Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Immunoassay and ultrastructural localization of isopentenyladenine and related cytokinins using monoclonal antibodies

Two hybridoma cell lines, J40-IV-A1 and J40-IV-C4 were obtained from a fusion of spleen cells of Balb/c mice immunized against an isopentenyladenosine-bovine serum albumin conjugate with X63. Ag 8.653 myeloma cells. These hybrids secrete monoclonal antibodies of the immunoglobulin G (IgG) class and share high affinities and specificities to isopentenyladenine and isopentenyladenosine suitable for the detection of femtomole amounts of these cytokinins in plant extracts by enzyme-linked immunosorbent assay (ELISA). One of the monoclonal antibodies (J40-IV-C4) has been employed to localize isopentenyladenine immunoreactivity in a cytokinin-over-producing mutant of the moss, Physcomitrella patens. After fixation and embedding at low temperature, immunoreactivity was visualized in protonemal filaments of the moss mutant by the use of indirect immunogold labelling. In the mutant, the labelling was predominantly in the wall of the protonemal cells. Neither the wild-type nor control treatments showed any labelling. The signficance of these observations is discussed with respect to the applicability of immunocytochemical techniques for the localization of low-molecular-weight compounds in plant tissue.Abbreviations ELISA enzyme linked immunosorbent assay - HPLC high-performance liquid chromatography - IP isopentenyladenine - IPA isopentenyladenosine - mAB monoclonal antibody - OVE cytokinin-over-producing mutant - RIA radioimmunoassay     

Immunocytochemical localisation of auxin-binding proteins in coleoptiles and embryos ofZea mays L.

Summary The auxin-binding protein ABP-1 was localised immunocytochemically in coleoptiles and immature embryos ofZea mays. Two primary polyclonal antibodies raised against ABP-1 and secondary antibodies were either labelled with FITC or 10 nm gold particles for light microscopy, and with 10 nm gold particles for transmission electron microscopy. Light microscopy revealed that ABP-1 was localised in the epidermal cells of etiolated maize coleoptiles, in subepidermal parenchymatic mesophyll cells of the coleoptile and in the companion cells of the vascular bundles. Most labelling was found in the cytoplasm, less in nuclei and vacuoles and cell walls appeared negative. The region of the plasma membrane exhibited prominent labelling. Embryos showed low labelling throughout their tissues just after excision, but after culture for 7 days intensive labelling was found in the epidermis of the scutellum. Quantitative electron microscopy confirmed that ABP-1 was present in the cytoplasm of epidermal, mesophyll, and companion cells of coleoptiles. Gold particles were neither found in cell walls nor in the cuticle. Areas with ER and dictyosomes within epidermal and mesophyll cells of coleoptiles had a denser labelling with gold particles than elsewhere. Labelling at the plasma membrane, being the site where the auxin binds to the ABP, was observed at low levels in all cells examined, which is due to the method applied. Epidermal cells of embryos cultured for 5 days exhibited high levels of gold particles in ER and nuclei, and lower levels in the cytoplasm. The distribution is only partly in accordance with the model in which ABP is thought to cycle through the plant cell from the ER via the Golgi system towards the plasma membrane.Abbreviations ABP-1 auxin-binding protein 1 - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - LM light microscopy - LR Write London resin white - PBS phosphate-buffered saline - PEG polyethylene glycol - TEM transmission electron microscopy     

Purification and immuno-electron microscopical characterization of crystalline inclusions from plant peroxisomes

Summary This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractions which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.Abbreviations ACOx acyl-CoA oxidase - BSA bovine serum albumin - EDTA ethylenediamine-tetraacetate - GDH glutamate dehydrogenase - GOx glycolate oxidase - KPB potassium phosphate buffer     

In situ ultrastructural localization of sugar-binding sites in lizard granulosa cell nuclei

Mannose-binding sites were detected at the ultrastructural level in nuclei of lizard granulosa cells in situ by means of mannosylated ferritin. When ultrathin sections of material embedded in glycol methacrylate were incubated with mannosylated ferritin, a strong labelling was observed over nucleoli, chromatin and the external leaflet of the nuclear envelope, but no labelling was detected in the perinuclear space except for nuclear pores. This labelling was clearly inhibited when sections were incubated in a solution containing both mannosylated ferritin and a sugar-related neoglycoprotein (mannosylated serum albumin). These ultrastructural data supporting the existence of nuclear lectin-like components in reptilian cells are in agreement with our previous findings about such components in nuclei isolated from mammalian cells. Owing to the unique organization of the granular component of the nucleoli in specialized granulosa cells, we are able to show that some of the mannose-binding sites are associated with ribosomal precursors.     

Identification of associating carbohydrate sequences with labelled oligosaccharides

The gelling subunit of alginate, the major cell-wall polysaccharide of brown algae, was used as a molecular marker for identification of this cell-wall carbohydrate subunit at the cellular level. Short polyguluronate chains were conjugated to fluorescein and used as a probe to identify the gelling regions of alginate in tissue sections from a brown alga. The specificity of the probe for gelling subunits was demonstrated by lack of cell-wall labelling in the absence of calcium, correlation between divalent-cation binding affinities of polyguluronate with labelling intensity, and lack of labelling by fluorescein-conjugated nongelling subunits. The probe labelling-pattern also differed from sulfated fucan distribution. Extracellular matrix and cell walls were labelled on sections of vegetative blade, stipe and reproductive frond of Fucus gardneri Silva. Probe labelling was rapid, being virtually complete within 5 min. Probe labelling in seawater differed markedly from labelling at lower ionic strength and is interpreted as reflecting alginategelling properties in natural conditions. High-and low-affinity binding sites are discussed in terms of gelling-subunit length and steric availability. Fluorescein-conjugated polygalacturonate, which also forms calcium dimers, labelled extracellular alginate by formation of mixed polygalacturonate-polyguluronate dimers. Binding by the alginate hybridization probe differs from nucleic-acid hybridization in divalent-cation bridging and the lack of both a conformational transition and polymer polarity.Abbreviations EDTA ethylenedinitrilo tetraacetic acid (ethylenediaminetraacetic acid) - NMR nuclear magnetic resonance spectroscopy     

Isolation and function of a low molecular weight protein of mung bean embryonic axes

A low molecular weight protein from dry mung bean (Vigna radiata) embryonic axes has been purified to near homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. It shows a molecular weight of about 12,000 in sodium dodecyl sulfate-polyacrylamide gels and a sedimentation coefficient of about 2 S in sucrose gradients. This protein occurs in greater amounts in dry axes than in dry cotyledons, and it dramatically disappears during early germination of the seed. Affinity chromatography tests do not indicate it as a trypsin inhibitor or as a glycoprotein. It is a water-soluble cytoplasmic protein exhibiting an amino acid composition characteristic of storage proteins with a high content of glutamic acid/glutamine. We suggest that it is a low molecular weight storage albumin.Abbreviations Asx aspartic acid/asparagine - BSA bovine serum albumin - Con A concanavalin A - EB extraction buffer - Glx glutamic acid/glutamine - HA hydroxylapatite - PB phosphate buffer - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Localization of mRNA for pea legumin:In situ hybridization using a biotinylated cDNA probe

Summary In situ hybridization has been used to locate mRNA, for the storage protein legumin, in cotyledon storage parenchyma tissue of developing pea (Pisum sativum L.) seeds. The mRNA was hybridized with a biotinylated probe of cDNA in pBR 322 and subsequently located by avidin conjugates. Avidin-rhodamine was used for fluorescence microscopy localization at a tissue/cellular level and avidin-peroxidase (with DAB) and avidin-ferritin compared for localization at an ultrastructural level. Specific fluorescence associated with avidin-rhodamine was distributed unevenly throughout the cytosol but the cell walls, starch grains, vacuoles and protein deposits were unstained. The sizes and distribution of the regions of higher labeling within the cytosol suggest an association with elements of the endomembrane system. Following DAB reaction of the specifically localized avidin-peroxidase most, although not all, stain product was associated with the endoplasmic reticulum. The ER-associated reaction product was also accumulated within the ER lumen.Avidin-ferritin was also localized both in the cytosol and in association with the endoplasmic reticulum, although was less readily visualized in cells with a conventional ultrastructural appearance.Localization of avidin-ferritin was more readily visualized in cells which had undergone some limited structural damage during specimen preparation. In such cases ferritin was also shown to be specifically associated with the transition vesicles and trans-face peripheral vesicles of some dictyosomes.     

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.     

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.     

Heterogeneity of sulphur content in the storage proteins of pea cotyledons

By means of crossed immunoelectrophoresis of the cotyledonary storage proteins of Pisum sativum L. it was shown that reduced accumulation of the legumin fraction, resulting from severe sulphur deficiency during growth, is accompanied by relative suppression of a quantitatively minor storage protein (Peak 3) shown previously by subunit analysis to be related to the vicilin series of holoproteins. The pattern of isotopic labelling of the storage proteins after injection of [³⁵S]methionine into the pedicel during seed development under normal nutritional conditions indicated that Peak-3 protein, like legumin, has a relatively high content of sulphur amino-acids. Like certain of the vicilin molecules carrying the determinants responsible for Peak-4, Peak-3 protein binds selectively to concanavalin A.     

Storage-protein hydrolysis and protein-body breakdown in germinatedZea mays L. seeds

Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein 1. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.     

Electron-microscopic demonstration of olfactory-marker protein with protein G-gold in freeze-substituted,Lowicryl K11M-embedded rat olfactory-receptor cells

Summary In this study electron-microscopic immunocytochemistry was used to localize olfactory marker protein in olfactory epithelia. Rat olfactory-epithelial samples were rapidly frozen, freeze-substituted with acetone, embedded at low temperatures with Lowicryl K11M and labelled on the sections with polyclonal antibodies raised against olfactory marker protein and with protein G conjugated to colloidal gold. Apart from the aforementioned use of acetone, substitution was carried out in the complete absence of chemical fixation, i.e., neither aldehydes nor OsO⁴ were used. This procedure resulted in localization concurrent with a good ultrastructural preservation. Olfactory-marker protein was present throughout the cytoplasmic compartments of dendrites and dendritic endings of olfactory-receptor cells, but it was not found in organelles such as mitochondria. Olfactory-marker protein was found only in dendriticendings of olfactory-receptor cells mature enough to have given rise to cilia, but these cilia displayed less labelling than dendrites and dendritic endings. Olfactory-marker protein was not found in apices and microvilli of neighboring olfactory-supporting cells.     

Separation of macromolecules by ultrafiltration: Removal of poly(ethylene glycol) from human albumin

Mixtures of albumin and poly(ethylene glycol) (PEG) were used to elucidate some of the factors which influence the separation of macromolecules by thin-channel ultrafiltration. Several membranes which readily passed PEG-4000 in the absence of protein were found to exhibit increased rejection of the synthetic polymer when albumin was added to the system. Based on a comparison of filtration flux and net sieving properties, the PM-30 membrane of Amicon was chosen for further characterization. The increased rejection of PEG-4000 in the presence of albumin was independent of albumin concentration between 1 and 100 mg/ml and persisted even after albumin was removed and the system flushed with water. Overnight incubation of the membrane with trypsin restored the original sieving properties, indicating that the ‘permanent’ effects were due to irreversible adsorption to the membrane. By measuring flux over a 10⁶-fold range of albumin concentration it was possible to resolve the effects of protein adsorption, a saturable process which occurs at low protein concentration (<0.01 mg/ml), from the effects of concentration polarization which occur at high protein concentration (>0.1 mg/ml). Only the former process has an effect on the net sieving properties in this system. In spite of the adverse effects of protein adsorption, it was still possible to obtain efficient removal of PEG-4000 from albumin. Exchange of approximately 5 vols. of solvent at room temperature resulted in a 10-fold reduction in the concentration of PEG in the sample, with no loss of albumin, and no formation of albumin dimers.     

Plant tyrosine decarboxylase can be strongly inhibited by l-α-aminooxy-β-phenylpropionate

The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of DNA and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1–1 g · ml^-1 tunicamycin, cell division and DNA synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear DNA content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells.Abbreviations TCA trichloroacetic acid - TM tunicamycin     

A naturally occurring molecular form of human plasma cholinesterase is an albumin conjugate

Human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) consists of four main molecular forms designated as C1, C2, C3 and C4 according to their electrophoretic mobility on gels. The major component, C4, is the tetrameric form; C1 and C3 are the monomeric and dimeric forms, respectively. The C2 form, which has an apparent free electrophoretic mobility higher than that of the three size isomers, and, moreover, a higher isoelectric point, was found to be a covalent conjugate between the cholinesterase monomer and serum albumin. This result is supported by the following arguments: the non-catalytic subunit of C2 was found to be a carbohydrate-free protein of apparent molecular mass 65 kDa that could not be labelled by diisopropylfluorophosphonate in the labelling conditions of esterases. It possesses a high affinity for a long-chain aliphatic ligand (a substituted octadecylamine) and for Cibacron blue F3 GA, and could be adsorbed on an immunoadsorbent for albumin. The two subunits of C2 are disulfide bridge linked; the active center of the cholinesterase subunit is partly masked by the albumin molecule. The conjugation reaction very likely occurs in the hepatic cell and not in plasma.     

The initiation of legumin synthesis in immature embryos of Pisum sativum L. grown in vivo and in vitro

A highly sensitive immunoassay has been used for the detection of a major storage protein, legumin, in embryos of Pisum sativum L.; with this technique nanogram quantities could be measured. In the two varieties tested, legumin could be detected in embryos in vivo, when they had attained a fresh weight of 2·10^-3 g and 3·10^-3 g, respectively. Contrary to earlier claims, embryos cultured in vitro were shown to be capable of initiating legumin synthesis. This capacity to initiate legumin synthesis was confirmed by two-dimensional isoelectric focusing-electrophoresis and fluorography; embryos harvested before initiation of legumin synthesis and cultured in radioactive medium were shown to have synthesized legumin subunits. The amounts of legumin and total protein synthesized per unit fresh weight were consistently greater in vitro than in equivalent embryos grown in vivo.Abbreviations ELISA Enzyme-linked immunosorbent assay - BSA bovine serum albumin - IgG immunoglobulin - SDS sodium dodecyl sulphate - DSP Pisum cv. Dark Skinned Perfection     

Alterations of the chlorophyll-protein pattern in chronically photoinhibited Chenopodium rubrum cells

When photoautotrophic Chenopodium rubrum L. culture cells were exposed to high photon flux densities for seven consecutive light periods a marked reduction in photochemical efficiency, chlorophyll (Chl) content and Chl a/b ratio occurred. These alterations were accompanied by distinct changes in the pigment and protein composition of the thylakoid membranes. In photosystem II (PSII) a reduction in the relative contents of proteins from the reaction center (D1 protein, D2 protein and Cyt b₅₅₉) and the inner antenna (CP43 and CP47) was observed. In agreement with the reduction in the Chl a/b ratio an increase in the relative content of the major light-harvesting complex of PSII (LHCII) could be demonstrated. The minor chlorophyll-proteins of PSII were only slightly affected but PSI (quantified as total complex) showed a reduction upon chronic photoinhibition. The changes in protein composition were accompanied by a drastic increase in the contents of lutein and the xanthophyll-cycle pigments and by a reduction in the β-carotene content. The effects on lutein and xanthophyll-cycle pigment content were most pronounced in stroma thylakoids. Here, an increase in LHCII (which harbours these pigments) was clearly detectable. Considering the pigment content of LHCII, the change in its apoprotein content was not large enough to explain the pigment changes.     

Non-expression of insulin-like growth factor-I receptor is associated with apoptosis: an ultrastructural study on rat ameloblasts

Insulin-like growth factor-I (IGF-I) is a pleiotrophic polypeptide which appears to have roles both as a circulating endocrine hormone and as a locally synthesized paracrine or autocrine tissue factor. IGF-I plays a major role in regulating the growth of cells in vivo and in vitro and initiates metabolic and mitogenic processes in a wide variety of cell types by binding to specific type I receptors in the plasma membrane. In this study, we report the distribution of IGF-I receptors in odontogenic cells at the ultrastructural level using the high resolution protein A-gold technique. In the pre-secretory stage, very little gold label was visible over the ameloblasts and odontoblasts. During the secretory stage the label was mostly seen in association with the cell membranes and endoplasmic reticulum of the ameloblasts. Lysosome-like elements in the post-secretory stage were labelled as well as multivesicular dense bodies. Very little labelling was encountered in the ameloblasts in the transitional stage, where apoptotic bodies were clearly visible. The maturation stage also exhibited labelling of the secretory-like granules in the distal surface. The presence of gold particles over the plasma membrane is an indication that IGF-I receptor is a membrane-bound receptor. Furthermore, the intracellular distribution of the label over the endoplasmic reticulum supports the local synthesis of the IGF-I receptor. The absence of labelling over the transitional ameloblasts suggests that the transitional stage may require the non-expression of IGF-I as a prerequiste or even a trigger for apoptosis. This revised version was published online in November 2006 with corrections to the Cover Date.