Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex

Abstract

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.

Figure 1.

CER1 Interacts with Itself, CER3, and CYTB5s in the SUY2H System. Yeast cells cotransformed with Cub-CER1 as bait and NubG-CER1, NubG-CER3, or NubG-CYTB5 isoforms as prey are able to grow on stringent media lacking His, Leu, Trp (−HTL), and adenine (−AHTL), and show β-galactosidase activity (X-Gal), whereas cells cotransformed with Cub-CER1 and empty prey vector (NubG) as negative control do not (OD₆₀₀ yeast cells: 10⁻¹; 10⁻²; 10⁻³; 10⁻⁴). [See online article for color version of this figure.]

Figure 2.

CER1 Physically Interacts with Itself, CER3, and CYTB5s in Arabidopsis Cells. (A) Relative light emission measurements of the different NterLUC-CER1/CterLUC-PREY pairs coexpressed in Arabidopsis seedlings indicate an interaction between CER1 and itself, CER3, and CYTB5s in Arabidopsis cells. At-DPL1 (LCB-1P lyase) and At-SUR2B (LCB hydroxylase) were used as negative control prey. Relative luciferase activity is expressed in quanta of light (QL)/mm². The data represent mean values with corresponding sd values (n ≥ 4; n = 2 for negative controls). (B) Confocal images of Arabidopsis thaliana cotyledons transiently cotransformed with GFP-CER1 and YFP-CYTB5s as indicated in the legend. YFP-CYTB5s are localized to the ER (left panel) where they colocalized with GFP-CER1 (central panel) in the merge (right panel). Bars in (B) = 10 μm.

Figure 3.

The Wild-Type Yeast Strain (INVSc1) Transformed with the Mutated SUR4 F262K/K266L (SUR4#) Protein Shows Production of VLC Fatty Acids and VLC Acyl-CoAs. (A) Comparison of the FAME profile of INVSc1 control strain (transformed with empty vector) with INVSur4# (INVSc1 transformed with Sur4#) shows that expression of SUR4# in wild-type yeast triggers an overaccumulation of C28 fatty acids as well as the synthesis of C30 and C32 fatty acids. Mean values (%) of percentage of total FAMEs are given with sd (3 ≤ n ≤ 14). Each FAME species is designated by carbon chain length and degrees of unsaturation. The C17:0 fatty acid was used as internal standard. Insert: mean values are expressed in μg per mg of dry weight (DW) of total fatty acids as FAMEs with sd (3 ≤ n ≤ 14). (B) The chromatogram of the INVSur4# strain acyl-CoA pool indicates the production of VLC acyl-CoAs (highlighted by stars). Each acyl-CoA is designated by carbon chain length and degrees of unsaturation. C17:0 acyl-CoA was used as internal standard. Intensity is expressed in counts per second (cps).

Figure 4.

Synthesis of VLC Alkanes in INVSur4# Yeast Coexpressing Various Combinations of CER1 Together with CER3, CYTB5-B, and LACS1. (A) The INVSur4# yeast coexpressing Arabidopsis CER1 and CER3 has the ability to synthesize nonacosane. Additional coexpression of CYTB5-B enhances nonacosane production. GC-MS traces of total fatty acyl chain analyses of INVSur4# cotransformed with denoted Arabidopsis transgenes or with corresponding empty vectors as control. (B) and (C) Quantitative analysis of nonacosane production shows that Arabidopsis LACS1 (B) and CYTB5-B ([B] and [C]) enhance alkane production when coexpressed together with CER1 and CER3 in INVSur4#. (B) Yeast cells were grown in stringent medium lacking His, Trp, Leu, and uracil. Mean values of nonacosane (ng/mg of dry weight [DW]) are given with sd (6 ≤ n ≤ 8). Significance was assessed by Student’s t test (***, P < 0.01). (C) Yeast cells were grown in stringent medium lacking Trp, Leu, and uracil. Mean values of nonacosane are expressed in ng per mg of dry weight with sd (n = 14). Significance was assessed by Student’s t test (***, P < 0.01). (D) The INVSur4# yeast coexpressing Arabidopsis CER1, CER3, CYTB5-B, and LACS1 produces VLC alkanes with chain lengths ranging from 27 to 31 carbon atoms. INVSur4# control strain (transformed with empty vectors) shows no alkane production. GC-FID traces of the hydrocarbon fractions after separation from total lipid extract by TLC. Docosane (20 μg) was used as internal standard.

Figure 5.

Functional Relevance of His-Rich Motifs in Arabidopsis CER1 and CER3. (A) Schematic representation of CER1 and CER3 primary protein structures indicating the positions of the three His-rich motifs and the WAX2 C-terminal domain. Residue number of the His replaced by an Ala is indicated. LHD, long hydrophobic domain. (B) Coexpression of CER1 His mutants together with CER3 and CYTB5-B in INVSur4# does not lead to nonacosane production, in contrast with coexpression of wild-type (WT) CER1 with CER3 and CYTB5-B. (C) Coexpression of CER3 His mutants together with CER1 in INVSur4# does not lead to modification in nonacosane production compared with coexpression of wild-type CER3 with CER1. GC-MS traces of total fatty acyl chain analyses of INVSur4# cotransformed with Arabidopsis transgenes as detailed in the legend or with corresponding empty vectors as control. Mean values of nonacosane quantities (%) relative to the wild-type CER1 or CER3 averages are given with sd (n = 5). ND, not detected. (D) Molecular and phenotypic characterization of His-mutated CER1-overexpressing Arabidopsis lines. Alkane amounts of wild-type Col-0, cer1-1, cer1-1:CER1, cer1-1:CER1H146A, and cer1-1:CER1H248A Arabidopsis lines are expressed as μg dm⁻² stem or leaf surface area (Top). The data represent means ± sd (n = 3). RT-PCR analysis of steady state CER1 transcripts in leaves of 6-week-old plants of the different lines compared with the wild-type plants as indicated (Bottom). The ACTIN2 gene was used as a constitutively expressed control. (E) CER1 His mutants interact with CER1 and CER3 in the SUY2H system. Yeast cells cotransformed with Cub-CER1, Cub-CER1H146A, Cub-CER1H161A, or Cub-CER1H248A as bait and NubG-CER1 or NubG-CER3 as prey are able to grow on stringent media lacking His, Leu, Trp (−HTL), and adenine (−AHTL) and show β-galactosidase activity, whereas cells cotransformed with each bait and empty prey vector (NubG) as negative control do not (OD yeast cells: 10⁻¹; 10⁻²; 10⁻³; 10⁻⁴). [See online article for color version of this figure.]

Figure 6.

Proposed Biochemical Model in Which Arabidopsis CER1 and CER3 Act Synergistically for VLC Alkane Synthesis. The results obtained in this study lead us to propose that the CER1 and CER3 proteins would form an enzymatic complex catalyzing the conversion of VLC acyl-CoAs to VLC alkanes. VLCFAs activated by the long-chain acyl CoA synthases (LACSs) in VLC acyl-CoAs would be used as precursors of VLC alkane synthesis. A mandatory CER1/CER3 heterodimer would efficiently catalyze a two-step reaction starting with the reduction of acyl-CoA to a potential intermediate aldehyde subsequently decarbonylated to alkane with the loss of one carbon potentially in carbon monoxide or formate as reported in cyanobacteria (Li et al., 2008). CYTB5s would interact with the di-iron catalytic core of CER1, providing electron(s) required for the decarbonylation reaction. [See online article for color version of this figure.]