Histochemical Journal 23, 483-489 (1991)
Catalytic histochemistry of acid and neutral hydrolases in plant seedlings R. GOSSRAU* Department of Anatomy, Free University of Berlin, Ko'nigin-Luise-Strasse 15, W-IO00 Berlin 33, Germany Received 18 December 1990 and in revised form 23 May 1991
Summary In contrast to human and animal tissues, little information is available on the activity, distribution and functional role of acid and neutral hydrolases in plant cells and tissues. Because it is known that these enzymes are relatively active during germination, they were analysed histochemically during this process using light microscope azo, azoindoxyl, indigogenic and tetrazolium methods. Proteases, glucosidases and glucuronidases could not be detected. Non-specific acid phosphatases were species-independent and showed considerable activities in aleuron and nutritional cells, in other cell types of cotyledon or endosperm tissue and in different types of embryonic cells. Acid glycosidases and non-specific esterases, in contrast, displayed a species-dependent activity and differences in localization. Of the glycosidases, c~-D-galactosidasewas the most active. Non-specific esterases, acid phosphatase and glucosaminidase were also present in the extracellular matrix. During germination, acid hydrolase activity either decreased or increased, depending on the seedling species and enzyme.
Introduction Both well-established and recently-developed light microscopical histochemical methods have contributed considerably to our present knowledge of many kinds of acid and neutral hydrolases. They have helped to elucidate the role of these enzymes in lysosomal and non-lysosomal digestive processes in healthy and diseased human cells and tissues, and also in animal cells and tissues during development and in the adult state (for references about glycosidases, see Gossrau et al., 1991; for proteases, Lojda et al., 1991; for non-specific acid phosphatases and esterases, Wohlrab & Gossrau, 1991). Far less information is available about the extracellular and intracellular localization, activity and function of these hydrolases in developing and adult plant cells and tissues (for references, see Benes et al., 1981; Gahan, 1984). This lack of knowledge is due, to some extent, to the uncritical use of histochemical methods in plant studies, e.g. the simultaneous azo coupling procedures for glycosidases using 6-bromo-2-naphthol or 1-naphthol substrates and stable diazonium salts, that create numerous artefacts (Lojda et al., 1979). In other cases, well established methods have not yet been applied to plant tissues, e.g. the indigogenic, tetrazolium or azoindoxyl techniques for various hydrolytic enzymes (Gahan, 1984). The recent and increasing number of biochemical * To whom all correspondence should be addressed. 0018-2214/91 $03.00 +.12 9 1991 Chapman & Hall
observations on the role of this group of enzymes (Bewley & Black, 1982, 1983) during normal and disturbed germination have not been considered sufficiently so far ink histochemical studies. Germinating seeds are said to be especially active for hydrolases involved in autolytic lysosomal and non-lysosomal food mobilization and other events (Libbert, 1987). Therefore, the aim of the present investigation was to analyse various neutral and acid proteases, glycosidases, phosphatases and non-specific esterases, which might participate in these processes in different plant seedlings during day 0-6 of germination. Both existing and newly-developed methods of catalytic histochemistry and various kinds of tissue pretreatment were used~
Material and methods Seeds, i.e. nutritional (endosperm, cotyledons) and embryonic tissue of mustard, lucerne (alfalfa), sojbean, cotton, Adzuki bean, clover and chick-pea (BIOCOSMA GmbH, Konstanz, Germany) were germinated at room temperature in a standard household plastic germinating chamber according to the instructions of the manufacturer (BIOCOSMA GmbH). The seedlings were harvested after 0-6 days of germination and fixed either in 1.5% glutaraldehyde or 4% formaldehyde, rinsed in Holt's solution according to Lojda et al., (1979), and mounted in pieces of meat as supporting tissue. Alternatively, the seedlings were mounted in this way unfixed on cork-plates, wrapped with
484 plastic foil, and frozen in liquid nitrogen. Sections 10 ~m thick, were cut in a cryostat (model 2800 N; Reichert-Jung, Nu~loch, Germany), mounted on glass slides coated with chromalum-gelatin and used unfixed. Other fresh sections were freeze-dried and coated with celloidin, mounted on semipermeable membranes or fixed in 4% formaldehyde or 1.5% glutaraldehyde, rinsed in tap water and transferred to distilled water as described by Lojda et al., (1979). Microsomal alanyl aminopeptidase (EC 3.4.11.2), glutamyl aminopeptidase (EC 3.4.11.7), 7-glutamyl transpeptidase (EC 2.3.2.2), dipeptidyI peptidases I (EC 3.4.14.1), II (EC 3.4.14.2) and IV (EC 3.4.14.5) were demonstrated using 4-methoxy-2-naphthylamine (MNA) substrates and Fast Blue B or hexazotized New Fuchsin for simultaneous coupling according to Lojda et al., (1979) and Gossrau (1981). Trypsin-type and cathepsin B-type proteases were investigated using MNA substrates and the same coupling agents as recommended by Gossrau (1981) and Sannes (1988). Non-specific acid phosphatases (EC 3.1.2.1) were visualized using (a) simultaneous azo methods with various naphthol AS phosphates, (b) the azoindoxyl simultaneous coupling technique with hexazotized Pararosaniline according to Lojda et aL, (1979), and (c) the tetrazolium salt procedure with menadiol diphosphate as given by Dikow and Gossrau (1990). To demonstrate acid [3-D-galactosidase (EC 3.2.1.23), c~glucosidase (EC 3.2.1.20), ~-D-glucosidase (EC 3.2.1.21), c~D-mannosidase (EC 3.2.1.24), C~-D-galactosidase (EC 3.2.1.22), ~-D-glucuronidase (EC 3.2.1.31) and N-acetyl-Dglucosaminidase (EC 3.2.1.30) and non-specific esterases (EC 3.1.1.1; 3.1.1.2; 3.1.1.6) the available azo, azoindoxyl and indigogenic methods with 1-naphthyl, naphthol ASBI, naphthol AS-D and indoxyl, 5-bromo-3-indoxyl and 5bromo-4-chloro-3-indoxyl substrates were used as prescribed by Lojda et al., (1979) and Gossrau and Lojda (1989, 1991). Incubations were performed at 37~ for 5, 10, 15, 30 and 60 min. Sections from the same tissue samples were stained with Haematoxylin and Eosin or Methylene Blue, with the Sudan Black reaction for lipids with and without acetone pretreatment, and the periodic acid-Schiff (PAS) reaction for carbohydrates with and without a-amylase pretreat-
GOSSRAU ment (Romeis, 1968). Substrate-free incubation media for the visualization of phosphatases and oxidases with lead and cerium capture methods were served for the detection of phosphate groups in germinating plant tissues as given by Gossrau (1990). Controls for the enzyme reactions were performed using either media without substrate or complete media to which appropriate inhibitors were added (Lojda et aI., 1979). All chemicals were of analytical grade and purchased from Bachem (Heidelberg, Germany), Serva (Heidelberg, Germany), Boehringer. (Mannheim, Germany), Chroma (Stuttgart, Germany) and Merck (Darmstadt, Germany).
