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.     

Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the β-glucosyl Yariv reagent (βglcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of βglcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with βglcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (>80%). However, without transfer to the AC medium, βglcY at concentrations higher than 20 μg ml⁻¹ inhibited cell division. No effect on cell survival nor cell division was observed with the α-galactosyl Yariv reagent. Staining of β-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of β-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without βglcY, while little or no β-1,3-glucan was stained by AB in protoplasts cultured with βglcY. These results suggest that AGPs and β-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.     

Localisation pattern of homogalacturonan and arabinogalactan proteins in developing ovules of the gymnosperm plant Larix decidua Mill

We have identified and characterised the temporal and spatial distribution of the homogalacturonan (HG) and arabinogalactan proteins (AGP) epitopes that are recognised by the antibodies JIM5, JIM7, LM2, JIM4, JIM8 and JIM13 during ovule differentiation in Larix decidua Mill. The results obtained clearly show differences in the pattern of localisation of specific HG epitopes between generative and somatic cells of the ovule. Immunocytochemical studies revealed that the presence of low-esterified HG is characteristic only of the wall of megasporocyte and megaspores. In maturing female gametophytes, highly esterified HG was the main form present, and the central vacuole of free nuclear gametophytes was particularly rich in this category of HG. This pool will probably be used in cell wall building during cellularisation. The selective labelling obtained with AGP antibodies indicates that some AGPs can be used as markers for gametophytic and sporophytic cells differentiation. Our results demonstrated that the AGPs recognised by JIM4 may constitute molecules determining changes in ovule cell development programs. Just after the end of meiosis, the signal detected with JIM4 labelling appeared only in functional and degenerating megaspores. This suggests that the antigens bound by JIM4 are involved in the initiation of female gametogenesis in L. decidua. Moreover, the analysis of AGPs distribution showed that differentiation of the nucellus cells occurs in the very young ovule stage before megasporogenesis. Throughout the period of ovule development, the pattern of localisation of the studied AGPs was different both in tapetum cells surrounding the gametophyte and in nucellus cells. Changes in the distribution of AGPs were also observed in the nucellus of the mature ovule, and they could represent an indicator of tissue arrangement to interact with the growing pollen tube. The possible role of AGPs in fertilisation is also discussed.     

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.     

Quantification of arabinogalactan proteins during in vitro morphogenesis induced by β-d-glucosyl Yariv reagent in Centaurium erythraea root culture

Arabinogalactan proteins (AGPs) are a family of highly glycosylated cell surface proteins located at the plasma membrane and plant cell wall. AGPs play important roles in plant growth and development. Yariv phenylglycoside (βGlcY), synthetic red-brown dye that specifically binds and precipitates AGPs, has been used for detection and quantification of AGPs in plant tissue. Graded concentrations of βGlcY (0–75 μM) were used to investigate the effect of this synthetic dye on induction of in vitro morphogenesis in Centaurium erythraea root culture on two nutrient media: ½MS and ½MS + IBA 1.0 μM. Regeneration of C. erythraea shoots on root explants was stimulated on both media supplemented with 25 μM βGlcY after 8 weeks in culture. Quantification of AGPs in different tissues of C. erythraea was determinate with single radial diffusion method. This work emphasizes clear effect of βGlcY on induction of morphogenesis in vitro in C. erythraea root culture.     

Occurrence of cell surface arabinogalactan-protein and extensin epitopes in relation to pericycle and vascular tissue development in the root apex of four species

Monoclonal antibodies recognizing two classes of developmentally regulated plant cell surface components – arabinogalactan-proteins (AGPs) and extensins – have been used to immunolabel cells at the root apices of four species with different characteristics of pericycle and vascular tissue development. Root apices of pea (Pisum sativum L.), radish (Raphanus sativus L.), carrot (Daucus carota L.) and onion (Allium cepa L.) were immunolabelled with the anti-AGP monoclonal antibodies JIM4 and JIM13 and anti-extensin monoclonal antibodies JIM11, JIM12, JIM19 and JIM20. All of these antibodies recognized subsets of pericycle cells in at least one, but never all, of these species. The restricted patterns of epitope occurrence also reflected vascular cell development. The differences in patterns of antibody recognition in the four species are discussed in relation to the possible roles of these cell surface molecules in cell differentiation and root patterning events. Received: 11 March 1997 / Accepted: 20 May 1997     

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.     

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.     

Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha

Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H₂O g⁻¹ dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H₂O g⁻¹ DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H₂O g⁻¹ DW, desiccation-tolerant cells that had been precultured were vitrified above 0°C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.     

Loss of arabinogalactan-proteins from the plasma membrane of NaCl-adapted tobacco cells

Cultured cells of tobacco (Nicotiana tabacum L.) adapted to 428 mM NaCl exhibited a reduced rate of cell enlargement, which is probably due to decreased cell-wall extensibility. Arabinogalactan-protein (AGP) has been implicated as a cell-wall-loosening factor (Schopfer 1990). Levels of plasma membrane and extracellular AGPs that react with Yariv reagent were measured and compared between NaCl-adapted and unadapted tobacco cells. Unadapted cells contained a very high level of AGPs on the plasma membrane, which amounted to 0.16 g·g^–1 membrane protein. In contrast, AGPs were virtually undetectable on the plasma membrane of NaCl-adapted cells. Accumulation of AGPs was also decreased in culture media of NaCl-adapted cells. These data support the hypothesis that AGPs participate in cell expansion. Possible mechanisms of the proposed cell-expansion role of AGPs are discussed.Abbreviations AGP arabinogalactan-protein - S₀, S₂₅ cells un-adapted, NaCl-adapted tobacco cells This work was supported in part by a Mcknight Foundation fellowship to J.K.Z. This is journal paper No. 13,569 of Purdue University Agricultural Experimental Station. The authors thank Dr. Eugene A. Nothnagel for the Yariv reagent gift and for helpful discussion. The authors also thank Glenda McClatchey for excellent technical assistance.     

