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.     

Arabinogalactan-protein epitope Gal4 is differentially regulated and localized in cell lines of hybrid fir (<Emphasis Type="Italic">Abies alba</Emphasis> × <Emphasis Type="Italic">Abies cephalonica</Emphasis>) with different embryogenic and regeneration potential

Arabinogalactan proteins (AGPs) are important proteoglycans regulating somatic embryogenesis in diverse plant species. Embryogenic cells of somatic embryos are covered by special extracellular cell wall layer called extracellular surface matrix network (ECMSN) at their early developmental stages. Here we show that highly embryogenic cell line AC78 of hybrid fir (Abies alba × Abies cephalonica) differs from very low-embryogenic cell line AC77 in the abundance, subcellular localization and deposition of subset of secreted AGPs. A specific AGP epitope containing Gal residues and reacting to Gal4 antibody is secreted and deposited into ECMSN, which covers the surface of the embryogenic cells showing high embryogenic and regeneration capacity in the cell line AC78. On the other hand, this Gal4 AGP epitope was not secreted and/or found on the surface of meristematic cells showing low embryogenic and regeneration capacity in the cell line AC77, as well as on the surface of non-embryogenic suspensor cells and callus cells in both cell lines AC77 and AC78. As a positive control, we have used another AGP epitope LM2 (containing glucuronic acid) showing no significant differences in these two Abies hybrid lines. This study defines specific AGPs containing β-(1→6)-galactotetraosyl group as a first molecular component of ECMSN covering embryogenic cells in gymnosperms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Purification and cloning of an arabinogalactan-protein from xylem of loblolly pine

 An arabinogalactan-protein (AGP) was purified from differentiating xylem of loblolly pine (Pinus taeda L.) and the N-terminal sequence used to identify a cDNA clone. The protein, PtaAGP3, was not coded for by any previously identified AGP-like genes. Moreover, PtaAGP3 was abundantly and preferentially expressed in differentiating xylem. The encoded protein contains four domains, a signal peptide, a cleaved hydrophilic region, a region rich in serine, alanine, and proline/hydroxyproline, and a hydrophobic C-terminus. It is postulated to contain a GPI (glycosylphosphatidylinositol) anchor site. If the protein is cleaved at the putative GPI anchor site, as has been observed in other classical AGPs, all but the Ser-Ala-Pro/Hyp-rich domain may be missing from the mature protein. Xylem-specific AGPs are hypothesized to be involved in xylem development. Received: 29 July 1999 / Accepted: 19 August 1999     

Involvement of arabinogalactan proteins in the control of cell proliferation of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> suspension cultures

Arabinogalactan proteins (AGPs) secreted by zucchini squash (Cucurbita pepo L.) cell cultures into the medium are implicated in cell proliferation. Conditioned medium derived from cell suspensions of squash cultivar Dundoo could enhance multiplication rate of slow-growing cell line Cx3005. To examine the role of AGPs, a precipitation assay was performed using Yariv reagent which binds selectively to AGPs. This AGP precipitation as well as proteinase application arrested cell division. However, chitinase treatment successfully increased embryogenic callus mass. A growth promotion was also obtained by arabinogalactan addition to the culture medium. Immunoblotting analysis using the MAC 207 anti-AGP monoclonal antibody showed high AGP expression in Dundoo cell cultures.     

Salt stress upregulates periplasmic arabinogalactan proteins: using salt stress to analyse AGP function

Arabinogalactan proteins (AGPs) are implicated in cell expansion by unknown mechanisms, thus AGP content and cell-expansion rate might be correlated. We used Yariv reagent to quantify release rates and distribution of AGP at the cell surface of tobacco BY-2 cells: plasma membrane (M); soluble periplasmic AGPs released by cell rupture (S); cell wall (W); and growth medium (Gsink). In contrast to earlier reports, we observed massive upregulation of AGPs in salt-stressed cells, and hence the absence of a simple, direct cause-and-effect relationship between growth rate and AGP release. There was a more subtle connection. A dynamic flux model, M-->S-->W-->Gsink, indicated that turnover was nondegradative, with little free diffusion of AGPs trapped in the pectic matrix of nonadapted cells where transmural migration of high molecular-weight AGPs occurred mainly by plug flow (apposition and extrusion). In contrast, however, an up to sixfold increased AGP release rate in the slower-growing salt-adapted cells indicated a greatly increased rate of AGP diffusion through a much more highly porous pectic network. We hypothesize that classical AGPs act as pectin plasticizers. This explains how beta-D-glycosyl Yariv reagents might inhibit expansion growth by crosslinking monomeric AGPs, and thus mimic an AGP loss-of-function mutation.     

Characterization of an arabinogalactan-protein from suspension culture of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Suspension cultures of Echinacea purpurea have been established in MS medium supplemented with 2,4-D and an arabinogalactan-protein (AGP) was purified from the secreted soluble polymers by precipitation with ethanol, followed by precipitation with β-glucosyl Yariv reagent. It revealed typical features of AGPs: a high amount of polysaccharide (90% w/w) with the dominating monosaccharides galactose and arabinose and some glucuronic acid, and a small protein moiety (10% w/w) with the main amino acids Ala, Hyp, Glx, Ser, Asx and Thr. Linkage- and NMR-analyses showed the polysaccharide part to be composed of a branched core-polysaccharide of 3-, 6- and 3,6-linked Galp residues with terminal Araf, Arap, Galp and GlcAp residues. Compared to an AGP from pressed juice of the aerial parts of Echinacea purpurea, differences particularly in terminal arabinose mono- and oligosaccharides in arabinogalactan (AG) side branches could be detected. Testing of different AGP-antibodies with both AGPs confirmed the results of the analytical investigations. Binding of AGPs from plant and cell cultures to LM2, a monoclonal AGP-antibody reacting with a GlcA containing epitope, was comparable. The reactivity of a monoclonal antibody raised against the AGP from the plant recognizing a galactan epitope was also nearly similar with both AGPs. In contrast, polyclonal antibodies raised against the AGP from the plant and directed against an Araf-containing epitope of the AG side branches showed nearly no cross reactivity with the AGP from cell culture.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

Arabinogalactan-protein epitopes in somatic embryogenesis of Daucus carota L.

