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.     

STRUCTURE OF THE PEAR LEAF CUTICLE WITH SPECIAL REFERENCE TO CUTICULAR PENETRATION

Study of the pear leaf cuticle (Pyrus communis L. ‘Bartlett‘), in both intact and enzymatically isolated forms, has revealed that the cuticular membrane is separated from the underlying epidermal cell wall by a layer of pectic substances which extend into but not through the membrane. A layer of embedded birefringent waxes occurs towards the outer surface of the cuticular membrane. Platelet-like epicuticular waxes are deposited on the outer surface. The upper cuticular membrane is astomatous. The lower epidermis is stomatous, and the outer cuticular membrane is continuous with that lining the substomatal cavity. The lower cuticular membrane is also generally thicker than the upper, and both the upper and lower cuticular membranes are thicker over veinal than over mesophyll tissue. The birefringence frequently is discontinuous over anticlinal walls and over veinal tissue. The lower cuticle appears to contain fewer embedded waxes (as indexed by birefringence) than the upper. Enzymatic isolation of the cuticular membrane from the underlying tissues does not appear to cause any discernible change in structure as viewed with a light microscope. These findings are discussed in light of current knowledge concerning penetration of foliar applied substances into the leaf.     

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.     

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.     

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.     

Equivalent pore radii of hydrophilic foliar uptake routes in stomatous and astomatous leaf surfaces – further evidence for a stomatal pathway

Foliar uptake pathways for hydrophilic solutes were studied by the analysis of co-uptake of ¹⁵N-labelled urea, NH₄⁺ or NO₃⁻ and ¹³C-labelled sucrose across leaf surfaces of various plant species. Uptake of N (y) and sucrose (x) were strongly correlated. Curvilinear regression revealed significantly positive intercepts with the y-axis indicating the involvement of a sucrose-excluding pathway consisting of small pores with radii <0.5 nm. Depending on plant species, N source, leaf side and aperture of stomata, these small pores accounted for 6–62% of total N uptake. Regression analysis revealed that in stomatous leaf surfaces of Vicia faba L., Coffea arabica L. and Prunus cerasus L., the remaining N uptake occurred via another pathway with an estimated average pore radius (r_P) greater than 20 nm. This is two orders of magnitude greater than previous estimations of cuticular r_P, indicating that this pathway, which was only found in stomatous leaf surfaces, was probably not located in the cuticle but at the surfaces of the stomatal pores. In astomatous leaf surfaces of C. arabica and Populus × canadensis Moench, average r_P was 2.0 and 2.4 nm, respectively, which is four to eight times larger than previous estimations of cuticular r_P. These results indicate that for polar solutes, the size exclusion limits of plant surfaces can be considerably larger than previously estimated. The far-reaching implications of these findings are discussed.     

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     

空气湿度与土壤水分胁迫对紫花苜蓿叶表皮蜡质特性的影响

选用2个抗旱性不同的紫花苜蓿品种,敖汉(强抗旱)和三得利(弱抗旱),设置空气湿度(45%-55%和75%-85%)和土壤水分胁迫(75%和35%田间持水量)处理,分析紫花苜蓿叶表皮蜡质含量、组分及晶体结构、气体交换参数、水势及脯氨酸含量的变化规律。结果表明,单独土壤水分胁迫时,紫花苜蓿叶表皮蜡质晶体结构及蜡质总量无显著变化;敖汉蜡质组分中烷类、酯类含量增加,醇类含量下降;三得利醇类含量下降,烷类、酯类含量变化不显著。低空气湿度胁迫时,两品种蜡质总量无显著变化,烷类和酯类含量显著增加,醇类含量显著下降,叶表皮片状蜡质晶体结构熔融呈弥漫性,扩大了对叶表面积的覆盖,其蒸腾速率显著低于正常湿度。复合胁迫处理时,叶表皮片状蜡质晶体结构继续呈弥漫性,烷类、酯类、未知蜡质组分含量均高于单独胁迫处理,醇类含量最低,而蜡质总量除三得利显著高于对照外,其余均无显著差异。紫花苜蓿叶表皮蜡质各组分含量(除醇类)及蜡质总量与光合速率呈显著负相关,与蒸腾速率无显著相关关系。蜡质总量与叶水势呈显著正相关。总体上,敖汉蜡质总量显著高于三得利,蜡质组分中烷类物质的增加有助于提高植株的抗旱性。在复合胁迫下,强抗旱品种主要通过气孔因素控制水分散失,而弱抗旱品种通过气孔和非气孔因素共同控制植物水分散失。     

Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces

The reaction of plants to environmental factors often varies with developmental stage. It was hypothesized, that also the cuticle, the outer surface layer of plants is modified during ontogenesis. Apple plantlets, cv. Golden Delicious, were grown under controlled conditions avoiding biotic and abiotic stress factors. The cuticular wax surface of adaxial apple leaves was analyzed for its chemical composition as well as for its micromorphology and hydrophobicity just after unfolding of leaves ending in the seventh leaf insertion. The outer surface of apple leaves was formed by a thin amorphous layer of epicuticular waxes. Epidermal cells of young leaves exhibited a distinctive curvature of the periclinal cell walls resulting in an undulated surface of the cuticle including pronounced lamellae, with the highest density at the centre of cells. As epidermal cells expanded during ontogenesis, the upper surface showed only minor surface sculpturing and a decrease in lamellae. With increasing leaf age the hydrophobicity of adaxial leaf side decreased significantly indicated by a decrease in contact angle. Extracted from plants, the amount of apolar cuticular wax per area unit ranged from only 0.9 microgcm(-2) for the oldest studied leaf to 1.5 microgcm(-2) for the youngest studied leaf. Differences in the total amount of cuticular waxes per leaf were not significant for older leaves. For young leaves, triterpenes (ursolic acid and oleanolic acid), esters and alcohols were the main wax components. During ontogenesis, the proportion of triterpenes in total mass of apolar waxes decreased from 32% (leaf 1) to 13% (leaf 7); absolute amounts decreased by more than 50%. The proportion of wax alcohols and esters, and alkanes to a lesser degree, increased with leaf age, whereas the proportion of acids decreased. The epicuticular wax layer also contained alpha-tocopherol described for the first time to be present also in the epicuticular wax. The modifications in the chemical composition of cuticular waxes are discussed in relation to the varying physical characteristics of the cuticle during ontogenesis of apple leaves.     

Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase

Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition- function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase beta-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C30, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.     

Giant cuticular pores in Eidothea zoexylocarya (Proteaceae) leaves

Ubiquitous, large diameter pores have not previously been adequately demonstrated to occur in leaf cuticles. Here we show conclusively that such structures occur in Eidothea zoexylocarya, a rainforest tree species of Proteaceae restricted to the Australian Wet Tropics. The pores are abundant, large-diameter apertures (～1 μm), that extend perpendicularly most of the way through the cuticle from the inside. They occur on both sides of the leaf, but are absent from the cuticle associated with stomatal complexes on the abaxial side. No such pores were found in any other species, including the only other species of Eidothea, E. hardeniana from New South Wales, and other species that have previously been purported to possess cuticular pores. To determine whether these pores made the cuticles more leaky to water vapor, we measured astomatous cuticular conductances to water vapor for E. zoexylocarya and seven other Proteaceae species of the Wet Tropics. Cuticular conductance for E. zoexylocarya was relatively low, indicating that the prominent pores do not increase conductance. The function of the pores is currently obscure, but the presence of both pores and an adaxial hypodermis in E. zoexylocarya but not E. hardeniana suggests evolution in response to greater environmental stresses in the tropics.     

Characterization of the components of plant cuticles in relation to the penetration of 2,4-D

Quantitative comparisons were made of the components of the cuticles of leaves of plantain, fat hen, dandelion, dock, chickweed and forget-me-not. Hydrocarbons and triterpenoids were prominent in the surface wax of plantain; esters and alcohols in the other surface waxes. Polar compounds predominated in the cuticular waxes. Cuticle development in plantain, dandelion and chickweed was similar, but the cutins differed in the relative proportions of hydroxy-fatty and fatty acid components. Sorption of 2,4-D by the cuticular membrane was inversely related to the amount of cuticular wax. Hydrocarbons and an aldehyde fraction isolated from surface wax most effectively reduced the penetration of water in a model system.     

