JXB Advance Access originally published online on December 12, 2003
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Journal of Experimental Botany, Vol. 55, No. 396, pp. 543-545, February 1, 2004
© 2004 Oxford University Press
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Functional characterization of the Arabidopsis thaliana orthologue of Tsc13p, the enoyl reductase of the yeast microsomal fatty acid elongating system
Received 3 September 2003; Accepted 7 November 2003
1 Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 21084, USA
2 Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
* To whom correspondence should be addressed: Fax: +1 301 295 3512. E-mail: tdunn{at}usuhs.mil
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Abstract |
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The protein encoded by the Arabidopsis At3g55360 gene was selected as a candidate for the enoyl reductase of the microsomal elongase system based on its homology to the Tsc13p protein of S. cerevisiae. The studies presented here demonstrate that heterologous expression of At3g55360 functionally complements the temperature-sensitive phenotype of a yeast tsc13 mutant that is deficient in enoyl reductase activity. Furthermore, AtTSC13 is shown to interact physically with the Elo2p and Elo3p components of the yeast elongase complex. At3g55360 apparently encodes the sole enoyl reductase activity associated with microsomal fatty acid elongation in Arabidopsis. Consistent with this conclusion, AtTSC13 is ubiquitously expressed in Arabidopsis.
Key words: Arabidopsis, elongase, enoyl-CoA reductase, fatty acid elongation, very long chain fatty acids, VLCFAs.
Microsomal fatty acid elongation generates the very long chain fatty acids (VLCFAs) that are critical components of several classes of lipids. Higher plants accumulate VLCFAs as components of triacylglycerols, cuticular waxes and sphingolipids. Plant VLCFAs are either saturated or monounsaturated, whereas animals accumulate polyunsaturated VLCFAs. C16/18 substrate fatty acids are lengthened by the sequential addition of C2 units in four successive biochemical reactions: condensation of malonyl-CoA with the acyl-CoA substrate, 3-ketoacyl-CoA reduction, 3-hydroxyacyl-CoA dehydration, and, finally, enoyl-CoA reduction. The elongating enzymes are organized in a complex referred to as the elongase (Cinti et al., 1992).
Genes encoding components of the elongase have been identified from a number of different species. In Arabidopsis, mutations in the FATTY ACID ELONGATION (FAE1) gene result in reduced levels of very long chain fatty acids in seed (James and Dooner, 1990; Kunst et al., 1992; Lemieux et al., 1990). Extensive characterization of FAE1 and the related KCS1 has confirmed that these genes encode condensing enzymes (Ghanevati and Jaworski, 2001; Roscoe et al., 2001; Todd et al., 1999). In higher plants the FAE1-like multigene family (there are 21 putative KCS genes, including FAE1, in the Arabidopsis genome) encodes enzymes responsible for the synthesis of a diverse range of VLCFAs (James et al., 1995). While the FAE1-like gene family appears to be unique to plants, the ELO-like genes, first identified in Saccharomyces cerevisiae (Oh et al., 1997), appear to encode a class of condensing enzymes that are present in fungi, mammals, and plants (Tvrdik et al., 2000).
Recently, genes encoding the 3-ketoreductase and enoyl-CoA reductase have been identified. The yeast YBR159 gene was found to be involved in heterologous synthesis of polyunsaturated VLCFAs (Beaudoin et al., 2002), and biochemical characterization confirmed its function as the 3-ketoreductase (Han et al., 2002). The Arabidopsis At1g67730 gene, which functionally complemented the yeast ybr159
mutant, showed homology to the maize gene Glossy8 (G8), which was required for the normal synthesis of cuticular waxes (Xu et al., 2002). The TSC13 gene encoding the enoyl-CoA reductase was identified in a genetic screen for mutants with defects in sphingolipid synthesis in yeast (Beeler et al., 1998). TSC13 was shown biochemically to encode the enoyl-CoA reductase and to be a physical component of the microsomal elongase, interacting with the yeast Elo2/3 proteins (Kohlwein et al., 2001).
A number of presumptive orthologues of TSC13 were identified from species including mammals, fungi (S. pombe), and Arabidopsis (Kohlwein et al., 2001). This work has now been extended to demonstrate that the Arabidopsis gene At3g55360 (designated hereafter AtTSC13) functions as a microsomal enoyl-CoA reductase. The AtTSC13 cDNA was PCR-amplified with the AT-Tsc13 up (5'-GGGCCCCTCGAGCAAGGTCACCGTCGTCTCC) and AT-Tsc13 down (5'-GGGCCCCTCGAGCTAAAGGAATGGAGG AAG) primer pair using a RIKEN Arabidopsis Full Length (RAFL) AtTSC13 cDNA (from the RIKEN Bioresource Center) as template. The amplified fragment was restricted with XhoI (sites underlined and in bold), purified using the Qiagen PCR purification kit, and ligated into the SalI site of pADH1, thereby generating a yeast plasmid constitutively expressing the HA-tagged AtTSC13 protein. Heterologous expression of the HA-tagged AtTSC13 protein in yeast rescued the ts-lethality of the tsc13–1 elo3 double mutant (Fig. 1A), indicating that AtTSC13 was very likely to encode the functional orthologue of the yeast TSC13 enoyl-CoA reductase.
