Introduction
============

Gamma-linolenic acid (γ-linolenic acid, GLA; C18:3, Δ^6,9,12^) is an essential fatty acid (EFA) of the omega-6 family and exists primarily in a few plant-based oils, such as evening primrose oil, blackcurrant oil, and borage oil ([@bib26]; [@bib2]). GLA was also found in considerable quantities in *Spirulina*, a cyanobacterium ([@bib34]), some species of *Primula* ([@bib23]*a*), and fungi ([@bib30]; [@bib12]). However, there is no GLA in the major oil crops ([@bib16]; [@bib23]), such as oilseed rape, soybean, peanut, and palm. GLA may be helpful in the prevention and/or treatment of diabetes, eye disease, osteoporosis, premenstrual syndrome (PMS), eczema, allergies, rheumatoid arthritis, high blood pressure, and heart disease ([@bib4]).

Δ^6^-desaturase is classified as an acyl-lipid desaturase that introduces a double bond between the pre-existing double bond and the carboxyl (front) end of the fatty acid ([@bib14]). It is critical for the metabolic conversion of linoleic acid (LA; C18:2 Δ^9,12^) and α-linolenic acids (ALA; C18:3 Δ^9,12,15^) into GLA and octadecatetraenoic acids (OTA; C18:4 Δ^6,9,12,15^), respectively ([@bib4]; [@bib16]). The cytochrome *b5* (Cyt *b5*) domain is essential for the borage Δ^6^-desaturase ([@bib23]*b*), and three conserved His boxes of the enzyme are also proved to be essential for its function ([@bib20]).

In the last 20 years, a number of Δ^6^-desaturase genes, from cyanobacterium ([@bib15]), fungi ([@bib19]), plants ([@bib26], 1999*a*; [@bib30]; [@bib41]), and animals ([@bib10]; [@bib27]), were isolated and characterized. Accumulation, function, and expression of Δ^6^-desaturase genes had been investigated in only a few higher plants synthesizing GLA. In *Borago officinalis*, GLA is accumulated up to 23.1% of the total fatty acids in mature seeds, and more than 8.0% in other tissues. The Δ^6^-desaturase gene is expressed but at different levels in all tissues: higher in seeds, young leaves, and roots, and lower in mature leaves and flowers ([@bib23]*c*). In *Anemone leveillei*, GLA and OTA are absent in seeds, but are readily detectable in leaves. The Δ^6^-desaturase gene is expressed at the highest level in leaves, a moderate level in seeds, and is completely absent in roots ([@bib41]). In *Primula,* different species accumulated varying levels of GLA and OTA. In the species with a high level of Δ^6^-fatty acids, GLA and OTA accumulated mainly in the leaves and seeds, and relatively little in roots ([@bib23]*a*).

It was recently reported that there were two nearly identical copies of the Δ^6^-desaturase gene, which differ by nine silent mutations, in the marine microalga *Glossomastix chrysoplasta* ([@bib5]). While, in the higher plants that had been investigated, there were no instances of several different active Δ^6^-desaturase genes being found in the same organism.

Δ^6^-desaturases from some organisms exhibited substrate selectivity. The Δ^6^-desaturase of moss (*Ceratodon purpureus*) preferred C16:1 Δ^9^ over C18:1 Δ^9^ ([@bib30]), whereas the Δ^6^-desaturase from *Mucor rouxii* had a strong preference for the C18:1 Δ^9^ ([@bib12]). In Boraginaceae, the Δ^6^-desaturase from Macaronesian *Echium gentianoides* preferred the LA substrate over the ALA substrate ([@bib3]). Δ^6^-desaturases from different *Primula* species had different substrate preferences; for example, the Δ^6^-desaturase from *Primula vialii* had a very strong preference for ALA compared with LA ([@bib21]), whereas that from *Primula cortusoides* mainly desaturated LA ([@bib22]).

The fatty acid-synthesizing and desaturation system had been widely known to exist in chloroplasts and the endoplasmic reticulum (ER) in higher plants. It had been confirmed by using the \[^14^C\]linoleoyl phosphatidylcholine that the microsomal membrane, prepared from the developing cotyledons, contained an active Δ^6^-desaturase, which catalysed the conversion of linoleate into γ-linolenate ([@bib33]). Since then, front-end desaturases were generally thought to locate in the ER ([@bib11]). To date, however, there has been no direct evidence for subcellular localization to locate plant Δ^6^-desaturases.

Blackcurrant seed oil, typically containing 15--20% GLA, is known to be one of the available natural sources of GLA ([@bib36]). However, until now, the Δ^6^-desaturase blackcurrant gene has not been reported.

