JXB Advance Access originally published online on November 22, 2006
Journal of Experimental Botany 2007 58(3):473-481; doi:10.1093/jxb/erl218
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Genetic and biochemical studies in yeast reveal that the cotton fibre-specific GhCER6 gene functions in fatty acid elongation
1National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China
2Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing 100871, China
3Biocenter Oulu and Department of Biochemistry, University of Oulu, PO Box 3000, FIN-90014, Finland
* To whom correspondence should be addressed. E-mail: qinym{at}pku.edu.cn
Received 19 June 2006; Revised 19 September 2006 Accepted 28 September 2006
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Abstract |
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3-Ketoacyl-CoA synthase catalyses the initial condensation reaction during fatty acid elongation using malonyl-CoA and long-chain acyl-CoA as substrates. Previously, it was reported that several genes encoding putative cotton 3-ketoacyl-CoA synthases were significantly up-regulated during early cotton fibre development. In this study, GhCER6 cDNA that contains an open reading frame of 1479 bp, encoding a protein of 492 amino acid residues homologous to the Arabidopsis condensing enzyme CER6, was isolated and cloned. In situ hybridization results demonstrated that GhCER6 mRNA was detected only in the elongating wild-type cotton fibre cells. When GhCER6 was transformed to the Saccharomyces cerevisiae elo3 deletion mutation strain that was deficient in the production of 26-carbon fatty acids and displayed a very slow-growth phenotype, the mutant cells were found to divide similarly compared with those of the wild-type cells. Further, heterologous expression of GhCER6 restored the viability of the S. cerevisiae haploid elo2 and elo3 double-deletion strain. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed that GhCER6 was enzymatically active since the yeast elo2 and elo3 double-deletion mutant expressing the cotton gene produced very-long-chain fatty acids that are essential for cell growth. The results suggest that GhCER6 encodes a functional 3-ketoacyl-CoA synthase.
Key words: Fatty acid elongation, Gossypium hirsutum, 3-ketoacyl-CoA synthase, MALDI-TOF-MS
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Introduction |
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Seed trichomes of Gossypium hirsutum are economically important in the modern textile industry. Cotton fibres are unicellular, linear structures derived from the epidermis of the ovule. Understanding how the metabolic pathways work in cotton fibres is important for the manipulation of the quality of their chemical constituents. It has been reported that the expression levels of several putative condensing enzymes, 3-ketoacyl-CoA synthases (KCSs), involved in cotton fatty acid elongation highly increased in fibre cells at 10 d post-anthesis (dpa) in comparison with their activities in 0 dpa ovules (Ji et al., 2003; Shi et al., 2006). Very-long-chain fatty acids (VLCFAs; fatty acids >C20) are widely distributed in nature. They are found in cuticular waxes, seed storage lipids, and membrane lipids of most plant species (reviewed in Kunst and Samuels, 2003).
In plants, VLCFAs are synthesized by the microsomal fatty acid elongation system, which consists of four sequential reactions. The first reaction is the condensation of malonyl-CoA with a long-chain acyl-CoA catalysed by KCS to yield carbon dioxide and 3-ketoacyl-CoA in which the acyl-CoA moiety has been elongated by two carbons. Subsequent reactions are reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA, dehydration to trans-2 enoyl-CoA, and a second reduction to yield the elongated acyl-CoA (reviewed in Dunn et al., 2004). A number of plant KCSs has been investigated. Simmondsia chinensis KCS (Lassner et al., 1996), Arabidopsis thaliana FAE (James et al., 1995; Millar and Kunst, 1997), Brassica napus FAE (Han et al., 2001), moss Marchantia polymorpha FAE (Kajikawa et al., 2003), and Nasturtium FAE (Mietkiewska et al., 2004) are seed-specific condensing enzymes that produce VLCFA precursors for biosynthesis of storage lipids. Arabidopsis thaliana KCS (Todd et al., 1999) and CER6/CUT1 (Millar et al., 1999; Fiebig et al., 2000; Hooker et al., 2002) participated in synthesis of VLCFAs mainly for cuticular wax production in shoots. Root-specific condensing enzymes were also characterized and implicated in VLCFA synthesis in suberin formation (Schreiber et al., 2000; Moon et al., 2004). Interestingly, most genes encoding condensing enzymes in higher plants share very low sequence similarity with three ELO genes identified from Saccharomyces cerevisiae (Toke and Martin, 1996; Oh et al., 1997). Elo1p is known to be involved in the elongation of C14 to C16, Elo2p is responsible for the elongation of fatty acids up to C24, and Elo3p is essential for the conversion of C24 to C26 (Toke and Martin, 1996; Oh et al., 1997). Yeast cells with a single ELO gene deletion are viable, but cells with both ELO2 and ELO3 genes deleted are synthetically lethal (Oh et al., 1997). Arabidopsis FAE1-like genes rescue the lethality of the yeast elo2 and elo3 double-deletion (elo2
elo3) mutant (Paul et al., 2006) and provide a good system to identify functional plant KCS genes.