Results I n d e p e n d e n t of the germination day, application of incubation media with and without substrates containing enzyme-specific inhibitors gave negative findings with the exception of the assays for non-specific phosphatases. These e n z y m e s were inhibited by fluoride a n d cupric ions but not by tartrate. After preincubation of sections with m-amylase, starch was absent, whereas cell wall material still reacted. Acetone pretreatment yielded negative findings with the Sudan Black procedure with the exception of vascular elements in the nutritional a n d embryonic tissue a n d the aleuron cells. Sections of formaldehyde-fixed total seedlings yielded the highest a m o u n t s of precisely localized stain. Sections of glutaraldehyde-fixed seedlings usually led to less final reaction product being formed, but its localization was equally precise. An exception was acid ~-D-galactosidase which generated more stain t h a n after formaldehyde-fixation. With other tissue pretreatments, the final reaction products were less precisely localized, and with the exception of the semipermeable m e m b r a n e technique, less were formed (unfixed sections, glutaraldehyde- and formaldehyde-fixed sections).
Figs 1 and 2. Demonstration of'phosphatases with naphthol AS-TR phosphate in a 2-day old mustard seedling. Fig. 1. Staining of nutritional cells (arrows). Fig. 2. Staining of rhizodermis (R), cortical (C) and endodermis cells (E). V = vascular bundle. Fig. 3. See Fig. 2. Performance of the reaction with naphthoI AS--BI phosphate, arrows low amounts of stain. Figs 4 and 5. Lucerne seedlings on day 2 of germination. Visualization of c~-D-galactosidase with the indigogenic method. Fig. 4. Indigo in the aleuron cells (thick short arrow) and groups of nutritional cells (arrows) of a cotyledon. V = vascular bundle. Fig. 5. Indigo (arrows) in cortical cells of the embryo. V = vascular bundle. Figs 6 and 7. ~-N-Acetyl-D-glucosaminidase activity (arrows) in a clover embryo of germination day 1. Fig. 6. V = vascular bundle, C = cortical cells. Fig. 7. R = rhizodermis cells, C = cortical cells. Fig. 8. Non-specific esterase reaction in special cells (arrows) around the vascular tissue (V) in a clover seedling of day 1. N = nutritional cells. Figs 9 and 10. Exoglycosidases (arrows) in certain vascular elements (V) of cotyledons on germination day 2, N = nutritional cells. Fig. 9. Visualization of a-D-mannosidase with the azoindoxyl procedure in sojbean. Fig. 10. Staining by acid 13-0galactosidase with the indigogenic technique in mustard. Fig. 11. Non-specific esterase activity (arrows) in chick pea nutritional tissue of germination day 2. S = starch granules, thick arrows cell wall staining. Primary magnification of all figures, x430.
Z
J7
ct~
o
q~
486 Depending on the enzyme, the quantity of stain increased with incubation time. The shortest incubation time required to obtain optimal staining was 15 min.
Day O, 1 or 2 of germination All proteases, ~-D-glucuronidase, and a- and f~-glucosidase could not be detected in any of the analysed seedling species between day nought and six of germination. In contrast, non-specific phosphatases yielded positive histochemical results in mustard, alfalfa, sojbean, cotton, clover, Adzuki bean and chick pea nutritional and embryonic tissue and showed a certain species-independence. However, the results were substrate-dependent: Naphthol AS-TR phosphate (Figs 1 and 2) delivered high amounts of stain, e.g. in the aleuron cells, most of the nutritional cells and in the rhizodermis, cortical and endodermis cells of mustard embryos. Similar findings were obtained with the AS-MX, AS-E, AS-GR and AS-LC type substrates and with 1-naphthyl phosphate, whereas naphthol AS--BI (Fig. 3), and especially 5-bromo-4chloro-3-indoxyl phosphate and menadiol diphosphate were hydrolysed more slowly or not at all. In contrast to the generally species-independent activities of non-specific acid phosphatases and the widespread occurrence of these enzymes, exoglycosidases such as a-D-mannosidase, a-D-galactosidase, acid ~-D-galactosidase and Nacetylglucosaminidase showed interspecies differences, were often limited to special cells and had a certain activity ratio. Cell-specific localization was also seen for the non-specific esterases. The indigogenic or azoindoxyl methods for a-Dgalactosidase, acid ~-D-galactosidase and a-Dmannosidase always delivered higher amounts of more precisely localized stain than procedures based on 1-naphthyl or naphthol AS-BI glycosides or the tetrazolium techniques. However, this last substrate type was best suited for O-N-acetylglucosaminidase. In comparison, R-D-galactosidase (Figs 4 and 5) was the most active exoglycosidase and generated large quantities of indigo in the aleuron and nutritional cells proper as well as in the embryonic rhizodermis and cortical cells and in the vascular elements primarily of alfalfa and dover; O-D-galactosidase and a-D-mannosidase yielded smaller amounts of indigo reaction product. Far less stain was generated by these glycosidases in the nutritional and embryonic tissue of the other seedlings. N-acetylglucosaminidase was only detectable in some nutritional and embryonic cells of clover (Figs 6 and 7). Non-specific esterases could best be demonstrated with 1-naphthyl acetate; far lower amounts of stain were generated when using 5-bromo-4-chloro-3indoxyl acetate or the 5-bromo-derivative and still
GOSSRAU less with naphthol AS-D and indoxyl acetate as substrates. In clover and sojbean nutritional tissue, very active non-specific esterases (Fig. 8) and a-D-mannosidase were present in cells around the vascular elements. In contrast to the other cell types, all hydrolases produced different amounts of stain in the vascular elements (Figs 9 and 10) of the nutritional and embryonic tissue. The intracellular localization of the stain generated by the phosphatases, glycosidases and non-specific esterases appeared to be identical in the nutritional and embryonic cells of one single seedling species, but showed interspecies differences. For example, in chick pea nutritional tissue (Figs 11 and 12) the final reaction product of all enzymes was generated in the peripheral cytoplasm. Reaction product was also generated by the glycosidases and non-specific esterases in mustard, lucerne and sojbean nutritional and embryonic cells. Acid phosphatase exhibited dual localization (c.f. Figs 1 and 2), its reaction product also appearing in the globoids of protein bodies. All the hydrolases investigated were present inside the plant seedling cells, whereas non-specific esterases (Fig. 13) and sometimes also non-specific acid phosphatases, and very rarely N-acetyl~D-glucosaminidase, were also seen in the extracellular matrix including the cell walls of the nutritional tissues, and especially the embryonic tissue, of all the seedling species studied. Control media (Figs 14 and 15) for the cerium and lead methods for the phosphatases and oxidases stained the globoids of the protein bodies. Starch granules (c.f. Figs 11 and 12) visualized by the PAS reaction were free of any enzymaticallygenerated reaction products. In contrast, positive sites revealed with Sudan Black (Fig. 16) contained stain produced by glycosidases, non-specific acid phosphatases and non-specific esterases.
