Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.)

The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, β- and OH-β-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of β- and OH-β-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, β-diketone existed in all varieties, but OH-β-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-β-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C₂₈ alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C₂₄ or C₂₆ depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days.     

植物角质层蜡质的化学组成研究综述

角质层是植物与外界的第一接触面,而角质层蜡质则是由位于角质层外的外层蜡质和深嵌在角质层中的内层蜡质两部分构成。植物角质层蜡质成分极其复杂,具有重要的生理功能。综述了有关植物角质层蜡质的化学组成信息,探讨了目前植物角质层蜡质化学成分研究中存在的一些问题,展望了角质层蜡质成分的研究前景。     

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 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     

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.     

Compositae Plants Differed in Leaf Cuticular Waxes between High and Low Altitudes

We sampled eight Compositae species at high altitude (3482 m) and seven species at low altitude (220 m), analyzed the chemical compositions and contents of leaf cuticular wax, and calculated the values of average chain length (ACL), carbon preference index (CPI), dispersion (d), dispersion/weighted mean chain length (d/N), and C₃₁/(C₃₁ + C₂₉) (Norm31). The amounts of total wax and compositions were significantly higher at high altitude than at low altitude, except for primary alcohol, secondary alcohol, and ketone. The main n‐alkanes in most samples were C₃₁, C₂₉, and C₃₃. Low altitude had more C₃₁ and C₃₃, whereas more C₂₉ occurred at high altitude. The ACL, CPI, d, d/N, and Norma 31 were higher at low altitude than at high altitude. The fatty acid and primary alcohol at low altitude contained more C₂₆ homologous than at high altitude. More short‐chain primary alcohols were observed at high altitude. At low altitude, the primary alcohol gave on average the largest amount, while it was n‐alkane at high altitude. These results indicated that the variations of leaf cuticular waxes benefited Compositae plants to adapt to various environmental stresses and enlarge their distribution.     

Changes in leaf cuticular waxes of sesame (Sesamum indicum L.) plants exposed to water deficit

Sesame (Sesamum indicum L.) is one of the most important oilseed crops, having seeds and oil that are highly valued as a traditional health food. The objective of this study was to evaluate leaf cuticular wax constituents across a diverse selection of sesame cultivars, and the responses of these waxes to drought-induced wilting. Water-deficit was imposed on 18 sesame cultivars by withholding irrigation for 15d during the post-flowering stage, and the effect on seed yield and leaf waxes compared with a well-watered control. Leaf cuticular waxes were dominated by alkanes (59% of total wax), with aldehydes being the next-most abundant class. Compared to well-irrigated plants, drought treatment caused an increase in wax amount on most cultivars, with only three cultivars having a notable reduction. When expressed as an average across all cultivars, drought treatment caused a 30% increase in total wax amount, with a 34% increase in total alkanes, a 13% increase in aldehydes, and a 28% increase in the total of unknowns. In all cultivars, the major alkane constituents were the C27, C29, C31, C33, and C35 homologs, whereas the major aldehydes were the C30, C32, and C34 homologs, and drought exposure had only minor effects on the chain length distribution within these and other wax classes. Drought treatments caused a large decrease in seed yield per plant, but did not affect the mean weight of individual seeds, showing that sesame responds to post-flowering drought by reducing seed numbers, but not seed size. Seed yield was inversely correlated with the total wax amount (-0.466*), indicating that drought induction of leaf wax deposition does not contribute directly to seed set. Further studies are needed to elucidate the ecological role for induction of the alkane metabolic pathway by drought in regulating sesame plant survival and seed development in water-limiting environments.     

Localization of the Transpiration Barrier in the Epi- and Intracuticular Waxes of Eight Plant Species: Water Transport Resistances Are Associated with Fatty Acyl Rather Than Alicyclic Components

