Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl-CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2-like family: CER2, CER26 and CER26-like . Expression pattern analysis showed that the three genes are differentially expressed in an organ- and tissue-specific manner. Using individual T–DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl-CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.
Terrestrial plants have developed a protective barrier, or cuticle, that controls the movement of water between the outer cell wall of the epidermis and the atmosphere adjacent to the plant (Shepherd and Wynne Griffiths, 2006). The cuticle is a rather thin membrane consisting of cutin, polysaccharides and associated solvent-soluble lipids, called cuticular waxes. Cutin is the main structural component of the cuticular matrix. It consists of a three-dimensional polymer of mostly C16 and C18 hydroxy fatty acids cross-linked by ester bonds (Beisson et al., 2012). Cuticular waxes are a complex mixture of very long chain (VLC) aliphatic lipids, triterpenoids and minor secondary metabolites, such as sterols and flavonoids (Buschhaus and Jetter, 2011). The physical and chemical properties of cuticular waxes determine vital functions for plants (Dominguez et al., 2011). Indeed, besides playing a major role in limiting uncontrolled water loss (Kosma et al., 2009), waxes protect plants against ultraviolet radiation and help to minimize deposits of dust, pollen and air pollutants (Kunst and Samuels, 2003). In addition, surface wax is believed to play important roles in plant defense against bacterial and fungal pathogens (Raffaele et al., 2009), and has been shown to participate in a variety of plant–insect interactions (Eigenbrode et al., 2000).
Wax aliphatic compounds are produced in epidermal cells via two different pathways: the alcohol-forming pathway, which produces VLC alcohols and esters (Rowland et al., 2006; Li et al., 2008), and the alkane-forming pathway leading to the formation of VLC alkanes and their derivatives (Greer et al., 2007; Bernard et al., 2012). The two metabolic routes share a pool of precursors consisting of VLC fatty acids (VLCFAs), with predominant chain lengths ranging from 26 to 34 carbons. VLCFAs result from the endoplasmic reticulum (ER)-associated acyl-CoA elongase activity, which is carried out by multi-enzymatic fatty acid elongase (FAE) complexes (von Wettstein-Knowles, 1982). Each elongation cycle catalyzes four successive reactions, generating an acyl chain extended by two carbons. The first step of elongation, which consists of the condensation of an acyl-CoA with a malonyl-CoA, is catalyzed by 3–ketoacyl-CoA synthase (KCS). The resulting 3–ketoacyl-CoA is then reduced by 3–ketoacyl-CoA reductase (KCR) into 3–hydroxyacyl-CoA, which is subsequently dehydrated by 3–hydroxacyl-CoA dehydratase (HCD) to form a trans-2,3-enoyl-CoA. In the final elongation step, the trans-2,3-enoyl-CoA is reduced by the trans-2,3-enoyl-CoA reductase (ECR), which yields an acyl-CoA elongated by two carbons. In plants, evidence suggests that multiple elongases with distinct chain-length specificity are likely to exist, producing the broad range in chain lengths of VLCFAs (von Wettstein-Knowles, 1982). The KCR, HCD and ECR enzymes are thought to have broad substrate specificity and to be shared by all FAE complexes (Kunst and Samuels, 2009). In contrast, KCS enzymes have been proposed to determine the chain length substrate specificity of each elongation reaction. Consistently, 21 putative KCS have been annotated in the Arabidopsis genome (Joubès et al., 2008), but to date only a few of them have been characterized in depth. In particular, although wax component chain lengths vary from C22 to C34, suggesting that VLCFA precursors would result from various elongase complexes, only four KCS (FDH, KCS1, HIC and CER6) with a potential role in wax precursor synthesis have been identified and analyzed in detail (Yephremov et al., 1999; Todd et al., 1999; Millar et al., 1999; Pruitt et al., 2000; Gray et al., 2000). Notably, based on expression patterns, mutant phenotypes and heterologous expression in yeast, CER6 was shown to have a major role in the production of fatty acids with 26 and 28 carbons (Millar et al., 1999; Fiebig et al., 2000; Tresch et al., 2012). Similar to the cer6 mutant, the Arabidopsis cer2 mutant shows a defect in all classes of wax compounds causing a 40% reduction of the total quantity of stem waxes compared with wild-type plants (Hannoufa et al., 1993). All components with more than 28 carbons were found to be depleted in the cer2 mutant, whereas compounds with 28 or less carbons were consistently over-accumulated, suggesting a block in the elongation of C28 VLCFAs. Based on these observations it has been proposed that CER2 encodes a component of the elongase (McNevin et al., 1993). Nevertheless, CER2 does not share sequence similarities with any of the elongase enzymes, suggesting that CER2 could instead function as a regulator of the elongase system specific for the last elongation steps (Jenks et al., 1995). CER2 was previously reported as being localized in the nucleus based on immunodetection using subcellular fractions, which would be consistent with a regulatory role (Xia et al., 1997); however, latterly it has been shown that CER2 is localized in the ER of young stem epidermal cells, i.e. at the site of VLCFA biosynthesis, thereby supporting the hypothesis of its role as a component of the elongase complex (Haslam et al., 2012). Finally, evidence has been provided for the role of CER2 in C28 elongation, as the co-expression of CER2 with CER6 in yeast resulted in the production of C30 fatty acids (Haslam et al., 2012).