In this research, GLA was detected mainly in seeds and rarely in other organs of blackcurrant. Three genes were cloned (*RnD6C*, *RnD6D*, and *RnD6E*) encoding Δ^6^-desaturases and two genes (*RnD8A*, *RnD8B*) encoding Δ^8^-sphingolipid desaturases in blackcurrant. RnD6C, RnD6D, and RnD6E could use both LA and ALA as substrate. RnD6C/D/E was located in the mitochondrion (MIT) in yeast and the ER in tobacco. *RnD6C* and *RnD6E* were expressed in all the tissues at a low level, whereas *RnD6D* was expressed at a high level but only in seeds. This research should provide a useful addition to the previously identified orthologues from other plants and may well prove to be useful for biotechnological applications. Furthermore, this research may also provide not only new insights into the evolutionary relationship of this class of desaturases but also some new clues as to the amino acid determinants which define the precise enzyme activity (i.e. either Δ^6^-desaturase or Δ^8^-sphingolipid desaturase).

Materials and methods
=====================

Plant materials
---------------

Blackcurrant (*Ribes nigrum* L. var. BRÖDTORP), a Finnish variety ([@bib40]) was used in this research.

Blackcurrant genomic DNA extraction and full-length Δ^6^-desaturase gene cloning
--------------------------------------------------------------------------------

Genomic DNA was extracted by a DNA extraction kit (Biomed-tech, Beijing, China) from young leaves of blackcurrant. The DNA fragments of putative cytochrome *b*5 fusion desaturase genes were amplified from the genomic DNA by using degenerated primers designed according to the sequence of conserved histidine boxes of known Cyt *b*5 fusion desaturases ([@bib16]; [@bib41]; [@bib21]). The primer sequences were: PL, 5′-TTGGGTGAAAGACCATCCA(T)GGT(A)GG-3′ PR, 5′-GGGAAACAAA(G)TGA(G)TGCTCAACCTG-3′. Five amplified products (about 1032 bp) were cloned into the pEASY-Blunt vector (TransGen Biotech, Beijing, China) and sequenced. The 5′ and 3′ extension of these fragments were obtained by the genomic walking method using the BD GenomeWalker™ Universal Kit (BD Biosciences, Palo Alto, CA, USA). Conserved genomic walking primers were: 5GSP1 (480), GACATTGTCTG(A/G)TAATGCCCAGAATC; 5GSP2 (270), CCTATAATCTTTGGATACATCTG(A/g)GA(C/t); 3GSP1(702), AGGAA(A/G)TTGGAGTTTGATGCTTTTGCTAGGTTC; 3GSP2(1078), CG(T/C)CTTGGATGGATTGGTTTCATGGTGG. The PCR products were cloned into the pEASY-Blunt vector (TransGen Biotech) and sequenced. Five sequences similar to 'front-end' Δ^6^-desaturase or Δ^8^-sphingolipid desaturase genes were obtained and named *RnD8A*, *RnD8B*, *RnD6C*, *RnD6D*, and *RnD6E*.

Yeast expression plasmid construction and transformation
--------------------------------------------------------

The five sequences similar to 'front-end' Δ^6^-desaturase or Δ^8^-sphingolipid desaturase genes were purified from 1% agarose gel, digested with the restriction enzymes *Hin*dIII and *Sac*I (the restriction sites of the enzymes had been designed in the primers), cloned into the corresponding sites of the yeast expression vector pYES2 (Invitrogen, UK), and sequenced. The five construct plasmids (pRnD8A, pRnD8B, pRnD6C, pRnD6D, and pRnD6E) were transformed into *S. cerevisiae* strain INV Sc 1 (Invitrogen) by using the lithium acetate method.

Characterization of genes with a Δ^8^-sphingolipid desaturase function in *S. cerevisiae*
-----------------------------------------------------------------------------------------

The RnD8A/B-transformed yeasts were grown in SC-U, containing 1% raffinose and 1% (w/v) tergitol NP-40 (Sigma, Germany), and induced with 2% (w/v) galactose. The yeast cells were cultivated for an additional 72 h at 20 °C, and then harvested and dried at 50 °C. 100 mg dried cells were used to prepare the long chain bases (LCBs) from glycerolipids for subsequent analysis by HPLC as previously described ([@bib8]) and phytosphinganine (t18:0) (Sigma) was used as the standard. Briefly, induced yeast cells (100 mg, dried weight) were grounded into a fine powder, and subjected to strong alkaline hydrolysis in 2 ml of 10% (w/v) aqueous Ba(OH)~2~ and 2 ml of dioxane for 16 h at 110 °C. After hydrolysis, 2 ml of 2% (w/v) ammonium sulphate was added, and the liberated sphingolipid long chain bases were extracted with 2 ml of diethylether. The upper phase was removed to a second tube, dried under nitrogen, and derivatized with *o*-phthaldialdehyde (OPA) (Invitrogen) as previously described ([@bib9]). Individual LCBs were separated by reversed phase HPLC (Waters 600E, USA) using Penomenex C18 column (250×4.6 mm, 5 μm). Elution was carried out at 1.5 ml min^−1^ with 20% solvent RA (5 mM potassium phosphate, pH 7), 80% solvent RB (100% methanol) for 7 min, increasing to 90% solvent RB by 15 min, returning to 80% solvent RB and re-equilibrating for 2 min ([@bib8]). Fluorescence was excited at 340 nm and detected at 455 nm.

