Leaf rust induced volatile organic compounds signalling in willow during the infection

Plants are known to emit volatile organic compounds (VOC) in response to various biotic or abiotic stresses. Although the VOC emission in the case of insect attacks is well described, there is only little known about the impact of pathogens on plant emission. In the present study, we used a willow-leaf rust system to describe the effects of a biotrophic fungal infection on the VOC emission pattern of willow leaves. We detected that isoprene emissions from rust-infected leaves decreased threefold compared to control. The total monoterpene emissions did not change although a stress-signalling compound (Z)-β-ocimene showed an increase in infected plants on several days. The infection also increased the emission of sesquiterpenes and lipoxygenase products (LOX) by factors of 175-fold and 10-fold, respectively. The volatile emission signals showed two clear peaks during the experiment. At 6, 7 and 12 days post-infection (dpi), the relative volatile emission signal increased to about sixfold compared to uninfected plants. These time points are directly connected to rust infection since at 6 dpi the first rust pustules appeared on the leaves and at 12 dpi necrosis had developed around several pustules. We present correlations between LOX and sesquiterpene emission signals, which suggest at least two different steps in eliciting the volatile emission.     

How specialized volatiles respond to chronic and short‐term physiological and shock heat stress in Brassica nigra

Brassicales release volatile glucosinolate breakdown products upon tissue mechanical damage, but it is unclear how the release of glucosinolate volatiles responds to abiotic stresses such as heat stress. We used three different heat treatments, simulating different dynamic temperature conditions in the field to gain insight into stress‐dependent changes in volatile blends and photosynthetic characteristics in the annual herb Brassica nigra (L.) Koch. Heat stress was applied by either heating leaves through temperature response curve measurements from 20 to 40 °C (mild stress), exposing plants for 4 h to temperatures 25–44 °C (long‐term stress) or shock‐heating leaves to 45–50 °C. Photosynthetic reduction through temperature response curves was associated with decreased stomatal conductance, while the reduction due to long‐term stress and collapse of photosynthetic activity after heat shock stress were associated with non‐stomatal processes. Mild stress decreased constitutive monoterpene emissions, while long‐term stress and shock stress resulted in emissions of the lipoxygenase pathway and glucosinolate volatiles. Glucosinolate volatile release was more strongly elicited by long‐term stress and lipoxygenase product released by heat shock. These results demonstrate that glucosinolate volatiles constitute a major part of emission blend in heat‐stressed B. nigra plants, especially upon chronic stress that leads to induction responses.     

Tomato linalool synthase is induced in trichomes by jasmonic acid

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato.     

Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata

After herbivore attack, plants launch a suite of direct and indirect defense responses that must be coordinated if plants are to realize a fitness benefit from these responses. Here we characterize the volatile emissions in the native tobacco plant, Nicotiana attenuata Torr. ex Wats., that are elicited by tobacco hornworm (Manduca sexta L.) attack and are known to function as attractants for parasitoids. To provide the first ecophysiological comparison of examples of both types of defense in the same species, we characterize the elicitation and signaling mechanisms, the resources required, and the potential costs and benefits of the volatile release and compare these traits with those of the well-described induced direct defense in this species, nicotine production. The release of (E)-β-ocimene, cis-α-bergamotene and linalool is dramatically induced within 24 h by application of methyl jasmonate (MeJA), caterpillar feeding, and the treatment of mechanical wounds with larval oral secretions (OS), but not by mechanical damage alone. Plants from different geographic locations produce volatile blends that differ in composition. The most consistently released component from all genotypes, cis-α-berga-motene, is positively related to the amount of MeJA and the level of wounding if OS are applied to the wounds. The volatile release is strongly light dependent, dropping to undetectable quantities during dark periods, even when temperatures are elevated to match those of the light period. Inhibitors of wound-induced jasmonate accumulation (salicylates and auxins), which are known to inhibit wound-induced nicotine production, do not inhibit the release of volatiles. By individually inducing different leaf positions with OS and, on other plants, excising them after induction, we demonstrate that the emission is largely a systemic, whole-plant response, which is maximally triggered when the second fully expanded leaf is induced. We conclude that while both are whole-plant, systemic responses that utilize recently acquired resources for their production and are activated by the jasmonate cascade, the elicitation of the volatile release exhibits greater tissue sensitivity and utilizes additional signaling components than does nicotine production. In contrast to the large investment of fitness-limiting resources required for induced nicotine production or the resources used in benzyl acetone release from flowers for pollinator attraction, the resource requirements for the volatile release are minor. Hence the argument that the volatile release incurs comparatively large physiological costs cannot be supported in this system. Received: 4 November 1999 / Accepted: 1 March 2000     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Identification of Pip4k2β as a mechanical stimulus responsive gene and its expression during musculoskeletal tissue healing

To investigate the mechano-transduction system of cells, we identified genes responsive to a cyclic mechanical stimulus. MC3T3.E1 cells were cultured on a computer-controlled vacuum-pump-operated device designed to provide a cyclic mechanical stimulus. A maximum elongation of 15% of membrane at 10 cycles/min (3 s extension followed by 3 s relax per cycle) was repeated for 48 h. By means of a differential display, the gene expression pattern of cells exposed to the stimulus was compared with that of unexposed cells. As a result, a gene fragment that was exclusively expressed in mechanically stressed cells was identified. By using expressed sequence tag walking together with the oligo-capping method, this gene was identified as phosphatidylinositol 4-phosphate 5-kinase type II β (initially known as Pip5k2β but now reclassified as Pip4k2β). The specific up-regulation of Pip4k2β upon mechanical stimulus was also confirmed by using another apparatus, viz. a computer-controlled linearized-stepping motor system. To examine the involvement of the cyclic mechanical stimulus in the regulation of Pip4k2β expression in musculoskeletal tissue, we created an Achilles tendon transection model in rabbits. The temporal expression of Pip4k2β was assessed by means of a quantitative reverse-transcribed polymerase chain reaction. In the gastrocnemius muscle, expression of Pip4k2β rapidly decreased 1 week after transection but was restored to normal levels at 4 weeks. In the Achilles tendon, however, expression remained decreased until 4 weeks after transection. We suggest that the expression of Pip4k2β can be used as a marker for cells receiving a suitable mechanical stimulus. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.     

Natural secretory products of human neural and microvessel endothelial cells

Neurons, glia, and endothelial cells of the cerebral microvasculature co-exist in intimate proximity in nervous tissues, and their homeostatic interactions in health, as well as coordinated response to injury, have led to the concept that they form the basic elements of a functional neurovascular unit. During the course of normal cellular metabolism, growth, and development, each of these brain cell types secrete various species of potentially neurotoxic peptides and factors, events that increase in magnitude as brain cells age. This article reviews contemporary research on the secretory products of the three primary cell types that constitute the neurovascular unit in deep brain regions. We provide some novel in vitro data that illustrate potentially pathogenic paracrine effects within primary cells of the neurovascular unit. For example, the pro-inflammatory cytokine interleukin (IL)-1β was found to stimulate amyloid-β (Aβ) peptide release from human neural cells, and human brain microvessel endothelial cells exposed to transient hypoxia were found to secrete IL-1β at concentrations known to induce Aβ42 peptide release from human neural cells. Hypoxia and excessive IL-1β and Aβ42 abundance are typical pathogenic stress factors implicated in the initiation and development of common, chronic neurological disorders such as Alzheimer's disease. These data support the hypothesis that paracrine effects of stressed constituent cells of the neurovascular unit may contribute to “spreading effects” characteristic of progressive neurodegenerative disorders.     

Epirrita autumnata induced VOC emission of silver birch differ from emission induced by leaf fungal pathogen

The production of volatile organic compounds (VOCs) through the activation of different signal-transduction pathways may be induced in various biotic and abiotic stress situations having importance e.g. in insect and disease resistance. We compared the emission of VOCs emitted from silver birch Betula pendula Roth (clones 4 and 80) twigs damaged either by larvae of Epirrita autumnata, or infected with pathogenic leaf spot causing fungus Marssonina betulae. We also analysed whether local herbivore damage can systemically induce the release of VOCs from the undamaged top of same sapling. The emissions of methylsalicylate (MeSA), (Z)-ocimene, (E)-β-ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and linalool were induced from the twigs after 72 h feeding damage by E. autumnata larvae. However, 48 h feeding damage did not induce rapid systemic release of VOCs from undamaged top leaves of the same twigs. Pathogen-infected birch twigs had significantly greater emission of (Z)-ocimene and (E)-β-ocimene than intact control twigs. The emission of DMNT was not significantly induced and MeSA was not found at all after pathogen infection, both being significantly different from herbivore damaged twigs. According to our results leaf fungal pathogen induces VOC emission profile differs from that of arthropod herbivore-damaged leaves, suggesting that birch is able to transmit parasite-specific information via VOC emissions to conspecifics and natural enemies of herbivores. Handling editor: Yvan Rahbé     

Localization of chitinolytic activities in Fagus sylvatica mycorrhizas

 Localization of chitinolytic activities in Fagus sylvatica (beech) mycorrhizas was examined using a range of fluorogenic 4-methylumbelliferyl [4-MU-(GlcNAc)_1–4] substrates in order to distinguish between exochitinase, endochitinase and β-N–acetylglucosaminidase activities. The validity of the technique was confirmed using onion epidermis cells. In the beech mycorrhiza, endochitinase activity was not detectable above background fluorescence. Exochitinase activity was detected in the fungal sheath and the Hartig net. β-N–Acetylglucosaminidase activity was also mainly associated with the fungal sheath and Hartig net. Individual fungal hyphae extending from these structures also showed substantial β-N–acetylglucosaminidase activity. The cortical cell walls of the host in the Hartig net region also fluoresced brightly. The localization of β-N–acetylglucosaminidase activity was confirmed using a chromogenic histochemical reagent, 5-bromo-4-chloro-3-indolyl-N–acetyl-β-d-glucosaminide (X-GlcNAc). Accepted: 5 December 1995     

Timing of induced volatile emissions in maize seedlings

Maize (Zea mays L.) releases specific volatiles in response to herbivory by caterpillars. These volatiles are known to serve as cues for parasitic wasps to locate the herbivores. In the present study the exact time of volatile emission after simulated herbivory (mechanical damage and treatment with caterpillar regurgitant) was measured for seedlings of the cultivars “Ioana Sweet Corn” and “LG11”. Odours were collected every 0.5 h for a total of 12 h. Typical “green leaf odours”, (Z)-3-hexenal, (E )-2-hexenal, (Z)-hexen-1-o1, and (Z)-3-hexen-1-yl acetate, were emitted immediately upon damage and their amounts dropped rapidly after the first collections. Several of the induced compounds were released within 2 h after treatment, while others (mainly sesquiterpenoids) started to be released after 4 h. The LG11 seedlings emitted several compounds (e.g. β-myrcene, (Z)-β-ocimene, benzyl acetate, β-caryophyllene, (E,E )-α-farnesene) that were not detected for Ioana. (E,E )-α-farnesene was continuously emitted by LG11 seedlings, even by undamaged plants. Timing of the release of volatile compounds that the two varieties had in common did not differ significantly, with the exception of indole for which the peak production was considerably earlier for LG11. These findings are discussed in the context of biosynthetic pathways and mechanisms involved in induced emissions of plant volatiles and the exploitation of the resulting odour by parasitoids and predators of herbivores. Received: 23 October 1997 / Accepted: 9 June 1998     

Interaction of pre-attack and induced monoterpene concentrations in host conifer defense against bark beetle-fungal complexes

Two pine species (Pinus resinosa, P. banksiana) responded to inoculation with fungi carried by bark beetles by rapidly increasing monoterpene concentrations at the entry site. Changes in total monoterpenes were more pronounced than changes in proportionate compositions. The extent and rate of host response was affected by fungal species, the viability of the inoculum, and host tree species. In general, host responses were highest to fungi that are phytopathogenic and consistently associated with the major bark beetles in the study region. Simple mechanical wounding cannot account for the observed allelochemical changes, as aseptic inoculations elicited only minor reactions. Similarly, inoculation with autoclaved inviable fungi generally elicited intermediate responses, suggesting that both structural and metabolic fungal properties are important. Responses by jack pine, P. banksiana, were generally more rapid and variable than those of red pine, P. resinosa. Dose-toxicity experiments with synthetic compounds demonstrated that monoterpene concentrations present in vivo only a few days after simulated attack are lethal to most beetles. Constitutive (pre-attack) monoterpene levels can also exert some toxicity. Because bark beetles engage in pheromone-mediated mass attacks that can deplete host defenses, constitutive monoterpene levels, while a necessary early phase of successful plant defense, appear insufficient by themselves. Such interactions between constitutive and induced defense chemistry may be important considerations when evaluating general theories of plant defense.     

Analysis of beta-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7 μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1 μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin. Received: 16 March 1999 / Accepted: 21 August 1999     

Isolation and characterization of a fungus <Emphasis Type="Italic">Aspergillus</Emphasis> sp. strain F-3 capable of degrading alkali lignin

A fungus strain F-3 was selected from fungal strains isolated from forest soil in Dalian of China. It was identified as one Aspergillus sp. stain F-3 with its morphologic, cultural characteristics and high homology to the genus of rDNA sequence. The budges or thickened node-like structures are peculiar structures of hyphae of the strain. The fungus degraded 65% of alkali lignin (2,000 mg l⁻¹) after day 8 of incubation at 30°C at pH 7. The removal of colority was up to 100% at 8 days. The biodegradation of lignin by Aspergillus sp. F-3 favored initial pH 7.0. Excess acid or alkali conditions were not propitious to lignin decomposing. Addition of ammonium l-tartrate or glucose delayed or repressed biodegradation activities. During lignin degradation, manganese peroxidase (28.2 U l⁻¹) and laccase (3.5 U l⁻¹)activities were detected after day 7 of incubation. GC-MS analysis of biodegraded products showed strain F-3 could convert alkali lignin into small molecules or other utilizable products. Strain F-3 may co-culture with white rot fungus and decompose alkali lignin effectively.     

Non-toluene-associated respiration in a Pseudomonas putida 54G biofilm grown on toluene in a flat-plate vapor-phase bioreactor

Non-toluene-associated respiration (NTAR) within a Pseudomonas putida 54G biofilm growing on toluene as sole external carbon source was evaluated using oxygen microelectrodes in a flat-plate vapor-phase biological reactor. Two fluorescent probes, 2,4-diamidino-2-phenylindole and 5-cyano-2,3-ditolyltetrazolium chloride, were used to evaluate the number of total and respiring cells respectively within the biofilm. Biofilm samples were also analyzed for viable and toluene-culturable cells by spread-plating on non-selective and selective media respectively. Fractions of viable stressed, respiring and non-respiring cells within the biofilm were evaluated. The NTAR rate was positively correlated with the fraction of viable stressed and non-respiring cells within the biofilm, which suggested the capability of some cells to grow at the expense of leakage and lysis products coming from injured and dead cells. This effect was more pronounced at higher toluene concentration. Results suggest that NTAR should be incorporated into mathematical models of biofilm reactors degrading volatile organic carbon compounds. Received: 4 January 1997 / Received revision: 20 March 1997 / Accepted: 27 March 1997     