Although CER2 is expressed in all aerial organs in Arabidopsis, the cer2 mutation does not affect leaf wax composition (Jenks et al., 1995; Negruk et al., 1996; Xia et al., 1996), suggesting that other proteins could be involved in VLCFA elongation in leaves. CER2 is a member of the BAHD superfamily of acyl-CoA-dependent acyltransferases, and clusters in group V with two other proteins, At4g13840 (herein called CER26, which shows 32.2% identity with CER2) and its close homologue At3g23840 (herein called CER26-like, which shows 35.7% identity with CER2 and 64% identity with CER26; Figure S1; Yu et al., 2009). In order to better understand the elongation process leading to the formation of VLC wax precursors in leaves, the CER2-like family members were characterized here. A detailed molecular analysis showed that CER2 and CER26 have overlapping expression patterns in leaves, whereas CER26 expression is excluded from the stem. Insertional mutants for each of the three genes and CER26 overexpressing lines were identified and characterized at the biochemical level, showing that CER26 is involved in the elongation of VLCFAs longer than 30 carbons, and has overlapping function with CER2 in leaves. Altogether, our results demonstrate that CER26, like CER2, is involved in the formation of VLC fatty acids, and support its role as an element of the fatty acid elongation machinery for wax synthesis.
Expression profiling in Arabidopsis organs of the CER2-like genes
The organ distribution of CER2, CER26 and CER26-like was determined by real-time PCR in seedlings, roots, stems, cauline and rosette leaves, flowers and siliques (Figure 1a). The three genes showed a typical wax-associated expression pattern, as they were found expressed in aerial vegetative and reproductive organs, but not in roots. CER2 was expressed in all aerial organs, and it was notably the only gene of the cluster expressed in stems. Indeed, compared with CER2, CER26 was found highly expressed in leaves, but no transcripts were detected in stems. Furthermore, CER26 was weakly expressed in flowers and siliques. CER26-like was mainly expressed in flowers and expressed at very low levels in all other tissues. These results are in agreement with microarray data available in the Genevestigator gene expression database (http://www.genevestigator.ethz.ch).
To investigate the cell-type expression pattern of the three genes, we generated transgenic Arabidopsis lines expressing the β–glucuronidase (GUS) reporter gene under the control of each of the three promoter sequences (Figure 1b). Several homozygous reporter lines showed a similar expression pattern, which was consistent with real-time reverse transcription (RT)-mediated PCR analysis. In young leaves, CER26 expression was detected in epidermal cells and trichomes (Figure 1b). Under the control of the CER2 promoter, GUS expression was detected in leaf epidermal cells and the signal was also specifically localized in the guard cells (Figure 1b). CER2 expression was detected specifically in the epidermis of the elongated floral stems (Figure 1b). Both CER26 and CER2 promoter activities were detected in siliques, where the expression of CER26 was found mostly expressed in the pod dehiscence zones, whereas CER2 was localized in the pod epidermis (Figure 1b). In young flowers, CER2 promoter activity was detected in the pistil, the petals and the sepals (Figure 1b). CER26-like promoter activity was only detected in stamens at early stages of flower development (Figure 1b). These results show that CER2, CER26 and CER26-like are differentially expressed at the tissue and organ level throughout plant development, suggesting that they could perform different or complementary functions in wax biosynthesis.
Molecular identification of Arabidopsis mutants
To understand the role of the CER2-like proteins in the production of cuticular waxes, we selected the cer26 allele (SALK_087857 in the Col–0 ecotype), in which the CER26 gene was disrupted in the intronic region (at nucleotide 480 relative to the start codon) (Figure 2a). We also identified a cer26-like mutant (SAIL_3_F11 in the Col–0 ecotype), which showed a T–DNA insertion in the first exon of the gene (at nucleotide 200 relative to the start codon). As previously reported, cer2 mutants were in the genetic background Ler–0 (Koornneef et al., 1989) or WS (McNevin et al., 1993), and we selected the insertional cer2 mutant (SALK_0844439) in the Arabidopsis ecotype Col–0 for comparison with cer26 and cer26-like mutants (Figure 2a). The T–DNA insertion in cer2 disrupted the second exon of the CER2 gene (at nucleotide 814 relative to the start codon). The T–DNA insertions were confirmed by PCR on genomic DNA (Figure 2b). Given that CER2 and CER26 have overlapping expression pattern in leaves, we hypothesized that the absence of any wax phenotype in leaves of the cer2 mutant could result from a potential compensatory function of CER26. To test this hypothesis we further generated a cer2 cer26 double mutant by crossing the cer2 and cer26 lines. Gene expression was analyzed by semi-quantitative RT-PCR in leaves and stems of the cer2, cer26 and cer2 cer26 mutants, and in flower transcripts for the cer26-like line (Figure 2c). No corresponding full-length transcripts were detected in the different alleles. This observation was confirmed by qRT–PCR in vegetative or reproductive organs of the different single mutants and the cer2 cer26 double mutant (Figure 2d). It is worth noting that the absence of expression of CER2, CER26, CER26-like or both CER2 and CER26 did not lead to compensatory overexpression of the remaining members of the gene family in the single or double mutants (Figure 2d).
