Responses of the species complex Fallopia × bohemica to single-metal contaminations to Cd, Cr or Zn: growth traits, metal accumulation and secondary metabolism.

Plant responses to heavy metals and their storage constitute a crucial step to understand the environmental impacts of metallic trace elements (MTEs). In controlled experiments, we previously demonstrated the tolerance and resilience of Japanese knotweed to soil artificial polymetallic contamination. Using the same experimental design, we tested here the effect of three individual MTEs on Fallopia × bohemica performance traits. Rhizome fragments from three different sites (considered as distinct morphotypes) were grown in a greenhouse for 1 month on a prairial soil artificially contaminated with either Cd, Cr (VI) or Zn at concentrations corresponding to relatively highly polluted soils. Our results confirmed the high tolerance of Bohemian knotweed to metal stress, though, plant response to MTE pollution was dependant on MTE identity. Bohemian knotweed was stimulated by Cr (VI) (increased root and aerial masses), did not display any measurable change in performance traits under Cd at the high dose of 10 mg kg-1, and uptook all MTEs in its rhizome, but only Zn was transferred to its aerial parts. We also highlighted changes in root secondary metabolism that were more accentuated with Zn, including the increase of anthraquinone, stilbene and biphenyl derivatives. These results compared to multi-contamination experiments previously published suggest complex interactions between metals and plant, depending principally on metal identity and also suggest a potential role of soil microbes in the interactions.

Identification of B-type procyanidins in Fallopia spp. involved in biological denitrification inhibition.

Nitrogen (N) is considered as a main limiting factor in plant growth, and nitrogen losses through denitrification can be responsible for severe decreases in plant productivity. Recently, it was demonstrated that Fallopia spp. is responsible for biological denitrification inhibition (BDI) through the release of unknown secondary metabolites. Here, we investigate the secondary metabolites involved in the BDI of Fallopia spp. The antioxidant, protein precipitation capability of Fallopia spp. extracts was measured in relation to the aerobic respiration and denitrification of two bacteria (Gram positive and Gram negative). Proanthocyanidin concentrations were estimated. Proanthocyanidins in extracts were characterized by chromatographic analysis, purified and tested on the bacterial denitrification and aerobic respiration of two bacterial strains. The effect of commercial procyanidins on denitrification was tested on two different soil types. Denitrification and aerobic respiration inhibition were correlated with protein precipitation capacity and concentration of proanthocyanidins but not to antioxidant capacity. These proanthocyanidins were B-type procyanidins that inhibited denitrification more than the aerobic respiration of bacteria. In addition, procyanidins also inhibited soil microbial denitrification. We demonstrate that procyanidins are involved in the BDI of Fallopia spp. Our results pave the way to a better understanding of plant-microbe interactions and highlight future applications for a more sustainable agriculture.

Allelopathic effect of a native species on a major plant invader in Europe.

Biological invasions have become a major global issue in ecosystem conservation. As formalized in the "novel weapon hypothesis", the allelopathic abilities of species are actively involved in invasion success. Here, we assume that allelopathy can also increase the biotic resistance of native species against invasion. We tested this hypothesis by studying the impact of the native species Sambucus ebulus on the colonization of propagules of the invasive species Fallopiaxbohemica and the subsequent development of plants from these. Achenes and rhizome fragments from two natural populations were grown in a greenhouse experiment for 50 days. We used an experimental design that involved "donor" and "target" pots in order to separate resource competition from allelopathy. An allelopathic treatment effect was observed for plant growth but not for propagule establishment. Treatment affected, in particular, the growth of Fallopia plants originating from achenes, but there was less influence on plants originating from rhizomes. By day 50, shoot height had decreased by 27% for plants originating from rhizomes and by 38% for plants originating from achenes. The number of leaves for plants originating from achenes had only decreased by 20%. Leaf and above- and below-ground dry masses decreased with treatment by 40, 41 and 25% for plants originating from rhizomes and 70, 61 and 55% for plants originating from achenes, respectively. S. ebulus extracts were analysed using high-performance chromatography, and the choice of test molecules was narrowed down. Our results suggest native species use allelopathy as a biotic containment mechanism against the naturalization of invasive species.

Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites.

Previous studies on the effect of secondary metabolites on the functioning of rhizosphere microbial communities have often focused on aspects of the nitrogen (N) cycle but have overlooked biological denitrification inhibition (BDI), which can affect plant N-nutrition. Here, we investigated the BDI by the compounds of Fallopia spp., an invasive weed shown to be associated with a low potential denitrification of the soil. Fallopia spp. extracts were characterized by chromatographic analysis and were used to test the BDI effects on the metabolic and respiratory activities of denitrifying bacteria, under aerobic and anaerobic (denitrification) conditions. The BDI of Fallopia spp. extracts was tested on a complex soil community by measuring denitrification enzyme activity (DEA), substrate induced respiration (SIR), as well as abundances of denitrifiers and total bacteria. In 15 strains of denitrifying bacteria, extracts led to a greater BDI (92%) than respiration inhibition (50%). Anaerobic metabolic activity reduction was correlated with catechin concentrations and the BDI was dose dependent. In soil, extracts reduced the DEA/SIR ratio without affecting the denitrifiers: total bacteria ratio. We show that secondary metabolite(s) from Fallopia spp. inhibit denitrification. This provides new insight into plant-soil interactions and improves our understanding of a plant's ability to shape microbial soil functioning.

Invasive knotweeds are highly tolerant to salt stress.

Japanese knotweed s.l. are some of the most invasive plants in the world. Some genotypes are known to be tolerant to the saline concentrations found in salt marshes. Here we focus on tolerance to higher concentrations in order to assess whether the species are able to colonize and establish in highly stressful environments, or whether salt is an efficient management tool. In a first experiment, adult plants of Fallopia japonica, Fallopia × bohemica and Fallopia sachalinensis were grown under salt stress conditions by watering with saline concentrations of 6, 30, 120, or 300 g L(-1) for three weeks to assess the response of the plants to a spill of salt. At the two highest concentrations, their leaves withered and fell. There were no effects on the aboveground parts at the lowest concentrations. Belowground dry weight and number of buds were reduced from 30 and 120 g L(-1) of salt, respectively. In a second experiment, a single spraying of 120 g L(-1) of salt was applied to individuals of F. × bohemica and their stems were clipped to assess the response to a potential control method. 60 % of the plants regenerated. Regeneration was delayed by the salt treatment and shoot growth slowed down. This study establishes the tolerance of three Fallopia taxa to strong salt stress, with no obvious differences between taxa. Their salt tolerance could be an advantage in their ability to colonize polluted environments and to survive to spills of salt.

Scent evolution in Chinese roses.

The phenolic methyl ether 3,5-dimethoxytoluene (DMT) is a major scent compound of many modern rose varieties, and its fragrance participates in the characteristic "tea scent" that gave their name to Tea and Hybrid Tea roses. Among wild roses, phenolic methyl ether (PME) biosynthesis is restricted to Chinese rose species, but the progenitors of modern roses included both European and Chinese species (e.g., Rosa chinensis cv Old Blush), so this trait was transmitted to their hybrid progeny. The last steps of the biosynthetic pathways leading to DMT involve two methylation reactions catalyzed by the highly similar orcinol O-methyltransferases (OOMT) 1 and 2. OOMT1 and OOMT2 enzymes exhibit different substrate specificities that are consistent with their operating sequentially in DMT biosynthesis. Here, we show that these different substrate specificities are mostly due to a single amino acid polymorphism in the phenolic substrate binding site of OOMTs. An analysis of the OOMT gene family in 18 species representing the diversity of the genus Rosa indicated that only Chinese roses possess both the OOMT2 and the OOMT1 genes. In addition, we provide evidence that the Chinese-rose-specific OOMT1 genes most probably evolved from an OOMT2-like gene that has homologues in the genomes of all extant roses. We propose that the emergence of the OOMT1 gene may have been a critical step in the evolution of scent production in Chinese roses.

An allelochemical from Myrica gale with strong phytotoxic activity against highly invasive Fallopia x bohemica taxa.

We report the identification of the allelochemical 3-(1-oxo-3-phenylpropyl)-1,1,5-trimethylcyclo-hexane-2,4,6-trione, known as myrigalone A, from the fruits and leaves of Myrica gale. The structure of the compound was confirmed by high-resolution techniques (UV, MS and NMR analysis). The compound is phytotoxic towards classical plant species used for allelochemical assays and also against Fallopia x bohemica, a highly invasive plant. Application of either powdered dry leaves or dry fruits of M. gale also showed in vitro phytotoxic activity. We hypothesize that M. gale could be used as a green allelopathic shield to control Fallopia x bohemica invasion, in addition to its potential use as an environmentally friendly herbicide.

Root fungal endophytes: identity, phylogeny and roles in plant tolerance to metal stress.

Metal trace elements accumulate in soils mainly because of anthropic activities, leading living organisms to develop strategies to handle metal toxicity. Plants often associate with root endophytic fungi, including nonmycorrhizal fungi, and some of these organisms are associated with metal tolerance. The lack of synthetic analyses of plant-endophyte-metal tripartite systems and the scant consideration for taxonomy led to this review aiming (1) to inventory non-mycorrhizal root fungal endophytes described with respect to their taxonomic diversity and (2) to determine the mutualistic roles of these plant-fungus associations under metal stress. More than 1500 species in 100 orders (mainly Hypocreales and Pleosporales) were reported from a wide variety of environments and hosts. Most reported endophytes had a positive effect on their host under metal stress, but with various effects on metal uptake or translocation and no clear taxonomic consistency. Future research considering the functional patterns and dynamics of these associations is thus encouraged.

Effects of the Denitrification Inhibitor "Procyanidins" on the Diversity, Interactions, and Potential Functions of Rhizosphere-Associated Microbiome.