Plant cuticular waxes play a crucial role in limiting nonstomatal water loss. The goal of this study was to localize the transpiration barrier within the layered structure of cuticles of eight selected plant species and to put its physiological function into context with the chemical composition of the intracuticular and epicuticular wax layers. Four plant species (Tetrastigma voinierianum, Oreopanax guatemalensis, Monstera deliciosa, and Schefflera elegantissima) contained only very-long-chain fatty acid (VLCFA) derivatives such as alcohols, alkyl esters, aldehydes, and alkanes in their waxes. Even though the epicuticular and intracuticular waxes of these species had very similar compositions, only the intracuticular wax was important for the transpiration barrier. In contrast, four other species (Citrus aurantium, Euonymus japonica, Clusia flava, and Garcinia spicata) had waxes containing VLCFA derivatives, together with high percentages of alicyclic compounds (triterpenoids, steroids, or tocopherols) largely restricted to the intracuticular wax layer. In these species, both the epicuticular and intracuticular waxes contributed equally to the cuticular transpiration barrier. We conclude that the cuticular transpiration barrier is primarily formed by the intracuticular wax but that the epicuticular wax layer may also contribute to it, depending on species-specific cuticle composition. The barrier is associated mainly with VLCFA derivatives and less (if at all) with alicyclic wax constituents. The sealing properties of the epicuticular and intracuticular layers were not correlated with other characteristics, such as the absolute wax amounts and thicknesses of these layers.The plant cuticle is one of the major adaptations of vascular plants for life in the atmospheric environment. Accordingly, the primary function of cuticles is to limit nonstomatal water loss and, thus, to protect plants against drought stress (Burghardt and Riederer, 2006). However, plant cuticles also play roles in minimizing the adhesion of dust, pollen, and spores (Barthlott and Neinhuis, 1997), protecting tissues from UV radiation (Krauss et al., 1997; Solovchenko and Merzlyak, 2003), mediating biotic interactions with microbes (Carver and Gurr, 2006; Leveau, 2006; Hansjakob et al., 2010, 2011; Reisberg et al., 2012) as well as insects (Eigenbrode and Espelie, 1995; Müller and Riederer, 2005), and preventing deleterious fusions between different plant organs (Tanaka and Machida, 2013).Cuticles are composite (nonbilayer) membranes consisting of an insoluble polymer matrix and solvent-soluble waxes. The polymer matrix (MX) is mainly made of the hydroxy fatty acid polyester cutin (Nawrath, 2006) and also contains polysaccharides and proteins (Heredia, 2003). In contrast, cuticular waxes are complex mixtures of aliphatic compounds derived from very-long-chain fatty acids (VLCFAs) with hydrocarbon chains of C₂₀ and more (Jetter et al., 2007). Wax quantities and compositions vary greatly between plant species and, in many cases, even between organs and developmental stages. Diverse VLCFA derivatives can be present, including free fatty acids, aldehydes, ketones, primary and secondary alcohols, alkanes, and alkyl esters. Besides, the cuticular waxes of many plant species also contain cyclic compounds such as triterpenoids and aromatics.In order to characterize the physiological function of cuticular waxes, methods have been developed for the isolation of astomatous cuticles and the measurement of transpiration rates under exactly controlled conditions, so that well-defined physical transport parameters such as permeances and resistances can be determined and compared across species and organs (Schönherr and Lendzian, 1981; Kerstiens, 1996; Riederer and Schreiber, 2001; Lendzian, 2006). With these methods, it was demonstrated that the cuticular water permeance increases by up to 3 orders of magnitude upon wax removal, thus showing the central role of waxes as a transpiration barrier (Schönherr, 1976). Permeances for water determined so far with astomatous isolated leaf cuticular membranes (CMs) or in situ leaf cuticles range over 2.5 orders of magnitude, from 3.63 × 10⁻⁷ m s⁻¹ (Vanilla planifolia) to 7.7 × 10⁻⁵ m s⁻¹ (Maianthemum bifolium; Riederer and Schreiber, 2001).The species-dependent differences of both wax composition and permeance led to a search for correlations between cuticle structure and function. If such a structure-function relationship could be established, then it would become possible to select or alter wax composition in order to improve cuticle performance in crop species (Kosma and Jenks, 2007). However, all attempts to understand cuticle permeance based on cuticle composition have failed so far: correlations between wax amounts and permeances could not be established, contrary to the common assumption that thicker wax layers must provide better protection against desiccation (Schreiber and Riederer, 1996; Riederer and Schreiber, 2001). Similarly, a correlation between wax quality (i.e. the relative portions of its constituents) and permeance could also not be established to date (Burghardt and Riederer, 2006). It is not clear how certain wax components contribute to the vital barrier function of the cuticle.Previous attempts to establish wax structure-function relationships may have failed because only bulk wax properties were studied and important effects of substructures were averaged out. However, distinct compartments of wax exist within the cuticle, most prominently as a layer of intracuticular wax embedded within the MX and a layer of epicuticular wax deposited on the outer surface of the polymer (Jeffree, 2006). Over the last years, methods have been developed that allow the selective removal of epicuticular wax by adhesive surface stripping, followed by equally selective extraction of intracuticular wax (Jetter et al., 2000; Jetter and Schäffer, 2001). Chemical analyses showed that, for most plant species investigated to date, both wax layers have distinct compositions (Buschhaus and Jetter, 2011). The most pronounced differences between the layers were found for the triterpenoids, which were localized predominantly (or even exclusively) in the intracuticular wax. These findings raised the possibility that the chemically distinct wax layers might also have distinct functions, leading back to the long-standing question of whether the water barrier function is exerted by the intracuticular and/or the epicuticular wax. There are only scant data to answer this question so far, mainly because methods allowing a distinction between epicuticular and intracuticular waxes were established only recently. Using these sampling techniques, it was recently found that, for leaves of Prunus laurocerasus, the epicuticular wax layer does not contribute to the transpiration barrier (Zeisler and Schreiber, 2016). In contrast, it had been reported that removal of the epicuticular wax layer from tomato (Solanum lycopersicum) fruit caused an approximately 2-fold increase in transpiration, suggesting that, in this species, the epicuticular layer constitutes an important part of the barrier (Vogg et al., 2004). Based on these conflicting reports, it is not clear to what extent the intracuticular or the epicuticular waxes contribute to the sealing function of the plant skin.The goal of this study was to localize the transpiration barrier within the cuticular membrane of selected plant species and to put the physiological function into context with the chemical composition of both the epicuticular and intracuticular wax layers. To this end, we selected eight species from which leaf cuticles could be isolated and methods for step-wise wax removal could be applied without damaging the cuticle. Preliminary studies had shown that the adaxial cuticles on leaves of Citrus aurantium (Rutaceae), Euonymus japonica (Celastraceae), Clusia flava (Clusiaceae), Garcinia spicata (Clusiaceae), Tetrastigma voinierianum (Vitaceae), Oreopanax guatemalensis (Araliaceae), Monstera deliciosa (Araceae), and Schefflera elegantissima (Araliaceae) were astomateous and showed wide chemical diversity. Therefore, these eight species were selected to address the following questions: (1) What are the amounts of epicuticular and intracuticular waxes? (2) Do compositional differences exist between the layers? (3) Where are the cuticular triterpenoids located? (4) How much do the epicuticular and intracuticular waxes contribute to the transpiration barrier? (5) Is the barrier associated with certain components of the intracuticular or epicuticular waxes?     