The cer2 and cer2 cer26 mutants only show glossy stems
To define the growth characteristics of the different lines, the plants were cultivated in the automated phenotyping platform PHENOPSIS, which was designed for the strict control of environmental conditions (Granier et al., 2006). Wild-type (Col–0), cer2, cer26 and cer2 cer26 growth was phenotyped by measuring several morphological and anatomical traits in optimal plant growth conditions (Table S1). When compared with the wild-type line, the cer2, cer26 and cer2 cer26 mutants did not show significantly different leaf growth variables, i.e. rosette leaf number, rosette leaf area, and the area and thickness of leaf 6. Moreover, fresh and dry weights of rosettes at bolting were not significantly modified compared with those of wild-type rosettes. A detailed analysis of epidermal cells on the adaxial side of leaf 6 in cer2, cer26 and cer2 cer26 mutants, compared with wild-type plants, did not reveal any differences. The cer2, cer26 and cer2 cer26 lines were similar in all aspects to wild-type plants (Figure 3a), except for the bright-green stem phenotype exhibited by the cer2 and cer2 cer26 mutants (Figure 3b). This phenotype, typically associated with wax-deficient mutants, indicates qualitative and/or quantitative modifications in the production of cuticular waxes on the stem surface. These observations were confirmed by scanning electron microscopy analysis of young stem surfaces of wild-type, cer2, cer26 and cer2 cer26 lines (Figure 3c). The CER2 inactivation in cer2 and cer2 cer26 lines led to the complete disappearance of the wax crystals formed on the stem surface, whereas the stem surface of the cer26 line was similar to that of wild-type plants.
Arabidopsis cer2 and cer26 mutants show altered cuticular wax composition
In order to assess the deregulating effect of CER2, CER26 and CER26-like expression on cuticular waxes, the cuticular wax composition of stems and leaves of the different lines: Col–0 (wild type, WT), cer2, cer26, cer26-like and cer2 cer26 was analyzed in detail (Figures 4 and S2; Tables 1 and 2).
Table 1. Cuticular wax composition of inflorescence stems of Arabidopsis Col–0 (WT), cer2, cer26, cer2 cer26 and cer26R
Mean values (μg dm−2) of total wax loads and coverage of individual compound classes are given with SDs (n = 4). The sums include shorter chain length constituents not presented in Figures 4 and 5.
1 907.7 ± 82.4
815.6 ± 37.3
177.7 ± 7.9
421.9 ± 6.7
246.7 ± 7.6
10.9 ± 0.8
100.0 ± 30.3
20.4 ± 2.5
114.4 ± 4.9
1 182.8 ± 53.1
122.3 ± 11.9
15.2 ± 0.7
25.0 ± 4.0
583.6 ± 67.5
38.3 ± 6.2
154.0 ± 20.7
80.3 ± 10.0
164.1 ± 22.6
1 811.4 ± 53.1
776.9 ± 65.0
162.2 ± 11.5
391.6 ± 15.9
226.9 ± 15.4
14.4 ± 1.2
92.8 ± 11.9
30.0 ± 2.4
116.7 ± 10.1
1 108.9 ± 98.7
124.4 ± 20.5
21.8 ± 8.4
18.4 ± 9.5
546.4 ± 71.4
31.1 ± 2.9
124.5 ± 12.0
47.2 ± 6.7
195.2 ± 19.4
1 686.3 ± 112.4
770.5 ± 69.5
135.3 ± 14.0
313.8 ± 9.7
228.4 ± 16.7
14.2 ± 4.7
41.5 ± 13.4
29.1 ± 6.0
153.4 ± 8.3
Table 2. Cuticular wax composition of rosette leaves of Arabidopsis Col–0 (WT), cer2, cer26, cer2 cer26 and cer26R
Mean values (μg dm−2) of total wax loads and coverage of individual compound classes are given with SDs (n = 4). The sums include shorter chain length constituents not presented in Figures 4 and 5.
