Immunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase or immunoglobulin G-gold as markers were used for the localization of cytokinins (CKs) in two isogenic lines, Craigella (C) and Craigella lateral suppressor (Cls), of tomato Lycopersicon esculentum Mill. Terminal buds, nodes, hypocotyl segments and root tips were submitted to a periodate-borohydride procedure, to obtain the coupling of isopentenyladeosine and zeatin riboside to cellular proteins, followed by a fixative step with a paraformaldehyde and glutaraldehyde mixture. Enzyme-linked immunosorbent assay tests performed on ovalbumin-coated microtitration plates have shown that this method was effective for CK riboside and base coupling to proteins. Paraffin-wax- or Spurr's-resin-embedded sections were cleared of wax or resin before incubation with anti-zeatin riboside or anti-isopentenyladenosine antibodies. The procedure was thoroughly investigated and many controls were done in order to eliminate artefacts. The immunostaining patterns observed along the plants showed a basipetally decreasing gradient of CKs along the stem and in the roots. Immunolabelling was higher in the actively growing regions of the stem bud and root apices. Terminal buds of Cls appeared to be less immunoreactive than C, whereas no differences were detected in root-tip immunolabelling. The staining patterns are consistent with the idea that root and bud apices have a different CK metabolism. The absence of axillary bud formation in Cls is correlated with low CK levels in the organogensis sites.Abbreviations C Craigella, isogenic line - CK cytokinin - Cls Craigella lateral suppressor - EDC 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride - ELISA enzyme-linked immunosorbent assay - 2iP isopentenyladenine - 2iPA isopentenyladenosine - PAP peroxidase-anti-peroxidase - PFAG paraformaldehyde/glutaraldehyde mixture - Z zeatin - ZR zeatin riboside     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Monoclonal antibodies for the analysis and purification of isopentenyladenine cytokinins

A series of monoclonal antibodies (McAb) have been generated which show a high affinity for the cytokinin, isopentenyladenosine (2iPA). The McAb show different specificities to a range of cytokinins in a radioimmunoassay and are suitable for the analysis of isopentenyladenine (2iP) and related compounds in plant extracts. One McAb, MAC 160, has a high cross-reactivity with several cytokinins and is a good candidate for use in group separation or the analysis of a number of cytokinins simultaneously after separation. When coupled to Sepharose 4B affinity support, the McAb can also be used as immunoaffinity matrices to purify and resolve cytokinins from plant extracts. The McAb have been used to demonstrate the presence of concentrations of 2iP in tissues of the moss, Physcomitrella patens which exceed those in its culture medium.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Monoclonal antibodies to sperm surface antigens of the brown alga Fucus serratus exhibit region-, gamete-, species- and genus-preferential binding

A panel of twelve monoclonal antibodies (MAbs), designated FS1 to FS12, have been raised against surface antigens of Fucus serratus sperm. The antibodies were selected on the basis that they show region-, gamete-, species- or genus-preferential binding. Indirect immunofluorescence shows that the antigens bound by the MAbs are distributed non-randomly over the cell surface. Seven MAbs (FS1, FS3, FS4, FS6, FS8, FS9, FS10) bind antigens located primarily on the cell body, while the others (FS2, FS5, FS7, FS11, FS12) bind antigens located primarily on the anterior flagellum. Of the MAbs that label the anterior flagellum, FS2, FS5, FS7 and FS12 form a halo at the perimeter of the flagellum. Electron microscopic-immunogold studies indicate that the halo results from labelling of the mastigonemes, as opposed to the flagellar plasmamembrane. Gamete-preferential binding of antibodies was detected using an enzyme-linked immunosorbent assay with egg membrane vesicles. Eight of the MAbs bind sperm antigens not common to eggs, though FS2, FS4, FS5 and FS9 bind antigens present on both sperm and eggs. In studies of species- and genus-specificity FS2, FS3, FS5, FS6, FS7, FS8, FS10, FS11 and FS12 exhibit genus-preferential binding, labelling sperm of F. serratus and F. vesiculosus more intensely than that of Ascophyllum nodosum. Only FS10 showed marked species-preferential binding, labelling sperm of F. serratus much more intensely than that of F. vesiculosus.Abbreviations Au-GAMIG gold-conjugated goat anti-mouse immunoglobulin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - FITC-RAMIG fluorescein-isothiocyanate-conjugated rabbit anti-mouse immunoglobulin - IIF indirect immunofluorescence - MAb monoclonal antibody     

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Avena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - McAb monoclonal antibody(ies) - PBS phosphate-buffered saline - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - PMSF phenylmethylsulphonylfluoride     

Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.)