Characterization of genes with Δ^6^-desaturase function in *S. cerevisiae*
--------------------------------------------------------------------------

Heterologous expression of *RnD6C/D/E* was induced under transcriptional control of the yeast *GAL1* promoter. Yeast cultures were grown to logarithmic phase at 30 °C in SC-U containing 1% (w/v) raffinose, 0.67% (w/v) yeast nitrogen and 0.1% (w/v) tergitol NP-40 (sigma), supplemented with 0.5 mM LA (Sigma). The cells were induced by the addition of 2% (w/v) galactose and cultivated for an additional 72 h at 20 °C. Subsequently, cells were harvested by centrifugation, and washed three times with sterile distilled water. The cells were dried and ground into a fine powder. Then the fatty acid analysis was performed using the following method.

Plant plasmid construction and transformation of *Arabidopsis thaliana*
-----------------------------------------------------------------------

*RnD6C/D/E* cDNA under the control of the *duplicated CaMV 35S* (*D35S*) promoter and *nos* terminator were constructed in the binary vector pGI0029 (John Innes Centre, UK). The inserts were sequenced to ensure that undesired mutations were not introduced during the PCR reactions. *Agrobacterium tumefaciens* strain LBA4404 containing the helper plasmid pSoup (John Innes Centre, UK) was used as the host of the pGI0029 vectors containing the Δ^6^-desaturase gene. *Arabidopsis thaliana* (ecotype Columbia) was used for plant transformation with the floral dip method ([@bib1]). T~1~ seeds were plated out on selective media containing kanamycin (50 mg l^−1^), and then the selected transformed plants were transferred to soil. The harvested seeds were selected for another two generations, and the fixed transformants were selected.

Fatty acid analysis
-------------------

Cellular fatty acid was extracted by incubating 50 mg yeast powder or 200 mg fresh leaves of transformed *A. thaliana* or different blackcurrant tissues (young root, young stem, leaves, flowers, ripening exocarp, and ripening seeds) in 3 ml of 7.5% (w/v) KOH in methanol for saponification at 70 °C for 3 h. After the pH was adjusted to 2.0 with HCl, the fatty acid was subjected to methyl-esterification with 2 ml 14% (w/v) boron trifluoride in methanol at 70 °C for 1.5 h. Then 1 ml of 0.9% (w/v) NaCl was added and mixed well. Subsequently, fatty acid methyl esters (FAME) were extracted with 2 ml hexane. The upper phase was removed to a second tube, dried under nitrogen and dissolved in acetic ether. Qualitative analysis of FAME was performed by GC-MS (gas chromatography-mass spectrometry, TurboMass, PerkinElmer, USA) with the capillary column (BPX-70, 30 m×0.25 mm×0.25 μm). FAME were analysed and identified by the comparison of their peaks with those of standards: LA, ALA, and GLA (Sigma).

Subcellular localization of Δ^6^-desaturase in yeast
----------------------------------------------------

To determine its subcelluar location, *RnD6C/D/E* had been cloned in-frame at the 5′ end and 3′ end with the *GFP* gene in the pYES2 vector and expressed in the yeast strain INV Sc 1. Live yeast cells were incubated with MitoTracker^Red^ and/or ER-tracker^Blue^ as described in experimental protocol of 'MitoTracker^®^ and MitoFluor™ Mitochondrion-Selective Probes' and 'ER-Tracker™ Dyes for Live-Cell Endoplasmic Reticulum Labeling' (Invitrogen). The localization of RnD6C/D/E-GFP was determined by using a fluorescence microscope (Olympus, Tokyo, Japan). The desaturation function of the fused proteins was identified by GC-MS.

