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     

Effects of Yariv phenylglycosides onRosa cell suspensions: Evidence for the involvement of arabinogalactan-proteins in cell proliferation

Treatment of ‘Paul's Scarlet rose (Rosa sp.) cell suspensions with β-D-glucosyl Yariv phenylglycoside (β-D-Glc)₃, a chromophoric molecule that selectively binds arabinogalactan-proteins (AGPs), caused inhibition of cell growth in a concentration-dependent manner, with complete inhibition of growth occurring at 50 μM (β-D-Glc)₃ in the culture medium. Growth was not inhibited by either α-D-galactosyl or β-D-mannosyl Yariv phenylglycosides which do not bind AGPs. Staining of cells with fluorescein diacetate indicated that (β-D-Glc)₃ did not affect cell viability. Upon transfer of 50 μM (β-D-Glc)₃-treated cells to control conditions, cell growth recovered with a time-course similar to that of control cells. Cell sizes in control and (β-D-Glc)₃-treated cultures were similar, indicating that the mechanism of growth inhibition by (β-D-Glc)₃ involved suppression of cell division. Two different analyses of (β-D-Glc)₃-treated cells both showed that approximately 95% of the bound (β-D-Glc)₃ was in the cell wall. Molecules that bound (β-D-Glc)₃ were extracted from the cell wall and were identified as AGPs, as judged by their carbohydrate and amino acid compositions.     

A role for arabinogalactan-proteins in root epidermal cell expansion

Arabinogalactan-proteins (AGPs) are abundant plant proteoglycans that react with (β-d-Glc)₃ but not (β-d-Man)₃ Yariv reagent. We report here that treatment with (β-d-Glc)₃ Yariv reagent caused inhibition of root growth of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, the treated roots exhibited numerous bulging epidermal cells. Treatment with (β-d-Man)₃ Yariv reagent did not have any such effects. These results indicate a role for AGPs in root growth and control of epidermal cell expansion. Because treatment with (β-d-Glc)₃ Yariv reagent phenocopies the reb1 (root epidermal cell bulging) mutant of Arabidopsis, AGPs were extracted from the reb1-1 mutant and compared with those of the wild type. The reb1-1 roots contained an approximately 30% lower level of AGPs than the wild type. More importantly, while the profile of AGPs from wild-type roots showed two major peaks upon crossed electrophoresis, the profile of AGPs from reb1-1 roots exhibited only one of the major peaks. Therefore, the reb1 phenotype appears to be a result of defective or missing root AGPs. Taken together, this pharmacological and genetic evidence strongly indicates a function of AGPs in the control of root epidermal cell expansion. Received: 13 February 1997 / Accepted: 1 April 1997     

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

 Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474' (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed. Received: 26 August 1999 / Accepted: 28 January 2000     

Roles of arabinogalactan proteins in cotyledon formation and cell wall deposition during embryo development of <Emphasis Type="Italic">Arabidopsis</Emphasis>

Arabinogalactan proteins (AGPs) are a class of highly glycosylated, widely distributed proteins in higher plants. In the previous study, we found that the green fluorescence from JIM13-labeled AGPs was mainly distributed in embryo proper and the basal part of suspensor but gradually disappeared after the torpedo-stage embryos in Arabidopsis. And (β-d-Glc)₃ Yariv phenylglycoside (βGlcY), a synthetic reagent that specifically binds to AGPs, could inhibit embryo development. In this study, as a continuous work, we investigated the AGP functions in embryo germination, cotyledon formation, and cell wall deposition in Arabidopsis embryos by using immunofluorescent, immunoenzyme, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) techniques. The results showed that 50 μM βGlcY caused inhibition of embryo germination, formation of abnormal cotyledon embryos, and disorder of cotyledon vasculature. Compared with the normal embryos in vitro and in vivo, the AGPs and pectin signals were quite weaker in the whole abnormal embryos, whereas the cellulose signal was stronger in the shoot apical meristem (SAM) of abnormal embryo by calcofluor white staining. The FTIR assay demonstrated that the cell wall of abnormal embryos was relatively poorer in pectins and richer in cellulose than those of normal embryos. By TEM observation, the SAM cells of the abnormal embryos had less cytoplasm, more plastid and starch grains, and larger vacuole than that of normal embryos. These results indicated that AGPs may play roles in embryo germination, cotyledon formation, cell wall cellulose and pectin deposition, and cell division potentiality during embryo development of Arabidopsis.     

