Potassium fertilisation reduces radiocesium uptake by Japanese cypress seedlings grown in a stand contaminated by the Fukushima Daiichi nuclear accident.

We analysed suppressive effects of potassium (K) fertilisation on radiocesium (137Cs) uptake by hinoki cypress (Chamaecyparis obtusa) seedlings from soils contaminated after the Fukushima Daiichi Nuclear Power Plant accident. Three-year-old seedlings were planted in a clear-cut forest (ca. 4 ha) during June-July 2014, and potassium chloride fertiliser (83 kg K ha-1) was applied twice (August 2014 and April 2015). 137Cs concentrations in the needles in the fertilised plots were one-eighth of those in the control (unfertilised) plots at the end of the second growing season (October 2015). Our results clearly indicated that K fertilisation reduced radiocesium transfer from soil to planted cypress seedlings. A linear mixed model analysis revealed that 137Cs concentrations in the needles were significantly affected by 137Cs inventory in the soil (Bq m-2) adjacent to the sampled seedlings, exchangeable K concentrations in surface mineral soils (0-5 cm) and fertilisation. The exchangeable K concentrations in surface soils in October 2015 did not differ from those in August 2014 (before fertilisation) in the fertilised plots and in the control plots. These results suggested that the levels of exchangeable K would temporarily increase by fertilisation during the growing season, and radiocesium uptake by tree roots was suppressed.

Airborne Monoterpenes Emitted from a Cupressus lusitanica Cell Culture Induce a Signaling Cascade that Produces ß-Thujaplicin.

A cell culture of Cupressus lusitanica was used to investigate the reaction of a plant to certain airborne chemicals. Compared with laboratory and field methods using intact plants or tissues, a cell culture is advantageous because it is not affected by environmental factors, and the experiments are easier to reproduce. When exposed to an elicitor, our cell line produces 10 monoterpenes and ß-thujaplicin, which is a strong phytoalexin. These monoterpenes are emitted into the vapor phase and are expected to play a role in airborne signaling. In the present study, the cells were exposed to monoterpene vapors, and the volatiles present in the culture flasks were monitored. When the culture cells were exposed to low doses of sabinene, we detected γ-terpinene and p-cymene. After exposure to γ-terpinene, we found p-cymene and terpinolene, whereas p-cymene exposure resulted in terpinolene emission. By contrast, the other seven monoterpenes we investigated did not induce any emissions of other monoterpenes. These results strongly suggest that in C. lusitanica a signaling cascade exists that starts with the emission of sabinene and moves to γ-terpinene, p-cymene, and finally to terpinolene, which accelerates the production of the phytoalexin ß-thujaplicin.

Batch and dynamic biosorption of basic dyes from binary solutions by alkaline-treated cypress cone chips.

A simple alkaline pre-treatment of Cupressus sempervirens cone chips was performed to improve their biosorption capacity towards methylene blue and rhodamine B from aqueous solutions, in batch and continuous modes. Biosorption kinetics were determined from single and binary dyes solutions, and properly described by the pseudo-second-order rate model. Experimental single-dye equilibrium isotherms fitted the Langmuir-Freundlich model, with maximum biosorption capacities of 0.68mmol/g for methylene blue and 0.50mmol/g for rhodamine B. Single-dye dynamic biosorption showed that breakthrough time for methylene blue biosorption was almost four times longer than for rhodamine B and that the alkaline modification of the chips greatly improved the biosorption performance. Competitive dynamic biosorption demonstrated the preference of the modified cone chips for biosorbing methylene blue, confirmed by the exit concentration overshoots obtained in the breakthrough curves of rhodamine B.

Volatile and non-volatile monoterpenes produced by elicitor-stimulated Cupressus lusitanica cultured cells.

Elicitor treatment initiates defense responses in cultured Cupressus lusitanica cells. In order to investigate the defense mechanism with a yeast extract elicitor, we carried out solid-phase microextraction coupled with gas chromatography for monoterpene analysis. Ten hydrocarbon monoterpenes, including high amounts of sabinene and limonene, were detected in the gas phase of the elicitor-treated cell cultures. Six oxidized monoterpenes including beta-thujaplicin were also detected in the ether extract of the cells and the medium. Time-course profiles of volatile monoterpenes showed that one group of hydrocarbon monoterpenes was maximized on the second day after elicitation, while the other group was maximized on the third day. There were no oxidized monoterpenes that are structurally related to sabinene and limonene in the gas phase or cell extracts, suggesting that these compounds are produced exclusively for emission. Other monoterpenes, which are produced during later stages of elicitation, are metabolized into more complex compounds such as oxidized monoterpenes, including beta-thujaplicin. Although terpinolene synthase was the principal monoterpene synthase in these cell cultures, terpinolene was detected only as a minor compound in the gas phase. The time course for terpinolene synthase activity coincided with beta-thujaplicin biosynthesis. Thus, most of the terpinolene is metabolized rapidly to oxidized terpenes such as beta-thujaplicin rather than emitted.

