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Debra Mohnen - One of the best experts on this subject based on the ideXlab platform.
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evolving views of pectin biosynthesis
Annual Review of Plant Biology, 2013Co-Authors: Melani A Atmodjo, Debra MohnenAbstract:Recent progress in the identification and characterization of pectin biosynthetic proteins and the discovery of pectin domain–containing proteoglycans are changing our view of how pectin, the most complex family of plant cell wall polysaccharides, is synthesized. The functional confirmation of four types of pectin biosynthetic glycosyltransferases, the identification of multiple putative pectin glycosyl- and methyltransferases, and the characteristics of the GAUT1:GAUT7 Homogalacturonan biosynthetic complex with its novel mechanism for retaining catalytic subunits in the Golgi apparatus and its 12 putative interacting proteins are beginning to provide a framework for the pectin biosynthetic process. We propose two partially overlapping hypothetical and testable models for pectin synthesis: the consecutive glycosyltransferase model and the domain synthesis model.
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The Arabidopsis irregular xylem8 Mutant Is Deficient in Glucuronoxylan and Homogalacturonan, Which Are Essential for Secondary Cell Wall Integrity
The Plant Cell, 2007Co-Authors: Staffan Persson, Debra Mohnen, Michael G Hahn, Kerry Hosmer Caffall, Glenn Freshour, Matthew T. Hilley, Stefan Bauer, Patricia Poindexter, Chris SomervilleAbstract:The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 ( irx8 ) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a Homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and Homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8 , we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and Homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.
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functional identification of an arabidopsis pectin biosynthetic Homogalacturonan galacturonosyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Jason D Sterling, Melani A Atmodjo, Sarah Eichler E Inwood, V Kumar S Kolli, Heather F Quigley, Michael G Hahn, Debra MohnenAbstract:Galacturonosyltransferases (GalATs) are required for the synthesis of pectin, a family of complex polysaccharides present in the cell walls of all land plants. We report the identification of a pectin GalAT (GAUT1) using peptide sequences obtained from Arabidopsis thaliana proteins partially purified for Homogalacturonan (HG) α-1,4-GalAT activity. Transient expression of GAUT1 cDNA in the human embryonic kidney cell line HEK293 yielded uridine diphosphogalacturonic acid:GalAT activity. Polyclonal antibodies generated against GAUT1 immunoabsorbed HG α-1,4-GalAT activity from Arabidopsis solubilized membrane proteins. blast analysis of the Arabidopsis genome identified a family of 25 genes with high sequence similarity to GAUT1 and homologous genes in other dicots, in rice, and in Physcomitrella. Sequence alignment and phylogenetic Bayesian analysis of the Arabidopsis GAUT1-related gene family separates them into four related clades of GAUT and GAUT-like genes that are distinct from the other Arabidopsis members of glycosyltransferase family 8. The identification of GAUT1 as a HG GalAT and of the GAUT1-related gene family provides the genetic and biochemical tools required to study the function of these genes in pectin synthesis.
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Development of a filter assay for measuring Homogalacturonan: α-(1,4)-Galacturonosyltransferase activity
Analytical Biochemistry, 2005Co-Authors: Jason D Sterling, Jason A. Lemons, Ivy F. Forkner, Debra MohnenAbstract:Abstract α-(1,4)-Galacturonosyltransferases (GalATs) catalyze the addition of (1,4)-linked α- d -galacturonosyl residues onto the nonreducing end of Homogalacturonan chains. The nucleotide-sugar donor for the enzymatic reaction is uridine diphospho- d -galactopyranosyluronic acid (UDP- d -GalpA). Many GalAT activity assays are based on the incorporation of d -[14C]GalpA from UDP- d -[14C]GalpA onto exogenously added Homogalacturonan acceptors. Reactions based on this method can be time-consuming because multiple labor-intensive centrifugations and washes with organic solvents are required to remove the unincorporated UDP- d -[14C]GalpA from the 14C-labeled products. Here we report the development of an alternative GalAT filter assay based on the ability of Homogalacturonan to bind to cetylpyridinium chloride (CPC). GalAT assay reaction products made using radish (Raphanus sativus) microsomal membranes or solubilized proteins from tobacco (Nicotiana tabacum L. cv. Samsun) and Arabidopsis thaliana (cv. Columbia) were spotted onto Whatman 3MM paper treated with 2.5% (w/v) CPC. Unincorporated UDP- d -[14C]GalpA was selectively removed from the filters by washing with 150–250 mM NaCl. The versatility of this assay is demonstrated by using it to identify GalAT activity in fractions obtained during the partial purification of tobacco GalAT by SP Sepharose cation exchange chromatography and by detecting the GalAT-catalyzed incorporation of d -[14C]GalpA onto endogenous acceptors from Arabidopsis membranes.
