Rheological Properties of Enzymatically Isolated Tomato Fruit Cuticle

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

Composition differences between epicuticular and intracuticular wax substructures: how do plants seal their epidermal surfaces?

The protective wax coating on plant surfaces has long been considered to be non-uniform in composition at a subcellular scale. In recent years, direct evidence has started to accumulate showing quantitative compositional differences between the epicuticular wax (i.e. wax exterior to cutin that can be mechanically peeled off) and intracuticular wax (i.e. wax residing within the mechanically resistant layer of cutin) layers in particular. This review provides a first synthesis of the results acquired for all the species investigated to date in order to assign chemical information directly to cuticle substructures, together with an overview of the methods used and a discussion of possible mechanisms and biological functions. The development of methods to probe the wax for z-direction heterogeneity began with differential solvent extractions. Further research employing mechanical wax removal by adhesives permitted the separation and analysis of the epicuticular and intracuticular wax. In wild-type plants, the intracuticular (1-30 μg cm(-2)) plus the epicuticular wax (5-30 μg cm(-2)) combined to a total of 8-40 μg cm(-2). Cyclic wax constituents, such as triterpenoids and alkylresorcinols, preferentially or entirely accumulate within the intracuticular layer. Within the very-long-chain aliphatic wax components, primary alcohols tend to accumulate to higher percentages in the intracuticular wax layer, while free fatty acids and alkanes in many cases accumulate in the epicuticular layer. Compounds with different chain lengths are typically distributed evenly between the layers. The mechanism causing the fractionation remains to be elucidated but it seems plausible that it involves, at least in part, spontaneous partitioning due to the physico-chemical properties of the wax compounds and interactions with the intracuticular polymers. The arrangement of compounds probably directly influences cuticular functions.     

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8 / LACS1 , one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C₂₄) were elevated more than 155%. The C₁₆ cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C₁₈ monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C₂₀–C₃₀, with highest activity for C₃₀ acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C₁₆ but not C₁₈ cutin monomers are reduced in lacs1 , and C₁₆ acids are the next most preferred acid (behind C₃₀) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C₁₆ monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C₁₆) fatty acids for cutin synthesis.     

Biomechanics of isolated tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin matrix and polysaccharides

The mechanical characteristics of the cuticular membrane (CM), a complex composite biopolymer basically composed of a cutin matrix, waxes, and hydrolysable polysaccharides, have been described previously. The biomechanical behaviour and quantitative contribution of cutin and polysaccharides have been investigated here using as experimental material mature green and red ripe tomato fruits. Treatment of isolated CM with anhydrous hydrogen fluoride in pyridine allowed the selective elimination of polysaccharides attached to or incrusted into the cutin matrix. Cutin samples showed a drastic decrease in elastic modulus and stiffness (up to 92%) compared with CM, which clearly indicates that polysaccharides incorporated into the cutin matrix are responsible for the elastic modulus, stiffness, and the linear elastic behaviour of the whole cuticle. Reciprocally, the viscoelastic behaviour of CM (low elastic modulus and high strain values) can be assigned to the cutin. These results applied both to mature green and red ripe CM. Cutin elastic modulus, independently of the degree of temperature and hydration, was always significantly higher for the ripe than for the green samples while strain was lower; the amount of phenolics in the cutin network are the main candidates to explain the increased rigidity from mature green to red ripe cutin. The polysaccharide families isolated from CM were pectin, hemicellulose, and cellulose, the main polymers associated with the plant cell wall. The three types of polysaccharides were present in similar amounts in CM from mature green and red ripe tomatoes. Physical techniques such as X-ray diffraction and Raman spectroscopy indicated that the polysaccharide fibres were mainly randomly oriented. A tomato fruit CM scenario at the supramolecular level that could explain the observed CM biomechanical properties is presented and discussed.     

Attenuated total reflectance spectroscopy of plant leaves: a tool for ecological and botanical studies

Attenuated total reflectance (ATR) spectra of plant leaves display complex absorption features related to organic constituents of leaf surfaces. The spectra can be recorded rapidly, both in the field and in the laboratory, without special sample preparation. This paper explores sources of ATR spectral variation in leaves, including compositional, positional and temporal variations. Interspecific variations are also examined, including the use of ATR spectra as a tool for species identification. Positional spectral variations generally reflected the abundance of cutin and the epicuticular wax thickness and composition. For example, leaves exposed to full sunlight commonly showed more prominent cutin- and wax-related absorption features compared with shaded leaves. Adaxial vs. abaxial leaf surfaces displayed spectral variations reflecting differences in trichome abundance and wax composition. Mature vs. young leaves showed changes in absorption band position and intensity related to cutin, polysaccharide, and possibly amorphous silica development on and near the leaf surfaces. Provided that similar samples are compared (e.g. adaxial surfaces of mature, sun-exposed leaves) same-species individuals display practically identical ATR spectra. Using spectral matching procedures to analyze an ATR database containing 117 individuals, including 32 different tree species, 83% of the individuals were correctly identified.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

