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Birgit Classen - One of the best experts on this subject based on the ideXlab platform.
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characterization of a yariv precipitated Arabinogalactan Protein from fruits of rye secale cereale l
Die Pharmazie, 2013Co-Authors: E Goellner, W Blaschek, Birgit ClassenAbstract:: An Arabinogalactan-Protein (AGP) with a molecular mass of 110 kDa was isolated from whole grain of rye (Secale cereale L.) by double precipitation with (beta-D-Glc)3-Yariv-phenylglycoside (3GlcY) and its structure was analyzed. The AGP consists of a hydroxyproline-rich Protein backbone of about 7 % and an Arabinogalactan moiety of about 93%. By alkaline hydrolysis, hydroxyproline was identified as the main amino acid responsible for the binding between the Protein and the carbohydrate subunits via an O-glycosidic linkage. The Arabinogalactan moieties are highly branched consisting of 1,3-linked Galp residues, some of them linked in position 6 to 1,6-Galp side chains, terminating in Araf residues. With regard to its structure, the rye AGP is comparable to other cereal AGPs like those from oat or wheat grain.
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An Arabinogalactan-Protein from whole grain of Avena sativa L. belongs to the wattle-blossom type of Arabinogalactan-Proteins
Journal of Cereal Science, 2011Co-Authors: Esther M Gollner, Wolfgang Blaschek, Satoshi Kaneko, Hitomi Ichinose, Birgit ClassenAbstract:An Arabinogalactan-Protein (AGP) from whole grain of oat (Avena sativa L) has been isolated for the first time by double precipitation with beta-glucosyl Yariv reagent and characterized with regard to its polysaccharide and Protein part. The large carbohydrate moiety is rich in galactose (63.0 w/w) and arabinose (32.8 w/w) and free of uronic acids. Linkage analysis of AGP and its products obtained by partial acid hydrolysis as well as enzymatic degradation with specific recombinant enzymes revealed that the carbohydrate moiety consists of a 1,3-Galp backbone and is linked in position 6 to short 1,6-Galp chains, terminating in Aral In the Protein part, high contents of alanine, hydroxyproline and serine could be found which are typical for AGPs. The molecular mass of AGP was determined to be 83 kDa. The carbohydrate moieties, released by alkaline degradation of the Protein part, had a size of about 7 kDa. Consequently, the overall structure of the AGP from oat could be assigned to be consistent with the wattle-blossom model of AGPs. (C) 2011 Elsevier Ltd. All rights reserved.
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structural investigations on Arabinogalactan Protein from wheat isolated with yariv reagent
Journal of Agricultural and Food Chemistry, 2010Co-Authors: Esther M Gollner, W Blaschek, Birgit ClassenAbstract:For the first time a pure Arabinogalactan-Protein (AGP) could be isolated from whole grain of wheat (Triticum aestivum L.) by performing a double precipitation with β-glucosyl Yariv reagent. The putative bioactive AGP has been characterized with regard to its polysaccharide and Protein parts. Analytical investigations by GLC-MS and 13C NMR revealed a carbohydrate moiety consisting of a 1,3-Galp backbone, linked in position 6 to short 1,6-Galp-chains, terminating in Araf. In the Protein part, a high content of hydroxyproline has been found, probably responsible for linkage between Protein and polysaccharide moieties. The molecular mass of AGP has been determined by size exclusion chromatography with laser light scattering detection and found to be 125 kDa. Alkaline hydrolysis of the Protein resulted in single carbohydrate moieties with a molecular mass of about 20 kDa, indicating that AGP from whole grain of wheat belongs to the wattle blossom type of AGPs.
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Binding studies of an Arabinogalactan-Protein from Echinacea purpurea to leucocytes.
Phytomedicine : international journal of phytotherapy and phytopharmacology, 2005Co-Authors: S. Thude, Birgit Classen, Wolfgang Blaschek, D. Barz, H. ThudeAbstract:Flow cytometric investigations show binding of an isolated Arabinogalactan-Protein (AGP) from pressed juice of the aerial parts of Echinacea purpurea to the cell surface of human leucocytes. AGP demonstrates binding to lymphocytes, monocytes and granulocytes of different donors (n=8). Competition assays with two antibodies, directed against CD4 and CD8, revealed no interaction of AGP with these receptors, leading to the conclusion that binding of AGP to leucocytes is mediated via other structures.
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An acidic Arabinogalactan-Protein from the roots of Baptisia tinctoria.
Planta medica, 2005Co-Authors: Maren Wack, Birgit Classen, Wolfgang BlaschekAbstract:An acidic Arabinogalactan-Protein (AGP) isolated from an aqueous extract of the roots of wild indigo [ Baptisia tinctoria (L.) R. Br.] by precipitation with beta-glucosyl Yariv reagent consists of L-arabinose (34.0 %) and D-galactose (58.7 %) (1 : 1.7), minor amounts of D-glucuronic acid (4.0 %) and traces of D-rhamnose (2.3 %) and D-glucose ( 1.0 x 10 (7) Da, respectively. Both fractions show corresponding carbohydrate compositions and structural features with regard to the carbohydrate moiety. Mild acid hydrolysis of the AGP leads to loss of terminal arabinofuranosyl units. The residual galactan backbone does not react with the Yariv reagent in gel diffusion tests while the total AGP still interferes with the reagent following reduction of terminal glucuronic acid residues.
Marcia J Kieliszewski - One of the best experts on this subject based on the ideXlab platform.
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An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an Arabinogalactan Protein.
The Plant cell, 2013Co-Authors: Li Tan, John Glushka, Utku Avci, Marcia J Kieliszewski, Michael G Hahn, Sivakumar Pattathil, David Baldwin, Xiang Zhu, Ronald Orlando, Stefan Eberhard, Jeffrey S Miller, Chunhua Yuan, Charles Pham, Zhangying Hao, Clayton Warder, Ashley Darvill, Debra MohnenAbstract:Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% Protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoProtein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana Arabinogalactan Protein (AGP) encoded by hydroxyproline-rich glycoProtein family Protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the Arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN Arabinogalactan Protein1 (APAP1), is contrary to prevailing cell wall models that depict separate Protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.
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human growth hormone expressed in tobacco cells as an Arabinogalactan Protein fusion glycoProtein has a prolonged serum life
Transgenic Research, 2010Co-Authors: Shigeru Okada, Kenneth J Goodrum, Li Tan, John J Kopchick, Marcia J KieliszewskiAbstract:Therapeutic Proteins with molecular weights lower than 40 kDa often have short serum half-lives due to their susceptibility to serum proteases and rapid renal clearance. Chemical derivatization, such as PEGylation, or expression as serum albumin fusions increases molecular mass and overcome these problems but at the expense of decreased bioactivity. Here we applied a new method that yields biologically potent recombinant human growth hormone (rhGH) with increased serum half-life when expressed as an Arabinogalactan-Protein (AGP) in tobacco BY-2 cells. Thus, rhGH was expressed with 10 repeats of the AGP glycomodule Ser-Hyp (SO) at the C-terminus (rhGH-(SO)10). We also expressed rhGH as an AGP-enhanced green fluorescent Protein (EGFP) fusion, designated rhGH-(SO)10-EGFP, to assess the cellular distribution of the glycoProtein, which was mainly extracellular. Recombinant hGH-(SO)10 bound the hGH receptor with an affinity similar to that of a rhGH standard, stimulated the same intracellular signaling pathway as hGH, but possessed an in vivo serum half-life more than sixfold that of the hGH control. Furthermore, rhGH-(SO)10 gave a 500 fold greater secreted yield than the non-glycosylated control rhGH that was also targeted for secretion. Detailed analysis of the rhGH-(SO)10 glycans indicated a conserved structure with relatively little microheterogeneity and an average size of 25 monosaccharide residues. These results were consistent with earlier work expressing interferon α2b as an AGP chimera and further demonstrate the feasibility of this approach to the production of long-acting, biologically potent therapeutic Proteins by plant cells.
