Compositional, structural and functional cuticle analysis of Prunus laurocerasus L. sheds light on cuticular barrier plasticity.

Barrier properties of the hydrophobic plant cuticle depend on its physicochemical composition. The cuticular compounds vary considerably among plant species but also among organs and tissues of the same plant and throughout developmental stages. As yet, these intraspecific modifications at the cuticular wax and cutin level are only rarely examined. Attempting to further elucidate cuticle profiles, we analysed the adaxial and abaxial surfaces of the sclerophyllous leaf and three developmental stages of the drupe fruit of Prunus laurocerasus, an evergreen model plant native to temperate regions. According to gas chromatographic analyses, the cuticular waxes contained primarily pentacyclic triterpenoids dominated by ursolic acid, whereas the cutin biopolyester mainly consisted of 9/10,ω-dihydroxy hexadecanoic acid. Distinct organ- and side-specific patterns were found for cuticular lipid loads, compositions and carbon chain length distributions. Compositional variations led to different structural and functional barrier properties of the plant cuticle, which were investigated further microscopically, infrared spectroscopically and gravimetrically. The minimum water conductance was highlighted at 1 × 10-5 m s-1 for the perennial, hypostomatous P. laurocerasus leaf and at 8 × 10-5 m s-1 for the few-month-living, stomatous fruit suggesting organ-specific cuticular barrier demands.

Building a Barrier: The Influence of Different Wax Fractions on the Water Transpiration Barrier of Leaf Cuticles.

Waxes are critical in limiting non-stomatal water loss in higher terrestrial plants by making up the limiting barrier for water diffusion across cuticles. Using a differential extraction protocol, we investigated the influence of various wax fractions on the cuticular transpiration barrier. Triterpenoids (TRPs) and very long-chain aliphatics (VLCAs) were selectively extracted from isolated adaxial leaf cuticles using methanol (MeOH) followed by chloroform (TCM). The water permeabilities of the native and the solvent-treated cuticles were measured gravimetrically. Seven plant species (Camellia sinensis, Ficus elastica, Hedera helix, Ilex aquifolium, Nerium oleander, Vinca minor, and Zamioculcas zamiifolia) with highly varying wax compositions ranging from nearly pure VLCA- to TRP-dominated waxes were selected. After TRP removal with MeOH, water permeability did not or only slightly increase. The subsequent VLCA extraction with TCM led to increases in cuticular water permeabilities by up to two orders of magnitude. These effects were consistent across all species investigated, providing direct evidence that the cuticular transpiration barrier is mainly composed of VLCA. In contrast, TRPs play no or only a minor role in controlling water loss.

Cuticular wax coverage and its transpiration barrier properties in Quercus coccifera L. leaves: does the environment matter?

Plants prevent uncontrolled water loss by synthesizing, depositing and maintaining a hydrophobic layer over their primary aerial organs-the plant cuticle. Quercus coccifera L. can plastically respond to environmental conditions at the cuticular level. When exposed to hot summer conditions with high vapour-pressure deficit (VPD) and intense solar radiation (Mediterranean atmospheric conditions; MED), this plant species accumulates leaf cuticular waxes even over the stomata, thereby decreasing transpirational water loss. However, under mild summer conditions with moderate VPD and regular solar radiation (temperate atmospheric conditions; TEM), this effect is sharply reduced. Despite the ecophysiological importance of the cuticular waxes of Q. coccifera, the wax composition and its contribution to avoiding uncontrolled dehydration remain unknown. Thus, we determined several leaf traits for plants exposed to both MED and TEM conditions. Further, we qualitatively and quantitatively investigated the cuticular lipid composition by gas chromatography. Finally, we measured the minimum leaf conductance (gmin) as an indicator of the efficacy of the cuticular transpiration barrier. The MED leaves were smaller, stiffer and contained a higher load of cuticular lipids than TEM leaves. The amounts of leaf cutin and cuticular waxes of MED plants were 1.4 times and 2.6 times higher than that found for TEM plants, respectively. In detail, MED plants produced higher amounts of all compound classes of cuticular waxes, except for the equivalence of alkanoic acids. Although MED leaves contained higher cutin and cuticular wax loads, the gmin was not different between the two habitats. Our findings suggest that the qualitative accumulation of equivalent cuticular waxes might compensate for the higher wax amount of MED plants, thereby contributing equally to the efficacy of the cuticular transpirational barrier of Q. coccifera. In conclusion, we showed that atmospheric conditions profoundly affect the cuticular lipid composition of Q. coccifera leaves, but do not alter its transpiration barrier properties.

