Dual transcriptomic & metabolomic analysis of elicited flax sheds light on the kinetics of immune defense activation against the biotrophic pathogen Oidium lini.

Flax (Linum usitatissimum) grown under controlled conditions displayed genotype-dependent resistance to powdery mildew (Oidium lini) following COS-OGA (comprising chitosan- and pectin-derived oligomers) elicitor application. The present study reveals a two-step immune response in plants preventively challenged with the elicitor: an initial, rapid response characterized by the transcription of defense genes whose protein products act in contact with or within the cell wall, where biotrophic pathogens initially thrive, followed by a prolonged activation of cell wall peroxidases and accumulation of secondary metabolites. Thus, dozens of genes encoding membrane receptors, pathogenesis-related proteins, and wall peroxidases were initially overexpressed. Repeated COS-OGA treatments had a transient effect on the transcriptome response while cumulatively remodeling the metabolome over time, with a minimum of two applications required for maximal metabolomic shifts. Secondary metabolites, in particular terpenoids and phenylpropanoids, emerged as major components of this secondary defense response alongside pathogenesis-related proteins and wall peroxidases. The sustained accumulation of secondary metabolites, even after cessation of elicitation, contrasted with the short-lived transcriptomic response. Wall peroxidase enzyme activity also exhibited cumulative effects, increasing strongly for weeks after a third elicitor treatment. This underscores the plasticity of the plant immune response in the face of a potential infection, and the need for repeated preventive applications to achieve the full protective potential of the elicitor.

Modulation of plant plasma membrane structure by exogenous fatty acid hydroperoxide is a potential perception mechanism for their eliciting activity.

Oxylipins are lipid-derived molecules that are ubiquitous in eukaryotes and whose functions in plant physiology have been widely reported. They appear to play a major role in plant immunity by orchestrating reactive oxygen species (ROS) and hormone-dependent signalling pathways. The present work focuses on the specific case of fatty acid hydroperoxides (HPOs). Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this study, the potential of 13(S)-hydroperoxy-(9Z, 11E)-octadecadienoic acid (13-HPOD) and 13(S)-hydroperoxy-(9Z, 11E, 15Z)-octadecatrienoic acid (13-HPOT), as plant defence elicitors and the underlying mechanism of action is investigated. Arabidopsis thaliana leaf resistance to Botrytis cinerea was observed after root application with HPOs. They also activate early immunity-related defence responses, like ROS. As previous studies have demonstrated their ability to interact with plant plasma membranes (PPM), we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. The relationship between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.

Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes.

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

Mutations in chicory FEH genes are statistically associated with enhanced resistance to post-harvest inulin depolymerization.

KEY MESSAGE: Nucleotidic polymorphisms were identified in fructan exohydrolases genes which are statistically associated with enhanced susceptibility to post-harvest inulin depolymerization. Industrial chicory (Cichorium intybus L.) root is the main commercial source of inulin, a linear fructose polymer used as dietary fiber. Post-harvest, inulin is depolymerized into fructose which drastically increases processing cost. To identify genetic variations associated with enhanced susceptibility to post-harvest inulin depolymerization and related free sugars content increase, we used a candidate-gene approach focused on inulin and sucrose synthesis and degradation genes, all members of the family 32 of glycoside hydrolases (GH32). Polymorphism in these genes was first investigated by carrying out EcoTILLING on two groups of chicory breeding lines exhibiting contrasted response to post-harvest inulin depolymerization. This allowed the identification of polymorphisms significantly associated with depolymerization in three fructan exohydrolase genes (FEH). This association was confirmed on a wider panel of 116 unrelated families in which the FEH polymorphism explained 35 % of the post-harvest variance for inulin content, 36 % of variance for sucrose content, 18 % for inulin degree of polymerization, 23 % for free fructose content and 22 % for free glucose content. These polymorphisms were associated with significant post-harvest changes of inulin content, inulin chain length and free sugars content.

Chitosan oligosaccharides modulate the supramolecular conformation and the biological activity of oligogalacturonides in Arabidopsis.

