Cloning and transcriptional analysis of Crepis alpina fatty acid desaturases affecting the biosynthesis of crepenynic acid.

Crepis alpina acetylenase is a variant FAD2 desaturase that catalyses the insertion of a triple bond at the Delta12 position of linoleic acid, forming crepenynic acid in developing seeds. Seeds contain a high level of crepenynic acid but other tissues contain none. Using reverse transcriptase-coupled PCR (RT-PCR), acetylenase transcripts were identified in non-seed C. alpina tissues, which were highest in flower heads. To understand why functional expression of the acetylenase is limited to seeds, genes that affect acetylenase activity by providing substrate (FAD2) or electrons (cytochrome b5), or that compete for substrate (FAD3), were cloned. RT-PCR analysis indicated that the availability of a preferred cytochrome b5 isoform is not a limiting factor. Developing seeds co-express acetylenase and FAD2 isoform 2 (FAD2-2) at high levels. Flower heads co-express FAD2-3 and FAD3 at high levels, and FAD2-2 and acetylenase at moderate levels. FAD2-3 was not expressed in developing seed. Real-time RT-PCR absolute transcript quantitation showed 10(4)-fold higher acetylenase expression in developing seeds than in flower heads. Collectively, the results show that both the acetylenase expression level and the co-expression of other desaturases may contribute to the tissue specificity of crepenynate production. Helianthus annuus contains a Delta12 acetylenase in a polyacetylene biosynthetic pathway, so does not accumulate crepenynate. Real-time RT-PCR analysis showed relatively strong acetylenase expression in young sunflowers. Acetylenase transcription is observed in both species without accumulation of the enzymatic product, crepenynate. Functional expression of acetylenase appears to be affected by competition and collaboration with other enzymes.