162.3 ± 4.7
93.1 ± 3.5
3.2 ± 0.3
14.7 ± 1.1
9.9 ± 0.9
23.2 ± 2.0
16.1 ± 0.8
2.1 ± 0.2
172.3 ± 5.5
97.0 ± 4.2
3.5 ± 0.2
16.2 ± 2.0
9.9 ± 0.8
25.8 ± 1.6
16.2 ± 0.8
3.7 ± 0.5
162.1 ± 6.6
83.8 ± 5.4
2.8 ± 0.3
14.9 ± 1.7
10.9 ± 1.7
34.3 ± 3.3
13.9 ± 1.7
1.6 ± 0.1
92.6 ± 3.2
31.2 ± 1.4
2.7 ± 0.3
15.5 ± 0.8
9.4 ± 0.8
15.4 ± 1.2
16.5 ± 2.2
1.9 ± 0.2
167.8 ± 4.8
98.2 ± 3.9
2.4 ± 0.3
14.3 ± 0.9
9.1 ± 0.4
24.0 ± 0.9
17.3 ± 1.7
2.7 ± 0.1
In stems, the wax load of cer2 and cer2 cer26 was strongly reduced compared with wild-type plants (an average 38% decrease), whereas neither the wax load nor the wax composition of cer26 was significantly affected (Figure 4; Table 1). The decrease measured in cer2 and cer2 cer26 was largely the result of reduced levels in the three major components of stem waxes: the C29 alkane and its derivatives C29 2°–alcohol and C29 ketone. The CER2 inactivation further affected all wax components according to a specific carbon chain length. Indeed, the level of components with more than 28 carbons was dramatically decreased (C31 alkane, C30 1°–alcohol, C30 aldehyde), whereas the level of components with 28 or less carbons was increased (C27 alkane, C27 2°–alcohol, C26 and C28 1°–alcohols, C26 and C28 aldehydes, C26 and C28 VLCFAs), together with the level of C42 and C44 esters, which contain C26 and C28 esterified alcohols. In accordance with previous studies of cer2 mutants (Jenks et al., 1995; Negruk et al., 1996; Xia et al., 1996; Haslam et al., 2012), these results suggest that a defective CER2 blocks the formation of wax compounds with more than 28 carbons in stems.
In leaves, the wax load of the single cer2 and cer26 mutants was almost not affected when compared with wild-type plants (Table 2), whereas the wax load of the double cer2 cer26 mutant was considerably reduced (43% decreased). Whereas the wax profile of cer2 was comparable with that of the wild-type plants, the wax composition of cer26 was substantially modified (Figure 4; Table 1). The level of components with 30 or less carbons was increased (C29 alkane, C30 aldehyde, C30 VLCFA and C30 1°–alcohol), whereas the level of components with more than 30 carbons was decreased (C31, C33 and C35 alkanes, C32 aldehyde, and C32 1°–alcohol), suggesting a block in the synthesis of wax components with more than 30 carbon atoms. Because of this modified wax profile, the mutant can be associated with the large family of eceriferum (cer) mutants that are characterized by a modification of the quantity and/or quality of epicuticular waxes onto stems or leaves. To date, 25 eceriferum mutants have been described (Rashotte et al., 2004; Goodwin et al., 2004), and therefore we named this new mutant cer26.
The double cer2 cer26 mutant showed a novel wax phenotype. The level of components with more than 28 carbons was largely reduced (C29, C31, C33 and C35 alkanes, C30 VLCFAs, C30 and C32 1°–alcohols, C30 and C32 aldehydes), whereas the level of components with 28 or less carbon atoms was increased (C27 alkane, C28 1°–alcohol, C28 aldehyde, C28 VLCFA). These results demonstrate a role for CER2 in the formation of wax compounds with more than 28 carbons, and suggest that CER26 could compensate for the loss of CER2 activity in cer2 leaves.
The inactivation of CER26-like did not lead to any modification in the wax composition of stems, leaves or even flowers in which the gene is specifically expressed (Figure S2). Therefore, the CER26-like gene was not further characterized.
Ectopic expression of CER26 in the stem altered cuticular wax organization
To further understand CER26 function in wax metabolism, the cer26 allele was complemented with a construct harboring the CER26 gene under the control of Pro35S and, among several obtained lines that showed a similar phenotype, one line was selected for deeper analysis (cer26R). T–DNA insertions were confirmed by PCR on genomic DNA (Figure 5a), and gene expression was analyzed by qRT-PCR in leaves (Figure 5b). No transcripts were detected in cer26 compared with wild-type plants, whereas the rescued cer26 line exhibited a CER26 transcript level almost similar to that of wild-type plants.
The cuticular wax composition was determined in the stems and leaves of the different lines: Col–0 (WT), cer26 and cer26R (Figure 5c; Tables 1 and 2). CER26 expression in the cer26 background rescued the correct wax biosynthesis in leaves (Figure 5c; Table 2). Indeed, compared with the cer26 mutant, the level of all compounds with 30 or less carbons (C29 alkane, C30 aldehyde and C30 1°–alcohol) was reduced, whereas the level of the components with more than 30 carbons was increased (C31, C33 and C35 alkanes, C32 aldehyde, and C32 1°–alcohol), thus confirming the role of CER26 in the elongation of VLCFAs longer than 30 carbons. CER26 ectopic expression in stems reduced the wax load compared with wild-type plants (12% decreases; Table 1). This decrease was mainly caused by a slight reduction of the three major components of the stem waxes: C29 alkane, C29 2°–alcohol and C29 ketone (Figure 5c). Strikingly, the stems of the CER26R line showed a large increase of the C31 and C33 alkane levels compared with wild-type plants (increases of 1883 and 404%, respectively). This phenotype shows that ectopic expression of CER26 in stems leads to the production of components with more than 30 carbons, and therefore supports the hypothesis that CER26 could have a role in the elongation process of VLCFA with 30 or more carbon atoms.
