Planta (Berl.) 97, 93--105 (1971) 9 by Springer-Verlag 1971

Leaf Microbodies (Peroxisomes) and Catalase Localization in Plants Differing in their Photosynthetic Carbon Pathways* JoE H. HILLIARD, V. E. GlCACEN, and S. H. W~ST Department of Agronomy, University of Florida, Gainesville Received October 30 / December 26, 1970

Summary. The tropical grasses sugarcane (Saccharum o]/icinarum) and pangolagrass (Digitaria decumbens) contained fewer leaf microbodies than temperate orchardgrass (Dactylis glomerata). Leaf microbodies were seen in both the mesophyll and bundle sheath cells of tropical grasses The fibrous elements in the microbodies of tropical grasses differed from those of the temperate grass. Catalase was predominantly localized in the microbodies of leaf cells (3,3'-diaminobenzidine method). The site of greatest catalase activity appeared to be the fibrous and/or crystalline inclusions within the microbodies. The low rates of photorespiration noted in tropical grasses do not appear to be due to the complete absence of the necessary organelles. Introduction

Higher plants can be placed into two groups according to their pathways of photosynthetic C02 fixation. The "C a'' plants (temperate grasses and many others) fix C02 by the pentose cycle, have rates of photosynthesis sensitive to 02 concentrations, and have high C02 compensation points (as a rule, above 40 ppm C02). The "C4" plants (mostly tropical grasses) fix CO2 by the C4-dicarboxylic acid cycle as well as by the pentose cycle, have photosynthetic rates insensitive to 02 concentration, and have low CO2 compensation points (less than 10 ppm C02) (Krenzer and Moss, 1969; Stoy, 1969). The tropical grasses possess much lower rates of photorespiration than temperate grasses. Tolbert and coworkers have found a leaf organelle, the peroxisome, which contains enzymes of the photorespiratory pathway, and have suggested that this organelle is the site of photorespiration (Tolbert et al., 1968, 1969; Kisaki and Tolbert, 1969). A survey of several plants * Cooperative investigations of the University of Florida and Crops Research Division, Agricultural Research Service, U. S. Department of Agriculture. Trade names are mentioned for clarity and do not imply endorsement of products by the U. S. Department of Agriculture. Journal Series No. 3758 of the Florida Agricultural Experiment Station. 7

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showed t h a t peroxisomes could be isolated in g r eat er n u m b e r s from Ca p l a n t s t h a n f r o m Ca plants. A l t h o u g h t h e h o m o g e n a t e s f r o m a tropical grass (sugarcane) c o n t a i n e d only 2 - 5 % as m u c h a c t i v i t y of t h e e n z y m e s of t h e p h o t o r e s p i r a t o r y p a t h w a y as t h e t e m p e r a t e grasses, the a m o u n t s p r e s e n t in the tropical grasses were substantial. Th e e n z y m e catalase has been localized in leaf microbodies of tobacco, a t e m p e r a t e species. These mierobodies are believed to be identical to t h e peroxisomes isolated f r o m leaf h o m o g e n a t e s b y T o l b e r t ' s group (Frederick an d N e w c o m b , 1969a, b). Th er e is little i n f o r m a t i o n , however, a b o u t t h e occurrence an d u l t r a s t r u c t u r e of leaf microbodies f r o m tropical grasses. Th e purpose of this s t u d y was to c o m p a r e t h e occurrence a n d a p p e a r a n c e of leaf microbodies of tropical a n d t e m p e r a t e grasses. W e also c o m p a r e d the localizat i o n of cabalase in leaf cells of t h e t w o t y p e s of plants.

