A new technique for measurement of water permeability of stomatous cuticular membranes isolated from Hedera helix leaves

Transpiration of cuticular membranes isolated from the lower stomatous surface of Hedera helix (ivy) leaves was measured using a novel approach which allowed a distinction to be made between gas phase diffusion (through stomatal pores) and solid phase diffusion (transport through the polymer matrix membrane and cuticular waxes) of water molecules. This approach is based on the principle that the diffusivity of water vapour in the gas phase can be manipulated by using different gases (helium, nitrogen, or carbon dioxide) while diffusivity of water in the solid phase is not affected. This approach allowed the flow of water across stomatal pores ('stomatal transpiration') to be calculated separately from the flow across the cuticle (cuticular transpiration) on the stomatous leaf surface. As expected, water flux across the cuticle isolated from the astomatous leaf surface was not affected by the gas composition since there are no gas-filled pores. Resistance to flux of water through the solid cuticle on the stomatous leaf surface was about 11 times lower than cuticular resistance on the astomatous leaf surface, indicating pronounced differences in barrier properties between cuticles isolated from both leaf surfaces. In order to check whether this difference in resistance was due to different barrier properties of cuticular waxes on both leaf sides, mobility of 14C-labelled 2,4-dichlorophenoxy-butyric acid 14C-2,4-DB) in reconstituted cuticular wax isolated from both leaf surfaces was measured separately. However, mobility of 14C-2,4-DB in reconstituted wax isolated from the lower leaf surface was 2.6 times lower compared with the upper leaf side. The significantly higher permeability of the ivy cuticle on the lower stomatous leaf surface compared with the astomatous surface might result from lateral heterogeneity in permeability of the cuticle covering normal epidermal cells compared with the cuticle covering the stomatal cell surface.     

Cuticular permeability of the three tree species Prunus Iaurocerasus L., Ginkgo biloba L. and Juglans regia L.: comparative investigation of the transport properties of intact leaves, isolated cuticles and reconstituted cuticular waxes

Cuticular transport properties of intact leaves, isolated cuticularmembranes and reconstituted cuticular waxes of the three treespecies Prunus laurocerasus L., Ginkgo biloba L. and Juglansregia L. were measured using six different ¹⁴C-labelled compounds,benzoic acid, salicylic acid, 2,4-dichlorophenoxy acid, metribuzin,4-nitrophenol, and atrazine. For the same compound and the samespecies, the permeance of the intact leaf and the isolated cuticlewas equal. This provides strong evidence demonstrating thattransport properties of cuticles are not altered during isolation.Additionally, diffusion coefficients of the ¹⁴C-labelled compoundsin isolated and subsequently reconstituted cuticular wax ofthe three tree species were measured. Permeances of intact leavesand isolated cuticles could be predicted from diffusion coefficients,wax/water partition coefficients and the thickness of the transport-limitingwax layer with a mean deviation of about 1.7. This providesevidence that transport properties of recrystallized cuticularwaxes do indeed reflect barrier properties of isolated cuticularmembranes and intact leaves with in situ waxes. Thus, it canbe concluded that the investigation of cuticular permeabilityusing the three independent experimental systems of differentcomplexity give comparable results. Finally, it was observedthat permeances and diffusion coefficients measured with P.laurocerasus were always significantly lower than those measuredwith G. biloba and J. regia. This is interpreted as an ecologicaladaptation of the respective species. The evergreen speciesP. laurocerasus must be more adapted to environmental stresssuch as drought and frost injury compared to the two deciduousspecies G. biloba and J. regia. Key words: Cuticular permeability, diffusion coefficient, leaf surface, permeance, plant cuticle, transport     

Effect of temperature on cuticular transpiration of isolated cuticular membranes and leaf discs

