Functional characterisation of two cytochrome�b5-fusion desaturases from Anemone leveillei: the unexpected identification of a fatty acid ?6-desaturase

Abstract

The Ranunculaceae are known to accumulate a wide range of unusual fatty acids in their seed lipids, and this variability has been advocated as a taxonomic marker. The Anemone species, Anemone leveillei L. and Anemone rivularis Buch.-Ham., have previously been reported to accumulate Delta5-desaturated fatty acids in their seed tissue [K. Aitzetmüller (1995) Plant Syst Evol 9:229-240]. Two cDNAs, AL1 and AL2, with similarity to plant cytochrome b5-fusion "front-end" desaturases were isolated from developing seeds of A. leveillei and their function identified by expression in Saccharomyces cerevisiae. AL2 was characterised as a sphingolipid long-chain-base Delta8-desaturase, while AL1 acted as a fatty acid desaturase. However, AL1 did not produce Delta5-desaturated fatty acids as expected; instead, when expressed in transgenic S. cerevisiae or Arabidopsis thaliana this enzyme was functionally characterised as a Delta6-desaturase. Northern analysis confirmed the expression of this gene in seed tissue and leaf tissue of A. leveillei, though Delta6-desaturated fatty acids were found to accumulate only in the leaf tissue. The unexpected characterisation of a Delta6-desaturase in A. leveillei has implications for the use of fatty acids in chemotaxonomic studies. This is also the first report of a higher-plant Delta6-desaturase from a family other than the Boraginaceae.

Identification and Functional Analysis of a Δ6-Desaturase Gene and the Effects of Temperature and Wounding Stresses on Its Expression inMicroula sikkimensisLeaves

“…32) Similarly, the gene was expressed at the highest levels in the leaf tissue of Anemone leveillei and at moderate levels in the seeds, but was not detectable in the roots. 14) In blackcurrant, three genes encoding Á6-desaturase showed different expression profiles. Two of them were expressed in all tissues at low levels, while the other was expressed only in the seeds.…”

“…8) Á6-Desaturase is the key enzyme catalyzing the conversion of linoleic acid (LA, 18:2 Á9;12 ) and -linolenic acid (ALA, 18:3 Á9;12;15 ) to GLA and OTA respectively. 9) Over the past few decades, Á6-desaturase genes have been isolated from various organisms and characterized, including genes from cyanobacterium, 10) fungi, 11,12) plants, 13,14) and animals. 15,16) Furthermore, gene expression analysis has indicated that the transcript levels of Á6-desaturase genes in fungi increase under low temperature treatment, suggesting that these genes are involved in cold acclimation, 17,18) but the effect of environmental stresses on Á6-desaturase gene expression and their roles in stress responses have not been determined for higher plants.…”

Cloning and Characterization of Unusual Fatty Acid Desaturases from Anemone leveillei: Identification of an Acyl-Coenzyme A C20 Δ5-Desaturase Responsible for the Synthesis of Sciadonic Acid

“…These primers were used for PCR amplification with cDNAs transcribed from total RNA isolated from developing seeds of A. leveillei using previously described protocols (Whitney et al, 2003). The PCR fragments of the expected length (420 bp) were cloned into TOPO TA vector (Invitrogen) and sequenced.…”

“…Fatty acids were extracted and methylated as described previously (Whitney et al, 2003). Methyl ester derivatives of total fatty acids extracted from leaves were analyzed by GC and GC-MS. For the determination of the double bond positions, FAMEs were converted to the DMOX derivatives (Fay and Richli, 1991).…”

“…We have previously attempted to identify the D 5 -desaturase responsible for the synthesis of SA from Anemone leveillei, and initially considered it likely that this enzyme was a cytochrome b 5 fusion front-end desaturase. Although A. leveillei contains several such desaturases, none of them was shown to be involved in the synthesis of SA (Whitney et al, 2003). Thus, we further investigated the biosynthetic pathway of this unusual fatty acid, hypothesizing that the D 5 -desaturases were members of the relatively uncharacterized plant ADS-like class.…”