Hydrolases during germination Proteases, ~- and f3-ghicosidases and ~-glucuronidase could not be detected up to day six of germination. The activities of other hydrolases changed, depending on the seed species and enzyme in nutritional and embryonic tissue during the germination process. Phosphatases, glycosidases and non-specific esterases were less active in mustard (Fig. 17) on day four of germination compared to those in alfalfa and sojbean. In sojbean nutritional cells, lipids (Fig. 18) were decreased and occasionally some cells showed an enhanced co-localized non-specific esterase activity (Fig. 19). In contrast, acid phosphatase activity increased in the nutritional cells of cotton cotyledons. The activities of the other two groups of hydrolytic enzymes did not appear to change.
Catalytic histochemistry of acid a n d neutral hydroLases in plant seedlings
487
Fig. 12 see Fig. 11 but non-specific acid phosphatase. Figs 13, 14, 15. Mustard seedling on germination day 0. Arrows reaction producL Fig. 13. Cell wall staining t~y non-specific esterases between embryonic cortical cells. Figs 14 and 15. Cotyledon. Fig. 14. Staining of globoids by lead ions. Fig. 15. see Fig. 14 but visualization with cerium ions. Fig. 16. Lipid staining (arrows) by Sudan Black in sojbean nutritional cells on day 2. Fig. 17. Mustard seedling on germination day 4 with decreased amounts of stain (arrows) generated by non-specific phosphatase. R = rhizodermis cells, C = cortical cells. Figs 18 and 19. Sojbean cotyledon with nutritional cells on day 5 of germination. Fig. 18. Visualization of decreased quantities of lipids with Sudan Black (arrows). Fig. 19. Increased staining intensity in certain nutritional cells (arrows) by non-specific esterase. Primary magnification of all figures, x430.
Discussion
Germination of various plant seeds is accompanied by the presence of certain, but not all, acid and neutral hydrolases for which histochemical m e t h o d s
exist; w h e n a hydrolase is present, it occurs mostly in both nutritional and embryonic tissue. The histochemical absence, but biochemicallyk n o w n presence of proteases (Matile, 1978; Bhatna-
488 gar & Sawhney, 1981; Bewley & Black, 1983; Libbert, 1987) in those tissues with high concentrations of proteins, e.g. cotton cotyledons and embryos (Vigil et al., 1989), which are said to be normally degraded by proteinases or endopeptidases and to a lesser extent by exopeptidases during germination to nourish the embryo and for other purposes (Bewley & Black, 1983; Fincher, 1989), might be explained by methodological reasons. For example, the proteinases are relatively sensitive to fixation and the diazonium salts used in simultaneous azo-coupling methods (Lojda et al., 1979, 1991). Possibly the wrong proteases were also analysed. Furthermore, endogenous protease inhibitors may be responsible for the discrepancies of histochemical and biochemical data: Such inhibitors are known to be present in plant cells during germination (Bewley & Black, 1983). Another problem to be resolved is whether proteases and their respective inhibitors occur in the same cells or cell compartments. Recent fluorometric assays of homogenates of fresh cotyledon or endosperm using the same substrates as used in histochemical determinations, have shown that in the absence of inhibitory diazonium salts Alaand CBZ-Ala-Arg-Arg naphthylamines are hydrolysed in mustard, lucerne, sojbean, clover and Adzuki beans during germination (unpublished observations). Similar methodological reasons might explain why some glycosidases, e.g. ~- and fi-glucosidases and ]3D-glucuronidase, could not be detected. They have been reported to be present in seedling tissues, because of the presence of ~- and ~-linked glucose and glucuronic acid in various glycoproteins or other glycocomponents and the participation of R-D-glucosidases in starch mobilization (Bewley & Black, 1983; Libbert, 1987). In contrast, assays with substrates for non-specific acid phosphatases revealed high overall activities in all plant seedlings examined. However, glycosidases and non-specific esterases were only highly active in some species. Moreover, acid phosphatase showed a dual localization, i.e. in protein bodies (Matile, 1969) and outside them. Since the same protein bodies contain phosphate groups, as revealed by the lead and cerium control media for phosphatases and oxidases (Gossrau, 1990), they may represent phytin (phytate, myoinositol-phosphate)-containing globoids, the common phosphate source also of germinating seeds (Bewley & Black, 1983). Therefore, the staining generated in globoids might be produced by phytases; normally, 2- and 6-phytases release inorganic phosphate from phytin and may co-operate with other phosphatases associated with myoinositol-phosphate metabolism and capable of hydrolysing non-specific phosphatase substrates (Loewus & Loewus, 1983). The absence of other
GOSSRAU hydrolases which should be present in the globoids (Bewley & Black, 1983; Loewus & Loewus, 1983) might be explained by their comparatively lower activity in these structures. This activity is abolished by the inhibitory effects of formaldehyde and auxiliary agents, e.g. diazonium salts and iron ions. Outside the protein bodies with globoids, but inside the respective cells, the staining produced by the acid phosphatase assays might be actually caused by this enzyme, as is the staining by the positively reacting glycosidases and non-specific esterases. Although it is not possible for methodological and optical reasons to determine the exact intracellular localization of all of these hydrolytic enzymes, they might be associated with lysosomes, dicytosomes of the Golgi apparatus, and possibly with the endoplasmic reticulum in the nutritional and embryonic cells and tissues, and in plasmodesmata of the embryo (Matile, 1969; Gahan, 1973; Bewley & Black, 1983). The staining observed in the extracellular matrix, primarily with methods for non-specific esterases and acid phosphatases, might be situated between the plasma membrane and the cell walls and in the cell walls themselves (Gahan, 1973, 1984) after ,these enzymes have been secreted by the corresponding nutritional and embryonic cells in contrast to nonsecreted glyeosidases. Their functional role in the extracellular matrix might be participation in the turnover or dissolution of cell-wall material or other compounds during germination (Chrispeels, 1976; Labavitch, 1981; Bewley & Black, 1983). However, for these processes, active glycosidases, which we could not demonstrate, would also be necessary. Inside the nutritional cells, the non-secreted hydrolases may be involved in autolysis, i.e. the breakdown or hydrolysis of certain macromolecules like glycoproteins, galactomannans and glyco- and phosphocomponents in glyco- and phospholipids. The non-specific esterases might act as acid lipase in lipolysis in order to nourish the growing embryo and for other purposes (Bewley & Black, 1983; Libbert, 1987; Fincher, 1989). Further, the intracellular hydrolases might possibly control the secretion of non-specific esterases and acid phosphatases. Less probably, the intracellular (non-secreted) hydrolases participate in heterolysis of endocytosed material, since endocytosis is said to be generally rare or even absent in plant nutritional and embryonic cells during germination (Libbert, 1987). Altogether, the non-specific acid phosphatases, glycosidases and non-specific esterases visualized in the present study should take part in intracellular and extracellular (matrix) digestion or hydrolysis processes (Gahan, 1973). In contrast to all other cells in nutritional and embryonic tissue, at least some vascular elements have a constant high hydrolase activity. It is possible
Catalytic histochemistry of acid and neutral hydrolases in plant seedlings that these heavily reacting elements represent companion cells or procambium cells which can hydrolyse a broad range of non-specific acid phosphatase and exoglycosidase substrates. Since the germination period coincides with the differentiation of vascular elements, which includes partial or total physiological cell death and reorganization as well as degradation processes, these enzymes might be involved in those events (Gahan, 1973; Evert, 1977). In conclusion, existing histochemical methods for non-specific phosphatases, non-specific esterases and glycosidases, and newly-developed procedures for glycosidases using indoxyl substrates have revealed the presence of these hydrolases in many germinating plants; however, proteases and other glycosidases were not found with these techniques. In the plant cells studied, the enzymes were either species-dependent or independent and were always present in both the nutritional and embryonic tissue. The most regular localization was found in differentiating vascular elements of the nutritional tissue. Although the exact intracellular and extracellular localization was not determined in this study, these acid hydrolases might be involved in phytin, glycoprotein, phospho- and glycolipid as well as in lipid metabolism and digestion processes respectively.
