFAD3-2 failed to recover the loss of C18:3 in fad3-2. Sequence comparison among known FAD3s revealed putative mutations in PfFAD3-2 which might cause the malfunction of PfFAD3-2. Besides, lesquerella accumulates a minor hydroxy fatty acid, densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid, C18:2-OH) at about 1%. C18:2-OH has been shown to be produced by a FAD3 in Arabidopsis (AtFAD3) using ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid, 18:1-OH) as substrate. To test if any of PfFAD3s is able to convert C18:1-OH to C18:2-OH, PfFAD3-1 or PfFAD3-2 was transferred into a CL37 Arabidopsis which already expresses a castor (Ricinus communis) fatty acid hrdroxylase FAH12 gene (RcFAH12) and consequently accumulates C18:1-OH and C18:2-OH at 13.7% and 3.4%, respectively. Among 43 transgenic CL37 lines expressing PfFAD3-1, C18:2OH level varied from 0.2–7.2%, and four of these lines exceeded to the background level of 3.4%. Whereas among 23 transgenic CL37 lines expressing PfFAD3-2, C18:2OH level ranged from 0.4–3.4%, none exceeding 3.4%. The results consist with our notion that PfFAD3-1, not PfFAD3-2, exerts FAD3 function which includes converting C18:1-OH to C18:2-OH. Factors limiting PfFAD3s function in CL37 are discussed.