Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

Pseudomonas PA14H7: Identification and Quantification of the 7-Hydroxytropolone Iron Complex as an Active Metabolite against Dickeya, the Causal Agent of Blackleg on the Potato Plant.

Soft rot Pectobacteriaceae (SRP), such as Pectobacterium and Dickeya, are phytopathogenic agents responsible for blackleg disease on several crops, such as potatoes, affecting the yield and depressing the seed production quality. However, neither conventional nor biocontrol products are available on the market to control this disease. In this study Pseudomonas PA14H7, a bacteria isolated from potato rhizosphere, was selected as a potential antagonist agent against Dickeya solani. In order to understand the mechanism involved in this antagonism, we managed to identify the main active molecule(s) produced by PA14H7. Cell-free supernatant (CFS) of PA14H7 cultures were extracted and analyzed using LC-MS, GC-MS, and NMR. We further correlated the biological activity against Dickeya solani of extracted CFS-PA14H7 to the presence of 7-hydroxytropolone (7-HT) complexed with iron. In a second time, we have synthesized this molecule and determined accurately using LC-UV, LC-MS, and GC-MS that, after 48 h incubation, PA14H7 released, in its CFS, around 9 mg/L of 7-HT. The biological activities of CFS-PA14H7 vs. synthetic 7-HT, at this concentration, were evaluated to have a similar bacteriostatic effect on the growth of Dickeya solani. Even if 7-HT is produced by other Pseudomonas species and is mostly known for its antibacterial and antifungal activities, this is the first description of its involvement as an effective molecule against pectinolytic bacteria. Our work opens the way for the comprehension of the mode of action of PA14H7 as a biocontrol agent against potato blackleg.

Secondary Metabolism Rearrangements in Linum usitatissimum L. after Biostimulation of Roots with COS Oligosaccharides from Fungal Cell Wall.

In vitro culture of flax (Linum usitatissimum L.) was exposed to chitosan oligosaccharides (COS) in order to investigate the effects on the growth and secondary metabolites content in roots and shoots. COS are fragments of chitosan released from the fungal cell wall during plant-pathogen interactions. They can be perceived by the plant as pathogen-associated signals, mediating local and systemic innate immune responses. In the present study, we report a novel COS oligosaccharide fraction with a degree of polymerization (DP) range of 2-10, which was produced from fungal chitosan by a thermal degradation method and purified by an alcohol-precipitation process. COS was dissolved in hydroponic medium at two different concentrations (250 and 500 mg/L) and applied to the roots of growing flax seedlings. Our observations indicated that the growth of roots and shoots decreased markedly in COS-treated flax seedlings compared to the control. In addition, the results of a metabolomics analysis showed that COS treatment induced the accumulation of (neo)lignans locally at roots, flavones luteolin C-glycosides, and chlorogenic acid in systemic responses in the shoots of flax seedlings. These phenolic compounds have been previously reported to exhibit a strong antioxidant and antimicrobial activities. COS oligosaccharides, under the conditions applied in this study (high dose treatment with a much longer exposure time), can be used to indirectly trigger metabolic response modifications in planta, especially secondary metabolism, because during fungal pathogen attack, COS oligosaccharides are among the signals exchanged between the pathogen and host plant.

Fabrication and Characterization of Nanocomposite Hydrogel Based on Alginate/Nano-Hydroxyapatite Loaded with Linum usitatissimum Extract as a Bone Tissue Engineering Scaffold.

In the current paper, we fabricated, characterized, and applied nanocomposite hydrogel based on alginate (Alg) and nano-hydroxyapatite (nHA) loaded with phenolic purified extracts from the aerial part of Linum usitatissimum (LOH) as the bone tissue engineering scaffold. nHA was synthesized based on the wet chemical technique/precipitation reaction and incorporated into Alg hydrogel as the filler via physical cross-linking. The characterizations (SEM, DLS, and Zeta potential) revealed that the synthesized nHA possess a plate-like shape with nanometric dimensions. The fabricated nanocomposite has a porous architecture with interconnected pores. The average pore size was in the range of 100-200 µm and the porosity range of 80-90%. The LOH release measurement showed that about 90% of the loaded drug was released within 12 h followed by a sustained release over 48 h. The in vitro assessments showed that the nanocomposite possesses significant antioxidant activity promoting bone regeneration. The hemolysis induction measurement showed that the nanocomposites were hemocompatible with negligible hemolysis induction. The cell viability/proliferation confirmed the biocompatibility of the nanocomposites, which induced proliferative effects in a dose-dependent manner. This study revealed the fabricated nanocomposites are bioactive and osteoactive applicable for bone tissue engineering applications.

Phenolic Profiling of Flax Highlights Contrasting Patterns in Winter and Spring Varieties.

Flax (Linum usitatissimum) is a plant grown in temperate regions either for its fiber or for its seeds, which are rich in the essential fatty acid omega-3. It is also well known as a source of medicinal compounds. The chemical composition of its leaves is currently poorly described. In order to fill this gap, we have conducted a comprehensive analysis of flax leaf metabolome. The exploration of the metabolome allowed the characterization of compounds isolated for the first time in flax leaves. These molecules were isolated by preparative HPLC and then characterized by NMR, LC-MS and standard analysis. This work extended our picture of C-glycosyl-flavonoids and coniferyl alcohol derivatives accumulated in flax. The follow-up of the content of these different metabolites via UPLC-MS revealed significant accumulation differences in spring and winter flax leaves. In particular, two methylated C-glycosylflavonoids (swertisin and swertiajaponin) were the most abundant phenolic compounds in winter flax whereas they were not detected in spring flax. This result suggests that these 2 compounds are involved in cold stress tolerance in flax.

Trehalose determination in linseed subjected to osmotic stress. HPAEC-PAD analysis: an inappropriate method.

Trehalose is a non-reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co-elution of trehalose in these two species by a trehalase assay and liquid chromatography-high resolution mass spectrometry analysis, gas chromatography-mass spectrometry (GC-MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC-PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC-MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (-0.30 MPa), the quantity (31.49 nmol g(-1) dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.

Seasonal Variation in Cell Wall Composition and Carbohydrate Metabolism in the Seagrass Posidonia oceanica Growing at Different Depths.

Posidonia oceanica is a common seagrass in the Mediterranean Sea that is able to sequester large amounts of carbon. The carbon assimilated during photosynthesis can be partitioned into non-structural sugars and cell-wall polymers. In this study, we investigated the distribution of carbon in starch, soluble carbohydrates and cell-wall polymers in leaves and rhizomes of P. oceanica. Analyses were performed during summer and winter in meadows located south of the Frioul archipelago near Marseille, France. The leaves and rhizomes were isolated from plants collected in shallow (2 m) and deep water (26 m). Our results showed that P. oceanica stores more carbon as starch, sucrose and cellulose in summer and that this is more pronounced in rhizomes from deep-water plants. In winter, the reduction in photoassimilates was correlated with a lower cellulose content, compensated with a greater lignin content, except in rhizomes from deep-water plants. The syringyl-to-guaiacyl (S/G) ratio in the lignin was higher in leaves than in rhizomes and decreased in rhizomes in winter, indicating a change in the distribution or structure of the lignin. These combined data show that deep-water plants store more carbon during summer, while in winter the shallow- and deep-water plants displayed a different cell wall composition reflecting their environment.

Pathogen Stopping and Metabolism Modulation Are Key Points to Linum usitatissimum L. Early Response against Fusarium oxysporum.

Fusarium oxysporum is the one of the most common and impactful pathogens of flax. Cultivars of flax that show resistance to this pathogen have previously been identified. To better understand the mechanisms that are responsible for this resistance, we conducted time-lapse analysis of one susceptible and one resistant cultivar over a two-week period following infection. We also monitored changes in some metabolites. The susceptible cultivar showed a strong onset of symptoms from 6 to 8 days after inoculation, which at this time point, was associated with changes in metabolites in both cultivars. The resistant cultivar maintained its height and normal photosynthetic capacity but showed a reduced growth of its secondary stems. This resistance was correlated with the containment of the pathogen at the root level, and an increase in some metabolites related to the phenylpropanoid pathway.

Isolation and Identification of Flavones Responsible for the Antibacterial Activities of Tillandsia bergeri Extracts.

Plants are an everlasting inspiration source of biologically active compounds. Among these medicinal plants, the biological activity of extracts from some species of the Tillandsia genus has been studied, but the phytochemistry of the hardy species Tillandsia bergeri remains unknown. The aim of the present study was to perform the first phytochemical study of T. bergeri and to identify the compounds responsible for the antibacterial activity of T. bergeri extracts. Soxhlet extraction of predried and grinded leaves was first performed using four increasing polarity solvents. A bio-guided fractionation was performed using agar overlay bioautography as a screening method against 12 Gram-positive, Gram-negative, sensitive, and resistant bacterial strains. The results showed the inhibition of Gram-positive methicillin-sensitive Staphylococcus aureus ATCC 29213 (MSSA), methicillin-resistant S. aureus N-SARM-1 (MRSA), and Staphylococcus caprae ATCC 35538 by the dichloromethane fraction. A phytochemical investigation led to the isolation and identification by high-resolution mass spectrometry and nuclear magnetic resonance of the two flavones penduletin and viscosine, responsible for this antibacterial activity. For viscosine, the minimum inhibitory concentration (MIC) value is equal to 128 µg/mL against MSSA and is equal to 256 µg/mL against MRSA and S. caprae. The combination of these compounds with vancomycin and cloxacillin showed a decrease in MICs of the antibiotics. Penduletin showed synergistic activity when combined with vancomycin against MSSA (FICI < 0.258) and S. caprae (FICI < 0.5). Thus, unexplored Tillandsia species may represent a valuable source for potential antibiotics and adjuvants.

Physiological and Biochemical Traits of Two Major Arabidopsis Accessions, Col-0 and Ws, Under Salinity.

Salinity affects plant growth and development as shown with the glycophyte model plant, Arabidopsis thaliana (Arabidopsis). Two Arabidopsis accessions, Wassilewskija (Ws) and Columbia (Col-0), are widely used to generate mutants available from various Arabidopsis seed resources. However, these two ecotypes are known to be salt-sensitive with different degrees of tolerance. In our study, 3-week-old Col-0 and Ws plants were treated with and without 150 mM NaCl for 48, 72, or 96 h, and several physiological and biochemical traits were characterized on shoots to identify any specific traits in their tolerance to salinity. Before salt treatment was carried out, a different phenotype was observed between Col-0 and Ws, whose main inflorescence stem became elongated in contrast to Col-0, which only displayed rosette leaves. Our results showed that Col-0 and Ws were both affected by salt stress with limited growth associated with a reduction in nutrient uptake, a degradation of photosynthetic pigments, an increase in protein degradation, as well as showing changes in carbohydrate metabolism and cell wall composition. These traits were often more pronounced in Col-0 and occurred usually earlier than in Ws. Tandem Mass Tags quantitative proteomics data correlated well with the physiological and biochemical results. The Col-0 response to salt stress was specifically characterized by a greater accumulation of osmoprotectants such as anthocyanin, galactinol, and raffinose; a lower reactive oxygen detoxification capacity; and a transient reduction in galacturonic acid content. Pectin degradation was associated with an overaccumulation of the wall-associated kinase 1, WAK1, which plays a role in cell wall integrity (CWI) upon salt stress exposure. Under control conditions, Ws produced more antioxidant enzymes than Col-0. Fewer specific changes occurred in Ws in response to salt stress apart from a higher number of different fascilin-like arabinogalactan proteins and a greater abundance of expansin-like proteins, which could participate in CWI. Altogether, these data indicate that Col-0 and Ws trigger similar mechanisms to cope with salt stress, and specific changes are more likely related to the developmental stage than to their respective genetic background.

Age-Dependent Metabolic Profiles Unravel the Metabolic Relationships Within and Between Flax Leaves (Linum usitatissimum).

Flax for oil seed is a crop of increasing popularity, but its cultivation needs technical improvement. Important agronomic traits such as productivity and resistance to stresses are to be regarded as the result of the combined responses of individual organs and their inter-communication. Ultimately, these responses directly reflect the metabolic profile at the cellular level. Above ground, the complexity of the plant phenotype is governed by leaves at different developmental stages, and their ability to synthesise and exchange metabolites. In this study, the metabolic profile of differently-developed leaves was used firstly to discriminate flax leaf developmental stages, and secondly to analyse the allocation of the metabolites within and between leaves. For this purpose, the concentration of 52 metabolites, both primary and specialized, was followed by gas chromatography (GC-) and liquid chromatography coupled to mass spectrometry (LC-MS) in alternate pairs of flax leaves. On the basis of their metabolic content, three populations of leaves in different growth stages could be distinguished. Primary and specialized metabolites showed characteristic distribution patterns, and compounds similarly evolving with leaf age could be grouped by the aid of the Kohonen self-organising map (SOM) algorithm. Ultimately, visualisation of the correlations between metabolites via hierarchical cluster analysis (HCA) allowed the assessment of the metabolic fluxes characterising different leaf developmental stages, and the investigation of the relationships between primary and specialized metabolites.

Flax phenolic compounds as inhibitors of lipid oxidation: Elucidation of their mechanisms of action.

Polyunsaturated fatty acids are particularly sensitive to the damages due to reactive oxygen species and lipid oxidation has been reported to be involved in the degradation of food as well as in the early stages of several diseases. Our objective was to study the mechanisms of action of flax (Linum usitatissimum) phenolic compounds to prevent membrane lipid oxidation. To do so, several biophysical techniques (oxidative stress, surface tension, fluorescence spectroscopy and HPLC) were used to investigate the ability of the compounds to prevent lipid oxidation and to interact with membranes. We evidenced a relationship between the structure and the antioxidant efficiency as aglycone compounds were significantly more efficient (p < 0.05) than glucoside compounds. In addition, our results revealed that aglycone lignans spontaneously penetrated the membrane contrary to aglycone hydroxycinnamic acids. To conclude, the comparison of the antioxidant efficiencies revealed that membrane inserted compounds better inhibited lipid oxidation than non-inserted compounds.

Kinetics of the incorporation of the main phenolic compounds into the lignan macromolecule during flaxseed development.

The main flax lignan, secoisolariciresinol diglucoside, is stored in a macromolecule containing other ester-bound phenolic compounds. In this study, NMR and HPLC-UV analyses were performed on flaxseeds harvested at different developmental stages to identify and quantify the main phenolic compounds produced during seed development. Extraction was carried out with or without alkaline hydrolysis to determine if these molecules accumulate in the lignan macromolecule and/or in a free form. Monolignol glucosides accumulate in a free form up to 9.85mg/g dry matter at the early developmental stages. Hydroxycinnamic acid glucosides and flavonoid accumulate (up to 3.18 and 4.07mg/g dry matter, respectively) in the later developmental stages and are ester-bound in the lignan macromolecule. Secosiolariciresinol diglucoside accumulates (up to 28.65mg/g dry matter) in the later developmental stages in both forms, mainly ester-bound in the lignan macromolecule and slightly in a free form.

Osmotic stress alters the balance between organic and inorganic solutes in flax (Linum usitatissimum).

Flax (Linum usitatissimum) is grown for its oil and its fiber. This crop, cultivated in temperate regions, has seen a renewed interest due to the presence of abundant molecules of interest for many applications. Little information is available about the behavior of flax during osmotic stress; yet this is considered a major stress that causes significant yield losses in most crops. To control the presence of this stress better, flax behavior was investigated following the application of osmotic stress and the response was examined by applying increasing concentrations of PEG 8000. This resulted in the reorganization of 32 metabolites and 6 mineral ions in the leaves. The analysis of these two types of solute highlighted the contrasting behavior between a higher metabolite content (particularly fructose, glucose and proline) and a decrease in mineral ions (especially nitrate and potassium) following PEG treatment. However, this reorganization did not lead to a greater accumulation of solutes, with the total amount remaining unchanged in leaves during osmotic stress.

Improved stability of TMS derivatives for the robust quantification of plant polar metabolites by gas chromatography-mass spectrometry.

Plant metabolite profiling is commonly carried out by GC-MS of methoximated trimethylsilyl (TMS) derivatives. This technique is robust and enables a library search for spectra produced by electron ionization. However, recent articles have described problems associated with the low stability of some TMS derivatives. This limits the use of GC-MS for metabolomic studies that need large sets of qualitative and quantitative analyses. The aim of this work is to determine the experimental conditions in which the stability of TMS derivatives could be improved. This would facilitate the analysis of the large-scale experimental designs needed in the metabolomics approach. For good repeatability, the sampling conditions and the storage temperature of samples during analysis were investigated. Multiple injections of one sample from one vial led to high variations while injection of one sample from different vials improved the analysis. However, before injection, some amino acid TMS derivatives were degraded during the storage of vials in the autosampler. Only 10% of the initial quantity of glutamine 3 TMS and glutamate 3 TMS and 66% of α-alanine 2 TMS was detected 48 h after derivatization. When stored at 4 °C until injection, all TMS derivatives remained stable for 12 h; at -20 °C, they remained stable for 72 h. From the integration of all these results, a detailed analytical procedure is thus proposed. It enables a robust quantification of polar metabolites, useful for further plant metabolomics studies using GC-MS.

Trehalose and plant stress responses: friend or foe?

The disaccharide trehalose is involved in stress response in many organisms. However, in plants, its precise role remains unclear, although some data indicate that trehalose has a protective role during abiotic stresses. By contrast, some trehalose metabolism mutants exhibit growth aberrations, revealing potential negative effects on plant physiology. Contradictory effects also appear under biotic stress conditions. Specifically, trehalose is essential for the infectivity of several pathogens but at the same time elicits plant defense. Here, we argue that trehalose should not be regarded only as a protective sugar but rather like a double-faced molecule and that further investigation is required to elucidate its exact role in stress tolerance in plants.