Plant Leaf Cuticle Research Articles

The Tea Plant Leaf Cuticle: From Plant Protection to Tea Quality.

Camellia sinensis (tea tree) is a perennial evergreen woody crop that has been planted in more than 50 countries worldwide; its leaves are harvested to make tea, which is one of the most popular nonalcoholic beverages. The cuticle is the major transpiration barrier to restrict nonstomatal water loss and it affects the drought tolerance of tea plants. The cuticle may also provide molecular cues for the interaction with herbivores and pathogens. The tea-making process almost always includes a postharvest withering treatment to reduce leaf water content, and many studies have demonstrated that withering treatment-induced metabolite transformation is essential to shape the quality of the tea made. Tea leaf cuticle is expected to affect its withering properties and the dynamics of postharvest metabolome remodeling. In addition, it has long been speculated that the cuticle may contribute to the aroma quality of tea. However, concrete experimental evidence is lacking to prove or refute this hypothesis. Even though its relevance to the abiotic and biotic stress tolerance and postharvest processing properties of tea tree, tea cuticle has long been neglected. Recently, there are several studies on the tea cuticle regarding its structure, wax composition, transpiration barrier organization, environmental stresses-induced wax modification, and structure–function relations. This review is devoted to tea cuticle, the recent research progresses were summarized and unresolved questions and future research directions were also discussed.

The distribution and retained amount of benzo[a]pyrene at the micro-zones of mangrove leaf cuticles: Results from a novel analytical method

Plant leaf cuticles play a critical role in the accumulation and transport of atmospheric polycyclic aromatic hydrocarbons (PAHs). The relationship between the distribution and retained amount of PAHs on the leaf cuticles and the leaves micro-zone structures is still unclear. In this study, a confocal microscopic fluorescence spectral analysis (CMFSA) system with a spatial resolution of 200 nm was established as a direct and noninvasive means to determine the microscopic distribution and quantify the retained amount of benzo[a]pyrene (B[a]P) at Aegiceras corniculatum (Ac), Kandelia obovata (Ko) and Avicennia marina (Am) leaf cuticle micro-zones (0.096 mm2). The linear ranges for the established method were 10–1900 ng spot−1 for Ac, 15–1700 ng spot−1 for Ko and 30–1800 ng spot−1 for Am, and the detection limits were 0.06 ng spot−1 for Ac, 0.06 ng spot−1 for Ko and 0.07 ng spot−1 for Am. Notably, B[a]P formed clusters and unevenly distributed at the leaf cuticles. On the adaxial cuticles, B[a]P was mainly accumulated unevenly along the epidermis cell wall, and it was also distinctively distributed in the secretory cells around salt glands for Ac and Am. On the abaxial leaf cuticles, B[a]P was concentrated in the salt glands and stomata apart from being unevenly distributed in the epidermis cell wall. Moreover, the amount of B[a]P retained presented a negative correlation with the polarity of leaf cuticles, which resulted in the amount of B[a]P retained on the adaxial leaf cuticles being significantly higher than that on abaxial leaf cuticles. Our results provide a potential in situ method for investigating the distribution and retained amount of PAHs at plant leaf surface micro-zones, which would contribute to further studying and understanding the mechanism of migration and transformation of PAHs by plant leaves from a microscopic perspective.

Will the climate of plant origins influence the chemical profiles of cuticular waxes on leaves of Leymus chinensis in a common garden experiment?

