PEROXIDASES IN TOBACCO ABSCISSION ZONE TISSUE

J. Cell Sci. 13, 591-601 (1973) 591Printed in Great Britain

PEROXIDASES IN TOBACCO ABSCISSION

ZONE TISSUE

I. FINE-STRUCTURAL LOCALIZATION IN CELL WALLSDURING ETHYLENE-INDUCED ABSCISSION

E. W. HENRY AND T. E. JENSENDepartment of Biological Sciences, Herbert H. Lehman College of the City Universityof Netv York, Bronx, Neiv York 10468, U.S.A.

SUMMARYThe fine-structural localization of peroxidases during ethylene-induced abscission of flower

pedicels of Nicotiana tabacum L. cv. 'Little Turkish' has been investigated. Peroxidase activityhas been localized in both the cell walls and intercellular spaces of ethylene-treated flowerpedicels which were fixed in glutaraldehyde, incubated in diaminobenzidine (DAB) mediumwith postfixation in 2 % osmium tetroxide. Peroxidase staining is present in the cell walls andintercellular spaces of control tissue but is not as intense as in ethylene-treated tissue. Increasedperoxidase staining is evident in the intercellular spaces and cell walls after 2 h of exposure toethylene and increases in intensity between 2 and 5 h. At 5 h, ethylene-induced abscissionoccurs. Fine-structural investigations revealed prominent staining in the middle-lamellar andperipheral areas of the cell walls in ethylene-treated tissue. The peroxidase staining appears tobe due to peroxidase as prior incubation with potassium cyanide gives a marked reduction in thestaining reaction. Incubation of the ethylene-exposed tissue in aminotriazole, a specific inhibitorof catalase, does not reduce peroxidase staining, except in the microbodies, which reportedlycontain catalase.

INTRODUCTION

Several ultrastructural studies have been made on naturally occurring abscission intomato and tobacco flower pedicels (Jensen & Valdovinos, 1967, 1968; Valdovinos &Jensen, 1968). According to recent evidence, ethylene may be considered a naturalregulator of abscission (Jackson & Osborne, 1970). There is increasing interest in therole of ethylene in the abscission process and Addicott (1970) has reviewed thefindings of several investigators concerning the relationship between ethylene and themechanism of abscission. A recent report showing an increase in rough endoplasmicreticula (RER) in ethylene-treated tissue was the first direct evidence for a change in acellular organelle being due to a specific physiological function of ethylene (Valdo-vinos, Jensen &Sicko, 1971)- A recent study of the ultrastructural changes of ethylene-treated tobacco flower pedicels, over a 5 h period, revealed a 30-fold increase in roughendoplasmic reticula (RER) and a loss of integrity of the microbody membranes after5 h of exposure to ethylene (Valdovinos, Jensen & Sicko, 1972). Peroxidases haverecently been demonstrated in the cell walls and wound vessel elements of Coleus(Hepler, Rice & Terranova, 1972). Ethylene may control the extensibility and growthof plant cells by acting as a regulator for the hydroxylation of certain cell wall proteins

592 E. W. Henry and T. E. Jensen

(Ridge & Osborne, 1971). Ethylene treatment causes an increase in peroxidase levels incytoplasmic and covalently bound wall proteins in etiolated seedlings of Pisum sativumvar. Alaska (Osborne, Ridge & Sargent, 1970). The largest increase of peroxidase andhydroxyproline occurs in the wall-bound proteins of the tissue undergoing expansion(Osborne et al. 1970). Accordingly, an investigation was undertaken to examine theultrastructural locations of peroxidases during ethylene-caused abscission, over a 5-hperiod.

MATERIALS AND METHODS

The tobacco plants were grown to the flowering stage with subsequent exposure or non-exposure to ethylene as previously described (Valdovinos et al. 1972).

Tissue segments containing the abscission zone were excised from the pedicels of the flowersat the beginning of the experiment and then at hourly intervals beginning 1 h after the initiationof ethylene (5 /tl/1.) exposure. The tissue was fixed immediately in 3 % glutaraldehyde (50%aqueous solution, Fisher) in o-i M phosphate buffer, pH 7-2, for 1 h at 4 °C. Other tissue seg-ments were collected and fixed for 2 h at room temperature in a mixture of 2 % glutaraldehydebuffered with 0-05 M collidine plus 0-06 M sucrose at pH 7-3-7'4. The tissue sections were thenrinsed for 1-5 h in collidine buffer. Other tissue sections were fixed in 2 % formaldehyde for2 h at 4 °C (Mollenhauer & Totten, 1970).