Mechanical role of the leaf sheath in rattans

Leaf sheaths of rattans are long, tubular and persistent and unlike many self-supporting palms, extend far from the apex of the plant. The mechanical role of the leaf sheath was investigated in eight rattan species of the subfamily Calamoideae. The main objective was to analyse its influence on the mechanical architecture and contribution to the climbing habit. Bending mechanical properties were measured along climbing axes before and after removal of leaf sheaths. Results were related to stem and leaf sheath geometry and mechanical properties. Contribution of the leaf sheath to axial flexural rigidity was high (c. 90%) in the early stages of growth and towards the apex of older climbing axes for all climbing palms tested. Senescence and loss of the leaf sheath strongly influenced axial stiffness. A nonclimbing species, Calamus erectus, showed a different mechanical architecture. Although lacking secondary growth, palms have been able to develop successful climbers with a mechanical architecture broadly analogous to, although developmentally different from, dicotyledonous lianas. The role of the leaf sheath in modulating mechanical properties during ontogeny ought not to be neglected in studies on monocotyledons, as it possibly contributed significantly to the ways in which different growth forms have evolved in the group.     

Cuticular permeance in relation to wax and cutin development along the growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaf

The developing leaf three of barley provides an excellent model system for the direct determination of relationships between amounts of waxes and cutin and cuticular permeance. Permeance of the cuticle was assessed via the time-course of uptake of either toluidine blue or ¹⁴C-labelled benzoic acid ([¹⁴C] BA) along the length of the developing leaf. Toluidine blue uptake only occurred within the region 0–25 mm from the point of leaf insertion (POLI). Resistance—the inverse of permeance—to uptake of [¹⁴C] BA was determined for four leaf regions and was lowest in the region 10–20 mm above POLI. At 20–30 and 50–60 mm above POLI, it increased by factors of 6 and a further 32, respectively. Above the point of emergence of leaf three from the sheath of leaf two, which was 76–80 mm above POLI, resistance was as high as at 50–60 mm above POLI. GC-FID/MS analyses of wax and cutin showed that: (1) the initial seven fold increase in cuticular resistance coincided with increase in cutin coverage and appearance of waxes; (2) the second, larger and final increase in cuticle resistance was accompanied by an increase in wax coverage, whereas cutin coverage remained unchanged; (3) cutin deposition in barley leaf epidermis occurred in parallel with cell elongation, whereas deposition of significant amounts of wax commenced as cells ceased to elongate.     

Relative humidity and temperature modify the mechanical properties of isolated tomato fruit cuticles

The mechanical properties of enzymatically isolated cuticular membrane (CM) from ripe tomato fruits were investigated at 10 to 45°C and relative humidity (RH) of 40 to wet. CM samples were stressed by uniaxial tension loads to determine their tensile modulus, E, breaking stress (strength), σ(max), and maximum elongation, ε(max). The CM stress-strain curves revealed a biphasic behavior when tested at RH values below wet conditions. In the first phase, CM responded to the loads by instantaneous extension with no further extension recorded until a further load was added: defined as pure elastic strain (E(e)). In the second phase, CM responded by instantaneous extension and by some additional time-dependent extension, defined as viscoelastic strain (E(v)). When CMs were submerged in aqueous solution (wet), the stress-strain curves were monophasic, with both elastic and viscoelastic strain. E(e) depended on RH and was higher than E(v), which was independent of RH. Temperature decreased E(e) and σ(max) of tomato fruit CM. Temperature response was not linear but consisted of two temperature-independent phases separated by a transition temperature. This transition zone has been related previously to the presence of a secondary phase transition in the cutin matrix of the tomato fruit CM.     