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high yields and extended serum half life of human interferon α2b expressed in tobacco cells as Arabinogalactan Protein fusions
Biotechnology and Bioengineering, 2007Co-Authors: Li Tan, Kenneth J Goodrum, Marcia J KieliszewskiAbstract:Therapeutic Proteins like human interferon a2 generally possess short serum half-lives due to their small size, hence rapid renal clearance, and susceptibility to serum proteases. Chemical derivatization, such as addi- tion of polyethylene glycol (PEG) groups overcomes both problems, but at the expense of greatly decreased bioactivity. We describe a new method that yields biologically potent interferon a2b (IFNa2) in high yields and with increased serum half-life when expressed as Arabinogalactan-Protein (AGP) chimeras in cultured tobacco cells. Thus IFNa2- AGPs targeted for secretion typically gave 350-1400-fold greater secreted yields than the non-glycosylated IFNa2 control. The purified AGP domain itself was not immuno- genic when injected into mice and only mildly so when injected as a fusion glycoProtein. Importantly, the AGP- IFNa2 chimeras showed up to a 13-fold increased in vivo serum half-life while the biological activity remained similar to native IFNa2. The use of Arabinogalactan glycomodules may provide a general approach to the enhanced production of therapeutic Proteins by plants. Biotechnol. Bioeng. 2007;97: 997-1008. 2007 Wiley Periodicals, Inc.
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overexpression of tomato leagp 1 Arabinogalactan Protein promotes lateral branching and hampers reproductive development
Plant Journal, 2004Co-Authors: Wenxian Sun, Marcia J Kieliszewski, Allan M. ShowalterAbstract:LeAGP-1 is a glycosylphosphatidylinositol (GPI)-anchored Arabinogalactan-Protein (AGP) in tomato (Lycopersicon esculentum). Patterns of mRNA expression and Protein localization for LeAGP-1 indicate that it likely functions in certain aspects of plant growth and development. To elucidate LeAGP-1 function(s), transgenic tomato plants expressing enhanced green fluorescent Protein (GFP) fused to LeAGP-1 [GFP-LeAGP-1] or two LeAGP-1 variants, one lacking the C-terminal GPI-anchor domain [GFP-LeAGP-1DeltaC] and the other lacking the lysine-rich domain [GFP-LeAGP-1DeltaK], under the control of the CaMV35S promoter were produced using Agrobacterium-mediated transformation. Transgenic T0 and T1 lines with high levels of both GFP-LeAGP-1 mRNA and Protein: (i) were significantly shorter; (ii) were highly branched; (iii) produced more flower buds, but most of these flowers did not mature, resulting in less fruit production; and (iv) produced seeds that were significantly smaller than normal seeds. Overexpression of LeAGP-1DeltaK had a similar or even more pronounced effect on plant vegetative and reproductive growth, while the effect of LeAGP-1DeltaC overexpression on plant reproduction was minimal. These results indicate that the GPI anchor is critical for LeAGP-1 function. As the phenotype of GFP-LeAGP-1 overexpressing transgenic plants is similar to that of cytokinin-overproducing plants, mRNA expression patterns of LeAGP-1 under different hormone treatments were examined. Cytokinins upregulated LeAGP-1 mRNA expression, while auxins and ABA inhibited LeAGP-1 mRNA expression. Based on these results, GPI-anchored LeAGP-1 most likely functions in plant growth and development in concert with auxin/cytokinin signaling.
Li Tan - One of the best experts on this subject based on the ideXlab platform.
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An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an Arabinogalactan Protein.
The Plant cell, 2013Co-Authors: Li Tan, John Glushka, Utku Avci, Marcia J Kieliszewski, Michael G Hahn, Sivakumar Pattathil, David Baldwin, Xiang Zhu, Ronald Orlando, Stefan Eberhard, Jeffrey S Miller, Chunhua Yuan, Charles Pham, Zhangying Hao, Clayton Warder, Ashley Darvill, Debra MohnenAbstract:Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% Protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoProtein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana Arabinogalactan Protein (AGP) encoded by hydroxyproline-rich glycoProtein family Protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the Arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN Arabinogalactan Protein1 (APAP1), is contrary to prevailing cell wall models that depict separate Protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.