Cuticular wax covering the leaf surface plays important roles in protecting plants from biotic and abiotic stresses. Understanding the way in which plant leaf cuticles reflect their growing environment could give an insight into plant resilience to future climate change. Here, we analyzed the variations of cuticular waxes among 59 populations of Leymus chinensis in a common garden experiment, aiming to verify how environmental conditions influence the chemical profiles of cuticular waxes. In total, eight cuticular wax classes were identified, including fatty acids, aldehydes, primary alcohols, alkanes, secondary alcohols, ketones, β‐diketones, and alkylresorcinols, with β‐diketones the predominant compounds in all populations (averaged 67.36% across all populations). Great intraspecific trait variations (ITV) were observed for total wax coverage, wax compositions, and the relative abundance of homologues within each wax class. Cluster analysis based on wax characteristics could separate 59 populations into different clades. However, the populations could not be separated according to their original longitudes, latitudes, annual temperature, or annual precipitation. Redundancy analysis showed that latitude, arid index, and the precipitation from June to August were the most important parameters contributing to the variations of the amount of total wax coverage and wax composition and the relative abundance of wax classes. Pearson's correlation analysis further indicated that the relative abundance of wax classes, homologues in each wax class, and even isomers of certain compound differed in their responses to environmental factors. These results suggested that wax deposition patterns of L. chinensis populations formed during adaptations to their long‐term growing environments could inherit in their progenies and exhibit such inheritance even these progenies were exported to new environments.

Leaf Cuticle Can Contribute to Non-Host Resistance to Poplar Leaf Rust

The plant leaf cuticle is a chemically complex but largely waxy outer shell that limits water loss and also prevents some pathogens from gaining access to internal mesophyll. Rust fungi are obligate parasites, and most bypass the cuticle by thigmotropically locating stomata, growing through the stomatal openings, and then parasitizing mesophyll cells with haustoria. It is thought that even non-hosts of a given rust fungus do not resist until their mesophyll cells are contacted in this way. In other words, it is thought that the cuticle plays no role in non-host resistance. Here, we tested the hypothesis that poplar leaf cuticles might contribute to non-host resistance to rust fungi by chemically impeding the germination and growth of urediniosporelings of Melampsora larici-populina. Following an initial survey in China of the resistance of 36 genotypes of various species and interspecific hybrids of Populus to M. larici-populina, we selected three genotypes for the initial test of hypothesis: (1) A Populus purdomii genotype that is fully susceptible; (2) a Populus deltoides cv. ‘I-69′ that is incompletely resistant (i.e., a resistant host); and (3) a Populus tomentosa genotype that is a non-host to M. larici-populina. Urediniospores were assayed for germination in extracts of the cuticles of the three genotypes. Germination was most reduced by the P. tomentosa non-host cuticular extracts that also reduced the growth of germ tubes to 36 times less than that in controls or in the extract of the susceptible P. purdomii. Four cuticular components were identified as putative defense compounds given greater concentrations in P. tomentosa than in P. purdomii: Aucubin, hexakis(trimethylsilyl) ether, catechol, 7,9-Di-tert-buty l-1-oxaspiro (4,5) deca-6, 9-diene-2,8-dione and trifluoroacetamide. These four compounds were then tested, and they reduced urediniospore germination and uredinial density in inoculations of normally susceptible P. purdomii with Melampsora larici-populina. Thus, the cuticle of P. tomentosa can contribute to pre-haustorial, non-host resistance to M. larici-populina.

Apparent penetration depth in attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy of Allium cepa L. epidermis and cuticle.

Over the past decades, ATR-FTIR has emerged as promising tool for the identification of plants at the genus and (sub-) species level through surface measurements of intact leaves. Theoretical considerations regarding the penetration depth of the evanescent wave into the sample and the thickness of plant leaf cuticles suggest that the structure and composition of the cuticle represent universal taxonomic markers. However, experimental evidence for this hypothesis is scarce. In the current contribution, we present results of a series of simple experiments on epidermal monolayers derived from the bulbs of Allium cepa L. (Amaryllidaceae) as a model system to study the effect of an IR active probe located beyond the theoretical penetration depth of the evanescent wave. We found that this probe had a significant influence on the ATR-FTIR spectra for up to 4 epidermal layers stacked on top of each other corresponding to a total thickness of around 60 μm, exceeding the theoretical penetration depth of the evanescent wave by a factor of around 20. Altogether, our data indicate a major discrepancy between theory and practice in ATR-FTIR spectroscopy in general and provide strong evidence that in general plant leaf spectra cannot be fully explained by the structure and composition of the cuticle alone.

A simple technique for assessing the cuticular diffusion of humic acid biostimulants

BackgroundExperimental determination of the extent and rate of transport of liquid humates supplied to plants is critical in testing physiological effects of such biostimulants which are often supplied as foliar sprays. Therefore, an original experimental method for the qualitative investigation and quantitative description of the penetration of humates through plant cuticles is proposed, tested, and evaluated.ResultsThe proposed method involves the isolation of model plant leaf cuticles and the subsequent in vitro evaluation of cuticular humate transport. The employed novel methodology is based on a simple diffusion couple arrangement involving continuous spectrophotometric determination of the amount of penetrated humate in a hydrogel diffusion medium. Prunus laurocerasus leaf cuticles were isolated by chemical and enzymatic treatment and the rate of cuticular penetration of a commercial humate (lignohumate) was estimated over time in quantitative and qualitative terms. Different rates of lignohumate transport were determined for abaxial and adaxial leaf cuticles also in relation to the different cuticular extraction methods tested.ConclusionsThe proposed methodology represents a simple and cheap experimental tool for the study on the trans-cuticular penetration of humic-based biostimulants.

Mathematical Modeling of Diffusion of a Hydrophilic Ionic Fertilizer in Plant Cuticles: Surfactant and Hygroscopic Effects.

The agricultural industry requires improved efficacy of sprays being applied to crops and weeds to reduce their environmental impact and increase financial returns. One way to improve efficacy is by enhancing foliar penetration. The plant leaf cuticle is the most significant barrier to agrochemical diffusion within the leaf. The importance of a mechanistic mathematical model has been noted previously in the literature, as each penetration experiment is dictated by its specific parameters, namely plant species, environmental conditions such as relative humidity and spray formulation including adjuvant addition. A mechanistic mathematical model has been previously developed by the authors, focusing on plant cuticle diffusion of calcium chloride through tomato fruit cuticles including pore swelling, ion binding and evaporation, along with the ability to vary the active ingredient concentration and type, relative humidity and plant species. Here we further develop this model to include adjuvant effects as well as the hygroscopic nature of deliquescent ionic solutions with evaporation on the cuticle surface. These modifications to a penetration and evaporation model provide a novel addition to the literature and allow the model to be applied to many types of evaporating ionic hygroscopic solutions on many types of substrates, not just plant cuticles. We validate our theoretical model results against appropriate experimental data, discuss key sensitivities and relate theoretical predictions to physical mechanisms. The important governing mechanisms influencing surfactant enhanced penetration of ionic active through plant cuticles were found to be aqueous pore radius, pore density, cuticle thickness and initial contact angle of the applied droplet; ion binding, relative humidity and evaporation including hygroscopic water absorption parameters for point of deliquescence. The sensitivity analysis indicated surfactants increase penetration by changing the point of deliquescence of a solution, which alters the water absorption and the initial contact angle, which alters the number of pores under the droplet. The results of the validation and sensitivity analysis imply that this model accounts for many of the mechanisms governing penetration in plant cuticles.

Cuticle surfaces of fossil plants as a potential proxy for volcanic SO2 emissions: observations from the Triassic\u2013Jurassic transition of East Greenland

Flood basalt volcanism has been implicated in several episodes of mass extinctions and environmental degradation in the geological past, including at the Triassic–Jurassic (Tr–J) transition, through global warming caused by massive outgassing of carbon dioxide. However, the patterns of biodiversity loss observed are complicated and sometimes difficult to reconcile with the effects of global warming alone. Recently, attention has turned to additional volcanic products as potential aggravating factors, in particular sulphur dioxide (SO2). SO2 acts both directly as a noxious environmental pollutant and indirectly through forming aerosols in the atmosphere, which may cause transient global dimming and cooling. Here, we present a range of morphological changes to fossil plant leaf cuticle surfaces of hundreds of Ginkgoales and Bennettitales specimens across the Tr–J boundary of East Greenland. Our results indicate that morphological structures of distorted cuticles near the Tr–J boundary are consistent with modern cuticle SO2-caused damage and supported by recent leaf-shape SO2 proxy results, thus identifying cuticle surface morphology as a potentially powerful proxy for SO2. Recording the timing and duration of SO2 emissions in the past may help distinguish between the driving agents responsible for mass extinction events and thus improve our understanding of the Earth System.

Nonlinear Porous Diffusion Modeling of Hydrophilic Ionic Agrochemicals in Astomatous Plant Cuticle Aqueous Pores: A Mechanistic Approach.

The agricultural industry requires improved efficacy of sprays being applied to crops and weeds in order to reduce their environmental impact and deliver improved financial returns. Enhanced foliar uptake is one means of improving efficacy. The plant leaf cuticle is known to be the main barrier to diffusion of agrochemicals within the leaf. The usefulness of a mathematical model to simulate uptake of agrochemicals in plant cuticles has been noted previously in the literature, as the results of each uptake experiment are specific to each formulation of active ingredient, plant species and environmental conditions. In this work we develop a mathematical model and numerical simulation for the uptake of hydrophilic ionic agrochemicals through aqueous pores in plant cuticles. We propose a novel, nonlinear, porous diffusion model for ionic agrochemicals in isolated cuticles, which extends simple diffusion through the incorporation of parameters capable of simulating: plant species variations, evaporation of surface droplet solutions, ion binding effects on the cuticle surface and swelling of the aqueous pores with water. We validate our theoretical results against appropriate experimental data, discuss the key sensitivities in the model and relate theoretical predictions to appropriate physical mechanisms. Major influencing factors have been found to be cuticle structure, including tortuosity and density of the aqueous pores, and to a lesser extent humidity and cuticle surface ion binding effects.

Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition.

Electron microscopy techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been invaluable tools for the study of the micromorphology of plant cuticles. However, for electron microscopy, the preparation techniques required may invariably introduce artefacts in cuticle preservation. Further, there are a limited number of methods available for quantifying the image data obtained through electron microscopy. Therefore, in this study, optical microscopy techniques were coupled with staining proceduresand, along with SEM were used to qualitatively and quantitatively assess the ultrastructure of plant leafcuticles. Leaf cryosections of Triticum aestivum (wheat), Zea mays (maize), and Lupinus angustifolius (lupin) were stained with either fat-soluble azo stain Sudan IV or fluorescent, diarylmethane Auramine O and were observed under confocal laser scanning microscope (CLSM). For all the plant species tested, the cuticle on the leaf surfaces could be clearly resolved in many cases into cuticular proper (CP), external cuticular layer (ECL), and internal cuticular layer (ICL). Novel image data analysis procedures for quantifying the epicuticular wax micromorphology were developed, and epicuticular waxes of L. angustifolius were described here for the first time. Together, application of a multifaceted approach involving the use of a range of techniques to study the plant cuticle has led to a better understanding of cuticular structure and provides new insights into leaf surface architecture.

Surfactant softening of plant leaf cuticle model wax – A Differential Scanning Calorimetry (DSC) and Quartz Crystal Microbalance with Dissipation (QCM-D) study

The aim was to quantify the softening effect that two surfactants (C10EO7 and C8G1.6) have on a plant leaf cuticle model wax. Effects on the thermotropic phase behavior and fluidity of the wax (C22H45OH/C32H66/H2O) were determined.The model wax is crystalline at ambient conditions, yet it is clearly softened by the surfactants. Both surfactants decreased the transition temperatures in the wax and the G″/G′ ratio of the wax film increased in irreversible steps following surfactant exposure. C10EO7 has a stronger fluidizing effect than C8G1.6 due to stronger interaction with the hydrophobic waxes.Intracuticular waxes (IW) comprise both crystalline and amorphous domains and it has previously been proposed that the fluidizing effects of surfactants are due to interactions with the amorphous parts. New data suggests that this may be a simplification. Surfactants may also absorb in crevices between crystalline domains. This causes an irreversible effect and a softer cuticle wax.

Ultrastructure of plant leaf cuticles in relation to sample preparation as observed by transmission electron microscopy.

