Phytochemical and Biological Investigation of Helianthemum nummularium, a High-Altitude Growing Alpine Plant Overrepresented in Ungulates Diets.

Helianthemum nummularium is a European shrub growing at high altitude where it copes with a high level of stress. It was found to be overexpressed in ungulates diets compared to more abundant surrounding plants. These elements combined with the fact that H. nummularium from the Alps has never been investigated prompted us to study the phytochemical composition of its aerial parts. The analysis of the polar extract allowed for the isolation of eight compounds: p-hydroxybenzoic acid, tiliroside, kaempferol, astragalin, quercetin, plantainoside B, quercetin-3-O-glucoside, and quercetin-3-O-glucuronide. We investigated the effect of the polar extract and isolated compounds on nuclear factor erythroid 2-related factor 2 transcription factor, which regulates the expression of a wide variety of cytoprotective genes. We found that the ethanolic extract activates the expression of nuclear factor erythroid 2-related factor 2 in a dose-dependent manner, whereas the pure compounds were much less active. The activation of the nuclear factor erythroid 2-related factor 2 pathway by the plant extract could pave the way for studies to promote healthy aging through protection of cells against oxidative stress. Moreover, the isolated compounds could be investigated alone or in combination in the perspective of making the link between the ungulate's preference for this plant and possible use of it for self-medication.

[Beech (Fagus sylvatica) germination and seedling growth under climatic and allelopathic constraints]. / Germination et croissance des plantules de hêtre (Fagus sylvatica) sous contraintes climatiques et allélopathiques.

Recruitment is a key process for forest sustainability, especially in warm margin of distribution area. The influence of climate (temperate or warm), of soil water availability, and of allelopathic interactions from different forest species have been tested on the germination of Fagus sylvatica in controlled climatic conditions. Germination rates of non-dormant Fagus seeds were improved by relatively warm temperatures (20°C), but reversibly stopped under heat constraint (27°C). The relative growth rate of Fagus seedlings was better under temperate climatic conditions. Foliar extracts of Hedera helix showed the highest allelopathic effect on Fagus recruitment, especially in temperate conditions. Our results suggest a limitation of Fagus recruitment in warm margin of its distribution area, and a modulation of recruitment success according to the identity of plant neighbourhood.

The Impact of Competition and Allelopathy on the Trade-Off between Plant Defense and Growth in Two Contrasting Tree Species.

In contrast to plant-animal interactions, the conceptual framework regarding the impact of secondary metabolites in mediating plant-plant interference is currently less well defined. Here, we address hypotheses about the role of chemically-mediated plant-plant interference (i.e., allelopathy) as a driver of Mediterranean forest dynamics. Growth and defense abilities of a pioneer (Pinus halepensis) and a late-successional (Quercus pubescens) Mediterranean forest species were evaluated under three different plant interference conditions: (i) allelopathy simulated by application of aqueous needle extracts of Pinus, (ii) resource competition created by the physical presence of a neighboring species (Pinus or Quercus), and (iii) a combination of both allelopathy and competition. After 24 months of experimentation in simulated field conditions, Quercus was more affected by plant interference treatments than was Pinus, and a hierarchical response to biotic interference (allelopathy < competition < allelopathy + competition) was observed in terms of relative impact on growth and plant defense. Both species modulated their respective metabolic profiles according to plant interference treatment and thus their inherent chemical defense status, resulting in a physiological trade-off between plant growth and production of defense metabolites. For Quercus, an increase in secondary metabolite production and a decrease in plant growth were observed in all treatments. In contrast, this trade-off in Pinus was only observed in competition and allelopathy + competition treatments. Although Pinus and Quercus expressed differential responses when subjected to a single interference condition, either allelopathy or competition, species responses were similar or positively correlated when strong interference conditions (allelopathy + competition) were imposed.

Differential allelopathic effects of Japanese knotweed on willow and cottonwood cuttings used in riverbank restoration techniques.

Using bioengineering techniques to restore areas invaded by Fallopia japonica shows promising results. Planting tree cuttings could allow both rapidly re-establishing a competitive native plant community and reducing F. japonica performance. However, F. japonica has been shown to affect native plant species through different mechanisms such as allelopathy. This article investigates the phytotoxic effect of F. japonica on the resprouting capacity and the growth of three Salicaceae species with potential value for restoration. An experimental design which physically separates donor pots containing either an individual from F. japonica or bare soil from target pots containing cuttings of Populus nigra, Salix atrocinerea or Salix viminali was used. Leachates from donor pots were used to water target pots. The effects of leachates were evaluated by measuring the final biomass of the cuttings. F. japonica leachates inhibited the growth of cuttings, and this effect is linked to the emission of polyphenol compounds by F. japonica. Leachates also induced changes in soil nitrogen composition. These results suggest the existence of allelopathic effects, direct and/or indirect, of F. japonica on the growth of Salicaceae species cuttings. However, the three species were not equally affected, suggesting that the choice of resistant species could be crucial for restoration success.

Plant insecticidal toxins in ecological networks.

Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversification. Anti-herbivore defences also negatively impact mutualistic partners, possibly leading to an ecological cost of toxin production. However, in other cases toxins can also be used by plants involved in mutualistic interactions to exclude inadequate partners and to modify the cost/benefit ratio of mutualism to their advantage. When considering the whole community, toxins have an effect at many trophic levels. Aposematic insects sequester toxins to defend themselves against predators. Depending on the ecological context, toxins can either increase insects' vulnerability to parasitoids and entomopathogens or protect them, eventually leading to self-medication. We conclude that studying the community-level impacts of plant toxins can provide new insights into the synthesis between community and evolutionary ecology.

The role of volatile organic compounds, morphology and pigments of globeflowers in the attraction of their specific pollinating flies.

• Floral scents and visual cues of the globeflower Trollius europaeus may play a key role in the attraction of Chiastocheta flies, involved in a highly specific nursery pollination mutualism. • Here, headspace collection and GC-MS were used to identify and quantify the volatile organic compounds emitted by the globeflower. • Scents are produced in three different floral parts by four structures: secretory glands and flat epidermis cells in the abaxial sepal epidermis, conical cells in the adaxial sepal epidermis, and pollen. The blend is made up of 16 compounds commonly found in floral scents. Geographical variation among populations is low compared with variation amongst individuals within populations. Electroantenno-graphic analyses revealed that six compounds emitted by both anthers and sepals are detected by Chiastocheta flies. Removing the anthers hidden inside the globe from flowers in the field decreased the number of fly visits to globeflowers. • A multivariate analysis of the effect of several floral traits on pollinator visitation rate conducted in the field showed that both floral scents and visual flower cues play a role in pollinator attraction. However, their relative roles and the intensity of the selective pressures exerted on floral traits by pollinators appear to vary in time and space.

Plant chemical defence: a partner control mechanism stabilising plant--seed-eating pollinator mutualisms.

BACKGROUND: Mutualisms are inherently conflictual as one partner always benefits from reducing the costs imposed by the other. Despite the widespread recognition that mutualisms are essentially reciprocal exploitation, there are few documented examples of traits that limit the costs of mutualism. In plant/seed-eating pollinator interactions the only mechanisms reported so far are those specific to one particular system, such as the selective abortion of over-exploited fruits. RESULTS: This study shows that plant chemical defence against developing larvae constitutes another partner sanction mechanism in nursery mutualisms. It documents the chemical defence used by globeflower Trollius europaeus L. (Ranunculaceae) against the seed-eating larvae of six pollinating species of the genus Chiastocheta Pokorny (Anthomyiidae). The correlative field study carried out shows that the severity of damage caused by Chiastocheta larvae to globeflower fruits is linked to the accumulation in the carpel walls of a C-glycosyl-flavone (adonivernith), which reduces the larval seed predation ability per damaged carpel. The different Chiastocheta species do not exploit the fruit in the same way and their interaction with the plant chemical defence is variable, both in terms of induction intensity and larval sensitivity to adonivernith. CONCLUSION: Adonivernith accumulation and larval predation intensity appear to be both the reciprocal cause and effect. Adonivernith not only constitutes an effective chemical means of partner control, but may also play a key role in the sympatric diversification of the Chiastocheta genus.

