Key Concepts:

Figure 1. Common phenotypes of epicuticular waxes in the Gramineae. (a) Leaf sheaths and leaves of field grown wildtype Bonus and cer mutant barley. (b, c) A dense coating of small lobed plates on wildtype leaves, which shed water, is characteristic for waxes with high concentrations of primary alcohols. (d, e) The small mounds of wax on leaves of cer‐j⁵⁹, blocked in synthesis of primary alcohols, retain water drops enabling germination of spores and leaching of nutrients. (f) A dense coating of long, hollow tubes (0.1–0.2 μm in diameter, up to 5 μm long) attributed to high concentrations of β‐diketones covers wildtype leaf sheaths that, in addition to the above mentioned functions, can protect against frost damage (Barber and Jackson, 1957). (g) Only thin plates appressed to the surface occur on cer‐a⁸ leaf sheaths lacking β‐diketones. (h) Two of the three types of wax structures that occur on leaf sheaths of sugar cane (Haas et al., 2001); massive compound rodlets attributed to aldehydes and the small lobed plates characteristic of primary alcohols (see b). Transmission electron microscopy of pre‐shadowed carbon replicas (b, d and g). Scanning electron microscopy (f and h).

Figure 2. Phenotypes of waxes. (a) The epicuticular wax on Arabidopsis stems with major amounts of alkanes, secondary alcohols and ketones displays a variety of shapes. (b) A transverse section of Arabidopsis cer5 mutant stems with a nonfunctional ABCG12 protein reveals accumulation of cytoplasmic lipid inclusions (arrows) unable to enter the plasmalemma. (c) A pre‐shadowed carbon replica of the wax secreted onto the cuticle surface of a leaf sheath of cer‐zw²⁸⁶ barley, having reduced amounts of β‐diketones and shorter tubes, discloses formation of thin plates/films (arrows) before self‐assembly (Koch et al., 2010) into tubes characteristic of β‐diketones. Visualised by cryo‐scanning (a), transmission (b) and scanning electron microscopy (c). (a) and (b) courtesy of Lacey Samuels, University of British Columbia, Canada.

Figure 3. Assembly of wax carbon skeletons is accomplished by several enzyme complexes: fatty acid synthase (FAS), fatty acid elongase [FAE; originally designated elongase (ELS)] and a type III polyketide synthase (PKS), the β‐ketoacyl‐coenzyme A (CoA) synthase (pkKCS). Plastid localised FAS (left) carries out a reiterated cycle of four reactions in which a joining enzyme first decarboxylates an activated (star) C₃‐substrate to give the activated C₂ donor (boxed) which is added to the initial acceptor, acetyl‐CoA. Otherwise acyl carrier protein (ACP) serves as activator for FAS. The β‐keto (O) group of the C₄ condensation product is reduced to a hydroxy (OH) group, then dehydrated and finally removed by another reduction yielding an elongated acyl chain serving as a primer for the next extension. Repeated cycles yield chains with 16 and 18 carbons. After removal of the ACP, transport to the endoplasmic reticulum and activation with CoA, the 16 and 18 acyl chains serve as primers for FAE complexes (left, blue) that construct chains with 20–34 carbons activated with CoA. Type I and II PKS complexes (centre) which use ACP activated acyl chains differ from FAEs in lacking one or more of the four enzymes participating in specific cycles so that the resulting carbon skeletons are decorated with Os, OHs and/or double bonds. pkKCSs (centre, red) lack both reductases and the dehydratase and therefore each elongation introduces an O into the growing chain. This is illustrated on the right where two pkKCS condensations yield a triketide intermediate with O groups on alternating carbons, that is, β to one another. During subsequent cycles in which all four enzymes participate (pink), the number of carbons between the initial two O groups and the most recently added one increases from three to y resulting in acyl chains with internal β‐diketo groups. The resulting pkKCS derived skeletons range from 10 to 34 carbons in length and are designated keto‐CoAs. In A. thaliana the FAE enzymes are ketoacyl‐CoA synthase (KCS), ketoacyl‐CoA reductase (KCR), hydroxyacyl‐CoA dehydratase (HCD) and enoyl‐CoA reductase (ECR) that are encoded, for example, by CER6, KCR1, PAS2 and CER10, respectively. R‐one carbon in the first FAS cycle of elongation and increases by two in every successive round thereafter. Two systems for naming the individual carbons in an acyl chain starting from the carboxy carbon (α, β and 1, 2, 3) are included FAS, FAE, PKS and pkPKS are enzyme complexes.

Figure 4. Origin of Arabidopsis wax aliphatics from the very long chain, acyl‐CoAs (C₂₆–C₃₀) constructed by fatty acid elongase (FAE) enzyme complexes (see Figure 3, blue). The decarb pathway (blue arrows) gives rise to aliphatic wax classes (blue) with odd carbon numbers (C₂₉). The OH is on carbon 14 or 15, the O on carbon 15. The reductive pathway (green arrows) yields even carbon (C₂₆–C₃₀) primary alcohols (green). Fatty acid moieties of the esters are predominantly C₁₆. The steps in which the CER1, CER3, CER4, CER8, MAH1 and WSD genes function or preliminary results infer function (enclosed in parentheses) are shown. MAH1? denotes apparent synthesis by MAH1 of ketones and many structurally related ketols and diols (see text). Green ? denotes that synthesis of primary alcohols potentially also occurs via an aldehyde intermediate (see text). In other plants the chain lengths of the FAE products entering the decarb and reductive pathways can differ; for example, in barley leaf waxes the former use primarily C₃₂ and the latter primarily C₂₆. Moreover, the chain length distributions of ester alcohols often match that of the primary alcohols.

Figure 5. pkKCSs construct carbon chains (top right) that are precursors to three‐branch pathways giving rise to aliphatic classes in many Gramineae waxes. One pkKCS extension followed by a decarboxylation gives methylketones (left side). These are reduced to alkan‐2‐ols (primarily C₁₃ and C₁₅) which are esterified with fatty acids from a FAE complex (Figure 4) to give alkan‐2‐ol esters (green). A second extension possibly by the same pkKCS leads to internal β‐Os (see Figure 3 for details). Subsequent loss of a carbon yields β‐diketones (primarily 14,16‐C₃₁, dominating many waxes) into which an OH can be introduced (primarily on carbon 25) and then oxidised to give O derivatives (right side, blue). Only the alkan‐2‐ol branch aliphatics are present in sorghum leaf wax (von Wettstein‐Knowles et al., 1984). Arrows indicate the direction of chain synthesis. R is 6 carbons for the C₁₃‐2‐ol and 10 for the 14,16‐β‐diketone. Three functions are known for the Cer‐cqu gene in barley: cer‐q mutants are defective in synthesis of all one‐ and two‐step pkKCS derived aliphatics inferring malfunction of a step common to both branches, cer‐c mutants are blocked in synthesis of all β‐diketone lipids with increases of alkan‐2‐ol esters whereas cer‐u mutants are impaired in hydroxylation of β‐diketones. Three extensions by a pkKCS, known as alkylresorcinol synthase (top), in the terminal elongation cycles, gives tetraketide‐CoA intermediates (C₂₀–C₃₄) that are precursors of minor wax components, 5‐alkylresorcinols (pink). The alkyl side chain on 1,3‐dihydroxybenzene ranges from 13 to 27 carbons. Carbons are numbered according to IUPAC nomenclature rules. Parentheses enclose intermediates not present in the waxes.