Biotransformation of β-myrcene to geraniol by a strain of Rhodococcus erythropolis isolated by selective enrichment from hop plants

The biocatalytic generation of high-value chemicals from abundant, cheap and renewable feedstocks is an area of great contemporary interest. A strain of Rhodococcus erythropolis designated MLT1 was isolated by selective enrichment from the soil surrounding hop plants, using the abundant triene β-myrcene from hops as a sole carbon source for growth. Resting cells of the organism were challenged with β-myrcene, and the major product of biotransformation was determined by mass spectrometric analysis to be the monoterpene alcohol geraniol. Controls demonstrated that the product was biogenic and that an aerobic environment was required. The ability to transform β-myrcene was shown to be restricted to cells that had been grown on this substrate as sole carbon source. Pre-incubation of cells with the cytochrome P450 inhibitors metyrapone or 1-aminobenzotriazole reduced geraniol production by 23% and 73% respectively, but reduction in activity was found not to correlate with the inhibitor concentration. A comparative analysis of insoluble and soluble cell extracts derived from cells of MLT1 grown on either β-myrcene or glucose revealed at least four proteins that were clearly overproduced in response to growth on β-myrcene. Mass spectrometric analysis of tryptic digests of three of these protein bands suggested their identities as an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, each of which has a precedented role in hydrocarbon metabolism clusters in Rhodococcus sp. and which may therefore participate in a β-myrcene degradation pathway in this organism.     

Production and regulation of lignocellulose-degrading enzymes of <Emphasis Type="Italic">Poria</Emphasis>-like wood-inhabiting basidiomycetes

The wood-decomposing fungal species Antrodia macra, A. pulvinascens, Ceriporiopsis aneirina, C. resinascens and Dichomitus albidofuscus were determined for production of laccase (LAC), Mn peroxidase (MnP), lignin peroxidase (LiP), endo-l,4-P-β-glucanase, endo-l,4-β-xylanase, cellobiohydrolase, 1,4-β-glucosidase and 1,4-β-xylosidase. The results confirmed the brown-rot mode of Antrodia spp. which did not produce the activity of LAC and MnP. The remaining species performed detectable activity of both enzymes while no strain produced LiP. Significant inhibition of LAC production by high nitrogen was found in all white-rot species while only MnP of D. albidofuscus was regulated in the same way. The endoglucanase and endoxylanase activities of white-rotting species were inhibited by glucose in the medium while those of Antrodia spp. were not influenced by glucose concentration. The regulation of enzyme activity and bio-mass production can vary even within a single fungal genus.     

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca²⁺ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca²⁺ concentration, which peaked within 2–2.5 min of treatment. The Ca²⁺ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca²⁺ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca²⁺ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca²⁺ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca²⁺ signals were inhibited by both the Ca²⁺ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca²⁺-channel inhibitor La³⁺. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca²⁺ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca²⁺ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca²⁺ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses. Received: 23 July 1998 / Accepted: 16 October 1998     

Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated<Emphasis Type="Italic"> Cupressus lusitanica</Emphasis> cell cultures