Cuticular transpiration was measured in the temperature range between 10 degrees C and 55 degrees C using tritiated water and five species (Vinca major L., Prunus laurocerasus L., Forsythia intermedia L., Citrus aurantium L., and Hedera helix L.). Cuticular water permeabilities measured with isolated cuticular membranes were not different from cuticular water permeabilities measured with leaf discs. Depending on the species cuticular water permeabilities increased by factors between 12 (V. major) to 264 (H. helix) when temperature was increased from 10 degrees C to 55 degrees C. Arrhenius plots (lnP versus 1/T) of all investigated species were characterized by phase transitions occurring in the temperature range of 30-39 degrees C. Activation energies for water permeability across plant cuticles below and above the midpoint of phase transition were calculated from Arrhenius plots. Depending on the species they varied between 26 (F. intermedia) to 61 kJ mol(-1) (H. helix) below the phase transition and from 67 (V. major) to 122 kJ mol(-1) (F. intermedia) above the phase transition. Since the occurrence of phase transitions always lead to significantly increased rates of cuticular transpiration it is argued that temperatures higher than 35 degrees C caused structural defects to the transport-limiting barrier of the plant cuticles of all species investigated.     

Effect of humidity on cuticular water permeability of isolated cuticular membranes and leaf disks

The effects of humidity on water permeability of astomatous, isolated cuticular membranes and leaf disks of Citrus aurantium L., Vinca major L., Prunus laurocerasus L., Hedera helix L. and Forsythia intermedia (Thunb.) Vahl. were investigated by a new method using 3H2O. With isolated cuticular membranes of P. laurocerasus the isotope method resulted in values similar to those obtained by a well-established gravimetric method. Cuticular water permeability significantly increased by factors of 2 to 3 when air humidities increased from 2 to 100%. Plots of permeances vs. air humidity were non-linear and the slope increased with increasing air humidity. Permeances of intact leaf disks showed a response to increasing humidity similar to those of isolated cuticular membranes. When cuticular water permeability was measured using wax-free, isolated polymer matrix membranes that had been methylated, the effect of air humidity was significantly suppressed compared to non-methylated polymer matrix membranes. From this observation it is concluded that non-esterified, free carboxyl groups present in the cutin polymer matrix significantly contribute to the effect of humidity on cuticular water permeability. These and other polar groups sorb water, which in turn increases the water permeability of polar domains of the cuticle. This humidity-sensitive, polar path of cuticular water permeability is arranged in parallel with the major, dominating and humidity-independent, non-polar path of cuticular water permeability formed by the lipophilic wax components of the cuticle. This conclusion is supported by the fact that cuticular transpiration can be increased by orders of magnitude upon (i) wax extraction, (ii) increase in temperature or (iii) the action of plasticizers, none of which influenced or only marginally influenced the permeability of inorganic ions penetrating plant cuticles across humidity-sensitive polar pores.     

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。     

Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis

BACKGROUND: The plant cuticle is an extracellular lipophilic biopolymer covering leaf and fruit surfaces. Its main function is the protection of land-living plants from uncontrolled water loss. In the past, the permeability of the cuticle to water and to non-ionic lipophilic molecules (pesticides, herbicides and other xenobiotics) was studied intensively, whereas cuticular penetration of polar ionic compounds was rarely investigated. RECENT PROGRESS: Recent work measuring cuticular penetration of inorganic and organic ions is presented; the effects of molecular size of ions, temperature, wax extraction, humidity and plasticizers strongly support the conclusion that ions penetrate cuticles via water-filled pores. The cuticle covering stomata and trichomes forms the preferential site of ion penetration. This indicates that cuticles possess a pronounced lateral heterogeneity: the largest fraction of the cuticle surface is covered by the lipophilic domains of cutin and wax, but to a certain extent polar domains are also present in the cuticle, which form preferential sites of penetration for polar compounds. THE FUTURE: The chemical nature of these polar domains awaits detailed characterization, which will be of major importance in agriculture and green biotechnology, since polar paths of diffusion represent the most important transport routes for foliar-applied nutrients. Furthermore, many compounds acting as inducers of gene expression in transgenic plants are ionic and need to penetrate the cuticle via polar paths in order to be active.     

A simple photometric device analysing cuticular transport physiology: surfactant effect on permeability of isolated cuticular membranes of Prunus laurocerasus L.