Two monoclonal antibodies (ZUM 15 and ZUM 18) directed against carrot (Daucus carota L.) seed arabinogalactan proteins (AGPs) were used to isolate specific AGP fractions. For both carrot and tomato (Lycopersicon esculentum Mill.) seed AGPs analyzed by crossedelectrophoresis, the ZUM 15 and ZUM 18 AGP fractions showed one identical peak. However, the Rf values for the two species were different: 0.82 for carrot seed AGPs and 0.52 for tomato seed AGPs. When the fractionated AGPs (carrot or tomato) were added to carrot cell lines they had a dramatic effect on the culture. One AGP fraction (ZUM 15 AGPs) was able to induce vacuolation of embryogenic cells. Those cells failed to produce embryos. The other AGP fraction (ZUM 18 AGPs) increased the percentage of embryognic cells from about 40% up to 80% within one week and this subsequently resulted in the formation of more embryos on hormone-free medium. This activity was higher than that of unfractionated carrot seed AGPs, while the optimum concentration was 50-fold lower. Since both ZUM 18 AGPs (carrot or tomato) yielded identical responses it can be concluded that neither the Rf value nor the source are essential for biological activity. The dose-response curve of ZUM 18 AGPs showed a sharp optimum. When the AGPs that also bound to the antibody ZUM 15 were removed, the dose-response curve of the remaining AGPs (containing only the ZUM 18 epitope, not the ZUM 15 epitope) resembled a saturation curve. Regardless of its concentration, the fraction in which AGP molecules contained both epitopes showed no appreciable embryogenesis-promoting activity. The biological activity of AGPs was therefore determined by the presence of embryogenesis-enhancing and-inhibiting epitopes. The inhibiting and enhancing epitopes can be located on separate molecules or one single AGP molecule.     

Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (β-D-glucosyl)₃ Yariv phenylglycoside [(β-D-Glc)₃] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(β-D-Glc)₃ complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall. Received: 14 August 1997 / Accepted: 9 September 1997     

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.     

Isolation and identification of glycosylphosphatidylinositol-anchored arabinogalactan proteins and novel beta-glucosyl Yariv-reactive proteins from seeds of rice (Oryza sativa)

Arabinogalactan proteins (AGPs) are highly glycosylated extracellular glycoproteins playing important roles in plant growth and development. We have previously reported the possibility that AGPs are involved in the induction of alpha-amylase by gibberellin (GA) in barley aleurone layers by using the beta-glucosyl Yariv reagent (beta-GlcY), which has been presumed to specifically bind AGPs. In this present study, we isolated beta-GlcY-reactive proteins from rice bran rich in aleurone cells. The N-terminal sequences of classical AGP and AG peptides were determined from hydrophilic fractions obtained by reversed phase HPLC. Interestingly, a novel non-specific lipid transfer protein-like protein (OsLTPL1) and a novel early nodulin-like protein (OsENODL1) were also identified in the more hydrophobic fractions from HPLC as beta-GlcY-reactive proteins. Expression analysis of the genes coding for these proteins was performed. While classical AGP, AG peptides and OsLTPL1 were expressed in various parts of rice, OsENODL1 showed temporally and spatially specific expression in the aleurone layers. This new beta-GlcY-reactive protein is a promising candidate for the extracellular signaling factors of GA action in cereal seeds. Furthermore, the possibility that proteins with the AG glycomodule might react with beta-GlcY may broaden the definition of AGPs.     

Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside

Arabinogalactan proteins (AGPs) are abundant complex macromolecules involved in both reproductive and vegetative plant growth. They are secreted at pollen tube tips in Lilium longiflorum. Here, we report the effect of the (beta-D-glucosyl)3 Yariv phenylglycoside, known to interact with AGPs, on pollen tube extension in several plant species. In Annona cherimola the Yariv reagent clearly inhibited pollen tube extension within 1-2 h of treatment, as demonstrated previously for L. longiflorum, but had no effect on Lycopersicon pimpinellifolium, Aquilegia eximia, and Nicotiana tabacum. With the monoclonal antibody JIM13 we also examined these same species for evidence that they secreted AGPs at their pollen tube tips. Only A. cherimola showed evidence of AGPs at the pollen tube tip as does lily. The Yariv reagent causes arrest of tube growth in both A. cherimola and lily, but its removal from the medium allows regeneration of new tip growth in both species. We show that the site of the new emerging tip in lily can be predicted by localization of AGP secretion. Labeling with JIM13 appeared on the flanks of the arrested tip 1 h after removal of the Yariv reagent from the growth medium. After 4 h, many of the Yariv reagent-treated pollen tubes had regenerated new pollen tubes with the tips brightly labeled by JIM13 and with a collar of AGPs left at the emergence site. During this recovery, esterified pectins colocalized with AGPs. Secretion at the site of the new tip may be important in the initial polarization event that occurs on the flanks of the arrested tube tip and results in a new pollen tube.     

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.