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. 2012 Jul 6;24(7):3106–3118. doi: 10.1105/tpc.112.099796

Reconstitution of Plant Alkane Biosynthesis in Yeast Demonstrates That Arabidopsis ECERIFERUM1 and ECERIFERUM3 Are Core Components of a Very-Long-Chain Alkane Synthesis Complex[C],[W]

Amélie Bernard a,b, Frédéric Domergue a,b, Stéphanie Pascal a,b, Reinhard Jetter c, Charlotte Renne d, Jean-Denis Faure d, Richard P Haslam e, Johnathan A Napier e, René Lessire a,b, Jérôme Joubès a,b,1
PMCID: PMC3426135  PMID: 22773744

Very-long-chain alkanes are major components of cuticular waxes, a protective layer covering aerial surfaces of plants. This article shows that the Arabidopsis thaliana CER1 protein interacts with the wax-associated CER3 protein and with the cytochrome b5 isoforms found in the endoplasmic reticulum, and that these proteins constitute the enzymatic complex catalyzing the redox-dependent plant alkane synthesis.

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.

INTRODUCTION

The aerial epidermis of terrestrial plants produces and secretes a mixture of very-long-chain (VLC) lipids that assemble into a waterproof barrier called the cuticle. Strategically located at the plant–air interface, the cuticle plays a critical role in the interactions between the plant and its environment. Besides its main function in preventing plant desiccation, the cuticle is also involved in plant protection from UV radiation, a self-cleaning process called the lotus effect, which limits the deposition of biotic and abiotic pollutants on plant surfaces, and protection against plant pathogens and insects (Shepherd and Wynne Griffiths, 2006). Furthermore, several lines of evidence have linked the cuticle to plant development processes, such as specialized epidermal cell differentiation and delimitation of organ boundaries (Javelle et al., 2011). The protective capacities of the cuticle are mainly based on physical and biochemical properties of its two highly hydrophobic components, cutin and cuticular waxes (Pollard et al., 2008; Domínguez et al., 2011). Overlying the epidermis cell wall, the first layer of the cuticle consists of cutin, a polyester of modified fatty acids and glycerol monomers, embedded with intracuticular waxes (Pollard et al., 2008; Li and Beisson, 2009); covering the cutin matrix, epicuticular waxes form the smooth or crystalloid outermost layer of the cuticle (Jeffree, 1996). Beyond the polymerized cutin, cuticular waxes mostly consist of a complex mixture of VLC aliphatic compounds, triterpenoids, as well as minor sterols and flavonoids (Buschhaus and Jetter, 2011). Aliphatic acyl chains of waxes derive from very-long-chain fatty acids (VLCFAs), with predominant chain lengths ranging from 26 to 32 carbons, resulting from the epidermal acyl-CoA elongase activity (Zheng et al., 2005; Bach et al., 2008; Joubès et al., 2008; Beaudoin et al., 2009). After elongation, acyl chains are processed according to two different biosynthetic routes: either the alcohol-forming pathway, yielding fatty alcohols and alkyl esters, or the alkane-forming pathway, generating fatty aldehydes, alkanes, and their derivatives (Samuels et al., 2008).

In the past 20 years, reverse genetic analyses of Arabidopsis thaliana mutants have provided significant advances in the understanding of wax metabolism (Kunst and Samuels, 2009). In fact, biosynthesis of compounds on the alcohol-forming pathway is now well described, because genes have been clearly identified, and the corresponding proteins have been functionally characterized (Rowland et al., 2006; Li et al., 2008). However, these approaches failed to uncover the enzymes involved in alkane synthesis, leaving the alkane-forming pathway poorly understood. Yet VLC alkanes dominate the wax mixture of many plant species, representing up to 70% of the total wax amount in Arabidopsis leaves, where they play critical roles in plant physiology, notably in pollen hydration and thus plant fertility, resistance to drought stress, and plant–pathogen interactions (Aarts et al., 1995; Kosma et al., 2009; Bourdenx et al., 2011). Moreover, because alkanes are the major constituents of liquid fuels, the discovery of the alkane biosynthetic machinery in plants would provide a toolbox of enzymes for the production of renewable hydrocarbon sources and the next generations of biofuels (Joyce and Stewart, 2011). Therefore, because of the importance of alkanes for both biotechnological purposes and plant physiological functions, the characterization of the plant VLC alkane–forming enzymes has been a challenging goal for decades (Jetter and Kunst, 2008; Kunst and Samuels, 2009).

