Nanotubules on plant surfaces: chemical composition of epicuticular wax crystals on needles of Taxus baccata L

Needles of Taxus baccata L. were covered with tubular epicuticular wax crystals varying in diameters (100 and 250 nm) and lengths (300-500 and 500-1000 nm) on the abaxial and adaxial surfaces, respectively. Various sampling protocols were employed to study the chemical composition of the needle waxes on three different levels of spatial resolution. First, a dipping extraction of whole needles yielded the total cuticular wax mixture consisting of very long chain fatty acids (21%), alkanediols (19%), phenyl esters (15%), and secondary alcohols (9%) together with small amounts of aldehydes, primary alcohols, alkanes, alkyl esters, and tocopherols. Second, waxes from both sides of the needle were sampled separately by brushing with CHCl3-soaked fabric glass. Both sides showed very similar qualitative composition, but differed drastically in quantitative aspects, with nonacosan-10-ol (18%) and alkanediols (33%) dominating the abaxial and adaxial waxes, respectively. Third, the epi- and intracuticular wax layers were selectively sampled by a combination of mechanical wax removal and brushing extraction. This provided direct evidence that the tubular wax crystals contained high percentages of nonacosane-4,10-diol and nonacosane-5,10-diol on the abaxial surface, and nonacosan-10-ol on the adaxial surface of the needles. Together with these compounds, relatively large amounts of fatty acids and smaller percentages of aldehydes, primary alcohols, alkyl esters, and alkanes co-crystallized in the epicuticular layer. In comparison, the intracuticular wax consisted of higher portions of cyclic constituents and aliphatics with relatively high polarity. The formation of the tubular crystals is discussed as a spontaneous physico-chemical process, involving the establishment of gradients between the epi- and intracuticular wax layers and local phase separation.     

Chemical induction of adventitious root formation in Taxus baccata cuttings

The effect of some auxins (IBA and NAA), phenolic compounds (phloroglucinol, gentisic acid and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin) have been examined for stimulatory effects on adventitious root formation in stem cuttings (current season's growth) of Taxus baccata L. In general lower concentration (0.25 mM) of both IBA and NAA was more effective in inducing rooting of cuttings taken from both male and female trees. The combined treatment of IBA+NAA (0.25 mM each) showed some success in cuttings from male trees only (55%, compared to 15% rooting in cuttings from female trees). Generally, the callus formation was quite high (70%) in all auxin treatments (alone or in combination). Among the phenolics, 40% rooting success was achieved with phloroglucinol only, while coumarin and gentisic acid were ineffective. The combined treatment of auxins and phenolics also failed to promote rooting. On the other hand, Bavistin was extremely effective for callusing (90%) as well as rooting (80%). The effectiveness of various compounds tested for rooting of young stem cuttings declined in the order: 0.25 mM IBA>0.05% Bavistin>0.25 mM NAA>1.25 mM IBA>15 mM phloroglucinol>IBA+NAA (0.25 mM each). In addition to the auxins, IBA and NAA that are widely used for commercial propagation, the auxin-like properties of the fungicide Bavistin could be exploited for adventitious rooting in T. baccata, and in other plant species.     

Increased inbreeding and strong kinship structure in Taxus baccata estimated from both AFLP and SSR data

