The Cer-cqu gene cluster determines three key players in a β-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes

Abstract

Aliphatic compounds on plant surfaces, called epicuticular waxes, are the first line of defense against pathogens and pests, contribute to reducing water loss and determine other important phenotypes. Aliphatics can form crystals affecting light refraction, resulting in a color change and allowing identification of mutants in their synthesis or transport. The present study discloses three such Eceriferum (cer) genes in barley - Cer-c, Cer-q and Cer-u - known to be tightly linked and functioning in a biochemical pathway forming dominating amounts of β-diketone and hydroxy-β-diketones plus some esterified alkan-2-ols. These aliphatics are present in many Triticeae as well as dicotyledons such as Eucalyptus and Dianthus. Recently developed genomic resources and mapping populations in barley defined these genes to a small region on chromosome arm 2HS. Exploiting Cer-c and -u potential functions pinpointed five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase and Cer-u is a P450 enzyme. All were highly expressed in pertinent leaf sheath tissue of wild type. A physical map revealed the order Cer-c, Cer-u, Cer-q with the flanking genes 101kb apart, confirming they are a gene cluster, Cer-cqu. Homology-based modeling suggests that many of the mutant alleles affect overall protein structure or specific active site residues. The rich diversity of identified mutations will facilitate future studies of three key enzymes involved in synthesis of plant apoplast waxes.

Keywords: Barley; Cer-cqu gene cluster; Hordeum vulgare; carboxylesterase; cytochrome P450; diketone synthase (DKS); epicuticular wax; esterified alkan-2-ols; hydroxy-β-diketones; lipase; plant apoplast; type III polyketide synthase (PKS).; β-diketone aliphatics.

Fig. 1.

Two elongation pathways. (A). The β-ketoacyl-CoA synthase (KCS) moiety of a fatty acid elongase (FAE) complex in the endoplasmic reticulum adds C₂-units to an acyl chain synthesized by fatty acid synthase (FAS) in a plastid. Both FAS and FAE are type II enzyme complexes as their components are coded for by distinct genes. Three subsequent reactions by the other FAE moieties [β-ketoacyl-CoA ketoreductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD), β-enoyl-CoA reductase (ECR)] remove the β-oxygen. Reiteration yields C₂₀-C₃₄ acyl chains that serve as precursors of the reductive (R) and decarb (-C) derived FAE aliphatics. *, CoA. (B) Deduced elongation pathway giving rise to a carbon skeleton decorated with two oxygens. Addition of two C₂-units to a β-ketoC₁₈-CoA by a chalcone synthase (CHS)-like polyketide synthase (PKS) results in an acyl chain with four oxygens. That on the β-carbon is then removed by the successive action of a KCR, an HCD and an ECR. Six additional elongations of the FAE type results in a C₃₂ acyl chain with oxygens on carbons 16 and 18. A decarb reaction yields the β-diketone, hentriacontan-14,16-dione.

Fig. 2.

Location of Cer-c, Cer-q and Cer-u on barley chromosome arm 2HS. (A) Comparison of introgression regions in three different barley near-isogenic lines (Druka et al., 2010). BW409 carries the gsh6.s allele, which is a mutation in the Cer-c gene (Franckowiak and Lundqvist, 2012). BW404 and BW411 contain gsh1.a (Cer-q) and gsh8.ag (Cer-u), respectively. Map positions follow the BARLEX Draft Genome Explorer (Colmsee et al., 2015). (B) Mapping of the Cer-c, Cer-q and Cer-u loci using six SNP markers. Recombination frequency for each marker is given as number of recombinants/total progeny. The F₂-mapping populations were made from crosses between the three near-isogenic lines and barley cultivars Bowman (Bo), Barke (Ba), Morex (M) and Quench (Q). (C) Cer-c, Cer-q and Cer-u were mapped between SNP markers 1_0718 and 1_1059. Six genes of unknown order were selected as Cer-c, Cer-q and Cer-u candidates for further analysis.

Fig. 3.