In the current study, a cDNA encoding a putative cotton KCS (GhCER6) was identified from developing cotton fibre cells that was able to genetically complement yeast elo2elo3 cells.
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Materials and methods |
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Plant material
Upland cotton (G. hirsutum L. cv. Xuzhou 142) and fuzzless-lintless mutant (Zhang and Pan, 1992) were grown in a greenhouse as previously reported by Ji et al. (2003). Cotton ovules were removed from the ovaries and fibre cells were carefully scraped from the epidermis of the ovules and immediately stored in liquid nitrogen.
Identification and cloning of GhCER6
Upland cotton (Gossypium hirsutum L. cv. Xuzhou 142) and its fuzzless-lintless (fl) mutant, originally discovered in the same cotton field in China (Zhang and Pan, 1992), were grown in a soil mixture in a fully automated greenhouse. Ovules at different growth stages were excised from bolls on the cotton plant. Total RNA samples were prepared from 1 g of cotton fibres harvested from 5–10 dpa ovules, using a modified hot borate method (Lu, 2002; Ji et al., 2003). The cDNA microarrays containing 11 692 uniESTs were fabricated at CapitalBio Corporation (Beijing, China) (Shi et al., 2006). The open reading frame of GhCER6 was amplified from cotton fibre cDNA using primers: 5'-CACCACAAAATGCCACATTTTTCTAACTCTGTC-3' (F1) and 5'-CAGCTTGACAACTTCAGGGATATG-3' (R1). The sequence ‘CACC’ at the beginning of each forward primer is required for cloning into pENTR TOPO® vector (Invitrogen) and the sequence ‘ACAAA’ is used to enhance gene expression in yeast. The constructed vector, pENTR TOPO-GhCER6, was verified by sequence analysis. The gene was transferred from pENTR TOPO to yeast expression vector pYTV (Gong et al., 2004) via the attLxattR reaction, using homologous sequences present on both vectors, resulting in pYTV-GhCER6. The GAL1 promoter was fused at the N-terminus and 3xFLAG, 6xHis, and 2xIgG were present to facilitate induction and detection of the fusion protein (Gong et al., 2004).
Semi-quantitative reverse transcription (RT)-PCR analysis
Total RNA was extracted from wild-type cotton roots, leaves, stems, and ovules, together with fibres at –3, 0, +3, +5, +10, +15, +20 dpa or from –3, 0, +10 dpa fuzzless-lintless (fl) mutant cotton ovules. Cotton cDNA was reverse-transcribed from 5 µg total RNA. Gene-specific primers were designed as 5'-GTCGTTCCGAGAGGTGGCGTG-3' (F2) and 5'-ACCCTGTCCCCTTTTTTCATCCTCC-3' (R2), to amplify the 493 bp fragment of GhCER6. The cotton ubiquitin gene UBQ7 (accession no. AY189972) was used as an internal control in each reaction. To analyse heterologous expression of GhCER6 in yeast mutant cells, RT-PCR was performed with the same primer pairs and the yeast actin gene, ACT1 (accession number NP_116614) was used as an internal control in parallel reactions.
Real time quantitative (QRT)-PCR analysis of GhCER6 expression in different cotton tissues
QRT-PCR was carried out using the SYBR green PCR kit (Applied Biosystems) in a DNA Engine Opticon–Continuous Fluorescence Detection System (MJ Research). GhCER6-specific primers F2 and R2 were used. A single 493 bp DNA fragment was produced using specimens from different cotton tissues and mutant cotton ovules. All QRT-PCRs were performed with the same parameters described previously (Qin et al., 2005). Samples were analysed in triplicate using independent RNA samples and were quantified by the comparative cycle threshold method (Wittwer et al., 1997).