4-Nitrophenol permeabilities of astomatous cuticular membranesisolated from the upper surface of Prunus laurocerasus L. leaveswere measured applying a newly developed photometric device.Isolated cuticles were mounted between donor and receiver compartmentsof a stainless steel transport chamber. 4-Nitrophenol was appliedas non-dissociated species in citric buffer at pH 3.0 in thedonor compartment and sampled as dissociated species in thereceiver compartment in borate buffer at pH 9.0. Permeances,calculated from steady-state rates of 4-nitrophenol permeation,ranged from 1.73 10^–10 m s^–1 up to 38.410^–10ms^–1. They were in the same order of magnitude comparedto published permeances obtained with a different method usingradiolabelled 4-nitrophenol and isolated cuticles of Citrusaurantium L. In the presence of the surfactant Brij 30, whichis a polydisperse alcohol ethoxylate, cuticular permeabilitiesincreased on average by a factor of 37. Cuticles, initiallyhaving the lowest permeabilities, exhibited the highest increaseof their permeabilities due to the surfactant and vice versa.This increase ofcuticular permeabilities in the presence ofa surfactant is interpreted as a plasticizing effect of thesurfactant molecules on the cuticular wax forming the cuticulartransport barrier. Furthermore, surfactant-induced increasesof cuticular permeabilities were reversible to a large extent.Permeabilities decreased again after the removal of Brij 30reaching final values about 6-times higher compared to the initialpermeabilities. This demonstrates that the surfactant and thepermeating molecule must be present simultaneously in the cuticlein order to enhance cuticular permeation. Possible applicationsof this simple photometric device analysing further aspectsof cuticular transport physiology are finally suggested. Key words: Cuticular transport, leaf surface, permeability, plant cuticle, surfactant     

Plant response to drought stress simulated by ABA application: Changes in chemical composition of cuticular waxes

Plant cuticles form the interface between epidermal plant cells and the atmosphere. The cuticle creates an effective barrier against water loss, bacterial and fungal infection and also protects plant tissue from UV radiation. It is composed of the cutin matrix and embedded soluble lipids also called waxes. Chemical composition of cuticular waxes and physiological properties of cuticles are affected by internal regulatory mechanisms and environmental conditions (e.g. drought, light, and humidity). Here, we tested the effect of drought stress simulation by the exogenous application of abscisic acid (ABA) on cuticular wax amount and composition. ABA-treated plants and control plants differed in total aboveground biomass, leaf area, stomatal density and aperture, and carbon isotope composition. They did not differ in total wax amount per area but there were peculiar differences in the abundance of particular components. ABA-treated plants contained significantly higher proportions of aliphatic components characterized by chain length larger than C₂₆, compared to control plants. This trend was consistent both between and within different functional groups of wax components. This can lead to a higher hydrophobicity of the cuticular transpiration barrier and thus decrease cuticular water loss in ABA-treated plants. At both ABA-treated and control plants alcohols with chain length C₂₄ and C₂₆ were predominant. Such a shift towards wax compounds having a higher average chain length under drought conditions can be interpreted as an adaptive response of plants towards drought stress.     

Review of sorption and diffusion of lipophilic molecules in cuticular waxes and the effects of accelerators on solute mobilities

Many agrochemicals are applied to the leaf surfaces of crop plants. Systemic chemicals have to penetrate through the cuticle, which forms an effective transport barrier. The barrier properties of cuticles are mainly due to the cuticular waxes deposited as partially crystalline aggregates on the outer surfaces of leaves. Substances increasing the mobilities of agrochemicals in cuticular waxes are called accelerators and it is shown that they act as plasticizers when absorbed by cuticular waxes. They decrease the barrier properties of the waxes and thus increase the mobilities of the agrochemicals through them. In order to analyse the efficiency of different accelerators, the sorption and mobility of both agrochemicals and accelerators within cuticular waxes was measured. Such information was used to establish correlations between the internal concentrations of accelerators and their mobility-enhancing effects on agrochemicals in the cuticle. This, in turn, allowed the determination and comparison of the intrinsic effects of different accelerators and to rationalize the effect of accelerators on the cuticular permeability of agrochemicals. Results describing the sorption (partition coefficients) and mobility (diffusion coefficients) of lipophilic organic molecules in reconstituted cuticular waxes from different plant species, and the effect of two different classes of accelerators (alcohol ethoxylates and n-alkyl esters), on the mobility of organic molecules are presented and discussed.     