Some plant secondary metabolites, such as procyanidins, have been demonstrated to cause biological denitrification inhibition (BDI) of denitrifiers in soils concomitantly with a gain in plant biomass. The present work evaluated whether procyanidins had an impact on the diversity of nontarget microbial communities that are probably involved in soil fertility and ecosystem services. Lettuce plants were grown in two contrasting soils, namely Manziat (a loamy sand soil) and Serail (a sandy clay loam soil) with and without procyanidin amendment. Microbial diversity was assessed using Illumina sequencing of prokaryotic 16S rRNA gene and fungal ITS regions. We used a functional inference to evaluate the putative microbial functions present in both soils and reconstructed the microbial interaction network. The results showed a segregation of soil microbiomes present in Serail and Manziat that were dependent on specific soil edaphic variables. For example, Deltaproteobacteria was related to total nitrogen content in Manziat, while Leotiomycetes and Firmicutes were linked to Ca2+ in Serail. Procyanidin amendment did not affect the diversity and putative activity of microbial communities. In contrast, microbial interactions differed according to procyanidin amendment, with the results showing an enrichment of Entotheonellaeota and Mucoromycota in Serail soil and of Dependentiae and Rozellomycetes in Manziat soil.

Does Biological Denitrification Inhibition (BDI) in the Field Induce an Increase in Plant Growth and Nutrition in Apium graveolens L. Grown for a Long Period?

Intensive agriculture uses a lot of nitrogen fertilizers to increase crop productivity. These crops are in competition with soil-denitrifying microorganisms that assimilate nitrogen in the form of nitrate and transform it into N2O, a greenhouse gas, or N2. However, certain plant species exude secondary metabolites, called procyanidins, which inhibit denitrifiers and increase the nitrate pool in the soil available for plant nutrition. This phenomenon is called biological denitrification inhibition. Previously, we showed that the addition of exogenous procyanidins to a lettuce crop induces denitrifier inhibition and increases nitrate content in the soil, affecting lettuce morphological traits. Here, the effects of procyanidin amendments in the field on a more long-term and nitrogen-consuming crop species such as celery were tested. The effects of procyanidin amendment on celery growth with those of conventional ammonium nitrate amendments were, therefore, compared. Denitrification activity, nitrate concentration, the abundance of denitrifying bacteria in the soil, and traits related to celery growth were measured. It was shown that the addition of procyanidins inhibits denitrifiers and increases the soil nitrate level, inducing an improvement in celery morphological traits. In addition, procyanidin amendment induces the lowest nitrogen concentration in tissues and reduces N2O emissions.

The Rosa genome provides new insights into the domestication of modern roses.

Roses have high cultural and economic importance as ornamental plants and in the perfume industry. We report the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. We generated a homozygous genotype from a heterozygous diploid modern rose progenitor, Rosa chinensis 'Old Blush'. Using single-molecule real-time sequencing and a meta-assembly approach, we obtained one of the most comprehensive plant genomes to date. Diversity analyses highlighted the mosaic origin of 'La France', one of the first hybrids combining the growth vigor of European species and the recurrent blooming of Chinese species. Genomic segments of Chinese ancestry identified new candidate genes for recurrent blooming. Reconstructing regulatory and secondary metabolism pathways allowed us to propose a model of interconnected regulation of scent and flower color. This genome provides a foundation for understanding the mechanisms governing rose traits and should accelerate improvement in roses, Rosaceae and ornamentals.

Tolerance of Japanese knotweed s.l. to soil artificial polymetallic pollution: early metabolic responses and performance during vegetative multiplication.

The expansion of invasive Japanese knotweed s.l. is of particular concern because of its aptitudes to rapidly colonize diverse environments, especially anthropized habitats generally characterized by their pollution with heavy metals. Whether the presence of heavy metals impacts the performance traits of this plant is a central question to better understand its invasive properties, though no controlled approach to assess these effects was yet reported. In this aim, we undertook greenhouse experiments where rhizome fragments of Japanese knotweed s.l. (Fallopia japonica and Fallopia × bohemica) were grown during 1 and 3 months, in a soil pot artificially polluted or not with heavy metals added in mixture (Cd, Cr, Pb, Zn). Our results showed that (i) the presence of heavy metals delayed rhizome regeneration and induced lowered plant part weights but did not affect plant height after 3 months; (ii) the effect of metals on the metabolic profiles of belowground part extracts was only detectable after 1 month and not after 3 months of growth, though it was possible to highlight the effect of metals independently of time and genotype for root extracts, and torosachrysone seemed to be the most induced compound; and (iii) the hybrid genotype tested was able to accumulate relatively high concentrations of metals, over or close to the highest reported ones for this plant for Cr, Cd and Zn, whereas Pb was not accumulated. These findings evidence that the presence of heavy metals in soil has a low impact on Fallopia sp. overall performance traits during rhizome regeneration, and has a rather stimulating effect on plant growth depending on pollution level.

Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI.