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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 b-amyrin synthase) to compare water loss with and without added cuticular triterpenoids in Arabidopsis leaf waxes. b-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.

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 C 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 C 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 C 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 C aliphatic chains.

A gradient exists between the composition of the epi- and intracuticular wax layers of Rosa canina leaves. This gradient may result from polarity differences, in part caused by differences in chain lengths. The outer wax layer accessible to the phyllosphere showed a unique composition of wax compounds. The ecological consequences from such a gradient may now be probed.

V6T 1Z1 (R.J.) 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 3 10 4 and 1.5 3 10 4 s m 21 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.

Summary• 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.

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