The Spatio-Temporal Distribution of Cell Wall-Associated Glycoproteins During Wood Formation in Populus

Plant cell wall associated hydroxyproline-rich glycoproteins (HRGPs) are involved in several aspects of plant growth and development, including wood formation in trees. HRGPs such as arabinogalactan-proteins (AGPs), extensins (EXTs), and proline rich proteins (PRPs) are important for the development and architecture of plant cell walls. Analysis of publicly available gene expression data revealed that many HRGP encoding genes show tight spatio-temporal expression patterns in the developing wood of Populus that are indicative of specific functions during wood formation. Similar results were obtained for the expression of glycosyl transferases putatively involved in HRGP glycosylation. In situ immunolabelling of transverse wood sections using AGP and EXT antibodies revealed the cell type specificity of different epitopes. In mature wood AGP epitopes were located in xylem ray cell walls, whereas EXT epitopes were specifically observed between neighboring xylem vessels, and on the ray cell side of the vessel walls, likely in association with pits. Molecular mass and glycan analysis of AGPs and EXTs in phloem/cambium, developing xylem, and mature xylem revealed clear differences in glycan structures and size between the tissues. Separation of AGPs by agarose gel electrophoresis and staining with beta-D-glucosyl Yariv confirmed the presence of different AGP populations in phloem/cambium and xylem. These results reveal the diverse changes in HRGP-related processes that occur during wood formation at the gene expression and HRGP glycan biosynthesis levels, and relate HRGPs and glycosylation processes to the developmental processes of wood formation

Rôle des extensines et leur glycosylation dans la défense racinaire

Extensins are cell wall glycoproteins involved in various biological processes including plant protection. However, their mode of action in plant immunity response is not clearly established and remains to be elucidated. Extensins are able to strengthen the cell wall, one of the first cellular barriers against pathogens, through intra- and intermolecular cross-links. This cross-linking is catalysed by specific peroxidase enzymes and requires a correct conformation of extensins conferred by their glycan moiety.This PhD project aimed to investigate the impact of extensin glycosylation in root defence and to characterize, as a preliminary study, the peroxidases potentially involved in the extensin crosslinking in Arabidopsis thaliana. Through immunocytochemistry techniques on mutants impaired with extensin glycosylation, we have revealed that a modulation of extensin distribution occurs in A. thaliana root in response to elicitation with the bacterial peptide, flagellin 22. We have also showed that extensin arabinosylation plays a major role, although probably indirect, in the root colonization by the pathogen oomycete Phytophthora parasitica. We have therefore elaborated a model proposing to illustrate the importance of extensin arabinosylation in the cell wall organization and architecture, modulating pathogen adhesion on root cells and influencing in fine root colonization.Les extensines sont des glycoprotéines pariétales appartenant à la famille des HRGPs (Hydroxy prolin-rich glycoproteins) impliquées dans plusieurs fonctions telles que la croissance, le développement et la défense des plantes contre les pathogènes. Toutefois, leur mode d’action dans la réponse immunitaire végétale n’est pas encore bien connu et reste à élucider. Les extensines interviennent dans le renforcement de la paroi, un des premiers remparts cellulaires contre les pathogènes, en se liant entre elles de manière intra- et intermoléculaire. Ce « cross-linking » est catalysé par des enzymes peroxydases spécifiques et nécessite une correcte conformation des extensines, laquelle est conférée par leur partie glycosylée.Dans ce projet de thèse, nous avons donc entrepris d’étudier l’impact de la glycosylation des extensines sur la défense racinaire et tenté de caractériser, de manière préliminaire, des peroxydases potentiellement impliquées dans le « cross-linking » chez Arabidopsis thaliana. Des techniques d’immunocytochimie réalisées sur une sélection de mutants affectés dans la glycosylation des extensines ont révélé une modulation de la distribution des extensines dans la racine d’A. thaliana en réponse à une élicitation avec un peptide bactérien, la flagelline 22. L’un des résultats majeurs de cette étude a été de montrer l’importance de l’arabinosylation des extensines dans la colonisation de la racine par l’oomycète pathogène Phytophthora parasitica. Ainsi, l’ensemble de ces résultats nous a permis d’élaborer un modèle proposant d’illustrer l’importance de l’arabinosylation des extensines dans l’organisation et l’architecture de la paroi, modulant ainsi l’adhésion du pathogène sur les cellules de la racine et influençant in fine la colonisation de cette dernière

Rôle des extensines et leur glycosylation dans la défense racinaire

Extensins are cell wall glycoproteins involved in various biological processes including plant protection. However, their mode of action in plant immunity response is not clearly established and remains to be elucidated. Extensins are able to strengthen the cell wall, one of the first cellular barriers against pathogens, through intra- and intermolecular cross-links. This cross-linking is catalysed by specific peroxidase enzymes and requires a correct conformation of extensins conferred by their glycan moiety.This PhD project aimed to investigate the impact of extensin glycosylation in root defence and to characterize, as a preliminary study, the peroxidases potentially involved in the extensin crosslinking in Arabidopsis thaliana. Through immunocytochemistry techniques on mutants impaired with extensin glycosylation, we have revealed that a modulation of extensin distribution occurs in A. thaliana root in response to elicitation with the bacterial peptide, flagellin 22. We have also showed that extensin arabinosylation plays a major role, although probably indirect, in the root colonization by the pathogen oomycete Phytophthora parasitica. We have therefore elaborated a model proposing to illustrate the importance of extensin arabinosylation in the cell wall organization and architecture, modulating pathogen adhesion on root cells and influencing in fine root colonization.Les extensines sont des glycoprotéines pariétales appartenant à la famille des HRGPs (Hydroxy prolin-rich glycoproteins) impliquées dans plusieurs fonctions telles que la croissance, le développement et la défense des plantes contre les pathogènes. Toutefois, leur mode d’action dans la réponse immunitaire végétale n’est pas encore bien connu et reste à élucider. Les extensines interviennent dans le renforcement de la paroi, un des premiers remparts cellulaires contre les pathogènes, en se liant entre elles de manière intra- et intermoléculaire. Ce « cross-linking » est catalysé par des enzymes peroxydases spécifiques et nécessite une correcte conformation des extensines, laquelle est conférée par leur partie glycosylée.Dans ce projet de thèse, nous avons donc entrepris d’étudier l’impact de la glycosylation des extensines sur la défense racinaire et tenté de caractériser, de manière préliminaire, des peroxydases potentiellement impliquées dans le « cross-linking » chez Arabidopsis thaliana. Des techniques d’immunocytochimie réalisées sur une sélection de mutants affectés dans la glycosylation des extensines ont révélé une modulation de la distribution des extensines dans la racine d’A. thaliana en réponse à une élicitation avec un peptide bactérien, la flagelline 22. L’un des résultats majeurs de cette étude a été de montrer l’importance de l’arabinosylation des extensines dans la colonisation de la racine par l’oomycète pathogène Phytophthora parasitica. Ainsi, l’ensemble de ces résultats nous a permis d’élaborer un modèle proposant d’illustrer l’importance de l’arabinosylation des extensines dans l’organisation et l’architecture de la paroi, modulant ainsi l’adhésion du pathogène sur les cellules de la racine et influençant in fine la colonisation de cette dernière

Role of extensins and their glycosylation in root defence

Les extensines sont des glycoprotéines pariétales appartenant à la famille des HRGPs (Hydroxyprolin-rich glycoproteins) impliquées dans plusieurs fonctions telles que la croissance, le développement et la défense des plantes contre les pathogènes. Toutefois, leur mode d’action dans la réponse immunitaire végétale n’est pas encore bien connu et reste à élucider. Les extensines interviennent dans le renforcement de la paroi, un des premiers remparts cellulaires contre les pathogènes, en se liant entre elles de manière intra- et intermoléculaire. Ce « cross-linking » est catalysé par des enzymes peroxydases spécifiques et nécessite une correcte conformation des extensines, laquelle est conférée par leur partie glycosylée. Dans ce projet de thèse, nous avons donc entrepris d’étudier l’impact de la glycosylation des extensines sur la défense racinaire et tenté de caractériser, de manière préliminaire, des peroxydases potentiellement impliquées dans le « cross-linking » chez Arabidopsis thaliana. Des techniques d’immunocytochimie réalisées sur une sélection de mutants affectés dans la glycosylation des extensines ont révélé une modulation de la distribution des extensines dans la racine d’A. thaliana en réponse à une élicitation avec un peptide bactérien, la flagelline 22. L’un des résultats majeurs de cette étude a été de montrer l’importance de l’arabinosylation des extensines dans la colonisation de la racine par l’oomycète pathogène Phytophthora parasitica. Ainsi, l’ensemble de ces résultats nous apermis d’élaborer un modèle proposant d’illustrer l’importance de l’arabinosylation des extensines dans l’organisation et l’architecture de la paroi, modulant ainsi l’adhésion du pathogène sur les cellules de la racine et influençant in fine la colonisation de cette dernière.Extensins are cell wall glycoproteins involved in various biological processes including plantprotection. However, their mode of action in plant immunity response is not clearly established and remains to be elucidated. Extensins are able to strengthen the cell wall, one of the first cellular barriers against pathogens, through intra- and intermolecular cross-links. This cross-linking is catalysed by specific peroxidase enzymes and requires a correct conformation of extensins conferred by their glycan moiety. This PhD project aimed to investigate the impact of extensin glycosylation in root defence and to characterize, as a preliminary study, the peroxidases potentially involved in the extensin crosslinking in Arabidopsis thaliana. Through immunocytochemistry techniques on mutants impaired withextensin glycosylation, we have revealed that a modulation of extensin distribution occurs in A. thaliana root in response to elicitation with the bacterial peptide, flagellin 22. We have also showed that extensin arabinosylation plays a major role, although probably indirect, in the root colonization by the pathogen oomycete Phytophthora parasitica. We have therefore elaborated a model proposing to illustrate the importance of extensin arabinosylation in the cell wall organization and architecture,modulating pathogen adhesion on root cells and influencing in fine root colonization

