Patterns of expression of the JIM4 arabinogalactan-protein epitope in cell cultures and during somatic embryogenesis in Daucus carota L.

Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP arabinogalactan protein - 2,4-D 2,4-di-chlorophenoxyacetic acid - J4e JIM 4 epitope - PEM proembryogenic mass We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions.     

Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).Abbreviations DEAE diethylaminoethyl - MAb monoclonal antibody - NEPHGE nonequilibrium pH gradient electrophoresis We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan.     

Formative and proliferative cell divisions,cell differentiation,and developmental changes in the meristem of Azolla roots

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

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.     

Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga,Fucus

Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immunoglobulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues by indirect immunofluorescence. Each antibody labelled tissues with a distinctive distribution pattern in cell walls and extracellular matrix regions, demonstrating that each antibody was specific for a different extracellular epitope (i.e., antigenic determinant). Most antibodies also labelled intracellularly on at least one cell type. Punctate, fibrous or clumped labelling was characteristic of individual antibodies and provided information related to carbohydrate structure and solubility. These antibodies are molecular probes for small regions on cell wall polymers and should be valuable in studies of cell wall synthesis, secretion, assembly and modification as well as carbohydrate fine structure and function.Abbreviations EDTA ethylenediaminetetraacetic acid - EIA enzyme immunoassay - Ig immunoglobulin (IgG, IgM and IgA are immunoglobulin types)     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

A xylogalacturonan epitope is specifically associated with plant cell detachment

A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified that is specifically associated with a plant cell separation process that results in complete cell detachment.Abbreviations DAA Days after anthesis - 2,4-D 2,4-Dichlorophenoxyacetic acid - ELISA Enzyme-linked immunosorbent assay - GalA Galacturonic acid - HGA Homogalacturonan - HPAEC High-performance anion-exchange chromatography - HPSEC High-performance size-exclusion chromatography - RG-I Rhamnogalacturonan-I - RG-II Rhamnogalacturonan-II - XGA Xylogalacturonan     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

Variations in the structure of neutral sugar chains in the pectic polysaccharides of morphologically different carrot calli and correlations with the size of cell clusters

Carrot (Daucus carota L.) embryogenic callus (EC) loses its embryogenic competence and becomes nonembryogenic callus (NC) during long-term culture. With the loss of embryogenic competence, the cell clusters become smaller and the extent of intercellular attachments is reduced. Pectic fractions prepared from EC and NC were separated into two subfractions by gel filtration. A difference in sugar composition between EC and NC was found only in the high-molecular-mass (ca. 1300 kDa) subfraction, and the ratio of the amount of arabinose to that of galactose (Ara/Gal) was strongly and positively correlated with the size of cell clusters in several different cultures. From the results of sugar-composition and methylation analyses, and the results of treatment with exo-arabinanase, models of the neutral sugar chains of pectins from EC and NC are proposed. Both neutral sugar chains are composed of three regions. The basal region is composed of linearly linked arabinan 5-Ara_f> moieties in both types of callus. The middle galactan region is composed of 6-linked galactose, some of which branches at the 3 and 4 positions, and this region is larger and more frequently branched in NC than in EC. Finally, the terminal arabinan region is composed of 5-linked arabinose, branched at the 3 position, and the size of the terminal arabinan is larger in EC than in NC. The significance of the neutral sugar chains of pectins in the interaction of cell wall components and intercellular attachment is discussed.Abbreviations Ara/Gal ratio (w/w) of the amount of arabinose to that of galactose - EC embryogenic callus - NC non-embryogenic callus - T-Ara_f terminal arabinose The authors are grateful to Dr. Naoto Shibuya of the National Institute of Agrobiological Resources for his gift of exo-arabinanase.     