Subcellular localization of Δ^6^-desaturase in tobacco
------------------------------------------------------

Wild-type *Nicotiana benthamiana* was grown as previously described ([@bib17]; [@bib39]). *A. tumefaciens* strain LBA4404 with plastids containing *RnD6C/D/E-GFP* or *GFP-RnD6C/D/E* and the strain carrying the p19 construct ([@bib38]) were grown at 28 °C in YEB supplemented with 50 mg l^−1^ kanamycin and 5 mg l^−1^ tetracycline to the stationary phase. Bacterial cells were precipitated by centrifugation at 5000 *g* for 15 min at room temperature and resuspended in 10 mM MgCl~2~ and 150 mg l^−1^ acetosyringone. Cells were left in this medium for 3 h and then infiltrated into the abaxial air spaces of 2--4-week-old *N. benthamiana* plants. The culture of *Agrobacterium* was brought to an optical density of 1.0 (OD~600~) to avoid toxicity ([@bib37]). Transient co-expression of the *RnD6C/D/E-GFP* or *GFP-RnD6C/D/E* and the mCherry-tagged ER marker ([@bib13]) and p19 constructs was at OD600 1.0 ([@bib38]). The localization of RnD6C/D/E tagged with GFP was determined under a confocal microscope (Leica TCS SP5, Leica Microsystems, Bensheim, Germany).

Gene expression analysis
------------------------

Total RNA from different tissues was extracted using a RNA extraction kit (Biomed-tech, Beijing, China). cDNA was synthesized from total RNA using a reverse transcriptional kit (Toyobo, Osaka, Japan). The RT-PCR primers for detecting our cloned genes from blackcurrant were listed in [Table 1](#tbl1){ref-type="table"}. The housekeeping actin gene was used as the control in the RT-PCR reactions. The reaction was denatured at 95 °C for 3 min, followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C, and then 7 min at 72 °C.

###### 

Primers (from 5′ to 3′) used in RT-PCR

  Primer name   Sequences
  ------------- ------------------------------
  RTDL94        GGTAAGGTTTACAATGTCACCC
  RTDR830       CGCTTTGAGAGCAACATG
  RTA&BL420     ATG(C/T)(T/G)TTGTGGAGGCTTA
  RTA&BR1077    AGCATGAAATATCTATAGTCCCTT
  RTC&EL322     GAAAGGGCATGGTGTTTTC
  RTC&ER864     CAA(A/G)AGTCTGTTGGGTACTTTTTT
  Actin L       CATCAGGAAGGACTTGTACGG
  Actin R       GATGGACCTGACTCGTCATAC

Cladistic analysis
------------------

Nucleotide sequences of the desaturases were aligned by using the program ClustalX 2.0.10 ([@bib35]) and manual adjustment. The phylogenetic tree was generated using the alignment output based on the Minimum evolution method ([@bib18]), as implemented in the Mega 4.1 ([@bib6]). The complete deletion option and the Jukes and Cantor metric were used, and confidence of the tree branches was checked by bootstrap generated from 1000 replicates.

Results
=======

Fatty acid composition in different blackcurrant tissues
--------------------------------------------------------

The fatty acid compositions from young roots and stems, flowers, mature leaves, ripening exocarp, and seeds of blackcurrant were determined by GC-MS. The results showed that all the detected tissues except ripening exocarp contained GLA and OTA. There was very little GLA (\<1.1% of total fatty acid) and OTA (\<0.4% of total fatty acid) in young roots and stems, flowers and mature leaves, but abundant GLA (\>12% of total fatty acid) and OTA (\>2.6% of total fatty acid) in ripening seeds ([Table 2](#tbl2){ref-type="table"}).

###### 

Fatty acid composition of different blackcurrant tissues

         Young root   Young stem   Leaf       Flower     Ripening exocarp   Ripening seeds
  ------ ------------ ------------ ---------- ---------- ------------------ ----------------
  16:0   32.3±0.7     30.4±0.7     17.2±0.6   34.7±1.5   35.0±0.2           10.1±0.0
  16:1   23.4±0.9     --           1.7±0.0    --         --                 --
  16:2   --           --           1.2±0.0    --         --                 --
  16:3   --           --           5.0±0.2    --         --                 --
  18:0   14.3±0.6     11.2±0.8     2.5±0.3    11.1±0.6   19.5±1.0           3.9±0.0
  18:1   6.5±0.2      2.9±0.3      1.7±0.1    9.9±0.5    4.6±0.3            12.6±0.2
  LA     16.7±0.3     30.8±1.2     10.7±0.4   24.6±1.3   22.2±0.8           42.7±0.1
  GLA    0.8±0.0      1.1±0.0      0.4±0.0    0.3±0.1    --                 12.7±0.2
  ALA    3.6±0.0      20.7±0.5     57.1±1.3   16.9±1.2   16.7±0.7           13.4±0.1
  OTA    0.4±0.0      0.9±0.0      0.4±0.0    0.3±0.0    --                 2.6±0.1

The levels of individual fatty acids were determined by GC-MS and expressed as mole% of the total. Values given represent means *±*SE obtained from three independent experiments.

