localization of Hydrogen Peroxide ... - Plant Physiology · electron-dense materials that can be...

Plant Physiol. (1995) 107: 501-506

localization of Hydrogen Peroxide Production in Pisum sativum 1. Using Epi-Polarization Microscopy to

Follow Cerium Perhydroxide Deposition'

Lan Liu2, Karl-Erik L. Eriksson, and Jeffrey F. D. Dean*

Department of Biochemistry and Molecular Biology, Center for Biological Resource Recovery, University of Ceorgia, Athens, Ceorgia 30602-7229

Cerium is becoming an increasingly popular reagent for histochemical localization of oxidases and phosphatases because it com- bines directly with reaction products to form fine precipitates of electron-dense materials that can be easily detected using transmission electron microscopy or laser confocal scanning microscopy. We used epi-polarization microscopy to detect cerium perhydroxide deposits formed when H,O, was produced by diamine oxidase in pea (Pisum sativum L.) epicotyls exposed to exogenous putrescine. Diamine oxidase activity was abundant in cortical cell walls but showed little, if any, association with vascular tissues. Maps of cerium deposition generated using scanning electron microscopy/x-ray microanalysis verified these observations. This study demonstrates the use of epi-polarization microscopy to follow cerium deposition, and the ready accessibility of this microscopy technique should facilitate more widespread use of cerium for plant histochemistry and cytochemistry.

The formation of active oxygen species, i.e. singlet oxygen, superoxide, and hydroxyl radicals, as well as H202, is a fundamental consequence of aerobic metabolism. Al- though these agents carry with them significant risk of oxidative damage to cehlar components against which cells have developed multilayered protective mechanisms (Scandalios, 1993; Foyer et al., 1994), the oxidative capaci- ties of active oxygen species have also been harnessed by plants for a variety of physiological processes. Of particu- lar recent interest has been the involvement of H20, in plant defense responses (Sutherland, 1991; Chen et al., 1993; Mehdy, 1994). In addition to its production during host-pathogen interactions, H,O, is commonly synthesized in response to environmental stresses (Murphy and Huerta, 1990; Okuda et al., 1991) and phytohormone treatment (Brightman et al., 1988). Although the exact roles played by active oxygen species in these various physiological processes are still sketchy, H,O, often appears to function as an oxidizer for coupling of such aromatic moi- eties in plant cell walls as cinnamic acids esterified to

Financia1 support was provided by U.S. Department of Energy

l'resent address: Department of Horticulture, Ohio State

* Corresponding author; e-mail jdeanxl8uga.cc.uga.edu; fax

grant No. DE-FG02-92ER20082.

University, Columbus, OH 43210-1096.

1-706-542-2222. 501

polysaccharides (Fry and Miller, 1989; Hartley and Ford, 19891, Tyr residues in structural proteins (Cooper and Varner, 1984; Bradley et al., 1992), and the monomeric precursors of lignin (Masuda et al., 1983; Church and Galston, 1988; Dean and Eriksson, 1992).

Progress in determining what enzymes are responsible for producing H,O, in plant cell walls has been slow, in part because of the difficulty in localizing sites of production. Histochemical localization of H20, production in cell walls has been reported by use of starch/KI reagent (Smith, 1970; Kaur-Sawhney et al., 1981; Olson and Varner, 1993; Schopfer, 1994), as well as by staining for peroxidase activity (Angelini and Federico, 1989). However, these techniques are indirect measurements of H,O, production and are limited to detection of H202 produced at the cut surface of tissue sections. Briggs et al. (1975) introduced the use of Ce3+ as a reagent for specific detection of H,Oz production, and this technique is now widely used for the histochemical detection of various oxidases and phosphatases (Kausch, 1987; Van Noorden and Frederiks, 1993; Halbhu- ber et al., 1994). Ce3+ penetrates into tissues, albeit slowly, and reacts directly with H,O, to form insoluble, finely divided deposits of Ce(1V) perhydroxide. The reflectance properties imparted to these deposits by the electron-dense cerium makes them easily detectable via electron or scanning laser confocal microscopy (Robinson and Batten, 1990); however, they have not previously been amenable to conventional light microscopy .

