Very long-chain phenylpropyl and phenylbutyl esters from Taxus baccata needle cuticular waxes

The cuticular wax of Taxus baccata L. needles was found to contain four different classes of long-chain esters that were identified by various chemical transformations with product assignment employing GC-MS. Homologous series of (1) 3-(4'-hydroxyphenyl)-propyl esters of C(20)-C(36) fatty acids, (2) 4-(4'-hydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids, (3) 3-(3',4'-dihydroxyphenyl)-propyl esters of C(20)-C(32) fatty acids, and (4) 4-(3',4'-dihydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids were identified. The four compound classes amounted to 0.1-3.6 micro g/cm(2) of needle surface area, corresponding to 0.2-7.6% of the wax mixture, respectively. While both phenylpropyl ester series had a maximum for the homolog containing tetracosanoic acid, in the phenylbutyl esters homologs containing eicosanoic and docosanoic acids predominated.     

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.     

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.     

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.     

核盘菌侵染对拟南芥表皮蜡质结构及化学组成的影响

以野生型拟南芥与蜡质突变体cer1、cer4为试验材料,通过研究核盘菌胁迫对拟南芥茎表皮蜡质结构及组分含量的影响,揭示核盘菌侵染与表皮蜡质的关系。扫描电镜结果显示,野生型拟南芥蜡质晶体以垂直于表面的杆状、块状结构为主;突变体cer1晶体类型以水平的松针状、块状结构为主;突变体cer4蜡质晶体以垂直片层结构为主。核盘菌胁迫下,拟南芥蜡质晶体结构及分布形态发生变化。蜡质层结构在核盘菌胁迫下表现为:杆状、松针状蜡质晶体减少—蜡质晶体熔融—表皮"囊状凸起"—表皮膜层破裂。这些结构变化有利于病菌突破角质层屏障而侵入到植株体内。色质谱分析结果显示:与野生型相比,cer1突变体烷、次级醇、酮类显著减少;cer4突变体表现为一级醇含量减少。接种核盘菌后,野生型拟南芥与蜡质突变体一级醇类显著增加(cer1增加不显著);烷类、次级醇类、酮类含量与蜡质总量均显著减少,表明蜡质前体物质在受到核盘菌胁迫后更多地通过酰基还原途径生成一级醇,从而减少了由脱羰基途径所生成的蜡质组分。核盘菌通过改变表皮蜡质晶体结构与化学组分分泌量来促进侵染。     

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.     

Slippery surfaces of carnivorous plants: composition of epicuticular wax crystals in<Emphasis Type="Italic"> Nepenthes alata</Emphasis> Blanco pitchers

Plants in the genus Nepenthes obtain a substantial nutrient supply by trapping insects in highly modified leaves. A broad zone of the inner surface of these pitchers is densely covered with wax crystals on which most insects lose their footing. This slippery wax surface, capturing prey and preventing its escape from the trap, plays a pivotal role in the carnivorous syndrome. To understand the mechanism of slipperiness, the present investigation aimed at an ultrastructural and physico-chemical characterization of the wax crystals in pitchers of N. alata Blanco. Scanning electron microscopy revealed that entire platelets protruded perpendicularly from the surface. Methods were developed that allowed the mechanical removal of wax crystals from the pitcher surface. It could be shown that the sampling was selective for the epicuticular wax, relevant for plant–insect interactions. The crystals consisted of a mixture of aliphatic compounds dominated by very-long-chain aldehydes. Triacontanal, at 43% the most abundant constituent, was largely responsible for crystal formation. Solubility data indicate that the Nepenthes crystals contained polymeric forms of this aldehyde. The resulting mechanical properties of the polymer crystals and the mechanism of slipperiness are discussed.Abbreviation SEM scanning electron microscopy     

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.     

Taxonomic significance of the epicuticular wax composition in species of the genus Clusia from Panama

