Identification and functional analysis of the genes encoding  6-desaturase from Ribes nigrum 

Abstract

Gamma-linolenic acid (γ-linolenic acid, GLA; C18:3 Δ6, 9, 12) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Δ6-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Δ6-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Δ6-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Δ6-desaturases that could use both α-linolenic acids (ALA; C18:3 Δ9,12,15) and linoleic acid (LA; C18:2 Δ9,12) precursors in vivo, whereas RnD8A/B were Δ8-sphlingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Δ6-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Δ6,9,12,15) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.

Isolation and Functional Characterisation of the Genes Encoding Δ8-Sphingolipid Desaturase from Brassica rapa

“…Genes encoding plant D 8 -sphingolipid desaturases have been identified from Arabidopsis thaliana, Brassica napus, Helianthus annuus, Borago officinalis, Aquilegia vulgaris, Nicotiana tabacum, Stylosanthes hamata and Ribes nigrum (Sperling et al, 1998(Sperling et al, , 2000(Sperling et al, , 2001Michaelson et al, 2002;García-Maroto et al, 2007;Ryan et al, 2007;Song et al, 2010;Moreno-Pérez et al, 2011). Expression of these cDNA sequences in vivo using S. cerevisiae as a heterologous host resulted in the biosynthesis of both cis-and trans-isomers of plant characteristic 4-hydroxysphing-8-enines, which do not present in wild-type yeast cells (Sperling and Heinz, 2003).…”

Cytochrome b 5 Coexpression Increases Tetrahymena thermophila Δ6 Fatty Acid Desaturase Activity in Saccharomyces cerevisiae

“…Although routine analysis of fatty acid methyl esters by GC is reliable, there are variants of ⌬6-like proteins with alternate activities (26). In addition, when a Tetrahymena cytochrome b 5 was coexpressed with Des6, high desaturase activity produced two uncharacterized minor peaks (Fig.…”

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

“…1), as reported for other front-end desaturases. 6,13) The glutamine residue of the third histidine box, QXXHH, might be very important to maintenance of the proper structure and functioning of desaturases, because the conversion of this residue to histidine and to isoleucine resulted in a loss of enzyme activity. 26) Random mutagenesis of the Á6-desaturase gene from Echium plantagineum indicated that the L14P mutation in cytochrome b 5 and the S301P mutation between histidine box II and histidine box III significantly reduced enzyme activity.…”

“…Two of them were expressed in all tissues at low levels, while the other was expressed only in the seeds. 13) Plant fatty acid desaturases are regulated by various environmental factors. 1) Much research suggests that temperature can regulate fatty acid desaturases at the transcriptional and post-transcriptional levels, as described for FAD2 in several plants, 3,5,33) for FAD6 in olive fruit, 34) for FAD3 in wheat roots, 2) and for FAD8 in Arabidopsis leaves, 35) but no effect of temperature on the expression of the Á6-desaturase gene has been demonstrated in higher plants.…”

“…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.…”