Habitat fragmentation can have severe genetic consequences for trees, such as increased inbreeding and decreased effective population size. In effect, local populations suffer from reduction of genetic variation, and thus loss of adaptive capacity, which consequently increases their risk of extinction. In Europe, Taxus baccata is among a number of tree species experiencing strong habitat fragmentation. However, there is little empirical data on the population genetic consequences of fragmentation for this species. This study aimed to characterize local genetic structure in two natural remnants of English yew in Poland based on both amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers. We introduced a Bayesian approach that estimates the average inbreeding coefficient using AFLP (dominant) markers. Results showed that, in spite of high dispersal potential (bird-mediated seed dispersal and wind-mediated pollen dispersal), English yew populations show strong kinship structure, with a spatial extent of 50–100 m, depending on the population. The estimated inbreeding levels ranged from 0.016 to 0.063, depending on the population and marker used. Several patterns were evident: (1) AFLP markers showed stronger kinship structure than SSRs; (2) AFLP markers provided higher inbreeding estimates than SSRs; and (3) kinship structure and inbreeding were more pronounced in denser populations regardless of the marker used. Our results suggest that, because both kinship structure and (bi-parental) inbreeding exist in populations of English yew, gene dispersal can be fairly limited in this species. Furthermore, at a local scale, gene dispersal intensity can be more limited in a dense population.     

Homologous very-long-chain 1,3-alkanediols and 3-hydroxyaldehydes in leaf cuticular waxes of Ricinus communis L

Surface extracts from primary leaves of Castor bean were found to contain 1.8 microg cm(-2) of cuticular waxes. The mixture comprised alkanes (C(26)-C(29)), primary alcohols (C(22)-C(38)), aldehydes (C(26) and C(28)), fatty acids (C(20)-C(34)) and triterpenoids (lupeol, beta- and alpha-amyrin). Besides, a series of n-alkane-1,3-diols was detected, with chain lengths ranging from C(22) to C(28), a strong predominance of even-numbered homologs, and a maximum for hexacosane-1,3-diol. Seven other compounds were assigned to a novel class of wax constituents and identified as homologous unbranched 3-hydroxyaldehydes ranging from C(22) to C(28). As the chain length distribution of this series closely paralleled the homolog pattern of 1,3-diols, it seems likely that both compound classes are biosynthetically related.     

Taxanes with C-5-amino-side chains from the needles of Taxus canadensis

Five taxanes with an amino-side chain on C-5 were identified for the first time in the needles of the Canadian yew, Taxus canadensis. Their structures were characterized as 2alpha,7beta,9alpha,10beta,13-pentaacetoxy-11beta-hydroxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),12-diene (1), 2alpha,9alpha-dihydroxy-10beta,13alpha-diacetoxy-5alpha-(3'-methylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (2), 2alpha17-dihydroxy-9alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (3), 2alpha-hydroxy-7beta,9alpha,10beta,13alpha-tetraacetoxy-5alpha-(2'-hydroxy-3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (4), and 9alpha-hydroxy-2alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (5) on the basis of 1D-, 2D-NMR spectroscopic data and high-resolution fast atom bombardment MS analyses. Metabolite (1) was isolated from the needles of the Canadian yew for the first time but had previously been detected in the stems of the Japanese yew, whereas taxanes (2-5) are only now reported. Metabolite (3) is the first reported nitrogen-containing taxane with a 17-hydroxyl substitution.     

Leaf age as a factor in anatomical and physiological acclimative responses of Taxus baccata L. needles to contrasting irradiance environments

To evaluate the acclimative ability of current-year and previous-year needles of a shade tolerant conifer Taxus baccata L. to contrasting irradiance conditions, seedlings were raised under 27% solar irradiance and at 3 years of age they were transferred to an experimental garden and grown for one season under full irradiance (HL), 18% irradiance (ML) or 5% irradiance (LL). Whereas previous year needles did not change anatomically, current year needles in HL were thicker and had a thicker palisade and spongy mesophyll, and greater leaf mass per area than ML or LL needles. LL needles had greater nitrogen concentration than HL needles irrespective of age but only previous year LL needles also had an increased N per area content, thanks to their lack of reduction in LMA. Adjustment of chlorophyll and carotenoid content occurred in both needle age classes with LL and ML needles having much higher concentrations but, in current year needles, only slightly higher per area content than HL needles. Chlorophyll a/b ratio was not affected by age or irradiance. These modifications had no significant effect on photosynthetic capacities, which did not significantly differ between the age classes in HL or LL treatment and between treatments. On the other hand, high growth irradiance resulted in a greater photochemical yield, photochemical quenching, apparent electron transport rate and inducible non-photochemical quenching in needles formed in the current season. In previous year needles, however, only inducible NPQ was enhanced by high irradiance with other parameters remaining identical among treatments. To test sensitivity to photoinhibition, at the end of the summer plants from the three irradiance levels were transferred to a HL situation and F _v/F _M was determined over the following 18 days. Sensitivity to photoinhibition was negatively related to growth irradiance and previous year needles were less photoinhibited than current year needles. Thus, differences in acclimation ability between needle age classes were most pronounced at the level of anatomy and light reactions of photosynthesis, both of which showed almost no plasticity in previous year needles but were considerably modified by irradiance in current year needles.     