Cerium has been used to localize a variety of plant oxidases and phosphatases (Thomas and Trelease, 1981; Vaughn et al., 1982; Kausch et al., 1983; Chauhan et al., 1991; Slocum and Furey, 19911, but widespread use of the technique appears to have been limited by the requirement for expensive and specialized microscopy equipment. The technique of epi-polarization microscopy has recently been popularized for the visualization of colloidal gold and silver deposits formed during immunolocalization or in situ hybridization experiments (De Mey, 1983; De Waele et al., 1988; van den Brink et al., 1990; Rapaport et al., 1992). In this study, we examined the tissue localization of DA0

Abbreviations: CF, cortical fiber; DAO, diamine oxidase; DIC, differential interference contrast microscopy; SE, scanning electron microscopy; TEM, transmission electron microscopy; XRMA, x-ray microanalysis.

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502 Liu et al. Plant Physiol. Vol. 107, 1995

(EC 1.4.3.6) in pea (Pisum sativum L.) epicotyls using epi-polarization microscopy to follow Ce(IV) perhydroxidedeposition. The specificity of this technique for observingcerium deposition was verified by SEM/XRMA (Kausch,1987). Our results demonstrate that sites of H2O2 produc-tion can be localized using the relatively inexpensive tech-nique of epi-polarization microscopy.

MATERIALS AND METHODS

Chemicals and Reagents

Pea (Pisum sativum L.) seeds were obtained at the localmarket. Embedding medium (JB-4) was from Poly-sciences, Inc. (Warrington, PA). All other reagents were ofthe highest purity possible and were used without furtherpurification.

Preparation of Plant Materials

Pea seedlings were grown in moist potting soil for 5 d inthe dark at 25°C as described by Slocum and Furey (1991).Segments (2 mm) cut from the second internode using adouble-edged razor blade were immediately put into reac-tion buffer containing 100 mM Hepes, pH 7.5, 50 mM 3-ami-no-l,2,4-triazole, and 5 mM CeCl3 for 1 h at room temper-ature. Segments were subsequently transferred to freshreaction buffer containing 10 mM putrescine. For controlreactions, segments were incubated in reaction buffer with-out putrescine or in 100 mM Hepes, pH 7.5. Segments wereincubated in these buffers for a total of 12 h, during whichtime the respective buffers were replaced with freshlymade buffer after 2 and 6 h of incubation. All buffers weremade with preboiled double-distilled water, and duringincubation the samples were agitated on a platform shaker(Kausch, 1987; Slocum and Furey, 1992).

At the end of the incubation, epicotyl segments werewashed twice in 100 mM sodium cacodylate buffer, pH 6.2,and once in 100 mM sodium cacodylate buffer, pH 7.0. Eachwash was for at least 15 min, after which the segmentswere fixed overnight at 4°C in cacodylate buffer, pH 7.0,containing 2% glutaraldehyde. After fixation, tissue seg-ments were rinsed in cacodylate buffer and dehydrated ina graded ethanol series to 100%. Dehydrated tissues wereinfiltrated and embedded in JB-4 embedding medium andsubsequently sectioned (5 /urn thickness).

Light Microscopy

Tissue sections were examined with an Axioskop micro-scope (Carl Zeiss, Hanover, MD) equipped with a 100-Wmercury incident lamp for epi-fluorescence photomicros-copy. Although epi-polarization microscopy is usually per-formed using a halogen source for illumination, we choseto adapt the existent mercury lamp to minimize manipu-lations of the light source. The epi-polarized light imageswere obtained by inserting a Zeiss polarization filter blockin the fluorescence filter block carrier positioned betweenthe mercury lamp and objective lenses. A UV filter, a

neutral-density heat protection filter (Omega Optical, Inc.,Brattleboro, VT), and an iris diaphragm were positionedbetween the mercury lamp and the polarization filter blockto protect the polarization filters and allow for contrastadjustment. Samples were routinely examined under epi-polarized light using XlO oculars and either X40 or xlOOoil-immersion objectives, because the high numeric aper-ture of these lenses greatly increased image contrast andresolution (De Waele et al., 1988). The results were re-corded with T-Max 400 black and white film (Kodak,Rochester, NY). Transmitted light photomicrographs weremade using filters for DIC microscopy.

SE/XRMA

After samples mounted on glass slides were examinedby epi-polarization and DIC microscopy, the slides weredipped into xylene to remove the glass coverslip. Slideswere subsequently trimmed, mounted on aluminum stubsusing double-sided adhesive carbon tape, and coated witha heavy layer of evaporated carbon. Coated samples wereexamined using a Philips (Eindhoven, The Netherlands)505 scanning electron microscope equipped with a Noran

Figure 1. Mk ro£>r<iph ol d pea epicotyl cross-sect ion stained withtoluidine blue as observed using DIC microscopy. Observed tissuetypes were denoted as follows: E, epidermis; C, cortical parenchyma;CF, cortical fiber; LT, leaf trace vascular tissue; V, central vasculartissue. Scale bar = 500 (nm. www.plantphysiol.orgon August 5, 2020 - Published by Downloaded from

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Hydrogen Peroxide Localization by Epi-Polarization Microscopy 503

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L. Figure 2. Tissue section from pea epicotyl tissue incubated with putrescine and Ce 3- to demonstrate localization of DAO. A single tissue section taken from 100/~m below the cut surface of an epicotyl segment was examined by DIC (A and D), epi-polarization (B and E), and SE/XRMA microscopy (C and F). A through C show an unlignified CF bundle, and D through E show a section of cortical parenchyma immediately below the epidermis. Tissues are labeled as in Figure 1, and arrowheads point to examples of cytoplasm not adjacent to cell walls• Scale bars = 50/.Lm.

Instruments Inc. (Middleton, WI) TN-5500 x-ray analyzer and an Edax International (Mahwah, NJ) energy-dispers ive x-ray spectrometric detector. The accelerating voltage was 20 kV with an emission current of 50 /zA. The angle between the sample and the detector was 25.5 ° with a tilt of 0 °, and the working distance was 29.9 mm. A 7.30-~.m beryl l ium window was used, and spectra were collected

for 400 s at a spot size of 100. Cerium peak identification and background definit ion were performed using the No- ran TN-5500 basic software with internal s tandards. X-ray map images were processed using the IPP program in the same software package. SE/XRMA work was performed at the Center for Advanced Ultrastructural Research, Univer- sity of Georgia.



504 Liu et ai. Plant Physiol. Vol. 107, 1995

RESULTS

Figure 1 shows a transmitted light image of a pea epicotyl cross-section stained with toluidine blue. The structural organization of these tissues was somewhat irregular, but generally apparent was a central stelar region containing primary vascular tissues surrounded by a broad area of cortical parenchyma cells. Embedded in the cortex were unlignified CFs, as well as leaf traces that diverged from the central vascular stele and contained lignified tracheary elements.

Epicotyl segments were incubated in buffer containing the DA0 substrate, putrescine, to increase H,O, production. Ce3+ was added to capture the H,O, as insoluble Ce(IV) perhydroxide, and 3-amino-1,2,4-triazole was added to inhibit catalase and peroxidase activity and thereby limit H,O, dismutation and consumption (Slocum and Furey, 1991). An unlignified CF in a typical cross- section taken 100 pm below the cut surface of an epicotyl segment was examined by DIC (Fig. 2A), epi-polarization (Fig. 2B), and SE/XRh4A microscopy (Fig. 2C). An area of the same cross-section containing both epidermal and cortical parenchyma cells was examined under identical con- ditions using the same three microscopic techniques (Fig. 2, D-F). Under epi-polarized light, refractive particles were seen in cytoplasmic remnants (arrowheads), but the heavi- est accumulations of refractive particles were immediately adjacent to the cortical cell walls (Fig. 2E). Very few refractive particles were associated with the walls of CF or epidermal cells.

SE/XRMA microscopy was used to verify that the refractive particles detected by epi-polarization microscopy contained cerium. Figure 3 depicts emission spectra for cortical cell walls incubated in the presence of Ce3+ with and without putrescine. Addition of putrescine led to a greater than 10-fold increase in the intensity of the cerium peaks.

- Putrescine

4000

8000 ',si + Putrescine

6000 -

I

4000 -

2000 -

O O 2 4 6 8 10

Emission Energy (ev)

Figure 3. Semiquantitative comparison of elemental x-ray spectral analyses from cortical parenchyma cell walls from pea epicotyls incuhated with Ce3+ in the presence and ahsence of exogenous putrescine. ev, Electronvolt.

The first peak (L, 4.84 electronvolts) was used to generate SE/XRMA maps, such as those shown in Figure 2, C and F. These maps indicated that the refractive particles observed by epi-polarization microscopy contained substantial cerium. Omission of putrescine from the incubation buffer had its greatest effect on the deposition of cerium in cortical cell walls, i.e. few if any refractive particles or cerium deposits could be detected in such walls by epi-polarization or SE/XRMA microscopy, respectively (data not shown). The little cerium deposition that occurred in CFs and vascular tissues did not appear to be much affected by the presence or absence of putrescine.