A leachate a day keeps the seedlings away: mowing and the inhibitory effects of Festuca paniculata in subalpine grasslands.

BACKGROUND AND AIMS: Is the release of allelochemicals by the dominant tussock grass Festuca paniculata responsible for its dominance by inhibiting growth of neighbour grasses in subalpine grasslands? As such a community is also structured by mowing practices, what could be the impact of mowing on allelopathy? METHODS: A design was used that isolated allelopathy from resource competition by separating donor plants (Festuca paniculata) from target plants (F. paniculata, Dactylis glomerata and Bromus erectus). Leachates from donor pots containing bare soil, unmown F. paniculata or mown F. paniculata continuously irrigated target pots containing seedlings. Activated carbon was added in half of the target pots to adsorb potential allelochemicals. C and N analyses of target potting soil were used to test for any effect of treatments on resources. Total phenol concentration was measured in the solutions flowing from donor to target pots. RESULTS: Festuca paniculata leachates inhibited seedling growth of D. glomerata and B. erectus. Inhibition was correlated with polyphenol concentration, and was not due to resource competition for nitrogen. Mowing the leaves of the donor plants did not significantly increase this inhibition. The activated carbon treatment was not conclusive as it inhibited the seedling growing under control pots with only bare soil. CONCLUSIONS: The results suggest that allelopathy may be at least partly responsible for F. paniculata dominance in subalpine meadows by inhibition of colonization by neighbouring species.

Plant chemical defense induced by a seed-eating pollinator mutualist.

Plant-seed parasite pollination mutualisms involve a specific pollinator whose larvae develop by consuming a fraction of the host plant seeds. These mutualisms are stable only if the plant can control seed destruction by the larvae. Here, we studied the chemical response of the European globeflower Trollius europaeus to infestation by an increasing number of Chiastocheta fly larvae. We used liquid chromatographic analysis to compare the content of phenolic compounds in unparasitized and parasitized fruits collected in two natural populations of the French Alps, and mass spectrometry and nuclear magnetic resonance to elucidate the structure of adonivernith, a C-glycosyl-flavone. This compound is present in many of the organs of T. europaeus, but not found in other Trollius species. Furthermore, it is overproduced in the carpel walls of parasitized fruits, and this induced response to infestation by fly larvae is density-dependent (increases with larval number), and site-dependent (more pronounced in the high-altitude site). Mechanical damage did not induce adonivernith production. This tissue-specific and density-dependent response of T. europaeus to infestation by Chiastocheta larvae might be an efficient regulation mechanism of seed-predator mutualist population growth if it decreases survival or growth of the larvae.

The evolutionary ecology of insect resistance to plant chemicals.

Understanding the diversity of insect responses to chemical pressures (e.g. plant allelochemicals and pesticides) in their local ecological context represents a key challenge in developing durable pest control strategies. To what extent do the resistance mechanisms evolved by insects to deal with the chemical defences of plants differ from those that have evolved to resist insecticides? Here, we review recent advances in our understanding of insect resistance to plant chemicals, with a special emphasis on their underlying molecular basis, evaluate costs associated with each resistance trait, and discuss the ecological and evolutionary significance of these findings.

Uptake and translocation of phytochemical 2-benzoxazolinone (BOA) in radish seeds and seedlings.