Plant cell walls undergo remodeling during growth and development and are the first target of many invading pathogens. Acidic pectin (homogalacturonans) binds calcium and forms chain dimers called egg boxes and even multimers at higher calcium ion concentrations. Chitosan, the deacetylated form of chitin produced by fungi when invading plant tissues, is a cationic polymer that can interact with negatively charged pectin. The interaction between chitosan oligomers (COS) and pectic egg boxes was investigated using 2F4, a monoclonal antibody specific for calcium-associated dimers of pectin. Depending on the size of the pectic molecules, the COS to pectin ratio, the degree of polymerization and the degree of acetylation of COS in the mixture, the calcium-induced egg box conformation of oligogalacturonides (OGA) was strongly stabilized or destroyed. The biological activity of COS-stabilized egg boxes was assayed on Arabidopsis cell suspensions. COS-OGA egg boxes strongly enhanced extracellular alkalinization and decreased potassium fluxes compared to control COS and OGA alone. Furthermore, OGA rescued Arabidopsis from cell death induced by higher concentrations of deacetylated COS. The stabilized COS-OGA egg boxes could constitute a combined emergency signal that informs plant cells on both cell wall degradation and pathogen presence, triggering a much stronger response than individual components alone.

Insight into cellular response of plant cells confined within silica-based matrices.

The encapsulation of living plant cells into materials could offer the possibility to develop new green biochemical technologies. With the view to designing new functional materials, the physiological activity and cellular response of entrapped cells within different silica-based matrices have been assessed. A fine-tuning of the surface chemistry of the matrix has been achieved by the in situ copolymerization of an aqueous silica precursor and a biocompatible trifunctional silane bearing covalently bound neutral sugars. This method allows a facile control of chemical and physical interactions between the entrapped plant cells and the scaffold. The results show that the cell-matrix interaction has to be carefully controlled in order to avoid the mineralization of the cell wall which typically reduces the bioavailability of nutrients. Under appropriate conditions, the introduction of a trifunctional silane (ca. 10%) during the preparation of hybrid gels has shown to prolong the biological activity as well as the cellular viability of plant cells. The relations of cell behavior with some other key factors such as the porosity and the contraction of the matrix are also discussed.

Chloroplast Electron Chain, ROS Production, and Redox Homeostasis Are Modulated by COS-OGA Elicitation in Tomato (Solanum lycopersicum) Leaves.

The stimulation of plant innate immunity by elicitors is an emerging technique in agriculture that contributes more and more to residue-free crop protection. Here, we used RNA-sequencing to study gene transcription in tomato leaves treated three times with the chitooligosaccharides-oligogalacturonides (COS-OGA) elicitor FytoSave® that induces plants to fend off against biotrophic pathogens. Results showed a clear upregulation of sequences that code for chloroplast proteins of the electron transport chain, especially Photosystem I (PSI) and ferredoxin. Concomitantly, stomatal conductance decreased by half, reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] content and reactive oxygen species production doubled, but fresh and dry weights were unaffected. Chlorophyll, ß-carotene, violaxanthin, and neoxanthin contents decreased consistently upon repeated elicitations. Fluorescence measurements indicated a transient decrease of the effective PSII quantum yield and a non-photochemical quenching increase but only after the first spraying. Taken together, this suggests that plant defense induction by COS-OGA induces a long-term acclimation mechanism and increases the role of the electron transport chain of the chloroplast to supply electrons needed to mount defenses targeted to the apoplast without compromising biomass accumulation.

Construction of 12 EST libraries and characterization of a 12,226 EST dataset for chicory (Cichorium intybus) root, leaves and nodules in the context of carbohydrate metabolism investigation.

BACKGROUND: The industrial chicory, Cichorium intybus, is a member of the Asteraceae family that accumulates fructan of the inulin type in its root. Inulin is a low calories sweetener, a texture agent and a health promoting ingredient due to its prebiotic properties. Average inulin chain length is a critical parameter that is genotype and temperature dependent. In the context of the study of carbohydrate metabolism and to get insight into the transcriptome of chicory root and to visualize temporal changes of gene expression during the growing season, we obtained and characterized 10 cDNA libraries from chicory roots regularly sampled in field during a growing season. A leaf and a nodule libraries were also obtained for comparison. RESULTS: Approximately 1,000 Expressed Sequence Tags (EST) were obtained from each of twelve cDNA libraries resulting in a 12,226 EST dataset. Clustering of these ESTs returned 1,922 contigs and 4,869 singlets for a total of 6,791 putative unigenes. All ESTs were compared to public sequence databases and functionally classified. Data were specifically searched for sequences related to carbohydrate metabolism. Season wide evolution of functional classes was evaluated by comparing libraries at the level of functional categories and unigenes distribution. CONCLUSION: This chicory EST dataset provides a season wide outlook of the genes expressed in the root and to a minor extent in leaves and nodules. The dataset contains more than 200 sequences related to carbohydrate metabolism and 3,500 new ESTs when compared to other recently released chicory EST datasets, probably because of the season wide coverage of the root samples. We believe that these sequences will contribute to accelerate research and breeding of the industrial chicory as well as of closely related species.