Isolation of putative Δ^6^-desaturase genes
-------------------------------------------

Using the genomic DNA walking method, five DNA sequences (1347 bp in length) were cloned and sequenced. They were very similar in DNA sequences (\>85% identity) and encoded five distinct deduced polypeptides of 448 amino acids. All of them had an *N*-terminal Cyt *b5* domain as the heme-binding region and three conserved histidine boxes which exist in all membrane-bound desaturases ([Fig. 1](#fig1){ref-type="fig"}) ([@bib28]). These five DNA sequences could be grouped into two classes. One included two sequences that showed high identities (\>69%) to the DNA sequence of Δ^8^-sphingolipid desaturase from borage ([@bib29]). They were designated as *RnD8A* and *RnD8B* and registered in GenBank as GU198924 and GU198925, respectively. The other contained three sequences showing slightly lower identities (\>60%) to the DNA sequence of Δ^6^-desaturase from borage. They were designated as *RnD6C*, *RnD6D*, and *RnD6E,* which were registered in GenBank as GU198926, GU198927, and GU198928, respectively. The deduced amino acid sequences of the RnD6C, RnD6D, and RnD6E proteins shared a 93% identity to each other and a 70% identity to the sequence of Δ^6^-desaturase from borage. And the predicted amino acid sequences of RnD8A and RnD8B shared a 92% identity to each other and 61% to the sequence of Δ^8^-sphingolipid desaturase from borage ([@bib31]).

![Alignment of deduced amino acid sequences of blackcurrant desaturases (RnD6A, RnD6B, RnD6C, RnD6D, and RnD6E), borage Δ^6^-desaturase (acc. no. AAC49700), and Δ^8^-sphingolipid desaturase (AAG43277) by using DNAMAN 6.0. Numbers on the right denote the number of amino acid residues. Residues identical for all sequences in a given position are in white text on a black background, and those identical in two of the three or similar in the three sequences are on a grey background. Characteristic regions of the N-terminal cyt *b5* motif are underlined and the conserved His boxes are indicated by black dots.](jexboterq051f01_ht){#fig1}

Functional identification of RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E in *Saccharomyces cerevisiae*
------------------------------------------------------------------------------------------------

To validate the putative Δ^6^-desaturase activity of RnD6C, RnD6D, and RnD6E, they were expressed in *S. cerevisiae*. The fatty acid compositions of transformants and the control were analysed by GC-MS, respectively. In the control, no conversion into non-native products was found by feeding potential fatty acid precursors LA ([Fig. 2](#fig2){ref-type="fig"}) or ALA (data not shown) to the yeast cells transformed with empty vector pYES2. By contrast, yeast cells transformed with RnD6C, RnD6D or RnD6E could utilize two precursor forms LA ([Fig. 2](#fig2){ref-type="fig"}) and ALA (data not shown), to produce a new product GLA ([Fig. 2](#fig2){ref-type="fig"}) or OTA (data not shown), respectively. There were no significant difference in Δ^6^-desaturase activity among RnD6C, RnD6D, and RnD6E.

![GC analysis of FAME of total lipids of *Saccharomyces cerevisiae.* (A) The total fatty acid in yeast INV Sc 1; (B) the total fatty acid in yeast INV Sc 1 with LA and GLA standard (Sigma); (C) the total fatty acid in induced yeast transformed with pYES2; (D, E, F) the total fatty acid in induced yeast transformed with RnD6C/D/E (arrows indicated the novel peak of GLA). All the GC-MS an alyses were repeated three times and representative results are shown.](jexboterq051f02_ht){#fig2}

To identify the functions of RnD8A and RnD8B, they were expressed in *S. cerevisiae* under the control of the *GAL1* promoter. Besides the phytosphinganine, *cis*- and *trans*-desaturated sphingoid bases were found in the transformed yeast cells with RnD8A or RnD8B by detecting their LCBs with HPLC ([Fig. 3](#fig3){ref-type="fig"}) using the method described in the Materials and methods ([@bib8]). The results indicated that RnD8A and RnD8B were Δ^8^-sphingolipid desaturases.

![Formation of phytosphingenines in yeast cells by heterologous expression of a Δ^8^-sphingolipid desaturase from blackcurrant. (A) The phytosphinganine (t18:0) (Sigma) as the standard. (B) The predominating LCBs from *Saccharomyces cerevisiae* cells (INV Sc 1) as the control harbouring the empty vector pYES2 is phytosphinganine (t18:0). (C, D) Formation of *cis*-(*Z*) and *trans*-(*E*) of phytosphingenine (t18:1) in yeast cells expressing pRnD8A/B. LCBs from yeast cells were converted into their OPA derivatives and analysed by reversed-phase HPLC.](jexboterq051f03_lw){#fig3}

Functional identification of *RnD6C*, *RnD6D*, and *RnD6E* in *Arabidopsis thaliana*
------------------------------------------------------------------------------------

The cDNAs of *RnD6C*, *RnD6D*, and *RnD6E* were individually expressed in *Arabidopsis thaliana*. The transgenic plants were verified by PCR using two primers targeting the internal sequence of the genes. The fatty acid profile of lipids from leaves of transgenic plants was determined. [Figure 4](#fig4){ref-type="fig"} showed the fatty acid compositions in leaves of 3-week-old seedling of T~3~ RnD6C transgenic plants. Compared with the wild type, the leaves of the transgenic plants had an altered fatty acid profile containing two additional peaks corresponding to GLA and OTA. There were no significant differences in the production efficiency of GLA and OTA among transgenic RnD6C, RnD6D, and RnD6E lines (data not shown).

![GC analysis of FAME of total lipids of *Arabidopsis thaliana* leaves transformed with RnD6C. (A) The total fatty acids of wild-type *A. thaliana* leaves; (B) the total fatty acids of *A. thaliana* leaves transformed with RnD6C (arrows indicate the novel peak of GLA and OTA). All the GC-MS analyses were repeated three times and representative results are shown.](jexboterq051f04_ht){#fig4}

Subcellular localization of RnD6C/D/E in *S. cerevisiae* and tobacco
--------------------------------------------------------------------

The Δ^6^-desaturase RnD6C/D/E was tagged with GFP in both the N-terminal and C-terminal, and expressed in *S. cerevisiae* and *N. benthamiana*. RnD6C/D/E led to similar results and the representative results are shown. The results showed that RnD6C/D/E was localized in mitochondria when introduced into yeast cells ([Fig. 5](#fig5){ref-type="fig"}) and ER in tobacco epidermal cells ([Fig. 6](#fig6){ref-type="fig"}).

![The localization of RnD6C/D/E tagged with GFP at the C-terminal end (A--C) and N-terminal end (D--F) in yeast cells and yeast cells INV Sc 1 with pYES2 (G--H) as the negative control. (A, D, G) Fluorescent images of yeast cells that expressed GFP and the negative control under an exciter filter of 485 nm. (B, E, H) Fluorescent images of yeast cells that were stained with MItotracker^Red^ (Invitrogen) under an exciter filter of 510 nm. (C, F, I) The merged image. (This figure is available in colour at *JXB* online.)](jexboterq051f05_3c){#fig5}

![The localization of RnD6C/D/E tagged with GFP in tobacco epidermal cells. The results shown are from transient transformed *Nicotiana benthamiana* epidermal cells. (A--C) RnD6C/D/E fused with GFP at the C-terminal end; (D--F) RnD6C/D/E fused with GFP at N-terminal end. (A, D) Fluorescent images of epidermal cells that expressed GFP under an exciter filter of 485 nm. (B, E) Fluorescent images of epidermal cells that expressed the endoplasmic reticulum marker ER-mCherry under an exciter filter of 510 nm. (C, F) The merged image. (This figure is available in colour at *JXB* online.)](jexboterq051f06_3c){#fig6}

The fused protein also showed Δ^6^-desaturase activity in yeast. However, RnD6C/D/E-GFP had a relatively lower level Δ^6^-desaturase activity than RnD6C/D/E and GFP-RnD6C/D/E and the representative results are shown in [Fig. 7](#fig7){ref-type="fig"}.

![GC analysis of FAME of total lipids of RnD6C/D/E tagged with GFP in *Saccharomyces cerevisiae*. The total fatty acid in induced yeast transformed with (A) RnD6C/D/E, (B) GFP-RnD6C/D/E, and (C) RnD6C/D/E-GFP (arrows indicated the novel peak of GLA).](jexboterq051f07_ht){#fig7}

Expression profiles of *RnD8A*, *RnD8B*, *RnD6C*, *RnD6D*, and *RnD6E*
----------------------------------------------------------------------

RT-PCR was used to investigate the expression profiles of *RnD8A*, *RnD8B*, *RnD6C*, *RnD6D*, and *RnD6E*. The sequence identity between *RnD8A* and *RnD8B* is 91.6%, and that between *RnD6C* and *RnD6E* is 97.1%. Thus, the expression of *RnD8A* and *RnD8B*, and that of *RnD6C* and *RnD6E*, were investigated together. The results showed that *RnD8A&RnD8B* and *RnD6C*&*RnD6E* were expressed in all the tissues at a low level, while *RnD6D* was expressed only in seeds at a high level ([Fig. 8](#fig8){ref-type="fig"}). Using genomic walking, a seed specific expression promoter upstream of *RnD6D* has been cloned and sequenced (data not shown).

