Glycosylation motifs that direct arabinogalactan addition to arabinogalactan-proteins

Hydroxyproline (Hyp)-rich glycoproteins (HRGPs) participate in all aspects of plant growth and development. HRGPs are generally highly O-glycosylated through the Hyp residues, which means carbohydrates help define the interactive molecular surface and, hence, HRGP function. The Hyp contiguity hypothesis predicts that contiguous Hyp residues are sites of HRGP arabinosylation, whereas clustered noncontiguous Hyp residues are sites of galactosylation, giving rise to the arabinogalactan heteropolysaccharides that characterize the arabinogalactan-proteins. Early tests of the hypothesis using synthetic genes encoding only clustered noncontiguous Hyp in the sequence (serine [Ser]-Hyp-Ser-Hyp)(n) or contiguous Hyp in the series (Ser-Hyp-Hyp)(n) and (Ser-Hyp-Hyp-Hyp-Hyp)(n) confirmed that arabinogalactan polysaccharide was added only to noncontiguous Hyp, whereas arabinosylation occurred on contiguous Hyp. Here, we extended our tests of the codes that direct arabinogalactan polysaccharide addition to Hyp by building genes encoding the repetitive sequences (alanine [Ala]-proline [Pro]-Ala-Pro)(n), (threonine [Thr]-Pro-Thr-Pro)(n), and (valine [Val]-Pro-Val-Pro)(n), and expressing them in tobacco (Nicotiana tabacum) Bright-Yellow 2 cells as fusion proteins with green fluorescent protein. All of the Pro residues in the (Ala-Pro-Ala-Pro)(n) fusion protein were hydroxylated and consistent with the hypothesis that every Hyp residue was glycosylated with arabinogalactan polysaccharide. In contrast, 20% to 30% of Pro residues remained non-hydroxylated in the (Thr-Pro-Thr-Pro)(n), and (Val-Pro-Val-Pro)(n) fusion proteins. Furthermore, although 50% to 60% of the Hyp residues were glycosylated with arabinogalactan polysaccharide, some remained non-glycosylated or were arabinosylated. These results suggest that the amino acid side chains of flanking residues influence the extent of Pro hydroxylation and Hyp glycosylation and may explain why isolated noncontiguous Hyp in extensins do not acquire an arabinogalactan polysaccharide but are arabinosylated or remain non-glycosylated.     

The lysine-rich arabinogalactan-protein subfamily in Arabidopsis: gene expression, glycoprotein purification and biochemical characterization

AtAGP17, AtAGP18 and AtAGP19 are homologous genes encoding three putative glycosylphosphatidylinositol (GPI)-anchored classical arabinogalactan-proteins (AGPs) in Arabidopsis. They are distinguished from other AGPs by a short, C-terminal lysine-rich region. Organ-specific expression of these genes was revealed by Northern blot analysis. AtAGP17 was strongly expressed in leaves and stems, and weakly expressed in flowers and roots; AtAGP18 was strongly expressed in flowers, and moderately expressed in roots, stems and young leaves; and AtAGP19 was strongly expressed in stems, moderately expressed in flowers and roots, and weakly expressed in young leaves. One of these genes, AtAGP17, was expressed and purified as a green fluorescent protein (GFP) fusion protein in transgenic tobacco cells using hydrophobic interaction chromatography, size exclusion chromatography and reverse phase high-performance liquid chromatography. The fusion (glyco)protein produced a characteristic AGP 'smear' with a molecular mass of 80-150 kDa when detected by Western blot analysis. Glycosyl composition and linkage analyses of purified GFP-AtAGP17 showed that carbohydrate accounted for approximately 86% of the molecule, with arabinose and galactose as major, and rhamnose and glucuronic acid as minor glycosyl residues and with 1,3,6-galactose, 1,4-glucuronic acid, 1,3-galactose and terminal arabinose as major linkages. GFP-AtAGP17 was also precipitated by beta-Yariv reagent, further confirming that AtAGP17 is a bona fide AGP. Confocal fluorescence microscopy of plasmolysed, transformed cells indicated that AtAGP17 is localized on the plasma membrane and in Hechtian strands. Hydroxyproline (Hyp) glycoside profiles of GFP-AtAGP17 in conjunction with the deduced protein sequence also served to corroborate the Hyp contiguity hypothesis, which predicts contiguous Hyp residues as attachment sites for arabinosides and clustered, non-contiguous Hyp residues as attachment sites for arabinogalactan polysaccharides.     

Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein

Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato ( Lycopersicon esculentum , cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco ( Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.     

NaAGP4 is an arabinogalactan protein whose expression is suppressed by wounding and fungal infection inNicotiana alata

Arabinogalactan proteins (AGPs) are proteoglycans secreted by plant cells that have been implicated in plant growth and development. Most AGPs cloned to date possess highly labile glycosylphosphatidylinositol (GPI) lipid anchors. These anchors transiently attach AGPs to the plasma membrane before they are released into the cell wall following GPI anchor hydrolysis. We have isolated and partially sequenced the protein core of an AGP purified from styles of Nicotiana alata. The protein sequence data were utilised to clone the AGP's gene, NaAGP4. This AGP shares about 78% sequence identity with the tomato AGP LeAGP-1. RNA gel blot analyses of different plant organs indicate that NaAGP4 is expressed in the same tissues and at similar levels as LeAGP-1. Furthermore, NaAGP4 like LeAGP-1 is rapidly suppressed by tissue wounding and by pathogen infection. We believe NaAGP4 and LeAGP-1 are the first described examples of orthologous AGPs from different plant species. In contrast, another AGP from N. alata, NaAGP1, is comparatively unaffected by wounding and pathogen infection, although this AGP is expressed in similar tissues and at similar levels as NaAGP4.     

Isolation, characterization and immunolocalization of a novel, modular tomato arabinogalactan-protein corresponding to the LeAGP-1 gene

Arabinogalactan-proteins (AGPs) are a family of hydroxy-proline-rich glycoproteins implicated to function in plant growth and development. This report focuses on a novel, modular AGP found in tomato, LeAGP-1, which was predicted by DNA cloning and herein verified at the protein level as a major AGP component. LeAGP-1 was isolated from tomato suspension-cultured cells and verified to be an AGP by precipitation with (beta-D-galactosyl)3 Yariv phenylglycoside and by amino acid composition analysis. Furthermore, LeAGP-1 was determined to correspond to LeAGP-1 clones based on three criteria: (1) amino acid composition identity, (2) amino acid sequence identity, and (3) specific immunoreactivity of glycosylated and deglycosylated LeAGP-1 with an antibody developed against the highly basic subdomain predicted from LeAGP-1 clones. The antibody was also used to immunolocalize LeAGP-1 in tomato to the cell surface of suspension-cultured cells, maturing metaxylem elements in young internodes and petioles, and stylar transmitting tissue cells. At the subcellular level, LeAGP-1 immunolocalized to the cell walls of these particular cells as well as to intercellular spaces between stylar transmitting tissue cells. LeAGP-1 now emerges as one of the most comprehensively studied AGPs in terms of (1) characterization at the genomic DNA, cDNA and protein levels, (2) known organ-specific and developmentally regulated mRNA expression patterns, (3) development of an antibody against a unique, peptide subdomain which specifically recognizes LeAGP-1 in its glycosylated and deglycosylated states, and (4) immunolocalization of a single, well-defined AGP molecule at the tissue and subcellular levels.     

Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGP Pc 2) and the other from suspension cultures of Nicotiana alata (AGP Na 2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGP Pc 2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGP Na 2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.     