Early biochemical analyses led to the proposal that alkanes could be produced from elongated acyl chains via intermediate aldehydes. This hypothesis was based on the observed conversion of C18 aldehyde into C17 alkane with the release of CO in pea (Pisum sativum) and a green algae (Botryococcus braunii), suggesting that a potential decarbonylation mechanism occurred (Cheesbrough and Kolattukudy, 1984; Dennis and Kolattukudy, 1991; Schneider-Belhaddad and Kolattukudy, 2000). Further characterization of the partially purified alkane synthesis activity in pea indicated that it requires metal ions and is inhibited by O2 and reducing agents, suggesting that the reaction would be redox-independent (Schneider-Belhaddad and Kolattukudy, 2000). Because VLC aldehydes are found in the pool of cuticular waxes, it was proposed that VLCFAs could be reduced by a fatty acid reductase into corresponding even-numbered aldehydes, and then an aldehyde decarbonylase (AD) could catalyze the conversion into VLC alkanes with the loss of one carbon (Samuels et al., 2008). However, definitive evidence for intermediate aldehydes has never been provided, and enzymatic activities were never assessed. Interestingly, this putative biochemical mechanism can be compared with the long-chain alkane biosynthesis in the cyanobacteria Synechococcus sp and Nostoc punctiforme. Indeed, the microbial mechanism was recently reported to proceed via a two-step pathway from acyl–Acyl Carrier Protein (ACP) precursors to an aldehyde intermediate and the hydrocarbon product involving two enzymes, an acyl reductase and an aldehyde decarbonylase (Schirmer et al., 2010; Das et al., 2011).

The analysis of the Arabidopsis eceriferum1 (cer1) mutant indicated the involvement of the corresponding protein in alkane biosynthesis. Indeed, the cer1 mutant is characterized by a nearly depleted load of alkanes and their derivates accompanied by a slight increase in aldehyde content (Koornneef et al., 1989; Aarts et al., 1995; Jenks et al., 1995; Bourdenx et al., 2011). Support for the central role of CER1 in alkane production was provided by the recent characterization of Arabidopsis transgenic lines, in which CER1 overexpression led to a large and specific accumulation of wax VLC alkanes (Bourdenx et al., 2011). Consistent with such an involvement in wax metabolism, CER1 expression was found confined to the epidermis of aerial organs and the CER1 protein located to the endoplasmic reticulum (ER), where wax biosynthesis takes place (Kamigaki et al., 2009; Bourdenx et al., 2011). CER1 is a membrane-bound protein that shares no significant sequence similarity with functionally characterized proteins, except for a short sequence containing a tripartite His-rich motif HX3H+HX2HH+HX2HH (Aarts et al., 1995). His-rich clusters are part of the di-iron catalytic core found in a large family of integral membrane enzymes involved in fatty acid metabolism (Shanklin and Cahoon, 1998). Therefore, it has been suggested that CER1 could have an enzymatic activity associated with alkane production (i.e., that CER1 could be the alkane-forming enzyme [Aarts et al., 1995]). This hypothesis is further supported by the characterization of the ADs from cyanobacteria, which belong to a family of soluble nonheme di-iron carboxylate enzymes (Schirmer et al., 2010). Nevertheless, the function of CER1 has never been provided, partly because of the lack of information about VLC alkane–forming activity at the molecular level, the unknown substrate, and the low solubility and unavailability of commercial VLC aldehydes. Furthermore, because expression of CER1 in heterologous systems did not lead to production of alkanes (Bourdenx et al., 2011), the CER1 function remains a crucial open question for the understanding of VLC alkane formation.

In this article, the CER1 protein was used as a starting point to reconstitute the plant alkane–forming pathway. To learn more about CER1 function, an exploratory screen was conducted to identify CER1 physical interaction partners. In both yeast and plant cells, CER1 was found to interact with the wax-associated protein CER3 and with the cytochrome b5 isoforms (CYTB5s) serving as ER-reducing components. Through an iterative approach performed in a yeast heterologous system producing VLC acyl-CoAs, we showed that a mandatory CER1/CER3 coexpression results in VLC alkane production, thus identifying the enzymatic components catalyzing plant alkane synthesis. Arabidopsis CYTB5s were found to increase alkane production, and analysis of point-mutated CER1 and CER3 proteins suggested that CYTB5s are specific CER1 cofactors, highlighting a potential redox-dependent CER1 activity for alkane synthesis. Taken together, our results elucidate the plant alkane–forming pathway and support a new model for alkane synthesis in which CER1 and CER3 constitute the core components of a multiprotein enzymatic complex catalyzing the redox-dependent conversion of VLC acyl-CoAs to VLC alkanes.

RESULTS

CER1 Physically Interacts with Itself, CER3, and the ER-Associated CYTB5 Isoforms

Because all previous attempts to characterize CER1 activity by heterologous expression had failed (Bourdenx et al., 2011), we hypothesized that protein partners might be needed for CER1 to function. To find such partners, CER1 potential interactions with proteins that are involved in wax synthesis but with an unknown function were first assessed. Even though several Arabidopsis cer mutants with decreased wax alkane amounts similar to cer1 mutants have been reported, only the CER2, CER3/WAX2, CER5/ABCG12, CER6/KCS6, CER7, CER8/LACS1, and CER10/ECR genes have been cloned (Samuels et al., 2008). Most of the corresponding proteins are involved in wax formation, but not directly in alkane biosynthesis, leaving CER3 as the only currently available candidate with a putative function in alkane formation (Chen et al., 2003). The potential physical interaction of CER1 with CER3 was tested using a split-ubiquitin yeast two-hybrid (SUY2H) assay with Cub-CER1 as bait and NubG-CER3 as prey (Figure 1). Unlike yeast cells coexpressing Cub-CER1 and NubG as a negative control, the yeast coexpressing Cub-CER1 and NubG-CER3 were able to grow on the different selective media and show β-galactosidase activity, indicating that CER3 interacts with CER1 (Figure 1). Analysis of the yeast coexpressing Cub-CER1 as bait and NubG-CER1 as prey showed that, in yeast, CER1 also interacts with itself, suggesting that CER1 could dimerize in plant cells.