The molecular aspects of phytochemical interactions between plants, especially the process of phytochemical translocation by the target plant, remain challenging for those studying allelopathy. 2-Benzoxazolinone (BOA) is a natural chemical produced by rye (Secale cereale) and is known to have phytotoxic effects on weed seeds and seedlings. The translocation of BOA into target plants has been poorly investigated. Therefore, the total absorption of [ring U 14C] BOA was estimated by oxidizing whole seedlings of Raphanus sativus cv. for 8 days and quantifying the radioactivity. Non-radiolabelled BOA in seedlings was also estimated by HPLC. BOA applied at 10(-3) M was readily taken up by germinated radish at a rate of 1556 nmol g(-1) FW. At these same concentrations, BOA reduced radish germination by 50% and caused a delay in radicle elongation. Exogenous BOA was responsible for the observed germination inhibition. At a concentration of 10(-5) M, BOA was taken up by germinated seeds (31 nmol g(-1) FW), but this quantity did not affect radish germination. Labelled BOA was not mineralized in the culture medium during seedling growth as no 14CO2 was recovered. Both 10(-3) and 10(-5) M BOA were translocated into radish organs, mainly into roots and cotyledons. These organs were then identified as potential physiological target sites. Cotyledons remained the target sink (44% of the total radioactivity). The kinetics of BOA uptake at 10(-3) and 10(-5) M in radish seedlings was identical: BOA accumulation was proportional to its initial concentration. A comparison between radioactivity and HPLC quantification for 10(-3) M BOA indicated that BOA (along with some metabolites) could effectively be recovered in radish organs using chromatography.

Potential toxic effect on aquatic fauna by the dwarf shrub Empetrum hermaphroditum.

The common evergreen dwarf shrub Empetrum hermaphroditum has influence on the functioning of boreal terrestrial ecosystems in northern Sweden. The negative effects of E. hermaphroditum are partly attributed to the production of the dihydrostilbene, batatasin-III, which is released from leaves and litter by rain and snowmelt. In this study, we investigated whether batatasin-III is carried by runoff into streams and lakes during the snowmelt period and whether it is also potentially hazardous to aquatic fauna. Sampling of water from streams and a lake for which the surrounding terrestrial vegetation is dominated by E. hermaphroditum was done during the snowmelt period in May 1993 and in 1998, and analyzed for batatasin-III. Using 24- and 48-hr standard toxicity tests, we analyzed toxicity to brown trout (Salmo trutta) alevins and juvenile water fleas (Daphnia magna). Toxicity (proportion of dead individuals) to trout was tested at pH 6.5 and compared with that of a phenol within a range of concentrations. In the toxicity (proportion of immobilized individuals) test on D. magna, the interactive effect of pH (pH 5.5-7.0) was included. Concentration of batatasin-III was generally higher in 1998 than in 1993 and showed peak levels during snowmelt. Concentration in ephemeral runnels > the lake > streams running through clear-cuts dominated by E. hermaphroditum > control streams lacking adjacent E. hermaphroditum vegetation. The maximum concentration of batatasin-III found was 1.06 mg l(-1). The proportion of dead yolk sac alevins increased significantly (P < 0.001) with increasing concentrations of batatasin-III and time of exposure. After 24 hr, EC50 was 10 mg l(-1). It was 2 mg l(-1) after 48 hr. The effect of phenol was negligible, indicating a specific phytotoxic effect of the bibenzyl structure of batatasin-III. The proportion of mobile D. magna became significantly smaller (P < 0.001) with increasing concentrations of batatasin-III, with decreasing pH, and with increasing exposure time. EC50 varied between 7 and 17 mg l(-1) at pH 5.5 and 7.0, respectively. After 24 hr EC50 decreased and was 2.5 at pH 5.5 and 12 mg l(-1) at pH 7.0. The levels of batatasin-III found in the field samples were below the lowest EC50 in acute toxicity tests. However, in view of the interactive effect of pH and exposure time, this study suggests that this stable plant metabolite may impose a lethal effect on the aquatic fauna in small streams.