Cuticular wax coverage and its transpiration barrier properties in Quercus coccifera L. leaves: does the environment matter?

Plants prevent uncontrolled water loss by synthesizing, depositing and maintaining a hydrophobic layer over their primary aerial organs-the plant cuticle. Quercus coccifera L. can plastically respond to environmental conditions at the cuticular level. When exposed to hot summer conditions with high vapour-pressure deficit (VPD) and intense solar radiation (Mediterranean atmospheric conditions; MED), this plant species accumulates leaf cuticular waxes even over the stomata, thereby decreasing transpirational water loss. However, under mild summer conditions with moderate VPD and regular solar radiation (temperate atmospheric conditions; TEM), this effect is sharply reduced. Despite the ecophysiological importance of the cuticular waxes of Q. coccifera, the wax composition and its contribution to avoiding uncontrolled dehydration remain unknown. Thus, we determined several leaf traits for plants exposed to both MED and TEM conditions. Further, we qualitatively and quantitatively investigated the cuticular lipid composition by gas chromatography. Finally, we measured the minimum leaf conductance (gmin) as an indicator of the efficacy of the cuticular transpiration barrier. The MED leaves were smaller, stiffer and contained a higher load of cuticular lipids than TEM leaves. The amounts of leaf cutin and cuticular waxes of MED plants were 1.4 times and 2.6 times higher than that found for TEM plants, respectively. In detail, MED plants produced higher amounts of all compound classes of cuticular waxes, except for the equivalence of alkanoic acids. Although MED leaves contained higher cutin and cuticular wax loads, the gmin was not different between the two habitats. Our findings suggest that the qualitative accumulation of equivalent cuticular waxes might compensate for the higher wax amount of MED plants, thereby contributing equally to the efficacy of the cuticular transpirational barrier of Q. coccifera. In conclusion, we showed that atmospheric conditions profoundly affect the cuticular lipid composition of Q. coccifera leaves, but do not alter its transpiration barrier properties.

The permeation barrier of plant cuticles: uptake of active ingredients is limited by very long-chain aliphatic rather than cyclic wax compounds.

BACKGROUND: The barrier to diffusion of organic solutes across the plant cuticle is composed of waxes consisting of very long-chain aliphatic (VLCA) and, to varying degrees, cyclic compounds like pentacyclic triterpenoids. The roles of both fractions in controlling cuticular penetration by organic solutes, e.g. the active ingredients (AI) of pesticides, are unknown to date. We studied the permeability of isolated leaf cuticular membranes from Garcinia xanthochymus and Prunus laurocerasus for lipophilic azoxystrobin and theobromine as model compounds for hydrophilic AIs. RESULTS: The wax of P. laurocerasus consists of VLCA (12%) and cyclic compounds (88%), whereas VLCAs make up 97% of the wax of G. xanthochymus. We show that treating isolated cuticles with methanol almost quantitatively releases the cyclic fraction while leaving the VLCA fraction essentially intact. All VLCAs were subsequently removed using chloroform. In both species, the permeance of the two model compounds did not change significantly after methanol treatment, whereas chloroform extraction had a large effect on organic solute permeability. CONCLUSION: The VLCA wax fraction makes up the permeability barrier for organic solutes, whereas cyclic compounds even in high amounts have a negligible role. This is of significance when optimizing the foliar uptake of pesticides. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of temperature on the cuticular transpiration barrier of two desert plants with water-spender and water-saver strategies.

The efficacy of the cuticular transpiration barrier and its resistance to elevated temperatures are significantly higher in a typical water-saver than in a water-spender plant growing in hot desert.

The ecophysiology of leaf cuticular transpiration: are cuticular water permeabilities adapted to ecological conditions?

When the stomata are closed under drought, the only route for water loss from the leaf interior to the atmosphere is across the cuticle. Thus, the extent of cuticular transpiration in relation to the reservoirs of water in the plant and the water acquisition from the soil determines the fitness and survival of the plant. It is, therefore, widely assumed that the cuticular water permeability of plants regularly experiencing drought is comparatively low and, thus, adapted to the environment. To test this hypothesis, 382 measurements of cuticular permeability from 160 species were extracted from the literature published between 1996 and 2017. The data had been produced either by using isolated cuticles and astomatous leaf sides or by measuring the minimum leaf conductance under conditions assumed to induce maximum stomatal closure. The species were assigned to 11 life form groups. Except for two particular cases (epiphytes, and climbers and lianas), the cuticular permeabilities of all groups either did not differ significantly or the available data did not allow a statistical test. In conclusion, present knowledge either does not support the hypothesis that ecological adaptions of cuticular water permeability exist or the available data are insufficient to test it.

Chemical Composition and Water Permeability of Fruit and Leaf Cuticles of Olea europaea L.

The plant cuticle, protecting against uncontrolled water loss, covers olive (Olea europaea) fruits and leaves. The present study describes the organ-specific chemical composition of the cuticular waxes and the cutin and compares three developmental stages of fruits (green, turning, and black) with the leaf surface. Numerous organ-specific differences, such as the total coverage of cutin monomeric components (1034.4 µg cm-2 and 630.5 µg cm-2) and the cuticular waxes (201.6 µg cm-2 and 320.4 µg cm-2) among all three fruit stages and leaves, respectively, were detected. Water permeability as the main cuticular function was 5-fold lower in adaxial leaf cuticles (2.1 × 10-5 m s-1) in comparison to all three fruit stages (9.5 × 10-5 m s-1). The three fruit developmental stages have the same cuticular water permeability. It is hypothesized that a higher weighted average chain length of the acyclic cuticular components leads to a considerably lower permeability of the leaf as compared to the fruit cuticle.

Effectiveness of cuticular transpiration barriers in a desert plant at controlling water loss at high temperatures.

Maintaining the integrity of the cuticular transpiration barrier even at elevated temperatures is of vital importance especially for hot-desert plants. Currently, the temperature dependence of the leaf cuticular water permeability and its relationship with the chemistry of the cuticles are not known for a single desert plant. This study investigates whether (i) the cuticular permeability of a desert plant is lower than that of species from non-desert habitats, (ii) the temperature-dependent increase of permeability is less pronounced than in those species and (iii) whether the susceptibility of the cuticular permeability barrier to high temperatures is related to the amounts or properties of the cutin or the cuticular waxes. We test these questions with Rhazya stricta using the minimum leaf water vapour conductance (gmin) as a proxy for cuticular water permeability. gmin of R. stricta (5.41 × 10(-5) m s(-1) at 25 °C) is in the upper range of all existing data for woody species from various non-desert habitats. At the same time, in R. stricta, the effect of temperature (15-50 °C) on gmin (2.4-fold) is lower than in all other species (up to 12-fold). Rhazya stricta is also special since the temperature dependence of gmin does not become steeper above a certain transition temperature. For identifying the chemical and physical foundation of this phenomenon, the amounts and the compositions of cuticular waxes and cutin were determined. The leaf cuticular wax (251.4 µg cm(-2)) is mainly composed of pentacyclic triterpenoids (85.2% of total wax) while long-chain aliphatics contribute only 3.4%. In comparison with many other species, the triterpenoid-to-cutin ratio of R. stricta (0.63) is high. We propose that the triterpenoids deposited within the cutin matrix restrict the thermal expansion of the polymer and, thus, prevent thermal damage to the highly ordered aliphatic wax barrier even at high temperatures.

Water loss from litchi (Litchi chinensis) and longan (Dimocarpus longan) fruits is biphasic and controlled by a complex pericarpal transpiration barrier.

MAIN CONCLUSION: In litchi and longan fruits, a specialised pericarp controls water loss by a protective system consisting of two resistances in series and two water reservoirs separated by a barrier. In the fruits of litchi (Litchi chinensis) and longan (Dimocarpus longan), the pericarp is solely a protective structure lacking functional stomata and completely enclosing the aril that is the edible part. Maintaining a high water content of the fruits is crucial for ensuring the economic value of these important fruit crops. The water loss rates from mature fruits were determined and analysed in terms of the properties of the pericarps. Water loss kinetics and sorption isotherms were measured gravimetrically. The pericarps were studied with microscopy, and cuticular waxes and cutin were analysed with gas chromatography and mass spectrometry. The kinetics of fruit water loss are biphasic with a high initial rate and a lower equilibrium rate lasting for many hours. The outer and inner surfaces of the pericarps are covered with cuticles. Litchi and longan fruits have a unique type of transpiration barrier consisting of two resistances in series (endo- and exocarp cuticles) and two reservoirs of water (aril and mesocarp). The exocarp permeability controls the water loss from fresh fruits while in fruits kept for an extended time at low relative humidity it is determined by the endo- and exocarp permeabilities. Permeances measured are within the range for typical fruit cuticles. The findings may be used to design optimal postharvest storage strategies for litchi and longan fruits.

Aqueous pathways dominate permeation of solutes across Pisum sativum seed coats and mediate solute transport via diffusion and bulk flow of water.

The permeability of seed coats to solutes either of biological or anthropogenic origin plays a major role in germination, seedling growth and seed treatment by pesticides. An experimental set-up was designed for investigating the mechanisms of seed coat permeation, which allows steady-state experiments with isolated seed coats of Pisum sativum. Permeances were measured for a set of organic model compounds with different physicochemical properties and sizes. The results show that narrow aqueous pathways dominate the diffusion of solutes across pea seed coats, as indicated by a correlation of permeances with the molecular sizes of the compounds instead of their lipophilicity. Further indicators for an aqueous pathway are small size selectivity and a small effect of temperature on permeation. The application of an osmotic water potential gradient across isolated seed coats leads to an increase in solute transfer, indicating that the aqueous pathways form a water-filled continuum across the seed coat allowing the bulk flow of water. Thus, the uptake of organic solutes across pea testae has two components: (1) by diffusion and (2) by bulk water inflow, which, however, is relevant only during imbibition.

pH-dependent permeation of amino acids through isolated ivy cuticles is affected by cuticular water sorption and hydration shell size of the solute.

The permeabilities of amino acids for isolated cuticular membranes of ivy (Hedera helix L.) were measured at different pH. Cuticular permeances were lowest for the zwitterionic form at pH 6, followed by the cationic form at pH 1. Highest permeances were obtained for the anionic form at pH 11. Permeances were not correlated with octanol/water partition coefficients and decreased at a given pH with increasing molar volume of the solute. This finding suggests that permeation takes place in the polar cuticular pathways. The effect of pH on the cuticular transport properties was analysed according to the porous membrane model considering the polyelectrolytic character of the cuticle in terms of porosity, tortuosity, and size selectivity of the aqueous cuticular pathway which is altered by pH. An increase of water content and permeability of the cuticular membrane was caused by the dissociation of weak acidic groups with increasing pH leading to a swelling of the cuticle induced by fixed negative charges. In addition, the pH-dependent size of the hydration shell of the amino acids was identified as a secondary factor explaining the variability of cuticular permeances.

Modelling the effects of alcohol ethoxylates on diffusion of pesticides in the cuticular wax of Chenopodium album leaves.

Cuticular waxes represent the first and, in most cases, the limiting barrier for foliar uptake of pesticides from solution. Sorption of pesticides in reconstituted cuticular wax (wax/water partition coefficients) of Chenopodium album L. and in isolated cuticular membranes (cuticle/water partition coefficients) of Prunus laurocerasus L. was determined. Diffusion coefficients of pesticides in reconstituted cuticular wax of C. album leaves were size-dependent, increasing with increasing molar volume. In the presence of alcohol ethoxylates, diffusion coefficients were enhanced by up to two orders of magnitude, and size selectivity was significantly decreased. The accelerating effect and the decrease in size selectivity were attributed to plasticisation of the cuticular wax by the alcohol ethoxylates increasing the fluidity in the wax. A free volume model adopted from polymer science was successfully applied to predict diffusion coefficients of pesticides on the basis of the transport properties of the wax (size selectivity and crystallinity), the molar volume of the diffusing compound and the accelerator concentration in the wax.