Materials and Methods Clones of two tropical grasses, pangolagrass (Digitaria decumbens Stent.) and sugarcane (Saccharum o//icinarum L.) and of the temperate orchardgrass (Dactylis glomerata L.) were maintained in pots containing a mixture of 50 % sandy loam and 50 % peat. The plants were kept in a greenhouse with a minimum temperature of 30 ~ l-ram 2 blocks of tissue from young but fully expanded leaves were cut while immersed in 2% glutaraldehyde (v/v) buffered at pH 7.4 with 0.1 M sodium cacodylafic. The tissue was fixed by placing it in the same glutaraldehyde solution at 4~ for 4 hr, then rinsed twice in buffer for 20 min. After fixation, the tissue was incubated at 37 ~ in a modified Novikoff and Goldfisher (1969) incubation medium as described by Frederick and Newcomb (1969b). This medium contains 3,3'diaminobenzidine (DA:B) and H20 ~. Control tissues were either incubated in the same solution minus DAB, or were held in buffer at 4 ~ during the 1-hr incubation period. The tissue was post-fixed in osmium tetroxide (2 %, w/v) in 0.1 M cacodylate buffer pH 7.4 for 12 hr at 4 ~ then washed twice in buffer, and dehydrated in an ethanol-acetone series. Mollenhauer's (1964) Epon-Araldite mixture No. 2 was used for embedding. Sections of the embedded tissue were cut with a Dupont diamond knife on a Sorvall Porter-Blum MT-2 ultramicrotome. The sections were placed on copper grids ~nd were left unstained or were post-stained in uranyl acetate (either methanolic: Stempak and Ward, 1964, or aqueous) followed by lead citrate (Reynolds, 1963). The sections were examined with an accelerating voltage of 50 kV on a Hitachi HS-8 or HU-11 E electron microscope. Microbodies were counted in randomly selected low-magnification mierographs of pangolagrass, sugarcane and orehardgrass. Frequency was calculated as the number of microbodies per mm 2 of section surface.

Results

Occurrence el Lea/ Microbodies. Leaf microbodies of similar app e a r a n c e were f o u n d in b o t h th e m e s o p h y ll a n d b u n d l e - s h e a t h cells of pangolagrass a n d sugarcane (Figs. 1-4). These mierobodies c o n t a i n e d

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Table. Size and /requency o] lea/ microbodies in orchardgrass, pangolagrass and sugarcane Species and tissue

Average microbody diameter (~)

Microbody frequency (raicrobodies/mm2)

Orchardgrass Mesophyll

1.10 (0.060)* 79800 (2700)

Pangolagrass Mesophyll Bundle sheath

0.61 (0.033) 9400 (870) 0.66 (0.058) 14300 (850)

Sugarcane Mesophyll Bundle sheath

0.66 (0.050) 1.1] (0.180)

4700 (900) 6700(2600)

* Standard error of the mean.

fibrous elements which were composed of globular subunits with a diameter of approximately 160 A (Fig. 1). Crystalline inclusions were not observed. Leaf cells of orehardgrass contained a greater number of microbodies per unit area than did leaf cells of the tropical grasses, and the microbodies in the temperate grass leaves were larger (Table). Fibrous elements (Fig. 9) were seen within the mierobody matrix of orchardgrass, positioned in a random manner. They were approximately 80 A across and had a repeating linear subunit of 160A. These fibrous elements may be related to the crystalline inclusions which are often seen in the microbody matrix (Fig. 10). The lattice period of the crystalline inclusions in orchardgrass w~s 130 A. Catalase Localization. When glutaraldehyde-fixed leaf tissues were incubated in DAB medium, an electron-dense reaction product was localized in the microbodies and ceil walls of all the species included in this study (Figs. 5, 7, 11-13; non-incubated controls in Figs. 6, 8, 14). The pieces of leaf tissue were not completely infiltrated by the DAB incubation medium. Localization of the reaction product was often quite heavy in the peripheral region of the tissue but light to virtually nonexistent in the central regions. The crystalline inclusions and the fibrous elements of the mierobodies stained heavily when apparently there was insufficient penetration of the DAB medium to allow for dark staining of the entire mierobody matrix (Figs. 12, 13), i.e. localization of catalase activity appeared to be preferential for the fibrous elements and the crystalline inclusions. 7*

Figs. 1-4. Leaf microbodies (MB) from tropical grasses: pangolagrass mesophyll (Fig. 1) and bundle sheath (Fig. 2) and sugarcane mesolohyll (Fig. 3) and bundle sheath (Fig. 4). Mierobodies are often seen near chloroplasts (C). The globular

subunits (arrow) of the fibrous elements within the tropical grass microbody matrix have a diameter of 160 •. Post-stained with uranyl acetate and lead citrate. l~ig. 1, • Fig. 2, • Fig. 3, • Fig. 4, •

Figs. 5-8. Leaf microbodies of tropical grasses contain eatMase. Incubation in DAB-medium darkened mierobodies in pangolagrass (Fig. 5) and sugarcane (Fig. 7).

Non-incubated controls Figs. 6 and 8. In sugarcane (Fig. 7) DAB also localizes in the cell wall due to peroxidase activity. These micrographs are from unstained tissue. Fig. 5, x 107000; Fig. 6, • Fig. 7, • 73500; Fig. 8, • 75300

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Figs. 9 and lO

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The leaf microbodies were generally found in close association with chloroplasts and mitochondria. This is consistent with the findings by Frederick and Newcomb (1969a).

