Biological and Chemical Characterization of Musa paradisiaca Leachate.

There is a growing demand for molecules of natural origin for biocontrol and biostimulation, given the current trend away from synthetic chemical products. Leachates extracted from plantain stems were obtained after biodegradation of the plant material. To characterize the leachate, quantitative determinations of nitrogen, carbon, phosphorus, and cations (K+, Ca2+, Mg2+, Na+), Q2/4, Q2/6, and Q4/6 absorbance ratios, and metabolomic analysis were carried out. The potential role of plantain leachates as fungicide, elicitor of plant defense, and/or plant biostimulant was evaluated by agar well diffusion method, phenotypic, molecular, and imaging approaches. The plant extracts induced a slight inhibition of fungal growth of an aggressive strain of Colletotrichum gloeosporioides, which causes anthracnose. Organic compounds such as cinnamic, ellagic, quinic, and fulvic acids and indole alkaloid such as ellipticine, along with some minerals such as potassium, calcium, and phosphorus, may be responsible for the inhibition of fungal growth. In addition, jasmonic, benzoic, and salicylic acids, which are known to play a role in plant defense and as biostimulants in tomato, were detected in leachate extract. Indeed, foliar application of banana leachate induced overexpression of LOXD, PPOD, and Worky70-80 genes, which are involved in phenylpropanoid metabolism, jasmonic acid biosynthesis, and salicylic acid metabolism, respectively. Leachate also activated root growth in tomato seedlings. However, the main impact of the leachate was observed on mature plants, where it caused a reduction in leaf area and fresh weight, the remodeling of stem cell wall glycopolymers, and an increase in the expression of proline dehydrogenase.

LAM2: An Unusual Laminaran Structure for a Novel Plant Elicitor Candidate.

Laminarans are of interest because they have been shown to induce various immune responses in animals and plants. These ß-D-glucans differ from each other by their branching rate, which is possibly responsible for their biological activities. In the present study, we characterized a laminaran fraction extracted from Laminaria hyperborea and named LAM2 using sugar composition and structural analyses (NMR). Then, we evaluated its activity as a potential plant elicitor in vitro on tomato seedlings using gene expression analysis and cell wall immunofluorescence labeling. Our study showed that LAM2 isolated from L. hyperborea is a succinylated laminaran which significantly enhanced the plant defense of tomato seedlings and induced cell wall modifications, suggesting a higher elicitor activity than the laminaran standard extracted from Laminaria digitata.

Black Poplar (Populus nigra L.) Root Extracellular Trap, Structural and Molecular Remodeling in Response to Osmotic Stress.

The root extracellular trap (RET) consists of root-associated, cap-derived cells (root AC-DCs) and their mucilaginous secretions, and forms a structure around the root tip that protects against biotic and abiotic stresses. However, there is little information concerning the changes undergone by the RET during droughts, especially for tree species. Morphological and immunocytochemical approaches were used to study the RET of black poplar (Populus nigra L.) seedlings grown in vitro under optimal conditions (on agar-gelled medium) or when polyethylene glycol-mediated (PEG6000-infused agar-gelled medium) was used to mimic drought conditions through osmotic stress. Under optimal conditions, the root cap released three populations of individual AC-DC morphotypes, with a very low proportion of spherical morphotypes, and equivalent proportions of intermediate and elongated morphotypes. Immunolabeling experiments using anti-glycan antibodies specific to cell wall polysaccharide and arabinogalactan protein (AGP) epitopes revealed the presence of homogalacturonan (HG), galactan chains of rhamnogalacturonan-I (RG-I), and AGPs in root AC-DC cell walls. The data also showed the presence of xylogalacturonan (XGA), xylan, AGPs, and low levels of arabinans in the mucilage. The findings also showed that under osmotic stress conditions, both the number of AC-DCs (spherical and intermediate morphotypes) and the total quantity of mucilage per root tip increased, whereas the mucilage was devoid of the epitopes associated with the polysaccharides RG-I, XGA, xylan, and AGPs. Osmotic stress also led to reduced root growth and increased root expression of the P5CS2 gene, which is involved in proline biosynthesis and cellular osmolarity maintenance (or preservation) in aerial parts. Together, our findings show that the RET is a dynamic structure that undergoes pronounced structural and molecular remodeling, which might contribute to the survival of the root tip under osmotic conditions.

