Cytochemical localization of microbodies inPuccinia graminis var.tritici

EXPERIMENTAL

MYCOLOGY

ytochemical GERD HANSSLER,

5,

209-216 (1981)

Localization

of Microbodies var. tritici

i

DIETERMOHLENBACHER, AND HANS-J• ACHIM

institut fiir Bialogie III (FJlatlzerzphysiologie),

Technische Hochschule Aachen. Bundesrepublik

Accepted for publication

Dei
November 18, 1980

HANSSLER, G., M~HLENBACHER, D., AND REISENER, H.-J. 1981. Cytochemicai localization of microbodies in Pucciniu graminis var. tritici. En;oerimentai Mycology 5, 209-216. Microbodies in uredospores and germ tubes of Puccinicr grclminis var. fritici reacted positively to the catalase test using 3,3’-diaminobenzidine and hydrogen peroxide in an alkaline medium. Reaction occurred following fixation in 2% glutaraldehyde in cacodylate buffer but was enhanced by reducir,g the glutaraldehyde concentration to l%, which is significantly less than is normally used. Use of formaldehyde fixative. either alone or in combination with glutaraldehyde, resulted in a complete absence of staining. This also occurred when phosphate buffer was snbstituted for cacodylate. A similar inactivation of the staining process was obtained wken the material was treated with 3amino-1,2.4-triazole. It is concluded that the microbodies in germinating uredospores and germ ?&es of P. gruminis var. tritici have functions similar to those of glyoxysomes in higher plants. INDEX DESCRIPTORS: Pucciniu graminis var. tr-irici: microbodies: cytochemistry: cataiase; diaminobenzidine.

Microbodies have been found in a wide range of cells from animals, higher plants, algae, and fungi. Morphologically they are characterized by a single bounding membrame, by a finely granular matrix of moderate electron opacity, and, frequently, by amorphous or crystalline inclusions. Biochemical studies have led to the concept of two classes of microbodies: peroxisomes and glyoxysomes. Peroxisomes play a role in glycolate metabolism and photorespiration (de Duve, 1969; Tolbert, 1971), whereas glyoxysomes, frequently associated with lipids (Vigil, 1970, 1973; endgen, 1973; Trelease et al., 1974; Maxwell et al., 1975; Laborda and Maxwell. 1976), house enzymes of the glyoxylate cycle and are involved in converting lipids to carbohydrates (Beevers, 1969; Tolbert, 1971). Catalase is present in both classes and has been considered a marker enzyme for microbodies. Therefore, tRe cytochemical demonstration of catalase activity by the oxidation of 3,3’-diaminobenzidine (DAB)” has been used to iden1 Abbreviation

Esed: DAB: 3,3’-diaminobenzidine.

tify microbodies in many o~ga~isrn~, It has been shown t at DAB is oxidized via the peroxidatic activity of catalase to a water-insoluble, lipid-insoluble ~srnio~bi~~~ polymer which demonstrates the site of enzymatic activity eligman et kl!., 1 This method has ided i~format~Q~ on the localization Q abase in animal and plant tissue which is in agreement wi results of biochemical fractionation Duve and Baudhuin, 8966; Vigil, However, with the exceptions of micrabodies in Candida tropicalis (Teranis al., 1974), Nnnsenuku polymorphic Dijken et nl., 1975), ~~~~tocl~d~ef~~ emer1975, 1979), Entophlyctis ~~~i~~~~i~~ owell, 1974. 19771, Coelomomyces punt 781, Poiyphugus euglenae (Powell, 19 ~o~o~leph~re~~~ sp. 980), and Phyto” phthora palmivora ~~h~l~p~~ et al., 4973, aH reported attempts failed to show a~~re~~a~~e DAB staining of fungal microbodies at the ultrastructmal level, although photomctsis. mentors fungi showed assay of extra@ts 0 ndgen, 1973; Maxwel: catalase activity ( 209 0147-5975:81/030209-Q8$02.0010 Copyright All rights

0 1981 by Academic Press, Inc. or reproductim in any form resrrve~2.

