Ontogenetic variations in the composition of peach leaf wax

The composition of the epicuticular waxes from the adaxial and abaxial surfaces of peach leaves varies considerably during one season's growth. Triterpenoid acids are major components 84–95% of the waxes from the youngest leaves but the proportions of these constituents decrease as the leaves expand. The waxes from the abaxial surfaces of fully expanded leaves consist primarily of hydrocarbons (C_22–C34) and triterpenoid acids, whereas the adaxial surface waxes also contain large proportions of primary alcohols (C₂₆-C₃₄) and esters (C₄₂-C₅₂). The latter include sitosteryl esters of hexacosanoic, octacosanoic and eicosanoic acids. Variations were also noted between fully expanded leaves of different ages, the abaxial surface waxes of the oldest leaves containing the highest proportions of hydrocarbons, whilst the wax from the adaxial surface of the corresponding leaves contained the largest amounts of esters, sitosterol and hydrocarbons.     

Hydrocarbons in leaf waxes of Saccharum and related genera

The composition of the n-alkanes in the leaf waxes of over 80 clones of Saccharum officinarum, S. edule, S. robustum, S. spontaneum and from a number of related species have been compared by GLC. The waxes contain predominantly odd alkanes, C₂₇–C₃₅, the major components being C₂₉ and C₃₁. In a number of clones, particularly of S. edule, a homologous series of alkenes was also present. No chemotaxonomic relationship could be derived from the compositions as the intraspecific variation was greater than the interspecific variations.     

Composition of leaf epicuticular waxes of Pteridium subspecies

The leaf epicuticular waxes of two subspecies of Pteridium consisted principally of alkyl esters (92 %; C₄₀–C₅₀) together with small amounts of n-alkanols (2 %; C₂₄–C₃₂) and hydrocarbons (2%; C₂₇–C₃₁). The esters comprised C₂₂–C₃₂ alkanols randomly combined with C₂₀ – C₂₄ fatty acids.     

Epicuticular waxes of two sorghum varieties

The epicuticular waxes of the two sorghum varieties Alliance A and SD 102 have been analyzed, after separation of the leaf blades from the sheaths. The major constituents were found to be free fatty acids but small amounts of esters, aldehydes, alcohols, n-alkanes and sterols were also detected. The typical chain lengths of aldehydes, free alcohols and free fatty acids were C₂₈ and C₃₀.     

Chemotaxonomic significance of n-alkane distributions from leaf wax in genus of Sinojackia species (Styracaceae)

Sinojackia is a Chinese endemic genus of Styracaceae, containing eight species. The taxonomy of one species, Sinojackia dolichocarpa (≡Changiostyrax dolichocarpa), is controversial. Here we investigate the distribution of leaf wax n-alkanes to clarify the chemotaxonomic position of S. dolichocarpa. Leaf samples of six Sinojackia species from Wuhan Botanical Garden in central China were collected during April, July, and December in 2010 to capture different developmental stages of the epicuticular waxes. Our results show that the waxes of S. dolichocarpa differ from the other five species by having a lower abundance of nC₃₁ but a higher abundance of nC₃₃. Although developmental variations of n-alkane distributions were observed during the sampling periods, cluster analysis based on the percentages of n-alkanes from C₂₇ to C₃₃ separates S. dolichocarpa from the other Sinojackia species. Based on these finding, we suggest S. dolichocarpa is a species independent of the Sinojackia genus.     

Leaf n‐Alkanes as Characters Differentiating Coastal and Continental Juniperus deltoides Populations from the Balkan Peninsula

The composition of the cuticular n‐alkanes isolated from the leaves of nine populations of Juniperus deltoides R.P.Adams from continental and coastal areas of the Balkan Peninsula was characterized by GC‐FID and GC/MS analyses. In the leaf waxes, 14 n‐alkane homologues with chain‐lengths ranging from C₂₂ to C₃₅ were identified. n‐Tritriacontane (C₃₃) was dominant in the waxes of all populations, but variations between the populations in the contents of all n‐alkanes were observed. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses) were used to investigate the diversity and variability of the cuticular‐leaf‐n‐alkane patterns of the nine J. deltoides populations. This is the first report on the n‐alkane composition for this species. The multivariate statistical analyses evidenced a high correlation of the leaf‐n‐alkane pattern with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon.     

The effect of the environment on the permeability and composition of Citrus leaf cuticles

The constituents of the soluble cuticular lipids (SCL) of the leaf blades of Citrus aurantium L. were identified by gas chromatography-mass spectrometry and quantified. Major components were 1-alkanols (C₂₄ to C₄₀), n-alkyl esters (C₃₆ to C₅₆), n-alkanoic acids (C₂₈ to C₃₄), n-alkanes (C₂₂ to C₄₀) and triterpenones, while n-alkanals (C₂₉ to C₃₈), sterols, and alkyl benzenes (molecular weights 260, 274 and 288) made minor contributions. Leaf age and side significantly affected the quantitative composition of SCL. Increased day temperature during the development of leaves led to decreased amounts per unit area of n-alkanes, 1-alkanols, n-alkanoic acids and n-alkyl esters while increased night temperatures resulted in increased amounts of n-alkanes n-alkanoic acids and 1-alkanols. Relative humidity had no effect on the amounts or composition of SCL. The permeability of cuticular membranes to water (described in part I of this paper) and the composition of SCL were not related. A model for the molecular structure of the transport-limiting barrier of plant cuticles and for the transport of water across it is proposed.Abbreviations CM cuticular membrane - GC gas chromatogra-phy - MS mass spectroscopy - TLC thin-layer (planar) chromatography - SCL soluble cuticular lipids The authors are indebted to Dr. R. Winkler and H. Krause, Laboratorium für Strukturchemie des Fachbereichs Chemie, Biologie und Geowissenschaften, Technische Universität München, FRG, for performing the GC-MS analyses and their valuable help in the identification of SCL constituents. This work has been supported by the Deutsche Forschungsgemeinschaft and the Bayerische Staatsministerium für Wissenschaft und Kunst.     

Arabidopsis ketoacyl‐CoA synthase 16 (KCS16) forms C36/C38 acyl precursors for leaf trichome and pavement surface wax

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β‐ketoacyl‐CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C₃₅ and C₃₇ alkanes derived from C₃₆ and C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss‐of‐function mutants were depleted of C₃₅ and C₃₇ alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C₃₆ and C₃₈ fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C₃₄ to C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra‐long compounds in adjacent pavement cells.     

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.     

Surface wax of Andreaea and Pogonatum species

The mosses Andreaea rupestris, Pogonatum aloides and P. urnigerum contain surface waxes in amounts of 0.05–0.12% dry wt. The waxes consisted of esters (C₃₈-C₅₄), primary alcohols (C₂₀-C₃₂), free fatty acids (C₁₆-C₃₀), and alkanes (C₂₁-C₃₁). Additionally, aldehydes (C₂₂-C₃₀) were major constituents in the wax of P. urnigerum. The classes and their chain length distributions in the surface waxes of these mosses are comparable to those of epicuticular waxes of higher plants.     

Glossy mutants of maize. VIII. Accumulation of fatty aldehydes in surface waxes of gl5 maize seedlings

In corn seedlings (Zea mays L.) homozygous for the mutation gl5, the surface waxes are characteristically altered. In this mutant the main wax constituents (83.5%) are aldehydes while in the normal waxes alcohols predominate (62.7%). Moreover, in the normal waxes aldehydes and alcohols are made up mainly of the C₃₂ term (99%), whereas in gl5 waxes the principal aldehyde is still C₃₂ (90.7%) but the free alcohol composition pattern is noticeably modified. Here the predominant terms are C₂₄, C₂₆, and C₂₈, with C₃₂ representing only 16.6% of the total. The results indicate that the mutant induces a block in the synthesis of fatty alcohols while accumulating fatty aldehydes, the substrates from which the alcohols originate.     

Biogeographic Variation of Foliar n‐Alkanes of Juniperus communis var. saxatilis Pallas from the Balkans

The composition of the epicuticular n‐alkanes isolated from the leaves of ten populations of Juniperus communis L. var. saxatilis Pallas from central (continental) and western (coastal) areas of the Balkan Peninsula was characterized by GC‐FID and GC/MS analyses. In the leaf waxes, 14 n‐alkane homologues with chain‐lengths ranging from C₂₂ to C₃₅ were identified. All samples were dominated by n‐tritriacontane (C₃₃), but differences in two other dominant n‐alkanes allowed separating the coastal from the continental populations. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses as well as the Mantel test) were deployed to analyze the diversity and variability of the epicuticular‐leaf‐n‐alkane patterns of the ten natural populations of J. communis var. saxatilis and their relation to different geographic and bioclimatic parameters. Cluster analysis showed a high correlation of the leaf‐n‐alkane patterns with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon. Several bioclimatic parameters related to aridity were highly correlated with this differentiation.     

Stomatal and pavement cell density linked to leaf internal CO2 concentration

Background and Aims

Stomatal density (SD) generally decreases with rising atmospheric CO₂ concentration, C_a. However, SD is also affected by light, air humidity and drought, all under systemic signalling from older leaves. This makes our understanding of how C_a controls SD incomplete. This study tested the hypotheses that SD is affected by the internal CO₂ concentration of the leaf, C_i, rather than C_a, and that cotyledons, as the first plant assimilation organs, lack the systemic signal.

Methods

Sunflower (Helianthus annuus), beech (Fagus sylvatica), arabidopsis (Arabidopsis thaliana) and garden cress (Lepidium sativum) were grown under contrasting environmental conditions that affected C_i while C_a was kept constant. The SD, pavement cell density (PCD) and stomatal index (SI) responses to C_i in cotyledons and the first leaves of garden cress were compared. ¹³C abundance (δ¹³C) in leaf dry matter was used to estimate the effective C_i during leaf development. The SD was estimated from leaf imprints.

Key Results

SD correlated negatively with C_i in leaves of all four species and under three different treatments (irradiance, abscisic acid and osmotic stress). PCD in arabidopsis and garden cress responded similarly, so that SI was largely unaffected. However, SD and PCD of cotyledons were insensitive to C_i, indicating an essential role for systemic signalling.

Conclusions

It is proposed that C_i or a C_i-linked factor plays an important role in modulating SD and PCD during epidermis development and leaf expansion. The absence of a C_i–SD relationship in the cotyledons of garden cress indicates the key role of lower-insertion CO₂ assimilation organs in signal perception and its long-distance transport.     

Hydrogen isotopic compositions of n-alkanes from terrestrial plants correlate with their ecological life forms

Stable hydrogen isotopic compositions (δD) of compound-specific biomarkers, such as n-alkanes from plant leaf waxes, can be used as a proxy for paleoclimatic change. However, the relationship between hydrogen isotopes of plant leaf wax and plant ecological life forms is not well understood. Here, we report the δD of n-alkanes from 34 modern terrestrial plants, including twenty-one C₃ plants and thirteen C₄ plants from northwestern China, determined using gas chromatography/thermal conversion/isotope ratio mass spectrometry. Our data show that the stable hydrogen isotopes are poorly correlated with the plant photosynthetic pathway (C₃ vs. C₄) and that they do not give clear regional precipitation signals. Together with a comparative analysis of published δD values from plant leaf waxes in other regions, we believe that the stable hydrogen isotope of plant leaf waxes is more closely related to ecological life forms of these terrestrial plants (i.e. tree, shrub, and grass). In general, the grasses have more negative δD values than the co-occurring trees and shrubs. Our findings suggest that the δD values of sedimentary leaf waxes from higher plants may record changes of a plant ecosystem under the influence of environmental alteration and imply that reconstruction of the paleoclimate using δD values from plant n-alkanes should be based upon specific plant taxa, and comparison should be made among plants with similar ecological life forms.     

Identification of In-Chain-Functionalized Compounds and Methyl-Branched Alkanes in Cuticular Waxes of Triticum aestivum cv. Bethlehem

In this work, cuticular waxes from flag leaf blades and peduncles of Triticum aestivum cv. Bethlehem were investigated in search for novel wax compounds. Seven wax compound classes were detected that had previously not been reported, and their structures were elucidated using gas chromatography-mass spectrometry of various derivatives. Six of the classes were identified as series of homologs differing by two methylene units, while the seventh was a homologous series with homologs with single methylene unit differences. In the waxes of flag leaf blades, secondary alcohols (predominantly C₂₇ and C₃₃), primary/secondary diols (predominantly C₂₈) and esters of primary/secondary diols (predominantly C₅₀, combining C₂₈ diol with C₂₂ acid) were found, all sharing similar secondary hydroxyl group positions at and around C-12 or ω-12. 7- and 8-hydroxy-2-alkanol esters (predominantly C₃₅), 7- and 8-oxo-2-alkanol esters (predominantly C₃₅), and 4-alkylbutan-4-olides (predominantly C₂₈) were found both in flag leaf and peduncle wax mixtures. Finally, a series of even- and odd-numbered alkane homologs was identified in both leaf and peduncle waxes, with an internal methyl branch preferentially on C-11 and C-13 of homologs with even total carbon number and on C-12 of odd-numbered homologs. Biosynthetic pathways are suggested for all compounds, based on common structural features and matching chain length profiles with other wheat wax compound classes.     

The distribution of leaf photosynthetic activity in a mixed grass-legume pasture canopy

The distribution of net photosynthetic activity of leaves was measured in a mixed grass (Setaria sphacelata var. sericea)-legume (Desmodium intortum) pasture stand using a method based on concurrent measurement of the rate of CO₂ exchange, and ¹⁴CO₂ dosing followed by rapid harvesting according to height strata. Comparisons were also made between plots which differed in the period of regrowth following defoliation. The usual superiority of leaf net photosynthetic rates of a C₄ grass, compared with C₃ legume leaves, was found in the upper, well illuminated strata. These rates were, however, much lower than those usually described for horizontally exposed leaves, primarily because leaves in the pasture stand were inclined to the horizontal. At greater depth in the canopies, the superiority of rates in the grass was less evident, and consequently the relative contributions of grass and legume to canopy photosynthesis became more dependent on their leaf area indices. Attention is drawn to the relative simplicity of the method for examining the contribution of leaves, which may differ according to species or position in the canopy, to productivity of the whole stand.     

The distribution of leaf photosynthetic activity in a mixed grass-legume pasture canopy

The distribution of net photosynthetic activity of leaves was measured in a mixed grass (Setaria sphacelata var. sericea)-legume (Desmodium intortum) pasture stand using a method based on concurrent measurement of the rate of CO₂ exchange, and ¹⁴CO₂ dosing followed by rapid harvesting according to height strata. Comparisons were also made between plots which differed in the period of regrowth following defoliation. The usual superiority of leaf net photosynthetic rates of a C₄ grass, compared with C₃ legume leaves, was found in the upper, well illuminated strata. These rates were, however, much lower than those usually described for horizontally exposed leaves, primarily because leaves in the pasture stand were inclined to the horizontal. At greater depth in the canopies, the superiority of rates in the grass was less evident, and consequently the relative contributions of grass and legume to canopy photosynthesis became more dependent on their leaf area indices. Attention is drawn to the relative simplicity of the method for examining the contribution of leaves, which may differ according to species or position in the canopy, to productivity of the whole stand.     