Arabinogalactan proteins are involved in cell aggregation of cell suspension cultures of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Arabinogalactan proteins (AGPs) are glycoproteins present at cell surfaces. Although exact functions of AGPs remain elusive, they are implicated in plant growth and development. The aim of this study was to evaluate the role of AGPs in the process of cell aggregation of Beta vulgaris L. suspension cultures. It was observed that B. vulgaris suspension cultures accumulated AGPs in parallel form to its cell growth. The AGPs maximum content in the stationary phase was 0.330 mg g⁻¹ dry weight (DW) in the cell wall (CW) and 1.534 mg g⁻¹ DW in the culture medium (CM), generating cell aggregates >500 μm (93.21% DW). The addition of tunicamycin (TM) caused a reduction of AGPs content in CW and CM of 46 and 64%, respectively. These changes were associated with inhibition of growth and the reduction of the cell aggregates >500 μm (50.0% DW). When TM was removed from the CM, cell growth, aggregation, and AGPs content on CW and CM were recovered. Precipitation of AGPs with Yariv reagent generated a reduction of 61.14% of AGPs content in CW and a total inhibition of AGPs secretion in CM. This Yariv treatment generated a reduction in the cell aggregates >500 μm of 51.31% of DW. When the Yariv reagent was removed from the culture, cells did not recover their AGPs accumulation. In addition, cell cultures did not recover their ability to grow and aggregate. These results indicate that AGPs are molecules required in the cellular aggregation process of B. vulgaris L. suspension cultures.     

Localization of an arabinogalactan protein epitope and the effects of Yariv phenylglycoside during zygotic embryo development of Arabidopsis thaliana

Summary. Arabinogalactan proteins (AGPs) are a class of highly glycosylated proteins widely distributed in higher plants and thought to be involved in plant growth and development. In the present paper, Western blotting with the monoclonal antibodies JIM4, JIM13, and LM2 showed that JIM13 reacted best with total protein extracts from flowers and siliques of Arabidopsis thaliana. This monoclonal antibody was therefore used as a probe to localize the AGP epitope in zygotic embryos at different developmental stages. Immunofluorescent labeling with JIM13 showed that AGPs were mainly distributed in the embryo proper and the top 1 to 2 cells and basal part of suspensors. The results of immunogold labeling confirmed the JIM13 epitope distribution in the different cells of the suspensor. AGP immunofluorescence was also observed at the shoot apex meristem during transition from the globular to the heart embryo stage, but this gradually disappeared after the torpedo stage. After (β-D-Glc)₃ Yariv phenylglycoside (βGlcY), a synthetic reagent that specifically binds to AGPs, was added to A. thaliana ovule culture medium, the survival rate and frequency of development of ovules at the zygote stage decreased in a concentration-dependent manner, with complete inhibition at 100 μM. The frequency of embryo differentiation from the globular stage to heart or later stages also decreased sharply. When βGlcY was removed 24 h after inoculation, the inhibitory effects were reversible in a concentration-dependent and time-dependent manner. The results show that βGlcY can inhibit embryo development and differentiation in A. thaliana, and the inhibitory effects are concentration dependent and reversible, indicating that AGPs are involved in embryo differentiation and shoot meristem formation. The possible roles of AGPs in A. thaliana zygotic embryo development are also discussed. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Induction of multinucleation by β-glucosyl Yariv reagent in regenerated cells from <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> protoplasts and involvement of arabinogalactan proteins in cell plate formation

Arabinogalactan proteins (AGPs) are abundant plant cell surface proteoglycans widely distributed in plant species. Since high concentrations of β-glucosyl Yariv reagent (βglcY), which binds selectively to AGPs, inhibited cell division of protoplast-regenerated cells of the liverwort Marchantia polymorpha L. (Shibaya and Sugawara in Physiol Plant 130:271–279, 2007), we investigated the mechanism underlying the inability of the cells to divide normally by staining nuclei, cell walls and β-1,3-glucan. Microscopic observation showed that the diameter of regenerated cells cultured with βglcY was about 2.8-fold larger than that of cells cultured without βglcY. The cells cultured with βglcY were remarkably multinucleated. These results indicated that βglcY did not inhibit mitosis but induced multinucleation. In the regenerated cells cultured with low concentrations of βglcY (5 and 1 μg ml⁻¹), the cell plate was stained strongly by βglcY, suggesting abundant AGPs in the forming cell plate. In these cell plates, β-1,3-glucan was barely detectable or not detected. In multinucleated cells, cell plate-like fragments, which could not reach the cell wall, were frequently observed and they were also stained strongly by βglcY. Our results indicated that AGPs might have an important role in cell plate formation, and perturbation of AGPs with βglcY might result in remarkable multinucleation in protoplast-regenerated cells of M. polymorpha. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Programmed cell death induced by (β-d-galactosyl)3 Yariv reagent in Nicotiana tabacum BY-2 suspension-cultured cells