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     

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     

A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of β-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana

Seedlings of Arabidopsis thaliana were germinated and grown in medium containing β-glucosyl Yariv reagent (βGlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of βGlcY on the seedlings was to reduce the overall growth of both the root and the shoot. βGlcY only accumulated in the root tissues and the reduced growth of the shoot appeared to be an indirect effect of impaired root growth. Reduced root growth was a consequence of a reduction in cell elongation during the postproliferation phase of elongation at the root apex and this was associated with extensive radial expansion of root epidermal cells. βGlcY penetrated roots as far as the endodermis and it is suggested that the interaction of βGlcY with AGPs in the load-bearing cell layers inhibited root elongation. When βGlcY was added to carrot suspension-cultured cells that had been induced to elongate rather than proliferate, cell elongation was inhibited. The AGP-unreactive α-galactosyl Yariv reagent (αGalY) had no biological activity in either of these systems.     

In vitro cardiomyogenic differentiation of adipose‐derived stromal cells using transforming growth factor‐β1

Transplanting stem cells differentiated towards a cardiac lineage can regenerate cardiac muscle tissues to treat myocardial infarction. In this study, we tested the hypothesis that transforming growth factor‐β1 (TGF‐β1) induces cardiomyogenic differentiation of adipose‐ derived stromal cells (ADSCs) in vitro. Rat ADSCs were cultured with TGF‐β1 (10 ng ml^?1) for 2 weeks in vitro. ADSCs cultured without TGF‐β1 served as a control. The mRNA expression of cardiac‐specific gene was induced by TGF‐β1, while the control culture did not show cardiac‐specific gene expression. Immunocytochemical analyses showed that a small fraction of ADSCs cultured with TGF‐β1 for 2 weeks stained positively for cardiac myosin heavy chain (MHC) and α‐sarcomeric actin. Flow cytometric analyses showed that the proportion of cells expressing cardiac MHC increased with TGF‐β1. However, no mesenchymal differentiation (e.g., osteogenic and adipogenic differentiation) was detected other than cardiomyogenic differentiation. These results showed that TGF‐β1 induce ADSC cardiomyogenic differentiation in vitro, which could be useful for myocardial infarction stem cell therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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     

Differenziamento biochimico della parete in Angiosperme e Gimnosperme

Abstract

Biochemical changes in Angiosperms and Gymnosperms cell-walls during cellular differentiation in xylem. - During differentiation of the vascular cambium in xylem cells there are changes on the biosynthesis of cell-wall polysaccharides in angiosperms and gymnosperms. Pectins are synthesized during primary growth only, whereas the synthesis of hemicelluloses and cellulose increases markedly during secondary thickening; simultaneously the synthesis of lignin begins. The cells can modulate the synthesis of cell-wall polysaccharides by an induction and repression or an activation and inactivation of certain key enzymes. It has been shown that the major control operating during xylem differentiation is exerted on polysaccharide synthases. Further controls are envisaged on the transport of nucleoside diphosphate sugars from the cytoplasmic pool to the endomembrane, on the transport of neoformed polysaccharides through the endomembrane flow, and at plasmalemma level. It has also been emphasized the role of cell-wall in autoregulating its growth and differentiation.     

Modulation of the Antigenic Phenotype of Human Melanoma Cells by Differentiation-inducing and Growth-suppressing Agents

Tumor cells often display alterations in their normal program of cellular differentiation. A promising approach for the treatment of cancer involves the induction of terminal differentiation and a loss of proliferative capacity in cancer cells. In human melanoma cells, the combination of mezerein (MEZ) and fibroblast interferon (IFN-β), results in a rapid and irreversible suppression of cell growth with a concomitant increase in the synthesis of melanin. The induction of terminal differentiation is associated with alterations in the expression of several cellular genes, including fibronectin, ISG-15 and ISG-54, and changes in the expression of specific cell surface antigens, including intercellular adhesion molecule-1 (ICAM-1) and HLA Class I antigens. In the HO-1 human melanoma cell line, induction of terminal differentiation by MEZ plus IFN-β results in an induction and/or increased expression of ICAM-1. HLA Class I antigens and HLA Class II antigens. IFN-β and MEZ alone can modulate expression of these antigens to a lower extent than does the combination of compounds. Induction of terminal differentiation and the irreversible suppression of cell growth is not a prerequisite for antigenic modulation in HO-1 cells. This is indicated by the inability of immune interferon (IFN-γ), a strong inducer of ICAM-1, HLA Class I antigens and HLA Class II antigens synthesis, or the combination of IFN-β plus IFN-γ which synergistically but reversibly suppresses HO-1 growth. to induce melanin synthesis or terminal differentiation in HO-1 cells. The inhibitor of protein kinase C, H-7, only marginally alters 72 hr growth suppression induced by MEZ or the interferons, used alone or in combination. In several experiments, H-7 only partially and variably inhibited the enhanced expression of ICAM-1, HLA Class I antigens and HLA Class II antigens in HO-1 cells treated with MEZ. IFN-β or IFN-γ, used alone or in various combinations. This model system will be useful in defining the biochemical, genomic and antigenic changes associated with the chemical induction of terminal differentiation and the loss of proliferative capacity in human melanoma cells.     

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.