![Expression profiles of Δ^6^-desaturases and Δ^8^-sphingolipid desaturases by RT-PCR in different tissues. (A) Expression levels in root, stem, leaf, flower, exocarp, and seed and relative transcript levels (normalized to *actin* mRNA) were determined by reverse transcriptase (RT)-PCR. (B) Seed RNA from different ripening stages: green (G), green and black (G&B), black and green (B&G), black (B) and relative transcript levels (normalized to actin mRNA) determined by reverse transcriptase (RT)-PCR. All the RT-PCR experiments were repeated three times, and representative results are shown. The *actin* gene for actin RNA was amplified as a control. The PCR products were separated by 2% (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining.](jexboterq051f08_ht){#fig8}

Discussion
==========

In a few previously studied higher plants, the profiles of the GLA and OTA accumulation were different. GLA is accumulated in mature seeds and in other tissues in *Borago officinalis* at a high level ([@bib23]*c*), but was absent in seeds and was at a high level in leaves in *Anemone leveillei* ([@bib41]). In some primula species, GLA and OTA were accumulated mainly in leaves and seeds, and were relatively low in roots ([@bib23]*a*). In blackcurrant, mature seeds ([@bib36]) and ripening seeds accumulate GLA and OTA at a high level, while other tissues contained very little GLA and OTA.

In *Borago officinalis*, *Anemone leveillei*, and some species of primula with high GLA, a single gene encoding Δ^6^-desaturase had been identified and characterized. In this research on blackcurrant, three genes encoding Δ^6^-desaturase (namely *RnD6C, RnD6D*, and *RnD6E*) had been cloned. Their desaturase activities were similar in yeast and *Arabidopsis*. However, their expression profiles were different. *RnD6C*&*RnD6E* was expressed in all the tissues at a low level, while *RnD6D* expressed only in seeds. *RnD6D* may mainly contribute to the accumulation of GLA and OTA in the seeds of blackcurrant.

It is widely believed that the Δ^6^-desaturases, which were only found in some plants, have evolved from Δ^8^-sphingolipid desaturases that existed in nearly all plants ([@bib7]; [@bib32]). Cladistic analysis of Δ^6^-desaturases and Δ^8^-sphingolipid desaturases genes ([Fig. 9](#fig9){ref-type="fig"}) revealed that Δ^6^-desaturases and Δ^8^-sphingolipid desaturase genes from the same plants clustered together, rather than being separated into two groups, for example, *Ribes nigrum* and *Anemone lendsquerelli.* The Δ^6^-desaturases and Δ^8^-sphingolipid desaturases genes of *Primula vialli* (*Pv*), and *Primula farinose* (*Pf*) clustered in two groups as the two species belong to the same genus. The only exception is *Borago officinalis* in which the genes were not clustered together due to the greater difference between the two desaturases than that in other plant species. The sequence identity of Δ^6^-desaturase and Δ^8^-sphingolipid desaturase in *Borago officinalis* is 58%, suggesting that the Δ^6^-desaturase and Δ^8^-sphingolipid desaturase separated much earlier in *Borago officinalis* than those in other species. The identity of Δ^6^-desaturases and Δ^8^-sphingolipid desaturases is more than 80% in blackcurrant and the distance between them is much shorter than that in other species. Furthermore, among the plant species with reported Δ^6^-desaturase genes, blackcurrant is the only one in which several functional genes or copies of Δ^6^-desaturase have been found. These genes have identities higher than 92% based on the amino acid sequences. The existence of three such genes may have resulted from a recent gene duplication event, as the delta desaturases were thought to be conserved in evolution until now ([@bib7]). It is speculated that Δ^6^-desaturase genes in blackcurrant may have evolved later than those in other species.