Two sides of a leaf blade: <Emphasis Type="Italic">Blumeria graminis</Emphasis> needs chemical cues in cuticular waxes of <Emphasis Type="Italic">Lolium perenne</Emphasis> for germination and differentiation

Plant surface characteristics were repeatedly shown to play a pivotal role in plant–pathogen interactions. The abaxial leaf surface of perennial ryegrass (Lolium perenne) is extremely glossy and wettable compared to the glaucous and more hydrophobic adaxial surface. Earlier investigations have demonstrated that the abaxial leaf surface was rarely infected by powdery mildew (Blumeria graminis), even when the adaxial surface was densely colonized. This led to the assumption that components of the abaxial epicuticular leaf wax might contribute to the observed impairment of growth and development of B. graminis conidia on abaxial surfaces of L. perenne. To re-assess this hypothesis, we analyzed abundance and chemical composition of L. perenne ab- and adaxial epicuticular wax fractions. While the adaxial epicuticular waxes were dominated by primary alcohols and esters, the abaxial fraction was mainly composed of n-alkanes and aldehydes. However, the major germination and differentiation inducing compound, the C₂₆-aldehyde n-hexacosanal, was not present in the abaxial epicuticular waxes. Spiking of isolated abaxial epicuticular Lolium waxes with synthetically produced n-hexacosanal allowed reconstituting germination and differentiation rates of B. graminis in an in vitro germination assay using wax-coated glass slides. Hence, the absence of the C₂₆-aldehyde from the abaxial surface in combination with a distinctly reduced surface hydrophobicity appears to be primarily responsible for the failure of normal germling development of B. graminis on the abaxial leaf surfaces of L. perenne.     

Homologous long-chain δ-lactones in leaf cuticular waxes of Cerinthe minor

A homologous series of eleven δ-lactones (1,5-alkanolides) was identified in cuticular waxes from leaves of Cerinthe minor L., six of them representing novel compounds. They accounted for 79% of the total coverage of 41 μg wax per cm² leaf area. Various chemical transformations with product identification by GC-mass spectrometry and GC-FTIR were employed to assign the structures. The chain-lengths of the δ-lactones ranged from C₂₂ to C₃₂ and even-numbered homologues were prevalent. Additionally, aldehydes (C₂₆–C₃₀), alkanes (C₂₃–C₂₉), primary alcohols (C₂₆–C₃₂), alkanoic acids (C₂₀–C₃₂), wax esters (C₄₀–C₅₆) and lupeol were detected.     

Changes of epicuticular wax induced by enhanced UV-B radiation impact on gas exchange in Brassica napus

The epicuticular wax covering on plant surface plays important roles in protecting plants against UV radiation. However, the role of epicuticular wax in affecting leaf gas exchange under enhanced ultraviolet-B (UV-B) radiation remains obscure. In the present study, different aged leaves of Brassica napus were used to analyze the responses of crystal structure and chemical constituents of epicuticular wax to UV-B radiation and the effects of such responses on gas exchange indices. Enhanced UV-B radiation significantly decreased the amount of esters in all leaves except the first leaf, amount of secondary alcohols in the second, third and fourth leaves, and amount of primary alcohols in the second and third leaves, while increased the amounts of ketones and aldehydes in the first leaf. Enhanced UV-B level had no significant effect on the amounts of alkanes and total wax in all leaves. Exposure to UV-B radiation resulted in wax fusion on adaxial leaf and stomata opening on abaxial leaf. Fusions of plates and rods on adaxial leaf surface covered most of the stomata, thereby influencing the photosynthesis in the upper mesophyll of leaves. Enhanced UV-B level significantly reduced the net photosynthesis rate (P _N) but increased the stomata conductance (g _s), concentrations of intercellular CO₂ (C _i ), and transpiration rate (E) in all leaves. Both UV-B radiation and the wax fusion induced by enhanced UV-B radiation resulted in different stomata status on abaxial and adaxial leaf surface, causing decrease of P _N, and increase of g _s, C _i and E in leaves.