We attempted to separate species of the genus Clusia according to the concentration of linear alkanes (C25 and C35), and the presence and diversity of terpenes in epicuticular wax extracts. We collected leaves of 15 Clusia species growing in mountain forests of Panama (Cerro Jefe 1007 m and Altos de Campana 800 m a.s.l.) and from cultivated plants at two lowland sites. Leaf surfaces were washed gently with hexane to extract epicuticular waxes, which were analyzed using gas chromatography and mass spectrometry. The predominant alkanes were C29, C31, and C33. In the extract the ratio C31/C29 was ≤1 in 6 of the 15 species analyzed: Clusia multiflora, Clusia peninsulae (Hammel ined.), Clusia stenophylla, Clusia liesneri, Clusia coclensis, and Clusia triflora. The concentrations of C29 and C33 were inversely related, the latter being above 10% in Clusia divaricata, Clusia pratensis, Clusia rosea, Clusia uvitana, and Clusia valerioi. Proportion of triterpenes was less than 5% in the species C. minor, C. pratensis, C. uvitana, C. rosea, Clusia cylindrica, C. divaricata, and C. valerioi. The rest contained squalene, and specific triterpenes such as β-amyrine in C. liesneri, betuline in Clusia osseocarpa, taraxerol in C. stenophylla, and lupeol in C. multiflora. The variety of triterpenes was higher in Clusia liesneri (5) and C. multiflora (3). The results suggest that groups of species can be distinguished within the genus according to the presence of terpenes and ratios of linear alkanes. These groups overlap with those generated by other classifications using morphology and nuclear ribosomal DNA.     

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.     

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

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

Cuticular wax composition of Salix varieties in relation to biomass productivity

The leaf cuticular waxes of six Salix clones (one Salix miyabeana, one Salix dasyclados, one Salix eriocephala, two Salix purpurea, and one interspecific hybrid of Salix eriocephala x interior) with different biomass productivities were characterized by gas chromatography-mass spectrometry. Total wax content ranged from 6.3 to 16.8 microg cm(-2), and two distinct patterns of wax were measured. The wax from leaves of S. dasyclados 'SV1' differed from all other clones and was dominated by fatty acids (42%), high concentrations of n-alkanes (25%) and n-alcohols (28%), with low n-aldehyde content (4%). All other clones produced cuticular wax dominated by n-alcohols (32-51%), particularly 1-hexacosanol, with fatty acids (14-37%) and n-aldehydes (19-26%) present in lower abundances. Clones of Salix grown under identical environmental conditions produce noticeably different amounts of cuticular wax. In contrast to previous studies of Salix, total wax content was independent of biomass productivity, measured as basal area, suggesting that wax production is not directly linked with woody biomass production by shrub willows under these site conditions.     

UV-B辐射增强对拟南芥表皮蜡质的影响

以野生型拟南芥、蜡质不同程度缺失突变体CER1、CER3、CER4、CER6、CER10、CER20及KCS1为试验材料,通过施加50μW/cm2、长达10 d的UV-B辐射,研究了拟南芥表皮蜡质晶体结构、组分及蜡质基因对UV-B辐射的响应机制。结果表明:UVB辐射增强改变了拟南芥表皮蜡质晶体结构,表皮蜡质松针状(CER1)、柱状、杆状(CER3、CER10与KCS1)晶体结构显著减少,球状蜡质晶体类型出现在CER6表面,无规则片状、膜状结构覆盖在KCS1与CER10茎表面。野生型拟南芥蜡质晶体结构类型无明显变化,但在部分区域积累了大量水平杆状、管状结构,增加了蜡质层厚度。UV-B辐射增强也改变了拟南芥表皮蜡质组分的分泌量。野生型在UV-B处理后一级醇、酸、醛含量显著上升,烷、次级醇及酮含量显著下降,蜡质总量增加不显著。一级醇含量的增加及酮和次级醇含量的减少在拟南芥各材料响应UV-B辐射中具有普遍性。UV-B辐射增强诱导了野生型CER3、CER4、KCS1基因表达的上调,其中CER4大量表达,促进了蜡质组分中一级醇、酸和醛含量的积累;CER1在UV-B处理后表达量下调,可能导致烷合成下游分支途径相关产物(烷类、次级醇及酮类)的减少。WIN1表达量的下调对蜡质总量没有显著影响。UV-B辐射增强使蜡质前体从烷合成分支途径更多地转向一级醇分支途径。     

Chemistry of Chinese yew, Taxus chinensis var. mairei

Taxus chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu is an evergreen tall tree ubiquitous to the southeastern region in China. The first chemical study on this species was published in 1987. Since then about 163 compounds including taxoids and non-taxane compounds were isolated from seeds, root, bark and leaves of this species. This review summarized the chemical investigation on T. chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu in the past 20 years. T. chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu was assigned as a variation of T. chinensis (Pilger) Rehd. in Flora of China. However, present chemotaxonomic data would suggest that these two plants might belong to two different chemotypes; further genetic investigation and comprehensive chemical studies on them are warranted to address this issue.     