Elicitation of Roots and AC-DC with PEP-13 Peptide Shows Differential Defense Responses in Multi-Omics.

The root extracellular trap (RET) has emerged as a specialized compartment consisting of root AC-DC and mucilage. However, the RET's contribution to plant defense is still poorly understood. While the roles of polysaccharides and glycoproteins secreted by root AC-DC have started to be elucidated, how the low-molecular-weight exudates of the RET contribute to root defense is poorly known. In order to better understand the RET and its defense response, the transcriptomes, proteomes and metabolomes of roots, root AC-DC and mucilage of soybean (Glycine max (L.) Merr, var. Castetis) upon elicitation with the peptide PEP-13 were investigated. This peptide is derived from the pathogenic oomycete Phytophthora sojae. In this study, the root and the RET responses to elicitation were dissected and sequenced using transcriptional, proteomic and metabolomic approaches. The major finding is increased synthesis and secretion of specialized metabolites upon induced defense activation following PEP-13 peptide elicitation. This study provides novel findings related to the pivotal role of the root extracellular trap in root defense.

New Insights into Plant Extracellular DNA. A Study in Soybean Root Extracellular Trap.

exDNA is found in various organisms, including plants. However, plant exDNA has thus far received little attention related to its origin and role in the RET (root extracellular trap). In this study, we performed the first high-throughput genomic sequencing of plant exDNA from a Fabaceae with worldwide interest: soybean (Glycine max (L.) Merr.). The origin of this exDNA was first investigated in control condition, and the results show high-coverage on organelles (mitochondria/plastid) DNA relative to nuclear DNA, as well as a mix of coding and non-coding sequences. In the second part of this study, we investigated if exDNA release was modified during an elicitation with PEP-13 (a peptide elicitor from oomycete genus Phytophthora). Our results show that treatment of roots with PEP-13 does not affect the composition of exDNA.

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls.

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil-borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo-gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.

Identification of two compounds able to improve flax resistance towards Fusarium oxysporum infection.

Plants are surrounded by a diverse range of microorganisms that causes serious crop losses and requires the use of pesticides. Flax is a major crop in Normandy used for its fibres and is regularly challenged by the pathogenic fungus Fusarium oxysporum (Fo) f. sp. lini. To protect themselves, plants use "innate immunity" as a first line of defense level against pathogens. Activation of plant defense with elicitors could be an alternative for crop plant protection. A previous work was conducted by screening a chemical library and led to the identification of compounds able to activate defense responses in Arabidopsis thaliana. Four compounds were tested for their abilities to improve resistance of two flax varieties against Fo. Two of them, one natural (holaphyllamine or HPA) and one synthetic (M4), neither affected flax nor Fo growth. HPA and M4 induced oxidative burst and callose deposition. Furthermore, HPA and M4 caused changes in the expression patterns of defense-related genes coding a glucanase and a chitinase-like. Finally, plants pre-treated with HPA or M4 exhibited a significant decrease in the disease symptoms. Together, these findings demonstrate that HPA and M4 are able to activate defense responses in flax and improve its resistance against Fo infection.

A Novel In Vitro Tool to Study Cyst Nematode Chemotaxis.

This study presents a novel three-dimensional (3D) tool "3D in vitro choice" for chemotaxis assays with cyst nematodes. The original 3D in vitro choice was customized through digital printing. Freshly hatched second stage juveniles (J2s) of the cyst nematode Globodera pallida were used as the nematode model to illustrate chemo-orientation behavior in the 3D system. The efficiency and reliability of the 3D in vitro choice were validated with 2% Phytagel as navigation medium, in three biological assays and using tomato root exudates or potato root border cells and their associated mucilage as a positive attractant as compared with water. For each biological assay, J2s were hatched from the same population of a single generation glasshouse-cultured cysts. This novel easy to use and low-cost 3d-device could be a useful replacement to Petri dishes assays in nematode behavioral studies due to the ease of deposition of nematodes and test substances, coupled with its distinctive zones that allow for precision in choice making by the nematodes.