A protein associated with the mouse and rat hepatocyte junctional complex

Summary In this study we have examined a protein associated with bile canaliculi of mouse and rat hepatocytes that is detected by monoclonal antibody BG9.1. The protein is seen by indirect-immunofluorescence microscopy as 2 discrete parallel lines at the lateral borders of adjacent hepatocytes. This pattern is present during development in the day 13 fetal mouse liver. Electron microscopy with immuno-gold labeling indicated that the protein is associated with the cytoplasmic surface of junctional complexes located on either side of bile canaliculi. BG9.1 reacts with a protein of 192000 apparent molecular weight on immunoblots of plasma membrane isolated from mouse and rat hepatocytes. It has been reported that unlike most cellular components, tight junctions are not soluble in sodium deoxycholate. Extraction of isolated hepatic plasma membrane sheets with deoxycholate and other reagents did not eliminate the pattern seen by indirect-immunofluorescence microscopy and enhanced the intensity of reactions on immunoblots. BG9.1 also binds to the junctional-complex region in other epithelial cell types. These results indicate that BG9.1 detects a deoxycholate-insoluble protein associated with junctional complexes and suggests that the protein is a component of tight junctions.     

A proton nuclear magnetic resonance study of the physical changes in growing plant cell walls

Changes in broadline proton nuclear magnetic resonance parameters of cell walls during growth of etiolated hypocotyls of bean (Phaseolus vulgaris L.) indicate that cell wall structure becomes more rigid during development. Most of the changes are completed in the first 6 cm below cotyledon insertion and are correlated with increased restriction of proton movements in regions of dense polymer packing.Abbreviations FID free induction decay - M₂ second moment - M_2interpair interpair second moment - NMR nuclear magnetic resonance - T_1D dipolar relaxation time - T₂ spin-spin relaxation timeThis work was supported by grants from Natural Sciences and Engineering Research Council of Canada to A.L.M., I.E.P.T. and M. Bloom.     

Auxin-induced rapid changes in translatable mRNAs in tobacco cell suspension

When 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell suspensions, one normal and one transformed by Agrobacterium tumefaciens, were subcultured on hormone-lacking medium the stationary phase of the cell cycle was reached earlier than on medium containing 2,4-D. Addition of the auxin 2,4-D could restore cell division activity within 10–12 h for the most rapidly reacting cell line. The cell-division response was characterized as being auxin-specific and optimal with 2,4-D at 2.2 10^-6 M. Although the cell lines used showed different characteristics, both reacted with a rapid increase in at least three mRNA species within 1 or 2 h after 2,4-D application. Two, 2,4-D-induced protein spots, seen after in-vitro translation, had the same characteristics (MWs 35 kilodaltons (kDa) and 25 kDa with isoelectric points of 7.1 and 6.3, respectively) in both cell lines. Water-treated controls did not show alterations in the translatable mRNA populations. This indicates that the accumulation of the corresponding mRNAs is an early hormone-induced event. Since cell division is the only measurable reaction found after auxin application, cell systems as described here offer excellent possibilities for studying early auxin-induced changes at the molecular level preceding mitosis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - kDa kilodalton     

Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.     

The cellular basis of the elongation response in submerged deep-water rice

The cellular basis of internode elongation was studied in intact deep-water rice plants (Oryza sativa L. cv. Habiganj Aman II) and in isolated stem sections. In intact plants, growth was stimulated by submergence in water and by ethylene treatment. In isolated sections, growth was enhanced by submergence, by ethylene, and by exposure of the tissue to an atmosphere of 3% O₂, 91% N₂ and 6% CO₂ or 3% O₂, 91% N₂, 6% CO₂ and 1 l l^-1 C₂H₄ (by vol.). Under all these conditions, growth was localized in the intercalary meristem at the bases of the internodes. Autoradiography of [³H]thymidine-labeled tissue showed activation of cell division and longitudinal expansion of the intercalary meristem. Increased production of new cells and their subsequent elongation thus form the basis for the growth response to submergence and ethylene treatment in deep-water rice plants.