Applications of fluorochromes to pollen biology. II. The DNA probes ethidium bromide and Hoechst 33258 in conjunction with the callose-specific aniline blue fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

Applications of Fluorochjromes to Pollen Biology. Ii. The Dna Probes Ethidium Bromide and Hoechst 33258 in Conjunction with the Callose-Specific Aniline Blue Fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

Cytochemical Analysis of Callose Localization in Lilium longiflorum Pollen Tubes

Callose, a ß, 1–3 glucan as a component of plantcells has received sporadic attention. Here, we report an attemptto determine whether aniline blue and lacmoid are indeed specificfor visualizing callose. We also re-evaluate, based on a checkfor stain specificity, the localization of callose in elongatingLilium longiflorum, cv. ‘Ace’ pollen tubes. Specificityof these stains was checked by chemical and enzymatic extractionprocedures which solubilize proteins and polysaccharides. Resultsherein question the generally accepted validity of the fluorescent-anilineblue method for detecting callose. Lacmoid either possessesan affinity for both callose and protein or for callose as aglycoprotein. As for callose localization, the walls of thenon-growing region of the lily pollen tube contain callose,probably as a glycoprotein. Presence of the callosicglycoproteinin the wall of the growing tube-tip is dependent on tube length.Callose plugs exhibiting an affinity for aniline blue or lacmoidwere never seen. Phase-contrast microscopy revealed non-stainablewall ingrowths in fixed-tubes and free-moving cytoplasmic masseswithin living tubes.     

Effects of tissue-preparation-induced callose synthesis on est plasmodesma size exclusion limits

Plasmodesmata are often characterised by their size exclusion limit (SEL), which is the molecular weight of the largest dye, introduced by microinjection, that will move from cell to cell. In this study, we investigated whether commonly used techniques for isolation and manipulation of tissues, and microinjection of fluorescent dyes, affected the SEL, and whether any such effects could be ameliorated by inhibiting callose deposition. We examined young root epidermal cells of Arabidopsis thaliana and staminal hair cells of Tradescantia virginiana, two tissues often used in experiments on symplastic transport. Transport in root tips dissected from the main plant body and in stamen hairs removed from the base of the stamen filament was compared with transport in undissected roots and stamen hairs attached to the base of the filament, respectively. Tissues were microinjected with fluorescent dyes (457 Da to > 3 kDa) with or without prior incubation in the callose deposition inhibitors 2-deoxy-D-glucose or aniline blue fluorochrome. In both tissues, dissection reduced the SEL, which was largely prevented by prior incubation in 2-deoxy-D-glucose but not by incubation in aniline blue fluorochrome. Thus, standard methods for tissue preparation can cause sufficient callose deposition to reduce cell-to-cell transport, and this needs to be considered in studies employing microinjection. Introduction of the dyes by pressure injection rather than iontophoresis decreased the SEL in A. thaliana but increased it in T. virginiana, showing that these two injection techniques do not necessarily give identical results and that plasmodesmata in different tissues may respond differently to similar experimental procedures.     

植物小孢子母细胞减数分裂过程中胼胝质染色的新方法

利用改良苯酚品红-苯胺蓝压片法,观察小孢子母细胞减数分裂过程中胼胝质的动态变化。使用该方法简便、快速且省时,获得的照片颜色鲜艳,细胞质呈红色,染色体为深红色,胼胝质呈黄绿色荧光,对比明显,有三维效果。单用改良苯酚品红染液对新鲜材料进行压片,在蓝光激发下,细胞质与染色体呈红色荧光,染色体清晰。实验结果表明,改良苯酚品红染液可作为荧光染料代替DAPI及H33258等昂贵的核染料,从而降低实验成本。     

Occurrence and phylogenetic significance of cytokinesis-related callose in green algae, bryophytes, ferns and seed plants

 In order to investigate the occurrence of callose in dividing cells, we cultivated a selection of 30 organisms (the prokaryotic cyanobacterium Anabaena and eukaryotic green algae, bryophytes, ferns and seed plants) under defined conditions in the laboratory. Samples from these photoautotrophs, which are members of the evolutionary 'green lineage' leading from freshwater algae to land plants, were analysed by fluorescence microscopy. The β-1,3-glucan callose was identified by its staining properties with aniline blue and sirofluor. With the exception of the prokaryotic cyanobacterium, all of the eukaryotic organisms studied were capable of producing wound-induced callose. No callose was detected during cytokinesis of dividing cells of unicellular green algae (and Anabaena). However, in all of the multicellular green algae and land plants (embryophytes) investigated, callose was identified in newly made septae by an intense yellow fluorescence. The formation of wound callose was never detected in cells with callose in the newly formed septae. Additional experiments verified that no fixation-induced artefacts occurred. Our results show that callose is a regular component of developing septae in juvenile cells during cytokinesis in multicellular green algae and embryophytes. The implications of our results with respect to the evolutionary relationships between extant charophytes and land plants are discussed. Received: 15 September 2000 / Revision received: 23 October 2000 / Accepted: 23 October 2000     

