Constructing functional cuticles: analysis of relationships between cuticle lipid composition, ultrastructure and water barrier function in developing adult maize leaves

Abstract

Background and aims: Prior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant.

Methods: We examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features.

Key results: The water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development.

Conclusions: Cuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

Keywords: Zea mays; Cuticular wax; cuticle ontogeny; cuticle ultrastructure; cutin; leaf development; maize.

Fig. 1.

Cuticle maturation along the developmental gradient of a partially expanded, adult maize leaf. (A) Intact adult maize leaves (B73, leaf 8 at 50–60 cm length) stained with TBO for 1 or 2 h. Vertical lines indicate segments harvested for subsequent analysis of cuticle composition and ultrastructure. (B) Quantification of TBO penetration of the cuticle. Results at each position are expressed as absorbance readings at 630 nm normalized to surface area. (C) Cuticular evaporation rate, a measure of dehydration resistance, was measured for successive 2-cm segments. (D) Pavement cell surface areas. In (B–D), n = 3 leaves per time point, mean ± s.e.

Fig. 2.

Cuticular wax composition along the adult maize leaf developmental gradient. Changes in the accumulation of six classes of compounds present in chloroform-extracted wax mixtures (based on data shown in Supplementary data Table S1). Mean of four replicates and s.e. are reported.

Fig. 3.

Hydrocarbon and alkyl ester composition along the adult maize leaf developmental gradient. (A) Hydrocarbon (alkane and alkene) composition. (B) Alkyl ester composition (low-abundance components, namely C₃₈ and odd-carbon-number esters, are not shown). Mean of four replicates and s.e. are reported.

Fig. 4.

Lipid polyester monomer accumulation along the adult maize leaf developmental gradient. (A) Accumulation of the two main classes of cutin monomers present in maize leaf cutin compared with the total amount of monomers. (B) Representative profile of the maize mature leaf cutin monomer composition (portion between 20 and 22 cm from the leaf base). Means of four replicates and s.e. are reported.

Fig. 5.

B73 leaf pavement cell cuticle development visualized by TEM. (A) Pavement cell cuticle from a partially expanded leaf 8, 40–42 cm from the base, where leaf tissue is mature. Four distinct layers or zones are visible: a thin, darkly stained layer (white arrowhead) at the interface between the cell wall (CW) and cuticle, dark (asterisk) and light zones of the cuticle proper (C), and a darkly stained epicuticular layer (black arrowhead). Scale bar = 40 nm. (B) Thickness of pavement cell cuticles at the indicated positions along the developmental gradient of partially expanded B73 leaf 8. (C) Percentage of cuticle thickness at indicated positions occupied by the dark-staining inner layer of the cuticle proper. In (B) and (C), lower-case letters indicate significance groups identified by one-way ANOVA with the Tukey multiple comparisons post-test. (D–J) Representative images of pavement cell cuticles at the indicated positions along the developmental gradient of partially expanded B73 leaf 8. Scale bar = 100 nm.

Fig. 6.

Isolation and analysis of the epicuticular wax layer. (A) Mature adult maize leaf showing that the dark-staining external layer of the cuticle becomes detached in places (asterisks), demonstrating a loose association of this layer with the most internal part of the cuticle. (B) Stripping a mature adult maize leaf with gum arabic removes most of the dark-staining external layer. C, cuticle; CW, cell wall. Scale bars (A, B) = 100 nm. (C–G) Epicuticular, intracuticular and total wax composition in four segments, between 4 and 22 cm from the base, of the maize leaf 8 developmental gradient. Epicuticular waxes removed by adhesion to, and subsequent chloroform extraction from, gum arabic were compared with total wax extracted with chloroform from a matched sample; intracuticular waxes were inferred by calculating the difference between epicuticular and total waxes. Total wax coverage (C) and more abundant wax classes, namely alcohols (D), hydrocarbons (E), alkyl esters (F) and alicyclics (G), are shown for each fraction. Means of three replicates for chloroform-extracted waxes and of six replicates (from three adaxial and three abaxial) for epicuticular waxes and s.e. are reported. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.