Characterisation of two cold induced dehydrin genes from Cichorium intybus L.

Two dehydrin genes were identified from a Cichorium intybus EST database. They were among the most abundant sequences obtained from 10 cDNA libraries constructed from chicory roots grown under field conditions. The full length cDNA sequences, designated CiDHN1 and CiDHN2, were 1,176 and 1,055 bp long and encoded predicted polypeptides of 262 and 261 amino acids, respectively. The deduced CiDHN1 protein contains a S-segment and four lysine-rich consensus motifs (K-segments) which represent a typical SK(4) structure of dehydrins. The CiDHN2 sequence contains two Y motifs and two K-segments classifying CiDHN2 as Y(2)K(2)-type dehydrin. Southern-blotting analysis suggested that CiDHN1 and CiDHN2 are single copy genes. Northern-blotting analysis revealed that both CiDHN genes are expressed in roots and leaves, with seasonal variations in transcript accumulation. The effect of cold on the CiDHN1 and CiDHN2 transcript level was demonstrated. CiDHN1 and CiDHN2 promoter analysis revealed the presence of low temperature-responsive and ABA-responsive elements.

Systemic defense activation by COS-OGA in rice against root-knot nematodes depends on stimulation of the phenylpropanoid pathway.

Activation of induced plant resistance to control pests and diseases is regaining attention in the current climate where chemical pesticides are being progressively banned. Formulations of chitosan oligomers (COS) and pectin-derived oligogalacturonides (OGA), COS-OGA, have previously been described to induce resistance against fungal diseases in different crop plants. Here, we investigated their potential and mode-of-action as preventive measures to control root-knot nematode Meloidogyne graminicola infection in rice. The results show a significant reduction in root-galling and nematode development in rice plants that were treated through foliar application with the COS-OGA formulations FytoSol® and FytoSave® 24 h before nematode inoculation. Hormone measurements, gene expression analyses, corroborated by treatments on salicylic acid (SA) and jasmonic acid (JA)-mutants indicated that the systemic COS-OGA induced defense mechanism against nematodes is not based on SA or JA activation. However, phenylalanine ammonia lyase (PAL) gene expression in roots as well as enzymatic PAL activity in the shoots were significantly induced 24 h after foliar COS-OGA spraying in comparison with untreated plants. COS-OGA-induced systemic defense was abolished in the rice OsPAL4-mutant, demonstrating that COS-OGA-induced defense is dependent on OsPAL4 activation in rice plants.

Egg box conformation of oligogalacturonides: the time-dependent stabilization of the elicitor-active conformation increases its biological activity.

Circular dichroism spectrometry was used on oligogalacturonides (OGAs) and showed the existence of a calcium/sodium-induced conformational state that is intermediate between single-isolated chains and calcium-associated multimer chains. This conformation is interpreted as being egg box dimers. Using the 2F4 monoclonal antibody that specifically binds such an egg box dimer conformation of pectin, the stability of OGA dimers was investigated over a period of 24 hours. The extent to which egg box dimers were recognized by the antibody was dependent on the temperature and duration of preincubation of the OGA. This suggests a "maturation" process of the egg-box structure that consists in a progressive increase in the length of the junction sequences between two chains that slide along each other in order to form a maximum number of calcium bridges and dimer ends. The maturation of egg boxes induced both a significant increase in their binding to wall-associated kinase 1 (WAK1) and an increased extracellular alkalinization when applied to Arabidopsis thaliana cell suspensions. The chemical modification of the reducing end of the OGAs largely diminished their elicitating activity but did not hinder either dimerization or binding of these end-reduced egg boxes to WAK1. We conclude that there are at least two different perception systems for egg box dimers. One binds egg box junctions and the other binds egg box ends. The relevance of these results is discussed in terms of pectic signal perception and plant-pathogen interaction.