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Further evidence was provided by the demonstration that AtTSC13 physically interacted with the ELO protein components of the yeast elongase. A triple-myc tag was introduced at the N-terminus of AtTSC13 by replacing the coding sequence of the S. cerevisiae TSC13 gene with that of the AtTSC13 gene in the MYC-TSC13-426 plasmid (Kohlwein et al., 2001). This was accomplished by introducing SalI sites after the MYC tag and before the stop codon of the S. cerevisiae TSC13 gene in MYC-TSC13-426 by QuikChange mutagenesis (Stratagene, La Jolla, California). The SalI fragment carrying the yeast TSC13 gene was deleted and replaced with a PCR-generated SalI-ended fragment carrying the AtTSC13 cDNA. The myc-tagged AtTSC13 was co-expressed in yeast cells with HA-epitope tagged forms of either Elo2p or Elo3p (Kohlwein et al., 2001). Microsomal fractions were solubilized, and elongase components were immunoprecipitated. Immunoprecipitation with the HA antibody not only pulled down the HA-tagged Elo proteins, but also the myc-tagged AtTSC13 (Fig. 1B). This is a clear demonstration that AtTSC13 is in physical proximity to the yeast Elo proteins. Based on this co-IP data and the rescue of the tsc13–1elo3
mutant, AtTSC13 is very likely to be the Arabidopsis enoyl reductase of the microsomal fatty acyl elongase. As outlined above, the microsomal fatty acid elongase synthesizes VLCFAs, which are incorporated into a number of different classes of lipids. In yeast, loss of Tsc13p activity is associated with a decrease in the levels of VLCFAs found in sphingolipids. Higher plants accumulate many different types of VLCFA-containing lipids and, as mentioned earlier, there is a large family of FAE1-like condensing enzymes. However, this proliferation of activities does not appear to be represented in the other components of the elongase; there are only two 3-ketoreductase/G8 orthologues present in Arabidopsis (At1g67730/G8 and At1g24470) and AtTSC13 represents the only candidate enoyl-CoA reductase. Thus it seems likely that AtTSC13 is the sole enoyl-reductase component of the microsomal elongase. The expression pattern of AtTSC13 was analysed by northern blotting and it was determined that this transcript was present in all Arabidopsis tissues tested (leaves, stems, flowers, siliques, and roots) (Fig. 2). Upon prolonged exposure of the autoradiogram, the presence of transcripts in mature seeds (data not shown) was also identified. Consistent with these results, data in the Nottingham Arabidopsis Stock Centre’s Arabidopsis Affymetrix database (http://ssbdjc2.nottingham.ac.uk/narrays/experimentbrowse.pl; http://www.cbs.umn.edu/arabidopsis/) on the expression of At3g55360 confirms the presence of this transcript in a wide range of Arabidopsis tissues, including seedlings, lateral and primary roots, pollen, shoots, petioles, auxiliary buds, and suspension-cultured cells. Taken together with the authors’ northern blot data, this indicates the likelihood that AtTSC13 is ubiquitously expressed in Arabidopsis tissues. Therefore, AtTSC13 is likely to participate in the synthesis of most (if not all) VLCFAs. This would include the seed-specific VLCFAs found in storage triacylglcerols, the epidermal cuticular waxes and the sphingolipids.
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In conclusion, the sole orthologue of the elongase-associated enoyl-CoA reductase present in the Arabidopsis thaliana genome has been identified. It is predicted that AtTSC13 is a ubiquitous component of the multiple distinct elongases including those that depend on the FAE1-like family of condensing enzymes as well as those that utilize the ELO-like family of (putative) condensing enzymes. As such, it is also predicted to be an essential gene in Arabidopsis.
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Acknowledgements |
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Rothamsted Research receives grant-aided support from the BBSRC (UK). SG was the grateful recipient of a NFCI studentship. This work was also supported by NSF grants MCB-00(8100 and MCB-0(13433 and USUHS grant R071GW to TMD. The cDNA for At3g55360 was kindly provided by the RIKEN BioResource Center, Japan.
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