An overview on plant cuticle biomechanics

Plant biomechanics combines the principles of physics, chemistry and engineering to answer questions about plant growth, development and interaction with the environment. The epidermal-growth-control theory, postulated in 1867 and verified in 2007, states that epidermal cells determine the rate of organ elongation since they are under tension, while inner tissues are under compression. The lipid cuticle layer is deposited on the surface of outer epidermal cell walls and modifies the chemical and mechanical nature of these cell walls. Thus, the plant cuticle plays a key role in plant interaction with the environment and in controlling organ expansion. Rheological analyses indicate that the cuticle is a mostly viscoelastic and strain-hardening material that stiffens the comparatively more elastic epidermal cell walls. Cuticle stiffness can be attributed to polysaccharides and flavonoids present in the cuticle whereas a cutin matrix is mainly responsible for its extensibility. Environmental conditions such as temperature and relative humidity have a plasticizing effect on the mechanical properties of cuticle since they lower cuticle stiffness and strength.The external appearance of agricultural commodities, especially fruits, is of great economic value. Mechanical properties of the cuticle can have a positive or negative effect on disorders like fruit cracking, fungal pathogen penetration and pest infestation. Cuticle rheology has significant variability within a species and thus can be subjected to selection in order to breed cultivars resistant to pests, infestation and disorders.     

Surface lipids and plant defenses

The major function of the plant epidermis is to form the cuticle, a functional permeability barrier of the cell wall which prevents excessive water loss and the entry of harmful substances and pathogens into the host. This type of cell wall modification is mainly composed of a polyester matrix, cutin, and soluble waxes embedded in the matrix and deposited on the external surface. Cuticle-associated proteins may also be important. Recent observations are starting to reveal complex inter-relationships between cuticular lipids and immunity. This suggests that the cuticle is not simply a physical barrier, but a dynamic host defense with signaling circuits and effector molecules. Furthermore, these studies have also demonstrated that cuticular lipids and immunity may intersect in common pathways, although the significance of this is not fully understood. In this review, we examine the functions of the plant cuticle in host–pathogen interactions, and discuss the possibilities of integrating the membrane and cuticular glycerolipid biosynthesis.     

Fungal entomopathogens in a tritrophic context

Variation in plant quality has an important impact on insect growth and development and there is considerable evidence that plants can also influence an insect’s natural enemies. Here we discuss the potential for plant-mediated effects on fungal entomopathogens. Fungi differ from other insect pathogens in that they infect an insect directly through its cuticle. This means that they are particularly vulnerable to changes in microclimate and properties of the insect cuticle. Potential direct and indirect mechanisms for plant-mediated effects on fungal entomopathogens are discussed. It is clear from these studies that fungal entomopathogens could be affected by plant volatiles and plant surface chemistry. Plant secondary chemicals can also inhibit fungal growth, potentially protecting the insect herbivore. However, the site of action and the mechanism behind these effects in plant-based studies is not always clear. The implications for biocontrol using fungal entomopathogens are discussed.     

Studies on the host/parasite interface during the development of a pentastomid arthropod parasite in rodent intermediate hosts, with observations on protective surface membranes

The changing structure of the cuticle of the arthropod pentastomid parasite Porocephalus crotali, during growth to the infective stage in mouse and rattlesnake hosts, is described. The outermost cuticulin layer of the cuticle in instars II-VI is elevated to form a dense mat of epicuticular hairs. Since the VI larval cuticle is retained by the infective (VII) nymph as a protective sheath, effectively all stages in mice present a hairy surface to the host and this may constitute a physical barrier to inflammatory cells. The entire surface is overlain by a triple-track 'unit' membrane whose biophysical properties resemble those of a conventional plasma membrane, and there is evidence to suggest that this membrane is susceptible to immune attack. Under natural circumstances, epicuticular hairs entrap secretion, delivered to the cuticle via innumerable minute ducts which communicate with tegumental secretory cells termed subparietal cells (SPC). SPC synthesize lamellate droplets which unfold on the cuticle to constitute a layer of protective polymorphic vesicles. By contrast, infective nymphs in snakes possess a smooth cuticle and SPC membranous secretion is stacked over the entire surface, in sheets up to 20 deep. The function of the lipid and protein components of SPC secretion is discussed.     