![Phylogenetic analysis of Δ^6^-desaturases and Δ^8^-sphingolipid desaturase genes (ORF region) based on the multiple nucleotide sequence alignment program ClustalX and the Minimum evolution method. Organism abbreviations stand for: *Borago officinalis* (Bo), *Anemone lendsquerelli* (Al), *Camellia sinensis* (C[s),]{.smallcaps} *Nicotiana tabacum* (Nt), *Echium gentianoides* (Eg), *Arabidopsis thaliana* (At), *Brassica napus* (Bn), *Primula vialli* (Pv), *Primula farinosa* (Pf). GenBank accession numbers are: *BoD6/8* (U79010/AF133728), *AlD6/8* (AF536525/AF536526), *CsD6* (AY169402), *NtD8* (EF110692), *EgD6* (AY055117), *AtD8* (NM_116023), *BnD8* (AJ224160), *PvD6/8* (AY234127/ AY234126), *PfD6/8* (AAp23034/AY234124), *RnD8A/B* (GU198924/GU198925), and *RnD6C/D/E* (GU198926/GU198927/GU198928). Bootstrap values at nodes represent the percentages of times a clade appeared in 1000 replicates.](jexboterq051f09_lw){#fig9}

Δ^6^-desaturases from previously reported plants always exhibited different substrate selectivity. Some prefer LA ([@bib3]; [@bib22]) and some prefer ALA as the substrate ([@bib21]). In the present study, the three Δ^6^-desaturases, RnD6C, RnD6E, and RnD6D from blackcurrant can use both LA and ALA as the substrates, but their substrate selectivity needs to be confirmed by future studies.

Front-end desaturases are generally thought to locate in the ER, although the actual location is unknown ([@bib5]). Expression of GFP-tagged RnD6C/D/E in yeast and tobacco could facilitate the visualization of its exact location. The present study provides the unequivocal evidence that RnD6C/D/E is located in the ER in tobacco, but it is located in mitochondria when introduced into yeast.

The effect of the fusion of GFP with Δ^6^-desaturases on their enzyme activity has not been reported. Results from the present study indicate that the yeast cells containing RnD6C/D/E-GFP accumulated a lower level of GLA than those containing GFP-RnD6C/D/E or RnD6C/D/E and there were no significant differences between GFP-RnD6C/D/E and RnD6C/D/E ([Fig. 7](#fig7){ref-type="fig"}). It is shown that the GFP tagged with Δ^6^-desaturases in the N-terminal (GFP-RnD6C/D/E) had no effect on the activity of Δ^6^-desaturases, but tagged with C-terminal (RnD6C/D/E-GFP) could decrease the activity of Δ^6^-desaturases. This reduced activity may result from the alteration of the three-dimensional structure of the C-terminal when the protein is tagged with GFP at that end. The results suggested that the three-dimensional structure of the C-terminal of Δ^6^-desaturases should be important for their enzyme activity. However, the effect of the three-dimensional structure of the C-terminal of Δ^6^-desaturases on their enzyme activity is worthy of further investigation.

In conclusion, three genes encoding Δ^6^-desaturases (*RnD6C/D/E)* and two encoding Δ^8^-sphingolipid desaturases (*RnD8A/B)* from blackcurrant that were very similar in their sequences have been isolated and characterized. *RnD6C* and *RnD6E* was expressed in all the tissues at a low level, while *RnD6D* was expressed only in seeds. *RnD6D* could mainly contribute to the accumulation of GLA and OTA in blackcurrant seeds. To the authors' knowledge, this is the first report of the identification of several Δ^6^-desaturases in a single plant species and of finding a Δ^6^-desaturase gene expressed specifically in seeds. Blackcurrant Δ^6^-desaturases were confirmed to localize in the ER of plant cells and in the mitochondria of yeast cells. It is speculated that the evolution of Δ^6^-desaturase genes from Δ^8^-sphingolipid desaturase genes in blackcurrant occurred more recently than that in other species.

We thank Dr Chengcai Chu and Dr Yiqin Wang, Institute of Genetics and Developmental Biology, CAS, for their help with the HPLC analyses. This research was supported by project (No. 2008ZX08009-003) from the Ministry of Agriculture of China for transgenic research, project KSCX-YW-N-009 from the Chinese Academy of Sciences, and project 2006AA10A113 from the Ministry of Science and Technology of China.

GLA

:   γ-linolenic acid

EFA

:   essential fatty acid

ALA

:   α-linolenic acids

LA

:   linoleic acid

OTA

:   octadecatetraenoic acids

MIT

:   mitochondrion

ER

:   endoplasmic reticulum

FAME

:   fatty acid methyl esters

GC-MS

:   gas chromatography-mass spectrometry

Cyt *b*5

:   cytochrome b*5*

PMS

:   premenstrual syndrome

LCBs

:   long chair bases

OPA

:   o-phthaldialdehyde

GFP-

:   Green fluorescence protein tagged in C-terminal

-GFP

:   Green fluorescence protein tagged in N-terminal

[^1]: This work was carried out at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

[^2]: GenBank accession numbers: *RnD8A*, GU198924; *RnD8B*, GU198925; *RnD8C*, GU198926; *RnD8D*, GU198927; *RnD8E*, GU198928.
