Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.     

The effectiveness of cloning for the genetic improvement of Mexican white cypress Cupressus lusitanica (Mill.)

Two trials on Mexican cypress Cupressus lusitanica Miller in the North Island of New Zealand were assessed for diameter at breast height and at one site, subjective scores for branch size and stem canker (caused by Seiridium spp.) at age 6 from planting. The trials comprised 15 open-pollinated families, represented by both cloned and seedling progeny. Linear mixed model methodology, using a spatial model for the residuals, was applied to estimate genetic parameters. Estimated narrow-sense heritabilities were moderate to high for diameter at breast height (range from 0.46 to 0.62), stem canker (≈0.30) and branch size (range from 0.23 to 0.45) and did not appear to differ significantly between propagule types for all traits. Clonally replicated progeny led to an increase in accuracy of selection for additive genetic merit when compared with seedling testing, with the improvement being greater for traits with lower narrow-sense heritabilities. Estimated additive genetic correlations between cloned and seedling progeny were moderate to high (≥0.65) for diameter and branch size, indicating that selection decisions would not be substantially changed using either propagule type for progeny testing. All estimates of non-additive genetic variation based on the cloned progeny were non-significant. The use of spatial analysis was effective for diameter and branch size, but not for stem canker. No significant genotype by environment interaction was detected for diameter. Implications of the results for breeding and deployment of C. lusitanica are discussed.     

Production of beta-thujaplicin in Cupressus lusitanica suspension cultures fed with organic acids and monoterpenes.

Effects of some organic acids and monoterpenes on production of beta-thujaplicin were studied in Cupressus lusitanica suspension cultures. The fungal elicitor-induced biosynthesis of beta-thujaplicin was promoted by the feedings of malate, pyruvate, fumarate, succinate, and acetate. These results suggest some relationships between acetate/pyruvate metabolism and beta-thujaplicin biosynthesis, or between tricarboxylic acid cycle and beta-thujaplicin biosynthesis. Feedings of C. lusitanica suspension cultures with some monoterpenes inhibited elicitor-triggered beta-thujaplicin biosynthesis, but 2-carene and terpinyl acetate feedings significantly improved the beta-thujaplicin production of C. lusitanica suspension cultures. These results indicate a possible involvement of terpinyl acetate and 2-carene in beta-thujaplicin biosynthesis, as well as potential uses of these monoterpenes in large-scale beta-thujaplicin production.     

Differential metabolism of diastereoisomeric diterpenes by Preussia minima,found as endophytic fungus in Cupressus lusitanica

The plant diastereoisomeric diterpenes ent-pimara-8(14)-15-dien-19-oic acid, obtained from Viguiera arenaria, and isopimara-8(14)-15-dien-18-oic acid, isolated from Cupressus lusitanica, were distinctly functionalized by the enzymes produced in whole cell cultures of the fungus Preussia minima, isolated from surface sterilized stems of C. lusitanica. The ent-pimaradienoic acid was transformed into the known 7β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid, and into the novel diterpenes 7-oxo-8 β-hydroxy-ent-pimara-8(14)-15-dien-19-oic and 7-oxo-9β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acids. Isopimara-8(14)-15-dien-18-oic acid was converted into novel diterpenes 11α-hydroxyisopimara-8(14)-15-dien-18-oic acid, 7β,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, and 1β,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, along with the known 7β-hydroxyisopimara-8(14)-15-dien-18-oic acid. All compounds were isolated and fully characterized by 1D and 2D NMR, especially ¹³C NMR. The diterpene bioproduct 7-oxo-9β-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid is an isomer of sphaeropsidin C, a phytotoxin that affects cypress trees produced by Shaeropsis sapinea, one of the main phytopathogen of Cupressus. The differential metabolism of the diterpene isomers used as substrates for biotransformation was interpreted with the help of computational molecular docking calculations, considering as target enzymes those of cytochrome P450 group.     

Induced monoterpene and lignin production in mechanically stressed and fungal elicited cultured Cupressus lusitanica cells

Cultured Cupressus lusitanica cells induced by various stresses are thought to produce different complexes of defense chemicals to optimize defense. To compare the induced products of two stimulations, we investigated the emission of monoterpenes, biosynthesis of β-thujaplicin, and accumulation of lignin in mechanically stressed and fungal elicited cultured C. lusitanica cells. Both mechanical stress and fungal elicitor caused emission of qualitatively similar monoterpene blends indicating de novo biosynthesis of these compounds after stimulation, while mechanical stress alone is sufficient to induce fungal elicitor-related monoterpene emission. Sabinene and limonene were the dominant compounds over the time course in both volatile blends. Although the emitted volatile blends were qualitatively similar, the time course and the relative ratios of the constituents of the volatile blends differed with the type of stimulation. While fungal elicited cells produced significant amounts of β-thujaplicin over the 5-day time course, no β-thujaplicin was observed in the mechanically stressed cells. The production of β-thujaplicin was the main dissimilarity of the induced products of these two treatments, suggesting that synthesis of β-thujaplicin is not a general response to all types of stresses, but is a specific response and serves as a strong toxic compound against already invaded fungus. Significantly higher amounts of lignin accumulations were observed in the fungal elicited and mechanically stressed cells on the 5th day after induction. Based on these results, we suggest the composition of induced products was dependent on the method of stimulation.     

Variation and inheritance of resistance to cypress aphid, Cinara cupressi Buckton in Cupressus lusitanica Miller

Cupressus lusitanica seedlings from open-pollinated seeds of 18 families were inoculated with day-old first instar Cinara cupressi. Aphid survival was used to determine the genetic basis and inheritance of resistance to the insect. There was marked variation in aphid survival both between and within families. An individual-tree narrow-sense heritability of 0.76 ± 0.30 shows strong additive genetic control which could allow effective selection and breeding for resistance. Resistant parents produced resistant progeny while susceptible parents produced susceptible progeny. There were notable exceptions as some susceptible parents produced highly resistant progeny, indicating that they had acquired pollen from resistant neighbours. Recovery of aphid damaged trees is evident implying that care should be taken in selecting for resistance. The strong additive variance and potentially high heritability indicate that one cycle of selection may yield a resistant population, while intraspecific crossing may give better results. Implications of the results in a C. lusitanica breeding programme are discussed.     

Composition of essential oils from Cupressus lusitanica and a Xylariaceous fungus found on its leaves

A Xylariaceous endophytic fungus was isolated from leaves of Cupressus lusitanica. Essential oil was extracted from these two organisms using the hydrodistillation method and identified by gas chromatography-mass spectrometry and retention index. A variety of monoterpenes, sesquiterpenes and diterpenes were identified in the fungus and the plant, and some of them were co-produced by both organisms. The oil extracted from C. lusitanica was tested against three of the endophytic fungi found in its leaves and showed only slightly antifungal activity.     

Feedback regulation of beta-thujaplicin production and formation of its methyl ether in a suspension culture of Cupressus lusitanica

Suspension cell cultures of Cupressus lusitanica produce beta-thujaplicin, a tropolone found mostly in Cupressaceae heartwood. The factors controlling beta-thujaplicin accumulation in this cell culture system were investigated. Initial cell density of the cultures did not affect beta-thujaplicin levels, though initial addition of beta-thujaplicin suppressed its de novo production. When beta-thujaplicin accumulation reached a certain level (ca. 40 mg/l) in the medium, the cultures seemed to cease beta-thujaplicin production. However, beta-thujaplicin productivity was restored when the beta-thujaplicin-containing medium was exchanged for fresh medium; the formation of 2-methoxy-6-(methylethyl)cyclohepta-2,4,6-trien-1-one, an isomer of methylated beta-thujaplicin, in medium was also observed. These results suggest that beta-thujaplicin synthesis was regulated by product feedback mechanism in this cell line, and that excess accumulation of beta-thujaplicin is relieved by conversion of beta-thujaplicin to its methyl ether.     

The effect of abandoned plantations of Pinus patula and Cupressus lusitanica on soilsand regeneration of a tropical montane rain forest in Colombia

Species richness and diversity and soil physical and chemical properties were compared between a mature lower montane rain forest (LMRF) and abandoned 21-year-old plantations of introduced Pinus patula and Cupressus lusitanica in the central Andes of Colombia (2000m). In 0.1ha plots, species richness (number of species) and diversity (Shannon and Simpson indexes) were higher in the LMRF, followed by P. patula and C. lusitanica plantations. There were 56species and 30families of higher plants in the LMRF, 26species and 15families in the P. patula plantation, and 18species and 10families in the C. lusitanica plantation. There were no statistically significant differences (p<0.05) in soil physical or chemical properties between P. patula, C. lusitanica and native LMRF. Nevertheless, soil carbon, the C/N ratio and total bases tended to be higher in the native forest than in the plantations. Available P tended to be higher in the P. patula plantation. These results show that, although abandoned plantations can support some native species, particularly in the understorey, species richness is far from that of the native forest.     

Fine root dynamics and soil carbon accretion under thinned and un-thinned Cupressus lusitanica stands in, Southern Ethiopia

Background and aims

Forest management activities influences stand nutrient budgets, belowground carbon allocation and storage in the soil. A field experiment was carried out in Southern Ethiopia to investigate the effect of thinning on fine root dynamics and associated soil carbon accretion of 6-year old C. lusitanica stands.

Methods

Fine roots (≤2 mm in diameter) were sampled seasonally to a depth of 40 cm using sequential root coring method. Fine root biomass and necromass, vertical distribution, seasonal dynamics, annual turnover and soil carbon accretion were quantified.

