An Electron Microscopic Investigation into the Effect of EDTA on Plant Cell Wall

Klein, Shoshana; Ginzburg, B. Z.

doi:10.1083/jcb.7.2.335

Cited by 13 publications

(5 citation statements)

References 9 publications

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“…In spite of the brevity of the enzyme treatment, this and the subsequent squashing are capable of weakening or perforating the cell wall sufficiently to allow it to pass antibody molecules. As reported earlier by others using much higher concentrations and longer exposures to another Ca" chelator, EDTA (19,21), inclusion of 5 mM EGTA in all processing steps appears to aid in the cell separation, and results in a higher proportion of intact, undistorted cells after the squashing. Permeabilized, isolated, stabilized plant cells obtained in this way react with affinity-purified antibodies to porcine brain tubulin to reveal microtubule arrays throughout all stages of the cell cycle .…”

Section: Discussionsupporting

confidence: 67%

“…Inclusion of EGTA in the fixative did not have a noticeable effect on microtubule preservation, but made possible the isolation of undamaged cells from roots incubated with any of the enzymes. This is presumably a result of partial dissolution, by means of Ca" chelation, of middle lamella Ca" pectates that "cement" adjacent cell walls together (19,21). Weakening of intercellular bonding was enhanced when EGTA was included in buffer rinses as well as in the fixative and enzyme solutions, yielding cells such as seen in Fig.…”

Section: Isolation Of Cellsmentioning

confidence: 99%

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Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Wick

Seagull

Osborn

et al. 1981

The Journal of Cell Biology

242

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Microtubules participate as morphogenetic tools in two basic processes by which plants develop their characteristic forms : (a) production of new cells in specific sites and with specific initial shapes by partitioning of parental cells, and (b) further shaping of the progeny during their expansion and differentiation (8,18) . In respect of (a), microtubules are present in the mitotic spindle, where they develop in the absence of centrioles (14). Immediately before the division cycle they are deposited as a transitory "pre-prophase band" (PPB) (8,26,27), which in its positioning predicts the site and plane of the future cytokinesis. At telophase another microtubule system contributes to the organization of the phragmoplast, which contains the new partitioning wall, or cell plate. In respect of(b), there are many instances of congruence between the orientation of microtubules in the cell cortex during interphase ("interphase cortical arrays") and the orientation of currently deposited microfibrils of cell wall material (see 12 and 15 for recent summaries) . The inference is that the cell exerts geometrical control over its expansion by setting up specifically oriented microtubule arrays. These in turn guide wall deposition, thereby regulating the mechanical properties of the wall and determining its spatial reaction to the turgor forces that drive cell expansion.Until recently, the only available method for studying the various categories of microtubule array was electron microscopy. However, to gain detailed information on three-dimensional relationships from ultrathin sections, recourse to timeconsuming serial sectioning (12,13) ABSTRACT Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine . Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized . Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre-prophase bands, the mitotic spindle, and phragmoplast microtubules .The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.

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Section: Discussionsupporting

confidence: 67%

Section: Isolation Of Cellsmentioning

confidence: 99%

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Wick

Seagull

Osborn

et al. 1981

The Journal of Cell Biology

242

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“…This short wash has not removed the stain completely, but the difference is most striking. The effect described here seems to be essentially the same as that observed by Klein & Ginzburg (1960), though with the techniques then available it was not possible to see the details of individual microfibrils in thin sections. These results show clearly that the staining reaction is physical in nature, and there is therefore no reason to doubt that the stained structures are the cellulose microfibrils of the wall.…”

Section: R E S U L T S a N D Discussionsupporting

confidence: 60%

Electron microscopy of cellulose in entire tissue

Cox

Juniper²

1973

Journal of Microscopy

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SUMMARY Microfibrillar structures can be seen in ultrathin sections of cell walls which have been post‐stained with uranyl acetate and lead citrate. This stain is removable by washing, suggesting that a physical mechanism is involved. It is suggested that these structures are the cellulose microfibrils of the wall; they appear to be 3.5 nm in diameter and not fasciated into larger units. Freeze‐etching of untreated tissue supports this conclusion. These two techniques seem to have many advantages over methods previously used for the study of microfibril arrangement and structure.

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“…It has been reported that Versene has a disruptive effect upon the fine structure of plant cells (25), and the rat liver used for the present work responds in a similar way to this metalchelating agent. A m m o n i u m reineckate, on the other hand, improves the preservation of plant cells by reducing the extraction of phospholipid by formalin (26), probably through the formation of insoluble complexes with phospholipids (41).…”

Section: Discussionmentioning

confidence: 79%

Studies on Formalin Fixation for Electron Microscopy and Cytochemical Staining Purposes

Holt

Hicks

1961

The Journal of Cell Biology

220

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A study has been made of the preservation of fine structure, phospholipids, and the activity of acid phosphatase and esterase in rat liver fixed in various solutions containing 4 per cent formaldehyde. Examination of methacrylate-embedded preparations shows that calcium-containing fixatives result in poor preservation of fine structure, whereas veronal-treated or phosphate-buffered formalin gives excellent results if the tonicity of the solutions is suitably adjusted by addition of sucrose. Formol-phosphate, to which Versene has been added, causes deterioration of cellular morphology. Phospholipids are retained almost quantitatively in tissue fixed in formol-calcium, and in phosphate-, collidine-, or triethanolamine-buffered formalin. About 50 per cent of the activity of acid phosphatase and esterase are preserved after 24 hours exposure to these fixatives at 0–2°C, and the distributions of the enzymes and of phospholipids, as judged by cytochemical staining results, are not altered by any of these formalin solutions. Consideration of the morphological and biochemical integrity of the fixed tissue suggests that 4 per cent formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, and containing 7.5 per cent sucrose, is the most suitable of the fixatives for combined cytochemical staining and electron microscopical studies.

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An Electron Microscopic Investigation into the Effect of EDTA on Plant Cell Wall

Cited by 13 publications

References 9 publications

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Electron microscopy of cellulose in entire tissue

Studies on Formalin Fixation for Electron Microscopy and Cytochemical Staining Purposes

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