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Subcellular localization and topology of Homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells.
Planta, 1999Co-Authors: Florence Goubet, Debra MohnenAbstract:Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and inserted into the wall via exocytosis. The most abundant pectic polysaccharide, Homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase Homogalacturonan methyltransferase (HGA-MT). The subcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradients. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reticulum, was detected in the Golgi fraction. Homogalacturonan methyltransferase activity was not reduced by protease treatment of intact membranes or membranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was reduced by protease treatment of membranes permeabilized with 0.02% Triton X-100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and the lack of inhibition of HGA-MT activity by protease-treatment of intact membranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi.
J. Paul Knox - One of the best experts on this subject based on the ideXlab platform.
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An extended set of monoclonal antibodies to pectic Homogalacturonan.
Carbohydrate Research, 2009Co-Authors: Yves Verhertbruggen, Susan E. Marcus, Ash Haeger, José Juan Ordaz-ortiz, J. Paul KnoxAbstract:Three novel rat monoclonal antibodies, designated LM18, LM19 and LM20, were isolated from screens for binding to Arabidopsis thaliana seed coat mucilage. The binding of these antibodies to mucilage subject to enzyme and high pH pre-treatments and to a series of model Homogalacturonan-rich pectins with defined levels of methyl-esterification indicated their recognition of pectic Homogalacturonan epitopes. The binding capacities of these monoclonal antibodies to cell walls in sections of tobacco stem pith parenchyma were also differentially sensitive to equivalent treatments with high pH buffers and pectate lyase. The epitopes bound by these antibodies display some similarities and some differences to the epitopes recognized by the previously isolated and established pectic Homogalacturonan probes JIM5 and JIM7.
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QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects Homogalacturonan and xylan biosynthesis
Planta, 2005Co-Authors: Caroline Orfila, J. Paul Knox, Hoai Nam Truong, Susanne Oxenbøll Sørensen, Jesper Harholt, Naomi Geshi, Hazel Crombie, J. S. Grant Reid, Henrik Vibe SchellerAbstract:An insertion in the promoter of the Arabidopsis thaliana QUA1 gene ( qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of Homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002 ). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, Homogalacturonan α-1-4- D -galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4- D -xylan synthase, β-1-4- D -galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4- D -xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in Homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4- D -galacturonosyltransferase and β-1,4- D -xylan synthase activities.
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QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects Homogalacturonan and xylan biosynthesis
Planta, 2005Co-Authors: Caroline Orfila, J. Paul Knox, Hoai Nam Truong, Jesper Harholt, Naomi Geshi, Hazel Crombie, J. S. Grant Reid, Susanne Sørensen, Henrik Vibe SchellerAbstract:An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of Homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, Homogalacturonan alpha-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of beta-1-4-D-xylan synthase, beta-1-4-D-galactan synthase and beta-glucan synthase II activities were also measured in microsomal membranes. Of these, only beta-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in Homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on alpha-1,4-D-galacturonosyltransferase and beta-1,4-D-xylan synthase activities.
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Isolation and characterisation of the Homogalacturonan from type II cell walls of the commelinoid monocot wheat using HF-solvolysis.