植物小孢子母细胞减数分裂过程中胼胝质染色的新方法

利用改良苯酚品红-苯胺蓝压片法, 观察小孢子母细胞减数分裂过程中胼胝质的动态变化。使用该方法简便、快速且省时, 获得的照片颜色鲜艳, 细胞质呈红色, 染色体为深红色, 胼胝质呈黄绿色荧光, 对比明显, 有三维效果。单用改良苯酚品红染液对新鲜材料进行压片, 在蓝光激发下, 细胞质与染色体呈红色荧光, 染色体清晰。实验结果表明, 改良苯酚品红染液可作为荧光染料代替DAPI及H33258等昂贵的核染料, 从而降低实验成本。     

Deposition of Cytokinesis‐Related Callose in Riella helicophylla and Arabidopsis thaliana. Effects of Photolytically Altered Nifedipine1

Abstract: The cytokinesis‐related callose deposition in cell plates and juvenile cross walls of meristematic cells was investigated in the liverwort Riella helicophylla and seedlings of Arabidopsis thaliana. The β‐1,3‐glucan callose was detected by its specific staining properties with sirofluor and aniline blue by fluorescence microscopy. The photo‐labile calcium antagonist nifedipine (NIF) exerted a specific promotive effect when the substance was exposed to light. The nitroso derivative of photolysed NIF was found to be the active compound which was responsible for the enhancement in callose deposition. The nitroso derivative was isolated after photolysis of NIF by UV light (365 nm) and its structure was verified with ¹H‐nuclear magnetic resonance and infrared spectroscopy. The characteristic absorption maximum at 770 nm in dimethyl sulfoxide was employed to determine the concentration of the nitrosopyridine in solutions by use of the molar absorption coefficient of the isolated substance. In addition, the nitro derivative of nifedipine was prepared. This nitropyridine was ineffective with respect to the stimulation of callose deposition in dividing cells. The possible mechanism of this cytotoxic effect and its implications for symplastic growth in meristems is discussed.     

Fluorescent vital staining of plant sexual cell nuclei with DNA—specific fluorochromes and its application in gametoplast fusion

DNA－binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei.These were:the cytoplasmic streaming in pollen tubes whose nuclei were stined,the simultaneous visualization of fluorochromatic reaction and nucleus staining in isolated generative cells,and the capability of isolated.prestained generative or sperm cells to fuse with other protoplasts.The results confirmed that 4,6-diamidino-2-phenylindole(DAPI),Hoechst 33258 and mithramycin could be used as real vital stains,though their efficiency varied from case to case;among them DAPI showed best effect.The fluo rescent vital staining technique offered a useful means foridentification and selection of heterokaryons in gametoplast manipulation studies.     

Schistosoma mansoni: a diamidinophenylindole probe for in vitro death of schistosomula

The following fluorochromes were studied as probes for discrimination between living and dead Schistosoma mansoni schistosomula: ethidium bromide (EB), propidium iodide (PI), diamidinophenylindole (DAPI), and carboxyfluoresceine diacetate (C-FDA). While schistosomula stained with EB, PI, or C-FDA showed leakage of fluorochrome into the medium, this was not the case with DAPI. Dead schistosomula, which were stained with DAPI, showed an intense blue fluorescence, while living schistosomula were not stained even after prolonged incubation. In addition, the low DAPI concentration (1 microgram/ml) in the medium proved not to be toxic to the schistosomula, nor did it cause any background fluorescence. These properties make DAPI an ideal probe: the viability of S. mansoni schistosomula in cytotoxicity tests can be continuously monitored in tissue culture trays, using an inverted microscope with simultaneous transmitted light and incident fluorescent light illumination.     

Microsporogenesis and pollen development in Leymus chinensis with emphasis on dynamic changes in callose deposition

Leymus chinensis is an economically and ecologically important grass that exhibits low seed production. To better understand the causes of its low sexual reproductivity, the microsporogenesis and pollen development of this species were investigated, with emphasis on dynamic changes in callose deposition. A variety of histochemical stains were employed, including Heidenhain's hematoxylin, decolorized aniline blue, DAPI, and acetocarmine, along with a temporary mount method. Microsporogenesis and pollen development generally took place from June 12 to 26. The meiosis of microspore mother cells (MMCs) was of the successive type and the tetrad was isobilateral in shape. Mature pollen grains comprised two sperms and a vegetative nucleus. Callose initially appeared in the center of the anther locule at the premeiotic phase, and then gradually and unevenly deposited around the MMC before the commencement of meiosis. At the onset of meiosis, the accumulation of callose enclosing the MMC peaked, accompanied by the disappearance of callose in the center of the locule. At the dyad and tetrad stages, the dyads and tetrads were surrounded by callose wall and the microspores in the tetrads were isolated by a crossed cell plate composed of callose. Microspores just released from tetrads were still enclosed in callose wall, and then callose gradually disappeared in the pollen wall. Ultimately, callose almost completely disappeared from the walls of mature pollen grains. In the large numbers of sections observed, most of the cases of meiosis of the MMCs, pollen development, and callose dynamics were normal, with only a few abnormities observed. The results suggest that microsporogenesis, male gametogenesis, and callose dynamics during these processes are generally normal in this species, and that the callose wall plays an important role in the production of functional pollen grains. The small numbers of abnormities of these processes that occurred likely do not adversely affect the production of viable pollen grains. Therefore, microsporogenesis and pollen development may not be factors in the low seed production of L. chinensis.     