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.     

The Arabidopsis cer26 mutant,like the cer2 mutant,is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl‐CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2‐like family: CER2, CER26 and CER26‐like . Expression pattern analysis showed that the three genes are differentially expressed in an organ‐ and tissue‐specific manner. Using individual T–DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl‐CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.     

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.¹ A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer ''epicuticular'' layer as well as the blend consisting of the cutin biopolymer and ''intracuticular'' waxes.² Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Schäffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.^3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) ¹³C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS ¹³C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH₂O) chemical moieties.Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state ¹³C NMR reveals differences in the relative numbers of CHO and CH₂O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-μm length scale.     

Observation of an anisotropic texture inside the wax layer of insect cuticle

The outermost part of insect cuticles is very often covered with wax, which prevents desiccation and serves for chemical communication in many species. Earlier studies on cuticular waxes have mainly focused on their chemical composition revealing complex mixtures of lipids. In the absence of information on their physical organization, cuticular waxes have been considered isotropic. Here we report the presence of parallel stripes in the wax layer of the carapace of the scarab beetle, Chrysina gloriosa, with a textural periodicity of ca. 28 nm, as revealed by electron microscopy of transverse sections. Observations at oblique incidence argue for a layered organization of the wax, which might be related to a layer-by-layer deposition of excreted wax. Our findings may lay the foundation for further studies on the internal structure of cuticular waxes for other insects.     

Leaf cuticular waxes of potted rose cultivars as affected by plant development, drought and paclobutrazol treatments

The present work was carried out to evaluate how plant growth and cultural practices influence the amount and composition of cuticular waxes on leaves of rose cultivars. The total amount of cuticular wax per leaf area was higher for rose cultivar Apollo Parade than for Charming Parade. Both cultivars had waxes dominated by alkanes, with the major alkanes being the C₃₁ and C₃₃ homologues. Primary alcohols were the next most abundant constituent class, with C₂₆ as the dominant homologue. Compared with Charming Parade, Apollo Parade had higher proportions of its total wax load as primary alcohols but lower acids and aldehydes. The proportion of alkanes in the total load on these cultivars was similar. Commercially produced roses are routinely treated with paclobutrazol (PBZ) to retard growth. PBZ treatments caused a 10% increase in total wax load and changes in the proportions of certain wax constituents within 11 days of application. Notable was an increase in the total proportion of acids in the total load 25 days after PBZ application, primarily because of increased C₂₈ acids. An alternative method of retarding plant growth is production of roses under limited water availability. When Apollo Parade roses experienced periods of moderate drought stress during production, the wax load per leaf area increased 14 and 8% above control levels at 24 and 38 days after imposition of drought, respectively. Drought caused similar changes in the proportions of individual wax constituents as did PBZ application.     