Diterpenes and sesquiterpenes from the bark of Taxus yunnanensis

Two taxane-type diterpenes, 10beta-acetoxy-2alpha,5alpha,7beta,9alpha-tetrahydroxytaxa-4(20),11-dien-13-one and 2alpha-acetoxy-9alpha-benzoyloxy-5alpha,7beta,10beta,15-tetrahydroxy-11(15-->1)- abeotaxa-4(20),11-dien-13-one, and two new drimane-type sesquiterpenes, 1beta-acetoxy-7-drimen-11alpha-ol-12,11-lactone and 1beta-acetoxy-11,12-epoxy-6-drimen-8alpha,11alpha-diol, were isolated from the bark of Taxus yunnanensis together with 35 known taxane-type diterpenes, a known drimane-type sesquiterpene and a known flavanone.     

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.     

Transport properties of cuticular waxes of Fagus sylvatica L. and Picea abies (L.) Karst.: Estimation of size selectivity and tortuosity from diffusion coefficients of aliphatic molecules

Diffusion coefficients for thirteen lipophilic organic compounds in reconstituted waxes of Fagus sylvatica L. and Picea abies (L.) Karst. were measured to characterise the transport properties of the cuticular waxes that form the transport-limiting barrier of plant cuticles. Desorption kinetics (relative amounts desorbed versus time) were asymptotic, but could be linearized up to 50% desorption by plotting relative amounts desorbed versus the square root of time. Diffusion coefficients calculated from the slopes of the linear regressions ranged from 10^–22 to 10^–17 m²·s^–1 and decreased with increasing molecular size. This size dependency of diffusion coefficients was analysed, assuming an exponential dependence of the diffusion coefficients on molar volumes, which allowed cuticular transport properties to be related to the physical structure of the wax. Furthermore, the fact that the barrier properties of Fagus wax are less pronounced than those of Picea is interpreted as an ecological adaptation of the respective tree species to their habitats at the level of their cuticular transport barriers.Abbreviation D diffusion coefficient The authors gratefully acknowledge financial support by the Schwerpunkt Programm Baumphysiologie of the Deutsche Forschungsgemeinschaft.     

Foliar cuticular alkanes of Camarea (Malpighiaceae) and their taxonomic significance

Camarea is a South-American endemic genus comprising eight species. In the present work n-alkanes from foliar cuticular waxes of 23 specimens, representing seven species of Camarea were analyzed, aiming at establishing interspecific affinities and evaluating the usefulness of n-alkane distribution as species characteristic. The sampling included also specimens of Peixotoa reticulata and Janusia guaranitica (both Malpighiaceae). The results were used to obtain a phenogram indicating chemical affinities between species. The results are in agreement with morphological similarities among some Camarea species. Intraspecific variability was small, suggesting that n-alkane distribution may be useful for species characterization and establishment of links among Camarea species. The results support the recognition of Camarea triphylla as a synonym of Camarea axillaris and are not coherent with a hybrid condition of a population exhibiting morphological characteristics combining Camarea affinis and Camarea hirsuta, suggesting instead that the individuals analyzed belong either to Camarea hirsuta or a close species. Distribution of n-alkanes is inadequate to distinguish among Malpighiaceae genera: P. reticulata has n-alkane distribution similar to several Camarea species.     

