ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01502.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThis review considers some of the recent advances in the applications of molecular histochemical techniques to the analysis of the temporal and spatial patterns of gene expression associated with differentiation and development in plant tissues. A range of methods is outlined, and those for tissue preparation and the isotopic and non‐isotopic labelling of probes forin situhybridization studies are discussed, taking account of the various approaches which can be used for the localization of specifically bound probes.An overview of some successful applications of molecular histochemistry includes the elucidation of the roles of homeotic genes in floral biology, the ontogeny and differentiation of the photosynthetic apparatus, and aspects of seed development, including storage protein gene regulation during embryogenesi

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01503.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe use of fluorescent probes as indicator and tracer molecules is becoming an important aspect of plant cell biology. In many cases the dye, whether introduced directly into the cytosol or sequestered by the cell from its external environment, is preferentially transferred to the vacuole. In the light of increasing evidence for endocytosis in plant cells, the sequestration of high‐molecular‐weight fluorescent dextrans and the membrane‐impermeant dye Lucifer Yellow‐CH into the vacuole has been cited as evidence supporting the presence of a fluid‐phase endocytic pathway. In this review we consider these recent reports of vacuolar sequestration in the light of new evidence arising on the mechanisms underlying d

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01504.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYWe have used a multiple‐laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442‐nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC‐rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442‐ and 488‐nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi‐laser system and a multi‐line single‐laser instrument. This limited high‐resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre‐focusing the illumination. With this system we have imaged DAPI‐stained nuclei, callose in pollen tubes using Aniline Blue and the

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01505.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYHigh‐pressure frozen Golden Delicious apple leaves were cryosectioned at low temperature with diamond knives. Good cryosections were obtained by optimizing the cutting parameters, i.e. sectioning temperature, mechanical stability of the sample, and sectioning velocity. Cutting artefacts were minimized by reducing the electrostatic interactions between the knife surface and the cryosection. This was accomplished by sectioning the sample in the presence of an ionization electrode. The ionization device, with a primary voltage of 7–8 kV, produces positively and negatively charged nitrogen ions which neutralize the surface charges of the knife and the section. This minimizes the friction on the knife surface and results in ultrathin sections without crevasses or knife marks. Compression of the sections could be minimized, but not avoided, by reducing the knife angle to 30°. Improved contrast of the frozen‐hydrated sections was obtained with the Zeiss EM 902 energy‐filter microscope operated in the zero

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01506.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYCalcium plays a central role as a second messenger in plant and fungal cells and as such is involved in controlling numerous biological processes. Direct demonstration of signal‐response coupling via Ca2+requires the measurement and localization of changes in cytosolic free Ca2+, [Ca2+]i, during these processes in living cells. In recent years this has become possible with the introduction of a range of fluorescent dyes (e.g. Indo‐1 Fura‐2 and Fluo‐3) which have a high affinity and selectivity for free Ca2+. When used with recently developed microscope technologies (e.g. fluorescence ratio imaging or confocal scanning laser microscopy), subcellular localization and precise quantification of [Ca2+]idynamics in single cells can be achieved. This review describes the principles of [Ca2+]iimaging and measurement using fluorescent dyes, the equipment required to do it, the problems with botanical material and how they are being overcome, future developments for this approach in plant cell biology, and an entirely different strategy for the imaging and measurement of [Ca2+]iinvolving genetic transformation with the aequor

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01507.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYWe describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide‐field) fluorescence microscope.In situhybridization with probes to telomere and ribosomal rDNA sequences, combined with three‐dimensional (3‐D) microscopy, has been used to study interphase nuclei in root tissue ofPisum sativumandVicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in theViciatissues, suggesting specific binding interactions. rDNA labelling inP. sativumshows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3‐D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confoc

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01508.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe way in which transvacuolar strands radiating from the cell nucleus reorganize to form the phragmosome, within which division occurs, has been thoroughly studied in epidermal explants ofNautilocalyx lynchii.In recent years it has been established that the movement of the nucleus into the centre of large vacuolated cells such as these, in preparation for division, involves actin filaments. In the present study, the appearance and gradual reorganization of nucleus‐associated microtubules (NAMTs) over the premitotic period is described. Epidermal explants fluorescently labelled with anti‐tubulin were optically sectioned by confocal scanning laser microscopy, the sections reconstructed by an image processing computer and projected as rotating stereo pairs. This revealed that the NAMTs are a major component of the phragmosome, and that they change from a radiating to a planar distribution concomitantly with the ‘bunching’ of cortical MTs to form the pre‐prophase band. The continuity of the two sets of MTs indicates that the band contains newly polymerized microtubules. Other recent studies on the division of vacuolated cells are reviewed and factors affecting the alignment of the division plane are

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01509.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYAffinity methods (enzymes, lectins and antibodies used as specific probes) were applied in order to target cellulose and the major matrix components in cell walls of dicotyledons built up as helicoids. The probes were the enzyme cellobiohydrolase, CBH1, for cellulose, polyclonal antibodies and the lectin RCA (Ricinus communisagglutinin) for xyloglucans, and monoclonal antibodies JIM5 or JIM7 for homogalac‐turonan sequences (according to their degree of esterification). Observations were performed: (i)in muroand/or on heteromolecular fractions following controlled cell‐wall dissociation experiments; and (ii) at the light microscope level and/or at the electron miscroscope level by means of various visualization markers. Affinity labelling was complemented by subtractive cytochemistry and by the labelling of available anionic groups by means of cationic gold particles. Data confirm the importance of using a variety of probes, the combination of which allows the acquisition of convergent and complementary results. Concerning the particular case of helicoidal walls of elongating cells, it was shown that cellulose was always co‐localized with xyloglucans and homogalacturonan polymers in zones where the cholesteric order was well defined. Cellulose was always associated with compatible hemicellulose polymers capable of binding tightly. Moreover, residual charges were always present along the microfibrils, forming an anionic coat able to repel the adjacent cellulose microfibrils. A possible role of the heteromolecular association of xyloglucan and pectate as a surfactant allowing the cholesteric assembly is hypothe

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01510.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYDistinct polysaccharide fractions can be obtained from onion cell walls by sequential extraction methods. Pectins and xyloglucan molecules from these fractions have been imaged by electron microscopy and their lengths measured. Unexpectedly, instead of a simple distribution of lengths, both classes of matrix polysaccharide showed clear evidence of length periodicity, suggesting block polymer construction using subunits about 30 nm long, each containing a minimum of about sixty backbone sugar residues. These results are discussed in the light of our observation that the average hemicellulose cross‐link between cellulose microfibrils in the wall is also about 30 nm long, and that pectin forms a co‐extensive network with the cellulose/xyloglucan network. Using monoclonal antibodies to pectin we have further shown that even within a thin primary cell wall, containing only a few lamellae, distinct zones of pectin distribution are detectable, thus reinforcing our notion of the importance of domains in wall architecture. The chemical heterogeneity detected amongst pectins indicates that in a primary cell wall each lamella of cellulose microfibrils may be in a unique matrix environm

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01511.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY