Leaf metabolic traits reveal hidden dimensions of plant form and function.

The metabolome is the biochemical basis of plant form and function, but we know little about its macroecological variation across the plant kingdom. Here, we used the plant functional trait concept to interpret leaf metabolome variation among 457 tropical and 339 temperate plant species. Distilling metabolite chemistry into five metabolic functional traits reveals that plants vary on two major axes of leaf metabolic specialization-a leaf chemical defense spectrum and an expression of leaf longevity. Axes are similar for tropical and temperate species, with many trait combinations being viable. However, metabolic traits vary orthogonally to life-history strategies described by widely used functional traits. The metabolome thus expands the functional trait concept by providing additional axes of metabolic specialization for examining plant form and function.

Allelopathic Potential of Mangroves from the Red River Estuary against the Rice Weed Echinochloa crus-galli and Variation in Their Leaf Metabolome.

Mangroves are the only forests located at the sea-land interface in tropical and subtropical regions. They are key elements of tropical coastal ecosystems, providing numerous ecosystem services. Among them is the production of specialized metabolites by mangroves and their potential use in agriculture to limit weed growth in cultures. We explored the in vitro allelopathic potential of eight mangrove species' aqueous leaf extracts (Avicennia marina, Kandelia obovata, Bruguiera gymnorhiza, Sonneratia apetala, Sonneratia caseolaris, Aegiceras corniculatum, Lumnitzera racemosa and Rhizophora stylosa) on the germination and growth of Echinochloa crus-galli, a weed species associated with rice, Oryza sativa. Leaf methanolic extracts of mangrove species were also studied via UHPLC-ESI/qToF to compare their metabolite fingerprints. Our results highlight that A. corniculatum and S. apetala negatively affected E. crus-galli development with a stimulating effect or no effect on O. sativa. Phytochemical investigations of A. corniculatum allowed us to putatively annotate three flavonoids and two saponins. For S. apetala, three flavonoids, a tannin and two unusual sulfated ellagic acid derivatives were found. Some of these compounds are described for the first time in these species. Overall, A. corniculatum and S. apetala leaves are proposed as promising natural alternatives against E. crus-galli and should be further assessed under field conditions.

Amplified Drought Alters Leaf Litter Metabolome, Slows Down Litter Decomposition, and Modifies Home Field (Dis)Advantage in Three Mediterranean Forests.

In Mediterranean ecosystems, the projected rainfall reduction of up to 30% may alter plant-soil interactions, particularly litter decomposition and Home Field Advantage (HFA). We set up a litter transplant experiment in the three main forests encountered in the northern part of the Medi-terranean Basin (dominated by either Quercus ilex, Quercus pubescens, or Pinus halepensis) equipped with a rain exclusion device, allowing an increase in drought either throughout the year or concentrated in spring and summer. Senescent leaves and needles were collected under two precipitation treatments (natural and amplified drought plots) at their "home" forest and were left to decompose in the forest of origin and in other forests under both drought conditions. MS-based metabolomic analysis of litter extracts combined with multivariate data analysis enabled us to detect modifications in the composition of litter specialized metabolites, following amplified drought treatment. Amplified drought altered litter quality and metabolomes, directly slowed down litter decomposition, and induced a loss of home field (dis)advantage. No indirect effect mediated by a change in litter quality on decomposition was observed. These results may suggest major alterations of plant-soil interactions in Mediterranean forests under amplified drought conditions.

Amplified Drought and Seasonal Cycle Modulate Quercus pubescens Leaf Metabolome.

The intensification of summer drought expected with climate change can induce metabolism modifications in plants to face such constraints. In this experiment, we used both a targeted approach focused on flavonoids, as well as an untargeted approach, to study a broader fraction of the leaf metabolome of Quercus pubescens exposed to amplified drought. A forest site equipped with a rainfall exclusion device allowed reduction of natural rainfall by ~30% over the tree canopy. Leaves of natural drought (ND) and amplified drought (AD) plots were collected over three seasonal cycles (spring, summer, and autumn) in 2013 (the second year of rain exclusion), 2014, and 2015. As expected, Q. pubescens metabolome followed a seasonal course. In the summer of 2015, the leaf metabolome presented a shifted and early autumnal pattern because of harsher conditions during this year. Despite low metabolic modification at the global scale, our results demonstrated that 75% of Quercus metabolites were upregulated in springs when trees were exposed to AD, whereas 60 to 73% of metabolites (93% in summer 2015), such as kaempferols and quercetins, were downregulated in summers/autumns. Juglanin, a kaempferol pentoside, as well as rhododendrin derivatives, were upregulated throughout the year, suggesting an antioxidant ability of these metabolites. Those changes in terms of phenology and leaf chemistry could, in the end, affect the ecosystem functioning.

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.

Experimental assessment of the water quality influence on the phosphorus uptake of an invasive aquatic plant: biological responses throughout its phenological stage.

Understanding how an invasive plant can colonize a large range of environments is still a great challenge in freshwater ecology. For the first time, we assessed the relative importance of four factors on the phosphorus uptake and growth of an invasive macrophyte Elodea nuttallii (Planch.) St. John. This study provided data on its phenotypic plasticity, which is frequently suggested as an important mechanism but remains poorly investigated. The phosphorus uptake of two Elodea nuttallii subpopulations was experimentally studied under contrasting environmental conditions. Plants were sampled in the Rhine floodplain and in the Northern Vosges mountains, and then maintained in aquaria in hard (Rhine) or soft (Vosges) water. Under these conditions, we tested the influence of two trophic states (eutrophic state, 100 µg x l(-1) P-PO4(3-) and hypertrophic state, 300 µg x l(-1) P-PO4(3-)) on the P metabolism of plant subpopulations collected at three seasons (winter, spring and summer). Elodea nuttallii was able to absorb high levels of phosphorus through its shoots and enhance its phosphorus uptake, continually, after an increase of the resource availability (hypertrophic > eutrophic). The lowest efficiency in nutrient use was observed in winter, whereas the highest was recorded in spring, what revealed thus a storage strategy which can be beneficial to new shoots. This experiment provided evidence that generally, the water trophic state is the main factor governing P uptake, and the mineral status (softwater > hardwater) of the stream water is the second main factor. The phenological stage appeared to be a confounding factor to P level in water. Nonetheless, phenology played a role in P turnover in the plant. Finally, phenotypic plasticity allows both subpopulations to adapt to a changing environment.

Allelochemicals of Pinus halepensis as drivers of biodiversity in Mediterranean open mosaic habitats during the colonization stage of secondary succession.

The Mediterranean region is recognized as a global biodiversity hotspot. However, over the last 50 years or so, the cessation of traditional farming has given way to strong afforestation at the expense of open habitats. Pinus halepensis Miller, known to synthesize a wide range of secondary metabolites, is a pioneer expansionist species colonizing abandoned agricultural land that present high species richness. Here, laboratory bioassays were used to study the potential impact of P. halepensis on plant diversity through allelopathy, and the role of microorganisms in these interactions. Germination and growth of 12 target species naturally present in fallow farmlands were tested according to concentration of aqueous extracts obtained from shoots of young pines (aged about 5 years), with or without the presence of soil microorganisms (autoclaved or natural soil). Under the highest concentrations and autoclaved soil, more than 80 % of target species were germination and/or growth-inhibited, and only two species were non-sensitive. Under more natural conditions (lower extracts concentrations and natural soil with microorganisms), only 50 % of species were still inhibited, one was non-sensitive, and five were stimulated. Thus, microorganisms alter the expression of allelochemicals released into the ecosystem, which highlights their key role in chemical plant-plant interactions. The results of allelopathic experiments conducted in the lab are consistent with the community patterns observed in the field. These findings suggest that allelopathy is likely to shape vegetation composition and participate to the control of biodiversity in Mediterranean open mosaic habitats.

Use of urban composts for the regeneration of a burnt Mediterranean soil: a laboratory approach.

In Mediterranean region, forest fires are a major problem leading to the desertification of the environment. Use of composts is considered as a solution for soil and vegetation rehabilitation. In this study, we determined under laboratory conditions the effects of three urban composts and their mode of application (laid on the soil surface or mixed into the soil) on soil restoration after fire: a municipal waste compost (MWC), a compost of sewage sludge mixed with green waste (SSC) and a green waste compost (GWC). Carbon (C) and nitrogen (N) mineralisation, total microbial biomass, fungal biomass and soil characteristics were measured during 77-day incubations in microcosms. The impact of composts input on hydrological behaviour related to erodibility was estimated by measuring runoff, retention and percolation (i.e. infiltration) of water using a rainfall simulator under laboratory conditions. Input of composts increased organic matter and soil nutrient content, and enhanced C and N mineralisation and total microbial biomass throughout the incubations, whereas it increased sporadically fungal biomass. For all these parameters, the MWC induced the highest improvement while GWC input had no significant effect compared to the control. Composts mixed with soil weakly limited runoff and infiltration whereas composts laid at the soil surface significantly reduced runoff and increased percolation and retention, particularly with the MWC.

Purification processes involved in sludge treatment by a vertical flow wetland system: focus on the role of the substrate and plants on N and P removal.

Vertical-flow wetland systems were tested for treatment of liquid waste activated sludge with high content of organic compounds from a soft drink factory. A mesocosm experiment was carried out on planted and unplanted systems to understand the relative importance of substrate and plants in purification processes and to compare three species: Phragmites australis Cav., Typha latifolia L., or Iris pseudacorus L. All planted mesocosms performed better than unplanted mesocosms and Phragmites showed the highest efficiencies, both in volumes and loads, closely followed by Typha. Removal efficiencies were very high in all cases, and physical filtration by the organic substrate was identified as the main processes for nutrient removal (>50%). We showed that plants played direct and indirect roles such as nutrients uptake (up to 23% of the N for Phragmites), evapotranspiration reducing outflow volumes; or improvement of filtration by the root systems and stimulation of microbial activities (respiration rate was double compared to unplanted mesocosms).

Influence of plants on microbial activity in a vertical-downflow wetland system treating waste activated sludge with high organic matter concentrations.

The rhizosphere is a key zone for pollutant removal in treatment wetlands; therefore, studies on microbial activity may provide helpful information for a better understanding of purification processes. We studied microbial activity in a vertical-downflow constructed wetland system treating waste activated sludge with high organic matter concentrations, under Mediterranean climate. The aims of the work were to study the influence of (i) the presence of plants, (ii) the plant species (Phragmites australis Cav., Typha latifolia L., Iris pseudacorus L.), and (iii) the plant growth stage (plant senescence and plant fast growing stage) on total respiration rate and phosphatase activity in the substrate (intented here as the solid support on which the plants grow). The presence of plants had a positive influence on microbial activity, since substrate respiration and both acid and alkaline phosphatase activity were always higher in planted than in unplanted mesocosms. Among the three tested species, Phragmites was the one that most stimulated both substrate respiration rate and phosphatase activity, followed by Typha and Iris. These differences of microbial activity between mesocosms were corresponding to differences of removal efficiency. Substrate respiration and phosphatase activity were of similar magnitude at the two growth stages, while the stimulating effect of plants seemed to have been delayed and microbial activity showed higher fluctuations at plant fast growing stage than at plant senescence.

Direct and indirect impact of sewage sludge compost spreading on Quercus coccifera monoterpene emissions in a Mediterranean shrubland.

Monoterpene emissions of Quercus coccifera L. were repeatedly measured during the two years following the spreading of a sewage sludge compost at rates of 50 Mg ha⁻¹ and 100 Mg ha⁻¹, in a twelve-year-old post-fire Mediterranean shrubland. We also monitored the patterns of change in soil and leaf nutrient content, plant water potential, chlorophyll fluorescence, and plant growth. Compost spreading resulted in weak changes in leaf nutrient content and plant water status, and therefore no significant effect on monoterpene emissions at leaf scale, except during one summer sampling, probably related to advanced leaf maturity with the highest compost rate. However, compost increased plant growth, particularly the leaf biomass. The results suggest that compost spreading in Mediterranean shrublands has no strong short-term effect on Q. coccifera monoterpene emissions at leaf level, but may indirectly increase volatile organic compound fluxes at the stand scale, which may contribute to regional ozone pollution.

Variations in allelochemical composition of leachates of different organs and maturity stages of Pinus halepensis.

We investigated changes in the occurrence of allelochemicals from leachates of different Pinus halepensis organs taking into account the stages of pine stand age (i.e., young < 15-years-old, middle age +/- 30-years, and old > 60-years-old). GC-MS analysis of aqueous extracts revealed approx. 59 components from needles and roots. The major constituents were divided into different phytochemical groups-phenolics (50%), fatty acids (44%), and terpenoids. Further analyses were carried out to characterize the distribution of allelochemicals in different organs and P. halepensis successional stages. Roots and needles had two distinct chemical profiles, while needle leachates were composed mainly of oxygenated terpenoids (e.g., alpha-eudesmol, alpha-cadinol, and alpha-terpineol). Roots mainly contained fatty acids. Needles from young pine stands had the highest content of monoterpenes, suggesting their role as potential allelochemicals that could help young pine stands to establish. Pooling the different functional chemical groups showed that needles and, to a lesser extent, old roots, had higher chemical diversity than the roots of young and medium-aged pines. The highest diversity in phenolic constituents and fatty acids was in young needles (D(chem) = 2.38). Finally, caffeic acid, a compound that has allelopathic properties was found in aqueous extracts at high concentrations in both young needles and old roots. The role of this compound in mediation of biological interactions in P. halepensis ecosystem functioning is discussed.