Carbohydrate Research, 2003Co-Authors: Nicola Wiethölter, William G T Willats, J. Paul Knox, Barbara Graeßner, Manfred Mierau, Bruno M. MoerschbacherAbstract:In contrast to the typical type I cell wall of the dicot plants, the type II cell wall of the commelinoid monocot plants is known to be relatively poor in pectins. Assuming a critical role for the remaining pectins in terms of cell wall architecture and/or as a reservoir of signalling molecules, we have compared different protocols for the isolation of the main pectin polymer, Homogalacturonan, from wheat leaf cell walls. Pectin was detected in these cell walls immunochemically using the monoclonal antibodies JIM5 and JIM7, and biochemically by monosaccharide analysis. The Ca(++)-chelators CDTA and imidazole extracted a pectin rich fraction from isolated cell walls which was however contaminated with significant amounts of hemicelluloses. Pretreatment of the cell walls with anhydrous hydrogen fluoride at controlled low temperatures followed by HF/ether- and water-extraction prior to imidazole-extraction of pectins yielded a purer Homogalacturonan fraction. The near absence of rhamnosyl residues proved that the isolated Homogalacturonan fraction was free of rhamnogalacturonans. If HF-solvolysis was performed at -23 degrees C, the resulting Homogalacturonan had a degree of methyl esterification identical to that of the pectins in the initial wheat cell wall. The antibodies JIM5 and JIM7 as well as PAM1 and LM5 proved that the isolated Homogalacturonan had a low methyl ester content, was polymeric and free of galactan side chains. We can thus isolate native Homogalacturonan from the type II wheat cell walls with the original in muro pattern of methyl esterification still intact, to further investigate e.g., its degradability by plant or microbial pectic enzymes.
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Synthetic methyl hexagalacturonate hapten inhibitors of anti-Homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7.
Carbohydrate Research, 2003Co-Authors: Mads Hartvig Clausen, William G T Willats, J. Paul KnoxAbstract:A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-Homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens provides insight into the structures of the partially methyl-esterified pectin epitopes of these widely used monoclonal antibodies.
Henrik Vibe Scheller - One of the best experts on this subject based on the ideXlab platform.
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QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects Homogalacturonan and xylan biosynthesis
Planta, 2005Co-Authors: Caroline Orfila, J. Paul Knox, Hoai Nam Truong, Susanne Oxenbøll Sørensen, Jesper Harholt, Naomi Geshi, Hazel Crombie, J. S. Grant Reid, Henrik Vibe SchellerAbstract:An insertion in the promoter of the Arabidopsis thaliana QUA1 gene ( qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of Homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002 ). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, Homogalacturonan α-1-4- D -galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4- D -xylan synthase, β-1-4- D -galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4- D -xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in Homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4- D -galacturonosyltransferase and β-1,4- D -xylan synthase activities.
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QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects Homogalacturonan and xylan biosynthesis
Planta, 2005Co-Authors: Caroline Orfila, J. Paul Knox, Hoai Nam Truong, Jesper Harholt, Naomi Geshi, Hazel Crombie, J. S. Grant Reid, Susanne Sørensen, Henrik Vibe SchellerAbstract:An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of Homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, Homogalacturonan alpha-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of beta-1-4-D-xylan synthase, beta-1-4-D-galactan synthase and beta-glucan synthase II activities were also measured in microsomal membranes. Of these, only beta-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in Homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on alpha-1,4-D-galacturonosyltransferase and beta-1,4-D-xylan synthase activities.
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QUASIMODOl is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects Homogalacturonan and xylan
2005Co-Authors: Paul Knox, Henrik Vibe SchellerAbstract:An insertion in the promoter of the Arabid opsis thaliana QUAI gene (qual-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhe sion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of Homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link be tween QUAI and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qual-1 mutant tissue. Relative to the wild type, Homogalacturonan a-1-4-D-galacturonosyltransferase activity was consis tently reduced in qual-1 stems (by about 23% in microsomal and 33% in detergent-solubilized mem brane preparations). Activities of /)-1-4-o-xylan syn thase, s-1-4-D-galactan synthase and s-glucan synthase II activities were also measured in microsomal mem branes. Of these, only s-l-4-D-xylan synthase was af fected, and was reduced by about 40% in qual-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUAI mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in Homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUAI in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUAI in pectin and hemicellulose cell wall synthesis through affects on a-l,4-D-galacturonosyltransferase and s-1,4 D-xylan synthase activities.
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Pectin biosynthesis: a solubilized α1,4-galacturonosyltransferase from tobacco catalyzes the transfer of galacturonic acid from UDP-galacturonic acid onto the non-reducing end of Homogalacturonan
Planta, 1999Co-Authors: Henrik Vibe Scheller, Ron Lou Doong, Brent L. Ridley, Debra MohnenAbstract:A solubilized α1,4-galacturonosyltransferase (GalAT) from tobacco transfers galacturonic acid (GalA) residues from UDP-GalA onto oligogalacturonide (OGA) exogenous acceptors with degrees of polymerization greater than nine (R.L. Doong and D. Mohnen 1998, Plant J 13: 363–374). The solubilized GalAT has been identified as putative polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT, EC 2.4.1.43) based on its α1,4-galacturonosyltransferase activity and similar Km for UDP-GalA, pH optimum and Vmax to those of membrane-bound PGA-GalAT (R.L. Doong et al., 1995, Plant Physiol 109: 141–152). The direction of elongation of Homogalacturonan catalyzed by solubilized GalAT from microsomes of tobacco (Nicotiana tabacum L. cv. Samsun) cell suspensions has now been determined. Three different types of exogenous acceptor were used to study the direction of synthesis of Homogalacturonan: unmodified OGAs, OGAs derivatized by biotinylation at the reducing end, and OGAs containing a 4,5-unsaturated GalA at the non-reducing end. The unmodified OGAs and the OGAs modified at the reducing end functioned equally well as acceptors in the galacturonosyltransferase reaction. In contrast, OGAs with the 4,5-unsaturated residue at the non-reducing end were not acceptors for Homogalacturonan biosynthesis. These results show that Homogalacturonan biosynthesis by solubilized GalAT occurs via the addition of GalA to the non-reducing end of the polymer chain.
Jeanclaude Mollet - One of the best experts on this subject based on the ideXlab platform.
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biochemical and immunocytological characterizations of arabidopsis pollen tube cell wall
Plant Physiology, 2010Co-Authors: Flavien Dardelle, Arnaud Lehner, Yasmina Ramdani, Azeddine Driouich, Muriel Bardor, Patrice Lerouge, Jeanclaude MolletAbstract:During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified Homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified Homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.
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Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil
Plant Signaling and Behavior, 2010Co-Authors: Arnaud Lehner, Flavien Dardelle, Azeddine Driouich, Patrice Lerouge, Odile Soret-morvan, Jeanclaude MolletAbstract:Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well defined localization of cell wall epitopes with highly esterified Homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified Homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding 1) the ultrastructure of the pollen tube cell wall and 2) the immunolocalization of Homogalacturonan and arabinan epitopes in 16 h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.
Patrice Lerouge - One of the best experts on this subject based on the ideXlab platform.
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Identification of putative rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of bioinformatics approaches
PLoS ONE, 2012Co-Authors: Aline Voxeur, Aurélie André, Christelle Breton, Patrice LerougeAbstract:Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an a(1,4)-linked Homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of a-galacturonosyltransferases involved in the synthesis of a4-GalA oligogalacturonides present in both Homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.
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biochemical and immunocytological characterizations of arabidopsis pollen tube cell wall
Plant Physiology, 2010Co-Authors: Flavien Dardelle, Arnaud Lehner, Yasmina Ramdani, Azeddine Driouich, Muriel Bardor, Patrice Lerouge, Jeanclaude MolletAbstract:During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified Homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified Homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.
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Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil
Plant Signaling and Behavior, 2010Co-Authors: Arnaud Lehner, Flavien Dardelle, Azeddine Driouich, Patrice Lerouge, Odile Soret-morvan, Jeanclaude MolletAbstract:Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well defined localization of cell wall epitopes with highly esterified Homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified Homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding 1) the ultrastructure of the pollen tube cell wall and 2) the immunolocalization of Homogalacturonan and arabinan epitopes in 16 h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.
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The organization pattern of root border-like cells of Arabidopsis is dependent on cell wall Homogalacturonan.
Plant Physiology, 2009Co-Authors: Caroline Durand, Patrice Lerouge, Maïté Vicré-gibouin, Marie Laure Follet-gueye, Ludovic Duponchel, Myriam Moreau, Azeddine DriouichAbstract:Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less Homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in Homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.