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

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

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     

Cuticle lipids on heteromorphic leaves of Populus euphratica Oliv. growing in riparian habitats differing in available soil moisture

Populus euphratica is an important native tree found in arid regions from North Africa and South Europe to China, and is known to tolerate many forms of environmental stress, including drought. We describe cuticle waxes, cutin and cuticle permeability for the heteromorphic leaves of P. euphratica growing in two riparian habitats that differ in available soil moisture. Scanning electron microscopy revealed variation in epicuticular wax crystallization associated with leaf type and site. P. euphratica leaves are dominated by cuticular wax alkanes, primary‐alcohols and fatty acids. The major cutin monomers were 10,16‐diOH C_16:0 acids. Broad‐ovate leaves (associated with adult phase growth) produced 1.3‐ and 1.6‐fold more waxes, and 2.1‐ and 0.9‐fold more cutin monomers, than lanceolate leaves (associated with juvenile phase growth) at the wetter site and drier site, respectively. The alkane‐synthesis‐associated ECERIFERUM1 (CER1), as well as ABC transporter‐ and elongase‐associated genes, were expressed at much higher levels at the drier than wetter sites, indicating their potential function in elevating leaf cuticle lipids in the dry site conditions. Higher cuticle lipid amounts were closely associated with lower cuticle permeability (both chlorophyll efflux and water loss). Our results implicate cuticle lipids as among the xeromorphic traits associated with P. euphratica adult‐phase broad‐ovate leaves. Results here provide useful information for protecting natural populations of P. euphratica and their associated ecosystems, and shed new light on the functional interaction of cuticle and leaf heterophylly in adaptation to more arid, limited‐moisture environments.     

The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1

The cuticle coats the aerial organs of land plants and is composed of a cutin matrix embedded and overlayed with waxes. The Arabidopsis CER3 gene is important for cuticular wax biosynthesis and was reported to correspond to At5g02310 encoding an E3 ubiquitin ligase. Here, we demonstrate that CER3 is not At5g02310 and instead corresponds to WAX2/YRE/FLP1 (At5g57800), a gene of unknown function required for wax biosynthesis. CER3 protein has also been implicated in cutin production because strong cer3 alleles display organ fusions. Leaf cutin analysis of two cer3 alleles did not reveal significant differences in cutin load or composition, indicating that CER3 has no major role in leaf cutin formation.     

植物表皮蜡质与抗旱及其分子生物学

植物表皮蜡质是覆盖在植物最外层的一类有机混合物总称,它是植物自我防护的最后一道屏障,在植物生长发育过程中起重要作用。文章就植物表皮蜡质的成分、转运、晶体的形成和发育、形态结构以及环境变化对表皮蜡质的影响,特别是在蜡质与角质蒸腾、叶片水分利用效率和产量的关系以及蜡质的生理作用和合成过程的分子生物学研究进展方面做了系统综述,并对植物蜡质研究中存在的问题做了探讨。     

Diversity of cutinases from plant pathogenic fungi: differential and sequential expression of cutinolytic esterases by Alternaria brassicicola

Plant cuticles provide a protective layer that has to be penetrated by fungal pathogens. Evidence is provided for a differential and sequential induction of two classes of cutinolytic esterases by Alternaria brassicicola. Serine esterases with cutinolytic activities were expressed by conidia germinating on host surfaces. The enzymes were not induced by surface wax or cutin monomers. They were only expressed during initial (24 h) contact of conidia with cutin on host surfaces freed from wax, and with cutin in aqueous suspensions. In contrast, contact with cutin had no immediate effect on the expression of CUTAB1, a gene encoding two cutinase isozymes with crucial functions in the saprophytic utilization of cutin. Presence of a cutin monomer or prolonged exposure to cutin was required for the induction of CUTAB1 expression. The differential induction of cutinolytic esterases indicates a sequential recognition of cutin as a barrier to be penetrated and to be utilized as a carbon source in saprophytic stages.     

Leaf wax n-alkane δD values are determined early in the ontogeny of Populus trichocarpa leaves when grown under controlled environmental conditions

The stable hydrogen isotope ratios (δD) of leaf wax n-alkanes record valuable information on plant and ecosystem water relations. It remains, however, unknown if leaf wax n-alkane δD values record only environmental variation during the brief period of time of leaf growth or if leaf wax n-alkane δD values are affected by environmental variability throughout the entire lifespan of a leaf. To resolve these uncertainties, we irrigated Populus trichocarpa trees with a pulse of deuterium-enriched water and used compound-specific stable hydrogen isotope analyses to test if the applied tracer could be recovered from leaf wax n-alkanes of leaves that were at different stages of their development during the tracer application. Our experiment revealed that only leaf wax n-alkanes from leaves that had developed during the time of the tracer application were affected, while leaves that were already fully matured at the time of the tracer application were not. We conclude from our study that under controlled environmental conditions, leaf wax n-alkanes are synthesized only early in the ontogeny of a leaf. Our experiment has implications for the interpretation of leaf wax n-alkane δD values in an environmental context, as it suggests that these compounds record only a brief period of the environmental variability that a leaf experiences throughout its life.