Studies on water transport through the sweet cherry fruit surface: IX. Comparing permeability in water uptake and transpiration

Water uptake and transpiration were studied through the surface of intact sweet cherry (Prunus avium L.) fruit, exocarp segments (ES) and cuticular membranes (CM) excised from the cheek of sweet cherry fruit and astomatous CM isolated from Schefflera arboricola (Hayata) Hayata, Citrus aurantium L., and Stephanotis floribunda Brongn. leaves or from Lycopersicon esculentum Mill. and Capsicum annuum L. var. annuum Fasciculatum Group fruit. ES and CM were mounted in diffusion cells. Water (deionized) uptake into intact sweet cherry fruit, through ES or CM interfacing water as a donor and a polyethyleneglycol (PEG 6000, osmotic pressure 2.83 MPa)-containing receiver was determined gravimetrically. Transpiration was quantified by monitoring weight loss of a PEG 6000-containing donor (2.83 MPa) against dry silica as a receiver. The permeability coefficients for osmotic water uptake and transpiration were calculated from the amount of water taken up or transpired per unit surface area and time, and the driving force for transport. Permeability during osmotic water uptake was markedly higher than during transpiration in intact sweet cherry fruit (40.2-fold), excised ES of sweet cherry fruit (12.5- to 53.7-fold) and isolated astomatous fruit and leaf CM of a range of species (on average 23.0-fold). Partitioning water transport into stomatal and cuticular components revealed that permeability of the sweet cherry fruit cuticle for water uptake was 11.9-fold higher and that of stomata 56.8-fold higher than the respective permeability during transpiration. Increasing water vapor activity in the receiver from 0 to 1 increased permeability during transpiration across isolated sweet cherry fruit CM about 2.1-fold. Permeability for vapor uptake from saturated water vapor into a PEG 6000 receiver solution was markedly lower than from liquid water, but of similar magnitude to the permeability during self-diffusion of ³H₂O in the absence of osmotica. The energy of activation for self-diffusion of water across ES or CM was higher than for osmotic water uptake and decreased with increasing stomatal density. The data indicate that viscous flow along an aqueous continuum across the sweet cherry fruit exocarp and across the astomatous CM of selected species accounted for the higher permeability during water uptake as compared to self-diffusion or transpiration.     

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.     

Variations of cuticular wax in mulberry trees and their effects on gas exchange and post-harvest water loss

Though mulberry (Morus alba) tree shows great adaptations to various climate conditions, their leaf water status and photosynthesis are sensitive to climate changes. In the current study, seven widely planted mulberry cultivars in Chongqing, Southwest China, were selected to analyze leaf cuticular wax characteristics, gas exchange index, post-harvest leaf water status and their relationships, aiming to provide new theory in screening high resistant mulberry cultivars. Mulberry trees formed rounded cap-type idioblasts on the adaxial leaf surface. Film-like waxes and granule-type wax crystals covered leaf surfaces, varying in crystal density among cultivars. The stomatal aperture on the abaxial surface of cultivars with high wax amount was smaller than that of cultivars with low wax amount. The amount of total wax was negatively correlated with the net photosynthetic rate (P _N), transpiration rate (E) and stomatal conductance (g _s) and positively correlated with the moisture retention capacity. It suggested that both cuticular wax and stomatal factor might be involved in regulating water loss in mulberry leaves under field conditions. The variability in moisture retention capacity and cuticular wax characteristics might be important in evaluating and screening mulberry cultivars for increasing silk quality and silkworm productivity.     

The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: effects of a deficiency in a beta-ketoacyl-coenzyme A synthase (LeCER6)

Cuticular waxes play a pivotal role in limiting transpirational water loss across the primary plant surface. The astomatous fruits of the tomato (Lycopersicon esculentum) 'MicroTom' and its lecer6 mutant, defective in a beta-ketoacyl-coenzyme A synthase, which is involved in very-long-chain fatty acid elongation, were analyzed with respect to cuticular wax load and composition. The developmental course of fruit ripening was followed. Both the 'MicroTom' wild type and lecer6 mutant showed similar patterns of quantitative wax accumulation, although exhibiting considerably different water permeances. With the exception of immature green fruits, the lecer6 mutant exhibited about 3- to 8-fold increased water loss per unit time and fruit surface area when compared to the wild type. This was not the case with immature green fruits. The differences in final cuticular barrier properties of tomato fruits in both lines were fully developed already in the mature green to early breaker stage of fruit development. When the qualitative chemical composition of fruit cuticular waxes during fruit ripening was investigated, the deficiency in a beta-ketoacyl-coenzyme A synthase in the lecer6 mutant became discernible in the stage of mature green fruits mainly by a distinct decrease in the proportion of n-alkanes of chain lengths > C(28) and a concomitant increase in cyclic triterpenoids. This shift in cuticular wax biosynthesis of the lecer6 mutant appears to be responsible for the simultaneously occurring increase of water permeance. Changes in cutin composition were also investigated as a function of developmental stage. This integrative functional approach demonstrates a direct relationship between cuticular transpiration barrier properties and distinct chemical modifications in cuticular wax composition during the course of tomato fruit development.     