RNA in situ hybridization
Cotton ovules were collected from 2 dpa cotton flowers and fixed in formalin–acetic acid fixing solution. The fixed ovules were processed according to the protocol described by Ruan and Chourey (1998). The samples were infiltrated with paraffin. Tissue sections, 10-µm-thick, were cut and placed on dampened slides precharged with polylysine. Digoxigenin–11 UTP was incorporated into antisense or sense RNA probes according to the manufacturer's instructions (Roche). Hybridization and detection were carried out using the method of Ji et al. (2002).
Preparation of S. cerevisiae haploid elo3 and elo2 and diploid W1536 ELO3/elo3 and ELO2/elo2 strains
In order to prepare S. cerevisiae haploid strain W1536-5B elo3 (MATa; ade2, ade3, his3-11, his-3-12, trp1-1, ura3-1, elo3::kanMX4), PCR amplification of elo3::kanMX4 cassette from genomic DNA isolated from BY4741 elo3 (MATa; his31; leu20; met150; ura30; elo3::kanMX4, EUROSCARF) was performed using the primers in the flanking region of ScELO3, KoF1: 5'-CTGTGAATAAACAAAAGGTTGGCTTAC-3' and KoR1: 5'-ACTTGGACACTTTACAACATGCAAG-3'. The amplified 2.1 kb PCR product corresponding to the size of the elo3::kanMX4 cassette was isolated and purified from agarose gel and subsequently transformed into wild-type yeast cells W1536-5B (Kastaniotis et al., 2004). The cells carrying the elo3 deletion were selected on a G418 plate [1% (wt/v) yeast extract, 2% (wt/v) peptone, and 2% (wt/v) D-glucose] supplemented with 300 µg of geneticine ml–1.
The S. cerevisiae haploid strain W1536-8B elo2 (MAT; ade2, ade3, his3-11, his-3-12, trp1-1, ura3-1, elo2::kanMX4) was prepared in a similar way to W1536-5B elo3. The template was genomic DNA isolated from BY4742 elo2 (MAT; his31; leu20; lys20; ura30; elo2::kanMX4, EUROSCARF) and the flanking primers are KoF2: 5'-CGTGTAAATATGTCACATTTTATTTTTGTACG -3' and KoR2: 5'-CGTATGAAATTATTT GCGACCAAACTAAG-3'. The S. cerevisiae diploid strain W1536 ELO3/elo3, ELO2/elo2 (MAT a/
; ade2/ade2; ade3/ade3; can1-100/can1-100; his3-11,15/his3-11,15; leu2-3, 112/leu2-3, 112; trp1-1/trp1-1; ura3-1/ura3-1; ELO3/elo3::kanMX4, ELO2/elo2::kanMX4) was made by the first mating of W1536-5B elo3 transformed with pYES2 carrying URA3 marker with W1536-8B elo2 transformed with pTSV30A (Kastaniotis et al., 2004) carrying the LEU2 marker as well as the ADE3 gene, leading to the development of a red background colour in this strain. Diploids were then selected on a plate containing synthetic complete medium lacking both uracil and leucine (Sc-Ura-Leu), and finally the empty vectors were removed from the cells by growth on YPD after 20 generations. White colonies that were able to grow on an FOA plate [synthetic complete medium containing 2% (wt/v) D-glucose and 0.05% (wt/v) 5'-fluoroorotic acid] were selected, indicating loss of both pTSV30A and pYES2 plasmids.
Functional complementation of the yeast elo3 deletion by cotton GhCER6
The S. cerevisiae haploid strain W1536-5B elo3 was grown on YPD [1% (wt/v) yeast extract, 2% (wt/v) peptone, and 2% (wt/v) D-glucose] supplemented with 300 µg of geneticine ml–1. pYTV-GhCER6 was transformed into elo3 cells. Wild-type W1536-5B and elo3 cells were transformed with empty pYTV and used as controls. The transformants were selected on synthetic complete medium lacking uracil (SC-Ura) plates. The growth phenotypes of wild-type W1536-5B and elo3 cells transformed with pYTV, as well as the mutant cells transformed with pYTV-GhCER6, were examined on YPG containing 1% (wt/v) yeast extract, 2% (wt/v) peptone, and 2% (wt/v) galactose.