Compost may affect volatile and semi-volatile plant emissions through nitrogen supply and chlorophyll fluorescence.

The use of composted biosolids as an amendment for forest regeneration in degraded ecosystems is growing since sewage-sludge dumping has been banned in the European Community. Its consequences on plant terpenes are however unknown. Terpene emissions of both Rosmarinus officinalis (a terpene-storing species) and Quercus coccifera (a non-storing species) and terpene content of the former, were studied after a middle-term exposure to compost at intermediate (50tha(-1): D50) and high (100tha(-1): D100) compost rates, in a seven-year-old post-fire shrubland ecosystem. Some chlorophyll fluorescence parameters (Fv/Fm, ETR, Phi(PSII)), soil and plant enrichment in phosphorus (P) and nitrogen (N) were monitored simultaneously in amended and non-amended plots in order to establish what factors were responsible for possible compost effect on terpenes. Compost affected all studied parameters with the exception of Fv/Fm and terpene content. For both species, mono- and sesquiterpene basal emissions were intensified solely under D50 plots. On the contrary leaf P, leaf N levels reached in D50 were partly responsible of terpene changes, suggesting that optimal N conditions occurred therein. N also affected ETR and Phi(PSII) which were, in turn, robustly correlated to terpene emissions. These results imply that emissions of terpene-storing and non-storing species were under nitrogen and chlorophyll fluorescence control, and that a correct management of compost rates applied on soil may modify terpene emission rate of plants, which in turn has consequences in air quality and plant defense mechanisms.

Production and diversity of volatile terpenes from plants on calcareous and siliceous soils: effect of soil nutrients.

Fertilizer effects on terpene production have been noted in numerous reports. In contrast, only a few studies have studied the response of leaf terpene content to naturally different soil fertility levels. Terpene content, as determined by gas chromatography/mass spectrometry/flame ionization detector, and growth of Pinus halepensis, Rosmarinus officinalis, and Cistus albidus were studied on calcareous and siliceous soils under field conditions. The effect of nitrogen (N) and extractable phosphorus (P(E)) from these soils on terpenes was also investigated since calcareous soils mainly differ from siliceous soils in their higher nutrient loadings. Rich terpene mixtures were detected. Twenty-one terpenes appeared in leaf extracts of R. officinalis and C. albidus and 20 in P. halepensis. Growth of all species was enhanced on calcareous soils, while terpene content showed a species-specific response to soil type. The total monoterpene content of P. halepensis and that of some major compounds (e.g., delta-terpinene) were higher on calcareous than on siliceous soils. A significant and positive relationship was found between concentration of N and P(E) and leaf terpene content of this species. These findings suggest that P. halepensis may respond to an environment characterized by increasing soil deposition, by allocating carbon resources to the synthesis of terpene defense metabolites without growth reduction. Results obtained for R. officinalis showed high concentrations of numerous major monoterpenes (e.g., myrcene, camphor) in plants growing on calcareous soils, while alpha-pinene, beta-caryophyllene, and the total sesquiterpene content were higher on siliceous soils. Finally, only alloaromadendrene and delta-cadinene of C. albidus showed higher concentrations on siliceous soils. Unlike P. halepensis, soil nutrients were not involved in terpene variation in calcareous and siliceous soils of these two shrub species. Possible ecological explanations on the effect of soil type for these latter two species as well as the ecological explanation of rich terpene mixtures are discussed.

Can compost improve Quercus pubescens Willd establishment in a Mediterranean post-fire shrubland?

The aim of the study was to evaluate the effects of sewage sludge compost (control, 20 kg m(-2), 40 kg m(-2)) supplied to Quercus pubescens Willd seedlings planted in a post-fire calcareous site in Provence (France). Changes in soil properties, seedling survival, growth and nutrition were monitored 7 months, 1.5 years and 2.5 years after amendment, and possible trace metal contamination of soil and seedlings by compost was also evaluated. Compost improved overall soil fertility by increasing organic matter, cation exchange capacity, total N and exchangeable P, K, Mg and B concentrations, but 40 kg m(-2) induced a more significant and more durable effect than 20 kg m(-2). However, the compost had no effect on seedling survival and growth, but increased foliar P and B concentrations at 40 kg m(-2). No foliar contamination of seedlings by trace metals occurred, although amendment increased exchangeable Cu and Zn concentrations in soil. Compost P and exchangeable Cu and Zn concentrations could induce eutrophication and water pollution, and limit rates that can be applied without environmental hazard.