Figure 1.

Figure 1.

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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.]

To identify other CER1 partners, an exploratory screen was then conducted in the SUY2H system. Because CER1 expression is confined to the epidermis cells, where the entirety of wax metabolism takes place, a cDNA library was constructed in the SUY2H prey expression vector using RNA of epidermis peeled from Arabidopsis stems. The screen of the generated epidermis cDNA library with Cub-CER1 as bait isolated 320 clones, which were further selected for their ability to grow on the different stringent media as well as for their β-galactosidase activity. Furthermore, because CER1 locates to the ER, all candidate proteins that have been experimentally and unequivocally associated with subcellular compartments other than the ER and the cytosol were considered false positive (representing 20% of the clones). All together, this analysis led to the selection of 96 positive clones, in which 30% of the isolated proteins were found to be related to lipid metabolism. Interestingly, the screen notably isolated three CYTB5 proteins (CYTB5-B [At2g32720], CYTB5-C [At2g46650], and CYTB5-D [At5g48810]) as potential CER1 partners (representing up to 15% of the isolated clones). CYTB5s are ubiquitous hemoproteins that act as electron transfer components to nonheme irons associated with His clusters of ER-localized enzymes (Shanklin and Cahoon, 1998). Although early biochemical analyses proposed that the plant alkane–forming activity is redox-independent (Cheesbrough and Kolattukudy, 1984; Schneider-Belhaddad and Kolattukudy, 2000), the presence of His-clusters in the CER1 amino acid sequence suggests that CYTB5 proteins could be CER1 cofactors. Therefore, these candidate proteins were selected for further analyses. A targeted SUY2H assay confirmed the interaction of CYTB5-B, CYTB5-C, and CYTB5-D with CER1 and also indicated the interaction of CER1 with the fourth potentially ER-associated CYTB5-E isoform (Figure 1).

To test CER1 interaction with candidate proteins in plant cells, we performed an Arabidopsis transient split-luciferase assay (Van Leene et al., 2010). In this system, the firefly luciferase (LUC) protein is split into two halves, which are fused to the bait and prey proteins. In plant cells, coexpression of the fusion proteins will reconstitute LUC activity if, and only when, the proteins of interest interact. Cotyledons of Arabidopsis seedlings were cotransformed with constructs for expression of CER1 fused to the N-terminal part of the firefly LUC protein (NterLUC-CER1), and candidate partners fused to its C-terminal part (CterLUC-PREY). Two ER-localized proteins that are not implicated in wax production, At-DPL1 (LCB-1P lyase) and At-SUR2B (LCB hydroxylase), were used as negative control prey. Significant luciferase activity was observed only in seedlings expressing NterLUC-CER1 together with CterLUC-CER1, CterLUC-CER3, CterLUC-CYTB5-B, CterLUC-CYTB5-C, CterLUC-CYTB5-D, or CterLUC-CYTB5-E, indicating that CER1 interacts with all these proteins in Arabidopsis cells (Figure 2A).

Figure 2.

Figure 2.

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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.

Previous studies had shown that both CER1 and CER3 proteins are located to the ER (Kamigaki et al., 2009). Similarly, Arabidopsis CYTB5-B and CYTB5-D are known to be located to the ER (Maggio et al., 2007; Nagano et al., 2009), and based on sequence similarity, CYTB5-C and CYTB5-E were predicted also to be located there (Hwang et al., 2004). To verify this prediction, green fluorescent protein (GFP)-CER1 and yellow fluorescent protein (YFP)-CYTB5 fusion proteins were transiently coexpressed in Arabidopsis seedlings. Confocal microscopy analysis showed that CYTB5-B and CYTB5-D, as well as CYTB5-C and CYTB5-E, colocalized with CER1, illustrating how these proteins interact with CER1 in plant cells (Figure 2B).

Reconstitution of the VLC Alkane–Forming Pathway in Yeast

The finding that CER1 physically interacts with CER3 and CYTB5(s) suggested that these proteins could participate with CER1 in alkane production. To test this hypothesis, an iterative approach was conducted in yeast, an organism that does not naturally produce alkanes. Lipid analyses of engineered yeasts expressing CER1, CER3, and CYTB5-B genes in all single, double, and triple combinations never showed any difference when compared with the control yeast transformed with corresponding empty vectors. Our previous work had shown that overexpression of CER1 in Arabidopsis leads to a specific increase of alkanes with carbon chain lengths between 27 and 33 carbon atoms, suggesting that CER1 likely has chain length specificity for substrates with 28 to 34 carbon atoms (Bourdenx et al., 2011). However, wild-type yeast produces VLCFAs only up to C26 (with traces of C28) (Figure 3A). Therefore, the wild-type yeast strain was manipulated to generate a system where significant amounts of CER1 potential substrates were accessible. For that purpose, we used the mutated yeast elongase component SUR4 F262K/K266L (SUR4#), which had been shown to allow production of C28 and C30 fatty acids in sur4Δ mutant yeast (Denic and Weissman, 2007). Gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–tandem mass spectrometry analyses of fatty acid methyl esters (FAMEs) and acyl-CoAs, respectively, confirmed that SUR4# expression in wild-type yeast (engineered yeast hereafter referred to as INVSur4#) resulted in the production of substantial amounts of C28 and C30 as well as traces of C32 acyl compounds (Figures 3A and 3B).

Figure 3.

Figure 3.

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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).

When CER1 or CER3 alone was expressed in INVSur4#, alkanes could not be detected (Figure 4A). By contrast, when CER1 was expressed together with CER3 in INVSur4#, a new lipid was detected and identified as nonacosane (C29 alkane) by GC-MS (Figure 4A). No other modification of the fatty acid profile was observed (see Supplemental Figures 1A and 1D online), and no alkane was found in the yeast medium. Taken together, these results indicate that CER1 and CER3 are both necessary and sufficient for VLC alkane synthesis in VLCFA-producing yeast.

Figure 4.

Figure 4.

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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.

The engineered yeast system developed was then used to investigate the substrates used by CER1/CER3 to produce alkanes. Indeed, although it is well established that VLC alkanes derive from elongated acyl chains (Samuels et al., 2008), it is still unclear whether VLCFAs or acyl-CoAs are the true alkane precursors. To address this question, we used the Arabidopsis Long-Chain Acyl-CoA Synthetase1 (LACS1), which performs the conversion of fatty acids into their activated CoA thioesters with high in vitro specificity for C30 fatty acids (Lü et al., 2009). The coexpression of LACS1 together with CER1 and CER3 in INVSUR4# led to a 2-fold increase in nonacosane amount over the lines coexpressing only CER1 and CER3 (Figure 4B; see Supplemental Figures 1B to 1D online), supporting that VLC acyl-CoAs are precursors for VLC alkane synthesis.

To test the relevance of CER1 interaction with CYTB5s for alkane synthesis, CYTB5-B was then coexpressed with CER1 and CER3 in INVSur4#. When compared with the yeast INVSur4# coexpressing only CER1 and CER3, the additional presence of CYTB5-B led to an almost 2-fold increase in nonacosane production (Figures 4A to 4C; see Supplemental Figures 1A and 1D online), suggesting that CYTB5s are cofactors of a redox-dependent CER1/CER3 activity for VLC alkane synthesis in Arabidopsis.

CER1/CER3 alkane-forming activity was further characterized by scaling up yeast cultures and separating the hydrocarbon fraction from the total lipid extract on thin layer chromatography (TLC). Gas chromatography–flame ionization detection (GC-FID) and GC-MS analyses showed that the resulting fraction contained mainly nonacosane (C29) together with heptacosane (C27) and hentriacontane (C31) as well as traces of octacosane (C28) and triacontane (C30) (Figure 4D; see Supplemental Figure 2 online). Thus, the alkanes synthesized by CER1/CER3 in yeast match the chain length specificity observed in planta, as shown in Arabidopsis CER1-overexpressing lines (Bourdenx et al., 2011). Collectively, these results demonstrate that CER1 and CER3 constitute the enzymatic core components of plant alkane biosynthesis. Moreover, yeast cells coexpressing CER1/CER3 together with CYTB5-B and LACS1 produce an average of 86 ng of nonacosane per mg of dry weight. This yield corresponds to ∼1 and 64% of the amount produced by Arabidopsis stem (6900 ± 324 ng per mg of dry weight) or rosette leaves (135 ± 28 ng per mg of dry weight), respectively.

The CER1 His-Rich Motifs Are Essential for VLC Alkane Synthesis

His clusters have been shown to be essential for the catalytic activity of hydroxylase and desaturase enzymes, where they serve as ligands for a di-iron binding site mediating electron transfer from CYTB5s (Shanklin and Cahoon, 1998). Therefore, the functional relevance of the CER1 His clusters was assayed by generating mutated CER1 proteins in which a conserved His in each of the three motifs was replaced by an Ala (Figure 5A). When the CER1 mutants were coexpressed in INVSur4# together with CER3 and CYTB5-B, no alkanes were detected (Figure 5B; see Supplemental Figure 3 online), indicating that all three CER1 His-rich clusters are essential for alkane synthesis. To verify this result in planta, two CER1 His mutants were overexpressed in the Arabidopsis cer1-1 knockout mutant (cer1-1:CER1H146A; cer1-1:CER1H248A) and compared with a transgenic line overexpressing the wild-type CER1 gene (cer1-1:CER1). Although no CER1 transcripts were detected in the cer1-1 mutant, both His mutant lines and the cer1-1:CER1 line exhibited higher levels of transcripts than wild-type plants (Figure 5D). Gas chromatography analyses showed that overexpression of the wild-type CER1 in the cer1-1 background restored alkane biosynthesis, whereas overexpression of CER1H146A or CER1H248A was not able to rescue the wild-type phenotype (Figure 5D; see Supplemental Figures 4 and 5 and Supplemental Tables 1 and 2 online). Taken together, these results demonstrate that the His-rich motifs are essential for CER1 to function. Furthermore, SUY2H assays performed with CER1 mutated proteins as baits showed that the mutations in CER1 His clusters do not prevent CER1/CER1 or CER1/CER3 interactions (Figure 5E). These data indicate that the absence of alkane synthesis observed with the CER1 mutants is caused by a defect in CER1 activity rather than the incorrect formation of the CER1/CER3 complex, thereby demonstrating that CER1 His-rich motifs are part of the catalytic site of the enzyme.

Figure 5.

Figure 5.

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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.]

Interestingly, CER3 also contains a tripartite His cluster built around eight conserved histidines (Figure 5A) (Chen et al., 2003; Rowland et al., 2007). We further generated three CER3 mutants in which one conserved His in each of the three motifs (equivalent in position to those mutated in CER1) was replaced by an Ala (Figure 5A). Coexpression of CER3 mutated proteins together with CER1 in INVSur4# resulted in no obvious modification of nonacosane production as compared with CER3 wild-type coexpression (Figure 5C; see Supplemental Figure 6 online). Collectively, our findings suggest that the His clusters are part of the CER1 catalytic site but are not strictly required for CER3 activity.

DISCUSSION

CER1 and CER3 Functionally Interact to Produce VLC Alkanes

Even though strong evidence for Arabidopsis CER1 involvement in alkane biosynthesis had been provided (Aarts et al., 1995; Bourdenx et al., 2011), the function of this protein has never been elucidated. Because expression of CER1 alone is not sufficient to produce alkanes in heterologous systems, we speculated that CER1 might need protein partners for its activity. In this study, we report that CER1 physically interacts with CER3 in both yeast and Arabidopsis, suggesting that they associate to form an enzymatic complex. A strict expression of the CER1/CER3 complex in VLCFA-producing yeast leads to the synthesis of VLC alkanes, demonstrating that both proteins are required for the catalytic production of alkanes. Taken together, these results demonstrate that CER1 and CER3 are core components of the VLC alkane–forming enzymatic activity.

Toward the Understanding of the VLC Alkane–Forming Catalytic Activities

Based on early biochemical data (Cheesbrough and Kolattukudy, 1984; Schneider-Belhaddad and Kolattukudy, 2000) and on the analyses of Arabidopsis cer1 and cer3/wax2 mutant phenotypes (Aarts et al., 1995; Chen et al., 2003), a two-step pathway for VLC alkane synthesis was proposed, in which CER3 catalyzes the reduction of VLC acyl-CoAs generating intermediate fatty aldehydes that are subsequently decarbonylated by CER1 to VLC alkanes. Although it has been proposed that fatty aldehydes are intermediates of alkane formation in eukaryotes, such as insects and plants (Reed et al., 1994; Schneider-Belhaddad and Kolattukudy, 2000), the corresponding two-step pathway for long-chain alkane biosynthesis has been demonstrated only in cyanobacteria (Schirmer et al., 2010). In the microbial pathway, a fatty acyl-ACP reductase (FAAR) catalyzes the reduction of fatty acyl-ACPs to intermediate aldehydes, which are then used by an AD to produce alkanes (Schirmer et al., 2010; Das et al., 2011). Nevertheless, whereas the FAAR and AD from cyanobacteria do not share any common motifs with each other; Arabidopsis CER1 and CER3 are highly related. Sequence similarity and conserved intron positions in CER1 and CER3 indicate a common origin from the same ancestor gene (Sturaro et al., 2005). Furthermore, the two proteins share 35% identity (see Supplemental Figure 7 online), including elements analogous to both cyanobacterial proteins. Indeed, CER1 and CER3 both possess a conserved tripartite His motif serving as ligand for a di-iron active site cluster in the catalytic site of several fatty acid–related membrane-bound enzymes (Shanklin et al., 1997; Shanklin and Cahoon, 1998; Idkowiak-Baldys et al., 2003). His clusters constitute the conserved feature of integral membrane oxygenases and oxidases and are equivalent to the carboxylate bridge di-iron cluster of their functionally related soluble homologs. Therefore, even though the plant and microbial proteins do not share sequence similarity, both CER1 and CER3 resemble the cyanobacterial AD, which was found to be structurally and functionally related to nonheme di-iron enzymes (Das et al., 2011; Li et al., 2011). In addition, CER1 and CER3 possess the functionally uncharacterized WAX2 domain at their C termini (Figure 5A; see Supplemental Figure 7 online) that we also partially identified at the C terminus of the cyanobacterial FAAR protein (see Supplemental Figure 7 online). Interestingly, a mutation in the WAX2 domain of the CER3 protein was reported to severely inhibit wax synthesis in the Arabidopsis cer3-4 mutant, suggesting that this domain is required for plant alkane biosynthesis (Rowland et al., 2007). In our study, we showed that the CER3 His clusters are nonessential for alkane synthesis but are crucial for CER1 function in both Arabidopsis and yeast. This supports the hypothesis that CER1 acts as an AD. Conversely, CER3 would be essential for alkane production by providing fatty aldehydes or another yet to be described metabolic intermediate to CER1. Alternatively, it is plausible that the two proteins possess a weak capacity for both reactions, with CER3 acting preferentially as the fatty acyl reductase and CER1 as the AD. The finding that CER1 and CER3 interact in both yeast and Arabidopsis suggests that the two proteins could act synergistically (i.e., that heterodimerization of CER1 and CER3 is necessary for efficient activity of both proteins). Thus, in the absence of its partner, either CER1 or CER3 could homodimerize to perform weak conversion of acyl-CoAs to alkanes. This could explain the phenotypes of the cer3/wax2 and cer1 null mutants, which are not completely devoid of aldehydes and alkanes (Chen et al., 2003; Bourdenx et al., 2011) as well as the synergistic effect of both mutations on trichome development observed in the cer1 cer3 double mutant (Kurata et al., 2003). Technical limitations notwithstanding, further support for CER1/CER3 synergy is provided by the fact that no change in the fatty acyl composition was detected when only CER3 was expressed in yeast. In this context, significant amounts of aldehyde would be produced only when CER1 and CER3 are coexpressed in yeast but not detected, because they are converted into alkanes rather than accumulating. Thus, from our results, we propose a model in which a mandatory CER1/CER3 heterodimer would highly efficiently perform conversion of VLC acyl-CoAs to VLC alkanes (Figure 6).

Figure 6.

Figure 6.

Open in a new tab

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.]

CYTB5s as Electron Transfer Components for VLC Alkane Synthesis

In an attempt to identify new proteins involved in VLC alkane synthesis, we found that CER1 interacts with the entire subfamily of ER-associated Arabidopsis CYTB5s. CYTB5s act as electron transfer components to nonheme irons associated with His clusters of several ER-localized enzymes (Shanklin and Cahoon, 1998). Interestingly, coexpression of CYTB5-B together with CER1 and CER3 in yeast increased nonacosane production, suggesting an involvement of these proteins in VLC alkane formation in Arabidopsis. Because coexpression of CER1 and CER3 is sufficient for alkane synthesis in the engineered VLC acyl-CoA–synthesizing yeast, we hypothesize that reducing components must be provided by endogenous yeast CYTB5s. Mutagenesis analysis indicated that His-rich motifs are essential for CER1 activity but not for CER3, indicating that CYTB5s are likely to be cofactors specific for CER1 activity for VLC alkane formation. These results do not support the function of CER1 as a potentially redox-neutral decarbonylase proposed by early biochemical studies (Cheesbrough and Kolattukudy, 1984; Vioque and Kolattukudy, 1997; Schneider-Belhaddad and Kolattukudy, 2000). However, recent work on cyanobacterial alkane biosynthesis showed that, in vitro, long-chain aldehyde decarbonylation, catalyzed by a di-iron enzyme, strictly requires a reducing system to produce alkanes in both O2-dependent and -independent reactions (Das et al., 2011; Li et al., 2011). Based on these latest mechanisms and our data, we propose that CYTB5 act as a cofactor of redox-dependent CER1 activity in the CER1/CER3 VLC alkane–forming complex. It is interesting to note that reducing equivalents are apparently delivered to the cyanobacterial AD by the ferredoxin/ferredoxin reductase system (Schirmer et al., 2010) which is shared by several soluble nonheme di-iron enzymes, whereas, for the plant alkane–forming activity, this role could be played by the CYTB5s—and presumably the CYTB5 reductase—used by the ER membrane-bound nonheme di-iron enzymes (Sperling et al., 2003).

The early characterization of Arabidopsis cer1 and cer3 mutant phenotypes (Koornneef et al., 1989) pointed to the involvement of the corresponding proteins as components of wax metabolism. Nevertheless, the cloning of CER1 and CER3 was not sufficient to reveal their biochemical activity, leaving the VLC alkane synthesis unresolved. This study reports VLC alkane production in a heterologous yeast system, demonstrating the catalytic functions of CER1 and CER3 for VLC alkane production and thus identifying the enzymatic components of plant alkane synthesis. Further manipulations of the engineered yeast suggest a model for plant VLC alkane metabolism in which VLC acyl-CoAs would serve as precursors of a CYTB5s/CER1/CER3 complex for redox-dependent VLC alkane synthesis. The reconstitution of Arabidopsis VLC alkane biosynthesis in yeast provides an excellent tool for further understanding wax metabolism. Furthermore, production of VLC alkanes in yeast lays the foundation for conceptual and metabolic manipulation of this and other organisms toward large-scale production of tomorrow’s biofuels.

METHODS

Plant Material and Conditions

Arabidopsis thaliana plants (ecotype Columbia [Col-0]) were cultivated under optimal growth conditions as previously described (Joubès et al., 2008). The cer1-1 T-DNA insertion line (SALK_008544) and the cer1-1:CER1 transgenic line have been previously described (Bourdenx et al., 2011). Landsberg erecta Arabidopsis seedlings were used for transient split-luciferase assays as described in Van Leene et al. (2010).

Cloning and Transgenic Yeasts

Site-directed mutagenesis was performed as previously described (Taton et al., 2000) with minor modifications. His codons were replaced by the Ala codon GCT. Primers used for generation of mutation-containing fragments are detailed in Supplemental Tables 3 and 4 online. CER1, His-mutated CER1s, CER3, His-mutated CER3s, LACS1, and CYTB5-B open reading frames were amplified by PCR using primers detailed in Supplemental Table 4 online and cloned into yeast expression vectors as detailed in Supplemental Table 4 online. SUR4-F262A/K266L cloned in p416 MET25-FLAG3 was used as previously described (Denic and Weissman, 2007). Saccharomyces cerevisiae wild-type INVSc1 strain (MATa his3-D1 leu2 trp1-289 ura3-52) cells were transformed with the different combinations and grown on minimal medium agar plates lacking corresponding amino acid as indicated in Supplemental Table 5 and Supplemental Figure 1 online.

Cloning and Transgenic Plants

Stable transgenic plants were obtained as previously described (Bourdenx et al., 2011). Mutated CER1 open reading frames were cloned into the pDONR221 ENTRY vector by Gateway recombinational cloning technology and were subsequently transferred into the pH7WG2D destination vector (Karimi et al., 2002). Arabidopsis transient cotransformations were performed as previously described (Marion et al., 2008) with minor modifications. CER1, CYTB5-B, CYTB5-C, CYTB5-D, and CYTB5-E open reading frames were cloned into the pDONR221 ENTRY vector by Gateway recombinational cloning technology and were subsequently transferred into the pK7WGF2 (CER1) or pK7WGY2 (CYTB5s) destination vectors (Karimi et al., 2002).

Confocal Analysis

Arabidopsis thaliana cotyledons were transiently cotransformed with GFP-CER1 and YFP-CYTB5 as described above. Colocalization analyses have been performed as previously described (Chatre et al., 2009).

Gene Expression Analysis

RNA was extracted from Arabidopsis leaves or yeast cells using the RNeasy Plant Mini Kit (Qiagen). Purified RNA was treated with DNase I using the DNA-free kit (Ambion). For plant tissues, first-strand cDNA was prepared from 10 μg of total RNA with the SuperScript RT II kit (Invitrogen) and oligo(dT)18 according to the manufacturer’s instructions. For yeast cells, first-strand cDNA was prepared from 63 ng of total RNA with the SuperScript RT II kit (Invitrogen) and random primers according to the manufacturer’s instructions. A 1-μL aliquot of the total reaction volume (20 μL) was used as a template in RT-PCR amplification. The PCR amplification was performed with the gene-specific primers listed in Supplemental Table 4 online.

Lipid Analyses

Plant cuticular wax analyses were performed as previously described (Bourdenx et al., 2011). For fatty acyl chain analyses of individual yeast transformants, all transgenic yeasts were grown in 5 to 10 mL of appropriate liquid minimal medium (see Supplemental Table 5 online) supplemented with 2% Gal to induce expression of Arabidopsis transgenes (see Supplemental Figure 1D online). Starting from precultures with an OD600 of ∼0.017, expression cultures were grown for 4 to 6 d at 30°C. Yeast cells of 5 to 10 mL cultures were pelleted and washed in 2 mL NaCl (2.5%, w/v) before fatty acyl chain analyses. FAMEs were obtained by transmethylation at 85°C for 1 h 20 min of yeast cell sediments with 1 mL 0.5 M sulfuric acid in methanol containing 2% v/v dimethoxypropane and 50 μg of heptadecanoic acid (C17:0) as well as 10 μg of docosane (C22 alkane) as internal standards. After cooling, 1 mL NaCl 2.5% (w/v) was added, and fatty acyl chains, including alkanes, were extracted in 1 mL hexane. Extracts were washed with 1 mL 250 mM Tris, pH 8, in NaCl 0.9% (w/v) and then dried under a gentle stream of nitrogen. Samples were dissolved in 200 μL hexane and analyzed by GC-MS as previously described (Domergue et al., 2010). For TLC analysis, ∼2000 OD600 units were used to prepare FAMEs. The alkane fraction was separated from the extracts by TLC on silica gel plates (20 × 20 cm; Merck) using hexane as the mobile phase then scraped off and extracted in chloroform, concentrated, and analyzed on GC-FID and subsequent GC-MS as previously described (Domergue et al., 2010). For acyl-CoA composition analyses, one OD600 unit of transgenic yeasts was collected and used for acyl-CoA extraction and profiling as described (Larson and Graham, 2001).

Interaction Analyses

Split ubiquitin analysis was performed using the yeast two-hybrid system from DUALmembrane system (Dualsystems Biotech AG). The cDNA library was constructed following the manufacturer’s instructions (Evrogen) using 660 ng total RNA of epidermis peeled from Arabidopsis stems. The library had a complexity of 3.48 × 106 independent clones. For screening, 28 μg of the NubG-cDNA library was used for transformation of THY.AP4 yeast strain (MATa ura3 leu2 lexA-lacZ-TRP1 lexA-HIS3 lexA-ADE2) previously transformed with pBT3N:CER1. Yeast transformation efficiency was 1.25 × 105 clones/μg DNA, which indicates whole coverage of the library. Interactors were selected on −HTL medium supplemented with 0.8 mM 3-amino-1,2,4-triazole (Sigma-Aldrich) with further selection on −AHTL and were tested for β-galactosidase activity. For targeted analysis, CER1, CER3, and CYTB5s cDNA were amplified by PCR using SfiI restriction site–containing primers (see Supplemental Table 4 online) with subsequent orientated cloning of CER1 into pBT3N bait vector and CER1, CER3, and CYTB5s into pPR3N prey vector. THY.AP4 cells were cotransformed with pBT3N:CER1 and pPR3N:PREY or pPR3N empty vector as a negative control. Transformants were selected on −TL, and interactions were assayed on −HTL supplemented with 0.8 mM 3-amino-1,2,4-triazole and −AHTL. To measure β-galactosidase activity of lacZ with X-Gal, cells were grown on −TL and then covered with X-Gal–agarose buffer (0.5% agarose, 0.5 M phosphate buffer, pH 7.0, 0.002% X-Gal) and incubated at 37°C for 10 to 20 min. Split-luciferase analysis was performed as previously described (Van Leene et al., 2010). CER1, CER3, and CYTB5s open reading frames were cloned into the pDONR221 ENTRY vector by Gateway recombination cloning technology using primers detailed in Supplemental Table 4 online. DPL1 and SUR2 cloned in the pDONR221 were used as previously described (Marion et al., 2008). Open reading frames were subsequently transferred into appropriate destination vectors (Van Leene et al., 2010).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: CER1, At1g02205; CER3, At5g57800; Actin2, At1g49240; CYTB5-B, At2g32720; CYTB5-C, At2g46650; CYTB5-D, At5g48810; CYTB5-E, At5g53560; LACS1, At2g47240; ACT1, YFL039C; At-DPL1, At1g27980; At-SUR2B, At1g14290.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. FAME and Fatty Acyl-CoA Profiles of INVSur4# Yeast Cotransformed with Arabidopsis Transgene(s) CER1 and/or CER3 and/or CYTB5-B and/or LACS1 and/or Empty Vectors.

  • Supplemental Figure 2. Identification by GC-MS of the Different Alkanes Produced in the Transgenic INVSur4# Yeast Strain That Coexpressed Arabidopsis CER1, CER3, CYTB5-B, and LACS1.

  • Supplemental Figure 3. FAME Profiles of INVSur4# Cotransformed with CER3, CYTB5, and Wild-Type CER1 or CER1 His Mutants.

  • Supplemental Figure 4. Cuticular Wax Composition of Inflorescence Stems of Arabidopsis Col-0 (Wild-Type), cer1-1, cer1-1:CER1, cer1-1:CER1H146A, and cer1-1:CER1H248A Lines.

  • Supplemental Figure 5. Cuticular Wax Composition of Rosette Leaves of Arabidopsis Col-0 (Wild-Type), cer1-1, cer1-1:CER1, cer1-1:CER1H146A, and cer1-1:CER1H248A Lines.

  • Supplemental Figure 6. FAME Profiles of INVSur4# Yeast Cotransformed with CER1 and Wild-Type CER3 or CER3 His Mutants.

  • Supplemental Figure 7. Sequence Alignment of Arabidopsis CER1, CER3, and Cyanobacterial FAAR.

  • Supplemental Table 1. Inflorescence Stem Cuticular Wax Composition of Arabidopsis Col-0, cer1-1, cer1-1:CER1, cer1-1:CER1H146A, and cer1-1:CER1H248A.

  • Supplemental Table 2. Rosette Leaf Cuticular Wax Composition of Arabidopsis Col-0, cer1-1, cer1-1:CER1, cer1-1:CER1H146A, and cer1-1:CER1H248A.

  • Supplemental Table 3. Primers Used for Site-Directed Mutagenesis.

  • Supplemental Table 4. Yeast Expression Vectors and Primers Used for Cloning and PCR Analyses.

  • Supplemental Table 5. Transgenic Yeasts.

Supplementary Material

Supplemental Data
supp_24_7_3106__index.html (1.2KB, html)

Acknowledgments

We thank Owen Rowland of Carleton University, Canada, for vectors and stimulating discussions. We thank Su Melser of University of Bordeaux, France, for critical reviews of the article. We also thank Vladimir Denic of Harvard University for providing the p416 MET25-FLAG3:Sur4-F262A/K266L vector. Lipid analyses were carried out at Metabolome facility of Bordeaux (http://www.cgfb.u-bordeaux2.fr/en/metabolome). This study was funded by the Ministère de l’Enseignement Supérieur et de la Recherche (France) (doctoral fellowship for A.B.). This study received support from the Centre National de la Recherche Scientifique and the University Bordeaux Segalen. Rothamsted Research received grant-aided support from the Biotechnology and Biological Sciences Research Council. R.J. was supported by the Canada Research Chairs program, the Natural Sciences and Engineering Council, Canada, and the France-Canada Research Fund.

AUTHOR CONTRIBUTIONS

A.B., F.D., R.J., R.L., and J.J. designed research; A.B., F.D., S.P., and J.J. performed research; C.R., J.-D.F., R.P.H., and J.A.N. contributed analytic tools; A.B., F.D., and J.J. analyzed data; A.B., F.D., R.J., and J.J. wrote the article.

Glossary

VLC

very long chain

VLCFA

very-long-chain fatty acid

AD

aldehyde decarbonylase

ER

endoplasmic reticulum

SUY2H

split-ubiquitin yeast two-hybrid

GC-MS

gas chromatography–mass spectrometry

FAME

fatty acid methyl ester

TLC

thin layer chromatography

GC-FID

gas chromatography–flame ionization detection

FAAR

fatty acyl-ACP reductase

Col-0

ecotype Columbia

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