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] rapidly accumulates in elicited Cupressus lusitanica Mill. cultured cells by 4- to 5-fold over the control, and then it is metabolized. Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P₃ phosphatase activity declines at the beginning of elicitation and increases later. These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P₃ occur simultaneously and that the Ins(1,4,5)P₃ level may be regulated by both PtdIns(4,5)P₂–PLC and Ins(1,4,5)P₃ phosphatases. Studies on the properties of PLC and Ins(1,4,5)P₃ phosphatases indicate that PLC activity toward PtdIns(4,5)P₂ was optimal at a lower Ca²⁺ concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P₃ phosphatase activity is inhibited by high Ca²⁺ concentration. This suggests that Ins(1,4,5)P₃ biosynthesis and degradation may be regulated by free cytosolic Ca²⁺. In addition, a relationship between Ins(1,4,5)P₃ signaling and accumulation of a phytoalexin (-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P₃ accumulation by neomycin or LiCl affects elicitor-induced production of -thujaplicin. Moreover, ruthenium red inhibits elicitor-induced accumulation of -thujaplicin while thapsigargin alone induces -thujaplicin accumulation. These results suggest that Ca²⁺ released from intracellular calcium stores may mediate elicitor-induced accumulation of -thujaplicin via an Ins(1,4,5)P₃ signaling pathway, since it is widely accepted that Ins(1,4,5)P₃ can mobilize Ca²⁺ from intracellular stores. This work demonstrates an elicitor-triggered Ins(1,4,5)P₃ turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P₃ in elicitor-induced phytoalexin accumulation via a Ca²⁺ signaling pathway.Abbreviations Ins(1,4,5)P₃ Inositol-1,4,5-trisphosphate - Ins(1,4)P₂ Inositol-1,4-bisphosphate - Ins(4,5)P₂ Inositol-4,5-bisphosphate - Ins(1)P Inositol 1-phosphate - Ins(4)P Inositol 4-phosphate - PLC Phospholipase C - PtdIns Phosphatidylinositol - PtdIns(4,5)P₂ Phosphatidylinositol 4,5-bisphosphate - YE Yeast elicitor     

Biotransformation of menthol and geraniol by hairy root cultures of <Emphasis Type="Italic">Anethum graveolens</Emphasis>: effect on growth and volatile components

Two oxygen-containing monoterpene substrates, menthol or geraniol (25 mg l⁻¹), were added to Anethum graveolens hairy root cultures to evaluate the influence of the biotransformation capacity on growth and production of volatile compounds. Growth was assessed by the dissimilation method and by fresh and dry weight measurement. The volatiles were analyzed by GC and GC–MS. The total constitutive volatile component was composed, in more than 50%, by falcarinol (17–52%), apiole (11–24%), palmitic acid (7–16%), linoleic acid (4–9%), myristicin (4-8%) and n-octanal (2-5%). Substrate addition had no negative influence on growth. The relative amount of menthol quickly decreased 48 h after addition, and the biotransformation product menthyl acetate was concomitantly formed. Likewise, the added geraniol quickly decreased over 48 h alongside with the production of the biotransformation products. The added geraniol was biotransformed in 10 new products, the alcohols linalool, α-terpineol and citronellol, the aldehydes neral and geranial, the esters citronellyl, neryl and geranyl acetates and linalool and nerol oxides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Elevated β1,4-galactosyltransferase-I induced by the intraspinal injection of lipopolysaccharide

β1,4-Galactosyltransferase-I (β1,4-GalT-I) is one of the best studied glycosyltransferases. Previous studies demonstrated that β1,4-GalT-I was a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of β1,4-GalT-I deficient mice were impaired. In this study, we investigate the expression of β1,4-GalT-I in lipopolysaccharide (LPS)-induced neuroinflammatory processes. The results of this study demonstrated that β1,4-GalT-I was strongly induced by intraspinal administration of LPS. More than 90% galactose-containing glycans and β1,4-GalT-I were expressed in immune cells. The ELISA assay shows focal injection LPS also induces TNF-α alteration. Double staining indicated β1,4-GalT-I overlapped with TNF-α. Moreover, RT-PCR for β1,4-GalT-I mRNA showed that β1,4-GalT-I mRNA in microglia in vitro was affected in a dose- and time dependent manner in response to LPS or TNF-α stimulation. All these results indicated that the increase of β1,4-GalT-I might attribute to the effect of TNF-α excreting during inflammation. E-selectin, which ligand was modified by β1,4-GalT-I, was correlated with galactose-containing glycans following injecting LPS into spinal cord. We therefore suggest that β1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site. Aiguo Shen and Jianping Chen contributed equally to this work.