Summary
To protect plants against biotic and abiotic stress, the waxy cuticle must coat all epidermis cells. Here, two independent approaches addressed whether cell‐type‐specific differences exist ...between wax compositions on trichomes and other epidermal cells of Arabidopsis thaliana, possibly with different protection roles. First, the total waxes from a mutant lacking trichomes (gl1) were compared to waxes from wild type and a trichome‐rich mutant (cpc tcl1 etc1 etc3). In the stem wax, compounds with aliphatic chains longer than 31 carbons (derived from C32 precursors) increased in relative abundance in cpc tcl1 etc1 etc3 over gl1. Similarly, the leaf wax from the trichome‐rich mutant contained higher amounts of C32+ compounds as compared to gl1. Second, leaf trichomes were isolated, and their waxes were analyzed. The wax mixtures of the trichome‐rich mutant and the wild type were similar, comprising alkanes and alkenes as well as branched and unbranched primary alcohols. The direct analyses of trichome waxes confirmed that they contained relatively high concentrations of C32+ compounds, compared with the pavement cell wax inferred from analysis of gl1 leaves. Finally, the cell‐type‐specific wax compositions were put into perspective with expression patterns of wax biosynthesis genes in trichomes and pavement cells. Analyses of published transcriptome data (Marks et al., ) revealed that core enzymes involved in elongation of wax precursors to various carbon chain lengths are expressed differentially between epidermis cell types. By combining the chemical and gene expression data, we identified promising gene candidates involved in the formation of C32+ aliphatic chains.
Significance Statement
Surface properties of different epidermal cells differ. Cuticular wax analyses distinguish between the compositions of lipid mixtures coating Arabidopsis leaf trichomes and neighbouring pavement cells. Integrating wax compositions of different epidermal cell types with gene expression data shows that trichome cells have an autonomous wax biosynthesis machinery and gene candidates involved in the formation of aliphatic chains longer then C32 were identified.
Plants prevent dehydration by coating their aerial, primary organs with waxes. Wax compositions frequently differ between species, organs, and developmental stages, probably to balance limiting ...nonstomatal water loss with various other ecophysiological roles of surface waxes. To establish structure-function relationships, we quantified the composition and transpiration barrier properties of the gl1 mutant leaf waxes of Arabidopsis (Arabidopsis thaliana) to the necessary spatial resolution. The waxes coating the upper and lower leaf surfaces had distinct compositions. Moreover, within the adaxial wax, the epicuticular layer contained more wax and a higher relative quantity of alkanes, whereas the intracuticular wax had a higher percentage of alcohols. The wax formed a barrier against nonstomatal water loss, where the outer layer contributed twice as much resistance as the inner layer. Based on this detailed description of Arabidopsis leaf waxes, structure-function relationships can now be established by manipulating one cuticle component and assessing the effect on cuticle functions. Next, we ectopically expressed the triterpenoid synthase gene AtLUP4 (for lupeol synthase4 or β-amyrin synthase) to compare water loss with and without added cuticular triterpenoids in Arabidopsis leaf waxes. β-Amyrin accumulated solely in the intracuticular wax, constituting up to 4% of this wax layer, without other concomitant changes of wax composition. This triterpenoid accumulation caused a significant reduction in the water barrier effectiveness of the intracuticular wax.
The protective wax coating on plant surfaces has long been considered to be non-uniform in composition at a subcellular scale. In recent years, direct evidence has started to accumulate showing ...quantitative compositional differences between the epicuticular wax (i.e. wax exterior to cutin that can be mechanically peeled off) and intracuticular wax (i.e. wax residing within the mechanically resistant layer of cutin) layers in particular. This review provides a first synthesis of the results acquired for all the species investigated to date in order to assign chemical information directly to cuticle substructures, together with an overview of the methods used and a discussion of possible mechanisms and biological functions. The development of methods to probe the wax for z-direction heterogeneity began with differential solvent extractions. Further research employing mechanical wax removal by adhesives permitted the separation and analysis of the epicuticular and intracuticular wax. In wild-type plants, the intracuticular (1-30 μg cm⁻²) plus the epicuticular wax (5-30 μg cm⁻²) combined to a total of 8-40 μg cm⁻². Cyclic wax constituents, such as triterpenoids and alkylresorcinols, preferentially or entirely accumulate within the intracuticular layer. Within the very-long-chain aliphatic wax components, primary alcohols tend to accumulate to higher percentages in the intracuticular wax layer, while free fatty acids and alkanes in many cases accumulate in the epicuticular layer. Compounds with different chain lengths are typically distributed evenly between the layers. The mechanism causing the fractionation remains to be elucidated but it seems plausible that it involves, at least in part, spontaneous partitioning due to the physico-chemical properties of the wax compounds and interactions with the intracuticular polymers. The arrangement of compounds probably directly influences cuticular functions.
Previous research has shown that cuticular triterpenoids are exclusively found in the intracuticular wax layer of Prunus laurocerasus. To investigate whether this partitioning was species-specific, ...the intra- and epicuticular waxes were identified and quantified for the glossy leaves of Ligustrum vulgare, an unrelated shrub with similar wax morphology. Epicuticular wax was mechanically stripped from the adaxial leaf surface using the adhesive gum arabic. Subsequently, the organic solvent chloroform was used to extract the intracuticular wax from within the cutin matrix. The isolated waxes were quantified using gas chromatography with flame ionization detection and identified by mass spectrometry. The results were visually confirmed by scanning electron microscopy. The outer wax layer consisted entirely of homologous series of very-long-chain aliphatic compound classes. By contrast, the inner wax layer was dominated (80%) by two cyclic triterpenoids, ursolic and oleanolic acid. The accumulation of triterpenoids in the intracuticular leaf wax of a second, unrelated species suggests that this localization may be a more general phenomenon in smooth cuticles lacking epicuticular wax crystals. The mechanism and possible ecological or physiological reasons for this separation are currently being investigated.
The capacity of plants to minimize uncontrolled water loss is essential for survival in adverse and changing climatic conditions. In order to assess and compare the effectiveness of apoplastic ...barriers to water, the permeability of the barrier must first be quantified. Studies have accomplished this directly by quantifying tritium flux or indirectly by measuring the influx/efflux of water surrogates such as dyes, chlorophyll, and herbicides. Other studies have relied on comparative methods such as survival rates after drought. These methods rely on radioactive material, correlations, or qualitative comparisons. However, a quantitative method is necessary that directly measures water efflux and that allows easy comparisons within and between experiments, plant parts, plant species, and especially research laboratories. Here we outline in detail a gravimetric protocol first described by Schönherr and Lendzian (1981) that can be set up in less than half a day and completed in one to ten days depending on the plant barrier. This approach has been used in numerous studies on leaf and fruit cuticles and recently also on petals from cosmos (Cosmos bipinnatus; Buschhaus et al., 2015).
Cuticular waxes coat all primary aboveground plant organs as a crucial adaptation to life on land. Accordingly, the properties of waxes have been studied in much detail, albeit with a strong focus on ...leaf and fruit waxes. Flowers have life histories and functions largely different from those of other organs, and it remains to be seen whether flower waxes have compositions and physiological properties differing from those on other organs. This work provides a detailed characterization of the petal waxes, using Cosmos bipinnatus as a model, and compares them with leaf and stem waxes. The abaxial petal surface is relatively flat, whereas the adaxial side consists of conical epidermis cells, rendering it approximately 3.8 times larger than the projected petal area. The petal wax was found to contain unusually high concentrations of C(22) and C(24) fatty acids and primary alcohols, much shorter than those in leaf and stem waxes. Detailed analyses revealed distinct differences between waxes on the adaxial and abaxial petal sides and between epicuticular and intracuticular waxes. Transpiration resistances equaled 3 × 10(4) and 1.5 × 10(4) s m(-1) for the adaxial and abaxial surfaces, respectively. Petal surfaces of C. bipinnatus thus impose relatively weak water transport barriers compared with typical leaf cuticles. Approximately two-thirds of the abaxial surface water barrier was found to reside in the epicuticular wax layer of the petal and only one-third in the intracuticular wax. Altogether, the flower waxes of this species had properties greatly differing from those on vegetative organs.
The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β-ketoacyl-CoA synthase (KCS) enzyme that catalyses a crucial ...condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C
and C
alkanes derived from C
and C
acyl-CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss-of-function mutants were depleted of C
and C
alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C
and C
fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C
to C
acyl-CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra-long compounds in adjacent pavement cells.
Plants prevent dehydration by coating their aerial, primary organs with waxes. Wax compositions frequently differ between species, organs, and developmental stages, probably to balance limiting ...nonstomatal water loss with various other ecophysiological roles of surface waxes. To establish structure-function relationships, we quantified the composition and transpiration barrier properties of the
gl1
mutant leaf waxes of Arabidopsis (
Arabidopsis thaliana
) to the necessary spatial resolution. The waxes coating the upper and lower leaf surfaces had distinct compositions. Moreover, within the adaxial wax, the epicuticular layer contained more wax and a higher relative quantity of alkanes, whereas the intracuticular wax had a higher percentage of alcohols. The wax formed a barrier against nonstomatal water loss, where the outer layer contributed twice as much resistance as the inner layer. Based on this detailed description of Arabidopsis leaf waxes, structure-function relationships can now be established by manipulating one cuticle component and assessing the effect on cuticle functions. Next, we ectopically expressed the triterpenoid synthase gene
AtLUP4
(for lupeol synthase4 or β-amyrin synthase) to compare water loss with and without added cuticular triterpenoids in Arabidopsis leaf waxes. β-Amyrin accumulated solely in the intracuticular wax, constituting up to 4% of this wax layer, without other concomitant changes of wax composition. This triterpenoid accumulation caused a significant reduction in the water barrier effectiveness of the intracuticular wax.
In the petal waxes of Cosmos bipinnatus, homologous series of 1,2-diols and 1,3-diols ranging from C20 to C26 were identified. They were accompanied by primary and secondary monoacetates of C20–C24 ...1,2-diols. Display omitted .
► Structures of very-long-chain alkanediols and their monoacetates were identified. ► These were localized in cuticular waxes covering the petal surfaces of Cosmos bipinnatus. ► Their tissue surface coverage was quantified. ► Monoacetate isomers despite potential interconversion were distinguished and quantified.
Four uncommon classes of very-long-chain compounds were identified and quantified in the petal wax of Cosmos bipinnatus (Asteraceae). The first two were homologous series of alkane 1,2-diols and 1,3-diols, both ranging from C20 to C26. The upper and lower petal surfaces contained 0.11 and 0.09μg/cm2 of 1,2-diols, respectively. 1,3-Diols were present at quantities one order of magnitude less than the 1,2-diols. Both series had similar chain length distributions, with 6–20%, 59–73% and 20–31% of the C20, C22 and C24 diols, respectively. The other two compound classes were primary and secondary monoacetates of C20–C24 1,2-diols. The monoacetates had chain length profiles similar to the free 1,2-diols, and amounted to 0.04 and 0.09μg/cm2 on the adaxial and abaxial sides, respectively. Methods were developed to minimize acyl migration during monoacetate isomer analyses. The ratios of diol 1-acetates to diol 2-acetates averaged close to 3:5, and thus opposite to the chemical equilibrium ratio of 7:3.
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