Surprisingly, the cer26R line displays a bright-green stem phenotype (Figure 5d), which is usually associated with a depletion of wax crystals in cer mutants. In fact, scanning electron microscopy analyses showed that ectopic expression of CER26 in stems induced the formation of an amorphous wax layer instead of the typical crystalline organization. As the total wax level of the cer26R line is only slightly reduced compared with the WT, we hypothesize that the change in the structure of waxes would result from the qualitative modification of the wax composition.
cer2 and cer26 mutations specifically affect VLCFA-containing lipids
In plant cells, VLCFAs are used as precursors by multiple biochemical pathways leading to the formation of a wide variety of lipid molecules, such as phospholipids, sphingolipids, storage lipids, cutin, waxes and suberin. To further determine the role of CER2 and CER26 in VLCFA elongation, a global analysis of the VLCFA-containing lipids was performed in WT, cer2, cer26 and cer2 cer26 lines. The cutin polyester of stems and leaves from the different lines was extracted and depolymerized prior to the quantitative and qualitative analysis of cutin momoners. The different lines showed a similar cutin composition in both stem and leaf (Figure S3). Similarly, neither the suberin composition nor the seed lipid profile were found to be significantly affected in the different transgenic lines compared with the WT (Figures S4 and S5). Furthermore, the effect of cer2 and cer26 deregulation on the intracellular fatty acyl chain content was examined after the removal of the cuticular waxes of stems and leaves of the different lines. No modification of the general cellular fatty acyl chain profile was observed (Figure S6), except for slight modifications of the VLCFAs with chain length longer than 26 carbon atoms, which were in accordance with the changes observed in stem and leaf wax composition of the different transgenic lines.
Arabidopsis cer2 and cer26 mutants show altered acyl-CoA composition
The phenotype of cer2, cer26 and cer2 cer26 shows that the two proteins participate in the formation of VLC wax compounds, suggesting that they have a role in the terminal steps of fatty acid elongation. To test this hypothesis, the acyl-CoA pool of young stems and leaves of cer2, cer26 and cer2 cer26 lines was compared with the wild type (Figure 6). These analyses showed significant modifications of the acyl-CoA profiles, which were consistent with the changes observed in the wax profiles. Indeed, in stems both cer2 and cer2 cer26 acyl-CoA pools were affected (Figure 6a): the level of C26 acyl-CoA was largely increased, whereas C30 acyl-CoA was almost completely absent. Surprisingly, the level of C28 acyl-CoA was only slightly increased in the two lines compared with the WT. The acyl-CoA profile of cer26 stems was not modified compared with the wild-type profile. In cer2 rosette leaves, the acyl-CoA pool composition was not affected (Figure 6b). In rosette leaves of the cer26 mutant, the profile showed a large increase of C30 acyl-CoA. The cer2 cer26 double mutant showed a different profile, with a large increase of C28 acyl-CoA, accompanied by a slight reduction of C30 acyl-CoA. Collectively, these results demonstrate that CER2 and CER26 inactivation affects the VLCFA elongation process at specific stages.
CER26 encodes a novel protein involved in wax biosynthesis
As expected for cuticle-associated genes, CER2, CER26 and CER26-like expression was found to be restricted to the aerial part of plants. However, the three genes are differentially expressed at the tissue and organ level throughout plant development. CER2 and CER26 are highly expressed in leaves, but only CER2 transcripts were detected in stems. A defect in CER2 leads to a specific block in the synthesis of wax compounds with more than 28 carbons in stems. In common with cer2, the deregulation of CER26 affects all classes of wax compounds with a specific carbon chain length pattern, suggesting that CER26 acts on the synthesis of compounds with 30 or more carbons. In accordance with the CER26 expression pattern, this phenotype was observed in leaves, but not in stems. The involvement of CER26 in the synthesis of wax compounds with more than 30 carbons was further confirmed by the restoration of correct wax biosynthesis in plants overexpressing CER26 in the cer26 mutant background. Furthermore, ectopic expression of CER26 in stems shifts the stem wax composition to a leaf wax composition, further demonstrating that CER26 is implicated in the formation of wax compounds with more than C30 carbons. The cer26R line displays a shiny stem phenotype because of the disappearance of the typical crystalline microstructure normally found at the stem surface (Jenks et al., 2002). Little is known about the specific contribution of each of the wax compounds in the formation of wax crystals (Shepherd and Wynne Griffiths, 2006); however, the results presented in this study suggest that a tight balance in the alkane chain length is required for the correct microstructure of waxes.
As mentioned above, our results showed that the cer2 mutation does not impact wax biosynthesis in leaves, even though CER2 is expressed in all vegetative organs. Because CER2 and CER26 are both expressed in leaves, we hypothesized that CER26 could compensate for the loss of CER2 function in that tissue. Analyses of the cer2 cer26 mutant confirmed this hypothesis. The double cer2 cer26 mutant showed a leaf wax profile different from those of the single cer2 or cer26 mutants, indicating that CER2 and CER26 have overlapping functions in the synthesis of wax compounds with more than 28 carbons in leaves.
CER26 and CER2 inactivation specifically affects VLCFA-containing lipids
As the cer2 and cer26 phenotypes suggested that the corresponding proteins are involved in VLCFA elongation, the impact of a defect of CER2 and CER26 was also investigated in all other classes of VLFCA-containing lipids, i.e. cutin, suberin, TAGs and total intracellular fatty acyl chains. In all the lipid pools analyzed, no modification was observed on fatty acyl chains shorter than 26 carbons. In plants cells, VLCFAs with more than 26 carbons can be detected as trace levels in cutin; however, no significant modification of the composition of cutin was observed. VLCFAs can also be detected as trace levels in the total fatty acyl pool. Interestingly, the inactivation of CER26 and/or CER2 led to slight modifications of the total fatty acyl profile, in accordance with what was observed in waxes, indicating that CER2 and CER26 act specifically on the elongation of VLCFAs with more than 26 carbons.
CER26 and CER2 are critical components of the VLCFA elongation process
The global lipid analyses of the cer2, cer26 and cer2 cer26 mutants performed in our study indicated that CER2 and CER26 are likely to be involved in the formation of fatty acids with a specific chain-length pattern. Subsequent analysis of the acyl-CoA pools in the different mutant lines showed a pattern consistent with that observed in both waxes and intracellular fatty acyl chain composition. As observed in waxes, the block of C28 acyl-CoA elongation in the cer2 mutant is illustrated by a near disappearance of acyl-CoAs longer than 30 carbons, yet the C28 species are not over-accumulated. Conversely, the level of C26 species is largely increased, suggesting that CER2 could also have an impact on the elongation of C26 acyl-CoA to C28 acyl-CoA. Similarly, CER26, which has a clear role in the elongation of C30 acyl-CoA, is also likely to have a role in the elongation of C32 acyl-CoA because compounds with more than 32 carbons were found to be over-accumulated in stems ectopically expressing CER26. Unfortunately, this hypothesis could not be confirmed given the technical limitations of the current methods for acyl-CoA analysis, preventing the accurate detection of species longer than C30.
Towards the functional characterization of CER26
Based on the cer2 phenotype, a role of CER2 as a stem-specific element of the VLCFA elongase was proposed (Jenks et al., 1995; Negruk et al., 1996; Xia et al., 1996). We showed that CER2, together with the newly identified CER26 protein, is also involved in the elongation of VLCFAs in leaves. Nevertheless, the function of both proteins remains elusive. The combined analysis of wax and acyl-CoA pools in the different mutants showed that cer2 and cer26 have phenotypes similar to the KCS mutants. It would therefore be appealing to suggest that both CER2 and CER26 could encode condensing enzymes specific for the elongation of VLCFAs involved in wax biosynthesis. Nevertheless, the CER2-like proteins do not share sequence similarities with the Arabidopsis FAE1-like type of KCSs or the ELO-type of KCSs found in both Arabidopsis and yeast (Yu et al., 2009). Therefore, it is highly unlikely that CER2 and CER26 act as condensing enzymes. Alternatively, CER2 and CER26 could act as regulators of the activity of the elongase complexes generating VLCFAs. As such, they could facilitate the formation of VLC acyl-CoAs by stabilizing the FAE complexes, enhancing their activity or allowing the newly elongated acyl-CoA to be presented back to the KCS enzyme after an elongation cycle. These hypotheses suggest that CER2 and CER26 would be associated with ER membranes. However, CER2-like proteins are members of the BAHD superfamily of acyl-CoA-dependent acyltransferases, which are predicted to be soluble cytosolic enzymes (Yu et al., 2009), of which several have been experimentally shown to localize into the cytosol (Panikashvili et al., 2009; Rautengarten et al., 2012). Analyses of the primary sequences of the CER2-like proteins using prediction tools did not reveal any transmembrane domains, specific targeting signals or post-translational lipid modifications. CER2 was previously localized exclusively in the nucleus, based on immunodetection using subcellular fractions, which would be consistent with the early hypothesis of its regulatory role in the elongation process (Xia et al., 1997), but this does not fit with CER2 annotation as a cytosolic protein. GLOSSY2, the ortholog of CER2 from Zea mays (maize), was immunodetected in the cytoplasm fraction, and in a membrane fraction enriched with endoplasmic reticulum (ER) and mitochondria, suggesting that CER2 may be a cytosolic protein recruited to the ER for its activity (Velasco et al., 2002). This study is supported by the work of Haslam and co-workers who have shown, using a cer2 mutant functionally complemented with a