The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion

Most aspects of plant growth involve cell surface hydroxyproline (Hyp)-rich glycoproteins (HRGPs) whose properties depend on arabinogalactan polysaccharides and arabinosides that define the molecular surface. Potential glycosylation sites are defined by an O-Hyp glycosylation code: contiguous Hyp directs arabinosylation. Clustered non-contiguous Hyp directs arabinogalactosylation. Elucidation of this code involved a single species, tobacco (Nicotiana tabacum) BY-2 cells. However, recent work suggests species variation, perhaps tissue specific Hyp glycosylation. Thus, the extent to which the Hyp glycosylation code is 'global' needs testing. We compared the ability of distantly related Arabidopsis cell cultures to process putative HRGP glycosylation motifs encoded by synthetic genes. The genes included: repetitive Ser-Pro, Ser-Pro2, Ser-Pro4 and an analog of the tomato arabinogalactan-protein, LeAGP-1DeltaGPI. All were expressed as enhanced green fluorescent protein (EGFP) fusion glycoproteins, designated: AtSO-EGFP (O=Hyp), AtSO2-EGFP, AtSO4-EGFP and AtEGFP-LeAGP-1DeltaGPI, respectively. The Arabidopsis glycosylation patterns were essentially similar to those observed in Nicotiana: non-contiguous Hyp residues in AtSO-EGFP were glycosylated exclusively with arabinogalactan polysaccharides while contiguous Hyp in AtSO2-EGFP and AtSO4-EGFP was exclusively arabinosylated. Mixed contiguous and non-contiguous Hyp residues in AtEGFP-LeAGP-1DeltaGPI were also arabinosylated and arabinogalactosylated consistent with the code. However, slightly more arabinogalactosylated Hyp and less non-glycosylated Hyp in AtEGFP-LeAGP-1DeltaGPI than tobacco NtEGFP-LeAGP-1DeltaGPI suggested Arabidopsis prolyl hydroxylases have a slightly broader specificity. Arabidopsis Hyp-arabinogalactans differed from tobacco in decreased glucuronic acid content and lack of rhamnose. Yields of the EGFP fusion glycoproteins were dramatically higher than targeted EGFP lacking Hyp-glycomodules. This validates earlier suggestions that the glycosylation of proteins facilitates their secretion.     

Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum

Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glycosylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger arabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that sequence-dependent glycosylation of small peptide motifs results in glycomodules. These small functional units in combination with other repetitive peptide modules define the properties of HRGPs. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered noncontiguous Hyp residues. To determine the minimum level of Hyp contiguity that directs arabinosylation, we designed a series of synthetic genes encoding repetitive (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n). A signal sequence targeted these endogenous substrates to the endoplasmic reticulum/Golgi for post-translational proline hydroxylation and glycosylation in transformed Nicotiana tabacum cells. The fusion glycoproteins also contained green fluorescence protein, facilitating their detection and isolation. The (Ser-Pro(2))(n) and (Ser-Hyp(4))(n) fusion glycoproteins yielded Hyp-arabinosides but no Hyp-polysaccharide. The motif (Ser-Pro(3))(n) was incompletely hydroxylated, yielding mixed contiguous/noncontiguous Hyp and a corresponding mixture of Hyp-arabinosides and Hyp-polysaccharides. These results plus circular dichroic spectra of the glycosylated and deglycosylated (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n) modules corroborate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation is indeed sequence-driven.     

AtAGP18 is localized at the plasma membrane and functions in plant growth and development

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins (HRGPs). AtAGP17, 18 and 19 comprise the lysine-rich classical AGP subfamily in Arabidopsis. Overexpression of GFP–AtAGP17/18/19 fusion proteins in Arabidopsis revealed localization of the fusion proteins on the plant cell surface of different organs. Subcellular localization of the fusion proteins at the plasma membrane was further determined by plasmolysis of leaf trichome cells. To elucidate AtAGP17/18/19 function(s), these AGPs were expressed without the green fluorescent protein (GFP) tag under the control of 35S cauliflower mosaic virus promoter. In contrast to AtAGP17/AtAGP19 overexpressors which showed phenotypes identical to wild-type plants, AtAGP18 overexpressors displayed several phenotypes distinct from wild-type plants. Specifically, these overexpressors had smaller rosettes and shorter stems and roots, produced more branches and had less viable seeds. Moreover, these AtAGP18 overexpressors exhibited similar phenotypes to tomato LeAGP-1 overexpressors, suggesting these two AGP genes may have similar function(s) in Arabidopsis and tomato.     

Molecular interactions of arabinogalactan proteins with cortical microtubules and F-actin in Bright Yellow-2 tobacco cultured cells

Arabinogalactan proteins (AGPs), a superfamily of plant hydroxyproline-rich glycoproteins, are present at cell surfaces. Although precise functions of AGPs remain elusive, they are widely implicated in plant growth and development. A well-characterized classical tomato (Lycopersicon esculentum) AGP containing a glycosylphosphatidylinositol plasma membrane anchor sequence was used here to elucidate functional roles of AGPs. Transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells stably expressing green fluorescent protein (GFP)-LeAGP-1 were plasmolysed and used to localize LeAGP-1 on the plasma membrane and in Hechtian strands. Cytoskeleton disruptors and beta-Yariv reagent (which binds and perturbs AGPs) were used to examine the role of LeAGP-1 as a candidate linker protein between the plasma membrane and cytoskeleton. This study used a two-pronged approach. First, BY-2 cells, either wild type or expressing GFP-microtubule (MT)-binding domain, were treated with beta-Yariv reagent, and effects on MTs and F-actin were observed. Second, BY-2 cells expressing GFP-LeAGP-1 were treated with amiprophosmethyl and cytochalasin-D to disrupt MTs and F-actin, and effects on LeAGP-1 localization were observed. beta-Yariv treatment resulted in terminal cell bulging, puncta formation, and depolymerization/disorganization of MTs, indicating a likely role for AGPs in cortical MT organization. beta-Yariv treatment also resulted in the formation of thicker actin filaments, indicating a role for AGPs in actin polymerization. Similarly, amiprophosmethyl and cytochalasin-D treatments resulted in relocalization of LeAGP-1 on Hechtian strands and indicate roles for MTs and F-actin in AGP organization at the cell surface and in Hechtian strands. Collectively, these studies indicate that glycosylphosphatidylinositol-anchored AGPs function to link the plasma membrane to the cytoskeleton.     

Identification of three potent hydroxyproline O‐galactosyltransferases in Arabidopsis

Arabinogalactan proteins (AGPs) are plant‐specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O‐galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O‐galactosyltransferases, such that their disruption produces phenocopies of AGP‐deficient mutants, remain to be identified. Here, we purified and identified three potent Hyp O‐galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss‐of‐function analysis indicated that approximately 90% of the endogenous Hyp O‐galactosylation activity is attributable to these three enzymes. AGP14 expressed in the triple mutant migrated much faster on SDS‐PAGE than when expressed in wild‐type, confirming a considerable decrease in levels of glycosylation of AGPs in the mutant. Loss‐of‐function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp‐linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell‐to‐cell communication during plant growth and development.     

Different arabinogalactan proteins are present in carrot (Daucus carota) cell culture medium and in seeds

Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot ( Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively high levels of glucuronic acid and rhamnose. In contrast the seed AGPs only contained arabinose and galactose-rich AGP fractions that had low levels of glucuronic acid. Linkage analysis on all fractions showed that most of the arabinose residues were terminally linked and that almost all galactose was present in the 1,3-, 1,6- and 1,3,6- form. The strongly branched type II arabinogalactans are characteristic of the carbohydrate part of AGPs. AGP characteristic amino acid residues as Hyp, Pro, Glx, Ser, Gly, Asx, Ala, Leu and Thr were detected in three different fractions.     

The latest hype on Hyp-O-glycosylation codes

Contemporary glycobiology reflects the intense interest in glycoproteins and their biological roles. Addition of saccharides by N- or O-glycosylation is precise rather than random and forms a uniquely interactive molecular surface. We designate these well conserved glycomotifs as glycomodules to emphasize their functional significance. Thus, elucidation of the glycosylation codes that determine saccharide addition is a significant goal. The focus here is on the Hyp O-glycosylation of cell wall proteins. This involves two consecutive posttranslational modifications, proline hydroxylation and glycosylation. Peptide sequence rather than conformation seems to determine these modifications. Hyp glycosylation occurs in two distinct modes: Hyp arabinosylation and Hyp galactosylation. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered non-contiguous Hyp. Elucidation of Hyp glycosylation codes involves the design and expression of putative glycomotifs as simple repetitive peptides. Thus, repetitive (Ser-Hyp), directed Hyp galactosylation resulting in the exclusive addition of arabinogalactan polysaccharide to all the non-contiguous Hyp residues. and a new AGP. Another repetitive peptide from gum arabic glycoprotein, containing both contiguous and non-contiguous Hyp, directed both modes of Hyp glycosylation. Furthermore, expression of the (Ser-Hypx)n series confirmed the arabinosylation of contiguous Hyp. Thus, the Hyp contiguity hypothesis is a useful predictive tool in the functional genomics toolbox.     