CAGEs are Golgi-localized GT31 enzymes involved in cellulose biosynthesis in Arabidopsis

Cellulose is the main structural component in the plant cell walls. We show that two glycosyltransferase family 31 (GT31) enzymes of Arabidopsis thaliana, here named cellulose synthesis associated glycosyltransferases 1 and 2 (CAGE1 and 2), influence both primary and secondary cell wall cellulose biosynthesis. cage1cage2 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. Single and double cage mutants also show increased sensitivity to the cellulose biosynthesis inhibitor isoxaben. The cage1cage2 phenotypes were associated with an approximately 30% reduction in cellulose content, an approximately 50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels in stems and reduced cellulose biosynthesis rate in seedlings. CESA transcript levels were not significantly altered in cage1cage2 mutants, suggesting that the reduction in CESA levels was caused by a post-transcriptional mechanism. Both CAGE1 and 2 localize to the Golgi apparatus and are predicted to synthesize β-1,3-galactans on arabinogalactan proteins. In line with this, the cage1cage2 mutants exhibit reduced levels of β-Yariv binding to arabinogalactan protein linked β-1,3-galactan. This leads us to hypothesize that defects in arabinogalactan biosynthesis underlie the cellulose deficiency of the mutants

The Spatio-Temporal Distribution of Cell Wall-Associated Glycoproteins During Wood Formation in Populus

Plant cell wall associated hydroxyproline-rich glycoproteins (HRGPs) are involved in several aspects of plant growth and development, including wood formation in trees. HRGPs such as arabinogalactan-proteins (AGPs), extensins (EXTs), and proline rich proteins (PRPs) are important for the development and architecture of plant cell walls. Analysis of publicly available gene expression data revealed that many HRGP encoding genes show tight spatio-temporal expression patterns in the developing wood of Populus that are indicative of specific functions during wood formation. Similar results were obtained for the expression of glycosyl transferases putatively involved in HRGP glycosylation. In situ immunolabelling of transverse wood sections using AGP and EXT antibodies revealed the cell type specificity of different epitopes. In mature wood AGP epitopes were located in xylem ray cell walls, whereas EXT epitopes were specifically observed between neighboring xylem vessels, and on the ray cell side of the vessel walls, likely in association with pits. Molecular mass and glycan analysis of AGPs and EXTs in phloem/cambium, developing xylem, and mature xylem revealed clear differences in glycan structures and size between the tissues. Separation of AGPs by agarose gel electrophoresis and staining with β-D-glucosyl Yariv confirmed the presence of different AGP populations in phloem/cambium and xylem. These results reveal the diverse changes in HRGP-related processes that occur during wood formation at the gene expression and HRGP glycan biosynthesis levels, and relate HRGPs and glycosylation processes to the developmental processes of wood formation

Extensin, an underestimated key component of cell wall defence?

International audienceBackground: Extensins are plant cell wall hydroxyproline-rich glycoproteins known to be involved in cell wall reinforcement in higher plants, and in defence against pathogen attacks. The ability of extensins to form intra- and intermolecular cross-links is directly related to their role in cell wall reinforcement. Formation of such cross-links requires appropriate glycosylation and structural conformation of the glycoprotein.Scope: Although the role of cell wall components in plant defence has drawn increasing interest over recent years, relatively little focus has been dedicated to extensins. Nevertheless, new insights were recently provided regarding the structure and the role of extensins and their glycosylation in plant-microbe interactions, stimulating an interesting debate from fellow cell wall community experts. We have previously revealed a distinct distribution of extensin epitopes in Arabidopsis thaliana wild-type roots and in mutants impaired in extensin arabinosylation, in response to elicitation with flagellin 22. That study was recently debated in a Commentary by Tan and Mort (Tan L, Mort A. 2020. Extensins at the front line of plant defence. A commentary on: 'Extensin arabinosylation is involved in root response to elicitors and limits oomycete colonization'. Annals of Botany 125: vii-viii) and several points regarding our results were discussed. As a response, we herein clarify the points raised by Tan and Mort, and update the possible epitope structure recognized by the anti-extensin monoclonal antibodies. We also provide additional data showing differential distribution of LM1 extensin epitopes in roots between a mutant defective in PEROXIDASES 33 and 34 and the wild type, similarly to previous observations from the rra2 mutant defective in extensin arabinosylation. We propose these two peroxidases as potential candidates to specifically catalyse the cross-linking of extensins within the cell wall.Conclusions: Extensins play a major role within the cell wall to ensure root protection. The cross-linking of extensins, which requires correct glycosylation and specific peroxidases, is most likely to result in modulation of cell wall architecture that allows enhanced protection of root cells against invading pathogens. Study of the relationship between extensin glycosylation and their cross-linking is a very promising approach to further understand how the cell wall influences root immunity

Root defense, role of cell extracellular matrix

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Root defense, role of cell extracellular matrix

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Contributions to Arabinogalactan Protein Analysis

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