Feulgen-positive staining of the cell nuclei in fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae

The Feulgen reaction and the staining of preparations with two DNA-specific fluorochromes, Hoechst 33258 and 4',6-diamidino-2-fenilindol (DAPI), were used to study the preservation of DNA in the fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae, Paramyrtacicarpus plurilocularis and Paramyrtaciphyllum agapovii collected in Yakutia (Siberia, Russia). It was shown that chromatin structures of the fossilized plants form stable red-purple complexes with the Schiff's fuchsin sulphuric acid reagent in situ . This coloration is specific for DNA, in particular, for the deoxyribose residues. It means that the cell nuclei of these 53–55 Myr old plants preserve a part of the deoxyribose backbone of DNA molecules. On the other hand, there was no, or only a very weak, staining of the cell nucleus with fluorochromes DAPI or Hoechst 33258, which specifically bind to the double-stranded DNA and do not bind to either the single-stranded DNA or RNA molecules. The stainability of fossil plant cell preparations with alcian blue shows that there are also polysaccharides containing carboxyl groups in the cell walls of fossilized leaf and fruit tissues of the Lower Eocene Myrtaceae.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 315–321.     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.     

THE PRESENCE OF CALLOSE IN THE PRIMARY ZYGOTE WALL OF CHLAMYDOMONAS MONOICA AND THE EFFECTS OF ITS DEGRADATION ON ZYGOTE DEVELOPMENT

Chlamydomonas monoica constructs a temporary primary wall around its developing zygotes. This study aimed to confirm callose as a component of the primary wall, as well as to note the effects of primary wall degradation on zygote development. Glucanase, specific for the β-1,3 glycosidic bonds comprising callose, was added to mating media at concentrations ranging from 5 to 1 mg ml⁻¹ and light microscope observations were made as the zygotes developed. The overall health of the zygotes was assessed by comparing their ability to germinate after exposure to chloroform vapors. The bright staining of the primary wall with aniline blue, specific for β-1,3 polysaccharides, suggested the presence of callose. This was further supported by the adverse effects of glucanase on zygote development. After mating, declining levels of intact zygotes were found as their maturation continued, and dead immature zygotes accumulated in the treated cultures. Twelve days after mating, when the zygotes were plated for germination, fully mature zygotes were identified in only the lowest of the six enzyme concentrations. In addition, germinating zygotes from the treated cultures showed increased sensitivity to killing by chloroform vapors relative to untreated zygotes. These results suggest that callose is a key component in the primary zygote wall, and that its degradation negatively affects zygote maturation. Electron microscopy will be used to help determine whether structural defects in the primary wall occur as a result of glucanase treatment, and whether such defects affect secondary zygospore wall assembly.     

Combined Bright Field and Fluorescence Staining of Paraffin Sections for Studying the Fertilization Process in Plants

PAS-toluidine blue O—aniline blue staining of paraffin sections allows study of histological and cytological detail while retaining aniline blue induced fluorescence in all “callose sites”. Because most autofluorescence is eliminated by the PAS-toluidine blue prestaining, the detail and contrast of the fluorescence image is superior to slides stained in aniline blue alone. Slides are stained by the PAS reaction, 0.03% toluidine blue O, alkaline 0.005% aniline blue, and mounted directly in aqueous mounting medium.     

Cytological indication of the involvement of calcium and calcium-related proteins in the early responses ofBryonia dioica to mechanical stimulus

Summary The distribution of membrane-bound calium, activated calmodulin, and callose synthesis was visualized inBryonia dioica internodes before and after mechanical stimulus, using fluorescent probes, respectively, chlorotetracycline, fluphenazine, and aniline blue. Bright chlorotetracycline fluorescence remains localized in the plasma membrane of control cells, 30 s after stimulation calcium left the plasmalemma. A delocalization of activated calmodulin was observed after wounding and deposition of callose, which could not be detected before, appeared in the same times in most cells. The callose formation and the decrease in membrane-associated calcium suggest a rapid influx of calcium in the cytosol and an intervention of this ion in the cascade of the early events underlyingBryonia dioica thigmomorphogenesis.Abbreviation CTC chlorotetracycline