The Inhibitor of wax 1 locus (Iw1) prevents formation of β‐ and OH‐β‐diketones in wheat cuticular waxes and maps to a sub‐cM interval on chromosome arm 2BS

Glaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, two dominant genes lead to non‐glaucous phenotypes: Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact extent (beyond visual assessment) by which these genes affect the composition and quantity of cuticular wax is unclear. To describe the Iw1 locus we used a genetic approach with detailed biochemical characterization of wax compounds. Using synteny and a large number of F₂ gametes, Iw1 was fine‐mapped to a sub‐cM genetic interval on wheat chromosome arm 2BS, which includes a single collinear gene from the corresponding Brachypodium and rice physical maps. The major components of flag leaf and peduncle cuticular waxes included primary alcohols, β‐diketones and n‐alkanes. Small amounts of C19–C27 alkyl and methylalkylresorcinols that have not previously been described in wheat waxes were identified. Using six pairs of BC₂F₃ near‐isogenic lines, we show that Iw1 inhibits the formation of β‐ and hydroxy‐β‐diketones in the peduncle and flag leaf blade cuticles. This inhibitory effect is independent of genetic background or tissue, and is accompanied by minor but consistent increases in n‐alkanes and C₂₄ primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near‐isogenic lines differing at Iw1.     

The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function

The cuticle covers the aerial epidermis of land plants and plays a primary role in water regulation and protection from external stresses. Remarkable species diversity in the structure and composition of its components, cutin and wax, have been catalogued, but few functional or genetic correlations have emerged. Tomato (Solanum lycopersicum) is part of a complex of closely related wild species endemic to the northern Andes and the Galapagos Islands (Solanum Sect. Lycopersicon). Although sharing an ancestor <7 million years ago, these species are found in diverse environments and are subject to unique selective pressures. Furthermore, they are genetically tractable, since they can be crossed with S. lycopersicum, which has a sequenced genome. With the aim of evaluating the relationships between evolution, structure and function of the cuticle, we characterized the morphological and chemical diversity of fruit cuticles of seven species from Solanum Sect. Lycopersicon. Striking differences in cuticular architecture and quantities of cutin and waxes were observed, with the wax coverage of wild species exceeding that of S. lycopersicum by up to seven fold. Wax composition varied in the occurrence of wax esters and triterpenoid isomers. Using a Solanum habrochaites introgression line population, we mapped triterpenoid differences to a genomic region that includes two S. lycopersicum triterpene synthases. Based on known metabolic pathways for acyl wax compounds, hypotheses are discussed to explain the appearance of wax esters with atypical chain lengths. These results establish a model system for understanding the ecological and evolutionary functional genomics of plant cuticles.     

The cuticles of some Angiosperm leaves and fruits

The cuticles of twenty-four species from a wide range of mono- and di-cotyledonous plants were examined by chemical methods. The cuticles differ markedly in the amount and composition of the surface wax, in the thickness of the cuticular membrane, and in the content and composition of the cutin of the membrane. Fruits usually have heavier wax deposits and much thicker membranes than leaves. No direct relationship exists between surface waxiness and thickness of the membrane. Alkanes and primary alcohols are prominent in many of the surface waxes; triterpenoids occur less frequently. The cutin content of the membrane varies considerably; a delicate membrane tends to have a low content of cutin in which fatty acids are prevalent, and a well-developed membrane a higher content of cutin more rich in hydroxy-fatty acids. 10,16-Dihydroxyhexadecanoic acid is often an important constituent of cutin; 9,10,18-trihydroxyoctadecanoic acid is most prominent in the cutin of thicker membranes. The possible influence of the variations in cutin acids upon the structure of cutin and the taxonomic implications of wax and cutin composition are discussed.     

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

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

OsGL1-3 is Involved in Cuticular Wax Biosynthesis and Tolerance to Water Deficit in Rice

Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3–GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C₃₀–C₃₂ aldehydes and C₃₀ primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance.