Guillaume Meiffren

Plant secondary metabolite profiling evidences strain-dependent effect in the Azospirillum-Oryza sativa association

Azospirillum is a plant growth-promoting rhizobacterium (PGPR) able to enhance growth and yield of cereals such as rice, maize and wheat. The growth-promoting ability of some Azospirillum strains appears to be highly specific to certain plant species and cultivars. In order to ascertain the specificity of the associative symbiosis between rice and Azospirillum, the physiological response of two rice cultivars, Nipponbare and Cigalon, inoculated with two rice-associated Azospirillum was analyzed at two levels: plant growth response and plant secondary metabolic response. Each strain of Azospirillum (Azospirillum lipoferum 4B isolated from Cigalon and Azospirillum sp. B510 isolated from Nipponbare) preferentially increased growth of the cultivar from which it was isolated. This specific effect is not related to a defect in colonization of host cultivar as each strain colonizes effectively both rice cultivars, either at the rhizoplane (for 4B and B510) and inside the roots (for B510). The metabolic profiling approach showed that, in response to PGPR inoculation, profiles of rice secondary metabolites were modified, with phenolic compounds such as flavonoids and hydroxycinnamic derivatives being the main metabolites affected. Moreover, plant metabolic changes differed according to Azospirillum strain×cultivar combinations; indeed, 4B induced major secondary metabolic profile modifications only on Cigalon roots, while B510, probably due to its endophytic feature, induced metabolic variations on shoots and roots of both cultivars, triggering a systemic response. Plant secondary metabolite profiling thereby evidences the specific interaction between an Azospirillum strain and its original host cultivar.

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 plants ability to shape microbial soil functioning.

The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites

The aim of this study was to determine (i) whether plant nutritional strategy affects the composition of primary metabolites exuded into the rhizosphere and (ii) the impact of exuded metabolites on denitrification activity in soil. We answered this question by analysing primary metabolite content extracted from the root-adhering soil (RAS) and the roots of three grasses representing different nutrient management strategies: conservative (Festuca paniculata), intermediate (Bromus erectus) and exploitative (Dactylis glomerata). We also investigated the impact of primary metabolites on soil microbial denitrification enzyme activity without carbon addition, comparing for each plant RAS and bulk soils. Our data show that plant nutritional strategy impacts on primary metabolite composition of root extracts or RAS. Further we show, for the first time, that RAS-extracted primary metabolites are probably better indicators to explain plant nutrient strategy than root-extracted ones. In addition, our results show that some primary metabolites present in the RAS were well correlated with soil microbial denitrification activity with positive relationships found between denitrification and the presence of some organic acids and negative ones with the presence of xylose. We demonstrated that the analysis of primary metabolites extracted from the RAS is probably more pertinent to evaluate the impact of plant on soil microbial community functioning.

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.

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.

Invasive Fallopia × Bohemica Interspecific Hybrids Display Different Patterns in Secondary Metabolites

Abstract Plant secondary metabolites are important traits that can benefit an invasive plant in its new environment. In the case of rapid evolution in the invaded area, the chemical weapons of introduced plants may diversify, and novel combinations or extreme concentrations of these secondary metabolites may be expressed. The invasive Fallopia species complex (F. japonica, F. sachalinensis, and the F. × bohemica interspecific hybrids) is a good model to assess how chemical traits can vary during post-introduction evolution. We analyzed and compared the composition of secondary metabolite extracts in F. × bohemica hybrids and in the parental species grown in the introduced area. HPLC-DAD profiles were obtained for each Fallopia species, and the main peaks of the HPLC chromatograms represent phenolic compounds. Analyses based on secondary metabolite profiles showed that F. × bohemica hybrids are closer to F. japonica. The F. × bohemica hybrids expressed the compound families described in F. japonica and F. sachalinensis, with quantitative variations between them. Hybrid chemical cocktails showed a diversification of chemical weapons. Furthermore, transgressive segregation was observed. Three dianthrones were identified for the first time in a Fallopia species and were more highly expressed in F. japonica and F. × bohemica hybrids. These results suggest an evolution in the chemical traits of Fallopia taxa in invaded areas such that certain genotypes may well have acquired new chemical cocktails resulting from post-introduction hybridizations.