CER2-GFP fusion protein, that CER2 is localized to ER membranes in young epidermal cells at the apex of Arabidopsis stems (Haslam et al., 2012). Similarly, we generated a cer26 mutant functionally complemented with a YFP-CER26 fusion protein. Unfortunately, although transgenic plants with a normal wax phenotype were obtained and the fusion protein was detected in leaf extract by western blot using an antibody against the YFP, we were not able to localize the protein elsewhere other than in the cytosol of epidermal cells (Figure S7). We hypothesize that, because the CER2 association with the ER was only observed in the epidermal cells of the stem apex (the active site of wax synthesis components), the targeting of CER2-like proteins to ER membranes potentially requires interaction with microsomal proteins involved in VLCFA elongation. As the production of waxes in leaves is 10 times less important than in stems, the level of proteins involved in the elongation of C30 and C32 VLCFAs, which are potential targets of CER26, could be not sufficient to allow the targeting of the overexpressed CER26 protein to ER membranes that might be visualized using confocal microscopy. In such a model the tight control of CER26 overexpression at the tissue and the cellular level would be necessary to allow the correct localization of the protein. Further investigation must be performed to better understand this mechanism, and protein–protein interaction analysis might provide conclusive evidence that the ER localization of CER26 requires interaction with wax-associated membrane proteins, and notably, members of the FAE complexes.
Little is known about the regulation of elongase complexes throughout kingdoms. To date, the Arabidopsis PAS1 protein is the only example of an FAE complex regulator (Roudier et al., 2010). Similar to a loss of one of the elongase core components, a defect in PAS1 was shown to reduce all VLCFAs in TAGs and sphingolipids. PAS1 is a member of the immunophilin family of chaperones that are known to target protein complexes and regulate their assembly or activity. Although the phenotype of cer2 and cer26 shows some similarities with the phenotype of pas1, it is not likely that the proteins perform the same function. Indeed, the primary protein structure of CER2 and CER26 does not share similarities with PAS1. Moreover, the cer2 and cer26 mutants are only affected in VCLFAs with more than 26 carbons, showing that unlike PAS1, CER2 and CER26 are not involved in the regulation of all FAE complexes.
CER2 and CER26, together with CER26-like, have been annotated as BAHD acyltransferases (Yu et al., 2009), which are characterized by the presence of a conserved catalytic HXXXDG motif near the center position of the enzymes, and a second highly conserved structural C–terminal DFGWG motif. CER2 and CER26 proteins perform similar functions in the elongation process. Nevertheless, the HXXXDG motif was found to be conserved in CER2, but not in CER26, which contains a divergent NXXXDG motif, and neither CER26 nor CER2 contain the DFGWG motif, thus strongly questioning their putative function as acyltransferases. This hypothesis was recently confirmed by functional complementation of the cer2 mutant phenotype with mutated forms of CER2, in which the histidine residue of the HXXXDG motif was replaced by an alanine or an asparagine, demonstrating that this motif is not essential for the activity of the protein, and suggesting that CER2-like proteins could have a distinct biochemical function (Haslam et al., 2012).
As mentioned above, even though wax biosynthesis has been extensively studied, no KCS enzymes involved in the elongation of acyl chains longer than 30 carbons have been identified so far. In particular, yeast heterologous expression of CER6 was found to produce only C28 fatty acids (Haslam et al., 2012; Tresch et al., 2012), whereas expression of its close homologue CER60 led to the formation of C28 and C30 fatty acids (Trenkamp et al., 2004). This indicates that CER2 and CER26 are not strictly required for the activity of plant KCSs. This idea is further supported by the fact that the production of VLCFAs is not totally abolished in the cer2 cer26 double mutant. Nevertheless, the expression of CER60 produces only traces of C30, suggesting that the activity of this enzyme is not optimal in these conditions (Trenkamp et al., 2004). Therefore, we hypothesize that the function of CER2 and CER26 is to facilitate the activity of the KCSs specific for VLCFA formation. This hypothesis is supported by recent findings that show that co-expression of CER6 and CER2 in yeast led to the formation of C30 fatty acids (Haslam et al., 2012). In this context, the systematic co-expression of CER26 with plant KCSs in yeast should allow the identification of the KCS(s) enzyme(s) involved in the synthesis of VLCFAs with more than 30 carbons used as wax precursors.
Plant material and growth conditions
Arabidopsis thaliana (L.) Heynh (ecotype Columbia–0) was used in all experiments. The following T–DNA insertion lines were obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org): SALK_084443 (cer2), SAIL_3_F11 (cer26-like) and SALK_087857 (cer26). Arabidopsis plants were grown under controlled conditions, as previously described (Joubès et al., 2008). Measurements of leaf growth variables in control and soil water deficit conditions were performed with the PHENOPSIS automated platform, as previously described (Bourdenx et al., 2011).