In vitro characterization of the homogalacturonan-binding domain of the wall-associated kinase WAK1 using site-directed mutagenesis.

Wall-associated kinase 1--WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell wall in Arabidopsis thaliana (L.) HEYNH. In a previous paper [Decreux, A., Messiaen, J., 2005. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol. 46, 268-278], we showed that a recombinant peptide expressed in yeast corresponding to amino acids 67-254 of the extracellular domain of WAK1 specifically interacts with commercial non-methylesterified homogalacturonic acid, purified homogalacturonans from Arabidopsis and oligogalacturonides in a calcium-induced conformation. In this report, we used a receptor binding domain sequence-based prediction method to identify four putative binding sites in the extracellular domain of WAK1, in which cationic amino acids were selected for substitution by site-directed mutagenesis. Interaction studies between mutated forms of WAK1 and homogalacturonans allowed us to identify and confirm at least five specific amino acids involved in the interaction with homogalacturonan dimers and multimers. The presence of this homogalacturonan-binding domain within the extracellular domain of WAK1 is discussed in terms of cell wall architecture and signal transduction.

Plant immunity induced by COS-OGA elicitor is a cumulative process that involves salicylic acid.

Plant innate immunity offers considerable opportunities for plant protection but beside flagellin and chitin, not many molecules and their receptors have been extensively characterized and very few have successfully reached the field. COS-OGA, an elicitor that combines cationic chitosan oligomers (COS) with anionic pectin oligomers (OGA), efficiently protected tomato (Solanum lycopersicum) grown in greenhouse against powdery mildew (Leveillula taurica). Leaf proteomic analysis of plants sprayed with COS-OGA showed accumulation of Pathogenesis-Related proteins (PR), especially subtilisin-like proteases. qRT-PCR confirmed upregulation of PR-proteins and salicylic acid (SA)-related genes while expression of jasmonic acid/ethylene-associated genes was not modified. SA concentration and class III peroxidase activity were increased in leaves and appeared to be a cumulative process dependent on the number of sprayings with the elicitor. These results suggest a systemic acquired resistance (SAR) mechanism of action of the COS-OGA elicitor and highlight the importance of repeated applications to ensure efficient protection against disease.

Identification of a cytotoxic molecule in heat-modified citrus pectin.

Modified forms of citrus pectin possess anticancer properties. However, their mechanism of action and the structural features involved remain unclear. Here, we showed that citrus pectin modified by heat treatment displayed cytotoxic effects in cancer cells. A fractionation approach was used aiming to identify active molecules. Dialysis and ethanol precipitation followed by HPLC analysis evidenced that most of the activity was related to molecules with molecular weight corresponding to low degree of polymerization oligogalacturonic acid. Heat-treatment of galacturonic acid also generated cytotoxic molecules. Furthermore, heat-modified galacturonic acid and heat-fragmented pectin contained the same molecule that induced cell death when isolated by HPLC separation. Mass spectrometry analyses revealed that 4,5-dihydroxy-2-cyclopenten-1-one was one cytotoxic molecule present in heat-treated pectin. Finally, we synthesized the enantiopure (4R,5R)-4,5-dihydroxy-2-cyclopenten-1-one and demonstrated that this molecule was cytotoxic and induced a similar pattern of apoptotic-like features than heat-modified pectin.

Loss of function of 1-FEH IIb has more impact on post-harvest inulin degradation in Cichorium intybus than copy number variation of its close paralog 1-FEH IIa.

