Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

Dynamic turnover of arabinogalactan proteins in cultured Arabidopsis cells

Arabinogalactan proteins (AGPs) are very large proteoglycans thought to have more of a signaling than a structural role when secreted into the plant cell wall. AGPs are also the first known family of abundant plant proteins synthesized with glycosylphosphatidylinositol(GPI) anchors. Nascent cellular Arabidopsis AGPs, still bearing an intact GPI anchor, and AGPs copiously discharged into the culture medium after phospholipase-cleavage of their anchor were each represented by more than 15 seemingly homologous molecular species of increasing size. In washed cells ³H-ethanolamine was slowly incorporated into each AGP’s GPI anchor via phosphatidylethanolamine. Pulse labeling of AGPs by ³H-acetate and by ³H-galactose was much more rapid, allowing labeled AGP detection in the growth medium within 1 h. HPLC analysis of the radiolabel distribution in AGPs secreted within 1–8 h revealed a sharp preference for the larger molecular species. After several hours a population of smaller radioactive AGP species began to appear in the medium. Following certain manipulations of the cells newly secreted AGP species measured by HPLC on a relative mass basis formed a pattern surprisingly different from the radioactivity pattern, although larger species still dominated. Thus Arabidopsis cells appear capable of releasing higher mass AGP species apparently stored in cell wall sites along with a unique mixture of freshly synthesized AGPs in combinations potentially active in signaling.     

Using genomic resources to guide research directions. The arabinogalactan protein gene family as a test case

Arabinogalactan proteins (AGPs) are extracellular hydroxyproline-rich proteoglycans implicated in plant growth and development. The protein backbones of AGPs are rich in proline/hydroxyproline, serine, alanine, and threonine. Most family members have less than 40% similarity; therefore, finding family members using Basic Local Alignment Search Tool searches is difficult. As part of our systematic analysis of AGP function in Arabidopsis, we wanted to make sure that we had identified most of the members of the gene family. We used the biased amino acid composition of AGPs to identify AGPs and arabinogalactan (AG) peptides in the Arabidopsis genome. Different criteria were used to identify the fasciclin-like AGPs. In total, we have identified 13 classical AGPs, 10 AG-peptides, three basic AGPs that include a short lysine-rich region, and 21 fasciclin-like AGPs. To streamline the analysis of genomic resources to assist in the planning of targeted experimental approaches, we have adopted a flow chart to maximize the information that can be obtained about each gene. One of the key steps is the reformatting of the Arabidopsis Functional Genomics Consortium microarray data. This customized software program makes it possible to view the ratio data for all Arabidopsis Functional Genomics Consortium experiments and as many genes as desired in a single spreadsheet. The results for reciprocal experiments are grouped to simplify analysis and candidate AGPs involved in development or biotic and abiotic stress responses are readily identified. The microarray data support the suggestion that different AGPs have different functions.     

Arabinogalactan proteins are required for apical cell extension in the moss Physcomitrella patens

Cell biological, structural, and genetic approaches have demonstrated the presence of arabinogalactan proteins (AGPs) in the moss Physcomitrella patens and provided evidence for their function in cell expansion and specifically in the extension of apical tip-growing cells. Inhibitor studies indicated that apical cell expansion in P. patens is blocked by synthetic AGP binding beta-glucosyl Yariv reagent (betaGlcYR). The anti-(1-->5)-alpha-L-arabinan monoclonal antibody LM6 binds to some AGPs in P. patens, to all plasma membranes, and to the cell wall surface at the most apical region of growing protonemal filaments. Moreover, LM6 labeling of cell walls at the tips of apical cells of P. patens was abolished in the presence of betaGlcYR, suggesting that the localized movement of AGPs from the plasma membrane to the cell wall is a component of the mechanism of tip growth. Biochemical and bioinformatic analyses were used to identify seven P. patens ESTs encoding putative AGP core proteins from homology with Arabidopsis thaliana, Brassica napus, and Oryza sativa sequences and from peptide fragments isolated from betaGlcYR-precipitated AGPs. Gene knockout by homologous recombination of one of these genes, P. patens AGP1, encoding a classical AGP core protein, resulted in reduced cell lengths in protonemal filaments, indicating a role for AGP1 in apical cell expansion in P. patens.     

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.     

Carrot arabinogalactan proteins are interlinked with pectins

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI-TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.     

The cytological changes of tobacco zygote and proembryo cells induced by beta-glucosyl Yariv reagent suggest the involvement of arabinogalactan proteins in cell division and cell plate formation

ABSTRACT: BACKGROUND: In dicotyledonous plant, the first asymmetric zygotic division and subsequent several cell divisions are crucial for proembryo pattern formation and later embryo development.. Arabinogalactan proteins (AGPs) are a family of extensively glycosylated cell surface proteins that are thought to have important roles in various aspects of plant growth and development, including embryogenesis. Previous results from our laboratory show that AGPs are concerned with tobacco egg cell fertilization and zygotic division. However, how AGPs interact with other factors involved in zygotic division and proembryo development remains unknown. RESULTS: In this study, we used the tobacco in vitro zygote culture system and series of meticulous cell biology techniques to investigate the roles of AGPs in zygote and proembryo cell division. For the first time, we examined tobacco proembryo division patterns detailed to every cell division. The bright-field images and statistical results both revealed that with the addition of an exogenous AGPs inhibitor, beta-glucosyl Yariv (beta-GlcY) reagent, the frequency of aberrant division increased remarkably in cultured tobacco zygotes and proembryos, and the cell plate specific locations of AGPs were greatly reduced after beta-GlcY treatment. In addition, the accumulations of new cell wall materials were also significantly affected by treating with beta-GlcY. Detection of cellulose components by Calcofluor white stain showed that strong fluorescence was located in the newly formed wall of daughter cells after the zygotic division of in vivo samples and the control samples from in vitro culture without beta-GlcY treatment; while there was only weak fluorescence in the newly formed cell walls with beta-GlcY treatment. Immunocytochemistry examination with JIM5 and JIM7 respectively against the low- and high-esterified pectins displayed that these two pectins located in opposite positions of zygotes and proembryos in vivo and the polarity was not affected by beta-GlcY. Furthermore, FM4-64 staining revealed that endosomes were distributed in the cell plates of proembryos, and the localization pattern was also affected by beta-GlcY treatment. These results were further confirmed by subsequent observation with transmission electron microscopy. Moreover, the changes to proembryo cell-organelles induced by beta-GlcY reagent were also observed using fluorescent dye staining technique. CONCLUSIONS: These results imply that AGPs may not only relate to cell plate position decision, but also to the location of new cell wall components. Correlated with other factors, AGPs further influence the zygotic division and proembryo pattern establishment in tobacco.     