Each individual 2-mm section of flower pedicel tissue was cut into 4 smaller sections beforetransfer to the diaminobenzidine incubation staining medium of Novikoff & Goldfischer (1969).

The incubation medium contained 10 mg of 3,3'-diaminobenzidine tetrachloride (DAB,Sigma), o-i ml of 3 % hydrogen peroxide and 5 ml of 2-amino-2-methyl-i,3-propandiol buffer(005 M, pH 90).

Sections of flower pedicel tissue, fixed and sliced as described above, were incubated for1 h at 37 °C in one of the following:

(a) complete DAB staining medium; (6) complete medium minus DAB; and (c) completeDAB medium minus hydrogen peroxide. The tissue sections were then rinsed several times in0-05 M propandiol buffer, pH 9-0. For inhibition studies, the tissue sections were preincubatedfor 1 h at 37 CC in either 0-02 M 3-amino-i,2,4-triazole (Aldrich) or 0-02 M potassium cyanide.The tissue segments were then rinsed several times in 005 M propandiol buffer, pH 9'O. Afterpreincubation, the tissue sections were incubated for 1 h at 37 °C in complete DAB mediumcontaining either 0-02 M 3-amino-i,2,4-triazole or 0-02 M potassium cyanide. The respectivetissue sections were then rinsed several times in 0-05 M propandiol buffer, pH 9 0 . The DABincubation procedure was carried out for all tissue segments at separate ionic strengths of pH 9-0and pH 6-o (Novikoff & Goldfischer, 1969).

Tissue sections were postfixed with 2 % OsO4 in o-oi M phosphate buffer, pH 7-2, for 1 h.Tissue sections, previously treated with formaldehyde, were fixed in 1 % OsO4 for 1 h at 4 °C(Mollenhauer & Totten, 1970). Dehydration was carried out in a graded ethanol series, followedby treatment with propylene oxide, and embedded in Epon 812 (Luft, 1961).

Tissue samples selected for sectioning were taken at various depths in the cortical tissue of theabscission zone area. Sections were cut on an LKB ultramicrotome using a diamond knife, andcollected on 300-mesh copper grids. Sections were examined without counter stain or withpost-staining in lead or uranyl salts, separately, or in combination. The prepared sections wereexamined with an Hitachi HU 11E electron microscope using 75 kV.

RESULTS

A typical area within the abscission zone of tobacco flower pedicel tissue is illustratedin a light-microscopic view of a thick section (approximately 1 fim) of 5-h ethylene-treated and non-DAB-incubated control tissue (Fig. 1). It shows the area of separation(arrows) with cortical cells on either side of the zone of separation (Fig. 1).

Peroxidases during induced abscission 593

Cell walls

Control tissue (not treated with ethylene), incubated in medium without DAB, showsan absence of DAB-staining reaction in cell wall areas (Fig. 6). Non-ethylene-treatedtissue incubated in DAB medium has peroxidase staining throughout the cell wall,extending to the periphery towards the plasma membrane on each side of the centralmiddle-lamellar area (Fig. 2). The peroxidase staining reaction product is of a finegranular consistency.

Cortical tissue of the abscission zone, exposed to 3 h of ethylene treatment andincubated in DAB medium, demonstrates increased concentration of peroxidasestaining towards the middle-lamellar areas of the cell wall, with the staining con-tinuing into the intercellular spaces (Fig. 4). The peroxidase staining is of a coarsegranular consistency (Fig. 4). Abscission zone tissue that has been treated with ethy-lene for 5 h and incubated in DAB medium shows a heavy concentration of peroxidasestaining in the middle-lamellar portions of the cell wall, along the line of separation inthe ethylene-induced abscission tissue (Fig. 3). The peroxidase staining appears to beof a coarse granular consistency. When the inhibitor potassium cyanide is present inthe DAB medium the cell walls of ethylene-treated cortical tissue show an almostcomplete absence of peroxidase staining at 3 h (Fig. 5). Not all the cell walls of aparticular ethylene-treated tissue sample stain in DAB medium.

Intercellular spaces

Non-ethylene-treated cortical abscission zone tissue incubated in DAB mediumshows peroxidase staining in the intercellular spaces, with some extending into the cellwalls, exhibiting greater density towards the middle-lamellar portions of the walls(Fig. 7). In 5-h ethylene-treated abscission zone tissue, incubated in DAB medium,the peroxidase staining takes on a more coarse granular appearance (Fig. 8), and in5-h samples, the intercellular spaces have peroxidase staining with the coarse granu-larity extending into the middle-lamellar portions of the cell walls of the abscising cells(Fig. 3). Incubation of control, non-ethylene-treated cortical tissue at pH 6-o, ratherthan at the normal pH of 9-0, markedly inhibits peroxidase staining in the intercellularspaces and cell walls (Fig. 9).

DISCUSSION

The DAB standard incubation procedure has been used in studies of plant(Czaninski & Catesson, 1969; Poux, 1969) and animal (Behnke, 1969; Fahimi, 1968,1969, 1970) cells to demonstrate sites of endogenous peroxidase activity at theultrastructural level. It is our opinion that all of the staining observed in the cell wallsand intercellular spaces of tissue incubated in the standard DAB medium, is due toperoxidase-catalysed oxidation of DAB, followed by reaction of the newly oxidizedDAB with OsO4, which results in the formation of an electron-opaque product(Seligman, Karnovsky, Wasserkrug & Hanker, 1968). If hydrogen peroxide, one of thesubstrates, is not present in the standard DAB incubation medium there is almost a

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complete loss of staining due to catalase (Hepler et al. 1972). The use of the inhibitorpotassium cyanide, a generalized inhibitor of heme-containing enzymes such as per-oxidase, with the DAB incubation medium, results in greatly reduced staining (Vigil,1969, 1970; Hepler et al. 1972). Aminotriazole, a specific inhibitor of catalase, does notprevent peroxidase staining when present in the DAB incubation medium, with animaltissue cells (Fahimi, 1968, 1969, 1970), in biochemical assay (Heim, Appleman &Pyform, 1956; Margoliash & Novogrodsky, 1958; Margoliash, Novogrodsky &Schejter, i960) or with plant tissue (Frederick & Newcomb, 1969; Vigil, 1969, 1970;Hepler et al. 1972). Earlier studies on plant and animal tissues support our contentionthat the staining observed in our investigations is due to a heme-containing protein(peroxidase) and not to catalase, except for the staining of microbodies, which areknown to contain catalase (Tolbert et al. 1968; Frederick & Newcomb, 1969; Vigil,1969). The presence in the tissue of other heme-containing proteins such as cytochromeoxidase (Seligman et al. 1968) and myoglobin (Goldfischer, 1967) has to be taken intoconsideration. However, myoglobin is not known to be present in our particular typeof plant tissue and the mitochondria should show a positive staining reaction if cyto-chrome oxidase is the responsible agent but the mitochondria do not stain in the normalincubation at pH 9-0 and stain only at an acidic pH of 6-o (Novikoff & Goldfischer,1969).

In studies of wound vessel members of Coleus peroxidase activity has been localizedin the cell walls and cytoplasm of wound vessel elements (Hepler et al. 1972) using thestandard DAB incubation medium.

The cell wall degradative processes in tobacco are attributable to a deficiency ofauxin (IAA) within the tissue (Valdovinos & Jensen, 1968). Earlier studies havedemonstrated that IAA production by ovaries of unpollinated tobacco flowers occursat a very low level (Muir, 1942, 1947); the treatment of such flowers with IAA retardsthe onset of abscission (Yager & Muir, 1958).

Indoleacetic acid oxidase (IAA oxidase) is a light-activated flavoprotein enzymelinked with peroxidase (Galston & Baker, 1951). IAA oxidase is involved in limitingthe levels of IAA by participating in IAA destruction (Galston & Dalberg, 1954).

Earlier studies of etiolated pea shoots (Pisum sativum variety 'Alaska') indicate thatethylene treatment greatly increased the levels of wall-bound hydroxyproline,especially in young tissue (Ridge & Osborne, 1970). It is possible that ethyleneincreases the cytoplasmic hydroxylation of proline, resulting in an increase in certainhydroxyproline-rich wall proteins (peroxidases) according to Ridge & Osborne (1971).It was suggested that hydroxyproline-rich wall protein possesses enzyme activity andthe levels of hydroxyproline-rich proteins in cell walls may be the determining factor incell growth and cell wall extensibility (Ridge & Osborne, 1971). Exposure to ethyleneresults in significant changes in peroxidase activity in several tissues of seedling andolder vegetative cotton plants (Morgan & Fowler, 1972). In addition, it was observedthat these results were very similar to the changes that occurred in indoleaceticacid oxidase when identical or similar plants were treated with ethylene (Morgan& Fowler, 1972).