Gum arabic glycoprotein contains glycomodules of both extensin and arabinogalactan-glycoproteins

Gum arabic glycoprotein (GAGP) is a large molecular weight, hydroxyproline-rich arabinogalactan-protein (AGP) component of gum arabic. GAGP has a simple, highly biased amino acid composition indicating a repetitive polypeptide backbone. Previous work (Qi, W., Fong, C., Lamport, D.T.A., 1991. Plant Physiology 96, 848), suggested small (approximately 11 residue) repetitive peptide motifs each with three Hyp-arabinoside attachment sites and a single Hyp-arabinogalactan polysaccharide attachment site. We tested that hypothesis by sequence analysis of the GAGP polypeptide after HF-deglycosylation. A family of closely related peptides confirmed the presence of a repetitive 19-residue consensus motif. However, the motif: Ser-Hyp-Hyp-Hyp-Thr-Leu-Ser-Hyp-Ser- Hyp-Thr-Hyp-Thr-Hyp-Hyp-Leu-Gly-Pro-His, was about twice the size anticipated. Thus, judging by Hyp-glycoside profiles of GAGP, the consensus motif contained six Hyp-arabinosides rather than three and two Hyp-polysaccharides rather than one. We inferred the glycosylation sites based on the Hyp contiguity hypothesis which predicts arabinosides on contiguous Hyp residues and arabinogalactan polysaccharides on clustered non-contiguous Hyp residues, i.e. the GAGP motif would consist of arabinosylated contiguous Hyp blocks flanking two central Hyp-polysaccharides. We predict this rigidifies the glycoprotein, enhances the overall symmetry of the glycopeptide motif, and may explain some of the remarkable properties of gum arabic.     

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [¹⁴C]Gal from UDP-[¹⁴C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)₇ and anhydrous hydrogen fluoride-deglycosylated d(AO)₅₁. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [¹⁴C]Gal to (AO)₇, and the resulting product co-eluted with (AO)₇ by reverse-phase HPLC. Acid hydrolysis of the [¹⁴C]Gal-(AO)₇ product released ¹⁴C-radiolabel as Gal only. Base hydrolysis of the [¹⁴C]Gal-(AO)₇ product released a ¹⁴C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg²⁺ or Mn²⁺ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.     

Developmental expression and perturbation of arabinogalactan-proteins during seed germination and seedling growth in tomato

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins present throughout the plant kingdom. A synthetic chemical reagent, ( β - d -Gal)₃ Yariv reagent, specifically binds AGPs and can be used for histochemical staining, isolating and probing the function of AGPs. Here, the role of AGPs in tomato ( Lycopersicon esculentum Mill. cv. UC82B) seed germination and seedling growth was examined by following expression of AGPs during these events and by treatment with ( β - d -Gal)₃ Yariv to perturb AGP function. AGP expression changed during germination and seedling development both quantitatively and qualitatively as revealed by analysis of total AGP content, crossed electrophoresis patterns, RNA blots using LeAGP-1 probe, and western blots with LeAGP-1, JIM13, and MAC207 antibodies. ( β - d -Gal)₃ Yariv treatment of seeds and developing seedlings did not affect percent seed germination, but markedly inhibited seedling growth in roots and to a lesser degree in shoots. Root growth inhibition encompassed reductions in overall root length, epidermal root cell elongation, root cell numbers and root hair formation. This growth inhibition was reversible following removal of ( β - d -Gal)₃ Yariv. In a related experiment, water uptake by tomato seedlings was greatly inhibited by ( β - d -Gal)₃ Yariv treatment. Based on these experiments, AGPs are clearly associated with tomato seedling development and likely to function in root growth, more specifically in cell elongation, cell proliferation, root hair formation and water uptake.     

Focus Issue on Plant Cell Walls: A Bioinformatics Approach to the Identification,Classification, and Analysis of Hydroxyproline-Rich Glycoproteins

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development. This superfamily consists of three members: hyperglycosylated arabinogalactan proteins (AGPs), moderately glycosylated extensins (EXTs), and lightly glycosylated proline-rich proteins (PRPs). Hybrid and chimeric versions of HRGP molecules also exist. In order to “mine” genomic databases for HRGPs and to facilitate and guide research in the field, the BIO OHIO software program was developed that identifies and classifies AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs from proteins predicted from DNA sequence data. This bioinformatics program is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs. HRGPs identified by the program are subsequently analyzed to elucidate the following: (1) repeating amino acid sequences, (2) signal peptide and glycosylphosphatidylinositol lipid anchor addition sequences, (3) similar HRGPs via Basic Local Alignment Search Tool, (4) expression patterns of their genes, (5) other HRGPs, glycosyl transferase, prolyl 4-hydroxylase, and peroxidase genes coexpressed with their genes, and (6) gene structure and whether genetic mutants exist in their genes. The program was used to identify and classify 166 HRGPs from Arabidopsis (Arabidopsis thaliana) as follows: 85 AGPs (including classical AGPs, lysine-rich AGPs, arabinogalactan peptides, fasciclin-like AGPs, plastocyanin AGPs, and other chimeric AGPs), 59 EXTs (including SP₅ EXTs, SP₅/SP₄ EXTs, SP₄ EXTs, SP₄/SP₃ EXTs, a SP₃ EXT, “short” EXTs, leucine-rich repeat-EXTs, proline-rich extensin-like receptor kinases, and other chimeric EXTs), 18 PRPs (including PRPs and chimeric PRPs), and AGP/EXT hybrid HRGPs.The genomics era has produced vast amounts of biological data that await examination. In order to “mine” such data effectively, a bioinformatics approach can be utilized to identify genes of interest, subject them to various in silico analyses, and extract relevant biological information on them from various public databases. Examination of such data produces novel insights with respect to the genes in question and can be used to facilitate and guide further research in the field. Such is the case here, where bioinformatics tools were developed to identify, classify, and analyze members of the Hyp-rich glycoprotein (HRGP) superfamily encoded by the Arabidopsis (Arabidopsis thaliana) genome.HRGPs are a superfamily of plant cell wall proteins that are subdivided into three families, arabinogalactan proteins (AGPs), extensins (EXTs), and Pro-rich proteins (PRPs), and extensively reviewed (Showalter, 1993; Kieliszewski and Lamport, 1994; Nothnagel, 1997; Cassab, 1998; José-Estanyol and Puigdomènech, 2000; Seifert and Roberts, 2007). However, it has become increasingly clear that the HRGP superfamily is perhaps better represented as a spectrum of molecules ranging from the highly glycosylated AGPs to the moderately glycosylated EXTs and finally to the lightly glycosylated PRPs. Moreover, hybrid HRGPs, composed of HRGP modules from different families, and chimeric HRGPs, composed of one or more HRGP modules within a non-HRGP protein, also can be considered part of the HRGP superfamily. Given that many HRGPs are composed of repetitive protein sequences, particularly the EXTs and PRPs, and many have low sequence similarity to one another, particularly the AGPs, BLAST searches typically identify only a few closely related family members and do not represent a particularly effective means to identify members of the HRGP superfamily in a comprehensive manner.Building upon the work of Schultz et al. (2002) that focused on the AGP family, a new bioinformatics software program, BIO OHIO, developed at Ohio University, makes it possible to search all 28,952 proteins encoded by the Arabidopsis genome and identify putative HRGP genes. Two distinct types of searches are possible with this program. First, the program can search for biased amino acid compositions in the genome-encoded protein sequences. For example, classical AGPs can be identified by their biased amino acid compositions of greater then 50% Pro (P), Ala (A), Ser (S), and Thr (T), as indicated by greater than 50% PAST. Similarly, arabinogalactan peptides (AG peptides) are identified by biased amino acid compositions of greater then 35% PAST, but the protein (i.e. peptide) must also be between 50 and 90 amino acids in length. Likewise, PRPs can be identified by a biased amino acid composition of greater then 45% PVKCYT. Second, the program can search for specific amino acid motifs that are commonly found in known HRGPs. For example, SP₄ pentapeptide and SP₃ tetrapeptide motifs are associated with EXTs, a fasciclin H1 motif is found in fasciclin-like AGPs (FLAs), and PPVX(K/T) (where X is any amino acid) and KKPCPP motifs are found in several known PRPs (Fowler et al., 1999). In addition to searching for HRGPs, the program can analyze proteins identified by a search. For example, the program checks for potential signal peptide sequences and glycosylphosphatidylinositol (GPI) plasma member anchor addition sequences, both of which are associated with HRGPs (Showalter, 1993, 