Cuticular waxes from potato (Solanum tuberosum) leaves

The qualitative and quantitative compositions of leaf cuticular waxes from potato (Solanum tuberosum) varieties were studied. The principal components of the waxes were very long chain n-alkanes, 2-methylalkanes and 3-methylalkanes (3.1-4.6 microg cm(-2)), primary alcohols (0.3-0.7 microg cm(-2)), fatty acids (0.3-0.6 microg cm(-2)), and wax esters (0.1-0.4 microg cm(-2)). Methyl ketones, sterols, beta-amyrin, benzoic acid esters and fatty acid methyl, ethyl, isopropyl and phenylethyl esters were found for the first time in potato waxes. The qualitative composition of the waxes was quite similar but there were quantitative differences between the varieties studied. A new group of cuticular wax constituents consisting of free 2-alkanols with odd and even numbers of carbon atoms ranging from C25 to C30 was identified.     

Effect of the entomopathogenic fungus,Entomophthora muscae (Zygomycetes: Entomophthoraceae), on sex pheromone and other cuticular hydrocarbons of the house fly,Musca domestica

House fly (Musca domestica) males are highly attracted to dead female flies infected with the entomopathogenic fungus Entomophthora muscae. Because males orient to the larger abdomen of infected flies, both visual and chemical cues may be responsible for the heightened attraction to infected flies. Our behavioral assays demonstrated that the attraction is sex-specific-males were attracted more to infected females than to infected males, regardless of cadaver size. We examined the effect of E. muscae on the main component of the house fly sex pheromone, (Z)-9-tricosene, and other cuticular hydrocarbons including n-tricosane, n-pentacosane, (Z)-9-heptacosene, and total hydrocarbons of young (7 days old) and old (18 days old) virgin females. Young E. muscae-infected female flies accumulated significantly less sex pheromone and other hydrocarbons on their cuticular surface than uninfected females, whereas the cuticular hydrocarbons of older flies were unaffected by fungus infection. These results suggest that chemical cues other than (Z)-9-tricosene, visual cues other than abdomen size, or a combination of both sets of cues might be responsible for attraction of house fly males to E. muscae-infected females.     

Diterpene esters of aristolochic acids from Aristolochia pubescens

From the acetone and ethanol extracts of the tubercula of Aristolochia pubescens, two diterpene esters of aristolochic acids were isolated, together with 23 known compounds. The structures of the compounds were determined on the basis of spectroscopic analysis.     

Stigmastane derivatives and isovaleryl sucrose esters from Vernonia guineensis (Asteraceae)

Vernoguinoside, 16beta,22R;21,23S-diepoxy-3beta-O-beta-D-glucopyranosyloxy-21S,24-dihydroxy-5alpha-stigmasta-8,14-dien-28-one (1), a new stigmastane derivative, 16beta,22R;21,23S-diepoxy-21S,24-dihydroxy-5alpha-stigmasta-8,14-diene-3,28-dione (2) and two new sucrose esters, 1',3,3',4',6'-pentakis-O-(3-methylbutanoyl)-beta-D-fructofuranosyl alpha-D-glucopyranoside (3) and 1',2,3',6,6'-pentakis-O-(3-methylbutanoyl)-beta-D-fructofuranosyl alpha-D-glucopyranoside (4), have been isolated from the stem bark of Vernonia guineensis. The structures of the new compounds were determined on the basis of spectroscopic evidence.     

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。     

Essential fatty acids and phenolic acids from extracts and leachates of southern cattail (Typha domingensis P.)