DNA, RNA, cDNA preparation, real-time RT-PCR conditions and analysis
Genomic DNA was extracted from Arabidopsis leaves with the DNeasy Plant kit (Qiagen, http://www.qiagen.com) and RNA from Arabidopsis tissues with the RNeasy Plant mini kit (Qiagen). Purified RNA was treated with DNase I using the DNA-free kit (Ambion, now Invitrogen, http://www.invitrogen.com). First-strand cDNA was prepared from 1 μg of total RNA with the Superscript RT II kit (Invitrogen) and oligo(dT)18, according to the manufacturer's instructions. A 0.2–μl aliquot of the total reaction volume (20 μl) was used as a template in real-time RT-PCR amplification. The PCR amplification was performed with the gene-specific primers listed in Table S2. PCR efficiency ranged from 95 to 105%. All samples were assayed in triplicate wells. Real-time PCR was performed on iCycler™ (Bio-Rad, http://www.bio-rad.com). Samples were amplified in a 25–μl reaction containing 1x SYBR Green Master Mix (Bio-Rad) and 300 nM of each primer. The thermal profile consisted of one cycle at 95°C for 3 min and 30 s, followed by 40 cycles at 95°C for 30 s and at 58°C for 30 s. For each run, data acquisition and analysis was performed using iCycler™iQ 3.0 a (Bio-Rad). The transcript abundance in samples was determined using a comparative cycle threshold (Ct) method. The relative abundance of ACT2, EF–1α, eIF-4A–1, UBQ10 and PP2A mRNAs (Czechowski et al., 2005) in each sample was determined and used to normalize for differences of total RNA level according to the method described by Vandesompele et al. (2002). Semiquantitative RT-PCR analysis and PCR on genomic DNA were performed using Phire™ Hot Start DNA Polymerase (Finnzymes, http://diagnostics.finnzymes.fi).
Cloning and transgenic plants
Promoter sequences and open reading frames (ORFs) were amplified from Arabidopsis genomic DNA and from cDNA, respectively, using the primers listed in Table S2. The corresponding PCR fragments were cloned into the pDONR™221 ENTRY vector by GATEWAY® recombinational cloning technology, and subsequently transferred into the pKGWFS7, pK7WG2D and pH7WGY2 DESTINATION vectors (Karimi et al., 2002) by LR cloning. Constructs transferred into the Agrobacterium tumefaciens C58C1RifR strain harboring the plasmid pMP90 were used to generate stably transformed Arabidopsis plants using the floral-dip transformation method (Clough and Bent, 1998). Histochemical GUS analyses were performed as described by Joubès et al. (2008).
Cuticular wax analysis
Epicuticular waxes were extracted from leaves and stems by immersing tissues for 30 s in chloroform containing docosane as the internal standard. Extracts were derivatized and analyzed as described by Bourdenx et al. (2011). For epicuticular wax crystal formation on Arabidopsis stem surfaces, segments from the apical part of the stem were mounted onto stubs and viewed with an FEI Quanta 200 microscope used in environmental scanning electron microscopy (ESEM) mode.
Analysis of Acyl-CoA composition
Leaf and stem tissues from 5–week-old plants were collected and used for acyl-CoA extraction, as described by Larson and Graham (2001). Profiling was performed as described by Haynes et al. (2008).
Analysis of lipid polyester composition (cutin and suberin)
Fresh leaf and stem tissues and roots from 5–week-old plants were collected and immersed in hot isopropanol for 10 min at 80°C. After cooling, samples were extensively delipidated by extracting the soluble lipids, then dried and depolymerized as described by Domergue et al. (2010). Extraction, derivatization and analysis were performed as described by Domergue et al. (2010).
Analysis of total fatty acid composition
Total fatty acid from stems and leaves were transesterified overnight and analyzed as described by Mongrand et al. (1997).
Analysis of seed triacylglycerol composition
Seed triacylglycerol composition analysis was performed as described by Katavic et al. (1995).
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: CER2, At4g24510; CER26, At4g13840; CER26-like, At3g23840; CER1, At1g02205; Actin2, At1g49240; eIF4A-1, At3g13920; EF-1a, At5g60390; UBQ10, At4g05320; PP2A, At1g13320.
This work was supported by the Ministère de l'Enseignement Supérieur et de la Recherche (doctoral fellowship for AB), by the Centre National de la Recherche Scientifique and the University Bordeaux Segalen, and by the Biotechnology and Biological Sciences Research Council (grant support to Rothamsted Research). We thank the Salk Institute for Genomic Analysis Laboratory and the Arabidopsis Biological Resource Center for providing the sequence-indexed Arabidopsis T–DNA insertion lines. Scanning electron microscopy and confocal microscopy analyses were carried out at Bordeaux Imaging Center (http://www.bic.u-bordeaux2.fr). Lipid analyses were carried out at Metabolome facility of Bordeaux (http://www.cgfb.u-bordeaux2.fr/en/metabolome).