Acknowledgements I thank Ms B. Bochow for her expert technical assistance, Ms U. Sauerbier for the photographic work, Ms Irene Straatsburg for grammatical corrections and Ms A. Hechel for her patience with the preparation of the manuscript.
References K., I V A N O V , V. B. & H A D O C O V A , V. (1981) Glycosidases in the root tip. In Structure and Function of Plant Roots (edited by BROUWER,R.), pp. 137--9. The Hague, Boston, London: Martinus Nijhoff. BEWLEY, J. D. & BLACK, M. (1982) Physiology and Biochemistry of Seeds. Vol. 2. Berlin, Heidelberg, New York: Springer. BEWLEY, J. D. & BLACK, M. (1983) Physiology and Biochemistry of Seeds. Vol. 1. Berlin, Heidelberg, New York: Springer. B H A T N A G A R , S. P. & S A W H N E Y , V. (1981) Endosperm - its morphology, ultrastructure and histochemistry. Int. Rev. Cytol. 73, 55-t02. C~RISPEELS, M. I. (1976) Biosynthesis, intracellular transport, and secretion of extracellular macromolecules. Annu. Rev. Plant Physiol. 27, 19-38. mKOW, A. & GOSSRAU,R. (1990) Histochemical demonstration of non-specific esterases and non-specific acid phosphatases using menadiol substrates. Acta Histochem. 88, 167-74. BENES,
489
EVERT, R. F. (1977) Phloem structure and histochemistry. Annu. Rev. Plant Physiol. 28, 199-222. rlNCHER, G. G. (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu. Rev. Plant PhysioL Mol. Biol. 40, 305-46. GAHAN~ P. B. (1973) Plant lysosomes. In Lysosomes in Biology and Pathology (edited by DINGLE,J. T.), Vol. 3, pp. 6785. Amsterdam, London: North-Holland. G A H A N , P. B. (1984) Plant Histochemistry and Cytoehemistry. An Introduction. London, Orlando, San Diego, New York, Toronto, Montreal, Tokyo: Academic Press. GOSSRAU,R. (1981) Investigation of proteinases in the digestive tract using 4-methoxy-2-naphthylamine (MNA) substrates. J. Histochem. Cytochem. 29, 464-80. GOSSRAU, R. (1990) Cerium methods for the light microscopical demonstration of phosphatases and hydrogen-peroxide-generating oxidases in plant tissue. Trans. R. Microsc. Soc. (New Series) 1, 529-32. GOSSRAU, R. & L O J D A , Z. (1989) Histochemical detection of a-D-galactosidase with 5-Br-4-C1-3-indoxyl a-D-galactoside. Acta Histochem. 85, 213-22. GOSSRAU, R. & L O J D A , Z. (1991) Histochemical detection of c~-mannosidase in animal and plant tissue. Histochem. J. 23 (in press). GOSSRAU, R., L O J D A , Z. & S T O W A R D , P. J. (1991) Glycosidases. In Histochemistry: Theoretical and Applied (edited by S T O W A R D , P. J. & P E A R S E , A. G. E..)/ V o l . 3, 4th edn. pp. 241-79. Edinburgh, London: Churchill Livingstone. LABAVlTCH,J. M. (1981) Cell wall turnover in plant development. Annu. Rev. Plant Physiol. 32, 385-406. LIBBERT, E. (1987) Lehrbuch der Pflanzenphysiologie. 4. Aufl. Stuttgart, New York: Fischer. L O E W U S , F: A. & L O E W U S , M. W. (1983) Myo-inositol: Its biosynthesis and metabolism. Annu. Rev. Plant Physiol. 34, 137-61. L O J D A , Z., GOSSRAU, R. & S C H I E B L E R , T. H. (1979) Enzyme Histochemistry. A Laboratory Manual. Berlin, Heidelberg, New York: Springer. L O J D A , Z., GOSSRAU, R. & S T O W A R D , P, J. (1991) Proteases. In Histochemistry. Theoretical and Applied (edited by STOWARD,P. J. &PEARSE,A. G. E.), Vol. 3, 4th edn. pp. 281-335. Edinburgh, London: Churchill Livingstone. MATILE,P. H. (1969) Plant lysosomes. In Lysosomes in Biology and Pathology (edited by DINGLE, J. T. & FELL, ~f. B.), V o [ . 1, pp. 406-30. Amsterdam, London: North-Holland. MATILE,P. H. (1978) Biochemistry and function of vacuoles. Annu. Rev. Plant Physiol. 29, 193-213. ROMEIS, B. (1968) Mikroskopische Technik. Mfinchen: Oldenbourg. SANNES, P. L. (1988) The histochemical and cytochemical localization of proteases. Progr. Histochem. Cytochem. 18, 1--48. V I G I L , E. L., F R A Z I E R , L. C. & POOLEY, C. (1989) Effect of drought stress on protein body development in radicles of cotton embryos: A quantitative study with image analysis. Ceil Biol. Int. Reports 13, 47-64. WOHLRAB, F. a GOSSRAU, R. (1991) Katalytische Enzymhistochemie. Grundlagen und Methoden f~r die Etektronenmikroskopie. Jena: Fischer (in press).
Catalytic histochemistry of acid and neutral hydrolases in plant seedlings R. GOSSRAU* Department of Anatomy, Free University of Berlin, Ko'nigin-Luise-Strasse 15, W-IO00 Berlin 33, Germany Received 18 December 1990 and in revised form 23 May 1991
Summary In contrast to human and animal tissues, little information is available on the activity, distribution and functional role of acid and neutral hydrolases in plant cells and tissues. Because it is known that these enzymes are relatively active during germination, they were analysed histochemically during this process using light microscope azo, azoindoxyl, indigogenic and tetrazolium methods. Proteases, glucosidases and glucuronidases could not be detected. Non-specific acid phosphatases were species-independent and showed considerable activities in aleuron and nutritional cells, in other cell types of cotyledon or endosperm tissue and in different types of embryonic cells. Acid glycosidases and non-specific esterases, in contrast, displayed a species-dependent activity and differences in localization. Of the glycosidases, c~-D-galactosidasewas the most active. Non-specific esterases, acid phosphatase and glucosaminidase were also present in the extracellular matrix. During germination, acid hydrolase activity either decreased or increased, depending on the seedling species and enzyme.
Introduction Both well-established and recently-developed light microscopical histochemical methods have contributed considerably to our present knowledge of many kinds of acid and neutral hydrolases. They have helped to elucidate the role of these enzymes in lysosomal and non-lysosomal digestive processes in healthy and diseased human cells and tissues, and also in animal cells and tissues during development and in the adult state (for references about glycosidases, see Gossrau et al., 1991; for proteases, Lojda et al., 1991; for non-specific acid phosphatases and esterases, Wohlrab & Gossrau, 1991). Far less information is available about the extracellular and intracellular localization, activity and function of these hydrolases in developing and adult plant cells and tissues (for references, see Benes et al., 1981; Gahan, 1984). This lack of knowledge is due, to some extent, to the uncritical use of histochemical methods in plant studies, e.g. the simultaneous azo coupling procedures for glycosidases using 6-bromo-2-naphthol or 1-naphthol substrates and stable diazonium salts, that create numerous artefacts (Lojda et al., 1979). In other cases, well established methods have not yet been applied to plant tissues, e.g. the indigogenic, tetrazolium or azoindoxyl techniques for various hydrolytic enzymes (Gahan, 1984). The recent and increasing number of biochemical * To whom all correspondence should be addressed. 0018-2214/91 $03.00 +.12 9 1991 Chapman & Hall
observations on the role of this group of enzymes (Bewley & Black, 1982, 1983) during normal and disturbed germination have not been considered sufficiently so far ink histochemical studies. Germinating seeds are said to be especially active for hydrolases involved in autolytic lysosomal and non-lysosomal food mobilization and other events (Libbert, 1987). Therefore, the aim of the present investigation was to analyse various neutral and acid proteases, glycosidases, phosphatases and non-specific esterases, which might participate in these processes in different plant seedlings during day 0-6 of germination. Both existing and newly-developed methods of catalytic histochemistry and various kinds of tissue pretreatment were used~
Material and methods Seeds, i.e. nutritional (endosperm, cotyledons) and embryonic tissue of mustard, lucerne (alfalfa), sojbean, cotton, Adzuki bean, clover and chick-pea (BIOCOSMA GmbH, Konstanz, Germany) were germinated at room temperature in a standard household plastic germinating chamber according to the instructions of the manufacturer (BIOCOSMA GmbH). The seedlings were harvested after 0-6 days of germination and fixed either in 1.5% glutaraldehyde or 4% formaldehyde, rinsed in Holt's solution according to Lojda et al., (1979), and mounted in pieces of meat as supporting tissue. Alternatively, the seedlings were mounted in this way unfixed on cork-plates, wrapped with
484 plastic foil, and frozen in liquid nitrogen. Sections 10 ~m thick, were cut in a cryostat (model 2800 N; Reichert-Jung, Nu~loch, Germany), mounted on glass slides coated with chromalum-gelatin and used unfixed. Other fresh sections were freeze-dried and coated with celloidin, mounted on semipermeable membranes or fixed in 4% formaldehyde or 1.5% glutaraldehyde, rinsed in tap water and transferred to distilled water as described by Lojda et al., (1979). Microsomal alanyl aminopeptidase (EC 3.4.11.2), glutamyl aminopeptidase (EC 3.4.11.7), 7-glutamyl transpeptidase (EC 2.3.2.2), dipeptidyI peptidases I (EC 3.4.14.1), II (EC 3.4.14.2) and IV (EC 3.4.14.5) were demonstrated using 4-methoxy-2-naphthylamine (MNA) substrates and Fast Blue B or hexazotized New Fuchsin for simultaneous coupling according to Lojda et al., (1979) and Gossrau (1981). Trypsin-type and cathepsin B-type proteases were investigated using MNA substrates and the same coupling agents as recommended by Gossrau (1981) and Sannes (1988). Non-specific acid phosphatases (EC 3.1.2.1) were visualized using (a) simultaneous azo methods with various naphthol AS phosphates, (b) the azoindoxyl simultaneous coupling technique with hexazotized Pararosaniline according to Lojda et aL, (1979), and (c) the tetrazolium salt procedure with menadiol diphosphate as given by Dikow and Gossrau (1990). To demonstrate acid [3-D-galactosidase (EC 3.2.1.23), c~glucosidase (EC 3.2.1.20), ~-D-glucosidase (EC 3.2.1.21), c~D-mannosidase (EC 3.2.1.24), C~-D-galactosidase (EC 3.2.1.22), ~-D-glucuronidase (EC 3.2.1.31) and N-acetyl-Dglucosaminidase (EC 3.2.1.30) and non-specific esterases (EC 3.1.1.1; 3.1.1.2; 3.1.1.6) the available azo, azoindoxyl and indigogenic methods with 1-naphthyl, naphthol ASBI, naphthol AS-D and indoxyl, 5-bromo-3-indoxyl and 5bromo-4-chloro-3-indoxyl substrates were used as prescribed by Lojda et al., (1979) and Gossrau and Lojda (1989, 1991). Incubations were performed at 37~ for 5, 10, 15, 30 and 60 min. Sections from the same tissue samples were stained with Haematoxylin and Eosin or Methylene Blue, with the Sudan Black reaction for lipids with and without acetone pretreatment, and the periodic acid-Schiff (PAS) reaction for carbohydrates with and without a-amylase pretreat-
GOSSRAU ment (Romeis, 1968). Substrate-free incubation media for the visualization of phosphatases and oxidases with lead and cerium capture methods were served for the detection of phosphate groups in germinating plant tissues as given by Gossrau (1990). Controls for the enzyme reactions were performed using either media without substrate or complete media to which appropriate inhibitors were added (Lojda et aI., 1979). All chemicals were of analytical grade and purchased from Bachem (Heidelberg, Germany), Serva (Heidelberg, Germany), Boehringer. (Mannheim, Germany), Chroma (Stuttgart, Germany) and Merck (Darmstadt, Germany).