Results

Fine root biomass and necromass showed vertical and temporal variations. More than 70 % of the fine root mass was concentrated in the top 20 cm soil depth. Fine root biomass showed significant seasonal variation with peaks at the end of the major rainy season and short rainy season. Thinning significantly increased fine root necromass, annual fine root production and turnover. Mean annual soil carbon accretion, through fine root necromass, in the thinned stand was 63 % higher than that in the un-thinned stand.

Conclusions

The temporal dynamics in fine roots is driven by the seasonality in precipitation. Thinning of C. lusitanica plantation would increase soil C accretion considerably through increased fine root necromass and turnover.     

Intraspecific variation in host plant quality: the aphid Cinara cupressi on the Mexican cypress, Cupressus lusitanica

Abstract.

• 1 In Kenya, the aphid Cinara cupressi (Buckton) has an aggregated distribution across individuals of its host plant, the Mexican cypress, Cupressus lusitanica (Mill).
• 2 There are significant differences in host plant quality: C.cupressi nymphs transplanted onto cypress trees with low aphid infestations are significantly less likely to establish than aphids transplanted onto trees with high aphid infestations.
• 3 Given that some transplanted nymphs establish, the previous level of infestation does not influence aphid survival.
• 4 Evidence for local adaptation to individual plants was not found: nymphs transplanted to the trees from which their parents were collected neither established more readily nor showed lower mortality than those transplanted to other trees with similar levels of infestation. Therefore the variation in host plant quality has not led to the development of local adaptation to particular host plants.

     

Cupressus arizonica pollen wall zonation and in vitro hydration

The structure of Cupressus arizonica pollen at different degrees of hydration was examined by using cytochemical staining and light (LM) and scanning electron (SEM) microscopy. Most pollen grains are inaperturate and a minority are provided with an operculate pore enveloped by a concave annulus. Intine consists of: 1) a thin polysaccharidic outer layer, 2) a large polysaccharidic middle layer that is spongy and bordered by a mesh of large and branched fibrils, and 3) an inner cellulosic thick layer with callose concentrated on the inner side, which forms a shell around the protoplast. The protoplast is egg-shaped with PAS positive cytoplasm and prominent nucleus. Exine splits during hydration and is cast off according to three major steps: 1) the split opens like a mouth and the underlying intine is expelled by swelling like a balloon, 2) the protoplast enveloped by the inner intine is sucked in the outgrowing side, and 3) the backside of the intine gets rid of the exine shell. In water containing salts, exine is rapidly released and the middle intine may expand up to break the outer layer, with disgregation of the spongy material and release of the intine shell including the protoplast. In water lacking salts, the sporoderm hydration and breaking are negatively influenced by the population effect. Pollen when air dried after the exine release become completely flat owing to disappearance of the middle intine layer which may be restored by dipping pollen in water. The results are discussed in relation to the functional potentialities of the sporoderm.     

Chromium Biosorption from Cr(VI) Aqueous Solutions by Cupressus lusitanica Bark: Kinetics,Equilibrium and Thermodynamic Studies

The present study investigated the kinetics, equilibrium and thermodynamics of chromium (Cr) ion biosorption from Cr(VI) aqueous solutions by Cupressus lusitanica bark (CLB). CLB total Cr biosorption capacity strongly depended on operating variables such as initial Cr(VI) concentration and contact time: as these variables rose, total Cr biosorption capacity increased significantly. Total Cr biosorption rate also increased with rising solution temperature. The pseudo-second-order model described the total Cr biosorption kinetic data best. Langmuir´s model fitted the experimental equilibrium biosorption data of total Cr best and predicted a maximum total Cr biosorption capacity of 305.4 mg g^-1. Total Cr biosorption by CLB is an endothermic and non-spontaneous process as indicated by the thermodynamic parameters. Results from the present kinetic, equilibrium and thermodynamic studies suggest that CLB biosorbs Cr ions from Cr(VI) aqueous solutions predominantly by a chemical sorption phenomenon. Low cost, availability, renewable nature, and effective total Cr biosorption make CLB a highly attractive and efficient method to remediate Cr(VI)-contaminated water and wastewater.     

Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis

Beta-thujaplicin Is a natural troponoid with strong antifungal, antiviral, and anticancer activities. Beta-thujaplicin production in yeast elicitor-treated Cupressus lusitanica cell culture and its relationships with reactive oxygen species (ROS) and nitric oxide (NO) production and hypersensitive cell death were investigated. Superoxide anion radical (O2*-) induced cell death and inhibited beta-thujaplicin accumulation, whereas hydrogen peroxide (H2O2) induced beta-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O2*- induced programmed cell death, which can be blocked by protease inhibitors, protein kinase inhibitors, and Ca2+ chelators. Elicitor-induced NO generation was nitric oxide synthase (NOS)-dependent. Inhibition of NO generation by NOS inhibitors and NO scavenger partly blocked the elicitor-induced beta-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. Interaction among NO, H2O2, and O2*- shows that NO production and H2O2 production are interdependent, but NO and O2*- accumulation were negatively related because of coconsumption of NO and O2*-. NO- and O2*- -induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O2*- was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.