Discussion The results presented here indicate that tropicM grasses contain microbodies (peroxisomes), although they are less numerous than in the temperate grass examined (Table ; Figs. 1-4, 9). The leaf microbodies of the tropical grasses are, in general, smaller than the leaf microbodies seen in the temperate grass. The fibrous elements seen within the microbodies of the two types of grasses are different. Those in the tropical grasses have larger diameters and have globular subunits, as opposed to the linear subunits seen in orchardgrass leaf microbodies. CrystM]ine inclusions of the type seen in orchardgrass microbodies were not observed in sugarcane or pangolagrass. The "staining" of leaf microbodies with DAB in tropical as well as temperate grasses is taken as an indication of catalase activity (Tolbert etal., 1969; Frederick and Newcomb, 1969b). Substantial amounts of the DAB reaction product were locahzcd in the cell walls of sugarcane (Fig. 7). This is in contrast to the small amounts localized in the cell walls of the temperate grass used in this study (Fig. 13) and in another Ca plant studied by Frederick and Neweomb (1969b). However, the DAB locMized in the cell walls is probably due to peroxidase activity since catMase has not been found in cell walls. I t is nevertheless interesting to note the occurrence of differences in DAB staining between cell walls and microbodies. We detected different densities of localization of the DAB reaction product through the orchardgrass leaf tissue, presumably due to difficulties in penetration of the substrate. The earliest detectable localization occurred in the fibrous elements and the crystals (Figs. 12, 13), suggesting that these are the most active sites of catalase activity within the microbody. With more dense staining, we found the reaction product evenly distributed between the erystMs or fibers and the matrix (Fig. i 1). This indicates either that catMase activity is distributed throughout the microbody with the crystals or fibers being the most active sites, or that catMase is restricted to the crystals and fibers and that with the increase in the amount of substrate the reaction product becomes distributed throughout the mierobody. Figs. 9 and 10. Leaf mierobodies (MB) of temperate orchardgrass. The fibrous elements (Fig. 9, arrow) within the microbody matrix have a repeating structural unit of 160 A. CrystMloid inclusions (Fig. I0) with a lattice period of 130 A are frequently seen. Post-stMned with uranyl acetate and lead citrate. Fig. 9, x 60500; Fig. 10, x37200

Figs. 11-14. Orchardgrass leaf mierobodies contain catMase, as evidenced by darkening in DAB medium. W h e n the penetration of DAB medium is a t a high level, b o t h the matrix and the erystalloid inclusion are darkly stained '(Fig. 11, arrow). Lower levels of DAB penetration show t h a t the erystalloid inclusion

(Fig. 12) and the fibrous elements (Fig. 13, arrow) are preferrentiMly stained as compared to the non-incubated control (Fig. 14). The cell wall (CW) of orehardgrass does not localize DAB as heavily as that of sugarcane (Figs. 13, 7). Fig. 11 is unstained tissue; Figs. 12-14 post-stained with uranyl acetate and lead citrate. Fig. 11, • Fig. 12, • Fig. 13, • Fig. 14, •

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Microbodies were o b s e r v e d in b o t h t h e b u n d l e - s h e a t h a n d m e s o p h y l l cells of t h e t r o p i c a l grasses. Since the chloroplasts of these two t y p e s of cells differ in their p a t h w a y s of carbon f i x a t i o n (Slack et al., 1969; E d w a r d s et al., 1970), t h e r e l a t i o n s h i p of microbodies to chloroplasts is open to conjecture. I f mierobodics serve as a site for p h o t o r e s p i r a t i o n , t h e p o t e n t i a l for p h o t o r e s p i r a t i o n is p r e s e n t in b o t h t y p e s of cells, a n d t r o p i c a l grasses could p h o t o r e s p i r e . This is consistent w i t h a r e p o r t b y Bull (1969) t h a t 18-month-old sugarcane does e x h i b i t p h o t o r e s p i r a t i o n . H o w e v e r , t h e presence of catalase in t h e mierobodies does n o t p r o v e t h a t t h e y are i n v o l v e d o n l y in p h o t o r e s p i r a t i o n . The p o s s i b i l i t y r e m a i n s t h a t in t h e t r o p i c a l grasses m i e r o b o d i e s serve o t h e r functions, such as in the m e t a b o l i s m of lipids, glneoneogenesis, or other reactions involving small c a r b o n molecules. The finding of differences in t h e subs t r u c t u r e of t h e mierobodies in t r o p i c a l a n d t e m p e r a t e grasses, a l r e a d y p o i n t e d out, are suggestive of this idea, a n d so are t h e differences in

eatalase activity found in the two plant types. Although catalase activity was largest in the fibrous elements of the microbodics, the intensity of the DAB reaction in the tropical grasses was generally not as great as in the temperate orchard grass. The possibility that enzymes other than those i n v o l v e d in p h o t o r e s p i r a t i o n m a y also be localized in these organelles should be i n v e s t i g a t e d . Altogether, the presence of m i e r o b o d i e s in b o t h t e m p e r a t e a n d t r o p i c a l grasses c a n n o t be t a k e n as proof t h a t t h e organelles are f u n c t i o n a l l y identical. I n fact, t h e differences in degree of catalase a c t i v i t y (DAB reaction), s u b s t r u c t u r e , a n d r e l a t i v e size a n d d i s t r i b u t i o n i n d i c a t e t h a t basic differences in f u n c t i o n m a y exist b e t w e e n t h e mierobodies of t e m p e r a t e a n d t r o p i c a l grasses. We thank Dr. H. C. Aldrieh and his staff for the use of the facilities in the Biological Ultrastructure Laboratory at the University of Florida, and Sylvia Coleman for her assistance in the darkroom. This work was supported in part by an NDEA Fellowship Grant.

References Bull, T. A.: Photosynthetic effieiencies and photorespiration in Calvin cycle and Cd-diearboxylie acid plants. Crop Sci. 9, 726-729 (1969). Downton, W. J. S., Tregunna, E. B. : Fhotorespiration and glycolate metabolism : A re-examination and correlation of some previous studies. Plant Physiol. 43, 923-929 (1968). Edwards, G. E., Lee, S. S., Chen, T. M., Black, C. C. : Carboxylation reactions and photosynthesis of carbon compounds in isolated mesophyll and bundle sheath cells of Digitaria sanguinalis (L.) Scop. Biochem. biophys, l~es. Commun. 89, 389-395 (1970.) Frederick, S. E., Newcomb, E. It. : Microbody like organelles in leM ceils. Science 168, 1353-1355 (19699). -Cytoehemieal localization of catalase in leaf microbodies (poroxisomes). J. Cell Biol. 48, 343-353 (1969b).

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Kisaki, T., Tolbert, N . E . : Glycolate and glyoxylate metabolism by isolated peroxisomes or chloroplasts. Plant Physiol. 44, 242-250 (1969). Krenzer, E. G., Jr., Moss, D. N. : Carbon dioxide compensation in grasses. Crop Sci. 9, 619-621 (1969). l~Iollenhauer, H. H. : Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114 (1964). Novikoff, A. B., Goldfischer, S. : Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochom. 17, 675-680 (1969). Reynolds, E. S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208 213 (1963). Slack, C. R., Hatch, M. D., Goodchild, D. J. : Distribution of enzymes in mesophyll and parenchyma-sheath chloroplasts of maize leaves in relation to the C4dicarboxylic acid pathway of photosynthesis. Biochem. J. 114, 489 498 (1969). Stempak, J. G., Ward, R. T. : An improved staining method for electron microscopy. J. Cell Biol. 22, 697-701 (1964). Stoy, V. : Interrelationships among photosynthesis, respiration, and movement of carbon in developing crops. In: Physiological aspects of crop yield, p. 185-202, J. D. Eastin, F. A. Haskins, C. Y. Sullivan, C. H. M. van Bavel, eds. Madison, Wis. : Amer. Soc. Agron. and Crop Sei. Soc. Amer. 1969. Tolbert, N. E., Oeser, A., Kisaki, T., Hagoman, R. H., Yamazaki, R. K. : Peroxisomes from spinach ]eaves containing enzymes related to glycolate metabolism. J. biol. Chem. 243, 5179-5184 (1968). - - Yamazaki, R. K., Hageman, R. H., Kisaki, T. : A survey of plants for leaf peroxisomes. Plant Physiol. 44, 135 147 (1969). -

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Joe H. Hilliard Department of Agronomy 304 Newell Hall University of Florida Gainesville, Florida 32601, U.S.A.

V.E. Gracen's present address: Department of Plant Breeding and Biometry Cornell University Ithaca, New York 14850, U.S.A.