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

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% of seed oil which has industrial value. The remaining seed oil comprises mainly common fatty acids including α-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 DESATURASE3. 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 to 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 to 3.6%, showing that PfFAD3-2 failed to recover the loss of C18:3 in fad3-2. Sequence comparison among known FAD3s revealed putative variation in PfFAD3-2 which might cause the absence of PfFAD3-2. In addition, lesquerella accumulates a minor hydroxy fatty acid, densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid, C18:2OH) at about 1%. C18:2OH 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 either 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, C18:2-OH level varied from 0.2 to 7.2%, and four of these lines exceeded to the background level of 3.4% in CL37. Whereas among 23 transgenic CL37 lines expressing PfFAD3-2, C18:2-OH level ranged from 0.4 to 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.