210

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AND

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or a control mixture. The reaction mixture contained 9.8 ml 0.05 M propanediol buffer, pH 9.0, 0.2 ml 1% HzOz, and 20 mg diaminobenzidine (Sigma). The three control treaments were: (a) incubation for 30 min in 0.05 M propanediol buffer, pH 9.0 (no substrate); (b) incubation in the substrate reaction mixture in the presence of 0.02 or 0.04 M 3-amino-1,2,4-triazole (Sigma), the specific catalase inhibitor; and (c) preincubation for 30 min in propanediol buffer, pH 9.0, containing the inhibitor at a concentration of 0.02 M, and then incubation in the DAB medium containing 0.02 M aminotriazole. After incubation the slides were fixed for 90 min in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.1, rinsed for 30 min in water, and postfixed in 1% aqueous osmic acid for 1.5 h. After a brief rinse in distilled water, the specimens were dehydrated in a graded acetone series and embedded in Spurr’s resin. Thin secMATERIALS AND METHODS tions were cut with a diamond knife on a Uredospores of P. graminis var. tritici, Reichert Om U3 ultramicrotome. Sections Eriks. & Henn., race 32, obtained from were placed on Formvar-supported copper Prof. Dr. W. H. Fuchs, University G6t- grids and examined either unstained or tingen, were used in this experiment. The stained with aqueous uranyl acetate and uredospores (30 mg) were suspended in 30 Reynold’s lead citrate in a Zeiss EM 9s ml Freon 113 (Serva, Heidelberg, Ger- electron microscope at 60 kV. many), sprayed on microscope slides RESULTS coated with 2% agar (Difco Laboratories, Detroit, Mich.), and grown for 2 h in the Organelles which resemble the microdark at 22-24°C. To minimize loss of en- bodies previously described in many types zyme activity the slide cultures were fixed of animal cells (Hruban and Rechcigl, 1969), higher plant tissue (Frederick and for a short time (10 min) in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer Newcomb, 1969), and fungi (Maxwell et al., containing 0.2 M sucrose, pH 7.1, or in 0.1 1977) have been found in germinating M potassium phosphate buffer, pH 7.1. The uredospores of P. graminis var. tritici and procedure was also carried out using 2% in elongating germ tubes. They display the glutaraldehyde to determine the effect of features characteristic for microbodies of fixative concentration. In addition to other sources; a round to oval profile 0.3 to glutaraldehyde, formaldehyde (1 and 2%) 1.5 pm in diameter surrounded by a 6- to was employed. After fixation the material 8-nm-thick membrane and a matrix of modwas washed for 15 min in water, rinsed for erate electron density. Inclusions were 30 min in three changes of 0.05 M 2-aminonever observed (Fig. 1). Microbodies were 2-methyl-1,3-propanediol buffer (Sigma commonly associated with the endoplasmic Chemical Co., St. Louis, MO.), pH 9.0, and reticulum (Figs. 2-4), and a close proximthen incubated for 30 min at 37°C in the ity to nuciei is noted occasionally (Fig. 3). dark in either the complete reaction mixture Mitochondria may be close to but are not

et al., 1975). Glyoxylate

cycle enzymes have also been characterized for fungi (Graves et al., 1976). This has led to the hypothesis that during germination of fungal spores fungal microbodies might function in a way similar to that of glyoxysomes in the utilization of lipids during germination of fat-storing seeds (Mendgen, 1973; Beevers, 1979) and in the breakdown of lipids during germination of fern spores (Gemmrich, 1980). Therefore, the following studies were carried out on germinating uredospores, because the greatest utilization of lipids occurs during uredospore germination (Staples and Wynn, 1965). The results reported in this paper are part of a study on uredospores and germ tubes of Puccinia graminis var. tritici and show for the first time the presence of DAB staining for catalase in the microbodies of higher, filamentous fungi.

FIG. I Microbodies in a germ tube of Puccinia gruminis var. Iritici (~42,000). Abbreviations used in figures: ER, endoplasmic reticulum; Lb, lipid body: M. mitocbondrium; Mb, microbody; N, nucleus; SpW, spore wall, FIG.. 2, Thin section of a germ tube incubated in the DAB medium. Note deposition of electron-dense reaction product in microbodies (X56,000). FIG. 3. Thin section of uredospore incubated in the DAR medium + aminotriazole (40 mM). Reaction product is still present (~84,000). FIG. 4. Thin section of a germ tube preincubated in aminotriszole (20 mM) to inhibit catalase activity followed by incubation in the DAB medlmn + aminotriazole (20 mu). Reaction product is absent from microbodies and rnitochondrial membranes and cristae (x67.000).

212

HiiNSSLER,

MUHLENBACHER,

intimately associated with microbodies. Associations with lipids were observed frequently, and the microbody surface opposed to the lipid bodies conformed to the contour of these structures (Fig. 1). The cytochemical localization of catalase was attempted with the DAB procedure for detection of peroxidatic activity. After an incubation period of 30 min in complete reaction mixture, the microbodies as well as the mitochondrial cristae in the germ tube and in the uredospore gave a strong positive DAB reaction (Figs. 2 and 5). The outcome of the staining depended on the composition of the fixation mixture. Staining of the two organelles occurred only when glutaraldehyde was used as fixative and was superior with 1% glutaraldehyde. Formaldehyde or a mixture of formaldehyde and glutaraldehyde (1%/l%) abolished the staining. Not only the kind of aldehyde, but also the buffer system as part of the fixation medium had a striking influence on the outcome of the subsequent enzyme reaction. Both the microbodies and the intracristate space of the mitochondria were free of the dense oxidation product when the cacodylate buffer of the fixation mixture was replaced by potassium phosphate buffer in the presence of glutaraldehyde. Despite the presence of the catalase inhibitor 3-amino-1,2,4-triazole, a positive staining of microbodies (Fig. 3) and mitochondria with the DAB reaction mixture was obtained. When using an incubation medium lacking H,O, similar observations were made. No reaction product was observed when the material was preincubated for 30 min in 20 mM inhibitor in buffer, before incubation in the complete DAB medium containing 20 mM aminotriazole (Fig. 4). The absence of stain indicates that the cytochemical staining is the result of enzyme activity. DISCUSSION

Negative findings in a cytochemical test do not necessarily mean the absence of en-

AND

REISENER

zyme activity (Silverberg, 1975). They may be explained by unfavorable conditions such as inactivation by the fixative, inhibitory effects of ions, solubilization of the enzyme during penetration, or insufficient availability of reagents because of poor or reduced penetration. This seems to be true for the cytochemical demonstration of catalase via peroxidatic activity in fungal microbodies. This point was discussed by Maxwell et al. (1977), when they claimed that appropriate modifications of the DAB procedure would allow cytochemical demonstration of catalase activity in fungal microbodies. Methods developed for cells of higher plants or animals may not necessarily work in fungal systems. In general, plant catalase and peroxidase appear to be relatively stable and unaffected by aldehyde fixation (Hall and Sexton, 1972). In some cases prefixation with glutaraldehyde seems to be essential for peroxidative activity of catalase (van Dijken et al., 1975) or is reported to increase the activity (Roels et al., 1975). The concentrations of glutaraldehyde used varied from 0.125 to 6.0% and a mixture of glutaraldehyde and formaldehyde was employed (Mollenhauer and Totten, 1970; Powell, 1976). In our study only samples fixed with a low glutaraldehyde concentration (1%) showed an appreciable reaction. Specimens fixed in formaldehyde or in a mixture of the two aldehydes always remained unstained. This is in agreement with results obtained on microbodies of yeast (van Dijken, 1975); they did not stain when glutaraldehydeiformaldehyde or glutaraldehyde/acrolein mixtures were used. In contrast, microbodies of Entophlyctis gave a positive reaction after fixation in glutaraldehyde/formaldehyde (Powell, 1976, 1977, 1978). Philippi et al. (1975) commented on the concentration of glutaraldehyde. In their study on P. palmivora a positive DAB reaction was obtained only by using an unusually low percentage of glutaraldehyde for fixation, while higher concentrations (above 0.125%) rapidly inactivated the enzyme.

MICROBODIES

IN

P. graminis

var. trifici

214

HANSSLER,

MUHLENBACHER,

These findings could partly explain the absence of DAB reaction product in fungal microbodies. It is interesting to realize that attempts to stain fungal microbodies were only successful when low aldehyde concentrations were used (Mills and Cantino, 1975, 1979; Philippi et al., 1975) or when the fixation process was slowed down by low temperatures (van Dijken, 1975; Powell, 1976, 1977, 1979), whereas the DAB reaction failed in the case of higher concentrations of glutaraldehyde (Coffey et al., 1972; Wergin, 1972; Mendgen, 1973; Maxwell et al., 1975). It is not only aldehyde and its concentration that seem to influence the outcome of the DAB reaction in fungal microbodies. When we compared the effects of cacodylate-buffered glutaraldehyde with that of phosphate-buffered glutaraldehyde, only the first mixture allowed a positive DAB staining after incubation in the complete reaction mixture. It is very striking that in the successful attempts at staining fungal microbodies, cacodylate buffer has been employed, while the negative results-with one exception (Mendgen, 1973)-were obtained with phosphate-buffered glutaraldehyde. According to our results synergistic inhibition of catalase activity by glutaraldehyde and phosphate could have occurred in the experiments reported by Coffey et al. (1972), Wergin (1972), Bimpong and Hickman (1975), and Maxwell et al. (1975), while in the study by Mendgen (1973) the high glutaraldehyde concentration could have blocked the reaction. Thus, their results leave doubts that a positive staining of catalase actually is not feasible, because lowering of the glutaraldehyde concentration and replacement of phosphate buffer by cacodylate buffer would create proper conditions for the staining reaction. An explanation for the positive reaction in P. graminis in the presence of 3-amino1,2,4-triazole (Fig. 3) can be sought in a delayed penetration of the catalase inhibitor to the reaction sites. The positive catalase

AND

REISENER

test in the absence of exogenous H,O, can be explained by the presence of endogenous H,O, (Frederick and Newcomb, 1969). Since DAB can be used to demonstrate cytochrome oxidase activity (Seligman et al., 1968), the strong staining of mitochondrial cristae is probably due to cytochrome c oxidase activity. Microbodies frequently associated with lipid bodies have also been described in other fungi and in plants with fat-storing seeds in which microbody -lipid globule complexes are common. Here the microbodies (glyoxysomes) are involved in converting stored lipids to carbohydrates via succinate (Beevers, 1969; Tolbert, 1971). In fungi, microbodies might function in a similar manner during germination of fungal spores (Murray and Maxwell, 1974; Weber and Hess, 1974; Armentrout, 1976). Uredospores of rust do not require exogenous substrate for germination. It is well established that during this process the lipids in the lipid bodies are utilized (Frear and Johnson, 1961; Caltrider et al., 1963; Williams and Ledingham, 1964; Staples and Wynn, 1965). There is also evidence that P-oxidation of fatty acids, a necessary first step for utilization of lipids via the glyoxylate cycle, occurs in P. graminis (Reisener, 1976) and that isocitrate lyase and malate synthase, the key enzymes of the glyoxylate cycle, are present in uredospores (Caltrider et al., 1963). Therefore, it seems reasonable to suggest that microbodies in uredospores and germ tubes of P. graminis may be glyoxysomes or microbodies with glyoxysomal functions and that they play a central role in gluconeogenesis. ACKNOWLEDGMENTS The authors express their appreciation Maxwell and M. D. Maxwell for their helpful of the manuscript. REFERENCES

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