Results I n d e p e n d e n t of the germination day, application of incubation media with and without substrates containing enzyme-specific inhibitors gave negative findings with the exception of the assays for non-specific phosphatases. These e n z y m e s were inhibited by fluoride a n d cupric ions but not by tartrate. After preincubation of sections with m-amylase, starch was absent, whereas cell wall material still reacted. Acetone pretreatment yielded negative findings with the Sudan Black procedure with the exception of vascular elements in the nutritional a n d embryonic tissue a n d the aleuron cells. Sections of formaldehyde-fixed total seedlings yielded the highest a m o u n t s of precisely localized stain. Sections of glutaraldehyde-fixed seedlings usually led to less final reaction product being formed, but its localization was equally precise. An exception was acid ~-D-galactosidase which generated more stain t h a n after formaldehyde-fixation. With other tissue pretreatments, the final reaction products were less precisely localized, and with the exception of the semipermeable m e m b r a n e technique, less were formed (unfixed sections, glutaraldehyde- and formaldehyde-fixed sections).
Figs 1 and 2. Demonstration of'phosphatases with naphthol AS-TR phosphate in a 2-day old mustard seedling. Fig. 1. Staining of nutritional cells (arrows). Fig. 2. Staining of rhizodermis (R), cortical (C) and endodermis cells (E). V = vascular bundle. Fig. 3. See Fig. 2. Performance of the reaction with naphthoI AS--BI phosphate, arrows low amounts of stain. Figs 4 and 5. Lucerne seedlings on day 2 of germination. Visualization of c~-D-galactosidase with the indigogenic method. Fig. 4. Indigo in the aleuron cells (thick short arrow) and groups of nutritional cells (arrows) of a cotyledon. V = vascular bundle. Fig. 5. Indigo (arrows) in cortical cells of the embryo. V = vascular bundle. Figs 6 and 7. ~-N-Acetyl-D-glucosaminidase activity (arrows) in a clover embryo of germination day 1. Fig. 6. V = vascular bundle, C = cortical cells. Fig. 7. R = rhizodermis cells, C = cortical cells. Fig. 8. Non-specific esterase reaction in special cells (arrows) around the vascular tissue (V) in a clover seedling of day 1. N = nutritional cells. Figs 9 and 10. Exoglycosidases (arrows) in certain vascular elements (V) of cotyledons on germination day 2, N = nutritional cells. Fig. 9. Visualization of a-D-mannosidase with the azoindoxyl procedure in sojbean. Fig. 10. Staining by acid 13-0galactosidase with the indigogenic technique in mustard. Fig. 11. Non-specific esterase activity (arrows) in chick pea nutritional tissue of germination day 2. S = starch granules, thick arrows cell wall staining. Primary magnification of all figures, x430.
Z
J7
ct~
o
q~
486 Depending on the enzyme, the quantity of stain increased with incubation time. The shortest incubation time required to obtain optimal staining was 15 min.
Day O, 1 or 2 of germination All proteases, ~-D-glucuronidase, and a- and f~-glucosidase could not be detected in any of the analysed seedling species between day nought and six of germination. In contrast, non-specific phosphatases yielded positive histochemical results in mustard, alfalfa, sojbean, cotton, clover, Adzuki bean and chick pea nutritional and embryonic tissue and showed a certain species-independence. However, the results were substrate-dependent: Naphthol AS-TR phosphate (Figs 1 and 2) delivered high amounts of stain, e.g. in the aleuron cells, most of the nutritional cells and in the rhizodermis, cortical and endodermis cells of mustard embryos. Similar findings were obtained with the AS-MX, AS-E, AS-GR and AS-LC type substrates and with 1-naphthyl phosphate, whereas naphthol AS--BI (Fig. 3), and especially 5-bromo-4chloro-3-indoxyl phosphate and menadiol diphosphate were hydrolysed more slowly or not at all. In contrast to the generally species-independent activities of non-specific acid phosphatases and the widespread occurrence of these enzymes, exoglycosidases such as a-D-mannosidase, a-D-galactosidase, acid ~-D-galactosidase and Nacetylglucosaminidase showed interspecies differences, were often limited to special cells and had a certain activity ratio. Cell-specific localization was also seen for the non-specific esterases. The indigogenic or azoindoxyl methods for a-Dgalactosidase, acid ~-D-galactosidase and a-Dmannosidase always delivered higher amounts of more precisely localized stain than procedures based on 1-naphthyl or naphthol AS-BI glycosides or the tetrazolium techniques. However, this last substrate type was best suited for O-N-acetylglucosaminidase. In comparison, R-D-galactosidase (Figs 4 and 5) was the most active exoglycosidase and generated large quantities of indigo in the aleuron and nutritional cells proper as well as in the embryonic rhizodermis and cortical cells and in the vascular elements primarily of alfalfa and dover; O-D-galactosidase and a-D-mannosidase yielded smaller amounts of indigo reaction product. Far less stain was generated by these glycosidases in the nutritional and embryonic tissue of the other seedlings. N-acetylglucosaminidase was only detectable in some nutritional and embryonic cells of clover (Figs 6 and 7). Non-specific esterases could best be demonstrated with 1-naphthyl acetate; far lower amounts of stain were generated when using 5-bromo-4-chloro-3indoxyl acetate or the 5-bromo-derivative and still
GOSSRAU less with naphthol AS-D and indoxyl acetate as substrates. In clover and sojbean nutritional tissue, very active non-specific esterases (Fig. 8) and a-D-mannosidase were present in cells around the vascular elements. In contrast to the other cell types, all hydrolases produced different amounts of stain in the vascular elements (Figs 9 and 10) of the nutritional and embryonic tissue. The intracellular localization of the stain generated by the phosphatases, glycosidases and non-specific esterases appeared to be identical in the nutritional and embryonic cells of one single seedling species, but showed interspecies differences. For example, in chick pea nutritional tissue (Figs 11 and 12) the final reaction product of all enzymes was generated in the peripheral cytoplasm. Reaction product was also generated by the glycosidases and non-specific esterases in mustard, lucerne and sojbean nutritional and embryonic cells. Acid phosphatase exhibited dual localization (c.f. Figs 1 and 2), its reaction product also appearing in the globoids of protein bodies. All the hydrolases investigated were present inside the plant seedling cells, whereas non-specific esterases (Fig. 13) and sometimes also non-specific acid phosphatases, and very rarely N-acetyl~D-glucosaminidase, were also seen in the extracellular matrix including the cell walls of the nutritional tissues, and especially the embryonic tissue, of all the seedling species studied. Control media (Figs 14 and 15) for the cerium and lead methods for the phosphatases and oxidases stained the globoids of the protein bodies. Starch granules (c.f. Figs 11 and 12) visualized by the PAS reaction were free of any enzymaticallygenerated reaction products. In contrast, positive sites revealed with Sudan Black (Fig. 16) contained stain produced by glycosidases, non-specific acid phosphatases and non-specific esterases.
Hydrolases during germination Proteases, ~- and f3-ghicosidases and ~-glucuronidase could not be detected up to day six of germination. The activities of other hydrolases changed, depending on the seed species and enzyme in nutritional and embryonic tissue during the germination process. Phosphatases, glycosidases and non-specific esterases were less active in mustard (Fig. 17) on day four of germination compared to those in alfalfa and sojbean. In sojbean nutritional cells, lipids (Fig. 18) were decreased and occasionally some cells showed an enhanced co-localized non-specific esterase activity (Fig. 19). In contrast, acid phosphatase activity increased in the nutritional cells of cotton cotyledons. The activities of the other two groups of hydrolytic enzymes did not appear to change.
Catalytic histochemistry of acid a n d neutral hydroLases in plant seedlings
487
Fig. 12 see Fig. 11 but non-specific acid phosphatase. Figs 13, 14, 15. Mustard seedling on germination day 0. Arrows reaction producL Fig. 13. Cell wall staining t~y non-specific esterases between embryonic cortical cells. Figs 14 and 15. Cotyledon. Fig. 14. Staining of globoids by lead ions. Fig. 15. see Fig. 14 but visualization with cerium ions. Fig. 16. Lipid staining (arrows) by Sudan Black in sojbean nutritional cells on day 2. Fig. 17. Mustard seedling on germination day 4 with decreased amounts of stain (arrows) generated by non-specific phosphatase. R = rhizodermis cells, C = cortical cells. Figs 18 and 19. Sojbean cotyledon with nutritional cells on day 5 of germination. Fig. 18. Visualization of decreased quantities of lipids with Sudan Black (arrows). Fig. 19. Increased staining intensity in certain nutritional cells (arrows) by non-specific esterase. Primary magnification of all figures, x430.
Discussion
Germination of various plant seeds is accompanied by the presence of certain, but not all, acid and neutral hydrolases for which histochemical m e t h o d s
exist; w h e n a hydrolase is present, it occurs mostly in both nutritional and embryonic tissue. The histochemical absence, but biochemicallyk n o w n presence of proteases (Matile, 1978; Bhatna-
488 gar & Sawhney, 1981; Bewley & Black, 1983; Libbert, 1987) in those tissues with high concentrations of proteins, e.g. cotton cotyledons and embryos (Vigil et al., 1989), which are said to be normally degraded by proteinases or endopeptidases and to a lesser extent by exopeptidases during germination to nourish the embryo and for other purposes (Bewley & Black, 1983; Fincher, 1989), might be explained by methodological reasons. For example, the proteinases are relatively sensitive to fixation and the diazonium salts used in simultaneous azo-coupling methods (Lojda et al., 1979, 1991). Possibly the wrong proteases were also analysed. Furthermore, endogenous protease inhibitors may be responsible for the discrepancies of histochemical and biochemical data: Such inhibitors are known to be present in plant cells during germination (Bewley & Black, 1983). Another problem to be resolved is whether proteases and their respective inhibitors occur in the same cells or cell compartments. Recent fluorometric assays of homogenates of fresh cotyledon or endosperm using the same substrates as used in histochemical determinations, have shown that in the absence of inhibitory diazonium salts Alaand CBZ-Ala-Arg-Arg naphthylamines are hydrolysed in mustard, lucerne, sojbean, clover and Adzuki beans during germination (unpublished observations). Similar methodological reasons might explain why some glycosidases, e.g. ~- and fi-glucosidases and ]3D-glucuronidase, could not be detected. They have been reported to be present in seedling tissues, because of the presence of ~- and ~-linked glucose and glucuronic acid in various glycoproteins or other glycocomponents and the participation of R-D-glucosidases in starch mobilization (Bewley & Black, 1983; Libbert, 1987). In contrast, assays with substrates for non-specific acid phosphatases revealed high overall activities in all plant seedlings examined. However, glycosidases and non-specific esterases were only highly active in some species. Moreover, acid phosphatase showed a dual localization, i.e. in protein bodies (Matile, 1969) and outside them. Since the same protein bodies contain phosphate groups, as revealed by the lead and cerium control media for phosphatases and oxidases (Gossrau, 1990), they may represent phytin (phytate, myoinositol-phosphate)-containing globoids, the common phosphate source also of germinating seeds (Bewley & Black, 1983). Therefore, the staining generated in globoids might be produced by phytases; normally, 2- and 6-phytases release inorganic phosphate from phytin and may co-operate with other phosphatases associated with myoinositol-phosphate metabolism and capable of hydrolysing non-specific phosphatase substrates (Loewus & Loewus, 1983). The absence of other
GOSSRAU hydrolases which should be present in the globoids (Bewley & Black, 1983; Loewus & Loewus, 1983) might be explained by their comparatively lower activity in these structures. This activity is abolished by the inhibitory effects of formaldehyde and auxiliary agents, e.g. diazonium salts and iron ions. Outside the protein bodies with globoids, but inside the respective cells, the staining produced by the acid phosphatase assays might be actually caused by this enzyme, as is the staining by the positively reacting glycosidases and non-specific esterases. Although it is not possible for methodological and optical reasons to determine the exact intracellular localization of all of these hydrolytic enzymes, they might be associated with lysosomes, dicytosomes of the Golgi apparatus, and possibly with the endoplasmic reticulum in the nutritional and embryonic cells and tissues, and in plasmodesmata of the embryo (Matile, 1969; Gahan, 1973; Bewley & Black, 1983). The staining observed in the extracellular matrix, primarily with methods for non-specific esterases and acid phosphatases, might be situated between the plasma membrane and the cell walls and in the cell walls themselves (Gahan, 1973, 1984) after ,these enzymes have been secreted by the corresponding nutritional and embryonic cells in contrast to nonsecreted glyeosidases. Their functional role in the extracellular matrix might be participation in the turnover or dissolution of cell-wall material or other compounds during germination (Chrispeels, 1976; Labavitch, 1981; Bewley & Black, 1983). However, for these processes, active glycosidases, which we could not demonstrate, would also be necessary. Inside the nutritional cells, the non-secreted hydrolases may be involved in autolysis, i.e. the breakdown or hydrolysis of certain macromolecules like glycoproteins, galactomannans and glyco- and phosphocomponents in glyco- and phospholipids. The non-specific esterases might act as acid lipase in lipolysis in order to nourish the growing embryo and for other purposes (Bewley & Black, 1983; Libbert, 1987; Fincher, 1989). Further, the intracellular hydrolases might possibly control the secretion of non-specific esterases and acid phosphatases. Less probably, the intracellular (non-secreted) hydrolases participate in heterolysis of endocytosed material, since endocytosis is said to be generally rare or even absent in plant nutritional and embryonic cells during germination (Libbert, 1987). Altogether, the non-specific acid phosphatases, glycosidases and non-specific esterases visualized in the present study should take part in intracellular and extracellular (matrix) digestion or hydrolysis processes (Gahan, 1973). In contrast to all other cells in nutritional and embryonic tissue, at least some vascular elements have a constant high hydrolase activity. It is possible
Catalytic histochemistry of acid and neutral hydrolases in plant seedlings that these heavily reacting elements represent companion cells or procambium cells which can hydrolyse a broad range of non-specific acid phosphatase and exoglycosidase substrates. Since the germination period coincides with the differentiation of vascular elements, which includes partial or total physiological cell death and reorganization as well as degradation processes, these enzymes might be involved in those events (Gahan, 1973; Evert, 1977). In conclusion, existing histochemical methods for non-specific phosphatases, non-specific esterases and glycosidases, and newly-developed procedures for glycosidases using indoxyl substrates have revealed the presence of these hydrolases in many germinating plants; however, proteases and other glycosidases were not found with these techniques. In the plant cells studied, the enzymes were either species-dependent or independent and were always present in both the nutritional and embryonic tissue. The most regular localization was found in differentiating vascular elements of the nutritional tissue. Although the exact intracellular and extracellular localization was not determined in this study, these acid hydrolases might be involved in phytin, glycoprotein, phospho- and glycolipid as well as in lipid metabolism and digestion processes respectively.