PENETRATION OF 2,4-D IN RELATION TO CUTICLE THICKNESS

Cuticle thickness was measured, either by direct microscopic examination or as weight per unit area, for the astomatous cuticle from the upper leaf surface of nine species and from tomato fruit. Thickness ranged from 1.4 μm for peach leaf cuticle to 10.8 μm for oleander leaf cuticle, and the weight from 0.19 mg/cm² to 1.26 mg/cm², respectively. Cuticles were isolated by the pectinase method and permeability to 2,4-D was determined. There was no correlation between cuticle thickness and penetration of 2,4-D, either for non-dewaxed cuticles or after chloroform extraction of waxes. Penetration of 2,4-D was increased following wax removal, but there was no correlation between wax content and the magnitude of the increase. It is suggested that cutin and wax qualitative composition are probably more important than thickness in determining relative permeability of cuticle from different plant species to 2,4-D.     

Determination of diffusion coefficients of octadecanoic acid in isolated cuticular waxes and their relationship to cuticular water permeabilities

Transport properties of cuticular waxes from 40 different plant species were investigated by measuring desorption rates of ¹⁴C-labelled octadecanoic acid from isolated and subsequently reconstituted wax. Diffusion coefficients (D) of octadecanoic acid in reconstituted waxes, calculated from the slopes of the regression lines fitted to the linearized portions of desorption kinetics, ranged from 1.2 × 10^?19 m² s^?1 (Senecio kleinia leaf) to 2.9 × 10^?17 m² s^?1 (Malus cf. domestica fruit). Cuticular water permeabilities (cuticular transpiration) measured with intact cuticular membranes isolated from 24 different species varied from 1.7 × 10^?11 m s^?1 (Vanilla planifolia leaf) up to 2.1 × 10^?9 m s^?1 (Malus cf. domestica fruit), thus covering a range of more than 2 orders of magnitude. Cuticular water permeabilities were highly correlated with diffusion coefficients of octadecanoic acid in isolated cuticular wax of the same species. It is therefore possible to estimate cuticular barrier properties of stomatous leaf surfaces or of leaves where isolation of the cuticle is impossible by measuring D of octadecanoic acid in isolated waxes of these leaves.     

Water permeability of plant cuticles: permeance,diffusion and partition coefficients

Summary Using isolated cuticular membranes from ten woody and herbaceous plant species, permeance and diffusion coefficients for water were measured, and partition coefficients were calculated. The cuticular membranes of fruit had much higher permeance and diffusion coefficients than leaf cuticular membranes from either trees or herbs. Both diffusion and partition coefficients increased with increasing membrane thickness. Thin cuticles, therefore, tend to be better and more efficient water barriers than thick cuticles. We compared the diffusion coefficients and the water content of cuticles as calculated from transport measurements with those obtained from water vapor sorption. There is good to fair agreement for cuticular membranes with a low water content, but large discrepancies appear for polymer matrix membranes with high permeance. This is probably due to the fact that diffusion coefficients obtained from transport measurements on membranes with high permeance and water content are underestimated. Water permeabilities of polyethylene and polypropylene membranes are similar to those of leaf cuticular membranes. However, leaf cuticles have much lower diffusion coefficients and a much greater water content than these synthetic polymers. This suggests that cuticles are primarily mobility barriers as far as water transport is concerned.     

Water permeability of plant cuticles: The effect of temperature on diffusion of water

The effect of temperature on water permeability of plant cuticles (astomatous Citrus leaf cuticles) has been investigated. The Arrhenius plot (logarithm of the permeability coefficient vs. 1/temperature) has two linear portions that intersect at 44° C. Evidence is presented to show that this intersection represents the solid/liquid phase transition of cuticular lipids. As the Arrhenius plot has only one phase transition in the temperature range of 5 to 80° C, it appears that all soluble cuticular lipids in the cuticle are present as a homogeneous mixture rather than as individual layers differing in composition. This view is supported by electron spin resonance evidence showing homogenous distribution of spin label fatty acids. The original distribution of soluble cuticular lipids is irreversibly altered by heating cuticular membranes above the transition temperature. This is accompanied by an irreversible increase in water peremeability, demonstrating the importance of the structure of cuticular lipids with regard to cuticular permeability.Abbreviations CM cuticular membranes - MX polymer matrix - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid - J flux - ESR electron spin resonance - THO tritiated water     

Needle browning and death in the flagged crown of Abies mariesii in the timberline ecotone of the alpine region in central Japan

In the flagged crown, which is asymmetric growth formed by severe stresses during winter in alpine regions, needles of evergreen conifers often became brown and died in early spring, but did not in a cushion-shaped crown. Needle browning and death is thought to occur by increasing transpiration due to a thinner cuticle or mechanical damage to the cuticle by wind-born snow and ice particles. To confirm whether the needle browning and death in the flagged crown of Abies mariesii Mast., in the alpine region of Japan conform with this concept, we assessed mechanical damage of the needle cuticle in a timberline ecotone and evaluated the effect of cuticle thickness on cuticular resistance. Mechanical damage on needle cuticles of A. mariesii was not observed. In the cushion-shaped crown, epicuticular wax covered the cuticle and plugged stomatal antechambers. In the flagged crown, epicuticular wax was mostly absent. Cuticular resistance in the flagged crown was lower than that in the cushion-shaped crown. However, the cuticle in the flagged crown was thicker than that in the cushion-shaped crown. The needle browning and death in the flagged crown of A. mariesii occurred even though needle cuticles were not mechanically damaged. The thicker cuticle of the flagged crown may play a role in other stresses. To estimate desiccation stress in relation to the cuticle, we need to elucidate not only cuticular resistance and cuticle thickness, but also cuticle quality and structure.     

Thermodynamics of the water permeability of plant cuticles: characterization of the polar pathway

The water permeability of cuticles isolated from the leaves of 14 plant species was measured at temperatures from 10 degrees C to 55 degrees C at 5 K intervals. Permeances increased slightly with temperatures < or =35 degrees C and drastically in the higher temperature range. The data were analysed according to the Arrhenius formalism which led to distinct plots for the lower and higher temperature range, respectively. Activation energies of permeation for the lower temperature range were estimated to amount to 15.2-52.5 kJ mol(-1), at higher temperature activation energies ranged from 52.2-117.3 kJ mol(-1). This thermodynamics approach is used for further elucidating the pathway taken by water across the plant cuticle. Based on the results of this study it is hypothesized that the diffusion of water occurs along polysaccharide strands crossing the cuticle and that the transport properties of these polar pathways change with temperature.     

Analysis of the cuticular wax composition and ecophysiological studies in an arid plant - Ziziphus nummularia (Burm.f.) Wight & Arn.

Plants in arid regions are exposed to various abiotic stresses and the presence of the waxy cuticular layer acts as a defensive barrier, which consists mainly of long chain fatty acids, hydrocarbons and other derived compounds. Studies on the chemical composition and properties of cuticles of arid plants are scanty. The present study deals with the analysis of cuticular wax composition and effect of temperature on some ecophysiological parameters of an important arid plant Ziziphus nummularia. A total of 59 different wax compounds were detected from the leaf cuticle by capillary GC–MS. 4-Hydroxycyclohexanone, Heptacosane and 2,7-Dimethyloctane-3,5-dione were the dominant wax compounds in Z. nummularia. The variation of photosynthetic rate varied from 0.70 to 7.70 µmol CO2 m-2s-1 against the studied temperature range of 15–55 °C. The transpiration rate varies from 1.80 to 8.40 mmol H2O m-2s-1 within the temperature range of 15–55 °C. The quantum yield of photosystem II (Fv/Fm) also exhibited much variation due to the variation of temperature. The results clearly shows that Z. nummularia is highly adapted to restrict water loss and can tolerate high temperatures and can be considered as an appropriate species for vegetating the arid areas.     