Effect of provenance on free amino acid and chemical composition of Scots pine needles

Free amino acid (16 amino acids) and chemical composition (N, P, K, Mg, Ca, S, Fe, Mn, Cu, Zn) of Scots pine (Pinus sylvestris L.) needles were compared between six provenances in three different experimental areas. The main free amino acids in the needles were in the sequence of quantity; glutamic acid, glutamine, arginine and γ-aminobutyric acid. There were no significant differences in the concentrations of phenylalanine, γ-aminobutyric acid, methionine, proline and threonine in pine needles between the sites or between the provenances. Significant differences in the foliar concentrations of alanine and leucine were found between the sites and in the foliar concentrations of isoleucine, glutamine, glycine and tyrosine between the provenances. The concentrations of aspartic acid, glutamic acid, arginine and lysine were significantly affected by the sites and the provenances. The foliar nutrients, except copper, had statistically significant differences, both between the sites and between the provenances. Calcium did not differ between the provenances. This revised version was published online in June 2006 with corrections to the Cover Date.     

Essential oil composition of Hypericum L. species from Southeastern Serbia and their chemotaxonomy

The essential oils of the aerial parts of nine species of Hypericum (Hypericum barbatum, Hypericum hirsutum, Hypericum linarioides, Hypericum maculatum, Hypericum olympicum, Hypericum perforatum, Hypericum richeri, Hypericum rumeliacum and Hypericum tetrapterum), collected from different locations in Southeast Serbia, were obtained by steam distillation and analyzed by GC and GC–MS. The essential oils investigated were characterized by a high content of non-terpene compounds and a low content of monoterpenes. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and H. rumeliacum (section Drosocaprium) were similar and these oils were characterized by high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum differed from others by higher terpene content. A comparison was also carried out of the chemical composition of the essential oils from flower, leaf and stem of H. perforatum and it revealed that the highest concentration of non-terpene compounds was found in the flower and stem oil, while a high concentration of sesquiterpenes was characteristic for leaf oil. There were significant differences in the concentrations of the same compounds in the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location which could be explained by seasonal differences. All data were statistically processed with principal component analysis and cluster analysis. The main conclusion from the above data is that genetic and environmental factors both play a role in determining the composition of essential oils of the Hypericum species studied.     

Very long chain alkylresorcinols accumulate in the intracuticular wax of rye (Secale cereale L.) leaves near the tissue surface

Alkylresorcinols (ARs) are bioactive compounds occurring in many members of the Poaceae, likely at or near the surface of various organs. Here, we investigated AR localization within the cuticular wax layers of rye (Secale cereale) leaves. The total wax mixture from both sides of the leaves was found to contain primary alcohols (71%), alkyl esters (11%), aldehydes (5%), and small amounts (<3%) of alkanes, steroids, secondary alcohols, fatty acids and unknowns. A homologous series of ARs (3%) was identified by GC-MS and comparison with a synthetic standard of nonadecylresorcinol. The alkyl side chains of the wax ARs contained odd numbers of carbons ranging from C19 to C27, with a prevalence of C21, C23 and C25. Waxes from both sides of the leaf, analyzed separately in a second experiment, comprised the same compound classes in similar relative amounts and with similar homolog patterns. Finally, the epicuticular and intracuticular wax layers were sampled separately from the abaxial side of the leaf. While ARs accounted for 2% of the intracuticular wax, they were not detectable in the epicuticular wax. The intracuticular wax was also slightly enriched in steroids, whereas the epicuticular layer contained more primary alcohols. All other wax constituents were distributed evenly between both wax layers.     

Crystal structures and chemical composition of leaf surface wax depositions on the desert moss Syntrichia caninervis

The structures and composition of cuticular waxes deposited on the leaves of a typical desert moss, Syntrichia caninervis, were investigated. The wax crystals deposited on leaves shifted with leaf aging. The results of chemical analysis showed the main chemical components of the moss wax were fatty acids, alcohols and alkanes. Leaf aging increased the content of cuticular wax and the percentage of very long chain components, from 1150 μg g⁻¹ DW and 13.6% in younger leaves to 2640 μg g⁻¹ DW and 37.2% in aged leaves, respectively. Dehydration/hydration also augmented the wax content by 35.17% in juvenile leaves and by 1900% in lab-cultivated leaves after three-cycle treatments. Synthesis of hexadecanoic acid and tetracosane were predicted to be the first step of wax accumulation. The responses of cuticular waxes in crystal structure and chemical composition were recommended as a biomonitor for assessing the shift of ecological and environmental quality.