Programmed cell death (PCD) is involved in plant development and pathogen defence and can be triggered in vitro by several biotic and abiotic stimuli. In this report ( β - d -galactosyl)₃ Yariv reagent, a chemical that specifically binds to arabinogalactan-proteins (AGPs), completely inhibited cell growth and induced PCD in tobacco BY-2 suspension cultured cells. Analysis of DNA from these cells, by agarose gel electrophoresis, revealed a DNA ladder consisting of multimers of 140–170 bp, similar to apoptotic animal DNA internucleosomal fragmentation. Complementary morphological studies revealed additional PCD characteristics in the Yariv-treated BY-2 cells, including cell shrinkage and cytoplasmic condensation. These studies demonstrate the usefulness of BY-2 cells as a model plant PCD system and confirm a link between AGPs and PCD.     

The effect of arabinogalactan proteins on regeneration potential of juvenile citrus explants used for genetic transformation by Agrobacterium tumefaciens

A possible role of arabinogalactan proteins in control of shoot regeneration from stem explants of two citrus cultivars, Carrizo citrange and ‘Duncan’ grapefruit, was investigated. Treatment of explants with (β-d-Glc)₃ Yariv phenylglycoside, able to bind specifically to AGPs, led to a decrease of cumulative regeneration potential of both Carrizo citrange and ‘Duncan’ grapefruit. For Carrizo, lower cumulative regeneration potential on (β-d-Glc)₃ Yariv phenylglycoside-treated explants was the result of both lower number of shoots on the explants that had shoots (explant regeneration potential) and decreased percentage of explants with shoots. In the case of ‘Duncan’, treatment with (β-d-Glc)₃ Yariv phenylglycoside reduced cumulative regeneration potential only by lowering the percentage of explants with shoots, but it did not affect the number of shoots on the explants with shoots. Citrus explants treated with (α-d-Man)₃ Yariv phenylglycoside, which does not bind AGPs, responded similarly to untreated explants. Transformability of cells on the cut ends of explants was also lower for both cultivars following the treatment of explants with (β-d-Glc)₃ Yariv phenylglycoside. Our data suggest that arabinogalactan proteins play important role in processes controlling differentiation and genetic transformation of citrus cells by Agrobacterium.     

Localization of arabinogalactan-proteins in different stages of embryos and their role in cotyledon formation of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Arabinogalactan proteins (AGPs) have been implicated in plant development including sexual plant reproduction. In this paper, the expression of AGPs and the effects of β-glucosyl Yariv reagent (βGlcY, which binds arabinogalactan proteins) in embryo development and cotyledon formation were investigated. Immunofluorescence assay displayed that the expression of AGPs labeled with antibody JIM13 was developmentally regulated. In early stages, AGPs were evenly distributed in the whole embryo, except for a short polar expression in the basal suspensor cell. In the globular stage of embryo, AGPs were condensed in the embryo proper (EP), apex of the EP, and at the juncture of the EP and suspensor. In heart-shaped embryo, APGs were only present at the juncture of the EP and suspensor. Immunogold labeling assay showed that the strong expression of AGPs at the juncture of the EP and suspensor was localized in the cell wall. Provision of βGlcY to the in vitro ovule culture medium caused delayed growth of embryos, cotyledon defect and abnormal venation pattern. Consequently, βGlcY induced the death of defective seedlings with the characteristics of deformed or irregular single cotyledon. Our results suggested that AGPs play functional roles in embryo development, cotyledon formation and seedling morphology establishment in Nicotiana tabacum L.     

Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination

In lily, adhesion of the pollen tube to the transmitting-tract epidermal cells (TTEs) is purported to facilitate the effective movement of the tube cell to the ovary. In this study, we examine the components of the extracellular matrices (ECMs) of the lily pollen tubes and TTEs that may be involved in this adhesion event. Several monoclonal antibodies to plant cell wall components such as esterified pectins, unesterified pectins, and arabinogalactan-proteins (AGPs) were used to localize these molecules in the lily pollen tube and style at both light microscope (LM) and transmission electron microscope (TEM) levels. In addition, (-d-Glc)₃ Yariv reagent which binds to AGPs was used to detect AGPs in the pollen tube and style. At the LM level, unesterified pectins were localized to the entire wall in in-vivo- and in-vitro-grown pollen tubes as well as to the surface of the stylar TTEs. Esterified pectins occurred at the tube tip region (with some differences in extent in in-vivo versus in-vitro tubes) and were evenly distributed in the entire style. At the TEM level, esterified pectins were detected inside pollen tube cell vesicles and unesterified pectins were localized to the pollen tube wall. The in-vivo pollen tubes adhere to each other and can be separated by pectinase treatment. At the LM level, AGP localization occurred in the tube tip of both in-vivo- and in-vitro-grown pollen tubes and, in the case of one AGP probe, on the surface of the TTEs. Another AGP probe localized to every cell of the style except the surface of the TTE. At the TEM level, AGPs were mainly found on the plasma membrane and vesicle membranes of in-vivo-grown pollen tubes as well as on the TTE surface, with some localization to the adhesion zone between pollen tubes and style. (-d-Glc)₃ Yariv reagent bound to the in-vitro-grown pollen tube tip and significantly reduced the growth of both in-vitro- and in-vivo-grown pollen tubes. This led to abnormal expansion of the tube tip and random deposition of callose. These effects could be overcome by removal of (-d-Glc)₃ Yariv reagent which resulted in new tube tip growth zones emerging from the flanks of the arrested tube tip. The possible roles of pectins and AGPs in adhesion during pollination and pollen tube growth are discussed.Abbreviations AGP arabinogalactan-protein - ECM extracellular matrix - Glc glucose - MAbs monoclonal antibodies - LM light microscope - Man mannose - TEM transmission electron microscope - TTE transmitting tract epidermal cell The authors thank Michael Georgiady for assistance with the preparation of material for the TEM immunolocalization, Diana Dang for her help with the pectinase experiment, and Kathleen Eckard for assistance in all aspects of this study. The MAbs were the generous gifts of Dr. J.P. Knox. G.Y. Jauh thanks Dr. E.A. Nothnagel for assistance in making the Yariv reagent and for the gift of the control (-d-Man)₃ Yariv reagent. This work is in partial fulfilment of the dissertation requirements for a PhD degree in Botany and Plant Sciences for G.Y. Jauh at the University of California, Riverside. This work was supported by National Science Foundation grant 91-18554 and an R.E.U. grant to E.M.L.     

Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4

Embryogenic units of friable maize callus are formed as globular or oblong packets of tightly associated meristematic cells. These units are surrounded by conspicuous cell walls visible in light microscopy after staining with basic fuchsin. Transmission electron microscopy revealed that embryogenic cells are rich in endoplasmic reticulum, polysomes and small protein bodies, and that the outermost layer of their cell walls is composed of fibrillar material. Electron microscopy has also shown that this material covers the surface of embryogenic cells as a distinct layer which we denote as extracellular matrix surface network (ECMSN). Employing histochemical staining with β-glucosyl Yariv phenylglycoside, we localized arabinogalactan-proteins (AGPs) to the outer cell walls of embryogenic units including ECMSN. The most prominent staining was found in cell-cell junction domains. Large non-embryogenic callus cells were not stained with this AGP-specific dye. Immunofluorescence and silver-enhanced immunogold labelling using monoclonal antibody JIM4 has shown that the ECMSN of embryogenic cells is equipped with JIM4 epitope, while non-embryogenic callus cells are devoid of this epitope. We propose that some specific AGPs of the ECMSN might be relevant for cell-cell adhesion and recognition of embryogenic cells during early embryogenic stages, and that the JIM4 antibody can serve as an early marker of embryogenic competence in maize callus culture. Received: 13 March 1998 / Revision received: 6 June 1998 / Accepted: 1 July 1998     

Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement

Arabinogalactan proteins (AGPs) are a family of highly glycosylated, hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. (beta-D-glucosyl)3 and (beta-D-galactosyl)3 Yariv phenylglycosides, commonly known as Yariv reagents, specifically bind AGPs in a non-covalent manner. Here (beta-D-galactosyl)3 Yariv reagent was added to Arabidopsis thaliana cell suspension cultures and determined to induce programmed cell death (PCD) by three criteria: (i) DNA fragmentation as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of DNA 3'-OH groups; (ii) inter- nucleosomal DNA fragmentation as visualized by genomic Southern blotting; and (iii) structural changes characteristic of PCD including cytoplasmic shrinkage and condensation, chromatin condensation and nuclear membrane blebbing. These findings implicate AGP involvement in PCD in plants, presumably by perturbation of AGPs located at the plasma membrane-cell wall interface.     

