Concerted increases of FAE1 expression level and substrate availability improve and singularize the production of very-long-chain fatty acids in Arabidopsis seeds.

ABSTRACT

Our initial goal was to evaluate the contributions of high 181 phosphatidylcholine and the expression level of FAE1 to the accumulation of very-long-chain fatty acids (VLCFAs), which have wide applications as industrial feedstocks. Unexpectedly, VLCFAs were not improved by increasing the proportions of 181 in fad2-1 mutant, FAD2 artificial miRNA, and FAD2 co-suppression lines. Expressing Arabidopsis FAE1 resulted in co-suppression in 90% of transgenic lines, which was effectively released when it was expressed in the rdr6-11 mutant host. When FAE1 could be highly expressed, apart from its naturally preferred product, 201, other saturated and polyunsaturated VLCFAs also accumulated in seeds. We postulated that overabundant FAE1 might cause the diversified VLCFA profile. When FAE1 was highly expressed, knocking down FAD2 increased the content of 201, suggesting that the 181 availability in the acyl-CoA pool increased from the high 181-PC via acyl editing. Concurrent decreases of side products like 221 and 200 in these lines suggest that increasing availability of the preferred substrate could suppress the side elongation reactions and reverse the effect of VLCFA product diversification due to overabundant FAE1. Re-analysis of FAD2 knockdown lines indicated that increasing 181 led to a decrease of 221, which also supports the above hypothesis. These results demonstrate that 181 substrate could be increased by a downregulation of FAD2 and that a balance between the levels of enzyme and substrate may be crucial for engineering-specific VLCFA products.