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background  

Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton.     

Protecting against water loss: analysis of the barrier properties of plant cuticles

The cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other primary parts of higher plants. More than 100 mean values for water permeabilities determined with isolated leaf and fruit cuticles from 61 plant species are compiled and discussed in relation to plant organ, natural habitat and morphology. The maximum barrier properties of plant cuticles exceed that of synthetic polymeric films of equal thickness. Cuticular water permeability is not correlated to the thickness of the cuticle or to wax coverage. Relationships between cuticular permeability, wax composition and physical properties of the cuticle are evaluated. Cuticular permeability to water increases on the average by a factor of 2 when leaf surface temperature is raised from 15 degrees C to 35 degrees C. Organic compounds of anthropogenic and biogenic origin may enhance cuticular permeability. The pathway taken by water across the cuticular transport barrier is reviewed. The conclusion from this discussion is that the bulk of water diffuses as single molecules across a lipophilic barrier while a minor fraction travels along polar pores. Open questions concerning the mechanistic understanding of the plant cuticular transport barrier and the role the plant cuticle plays in ensuring the survival and reproductive success of an individual plant are indicated.     

Novel elasticity measurements reveal C. elegans cuticle stiffens with age and in a long-lived mutant

Changes in biomechanical properties have profound impacts on human health. C. elegans might serve as a model for studying the molecular genetics of mammalian tissue decline. Previously, we found that collagens are required for insulin signaling mutants' long lifespan and that overexpression of specific collagens extends wild-type lifespan. However, whether these effects on lifespan are due to mechanical changes during aging has not yet been established. Here, we have developed two novel methods to study the cuticle: we measure mechanical properties of live animals using osmotic shock, and we directly perform the tensile test on isolated cuticles using microfluidic technology. Using these tools, we find that the cuticle, not the muscle, is responsible for changes in the “stretchiness” of C. elegans, and that cuticle stiffness is highly nonlinear and anisotropic. We also found that collagen mutations alter the integrity of the cuticle by significantly changing the elasticity. In addition, aging stiffens the cuticle under mechanical loads beyond the cuticle's healthy stretched state. Measurements of elasticity showed that long-lived daf-2 mutants were considerably better at preventing progressive mechanical changes with age. These tests of C. elegans biophysical properties suggest that the cuticle is responsible for their resilience.     

A functional cutin matrix is required for plant protection against water loss

The plant cuticle, a cutin matrix embedded with and covered by wax, seals the aerial organ''s surface to protect the plant against uncontrolled water loss. The cutin matrix is essential for the cuticle to function as a barrier to water loss. Recently, we identified from wild barley a drought supersensitive mutant, eibi1, which is caused by a defective cutin matrix as the result of the loss of function of HvABCG31, an ABCG full transporter. Here, we report that eibi1 epidermal cells contain lipid-like droplets, which are supposed to consist of cutin monomers that have not been transported out of the cells. The eibi1 cuticle is fragile due to a defective cutin matrix. The rice ortholog of the EIBI1 gene has a similar pattern of expression, young shoot but not flag leaf blade, as the barley gene. The model of the function of Eibi1 is discussed. The HvABCG31 full transporter functions in the export of cutin components and contributed to land plant colonization, hence also to terrestrial life evolution.Key words: ABC transporter, cuticle, cuticular wax, drought resistance, inclusion     