Construction of the yeast elo3elo2 strain complemented by GhCER6
Saccharomyces cerevisiae diploid cells W1536 elo3elo2 was transformed with pYTV-GhCER6. The transformants were selected on an Sc-Ura plate, and sporulated on the plate containing 0.25% (wt/v) yeast extract, 1.5% (wt/v) potassium acetate, and 0.05% (wt/v) D-glucose as well as amino acids. After sporulation, asci were digested with zymolase (Seikagaku), and the tetrads were dissected using an MSM manual dissection microscope (Singer Instrument). Separated ascospores were grown on YPD plate for 5 d. The complemented double-deletion spore colonies were selected by replica plating on G418 and FOA plates. The positive candidate grew on a G418 plate but not on an FOA plate. The presence of the kanMX4 cassette in the locus of either ELO3 or ELO2 was further verified by PCR using specific flanking primers KoF3 (upstream of KoF1): 5'-GTCCACAAAGTGAAAAATTTTC-3' or KoF4 (upstream of KoF2): 5'-GGGAGGAGTT TTTAATTATAATTGTA-3' and reverse primer from kanMX4 cassette (kanR): 5'-CTGACCATCTCATCTGTAACA-3', which anneal inside the kanMX cassette. pYADE4-GhCER6 was constructed by PCR amplification of GhCER6 from cotton cDNA with primers: 5'-ATCGCGGATCCATGCCACATTTTTCTAACTCTGTC-3' (F3) and 5'-ACGCGTCGACCTACAGCTTGACAACTTCAGGG-3' (R3). The product was digested with BamHI and SalI, then ligated into pYADE4 carrying a TRP1 marker, resulting in a plasmid that allows restoration of growth of elo3elo2 cells transformed with pYTV-GhCER6 on an FOA plate.
MALDI-TOF-MS analyses of fatty acid composition
To determine the total fatty acid composition of yeast cells, fatty acid methyl esters from wild-type yeast cells and elo3 cells transformed with empty vector pYTV, as well as elo3 cells or elo2elo3 cells rescued by GhCER6 were prepared according to the method described by Trenkamp et al. (2004). Fatty acid methyl esters were subsequently hydrolysed with sodium hydroxide and sodium carboxylate [RCOONa, where R represents CH3(CH2)n] were produced and subjected to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) according to a protocol of Ayorinde et al. (2000). meso-Tetrakis (pentafluorophenyl) porohyrin (F20TPP) (Mid-Century Chemicals) was used as the matrix in all the reactions. A signal-to-noise ratio of 5:1 was used as the threshold noise level for assignment of sodicated molecules. For all experiments, the spectra consistently gave a peak resolution greater than 5000 counts over the whole mass range.
Other methods
Multiple amino acid sequence comparison of plant KCSs was performed using the CLUSTAL W method (Thompson et al., 1994) with default parameters.
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Results |
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Identification of a fibre-preferential and tissue-specific gene, GhCER6
GhCER6 was found specifically up-regulated during cotton fibre development after screening of the microarray (data not shown). The relative expression levels for the GhCER6 gene in various developmental stages were further analysed using data from six microarray hybridizations (Fig. 1A). GhCER6 mRNA increased to >7-fold in 10 dpa (at the point of fastest fibre-cell elongation) wild-type cotton ovules associated with fibres compared with that of –3 dpa ovules, and was found to remain at very low level in 10 dpa fl mutant ovules. RT-PCR analyses of GhCER6 expression patterns are given in Fig. 1B. These two datasets agreed with each other. QRT-PCR results using RNA samples prepared from different cotton tissues including root, leaves, stems, and ovules of both wild type and the fl mutant confirmed that GhCER6 transcripts accumulated only in wild-type fibre cells, but not in any other organs or tissue types (Fig. 1C).
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In situ hybridization of GhCER6 in a cross-section of upland cotton and fl mutant ovules
RNA in situ hybridization was performed using sections prepared from 2 dpa ovules of either wild-type cotton or the fl mutant. Again, GhCER6 mRNA was detected mainly in wild-type elongating fibre cells, whereas little hybridized signal was observed in non-differentiated epidermal outer and inner integument cells or when the sense-strand RNA was used as a probe (Fig. 2). GhCER6 mRNA was not detected in fl mutant ovules as well at the same time. When cotton ubiquitin gene 7 (UBQ7) was used to probe the cotton ovule sections, strong hybridizing signals were distributed evenly throughout the whole section, regardless of cell types (Fig. 2).