Release of an acid phosphatase activity during lily pollen tube growth involves components of the secretory pathway

Summary. An acid phosphatase (acPAse) activity was released during germination and tube growth of pollen of Lilium longiflorum Thunb. By inhibiting components of the secretory pathway, the export of the acPase activity was affected and tube growth stopped. Brefeldin A (1 μM) and cytochalasin D (1 μM), which block the production and transport of secretory vesicles, respectively, inhibited the acPase secretion. The Ca²⁺ channel blocker gadolinium (100 μM Gd³⁺) also inhibited acPase secretion and tube growth, whereas 3 mM caffeine, another Ca²⁺ uptake inhibitor, stimulated the acPase release, while tube growth was inhibited. The Yariv reagent (β-D-glucosyl)₃ Yariv phenylglycoside stopped tube growth by binding to arabinogalactan proteins of the tube tip cell wall but did not affect acPase secretion. A strong correlation between tube growth and acPase release was detected. The secreted acPase activity had a pH optimum at pH 5.5, a K _M of 0.4 mM for p-nitrophenyl phosphate, and was inhibited by zinc, molybdate, phosphate, and fluoride ions, but not by tartrate. In electrophoresis gels the main acPase activity was detected at 32 kDa. The conspicuous correlation between activity of the secretory pathway and acPase secretion during tube elongation strongly indicates an important role of the acPase during pollen tube growth and the secreted acPase activity may serve as a useful marker enzyme assay for secretory activity in pollen tubes Received July 25, 2001 Accepted January 15, 2002     

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.     

Protein changes during programmed cell death in tobacco

Programmed cell death (PCD) was induced by the Yariv reagent in Nicotiana tabacum cv. Bright Yellow-2 cell suspension. The analyses of proteins extracts by 2-D electrophoresis clearly show massive protein degradation which was mainly due to cysteine protease activity. In contrast, some proteins remained unchanged up to 72 h after PCD induction. Peptide mass fingerprints of these proteins, obtained by MALDI-TOF, identified calreticulin, heat shock protein (HSP) 60, HSP70, malate dehydrogenase and mitochondrial ATP synthase β-subunit.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> arabinogalactan proteins contain abundant terminal 3-<Emphasis Type="Italic">O</Emphasis>-methyl-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosyl residues not found in angiosperms

A biochemical investigation of arabinogalactan proteins (AGPs) in Physcomitrella patens was undertaken with particular emphasis on the glycan chains. Following homogenization and differential centrifugation of moss gametophytes, AGPs were obtained by Yariv phenylglycoside-induced precipitation from the soluble, microsomal membrane, and cell wall fractions. Crossed-electrophoresis indicated that each of these three AGP fractions was a mixture of several AGPs. The soluble AGP fraction was selected for further separation by anion-exchange and gel-permeation chromatography. The latter indicated molecular masses of ∼100 and 224 kDa for the two major soluble AGP subfractions. The AGPs in both of these subfractions contained the abundant (1,3,6)-linked galactopyranosyl residues, terminal arabinofuranosyl residues, and (1,4)-linked glucuronopyranosyl residues that are typical of many angiosperm AGPs. Unexpectedly, however, the moss AGP glycan chains contained about 15 mol% terminal 3-O-methyl-l-rhamnosyl residues, which have not been found in angiosperm AGPs. This unusual and relatively nonpolar sugar, also called l-acofriose, is likely to have considerable effects on the overall polarity of Physcomitrella AGPs. A review of the literature indicates that the capacity to synthesize polymers containing 3-O-methyl-l-rhamnosyl residues is present in a variety of bacteria, algae and lower land plants but became less common through evolution to the extent that this sugar has been found in only a few species of angiosperms where it occurs as a single residue on steroidal glycosides.