One limitation to using Ce3+ on intact tissues is that these ions diffuse relatively slowly through biological samples (Van Noorden and Frederiks, 1993; Halbhuber et al., 1994); for instance, Kausch (1987) reported that they pene- trate only 70 to 15 cell layers beyond the cut surface of tissue samples. Seria1 sections taken from pea epicotyl segments incubated with Ce3+ and putrescine, as well as from segments incubated in Ce3+ alone, showed fairly constant staining to depths between 100 and 200 pm (L. Liu, K.-E.L. Eriksson, J.F.D. Dean, unpublished data). However, although staining was difficult to detect below 200 pm in tissues not treated with putrescine, staining in putrescine- treated tissues could usually be seen to a depth of 300 pm.

A complication to using epi-polarization microscopy for the routine detection of H,O, production in plant tissues is the common presence of optically anisotropic structures, such as crystalline cellulose or calcium oxalate crystals (Lane, 1994), in the same tissues. This presented a partic- ular problem for detection of H,O, production in vascular tissues undergoing lignification because the thickened secondary walls of various vascular elements contained substantial crystalline cellulose (Fig. 4A, arrowhead). Thus, in tissue sections from an epicotyl segment treated with both Ce3+ and putrescine, the cell walls of tracheary elements (Fig. 48, arrowhead) scattered as much light as the. Ce(IV) perhydroxide deposits associated with surrounding cells. However, SE/XRMA of this tissue section clearly showed that the refractivity of the tracheary element walls was not due to significant cerium deposition (Fig. 4 0 .

DlSCUSSlON

Our results show that epi-polarization microscopy pro- vides a powerful tool for detecting Ce(1V) perhydroxide deposited at sites of H,O, production in plant tissues. To our knowledge this is the first report to describe the direct observation of Ce(1V) perhydroxide deposits using epi- polarization microscopy. Because the method can be used on thin sections (5-10 pm) of fixed tissues, it. enables researchers to quickly scan for tissue-specific deposition of cerium prior to using the same tissue sections for subcellular localization of cerium via SE or TEM. The ability of this technique to detect H,O, severa1 cell layers away from cut surfaces also lessens the potential for confusion of wound-induced H,O, production with deveIopmentaIIy regulated production.

The tissue structure in pea epicotyls is relatively simple, with few cells containing anisotropic materials. Only trac-



Hydrogen Peroxide Localization by Epi-Polarization Microscopy 505

Figure 4. H2O2 production in pea epicotyl tracheary elements. A leaftrace in a tissue section taken from 100 jtm below the cut surface ofa pea epicotyl segment incubated with Ce3+ and putrescine wasexamined using DIG (A), epi-polarization (B), and SE/XRMA micros-copy (C). The arrowheads point out lignified tracheary elements thatare highly refractive under epi-polarized illumination (B) but do notcontain significant deposits of cerium (C). Scale bars = 50 ;im.

heary elements, constituting the protoxylem, displayed sig-nificant refractivity due to crystalline cellulose in theirthickened secondary cell walls. The inability of epi-polar-ization microscopy to distinguish between cerium depositsand crystalline cellulose in secondary cell walls makesstudy of H2O2 production for lignin biosynthesis difficultwith this technique. This problem is even more pro-nounced in mature plant tissues, and appropriate experi-mental controls are critical for interpreting results (Halb-huber et al., 1994). However, in such cases, mapping thedistribution of elemental Ce with SE/XRMA can be used toclarify observations.