Key Message: The loss of mini-exon 2 in the 1-FEH IIb glycosyl-hydrolase results in a putative non-functional allele. This loss of function has a strong impact on the susceptibility to post-harvest inulin depolymerization. Significant variation of copy number was identified in its close paralog 1-FEH IIa, but no quantitative effect of copy number on carbohydrates-related phenotypes was detected. Inulin polyfructan is the second most abundant storage carbohydrate in flowering plants. After harvest, it is depolymerized by fructan exohydrolases (FEHs) as an adaptive response to end-season cold temperatures. In chicory, the intensity of this depolymerization differs between cultivars but also between individuals within a cultivar. Regarding this phenotypic variability, we recently identified statistically significant associations between inulin degradation and genetic polymorphisms located in three FEHs. We present here new results of a systematic analysis of copy number variation (CNV) in five key members of the chicory (Cichorium intybus) GH32 multigenic family, including three FEH genes and the two inulin biosynthesis genes: 1-SST and 1-FFT. qPCR analysis identified a significant variability of relative copy number only in the 1-FEH IIa gene. However, this CNV had no quantitative effect. Instead, cloning of the full length gDNA of a close paralogous sequence (1-FEH IIb) identified a 1028 bp deletion in lines less susceptible to post-harvest inulin depolymerization. This region comprises a 9 bp mini-exon containing one of the three conserved residues of the active site. This results in a putative non-functional 1-FEH IIb allele and an observed lower inulin depolymerization. Extensive genotyping confirmed that the loss of mini-exon 2 in 1-FEH IIb and the previously identified 47 bp duplication located in the 3'UTR of 1-FEH IIa belong to a single haplotype, both being statistically associated with reduced susceptibility to post-harvest inulin depolymerization. Emergence of these haplotypes is discussed.

Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

Negative purification method for the selection of specific antibodies from polyclonal antisera.

We developed a protocol to remove non-specific antibodies from polyclonal antisera by adsorption on non-target antigens immobilized on nitrocellulose membranes. This "negative" purification method is simple and provides better immunoreagents than the blocking of nonspecific antibodies in solution or the enrichment of specific antibodies on nitrocellulose membranes. For routine applications, this method is quicker and cheaper than the purification protocols based on selective precipitations and affinity chromatography.

Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA.

H2O2 acts as an important signaling molecule during plant/pathogen interactions but its study remains a challenge due to the current shortcomings in H2O2-responsive probes. In this work, ContPY1, a new molecular probe developed to specifically detect H2O2 was used to study the elicitation of Arabidopsis thaliana cells by a complex of chitosan oligomers (COS) and oligogalacturonides (OGA). The comparison of cell suspensions, protoplasts of cell suspensions and leaf protoplasts treated with different inhibitors gave indications on the potential sources of hydrogen peroxide in plant cells. The relative contribution of the cell wall, of membrane dehydrogenases and of peroxidases depended on cell type and treatment and proved to be variable. Our present protocol can be used to study hydrogen peroxide production in a large variety of plant species by simple protocol adaptation.

Development and characterization of microsatellite loci for the Moroccan endemic endangered species Argania spinosa (Sapotaceae).

PREMISE OF THE STUDY: Microsatellite loci were developed for the Moroccan endemic endangered species Argania spinosa with a combination of a typical library enrichment procedure and a 454 GS FLX Titanium-based high-throughput sequencing approach. • METHODS AND RESULTS: A genomic DNA library was enriched and further screened using (GA)15, (GTA)8, and (TTC)8 biotin-labeled probes coupled with chemi-luminescence detection. To increase simple sequence repeat (SSR) loci number, an ultra-high-throughput sequencing-based approach was used. Evaluation of all primer pairs was performed with labeled dUTP on an ABI 3130xl sequencer. Eleven polymorphic SSR loci were selected out of 79 SSR regions and extensively characterized on 150 individuals from eight populations. Total alleles ranged from six to 19 alleles per locus while expected heterozygosity ranged from 0.618 to 0.869. • CONCLUSIONS: The SSRs developed here will be used to further characterize the genetic diversity of A. spinosa across its distribution range, mainly in the southern part of Morocco and southwestern Algeria. They may also be transferable to other Sapotaceae species.

Validation of the boronate sensor ContPY1 as a specific probe for fluorescent detection of hydrogen peroxide in plants.

Studying the implication of hydrogen peroxide in biological processes in plants remains a challenge due to the current shortcomings of H2O2-responsive probes. The use of ContPY1, a new fluorescent probe, which is highly selective and sensitive for H2O2, was investigated. To validate the use of ContPY1 on plants, we have generated protocols employing cells suspensions and leaves, and measured specifically H2O2 production by plants using spectrofluorometry and microscopy.