Multiple xylosyltransferases heterogeneously xylosylate protein N-linked glycans in Chlamydomonas reinhardtii.

Nowadays, little information is available regarding the N-glycosylation pathway in the green microalga Chlamydomonas reinhardtii. Recent investigation demonstrated that C. reinhardtii synthesizes linear oligomannosides. Maturation of these oligomannosides results in N-glycans that are partially methylated and carry one or two xylose residues. One xylose residue was demonstrated to be a core ß(1,2)-xylose. Recently, N-glycoproteomic analysis performed on glycoproteins secreted by C. reinhardtii demonstrated that the xylosyltransferase A (XTA) was responsible for the addition of the core ß(1,2)-xylose. Furthermore, another xylosyltransferase candidate named XTB was suggested to be involved in the xylosylation in C. reinhardtii. In the present study, we focus especially on the characterization of the structures of the xylosylated N-glycans from C. reinhardtii taking advantage of insertional mutants of XTA and XTB, and of the XTA/XTB double-mutant. The combination of mass spectrometry approaches allowed us to identify the major N-glycan structures bearing one or two xylose residues. They confirm that XTA is responsible for the addition of the core ß(1,2)-xylose, whereas XTB is involved in the addition of the xylose residue onto the linear branch of the N-glycan as well as in the partial addition of the core ß(1,2)-xylose suggesting that this transferase exhibits a low substrate specificity. Analysis of the double-mutant suggests that an additional xylosyltransferase is involved in the xylosylation process in C. reinhardtii. Additional putative candidates have been identified in the C. reinhardtii genome. Altogether, these results pave the way for a better understanding of the C. reinhardtii N-glycosylation pathway.

Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum.

Although Phaeodactylum tricornutum is gaining importance in plant molecular farming for the production of high-value molecules such as monoclonal antibodies, little is currently known about key cell metabolism occurring in this diatom such as protein glycosylation. For example, incorporation of fucose residues in the glycans N-linked to protein in P. tricornutum is questionable. Indeed, such epitope has previously been found on N-glycans of endogenous glycoproteins in P. tricornutum. Meanwhile, the potential immunogenicity of the α(1,3)-fucose epitope present on plant-derived biopharmaceuticals is still a matter of debate. In this paper, we have studied molecular actors potentially involved in the fucosylation of the glycoproteins in P. tricornutum. Based on sequence similarities, we have identified a putative P. tricornutum GDP-L-fucose transporter and three fucosyltransferase (FuT) candidates. The putative P. tricornutum GDP-L-fucose transporter coding sequence was expressed in the Chinese Hamster Ovary (CHO)-gmt5 mutant lacking its endogenous GDP-L-fucose transporter activity. We show that the P. tricornutum transporter is able to rescue the fucosylation of proteins in this CHO-gmt5 mutant cell line, thus demonstrating the functional activity of the diatom transporter and its appropriate Golgi localization. In addition, we overexpressed one of the three FuT candidates, namely the FuT54599, in P. tricornutum and investigated its localization within Golgi stacks of the diatom. Our findings show that overexpression of the FuT54599 leads to a significant increase of the α(1,3)-fucosylation of the diatom endogenous glycoproteins.

Comparative in depth RNA sequencing of P. tricornutum's morphotypes reveals specific features of the oval morphotype.

Phaeodactylum tricornutum is the most studied diatom encountered principally in coastal unstable environments. It has been hypothesized that the great adaptability of P. tricornutum is probably due to its pleomorphism. Indeed, P. tricornutum is an atypical diatom since it can display three morphotypes: fusiform, triradiate and oval. Currently, little information is available regarding the physiological significance of this morphogenesis. In this study, we adapted P. tricornutum Pt3 strain to obtain algal culture particularly enriched in one dominant morphotype: fusiform, triradiate or oval. These cultures were used to run high-throughput RNA-Sequencing. The whole mRNA transcriptome of each morphotype was determined. Pairwise comparisons highlighted biological processes and molecular functions which are up- and down-regulated. Finally, intersection analysis allowed us to identify the specific features from the oval morphotype which is of particular interest as it is often described to be more resistant to stresses. This study represent the first transcriptome wide characterization of the three morphotypes from P. tricornutum performed on cultures specifically enriched issued from the same Pt3 strain. This work represents an important step for the understanding of the morphogenesis in P. tricornutum and highlights the particular features of the oval morphotype.

Heterologous expression of the N-acetylglucosaminyltransferase I dictates a reinvestigation of the N-glycosylation pathway in Chlamydomonas reinhardtii.

Eukaryotic N-glycosylation pathways are dependent of N-acetylglucosaminyltransferase I (GnTI), a key glycosyltransferase opening the door to the formation of complex-type N-glycans by transferring a N-acetylglucosamine residue onto the Man5GlcNAc2 intermediate. In contrast, glycans N-linked to Chlamydomonas reinhardtii proteins arise from a GnTI-independent Golgi processing of oligomannosides giving rise to Man5GlcNAc2 substituted eventually with one or two xylose(s). Here, complementation of C. reinhardtii with heterologous GnTI was investigated by expression of GnTI cDNAs originated from Arabidopsis and the diatom Phaeodactylum tricornutum. No modification of the N-glycans was observed in the GnTI transformed cells. Consequently, the structure of the Man5GlcNAc2 synthesized by C. reinhardtii was reinvestigated. Mass spectrometry analyses combined with enzyme sequencing showed that C. reinhardtii proteins carry linear Man5GlcNAc2 instead of the branched structure usually found in eukaryotes. Moreover, characterization of the lipid-linked oligosaccharide precursor demonstrated that C. reinhardtii exhibit a Glc3Man5GlcNAc2 dolichol pyrophosphate precursor. We propose that this precursor is then trimmed into a linear Man5GlcNAc2 that is not substrate for GnTI. Furthermore, cells expressing GnTI exhibited an altered phenotype with large vacuoles, increase of ROS production and accumulation of starch granules, suggesting the activation of stress responses likely due to the perturbation of the Golgi apparatus.

Root exudate of Solanum tuberosum is enriched in galactose-containing molecules and impacts the growth of Pectobacterium atrosepticum.

Background and aims Potato (Solanum tuberosum) is an important food crop and is grown worldwide. It is, however, significantly sensitive to a number of soil-borne pathogens that affect roots and tubers, causing considerable economic losses. So far, most research on potato has been dedicated to tubers and hence little attention has been paid to root structure and function. Methods In the present study we characterized root border cells using histochemical staining, immunofluorescence labelling of cell wall polysaccharides epitopes and observation using laser confocal microscopy. The monosaccharide composition of the secreted exudates was determined by gas chromatography of trimethylsilyl methylglycoside derivatives. The effects of root exudates and secreted arabinogalactan proteins on bacterial growth were investigated using in vitro bioassays. Key Results Root exudate from S. tuberosum was highly enriched in galactose-containing molecules including arabinogalactan proteins as major components. Treatment of the root with an elicitor derived from Pectobacterium atrosepticum, a soil-borne pathogen of potato, altered the composition of the exudates and arabinogalactan proteins. We found that the growth of the bacterium in vitro was differentially affected by exudates from elicited and non-elicited roots (i.e. inhibition versus stimulation). Conclusions Taken together, these findings indicate that galactose-containing polymers of potato root exudates play a central role in root-microbe interactions.

Determination of Multimodal Isotopic Distributions: The Case of a (15)N Labeled Protein Produced into Hairy Roots.