Ontogenetic and seasonal development of wax composition and cuticular transpiration of ivy (Hedera helix L.) sun and shade leaves

The ontogenetic and seasonal development of wax composition and cuticular transpiration of sun and shade leaves of ivy (Hedera helix L.) was analysed by investigating leaves varying in age between 4 and 202 d. It was discovered that the total amount of solvent-extractable wax was composed of two distinct fractions, separable by column chromatography: (i) a less polar or apolar monomeric wax fraction consisting of the typical linear, long-chain aliphatics usually described as cuticular wax components and (ii) a polar, oligomeric wax fraction consisting of primary alcohols and acids mostly esterified to C₁₂-, C₁₄- and C₁₆-ω-hydroxyfatty acids. The apolar wax fraction, which could be analysed directly by gas chromatography coupled with mass spectrometry (GC-MS), exhibited pronounced seasonal changes in composition. Wax amounts in the apolar fraction reached a maximum after about 30 d and gradually decreased again during the remaining period of the season investigated. In contrast, the polar wax fraction, which was analysable by GC-MS only after transesterification, rapidly increased early in the season, reaching a plateau after 40 d, and then remained constant during the rest of the season. Thus, total amounts of solvent-extractable cuticular waxes, which can be determined gravimetrically, will only be detected by GC-MS after fractionation and transesterification, a methodological approach rarely applied in the past in cuticular wax analysis. Additionally, investigation of the cutin polymer matrix after depolymerisation through transesterification, revealed that only those primary alcohols and acids forming an essential part of the apolar and the polar wax fractions were esterified during the investigated season and incorporated in increasing amounts into the cutin polymer matrix (matrix-bound wax fraction). Thus, it can be concluded that a complete analysis of cuticular wax of ivy and its seasonal development can only be achieved if all the relevant fractions (i) the less polar or apolar, (ii) the polar and (iii) the wax fraction bound to the cutin polymer matrix are investigated. Cuticular transpiration rapidly decreased within the first 30 d and essentially remained constant during the rest of the season. Thus, changes in cuticular water permeability were closely correlated with the most prominent changes in wax amounts and composition occurring during the first 30 d of ontogenetic leaf development. However, during the remainder of the year, up to 202 d, cuticular transport properties remained constant, although significant quantitative and qualitative changes in cuticular wax composition continued to occur. Thus, our study clearly demonstrated that there will be no simple relationship between chemical composition of cuticular waxes and transport properties of isolated ivy leaf cuticles. Received: 2 March 1998 / Accepted: 26 June 1998     

Effect of alterations in cuticular wax biosynthesis on the physicochemical properties and topography of maize leaf surfaces

The leaf surface properties of 11 cuticular wax mutants of maize were characterized, and this information was used to identify the quantitative relations among distinct leaf surface traits. Compared with the wild‐type maize, these mutants were reduced 3–24% in their leaf surface hydrophobicity, 20–88% in the mass of cuticular waxes on their leaves, and 52–94% in the percentage of planar leaf surface area covered with epicuticular crystalline waxes. They also differed in the presence and abundance of the epicuticular crystalline waxes in each of seven structural classes. With the exception of one mutant, the mass of cuticular waxes produced by these mutants was positively correlated with the number of epicuticular crystalline waxes per unit area on their leaves. Furthermore, an increase of 0·4 mg of cuticular wax per gram of leaf (dry weight) was associated with a 1% increase in leaf surface area covered by epicuticular crystalline waxes, and this 1% increase was associated with a 2° increase in the contact angle of a water droplet on the leaf surface. Linear differences in the leaf surface hydrophobicity were associated with exponential differences in the mass of the cuticular waxes produced. Quantitative knowledge of these leaf surface properties is highly relevant to the interactions of leaves with environmental factors such as microbes, insects, agricultural chemicals, and pollutants.