Cer-cqu candidate genes, barley BAC sequence and gene expression. (A) PCR amplification of six candidate genes using DNAs from barley cer-cqu triple mutants as templates. The inability to amplify MLOC_59804, MLOC_13397 and AK373499 from six mutants pinpointed these as Cer-c, Cer-q and Cer-u candidates. The number of primer pairs used with each gene is in parenthesis. +, gene amplified; -, gene not amplified. (B) Proposed order and gene models of the four genes identified on barley BAC HVVMRXALLrA0066C06. Regions encoding conserved domains are colored. Bar, 5kb. (C) Relative expression of the four genes based on RNA-seq analysis of barley flag leaf sheath (SRA PRJEB12101). Actin (MLOC_54382.1) is included as a reference gene. The dashed red line corresponds to a transcripts per million (tpm) value of 2, commonly used as the cut-off for real expression over noise.

Fig. 4.

Positions of identified mutations in the barley Cer-c, Cer-q and Cer-u genes. Most were point mutations leading to non-synonymous substitutions of amino acids (mutation names in black). Others included nonsense mutations (red), small deletions of 1–3bp (blue), or point mutations located upstream of the ATG start codon or in intron splice sites (green). Mutation cer-c.1329 (marked with an asterisk) contained two mutations in different parts of Cer-c. Further details are in Tables 1–3 and Supplementary Table S1. E, exon.

Fig. 5.

3D models of CER-C, CER-Q and CER-U. The proteins are represented in backbone cartoon with backbone colors representing functional sites in the cartoon models to the left and amino-acid residues affected by the mutations represented as yellow sticks in the models to the right. (A) CER-C represented in proposed homodimer form with subunits in black and white. To the left, the catalytic triad is represented in red sticks while the backbone of the residues forming the pockets associated with activity are colored purple (active site pocket) except for the catalytic residues in red; blue (substrate binding tunnel) and green (CoA-binding tunnel). (B) CER-Q with the lipase domain 1 in white and domain 2 in black. To the left, the active site residues are represented as red sticks and the backbone of the residues delineating the active site pocket are colored blue. (C) CER-U with heme included in the heme-binding pocket in red sticks. To the left, the backbone of the residues involved in heme binding are colored red while the substrate binding tunnel is colored blue. In the inserts the models are colored according to the predicted model error from blue (low error) to red (high error). PDB models and Pymol session files showing mutant names are accessible in the models (Supplementary Data S1–S3) and Pymol sessions (Supplementary Data S4–S6).

Fig. 6.

Proposed β-diketone synthase (DKS) polyketide pathway for synthesis of β-diketones, their derivatives and esterified alkan-2-ols. Fatty acid synthase (FAS) plus auxiliary enzymes (AE) in plastids (green) synthesize acyl chains (x=9–17) that are exported therefrom and presumed to enter the endoplasmic reticulum’s membrane (gray) where FAE derived aliphatics are synthesized. Here they are activated by CoA to form an acyl-CoA pool. Acyl editing transfers the acyl chains to a glycerolipid in a reversible reaction. Acyl-CoAs can serve as substrates for fatty acid elongase (FAE) complexes giving, for example, a fatty acid (y=17–21; pink, center left side). Given that CER-Q on the basis of homology is classified as a lipase, its acyl substrate is potentially esterified to a glycerolipid (top center). The CER-Q cleaved acyl chain activated by coenzyme A is elongated (+C₂) to give the β-ketoacyl compound that is the substrate for two pathways; (i) β-diketones and their derivatives (right side). The DKS (CER-C) introduces two oxygens into the acyl chain, which are followed by further elongations analogous to those carried out by FAE and loss of a carbon yields β-diketones (see Fig. 1). In barley CER-U a P450 hydroxylase inserts a hydroxyl group on carbon 25. (ii) Esterified alkan-2-ols (left side). Cleavage of CoA plus the carboxyl carbon yields methyl ketones that can be hydrolyzed to short alkan-2-ols, primarily with 13 and 15 carbons, for esterification with fatty acids originating from FAE. Mutants of Cer-u and Cer-c accumulate β-diketones and esterified alk-2-ols, respectively. The final aliphatics are transported to and through the apoplast (orange) onto its surface. Single arrows, known reaction(s); sequential arrows, hypothetical reaction(s).