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human growth hormone expressed in tobacco cells as an Arabinogalactan Protein fusion glycoProtein has a prolonged serum life
Transgenic Research, 2010Co-Authors: Shigeru Okada, Kenneth J Goodrum, Li Tan, John J Kopchick, Marcia J KieliszewskiAbstract:Therapeutic Proteins with molecular weights lower than 40 kDa often have short serum half-lives due to their susceptibility to serum proteases and rapid renal clearance. Chemical derivatization, such as PEGylation, or expression as serum albumin fusions increases molecular mass and overcome these problems but at the expense of decreased bioactivity. Here we applied a new method that yields biologically potent recombinant human growth hormone (rhGH) with increased serum half-life when expressed as an Arabinogalactan-Protein (AGP) in tobacco BY-2 cells. Thus, rhGH was expressed with 10 repeats of the AGP glycomodule Ser-Hyp (SO) at the C-terminus (rhGH-(SO)10). We also expressed rhGH as an AGP-enhanced green fluorescent Protein (EGFP) fusion, designated rhGH-(SO)10-EGFP, to assess the cellular distribution of the glycoProtein, which was mainly extracellular. Recombinant hGH-(SO)10 bound the hGH receptor with an affinity similar to that of a rhGH standard, stimulated the same intracellular signaling pathway as hGH, but possessed an in vivo serum half-life more than sixfold that of the hGH control. Furthermore, rhGH-(SO)10 gave a 500 fold greater secreted yield than the non-glycosylated control rhGH that was also targeted for secretion. Detailed analysis of the rhGH-(SO)10 glycans indicated a conserved structure with relatively little microheterogeneity and an average size of 25 monosaccharide residues. These results were consistent with earlier work expressing interferon α2b as an AGP chimera and further demonstrate the feasibility of this approach to the production of long-acting, biologically potent therapeutic Proteins by plant cells.
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high yields and extended serum half life of human interferon α2b expressed in tobacco cells as Arabinogalactan Protein fusions
Biotechnology and Bioengineering, 2007Co-Authors: Li Tan, Kenneth J Goodrum, Marcia J KieliszewskiAbstract:Therapeutic Proteins like human interferon a2 generally possess short serum half-lives due to their small size, hence rapid renal clearance, and susceptibility to serum proteases. Chemical derivatization, such as addi- tion of polyethylene glycol (PEG) groups overcomes both problems, but at the expense of greatly decreased bioactivity. We describe a new method that yields biologically potent interferon a2b (IFNa2) in high yields and with increased serum half-life when expressed as Arabinogalactan-Protein (AGP) chimeras in cultured tobacco cells. Thus IFNa2- AGPs targeted for secretion typically gave 350-1400-fold greater secreted yields than the non-glycosylated IFNa2 control. The purified AGP domain itself was not immuno- genic when injected into mice and only mildly so when injected as a fusion glycoProtein. Importantly, the AGP- IFNa2 chimeras showed up to a 13-fold increased in vivo serum half-life while the biological activity remained similar to native IFNa2. The use of Arabinogalactan glycomodules may provide a general approach to the enhanced production of therapeutic Proteins by plants. Biotechnol. Bioeng. 2007;97: 997-1008. 2007 Wiley Periodicals, Inc.
Cecile Albenne - One of the best experts on this subject based on the ideXlab platform.
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Arabinogalactan Protein 31 agp31 a putative network forming Protein in arabidopsis thaliana cell walls
Annals of Botany, 2014Co-Authors: May Hijazi, Elisabeth Jamet, Cecile Albenne, David Roujol, Huan Nguyenkim, Liliana Del Rocio Cisneros Castillo, Estelle SalandAbstract:Background and aims Arabinogalactan Protein 31 (AGP31) is a remarkable plant cell-wall Protein displaying a multi-domain organization unique in Arabidopsis thaliana: it comprises a predicted signal peptide (SP), a short AGP domain of seven amino acids, a His-stretch, a Pro-rich domain and a PAC (PRP-AGP containing Cys) domain. AGP31 displays different O-glycosylation patterns with Arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on the Pro-rich domain. AGP31 has been identified as an abundant Protein in cell walls of etiolated hypocotyls, but its function has not been investigated thus far. Literature data suggest that AGP31 may interact with cell-wall components. The purpose of the present study was to identify AGP31 partners to gain new insight into its function in cell walls. Methods Nitrocellulose membranes were prepared by spotting different polysaccharides, which were either obtained commercially or extracted from cell walls of Arabidopsis thaliana and Brachypodium distachyon. After validation of the arrays, in vitro interaction assays were carried out by probing the membranes with purified native AGP31 or recombinant PAC-V5-6xHis. In addition, dynamic light scattering (DLS) analyses were carried out on an AGP31 purified fraction. Key results It was demonstrated that AGP31 interacts through its PAC domain with galactans that are branches of rhamnogalacturonan I. This is the first experimental evidence that a PAC domain, also found as an entire Protein or a domain of AGP31 homologues, can bind carbohydrates. AGP31 was also found to bind methylesterified polygalacturonic acid, possibly through its His-stretch. Finally, AGP31 was able to interact with itself in vitro through its PAC domain. DLS data showed that AGP31 forms aggregates in solution, corroborating the hypothesis of an auto-assembly. Conclusions These results allow the proposal of a model of interactions of AGP31 with different cell-wall components, in which AGP31 participates in complex supra-molecular scaffolds. Such scaffolds could contribute to the strengthening of cell walls of quickly growing organs such as etiolated hypocotyls.
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characterization of the Arabinogalactan Protein 31 agp31 of arabidopsis thaliana new advances on the hyp o glycosylation of the pro rich domain
Journal of Biological Chemistry, 2012Co-Authors: May Hijazi, Jessica Durand, Carole Pichereaux, Frederic Pont, Elisabeth Jamet, Cecile AlbenneAbstract:Abstract Proteins are important actors in plant cell walls because they contribute to their architecture and their dynamics. Among them, hydroxyproline (Hyp)-rich glycoProteins constitute a complex family of O-glycoProteins with various structures and functions. In this study, we characterized an atypical Hyp-rich glycoProtein, AGP31 (Arabinogalactan Protein 31), which displays a multidomain organization unique in Arabidopsis thaliana, consisting of a short Arabinogalactan Protein (AGP) motif, a His stretch, a Pro-rich domain, and a C-terminal PAC (PRP-AGP containing Cys) domain. The use of various mass spectrometry strategies was innovative and powerful: it permitted us to locate Hyp residues, to demonstrate the presence of carbohydrates, and to refine their distribution over the Pro-rich domain. Most Hyp were isolated within repeated motifs such as KAOV, KSOV, K(PO/OP)T, K(PO/OP)V, T(PO/OP)V, and Y(PO/OP)T. A few extensin-like motifs with contiguous Hyp (SOOA and SOOT) were also found. The Pro-rich domain was shown to carry Gal residues on isolated Hyp but also Ara residues. The existence of new type Hyp-O-Gal/Ara-rich motifs not recognized by the β-glucosyl Yariv reagent but interacting with the peanut agglutinin lectin was proposed. In addition, the N-terminal short AGP motif was assumed to be substituted by Arabinogalactans. Altogether, AGP31 was found to be highly heterogeneous in cell walls because Arabinogalactans could be absent, Hyp-O-Gal/Ara-rich motifs of different sizes were observed, and truncated forms missing the C-terminal PAC domain were found, suggesting degradation in muro and/or partial glycosylation prior to secretion.
Robert Konieczny - One of the best experts on this subject based on the ideXlab platform.
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Arabinogalactan Protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in trifolium nigrescens viv
Plant Cell Tissue and Organ Culture, 2013Co-Authors: M Pilarska, Paul J Knox, Robert KoniecznyAbstract:The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of Arabinogalactan-Protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation.
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distribution of pectin and Arabinogalactan Protein epitopes during organogenesis from androgenic callus of wheat
Plant Cell Reports, 2007Co-Authors: Robert Konieczny, Joanna świerczynska, Andzej Z Czaplicki, J BohdanowiczAbstract:The distribution of several Arabinogalactan Protein and pectic epitopes were studied during organogenesis in androgenic callus of wheat. In cell wall of mature and degenerating parenchyma cells, the Arabinogalactan epitopes JIM4, JIM14, JIM16 or LM2 were expressed differently according to the cells location. LM2 was observed also in meristematic cells of regenerated shoot buds and leaves. Anti-pectin JIM7 labelled the wall of meristematic cells but fluorescence was strongest in outer walls of surface cells of callus and shoot buds coated by extracellular matrix surface network (ECMSN). During leaves growth the ECMSN disappeared, and JIM7 fluorescence decreased. JIM5 epitope was abundant in the cell walls lining the intercellular spaces of callus parenchyma and in tricellular junctions within regenerated buds and leaves.