2001; Youl et al., 1998; Sherrier et al., 1999; Svetek et al., 1999). Moreover, the program can identify repeated amino acid sequences within the sequence and has the ability to search for bias amino acid compositions within a sliding window of user-defined size, making it possible to identify HRGP domains within a protein sequence.Here, we report on the use of this bioinformatics program in identifying, classifying, and analyzing members of the HRGP superfamily (i.e. AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs) in the genetic model plant Arabidopsis. An overview of this bioinformatics approach is presented in Figure 1. In addition, public databases and programs were accessed and utilized to extract relevant biological information on these HRGPs in terms of their expression patterns, most similar sequences via BLAST analysis, available genetic mutants, and coexpressed HRGP, glycosyl transferase (GT), prolyl 4-hydroxylase (P4H), and peroxidase genes in Arabidopsis. This information provides new insight to the HRGP superfamily and can be used by researchers to facilitate and guide further research in the field. Moreover, the bioinformatics tools developed here can be readily applied to protein sequences from other species to analyze their HRGPs or, for that matter, any given protein family by altering the input parameters.Open in a separate windowFigure 1.Bioinformatics workflow diagram summarizing the identification, classification, and analysis of HRGPs (AGPs, EXTs, and PRPs) in Arabidopsis. Classical AGPs were defined as containing greater than 50% PAST coupled with the presence of AP, PA, SP, and TP repeats distributed throughout the protein, Lys-rich AGPs were a subgroup of classical AGPs that included a Lys-rich domain, and chimeric AGPs were defined as containing greater than 50% PAST coupled with the localized distribution of AP, PA, SP, and TP repeats. AG peptides were defined to be 50 to 90 amino acids in length and containing greater than 35% PAST coupled with the presence of AP, PA, SP, and TP repeats distributed throughout the peptide. FLAs were defined as having a fasciclin domain coupled with the localized distribution of AP, PA, SP, and TP repeats. Extensins were defined as containing two or more SP₃ or SP₄ repeats coupled with the distribution of such repeats throughout the protein; chimeric extensins were similarly identified but were distinguished from the extensins by the localized distribution of such repeats in the protein; and short extensins were defined to be less than 200 amino acids in length coupled with the extensin definition. PRPs were identified as containing greater than 45% PVKCYT or two or more KKPCPP or PVX(K/T) repeats coupled with the distribution of such repeats and/or PPV throughout the protein. Chimeric PRPs were similarly identified but were distinguished from PRPs by the localized distribution of such repeats in the protein. Hybrid HRGPs (i.e. AGP/EXT hybrids) were defined as containing two or more repeat units used to identify AGPs, extensins, or PRPs. The presence of a signal peptide was used to provide added support for the identification of an HRGP but was not used in an absolute fashion. Similarly, the presence of a GPI anchor addition sequence was used to provide added support for the identification of classical AGPs and AG peptides, which are known to contain such sequences. BLAST searches were also used to provide some support to our classification if the query sequence showed similarity to other members of an HRGP subfamily. Note that some AGPs, particularly chimeric AGPs, and PRPs were identified from an Arabidopsis database annotation search and that two chimeric extensins were identified from the primary literature as noted in the text.     

A protease/peptidase from culture medium of Flammulina velutipes that acts on arabinogalactan-protein

Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease.     

Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato

 Arabinogalactan-proteins (AGPs) are highly glycosylated cell surface proteins that are thought to function in plant growth and development. The developmentally regulated expression of LeAGP-1, a novel and major AGP in tomato, was examined in different organs and tissues of tomato (Lycopersicon esculentum Mill. cv. UC82B) plants with an anti-peptide antibody (i.e. the PAP antibody) directed specifically against the lysine-rich subdomain of the LeAGP-1 core protein. During cell differentiation in tomato plants, LeAGP-1 was associated with cell wall thickening and lignification of particular cell types. Specifically, LeAGP-1 was detected in secondary wall thickenings of maturing metaxylem and secondary xylem tracheary elements in roots and stems, and in thickened cell walls of phloem sieve elements. However, LeAGP-1 was also present in thin-walled, cortical parenchyma cells of seedling roots as well as thick-walled collenchyma cells in young stems, both of which are not lignified. Based on these observed patterns, possible roles for LeAGP-1 in plant growth and development are discussed. Received: 17 August 1999 / Accepted: 7 October 1999