A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4·10^-8 to 4·10^-7 M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5·10^-8 to 5·10^-7 M), higher concentrations (5·10^-6 M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - i6Ade isopentenyladenine - i6Ado isopentenyladenosine     

Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status

The effect of nitrogen on the cytokinin relations of Urtica dioica, the stinging nettle, has been investigated. The plants were grown in quartz sand and nutrient solutions providing levels of nitrate ranging from 1 to 22 mM. Nitrogen supply did not affect biomass production within the range of 3–15 mM NO ₃ ^- . However, the shoot: root ratio of biomass was significantly higher at 15 mM (standard plants) than at 3 mM (low-nitrogen plants) nitrate supply. The cytokinin patterns of the roots, stems and adult, as well as meristematic leaves of plants grown at these two levels of nitrate supply, were determined by means of high-performance liquid chromatography (HPLC) and immunoassays. Enzyme-linked immunosorbent assays (ELISAs) for zeatin riboside, dihydrozeatin riboside, isopentenyladenosine, benzyladenosine and o-hydroxybenzyladenosine enabled the quantification of 17 cytokinins, 13 of which were found in the various tissues of Urtica. trans-Zeatin and its conjugates were the predominant cytokinins in all examined samples. While the free base trans-zeatin and its O-glucoside were the major cytokinins in adult leaves, trans-zeatin riboside was prominent in the other tissues of at least the standard plants. Glucosides of the trans-zeatin type cytokinins were present only in lower amounts. However, considerable amounts of a compound, tentatively identified as cis-zeatin riboside-O-glucoside, were found, particularly in roots and meristematic leaves. Comparatively high amounts of trans-zeatin nucleotide as well as isopentenyladenosine phosphate were also demonstrated in these tissues. Analysis of the root-pressure exudates similarly showed trans-zeatin riboside and, at a lower concentration, trans-zeatin to be the only substantial components. In the low-nitrogen plants, shortage of nitrogen was manifest only in the roots; the nitrogen contents of the shoots did not respond to the nitrogen supply. Likewise, the total content of cytokinins in the shoots of the low-nitrogen plants equaled that of the standard-plant shoots, while it was lower by about 25% in the roots of the low-nitrogen plants. In the latter, the amounts of cytokinins exuded via the root-pressure fluid were also approximately 25% lower. Since the levels of only the trans-zeatin cytokinins in the roots showed a linear correlation with the shoot-to-root ratios, these cytokinins may play an important role in biomass partitioning in Urtica dioica.Abbreviations DHZ dihydrozeatin - ELISA enzyme-linked immunosorbent assay - -G glucoside - HPLC high-performance liquid chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - -N nucleotide (ribotide) - -OG O-glucoside - -R riboside - S/R shoot-to-root (ratio) - Z zeatin This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the SFB 137. The authors wish to thank Mrs. A. Fischbach for skilful technical assistence and Dr. Paul Ziegler (Lehrstuhl für Pflanzenphysiologie, University of Bayreuth, FRG) for linguistic suggestions.     

Comparative quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzyme-linked immunosorbent assay using the avidin-biotin system

In this report we describe an enzyme-linked immunosorbent assay (ELISA) for the quantitation of abscisic acid (ABA) in plant extracts. A microtitration plate is coated with an ABA-protein complex. The ABA, standard or sample, is then added to each well with a limiting quantity of rabbit anti-ABA antibodies. During the following incubation period, antibodies bind either to free or to bound ABA on the plates. After washing, bound antibodies are indirectly labelled in two steps by the means of biotinylated goat antirabbit immunoglobulin-G antibodies which act as a link between rabbit anti-ABA antibodies and an avidin-alkaline phosphatase complex. The relative enzyme activity bound is measured spectrophotometrically. The detection limit of this method is 5 pg ABA and the measuring range extends to 10 ng. Gas-liquid-chromatography controls, with an electron capture detector, show a good correlation with ELISA results obtained using extracts of Lycopersicon esculentum, Nicotiana tabacum and Pseudotsuga menziesii samples purified by high-performance liquid chromatography. This provides a good argument for the accuracy of the immunoenzymatic method. The indirect labelling of antibodies, with the avidin-biotin amplifying system, should make this technique suitable for the quantitation of other plant growth substances against which specific antibodies are available.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - GLC gas liquid chromatography - HPLC high-performance liquid chromatography - IgG Immunoglobulin G - PBS phosphate-buffered saline     

Isolation of two cytokinin metabolites from the rhizosphere of Norway spruce seedlings (Picea abies L. Karst.)

Roots of young Norway spruce seedlings were incubated under hydroculture conditions in a synthetic nutrient medium containing either ³H-isopentenyladenosine, isopentenyladenosine or zeatin riboside. When feeding with ³H-isopentenyladenosine a new radiaolabelled metabolite was found in the feeding solution as well as in root extracts. Isopentenyladenosine and zeatin riboside were metabolised and for both compounds an unknown metabolite was detected in the feeding solution. The metabolites were purified by solid phase extraction, HPLC and partially characterised. A major characteristic of the metabolites is their reactivity in the presence of NH₄OH, which results in the formation of the cytokinin bases isopentenyladenine or zeatin, respectively. UV-spectra and the chemical characteristics indicate that the new metabolites are closely related. The GC-MS analysis revealed, that the metabolites are true derivatives of isopentenyladenine and zeatin. The biogenesis of the new metabolites is discussed with regard to plant microbial interactions.Abbreviations Ck(s) = cytokinin(s) - GC-MS = gas chromatography-mass spectrometry - iP = isopentenyladenine - [9R]iP = isopentenyladenosine - [9G]iP = isopentenyladenine-9-glucoside - [9R-MP]iP = isopentenyladenosine-5-monophosphate - Z = trans-zeatin - [9R]Z = trans-zeatin riboside     

Growth retardation induced by nutritional deficiency or abscisic acid in Lemna gibba: The relationship between growth rate and endogenous cytokinin content

The relationship between endogenous cytokinin content and relative growth rate (RGR) was studied in cultures of Lemna gibba L. G3 supplied with daily doses of mineral nutrients that were increased exponentially over time. At the optimal level of nutrient supply the RGR was 30–35% day^-1. The RGR was regulated by adjusting the rate of nitrogen supply, or it was restricted by addition of 0.5 M abscisic acid (ABA). Another approach used to investigate the specific roles of nitrogen (N) and phosphorus (P), was to transfer optimally growing plants to media without N or P but otherwise complete. The plants were harvested at regular intervals for determination of the RGR and levels of cytokinins of the isopentenyladenosine (iPA) and zeatinriboside (ZR) types with an enzyme-linked immunosorbent assay (ELISA). Levels of both iPA- and ZR-type cytokinins decreased when nitrogen was applied to cultures in growth limiting amounts. The cytokinin levels decreased more rapidly than the RGR when either N or P was lacking in the medium, suggesting an early influence of nutrient availability on cytokinin levels which in turn may induce adaptive response by the plant. RGR retardation induced by ABA did not affect cytokinin levels during the first 4 days of the treatment, and the later effects were small. The experiments gave no indication that ABA is involved in the adaptation response of Lemna plants to nutritional stress.Abbreviations ABA - abscisic acid - BAP - benzylaminopurine - ELISA - enzyme-linked-immunosorbent-assay - iP - isopentenyladenine - iPA - isopentenyladenosine - PBS - phosphate-buffered saline - PVP - polyvinylpyrrolidone - RGR - relative growth rate - R_N - relative nitrogen addition rate - Z - trans-zeatin - ZR - trans-zeatin riboside     

Chicken egg yolk antibodies for cytokinin analysis

The production, isolation, and purification of specific chicken immunoglobulins (Igs) against three main groups of naturally occurring cytokinins are reported. The specific Igs directed against, respectively, zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine are extracted from the egg yolk and used in radioimmunoassays that allow the quantification in parallel of pmol of the cytokinins in plant extracts. As little as 50 fmol of zeatin riboside, 20 fmol of isopentenyladenosine, and 40 fmol of dihydrozeatin riboside can be detected. The levels of cytokinins measured in the radio-immunoassay correlate well with physicochemical analysis methods such as high performance liquid chromatography (HPLC) with UV spectrum detection and HPLC-coupled mass spectrometric detection. Cross-reactivity studies indicate that the assay is not affected by most of the structurally related compounds. The respective antibody preparations recognized zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine and the corresponding free bases. The results obtained when analyzing crude plant extracts are expressed as zeatin riboside equivalents, dihydrozeatin riboside equivalents, and isopentenyladenosine equivalents.Abbreviations B binding activity - B ₀ maximal binding - B ₁ unspecific binding - GC gas chromatography - HPLC high performance liquid chromatography - LC-MS HPLC-coupled mass spectrometry - MOPS 4-morpholinepropanesulfonic acid - RIA radioimmunoassay - TBS Tris-buffered saline - (diH)Z dihydrozeatin - (diH) [9R]Z dihydrozeatin riboside - iP isopentenyladenine - [9R]iP isopentenyladenosine - Z zeatin - [9R]Z zeatin riboside - [9G]iP isopentenyladenine-9-glucoside - [9R-5P]iP isopentenyladenosine-5-monophosphate     

Cross-reactivity of monoclonal antibodies against phytochrome from Zea and Avena

The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Avena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg · mol^-1 phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120–125 kg·mol^-1 from several ferns, a liverwort and mosses. This antibody (Z-3B1), which was localized within a 23.5-kg·mol^-1 section of Avena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical.Abbreviations ELISA enzyme-linked immunosorbent assay - McAb monoclonal antibody - PBS phosphate-buffered saline - Pfr (Pr) far-red-absorbing (red-absorbing) form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Identification and localisation of a nucleoporin-like protein component of the plant nuclear matrix

Salt-detergent extraction of purified plant nuclei yields a fraction enriched in putative structural proteins known as the nuclear matrix. Compared with mammalian nuclear matrices, which contain three major proteins, plant nuclear matrices are complex, containing at least 100 polypeptides. In order to characterise more fully the plant nuclear matrix we have used antibodies raised against both yeast (Saccharomyces cerevisiae) and mammalian (rat) nuclear pore proteins. We have shown that the nuclear matrix of carrot (Daucus carota L.) contains at least one nucleoporin-like protein of about 100 kDa which is immunologically related to both the yeast nuclear pore protein NSP1 and mammalian nucleoporins (p62). Antibody labelling of a variety of plant cells at the light-microscope and electron-microscope levels confirms that this antigen is located at the nuclear pores. This, to our knowledge, is the first identification of a nuclear pore protein in plants.Abbreviations IgG immunoglobulin G - kDa kilodaltons - DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothioganate The authors would like to thank Dr. E. Hurt (European Molecular Biology Laboratory, Heidelberg, FRG) for antibodies against yeast nucleoporins, and Dr. L. Davis (Whitehead Institute for Biomedical Research, Cambridge, Mass., USA) for the monoclonal antibodies MAb 414 & 350. We thank Brian Wells for useful advice on electron microscopy. We also thank Peter Scott, Andrew Davis, and Nigel Hannant for photography, and Sue Bunnewell for development and printing of electronmicrographs.     

High molecular mass glycoproteins associated with the siliceous scales and bristles of Mallomonas splendens (Synurophyceae) may be involved in cell surface development and maintenance

The elaborate scale case of Mallomonas splendens (Synurophyceae) consists of an overlapping arrangement of siliceous scales. In addition, siliceous bristles are attached to specialized base plate scales located at both the anterior and posterior ends of the cells. We have generated monoclonal antibodies against molecules associated with the scale case of M. splendens. One of these antibodies, designated MsS.H9, labelled a proteinaceous epitope of high-molecular-mass cell surface glycoproteins. Immunofluorescence and immunoelectron microscopy demonstrated that only two regions of M. splendens scale cases were labelled by MsS.H9, namely, the upper surface of the scales that contact neighboring scales and the bases of the bristles. Immunoelectron microscopy using thin sections of M. splendens cells showed these labelling sites corresponded to the amorphous material at the sites of scale-to-scale overlap and to a fibrillar complex located at scale-to-bristle attachment sites. Scales and bristles of M. splendens are formed within the cell, in silica deposition vesicles. Immunolabelling of cell sections containing developing scales and bristles showed that MsS.H9 labelling sites were present very early in the formation of these cell surface components. MsS.H9 labelling was also found associated with developing flagellar hairs whereas no labelling was detected on these structures after their deployment onto the flagellum. The location of MsS.H9 labelling sites strongly suggests that the molecule(s) recognized by the antibody plays a role in the adhesion of the individual components making up the scale case of M. splendens.Abbreviations CER chloroplast endoplasmic reticulum - ER endoplasmic reticulum - SDV silica deposition vesicle This work was supported by a grant from the Australian Research Council to R.W. We thank Dr. P. L. Beech for Fig. 13, Dr. L. Perasso for technical assistance and the Plant Cell Biology Group for the use of their monoclonal facilities.     