The evidence for ethylene acting as a regulator of the abscission process (Jackson &


Osborne, 1970), causing changes in the amount of rough endoplasmic reticula (RER)as observed by Valdovinos et al. (1971), and bringing about alterations in cellular finestructure (Valdovinos et al. 1972), suggests the further speculation that in ethylene-induced abscission, one of the effects of ethylene may be to alter the levels of endo-genous peroxidases, mediate the possible synthesis of new peroxidase isozymes, andeffect the release and mobilization of hydrolytic-type enzymes, to the areas of the cellsthat undergo dissolution during the process of abscission.

Further studies are in progress to ascertain the physiological levels and electro-phoretic character of the peroxidases present during ethylene-induced abscission.

The authors wish to thank Professor Jack G. Valdovinos for his kind help and suggestionsduring discussion of this research.

This research was supported in part by a Grant-in-Aid of Research Award from the Societyof the Sigma Xi.

Portions of this work were submitted by E. W. Henry to the graduate school, The CityUniversity of New York, New York, N.Y., as part of a Ph.D. thesis entitled: A Physiological andFine Structural Study of Peroxidases Within Cortical Cells of the Abscission Layer of Nicotianatabacum Flower Pedicels.

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FAHIMI, H. D. (1968). Cytochemical localization of peroxidatic activity in rat hepatic micro-bodies (peroxisomes). J. Histochem. Cytochem. 16, 547-550.

FAHIMI, H. D. (1969). Cytochemical localization of peroxidatic activity of catalase in rathepatic microbodies (peroxisomes). J. Cell Biol. 43, 275-288.

FAHIMI, H. D. (1970). The fine structural localization of endogenous and exogenous peroxidaseactivity in Kupffer cells of rat liver. J. Cell Biol. 47, 247-262.

FREDERICK, S. E. & NEWCOMB, E. H. (1969). Cytochemical localization of catalase in leafmicrobodiea (peroxisomes). J. Cell Biol. 43, 343-353.

GALSTON, A. W. & BAKER, R. S. (1951). Studies on the physiology of light action. III. Lightactivation of a flavoprotein enzyme by reversal of a naturally occurring inhibition. Am. J. Bot.38, 100-195.

GALSTON, A. W. & DALBERG, L. Y. (1954). The adaptive formation and physiological signi-ficance of indoleacetic acid oxidase. Am. J. Bot. 41, 373-380.

GOLDFISCHER, S. (1967). The cytochemical localization of myoglobin in striated muscle ofman and walrus. J. Cell Biol. 34, 398—403.

HEIM, W. G., APPLEMAN, D. & PYFROM, H. T. (1956). Effects of 3-amino-i,2,4,-triazoIe (AT)on catalase and other compounds. Am. J. Physiol. 186, 19-23.

HEPLER, P. K., RICE, R. M. & TERRANOVA, W. A. (1972). Cytochemical localization of peroxi-dase activity in wound vessel members of Coleus. Plant Physiol., Lancaster 50, 977-983.

JACKSON, M. A. & OSBORNE, D. J. (1970). Ethylene, the natural regulator of leaf abscission.Nature, Land. 225, 1019—1022.

JENSEN, T. E. & VALDOVINOS, J. G. (1967). Fine structure of abscission zones. I. Abscissionzones of the pedicels of tobacco and tomato flowers at anthesis. Planta 77, 298-318.

JENSEN, T. E. & VALDOVINOS, J. G. (1968). Fine structure of abscission zones. III. Cytoplasmicchanges in abscising pedicels of tobacco and tomato flowers. Planta 83, 303-313.

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MARGOLIASH, E. & NOVOCRODSKY, A. (1958). A study of the inhibition of catalase by 3-amino-1,2,4-triazole. Biochem. J. 68, 468-475.

MAKGOLIASH, E. A., NOVOGRODSKY, A. & SCHEJTER, A. (i960). Irreversible reaction of 3-amino-1,2,4-triazole and related inhibitors with the protein catalase. Biochem. J. 74, 339—348.

MOLLENHAUER, H. H. & TOTTEN, C. (1970). Studies on seeds. V. Microbodies, glyoxysomes,and ricinosomes of castor bean endosperm. Plant Physiol., Lancaster 46, 794—799.

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OSBORNE, D. J., RIDGE, I. & SARGENT, J. A. (1970). Ethylene and the growth of plant cells:Role of peroxidase and hydroxyproline-rich-proteins. In Plant Growth Substances (ed. D. J.Carr), pp. 534-542. New York: Springer-Verlag.

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VALDOVINOS, J. G. & JENSEN, T. E. (1968). Fine structure of abscission zones. II . Cell wallchanges in abscising pedicels of tobacco and tomato flowers. Planta 83, 295-302.

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(Received 5 December 1972)


Figs. 1-3. For legend see p. 598.


Fig. i. A light-microscopic view (thick section) of abscission zone cortical tissue,5-h ethylene-treated and non-DAB-treated, showing the area of separation (arrows)with cortical cells on either side. Glutaraldehyde-OsO4 fixation without post-staining.X3500.

Fig. 2. Portion of non-ethylene-treated abscission zone cortical tissue incubated inDAB medium. There is some peroxidase staining throughout the areas of the cellwalls (cio). The cell wall (cw) peroxidase staining is of a fine granular appearance.Fine granular phytoferritin (pf) granules are present in the matrix (cy) of the chloro-plast (ch). The thylakoids (th) and the single membrane-bound body (mb) of thechloroplast show DAB reaction product. The rough endoplasmic reticula (rer) andthe mitochondrion (m) do not show evidence of peroxidase staining. Plasmodesmata(pi) span the width of the cell walls but do not stain for peroxidase. Glutaraldehyde-OsO^ fixation with uranyl acetate and lead citrate post-staining, x 25 300.

Fig. 3. Portion of the cell wall (cw) of abscission zone cortical tissue treated withethylene for 5 h prior to incubation in DAB medium. The cell is abscising along themid-portion of the cell wall (arrows) and the peroxidase staining reaction is heavierand more concentrated along the line of separation in the middle lamellar (ml) region ofthe cell wall. Coarse granular deposits of the staining reaction are less concentratedthroughout the other areas of the cell wall. Glutaraldehyde-OsO4 fixation withoutpost-staining, x 50300.

Fig. 4. Portion of abscission zone cortical tissue treated with ethylene for 3 h priorto fixation in glutaraldehyde and subsequent incubation in DAB medium. Coarsegranular deposits of peroxidase reaction product are spread throughout the cell wall(cw), with particularly heavy concentrations towards the inner, middle-lamellar (ml)areas and in the intercellular spaces (i). The mitochondria (m) do not show DABreaction product. Glutaraldehyde-OsO4 fixation without post-staining, x 37000.Fig. 5. Abscission zone cortical tissue treated with ethylene for 3 h, subsequently fixed inglutaraldehyde and incubated in DAB medium plus potassium cyanide, shows markedinhibition of the DAB-staining reaction in the cell wall (civ), chloroplast thylakoids(th), mitochondria (m) and rough endoplasmic reticula (rer). Glutaraldehyde-OsO4fixation without post-staining, x 53000.Fig. 6. Abscission zone cortical tissue treated with ethylene for 5 h, subsequentlyfixed in glutaraldehyde and incubated in DAB medium plus potassium cyanide,shows marked inhibition of the DAB-staining reaction in the cell wall (ctv), chloroplastthylakoids (th), membrane-bound body (mb) and rough endoplasmic reticula (rer).Glutaraldehyde-Os04 fixation without post-staining, x 53000.


6oo E. W. Henry and T. E. Jensen

Fig. 7. Portion of control abscission zone cortical tissue, fixed in formaldehyde andincubated in DAB medium. Coarse granular reaction sites of peroxidase activity arepresent throughout the area of the intercellular space (i) and the cell walls (civ).Formaldehyde-OsO* fixation without post-staining, x 75 200.Fig. 8. Portion of abscission zone cortical tissue pretreated with ethylene for 5 h,subsequently fixed in glutaraldehyde and incubated in DAB medium. The intercellularspace (»') has coarse granular deposits of peroxidase reaction product and the staining isconfined to the middle-lamellar (ml) areas of the cell walls (cw) leading from the inter-cellular space. The thylakoids (th) of the chloroplast show evidence of the DAB reactionproduct, while the mitochondria (m) and rough endoplasmic reticula (rer) do not stain.Glutaraldehyde-OsO4 fixation with uranyl acetate post-staining, x 30700.Fig. 9. Portion of control abscission zone cortical tissue incubated in DAB medium atpH 60. There is appreciable inhibition of peroxidase staining in the intercellularspace (»') and cell walls (etc). Segments of rough endoplasmic reticula (rer) and a mito-chondrion (m) are present in the cytoplasm. Glutaraldehyde-OsOi fixation with uranylacetate post-staining, x 42 500.


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PEROXIDASES IN TOBACCO ABSCISSION ZONE TISSUE