Biochemical analysis of elastic and rigid cuticles of Cirsium horridulum

The cuticle is a complex structure of soluble lipids, lipid polymers and polysaccharides. In addition to its functions to reduce water loss and provide a protective barrier, its mechanical properties may be significant to plant growth and development. We investigated the cuticle of Cirsium horridulum Michx. because of its involvement in the thigmonastic contraction of staminal filaments. The staminal filaments and portions of the style are surrounded by a highly elastic cuticle in contrast to the rigid cuticle of the corolla and leaves. Our aim was to determine if the biochemical composition affected the elasticity of the cuticle. We discovered that the ratio of carbohydrates to lipids is 1:7 in floral parts but 2:1 in leaf cuticle. Esterified cutin components represented about 80% of the cuticle and di-hydroxyhexadecanoic acids were the major monomers of cutin, regardless of origin. The cutin of elastic tissues is characterized by a higher content of tri-hydroxy monomers than the cutin of rigid tissues. The data suggest that hydroxyl groups enhance the hydrophilic character of the cuticle and contribute to cuticular elasticity.     

Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy

Full appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato ( Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.     

Lipid melting and cuticular permeability: new insights into an old problem

The idea that the physical properties of cuticular lipids affect cuticular permeability goes back over 65 years. This proposal has achieved textbook status, despite controversy and the general lack of direct supporting evidence. Recent work supports the standard model, in which lipid melting results in increased cuticular permeability. Surprisingly, although all species studied to date can synthesize lipids that remain in a solid state at environmental temperatures, partial melting often occurs due to the deposition of lipids with low melting points. This will tend to increase water loss; the benefits may include better dispersal of lipids or other compounds across the cuticle or improved communication via cuticular pheromones. In addition, insects with high melting-point lipids are not necessarily less permeable at low temperatures. One likely reason is variation in lipid properties within the cuticle. Surface lipids differ from one region to another, and biophysical studies of model mixtures suggest the occurrence of phase separation between melted and solid lipid fractions. Lipid phase separation may have important implications for insect water balance and chemical communication.     

Rheological Properties of Enzymatically Isolated Tomato Fruit Cuticle

Rheological properties were determined for cuticular membranes (CMs) enzymatically isolated from mature tomato (Lycopersicon esculentum Mill. cv Pik Red) fruit. The cuticle responded as a viscoelastic polymer in stress-strain studies. Both CM and dewaxed CM expanded and became more elastic and susceptible to fracture when hydrated, suggesting that water plasticized the cuticle. Dewaxing of the CM caused similar changes in elasticity and fracturing, indicating that wax may serve as a supporting filler in the cutin matrix. Exposure of the cuticle to the surfactant Triton X-100 did not significantly affect its rheological properties.     

Unraveling the complex network of cuticular structure and function

A hydrophobic cuticle is deposited at the outermost extracellular matrix of the epidermis in primary tissues of terrestrial plants. Besides forming a protective shield against the environment, the cuticle is potentially involved in several developmental processes during plant growth. A high degree of variation in cuticle composition and structure exists between different plant species and tissues. Lots of progress has been made recently in understanding the different steps of biosynthesis, transport, and deposition of cuticular components. However, the molecular mechanisms that underlie cuticular function remain largely elusive.     

植物角质层生物学特性及水分渗透性研究进展

植物角质层作为植物体与外界环境的第一道保护屏障, 其最主要的功能是防止植物体过度失水。揭示植物角质层的生物学功能及其原理将为现代农业的发展以及仿生材料的开发应用提供科学指导。该文综述了植物角质层的生物学特性及其与水分渗透性关系的研究进展, 并展望了角质层水分渗透研究的应用前景。     

Spatial distribution of calcite and amorphous calcium carbonate in the cuticle of the terrestrial crustaceans Porcellio scaber and Armadillidium vulgare

The crustacean cuticle is an interesting model to study the properties of mineralized bio-composites. The cuticle consists of an organic matrix composed of chitin–protein fibres associated with various amounts of crystalline and amorphous calcium carbonate. It is thought that in isopods the relative amounts of these mineral polymorphs depend on its function and the habitat of the animal. In addition to the composition, the distribution of the various components should affect the properties of the cuticle. However, the spatial distribution of calcium carbonate polymorphs within the crustacean cuticle is unknown. Therefore, we analyzed the mineralized cuticles of the terrestrial isopods Armadillidium vulgare and Porcellio scaber using scanning electron-microscopy, electron probe microanalysis and confocal μ-Raman spectroscopic imaging. We show for the first time that the mineral phases are arranged in distinct layers. Calcite is restricted to the outer layer of the cuticle that corresponds to the exocuticle. Amorphous calcium carbonate is located within the endocuticle that lies below the exocuticle. Within both layers mineral is arranged in rows of granules with diameters of about 20 nm. The results suggest functional implications of mineral distribution that accord to the moulting and escape behaviour of the animals.     

The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR

Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.     

Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.     

Drosophila dumpy is a gigantic extracellular protein required to maintain tension at epidermal-cuticle attachment sites

BACKGROUND: Growth and morphogenesis during development depend both on patterning genes, which assign positional information, and on genes that regulate mechanical forces. The dumpy gene of the fruit fly Drosophila melanogaster is an example of the latter class, with mutant phenotypes affecting size and shape of the limbs, thoracic cuticle, trachea and mouthparts. RESULTS: The genetically complex dumpy locus was found to span over 100 kb and encode a gigantic 2.5 MDa extracellular matrix protein. Dumpy represents an extreme form of modular protein evolution, containing 308 epidermal growth factor (EGF) modules, interspersed with a new module class, DPY, and terminating in a crosslinking zona pellucida domain and membrane anchor sequence. We determined the three-dimensional structure of the DPY module by nuclear magnetic resonance (NMR) spectroscopy and found that it forms a disulphide-stabilised beta sheet motif, capable of linking end-to-end with EGF modules to form a fibre. Consistent with its cuticle phenotypes, dumpy is expressed at several sites of cuticle-epidermal cell attachment, including the trachea and the muscle tendon cells, which mediate anchorage of the muscles to the cuticle. CONCLUSIONS: The dumpy gene encodes a gigantic extracellular molecule that we predict to be a membrane-anchored fibre of almost a micrometer in length. Insertion and crosslinking of this fibre within the cuticle may provide a strong anchor for the underlying tissue, allowing it to maintain mechanical tension at sites under stress. This would explain its contribution to tissue morphogenesis through its regulation of mechanical properties.     

Decomposition and sorption characterization of plant cuticles in soil

The sorption of organic compounds by plant cuticular matter has been extensively investigated; however, little has been studied regarding the effect of plant cuticle degradation on their role in the sorption of organic compounds in soils. The sorption of phenanthrene was studied in soil samples which had been incubated for up to 9 months with three different types of plant cuticle isolated from tomato fruits, pepper fruits and citrus leaves. The main change in the diffuse reflectance Fourier-transform infrared (DRIFT) spectra during incubation of the cuticles was related to cutin decomposition. The peaks assigned to methyl and ethyl vibration and C=O vibration in ester links decreased with decomposition. In general, with all samples, the phenanthrene sorption coefficients calculated for the whole incubated soils (K _d) decreased with incubation time. In contrast, the carbon-normalized K _d values (K _oc) did not exhibit a similar trend for the different cuticles during incubation. The origin of the cuticle also affected the linearity of the sorption isotherms. With the tomato and citrus cuticle samples, the Freundlich N values were close to unity and were stable throughout incubation. However with the green pepper cuticle, the N values exhibited a significant decrease (from 0.98 to 0.70). This study demonstrates that the structural composition of the plant cuticle affects its biodegradability and therefore its ability to sorb organic compounds in soils. Of the residues originating from plant cuticular matter in soils, the cutan biopolymer and lignin-derived structures appear to play a dominant role in sorption as decomposition progresses. Responsible Editor: Alfonso Escudero.