Comparison of sorption and diffusion by pyridate and its polar metabolite in isolated cuticular wax of Chenopodium album and Hordeum vulgare.

Sorption and diffusion of the herbicide pyridate and its metabolite CL9673 were measured in reconstituted cuticular waxes isolated from Chenopodium album L. and Hordeum vulgare L. (cultivar Igri) leaves. The compounds have the same basic chemical structure, except that pyridate is characterized by a C8-alkyl chain bound via a thioester to the ionizable hydroxyl group of CL9673. Sorption of the weak acid CL9673 from aqueous solutions into cuticular waxes was pH-dependent, and the apparent wax/water partition coefficients decreased with increasing pH. Wax/water partition coefficients of pyridate were not dependent on pH, and they were about 4 orders of magnitude higher as compared to the nondissociated species of CL9673. Diffusion coefficients measured in reconstituted cuticular wax for CL9673 fitted established models predicting diffusion coefficients in relation to molar volumes. However, this was not the case with pyridate, which was characterized by a self-accelerating effect leading to diffusion coefficients, which were up to 2 orders of magnitude higher than predicted from the molar volume. This is a remarkable result since pyridate represents a compound combining the properties of an active ingredient and of a plasticizer.

Characterization of hydrophilic and lipophilic pathways of Hedera helix L. cuticular membranes: permeation of water and uncharged organic compounds.

The permeability of astomatous leaf cuticular membranes of Hedera helix L. was measured for uncharged hydrophilic (octanol/water partition coefficient log K(O/W) < or =0) and lipophilic compounds (log K(O/W) >0). The set of compounds included lipophilic plant protection agents, hydrophilic carbohydrates, and the volatile compounds water and ethanol. Plotting the mobility of the model compounds versus the molar volume resulted in a clear differentiation between a lipophilic and a hydrophilic pathway. The size selectivity of the lipophilic pathway was described by the free volume theory. The pronounced tortuosity of the diffusional path was caused by cuticular waxes, leading to an increase in permeance for the lipophilic compounds after wax extraction. The size selectivity of the hydrophilic pathway was described by hindered diffusion in narrow pores of molecular dimensions. A distinct increase in size selectivity was observed for hydrophilic compounds with a molar volume higher than 110 cm3 mol(-1). Correspondingly, the size distribution of passable hydrophilic pathways was interpreted as a normal distribution with a mean pore radius of 0.3 nm and a standard deviation of 0.02 nm. The increased permeance of the hydrophilic compounds by the removal of cuticular waxes was attributed to an increase in the porosity, a decrease in the tortuosity, and a widening of the pore size distribution. Cuticular transpiration resulted from the permeation of water across the hydrophilic pathway. The far-reaching implications of two parallel pathways for the establishment of correlations between cuticular structure, chemistry, and function are discussed.

Ecophysiological relevance of cuticular transpiration of deciduous and evergreen plants in relation to stomatal closure and leaf water potential.

The water permeability of the leaves of three deciduous plants (Acer campestre, Fagus sylvatica, Quercus petraea) and two evergreen plants (Hedera helix, Ilex aquifolium) was analysed in order to assess its role as a mechanism of drought resistance. Cuticular permeances were determined by measurement of the water loss through adaxial, astomatous leaf surfaces. Minimum conductances after complete stomatal closure were obtained by leaf drying curves. The comparison of the water permeabilities determined with these two experimental systems revealed good agreement in the case of Acer, Fagus, Quercus, and Ilex. For Hedera the minimum conductance was 3-fold higher than the cuticular permeance indicating a significant contribution of residual stomatal transpiration. The leaf water potential was measured as a function of water content and analysed by pressure-volume curves. The influence of water potential as a component of the driving force for transpirational water loss was assessed in order to identify modifications of the cuticular barrier by the leaf water content. The ecophysiological meaning of the water relations parameters describing transpiration under drought conditions (cuticular transpiration, minimum transpiration, residual stomatal transpiration, effect of leaf water content on transpiration) and the water relations parameters derived from pressure-volume curves (osmotic potential at full saturation, turgor loss point, bulk modulus of elasticity) are discussed with regard to adaptations for drought resistance.