We have been able to isolate several phytotoxic compounds from aqueous extracts and leachates of cattails (Typha domingensis) using activated charcoal as an absorbant, followed by successive extraction with organic solvents, analysis by GC/MS, and structural elucidation by NMR spectroscopy when possible. The phytotoxins were identified as essential fatty acids (linoleic acid and alpha-linolenic acid) and phenolic compounds of known phytotoxic activity (caffeic acid from the aqueous extracts; caffeic, p-coumaric, and gallic acid from the leachates). Both extracts and the phytotoxins in the extracts have the potential of inhibiting the growth and chlorophyll production of several ecologically relevant species.     

Taxol content in the bark of Himalayan Yew in relation to tree age and sex

Taxol content in the bark of Taxus baccata trees growing in a homogenous (uniform) environment at Jageshwar, District Almora in Central Himalaya has been quantified. The average taxol concentration in the bark of sampled trees was 0.0558+/-0.008% (of dry wt.) and was about 64% higher for male plants (averaged across tree age) in comparison to female trees. Maximum taxol content was recorded in the bark samples collected from trees of >110 yrs age. ANOVA indicates a significant difference in the taxol content of bark from trees of different ages, however, differences were not significant between sexes. Taxol was quantified by HPLC using a standard curve prepared with authentic taxol; the identification of bark taxol was confirmed by UV and mass spectrometry. The total taxol content of the bark of Taxus trees across an age series was found to range between 0.064 to 8.032 g/tree, and a tree of about 100 yrs age can yield 5.74 kg dry bark.     

Changes in the content and constituents of the cuticular wax of Picea abies (L.) Karst. in relation to needle ageing and tree decline in five European forest areas

Cuticular wax composition of healthy and and declining mature Norway spruce trees [Picea abies (L.) Karst.] was investigated in five European forest areas. The amount of extracted wax and the content of alkanes and secondary alcohols were analysed as a function of the factors sample area (five areas, detailed below), needle age (current year to 2 years) and decline class (Class O to Class 2). Using a GC-MS, alkanes from C₂₀ to C₃₁ and the following alcohols were quantified: 10-nonacosanol, 5,10-nonacosandiol, 4,10-nonacosandiol and the triterpenol 24-methylenecycloartanol. According to our results, the total wax content as well as the alkane and alcohol content of waxes show a large variation corresponding to sample area and needle age. Ageing caused a highly significant increase in alkane content and a highly significant decrease in total wax and alcohol content. The decline class significantly influenced only the content of the long chain alkane C₃₁ (increase), the secondary alcohol 10-nonacosanol (decrease), and the triterpenic alcohol (decrease). Total wax weight was not influenced by tree damage. Thus, according to our results, needle ageing and progressive tree damage are correlated to different changes in the examined parameters.     

Effect of mating stage on water balance, cuticular hydrocarbons and metabolism in the desert harvester ant, Pogonomyrmex barbatus

Water-loss rates increase after mating in queens of the harvester ant Pogonomyrmex barbatus (Formicidae: Myrmicinae), then increase again after the mated queens excavate an incipient nest. We determined the mechanistic basis for these increased water-loss rates by examining cuticular permeability, respiratory water loss, metabolic rates, and cuticular hydrocarbons for queens at three stages in the mating sequence: unmated alate queens, newly mated dealate queens, and mated queens excavated from their incipient nest. Both total water loss and cuticular transpiration increased significantly following mating, with cuticular transpiration accounting for 97% of the increased water loss. In contrast, metabolic rate and respiratory water loss were unaffected by mating stage. The total quantity of cuticular hydrocarbons did not vary by mating stage. However, relative amounts of four of the most abundant cuticular hydrocarbons did vary by mating stage, as did quantities of n-alkanes and methylalkanes. The general pattern was that percent composition of n-alkanes decreased through the mating sequence, while percent composition of methylalkanes increased over the same sequence. We discuss three mechanisms that might cause these post-mating increases in cuticular permeability. Our data support the hypothesis that part of this increase results from soil particles abrading the cuticle during the process of nest excavation.