Cuticular Pores and Transcuticular Canals in Diverse Fruit Varieties

Dewaxed thin-sectioned and dewaxed isolated mature fruit cuticlesrevealed the unequivocal presence in situ of visibly discrete,ubiquitous, cuticular pores or orifices concomitant with anticlinally-orientedtranscuticular canals in 51 varieties of fruit among 20 plantfamilies. More than 66 per cent of the fruit cuticles have poresand/or canals. No correlation exists between either fruit sizeor pore size and cuticle thickness. Dewaxed cuticles rangedfrom 1.25–22.5 µm in thickness. Canal lengths aredirectly related to cuticle thickness. Cuticular occlusionsof the epidermal cells were found in 76 per cent of the fruitsexamined. Evidence is provided by light microscopy photomicrographs. Fruit cuticles, cuticle morphology, cuticular pores, transcuticular canals     

Apple Fruit Cuticles and the Occurrence of Pores and Transcuticular Canals

Developmental studies were made on the dewaxed thin-sectionedapple fruit cuticles of 10 Malus sylvestris Mill, cultivarsfrom 4 weeks before anthesis through fruit maturation and harvest.Cuticular development appears to correlate well with the generalgrowth of the fruit. However, no correlation exists betweenfruit size and cuticle thickness. Cuticular pores were evidentas early as 1 week before anthesis and transcuticular canalsbecame evident by 1 week following anthesis. Dewaxed thin-sectioned,as well as isolated, mature fruit cuticles of 16 cultivars andfour crab-apples consistently revealed the distinct presenceof ubiquitous pores and canals. Evidence is provided by lightmicroscopy and scanning electron microscopy (SEM). Measurementswere taken of cuticle thicknesses during development and ofcuticular pore dimensions, and calculations were made of poreand canal numbers. Fruit size alone is not directly indicativeof total pore numbers per surface area. Canal lengths are directlyrelated to the developmental thickness of the cuticle. No correlationwas found between the thickness of the mature cuticle and eitherthe number of pores present or the pore diameters. Malus sylvestris Mill, apple fruit, morphology, cuticle, cuticular flanges, cuticular pores, transcuticular canals, ultrastructure     

Differences between water permeability of astomatous and stomatous cuticular membranes: effects of air humidity in two species of contrasting drought-resistance strategy

Cuticular water permeabilities of adaxial and abaxial leaf surfaces and their dependence on relative air humidity (RH) applied in long-term and short-term regimes have been analysed for Hedera helix, native in a temperate climate, and Zamioculcas zamiifolia, native in subtropical regions. The water permeability of cuticular membranes (CM) isolated from the adaxial (astomatous) and abaxial (stomatous) leaf sides was measured using a method which allowed the separation of water diffusion through the remnants of the original stomatal pores from water diffusion through the solid cuticle. The long-term effects of low (20-40%) or high (60-80%) RH applied during plant growth and leaf ontogeny ('growth RH') and the short-term effects of applying 2% or 100% RH while measuring permeability ('measurement RH') were investigated. With both species, water permeability of the solid stomatous CM was significantly higher than the permeability of the astomatous CM. Adaxial cuticles of plants grown in humid air were more permeable to water than those from dry air. The adaxial CM of the drought-tolerant H. helix was more permeable and more sensitive to growth RH than the adaxial CM of Z. zamiifolia, a species avoiding water stress. However, permeability of the solid abaxial CM was similar in both species and independent of growth RH. The lack of a humidity response in the abaxial CM is attributed to a higher degree of cuticular hydration resulting from stomatal transpiration. The ecophysiological significance of higher permeability of the solid stomatous CM compared to the astomatous CM is discussed.