Endogenous cytokinins during embryogenesis in an anise cell culture (Pimpinella anisum L.)

Endogenous levels of cytokinins in an anise cell culture were determined by the use of radioimmunoassay and gas chromatography-mass spectrometry in combination with single-ion monitoring, during proembryonal and embryonal development. In both cultures the highest cytokinin levels were correlated with logarithmic growth (embryonal: isopentenyladenosine, 4 ng g^-1 fresh weight [FW]; isopentenyladenine, 1.4 ng g^-1 FW; zeatin, 3.6 ng g^-1 FW; proembryonal: isopentenyladenosine, 58.3 ng g^-1 FW; isopentenyladenine, 7.9 ng g^-1 FW; zeatin 11.1 ng g^-1 FW). The proembryonic culture medium but not the embryonic culture medium contained isopentenyladenosine up to 28 pg ml^-1 during logarithmic growth. No correlation between different embryonic stages and the endogenous cytokinin level was obvious.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RIA radioimmunoassay - SIM single-ion monitoring     

Biochemical differentiation in the tobacco flower probed with monoclonal antibodies

We have isolated a series of monoclonal antibodies that react to antigens in flowers of Nicotiana tabacum L. (tobacco) displaying specificity or preferentiality in their cell and tissue distributions. We immunized mice with extracts from tobacco flowers and then screened the hybridomas by enzyme-linked immunosorbent assay (ELISA) against extracts from leaves, sepals, petals, stamens and pistils; twenty five were chosen from the total screened. The antigens detected by about half of the antibodies were periodate-sensitive, implying that the epitopes were carbohydrate. Competition ELISA assays were used to determine if any antibodies were reacting to the same epitopes. Western blot analysis showed that while some antibodies reacted to specific bands, the bulk either failed to react or reacted to multiple bands, consistent with a glyco-conjugate nature for many of the antigens. Analysis of the spatial pattern of antigen distribution within tobacco flowers by immunolocalization showed that some antibodies recognized epitopes that were limited to very specific cells and tissues. We used the immunolocalization technique to analyze a mutant with stigmoid anthers: an antibody recognizing a pistil transmitting-tract antigen also reacted to cells in stigmoid anthers. Our results with this antibody set imply that biochemical differentiation within the tobacco flower includes cell-and tissue-specific glyco-moeities, and also that similarities, at the biochemical level, exist between a normal floral organ and the abnormal organ in a phenotype with a developmental switch.Abbreviations ELISA enzyme-linked immunosorbent assay - Fg immunoglobulin - kDa kilodalton     

Monoclonal antibodies to surface glycoconjugates in Chlamydomonas eugametos recognize strain-specific O-methyl sugars

Monoclonal antibodies are described that are directed against cell surface components of the unicellular green alga Chlamydomonas eugametos. These antibodies recognize strain-specific epitopes which occur at the surface of vegetative and gametic cells. Two different groups of epitopes are distinguished that are never detectable together in one clonal cell culture. Evidence is presented showing that the antigenicity of cell surface molecules is a consequence of the presence of particular O-methylated sugars. Monoclonal antibodies reacting with one group of epitopes were studied in more detail, and immunoprecipitation and Western-blot studies showed that these epitopes can be arranged into four classes. The use of these monoclonal antibodies as strain-specific markers in light- and electron-microscopical techniques is illustrated.Abbreviations ELISA enzyme-linked immunosorbent assay - mt ^+/- mating type plus or minus - PAS periodic acid Schiff - Mab monoclonal antibody - PBS phosphate-buffered saline     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate     

Endogenous cytokinins in rose petals and the effect of exogenously applied cytokinins on flower senescence

In extracts from rose petals cytokinin activity was detected by Amaranthus bioassay in HPLC eluates corresponding to the standards: Z, ZR, 2iP and 2iPA; subsequently, the presence of two groups of endogenous cytokinins was confirmed by ELISA.Measurements of senesence indicators (cell sap osmolarity and conductivity) and observations of flower vase-life indicated that when the above cytokinins were applied as holding solutions they delayed flower senescence by 34–56% and prolonged rose longevity.Abbreviations B.H.T. 2.6-di-t-buytl-4-methyl phenol - ELISA Enzyme linked Immunosorbent Assay - HPLC High Performance Liquid Chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - Z trans-zeatin - ZR trans-zeatin riboside