Isotopic labeling is widely used in various fields like proteomics, metabolomics, fluxomics, as well as in NMR structural studies, but it requires an efficient determination of the isotopic enrichment. Mass spectrometry is the method of choice for such analysis. However, when complex expression systems like hairy roots are used for production, multiple populations of labeled proteins may be obtained. If the isotopic incorporation determination is actually well-known for unimodal distributions, the multimodal distributions have scarcely been investigated. Actually, only a few approaches allow the determination of the different labeled population proportions from multimodal distributions. Furthermore, they cannot be used when the number of the populations and their respective isotope ratios are unknown. The present study implements a new strategy to measure the (15)N labeled populations inside a multimodal distribution knowing only the peptide sequence and peak intensities from mass spectrometry analyses. Noteworthy, it could be applied to other elements, like carbon and hydrogen, and extended to a larger range of biomolecules.

Plant-specific glycosylation patterns in the context of therapeutic protein production.

While N-glycan synthesis in the endoplasmic reticulum (ER) is relatively well conserved in eukaryotes, N-glycan processing and O-glycan biosynthesis in the Golgi apparatus are kingdom specific and result in different oligosaccharide structures attached to glycoproteins in plants and mammals. With the prospect of using plants as alternative hosts to mammalian cell lines for the production of therapeutic glycoproteins, significant progress has been made towards the humanization of protein N-glycosylation in plant cells. To date, successful efforts in this direction have mainly focused on the targeted expression of therapeutic proteins, the knockout of plant-specific N-glycan-processing genes, and/or the introduction of the enzymatic machinery catalyzing the synthesis, transport and addition of human sugars. By contrast, very little attention has been paid until now to the O-glycosylation status of plant-made therapeutic proteins, which is surprising considering that hundreds of human proteins represent good candidates for Hyp-O glycosylation when produced in a plant expression system. This review describes protein N- and O-linked glycosylation in plants and highlights the limitations and advantages of plant-specific glycosylation on plant-made biopharmaceuticals.

Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER.

BACKGROUND: In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails. RESULTS: Here, using Arabidopsis glucosidase I as a model, we have identified two types of signals sufficient for the location of a type II membrane protein in the ER. A first signal is located in the luminal domain, while a second signal corresponds to a short amino acid sequence located in the cytosolic tail of the membrane protein. The cytosolic tail contains at its N-terminal end four arginine residues constitutive of three di-arginine motifs (RR, RXR or RXXR) independently sufficient to confer ER localization. Interestingly, when only one di-arginine motif is present, fusion proteins are located both in the ER and in mobile punctate structures, distinct but close to Golgi bodies. Soluble and membrane ER protein markers are excluded from these punctate structures, which also do not colocalize with an ER-exit-site marker. It is hypothesized they correspond to sites involved in Golgi to ER retrotransport. CONCLUSION: Altogether, these results clearly show that cytosolic and luminal signals responsible for ER retention could coexist in a same type II membrane protein. These data also suggest that both retrieval and retention mechanisms govern protein residency in the ER membrane. We hypothesized that mobile punctate structures not yet described at the ER/Golgi interface and tentatively named GERES, could be involved in retrieval mechanisms from the Golgi to the ER.

Production of recombinant proteins in suspension-cultured plant cells.

Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis thaliana. These systems are powerful tools for plant-made pharmaceuticals production in highly controlled conditions.

N-glycan trimming by glucosidase II is essential for Arabidopsis development.

Glucosidase II, one of the early N-glycan processing enzymes and a major player in the glycoprotein folding quality control, has been described as a soluble heterodimer composed of alpha and beta subunits. Here we present the first characterization of a plant glucosidase II alpha subunit at the molecular level. Expression of the Arabidopsis alpha subunit restored N-glycan maturation capacity in Schizosaccharomyces pombe alpha- or alphabeta-deficient mutants, but with a lower efficiency in the last case. Inactivation of the alpha subunit in a temperature sensitive Arabidopsis mutant blocked N-glycan processing after a first trimming by glucosidase I and strongly affected seedling development.

Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway.

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.