A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization)

Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.     

The classical arabinogalactan protein gene family of arabidopsis

Arabinogalactan proteins (AGPs) are extracellular proteoglycans implicated in plant growth and development. We searched for classical AGPs in Arabidopsis by identifying expressed sequence tags based on the conserved domain structure of the predicted protein backbone. To confirm that these genes encoded bona fide AGPs, we purified native AGPs and then deglycosylated and deblocked them for N-terminal protein sequencing. In total, we identified 15 genes encoding the protein backbones of classical AGPs, including genes for AG peptides-AGPs with very short backbones (10 to 13 amino acid residues). Seven of the AGPs were verified as AGPs by protein sequencing. A gene encoding a putative cell adhesion molecule with AGP-like domains was also identified. This work provides a firm foundation for beginning functional analysis by using a genetic approach.     

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.     

Localization of arabinogalactan proteins in the membrane system of etiolated hypocotyls of Phaseolus vulgaris L.

The subcellular distribution of arabinogalactan protein (AGP) in etiolated bean hypocotyls was studied by isopycnic density centrifugation on sucrose gradients at different Mg²⁺ concentrations. The distribution of hydroxyproline (a major amino acid in AGP) in the membrane-containing fractions indicated that hydroxyproline-containing proteins were associated with rough endoplasmic reticulum, possibly with the Golgi apparatus, and with the plasma membrane. Non-specific binding of hydroxyproline-containing molecules to membranes could be excluded. To detect AGPs, fractions obtained after isopycnic density centrifugation were isoelectrofocused on polyacrylamide gels, and the gels were stained with β-Gal-Yariv reagent. Bands appeared only at low pH values, where also most hydroxyproline was found. In the fractions at low densities (presumably membranefree), several bands were visible supporting the idea of the heterogeneous character of soluble AGP. The distribution of AGP in the membranous fractions strongly indicated that AGP was associated with the plasma membrane. Specific agglutination of protoplasts in the presence of β-Gal-Yariv reagent indicated that AGP was exposed at the outside of the cell membrane.     

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.     

An agarose gel electrophoresis method for the separation of arabinogalactan proteins

A method for resolving plasma membrane associated arabinogalactan proteins (AGPs) has been developed. Plasma membranes purified by aqueous polymer two-phase partitioning were first subjected to Triton X-114 fractionation. The resulting water phase contained all detectable plasma membrane-bound AGPs. Plasma membrane AGPs were then resolved in an SDS-agarose gel electrophoresis system (SDS-AGE). For separating plasma membrane AGP species of the same apparent molecular weight but with different net charge, a two-dimensional electrophoresis system was used, utilizing isoelectric focusing in an immobilized pH gradient in the first dimension and SDS-AGE in the second dimension. These methods enabled the separation of individual plasma membrane AGPs. In comparison, SDS-PAGE methods left AGPs as unresolved high molecular-weight smears. The methods described here may help to establish some basic features of AGPs, such as the number, organization, and protein and carbohydrate characteristics of plasma membrane AGPs, as well as the relationship between plasma membrane and extracellular AGPs.     

Arabinogalactan proteins in root and pollen-tube cells: distribution and functional aspects

BACKGROUND: Arabinogalactan proteins (AGPs) are complex proteoglycans of the cell wall found in the entire plant kingdom and in almost all plant organs. AGPs encompass a large group of heavily glycosylated cell-wall proteins which share common features, including the presence of glycan chains especially enriched in arabinose and galactose and a protein backbone particularly rich in hydroxyproline residues. However, AGPs also exhibit strong heterogeneities among their members in various plant species. AGP ubiquity in plants suggests these proteoglycans are fundamental players for plant survival and development. SCOPE: In this review, we first present an overview of current knowledge and specific features of AGPs. A section devoted to major tools used to study AGPs is also presented. We then discuss the distribution of AGPs as well as various aspects of their functional properties in root tissues and pollen tubes. This review also suggests novel directions of research on the role of AGPs in the biology of roots and pollen tubes.     

Arabinogalactan proteins in embryogenic and non-embryogenic callus cultures of Euphorbia pulcherrima

The arabinogalactan protein (AGP) fractions of embryogenic and non-embryogenic callus lines of Euphorbia pulcherrima Willd. ex. Klotzsch were analysed over a cultivation period of 9 weeks using the β -glucosyl Yariv reagent and an anti-AGP antibody (LM2). The amount of AGPs detected with the Yariv reagent increased in embryogenic cultures during the development of somatic embryos. The embryogenic and non-embryogenic callus contained different sets of AGPs characterized with the Yariv reagent and the LM2 monoclonal antibody. AGPs recognized by LM2 are localized primarily in the protodermal cells of globular somatic embryos. The development of somatic embryos of E. pulcherrima appears to be associated with the presence of particular AGPs.