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The GhCER6 gene is the AtCER6 homologue in cotton
Nucleotide sequence analysis revealed an open reading frame of 1479 bp encoding a polypeptide of 492 amino acids with a predicted molecular mass of 55 kDa. The sequence has been submitted to GenBank with the accession number DQ122189. As shown in Fig. 3, GhCER6 is closely related to Arabidopsis CER6 (86%) and CER60 (85%) at the deduced amino acid sequence level. Identity of GhCER6 with AtKCS1, AtFDH1, and AtFAE was on the level of 58%, 54%, and 52%, respectively. A highly conserved Cys220 was predicted to be the catalytically critical residue in the active site of GhCER6 (Fig. 3) based on its identity to the catalytic residue Cys223 of AtFAE1 (Ghanevati and Jaworski, 2002). Two likely transmembrane domains were found in the amino acid sequence of GhCER6 (Fig. 3), indicating that GhCER6 may be an integral membrane protein.
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GhCER6 is able to complement the yeast elo3 deletion mutant and the elo2 and elo3 double-deletion mutant
As shown in Fig. 4A, S. cerevisiae haploid elo3
cells were deficient in ELO3p activity, so that they grew at a much slower rate than wild-type cells. Mutant cells expressing GhCER6 displayed a significantly improved growth rate (Fig. 4A), suggesting that heterologous expression of GhCER6 was able to complement the genetic deficiency. In agreement with the spotting assay, elo3 cells expressing GhCER6 grew faster than the mutant cells in the early log phase when they were cultured in liquid media (Fig. 4B). RT-PCR analysis of elo3 cells transformed with pYTV-GhCER6 showed that GhCER6 mRNA was actively transcribed after galactose induction, but was barely detectable before the induction (Fig. 4C).
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The lethal elo2
elo3 double-deletion mutant was also used to show the essential biological function of the cotton enzyme. This mutant was obtained by mating opposite mating-type strains carrying each respective single deletion allele. The spores containing either single mutation were viable on YPG, G418, and FOA plates, and the spores carrying the double-deletion alleles could not grow on any plates (Fig. 5A). However, when diploid cells were transformed with the plasmid expressing GhCER6, viable spores grew on a G418 plate but not on an FOA plate (Fig. 5B). The genotype of this strain carrying the double-deletion cassettes was confirmed by PCR analysis (data not shown). The survival of these spores was found to depend on the presence of GhCER6, as demonstrated by the inability of the cells to grow on media containing 5-fluoroorotic acid, a compound that is converted to a toxic product by cells carrying the functional URA3 gene used as the genetic marker of the pYTV-GhCER6 plasmid (Fig. 5C). To show that the GhCER6 gene was essential for the survival of the double-mutant, diploid cells were also transformed with the pYADE4-GhCER6 plasmid that used a different marker gene. Spores carrying the double-deletion mutation were able to form colonies on FOA-containing media, indicating that GhCER6 but not the plasmid backbone was required for survival of the yeast double-mutant cells (Fig. 5C). Taken together, the present data indicate that the GhCER6 gene is able to complement the synthetic lethal phenotype caused by a double deletion of both ELO2 and ELO3 genes.
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MALDI-TOF-MS analyses of fatty acid composition in the yeast cells
The KCS activity of GhCER6 was determined by analysing the total VLCFA composition of the yeast cells using the MALDI-TOF-MS method. MALDI-TOF-MS of saponified lipids from wild-type cells transformed with empty pYTV vector showed (RCOONa+Na)+ of fatty acids: n-eicosanoic acid (C20:0), n-docosanoic acid (C22:0), n-tetracosenoic acid (C24:1), n-tetracosanoic acid (C24:0), n-hexacosenoic acid (C26:1), and n-hexacosanoic acid (C26:0), a normal fatty acid composition of yeast cells (Fig. 6A). Whereas, (RCOONa+Na)+ of fatty acids longer than C24 were not observed in the elo3
cells (Fig. 6B). When pYTV-GhCER6 was transformed into the mutant cells before induction by galactose, the spectrum of (RCOONa+Na)+ was the same as the one produced from elo3 cells (data not shown). After galactose induction, (RCOONa+Na)+ of fatty acids, n-hexacosenoic (C26:1) and n-hexacosanoic (C26:0), were detected in the mutant cells expressing GhCER6 (Fig. 6C). The spectrum of (RCOONa+Na)+ prepared from the elo2elo3 mutant cells rescued by GhCER6 was similar to the one observed in the wild-type yeast cells (Fig. 6D), indicating that GhCER6 is able to elongate the C16 acyl-CoA up to C26 acyl-CoA. In all cases, the experimental molecular weight for different fatty acids conjugated with the sodicated sodium carboxylates (RCOONa+Na)+ matched well with the theoretical value (<0.1 mass unit). A matrix peak corresponding to an m/z value of 531.30 was detected in all spectra (data not shown). The data indicated that GhCER6 was functionally expressed in the yeast mutant cells and that it catalysed the synthesis of C26 acyl-CoA by co-operating with the other three enzymes, 3-ketoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase, and trans-2-enoyl-CoA reductase, of the yeast fatty acid elongation system.
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Discussion |
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The elongation of fatty acids determines the amount and the overall chain length of fatty acid products and is controlled by the activity of KCS (Millar and Kunst, 1997). In cucumber seedlings, saturated VLCFAs including C20 to C26 were reported to account for at least 20% of the plasma membrane (Mattes and Böger, 2002). Several lines of evidence suggest that GhCER6 cDNA encodes a functional microsomal KCS (Figs 4, 6). Although the plant condensing enzymes share no sequence similarity with yeast ELO-type elongases that synthesize polyunsaturated and saturated fatty acids of sphingolipid in yeast (Oh et al., 1997), the growth rate of S. cerevisiae elo3
cells is recovered when the cells expressed the GhCER6 gene (Fig. 4). Further, expression of GhCER6 restores the viability of yeast haploid elo2 elo3 double-mutant cells (Fig. 5B). Analysis of fatty acids synthesized in the transgenic yeast strain indicates that functional complementation of both the elo3 deletion and double deletions by GhCER6 is caused by restoration of production of either C26 or VLCFAs ranging from C20 to C26 that play essential roles in the normal growth of the yeast cells. The finding that GhCER6 uses very-long-chain fatty acyl-CoA as the substrate is consistent with a previous study which found that AtCER6 is capable of elongating C22 and longer fatty acyl-CoAs in the Arabidopsis epidermal cells (Millar et al., 1999). GhCER6 was found to be highly transcribed at 10 dpa in elongating cotton fibre cells demonstrated by microarray analysis (Fig. 1A), RT-PCR data (Fig. 1B), and in situ hybridization (Fig. 2), indicating that the fatty acid elongation pathway is up-regulated during the development of cotton fibre. QRT-PCR showed that high accumulation of GhCER6 transcripts was only observed in wild-type cotton ovules associated with fibres at 10 dpa, and not in the other tissues including fl mutant ovules (Fig. 1C), indicating that GhCER6 expression is highly specific to the developing cotton fibre cells. The conclusion that VLCFAs and their derived lipids play important roles during cotton-fibre development is based on observations which included high expression of several cotton KCSs (Ji et al., 2003; Shi et al., 2006) and a number of cotton lipid-transfer protein genes (Ma et al., 1995; Feng et al., 2004), as well as the identification and characterization of two 3-ketoacyl-CoA reductases from cotton fibres (Qin et al., 2005). It is proposed that GhCER6 is a key condensing enzyme dedicated to the production of VLCFA precursors for the biosynthesis of sphingolipids that may play important roles in the elongation of cotton fibre cells. A similar conclusion was reached by Zheng et al. (2005) through functional characterization of the last enzyme in the fatty acid elongation pathway. The detailed mechanism of plant VLCFAs participating in the development of cotton fibre cells requires further investigation.
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Acknowledgements |
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This work was supported by grants from China National Basic Research Program (Grant 2004CB117302), National Natural Science Foundation of China (grant nos 30470171 and 30370085), the Sigrid Jusélius Foundation Finland, and the Academy of Finland. We thank Dr Jian-Guo Ji and Dr Wei Yang for MALTI-TOF-MS analysis.
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