The leaf cuticular ultrastructure of some plant species has been examined by transmission electron microscopy (TEM) in only few studies. Attending to the different cuticle layers and inner structure, plant cuticles have been grouped into six general morphological types. With the aim of critically examining the effect of cuticle isolation and preparation for TEM analysis on cuticular ultrastructure, adaxial leaf cuticles of blue-gum eucalypt, grey poplar, and European pear were assessed, following a membrane science approach. The embedding and staining protocols affected the ultrastructure of the cuticles analysed. The solubility parameter, surface tension, and contact angles with water of pure Spurr's and LR-White resins were within a similar range. Differences were however estimated for resin : solvent mixtures, since Spurr's resin is combined with acetone and LR-White resin is mixed with ethanol. Given the composite hydrophilic and lipophilic nature of plant cuticles, the particular TEM tissue embedding and staining procedures employed may affect sample ultrastructure and the interpretation of the results in physicochemical and biological terms. It is concluded that tissue preparation procedures may be optimised to facilitate the observation of the micro- and nanostructure of cuticular layers and components with different degrees of polarity and hydrophobicity.

Characterization of a plant leaf cuticle model wax, phase behaviour of model wax–water systems

We investigated the thermotropic phase behaviour of plant leaf intracuticular wax and two representatives of its main components, 1-docosanol (C22H45OH) and dotriacontane (C32H66), in dry and hydrated state. One objective was to obtain a model wax, which can be used to estimate formulations effects on cuticle diffusivity in vitro. The two wax components were chosen based on results from Gas Chromatography coupled to Mass Spectrometry analysis of cuticular wax. The wax was extracted from Clivia Miniata Regel leaves and contained 68% primary alcohols (C16–C32) and 16% n-alkanes (C21–C33). Differential Scanning Calorimetry, Polarized Light Microscopy and Small- and Wide Angle X-ray Diffraction were used to characterize the cuticular extract and the phase behaviour of the C22H45OH/C32H66/H2O model system. Four individual crystalline phases were discovered in the model wax–water system and eutectic melting occurred in both dry and hydrated state. The thermotropic transitions of the model wax occur within the broader transition region of the extracted leaf wax.

Effects of surfactants and thermodynamic activity of model active ingredient on transport over plant leaf cuticle

The main objective of this study was to investigate the mechanism of molecular transport across the cuticle of Clivia leaves. In vitro diffusion methodology was used to investigate the transport of a systemic fungicide, tebuconazole, over a model silicone membrane, enzymatically isolated cuticle membranes, and dermatomed leaves. It was shown that dermatomed leaves may replace enzymatically isolated cuticles. Furthermore, the effects of two surfactants, C10EO7 and C8G1.6, on the fungicide transport were investigated.Tebuconazole cuticle permeation was described using Fick's first law of diffusion, expressed by the thermodynamic activity of the solute in the membrane. A new method for calculation of diffusion coefficients in the membrane is proposed. To access the thermodynamic activity of the fungicide in the membranes, sorption isotherms of tebuconazole in the membrane materials studied were recorded. The thermodynamic activity of the fungicide in aqueous solutions was calculated from solubility data. For that purpose, the effect of surfactants on tebuconazole solubility was studied. The results show that addition of surfactants allows for higher concentrations of tebuconazole available for penetration. Nonetheless, at a fixed fungicide thermodynamic activity, all formulations produced the same flux over the silicone membrane independently on the fungicide concentration. This shows that the driving force across non-responding membranes is the gradient of thermodynamic activity, rather than the gradient of the fungicide concentration. In case of leaves, surfactants induced the same quantitative increase in both flux and diffusion coefficient of solute in the cuticle, while the cuticle-water partition coefficient was unaffected.