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.

Chemical and biological characterisation of sapinopyridione, a phytotoxic 3,3,6-trisubstituted-2,4-pyridione produced by Sphaeropsis sapinea, a toxigenic pathogen of native and exotic conifers, and its derivatives.

A phytotoxic trisubstituted 2,4-pyridione, named sapinopyridione, was isolated from the culture filtrates of Sphaeropsis sapinea, a fungal pathogen of conifers occurring world-wide. Three strains were isolated from two cypress species. Strain D-55 isolated from Cupressus sempervirens resulted high producer of sapinopyridione (12.3 mg l(-1)), whereas strain D-54 isolated from the same cypress species was low producer (1.1 mg l(-1)); strain D-50 isolated from C. macrocarpa was intermediate producer (5.4 mg l(-1)). Sapinopyridione was characterised by spectroscopic and chemical methods, as the 6-methyl-2-(2-methyl-1-oxobutyl)-1-oxa-5-azaspiro[2.5]oct-6-ene-4,8-dione. The structure was supported by the preparation of three key derivatives, whose phytotoxic and antimycotic activities were also tested on host plants and on three Seiridium species, virulent fungal agents of cypress canker disease. Some structure-activity relationships were identified for both phytoxicity and antifungal activities. These activities appear related to the presence of both pyridione and oxiran rings. Also the carbonyl group of the side chain seems to play a role into impart activity.

Oxidative stress in plant cell culture: a role in production of beta-thujaplicin by Cupresssus lusitanica suspension culture.

Oxidative stress is a common physiological stress that often challenges plants. Reactive oxygen species (ROS) are major factors in oxidative stress that significantly affect plant cell growth and secondary metabolism. Here we used beta-thujaplicin production by Cupressus lusitanica cell culture as an example to demonstrate the common occurrence of oxidative stress in cultivated plant cells and its effect on multiple aspects of cell culture process. C. lusitanica cells cultivated under Fe(2+) stress generate a significant level of ROS, and oxidative stress also occurs at late stages of C. lusitanica cell cultures under normal conditions. ROS production inhibited cell growth, induced lipid peroxidation and cell death, and enhanced ethylene and beta-thujaplicin production. It is demonstrated that Fe(2+) stress enhances ROS production via the Fenton reaction and promotes beta-thujaplicin production via ROS-induced lipid peroxidation that may activate cyclic oxylipin and ethylene pathways. Results further indicate that H(2)O(2) is a positive signal for beta-thujaplicin production, whereas superoxide anion radical (O(2) (- )) negatively affects beta-thujaplicin induction and strongly induces cell death. The study suggests that evaluating the oxidative stress and plant responses in a cell culture process is very necessary and important for understanding biochemical processes and for gaining the maximal productivity of target secondary metabolites.

Studies on structure-activity relationship of sphaeropsidins A-F, phytotoxins produced by Sphaeropsis sapinea f. sp. cupressi.

Six forms of sphaeropsidins (SA-SF), three- and tetra-cyclic unrearranged pimarane diterpenes produced by Sphaeropsis sapinea f. sp. cupressi, as well as eight derivatives obtained by chemical modification of SA-SC, were assayed for their bioactivity. The effect of each compound on plants which are host or non-host of the pathogen was investigated. Activity on some plant pathogenic fungi was also tested. Some structure-activity relationships have been identified for both phytotoxic and antifungal activity. It appears that the integrity of the tricyclic pimarane system, the preservation of the double bond C(8)-C(14), the tertiary hydroxyl group at C-9, the vinyl group at C-13, and the carboxylic group at C-10 as well as the integrity of the A-ring provide these molecules with non selective phytotoxic and antimycotic activity.

Sphaeropsidins D and E, two other pimarane diterpenes, produced in vitro by the plant pathogenic fungus Sphaeropsis sapinea f. sp. cupressi.

Two pimarane diterpenes structurally related to sphaeropsidins were isolated from the liquid culture of Sphaeropsis sapinea f. sp. cupressi, a plant pathogenic fungus causing a form of canker disease of Italian cypress (Cupressus sempervirens L.). The two metabolites, characterised by spectroscopic methods, were named sphaeropsidins D (0.40 mg l(-1)) and E (0.16 mg l(-1)). The same fungus produced sphaeropsidins A, B and C, sphaeropsidone and episphaeropsidone, which proved to be phytotoxic to cypress, and chlorosphaeropsidone and epichlorosphaeropsidone showing no phytotoxicity. Sphaeropsidin D assayed at 0.1 mg ml(-1) on severed cypress twigs caused leaf browning and necrosis on Cupressus macrocarpa, but no symptoms were observed on C. sempervirens and C. arizonica. Symptoms appeared in a period of time (6 days after toxin-treatment) shorter than that for sphaeropsidin A. Sphaeropsidin E assayed at 0.2 mg ml(-1) did not produce any symptom on the same cypress species tested with sphaeropsidin D.