We selected pea epicotyls for this initial study becauseDAO, a cell wall-localized H2O2-producing enzyme, hadbeen extensively studied in pea and other legumes(Federico and Angelini, 1986, 1991). Slocum and Furey(1991) examined the subcellular distribution of DAO in peaepicotyls and roots using TEM to detect Ce(IV) perhydrox-ide deposits formed in tissues incubated with putrescine.In that study, DAO activity in roots was localized primarilyto the middle lamella regions of cortical cell walls; how-ever, activity in the epicotyls was reported to be mostclosely associated with vascular parenchyma cells. Fromthese observations, Slocum and Furey (1991) concludedthat H2O2 generated by DAO might play an important rolein lignification of tracheary elements, in addition to facili-tating peroxidase-mediated cross-linking of other cell wallpolymers. Although we agree that DAO could provide anexcellent source of H2O2 for cell wall cross-linking reac-tions, our observations do not support a major role for thisenzyme in the lignification of vascular cell walls. We can-not explain the conflicting results from these two studies.As far as we can determine, the only differences betweenthe two studies were in the microscopy techniques usedand the use of 1 % osmium tetroxide to enhance the Ce(IV)perhydroxide deposits for TEM observation in the study bySlocum and Furey (1991). Although postfixation with os-mium tetroxide has been noted in some cases to lead tononspecific precipitation of electron-dense materials(Okada et al., 1986; Van Noorden and Frederiks, 1993), it isdifficult to see how such a phenomenon could explain thelack of staining in cortical cell walls of pea epicotyl notedby Slocum and Furey (1991). However, in the presentstudy, the electron-dense, refractile deposits observed incortical walls of such segments were verified to containcerium by elemental spectroscopy (Fig. 2F). Thus, it willlikely require immunolocalization or in situ hybridizationstudies of DAO expression to clarify the tissue localizationof this enzyme in pea epicotyls. On the other hand, immu-nolocalization studies of DAO in chickpea (Cicer arietinumL.) demonstrated significant immunoreactive protein incortical cell walls, but antibody binding appeared to behigher in xylem tissues (Angelini and Federico, 1989).

Epi-polarization microscopy is a technique that is adapt-able to any research microscope equipped for epi-illumi-nation, needing only the addition of a polarizing filter inthe incident beam and an analyzer (crossed polarizingfilter) in the reflected beam (Rapaport et al., 1992). Use ofthis technique to detect Ce(IV) perhydroxide deposits intissues producing H2O2 should make studies of H2O2 ac-cessible to more researchers.

ACKNOWLEDGMENTS

The authors thank Dr. Mark A. Farmer and the staff of theCenter for Advanced Ultrastructural Research for their assistancewith SE/XRMA experiments, as well as Dr. William E. Friedmanfor technical advice.

Received September 6, 1994; accepted October 24, 1994.Copyright Clearance Center: 0032-0889/95/107/0501/06. www.plantphysiol.orgon August 5, 2020 - Published by Downloaded from

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506 Liu et al. Plant Physiol. Vol. 107, 1995

LITERATURE ClTED

Angelini R, Federico R (1989) Histochemical evidence of polyamine oxidation and generation of hydrogen peroxide in the cell wall. J Plant Physiol 135: 212-217

Bradley DJ, Kjellbom P, Lamb CJ (1992) Elicitor- and wound- induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell 70 21-30

Briggs RT, Drath DB, Karnovsky ML, Karnovsky MJ (1975) Localization of NADH oxidase on the surface of human poly- morphonuclear leukocytes by a new cytochemical method. J Cell Biol 67: 566-586

Brightman AO, Barr R, Crane FL, Morre DJ (1988) Auxin-stimu- lated NADH oxidase purified from plasma membranes of soy- bean. Plant Physiol 86: 1264-1269

Chauhan E, Cowan DS, Hall JL (1991) Cytochemical localization of plasma membrane ATPase activity in plant cells. A comparison of lead and cerium-based methods. Protoplasma 165: 27-36

Chen Z, Silva H, Klessig DF (1993) Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 262 1883-1886

Church DL, Galston AW (1988) 4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements. Plant Physiol 88 679-684

Cooper JB, Vamer JE (1984) Cross-linking of soluble extensin in isolated cell walls. Plant Physiol 76: 414-417

Dean JFD, Eriksson K-EL (1992) Biotechnological modification of lignin structure and composition in forest trees. Holzforschung

De Mey J (1983) Colloidal gold probes in immunohistochemistry. In JM Polak, S Van Noorden, eds, Immunohistochemistry. Prac- tical Applications in Pathology and Biology. J. Wright and Sons, Bristol, UK, pp 82-112

De Waele M, Remans W, Segers E, Jochmans K, van Camp B (1988) Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy. J Histochem Cyto- chem 36: 679-683

Federico R, Angelini R (1986) Occurrence of diamine oxidase in the apoplast of pea epicotyls. Planta 167: 300-302

Federico R, Angelini R (1991) Polyamine catabolism in plants. In H Flores, R Angelini, eds, Biochemistry and Physiology of Poly- amines in I'lants. CRC I'ress, Boca Raton, FL, pp 41-56

Foyer CH, Descourvières P, Kunert KJ (1994) Protection against oxygen radicals: an important defence mechanism studied in transgenic plants. Plant Cell Environ 17: 507-523

Fry SC, Miller JG (1989) Toward a working model of the growing plant cell wall. Phenolic cross-linking reactions in the primary cell walls of dicotyledons. In NG Lewis, MG Paice, eds, Plant Cell Wall Polymers, ACS Symposium Series 399. American Chemical Society, Washington, DC, pp 33-46

Halbhuber K-J, Hulstaert CE, Feuerstein H, Zimmermann N (1994) Cerium as capturing agent in phosphatase and oxidase histochemistry-theoretical background and applications. Prog Histochem Cytochem 28 1-120

Hartley RD, Ford CW (1989) Phenolic constituents of plant cell walls and wall biodegradability. In NG Lewis, MG Paice, eds, Plant Cell Wall Polymers, ACS Symposium Series 399. American Chemical Society, Washington, DC, pp 135-145

Kaur-Sawhney R, Flores HE, Galston AW (1981) Polyamine oxidase in oat leaves. A cell wall-localized enzyme. Plant Physiol 68 494-498

46: 135-147

Kausch AP (1987) Cerium precipitation. In KC Vaughn, ed, Hand- book of Plant Cytochemistry, Vol 1. CRC Press, Boca Raton, FL,

Kausch AP, Wagner BL, Homer HT (1983) Use of cerium chloride technique and energy dispersive X-ray microanalysis in plant peroxisome identification. Protoplasma 118: 1-9

Lane BG (1994) Oxalate, germin, and the extracellular matrix of higher plants. FASEB J 8: 294-301

Masuda H, Fukuda H, Komamine A (1983) Changes in peroxidase isoenzyme patterns during tracheary element differentiation in a culture of single cells isolated from the mesophyll of Zinnia elegans. Z Pflanzenphysiol 112 417-426

Mehdy MC (1994) Active oxygen species in plant defense against pathogens. Plant Physiol 105 467-472

Murphy TM, Huerta AJ (1990) Hydrogen peroxide formation in cultured rose cells in response to W-C radiation. Physiol Plant

Okuda T, Matsuda Y, Yamanaka A, Sagisaka S (1991) Abrupt increase in the leve1 of hydrogen peroxide in leaves of winter wheat is caused by cold treatment. Plant Physiol 97: 1265-1267

Okada T, Robinson JM, Kamovsky MJ (1986) Cytochemical localization of acid phosphatase in striated muscle. Histochemis- try 85: 177-183

Olson, PD, Vamer JE (1993) Hydrogen peroxide and lignification. Plant J 4 887-892

Rapaport DH, Herman KG, LaVail MM (1992) Epi-polarization and incident light microscopy readily resolve an autoradio- graphic or heavy metal label from an obscuring background or second label. J Neurosci Methods 41: 231-238

Robinson JM, Batten BE (1990) Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy. J Histochem Cytochem 38: 315-318

Scandalios JG (1993) Oxygen stress and superoxide dismutases. Plant Physiol 101: 7-12

Schopfer P (1994) Histochemical demonstration and localization of H,O, in organs of higher plants by tissue printing on nitro- cellulose paper. Plant Physiol 104 1269-1275

Slocum, RD, Furey MJ 111 (1991) Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues. Planta 183: 443450

Smith TA (1970) Polyamine oxidase in higher plants. Biochem Biophys Res Commun 41: 1452-1456

Sutherland MW (1991) The generation of oxygen radicals during host plant responses to infection. Physiol Mo1 Plant Pathol 39

Thomas J, Trelease RN (1981) Cytochemical localization of glyco- late oxidase in microbodies (glyoxysomes and peroxisomes) of higher plant tissues with CeCl, technique. Protoplasma 108 39-53

van den Brink W, ven der Loos C, Volkers H, Lauwen R, van den Berg F, Houthoff H-J, Das PK (1990) Combined P-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization. J Histochem Cytochem 38:

Van Noorden CJF, Frederiks WM (1993) Cerium methods for light and electron microscopical histochemistry. J Microsc 171: 3-16

Vaughn KC, Duke SO, Duke SH, Henson CA (1982) Ultra- structural localization of urate oxidase in nodules of Sesbania exaltata, Glycine max, and Medicago sativa. Histochemistry 7 4

pp 25-36

7 8 247-253

70-93

325-329

309-318



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