Morphological Characteristics of Ginseng Leaves in High-Temperature Injury Resistant and Susceptible Lines of Panax ginseng Meyer

Plant leaf cuticle is related to the prevention of moisture loss, transpiration, and diffusion of light reflection. The purpose of this study was to examine the morphological characteristics of ginseng leaves in ginseng plants resistant and susceptible to high temperature injury (HTI) to be related with the leaf-burning. For the HTI resistant lines Yunpoong, high-temperature injury resistance (HTIR) 1, HTIR 2, and HTIR 3, and the HTI-susceptible line Chunpoong, the cuticle densities were 53.0%, 46.2%, 44.9%, 48.0%, and 17.0%; the adaxial leaf cuticle layers were 141.3, 119.7, 119.7, 159.4, and 85.0 nm in thickness; the abaxial leaf cuticle layers were 153.6, 165.8, 157.9, 199.6, and 119.4 nm in thickness; and the stomtal lengths were 21.7, 32.4, 29.4, 30.9, and 21.8 μm, respectively. All of these aspects suggest that HTI resistant lines have higher cuticle density, thicker adaxial and abaxial leaf cuticle layers, and longer of stomta length than the HTI-susceptible line, protecting leaves from moisture loss and excessive transpiration under high temperatures to be resistant against the leaf-burning.

Quantification of lateral heterogeneity in carbohydrate permeability of isolated plant leaf cuticles

In phyllosphere microbiology, the distribution of resources available to bacterial colonizers of leaf surfaces is generally understood to be very heterogeneous. However, there is little quantitative understanding of the mechanisms that underlie this heterogeneity. Here, we tested the hypothesis that different parts of the cuticle vary in the degree to which they allow diffusion of the leaf sugar fructose to the surface. To this end, individual, isolated cuticles of poplar leaves were each analyzed for two properties: (1) the permeability for fructose, which involved measurement of diffused fructose by gas chromatography and flame ionization detection (GC–FID), and (2) the number and size of fructose-permeable sites on the cuticle, which was achieved using a green-fluorescent protein (GFP)-based bacterial bioreporter for fructose. Bulk flux measurements revealed an average permeance P of 3.39 × 10−9 ms−1, while the bioreporter showed that most of the leaching fructose was clustered to sites around the base of shed trichomes, which accounted for only 0.37% of the surface of the cuticles under study. Combined, the GC–FID and GFP measurements allowed us to calculate an apparent rate of fructose diffusion at these preferential leaching sites of 9.15 × 10−7 ms−1. To the best of our knowledge, this study represents the first successful attempt to quantify cuticle permeability at a resolution that is most relevant to bacterial colonizers of plant leaves. The estimates for P at different spatial scales will be useful for future models that aim to explain and predict temporal and spatial patterns of bacterial colonization of plant foliage based on lateral heterogeneity in sugar permeability of the leaf cuticle.

Modelling sugar diffusion across plant leaf cuticles: the effect of free water on substrate availability to phyllosphere bacteria

We present a continuous model for the diffusion of sugars across intact plant leaf cuticles. It is based on the flow of sugars from a source, representing the leaf apoplast, to a sink, in the shape of a hemispherical drop of water on the outside of the cuticle. Flow is a function of the difference between sugar concentrations C(Source) and C(Sink) , permeability P of the cuticle, volume V(Sink) of the water drop, as well as its contact angle α with the cuticle surface. Using a bacterial bioreporter for fructose, and a two-compartment experimental set-up consisting of isolated cuticles of walnut (Juglans regia) carrying water droplets while floating on solutions with increasing concentrations of fructose, we determined a value of 1 × 10⁻⁶ m h⁻¹ for P. Using this value, we explored different scenarios for the leaching of sugars across plant leaf cuticles to reveal in quantitative terms how diffusion takes longer when V(Sink) increases, P decreases or α increases. Bacterial growth was modelled as a function of changes in P, α and V(Sink) and was consistent with observations or suggestions from the literature in relation to the availability of free water on leaves. These results are discussed in the light of bacteria as ecosystem engineers, i.e. with the ability to modify the plant leaf surface environment in favour of their own survival, e.g. by increasing cuticle leakage or leaf wetness. Our model represents a first step towards a more comprehensive model which will enhance our quantitative understanding of the factors that play a role in nutrient availability to bacterial colonizers of the phyllosphere, or plant leaf surface.