Acknowledgements I thank Ms B. Bochow for her expert technical assistance, Ms U. Sauerbier for the photographic work, Ms Irene Straatsburg for grammatical corrections and Ms A. Hechel for her patience with the preparation of the manuscript.
References K., I V A N O V , V. B. & H A D O C O V A , V. (1981) Glycosidases in the root tip. In Structure and Function of Plant Roots (edited by BROUWER,R.), pp. 137--9. The Hague, Boston, London: Martinus Nijhoff. BEWLEY, J. D. & BLACK, M. (1982) Physiology and Biochemistry of Seeds. Vol. 2. Berlin, Heidelberg, New York: Springer. BEWLEY, J. D. & BLACK, M. (1983) Physiology and Biochemistry of Seeds. Vol. 1. Berlin, Heidelberg, New York: Springer. B H A T N A G A R , S. P. & S A W H N E Y , V. (1981) Endosperm - its morphology, ultrastructure and histochemistry. Int. Rev. Cytol. 73, 55-t02. C~RISPEELS, M. I. (1976) Biosynthesis, intracellular transport, and secretion of extracellular macromolecules. Annu. Rev. Plant Physiol. 27, 19-38. mKOW, A. & GOSSRAU,R. (1990) Histochemical demonstration of non-specific esterases and non-specific acid phosphatases using menadiol substrates. Acta Histochem. 88, 167-74. BENES,
489
EVERT, R. F. (1977) Phloem structure and histochemistry. Annu. Rev. Plant Physiol. 28, 199-222. rlNCHER, G. G. (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu. Rev. Plant PhysioL Mol. Biol. 40, 305-46. GAHAN~ P. B. (1973) Plant lysosomes. In Lysosomes in Biology and Pathology (edited by DINGLE,J. T.), Vol. 3, pp. 6785. Amsterdam, London: North-Holland. G A H A N , P. B. (1984) Plant Histochemistry and Cytoehemistry. An Introduction. London, Orlando, San Diego, New York, Toronto, Montreal, Tokyo: Academic Press. GOSSRAU,R. (1981) Investigation of proteinases in the digestive tract using 4-methoxy-2-naphthylamine (MNA) substrates. J. Histochem. Cytochem. 29, 464-80. GOSSRAU, R. (1990) Cerium methods for the light microscopical demonstration of phosphatases and hydrogen-peroxide-generating oxidases in plant tissue. Trans. R. Microsc. Soc. (New Series) 1, 529-32. GOSSRAU, R. & L O J D A , Z. (1989) Histochemical detection of a-D-galactosidase with 5-Br-4-C1-3-indoxyl a-D-galactoside. Acta Histochem. 85, 213-22. GOSSRAU, R. & L O J D A , Z. (1991) Histochemical detection of c~-mannosidase in animal and plant tissue. Histochem. J. 23 (in press). GOSSRAU, R., L O J D A , Z. & S T O W A R D , P. J. (1991) Glycosidases. In Histochemistry: Theoretical and Applied (edited by S T O W A R D , P. J. & P E A R S E , A. G. E..)/ V o l . 3, 4th edn. pp. 241-79. Edinburgh, London: Churchill Livingstone. LABAVlTCH,J. M. (1981) Cell wall turnover in plant development. Annu. Rev. Plant Physiol. 32, 385-406. LIBBERT, E. (1987) Lehrbuch der Pflanzenphysiologie. 4. Aufl. Stuttgart, New York: Fischer. L O E W U S , F: A. & L O E W U S , M. W. (1983) Myo-inositol: Its biosynthesis and metabolism. Annu. Rev. Plant Physiol. 34, 137-61. L O J D A , Z., GOSSRAU, R. & S C H I E B L E R , T. H. (1979) Enzyme Histochemistry. A Laboratory Manual. Berlin, Heidelberg, New York: Springer. L O J D A , Z., GOSSRAU, R. & S T O W A R D , P, J. (1991) Proteases. In Histochemistry. Theoretical and Applied (edited by STOWARD,P. J. &PEARSE,A. G. E.), Vol. 3, 4th edn. pp. 281-335. Edinburgh, London: Churchill Livingstone. MATILE,P. H. (1969) Plant lysosomes. In Lysosomes in Biology and Pathology (edited by DINGLE, J. T. & FELL, ~f. B.), V o [ . 1, pp. 406-30. Amsterdam, London: North-Holland. MATILE,P. H. (1978) Biochemistry and function of vacuoles. Annu. Rev. Plant Physiol. 29, 193-213. ROMEIS, B. (1968) Mikroskopische Technik. Mfinchen: Oldenbourg. SANNES, P. L. (1988) The histochemical and cytochemical localization of proteases. Progr. Histochem. Cytochem. 18, 1--48. V I G I L , E. L., F R A Z I E R , L. C. & POOLEY, C. (1989) Effect of drought stress on protein body development in radicles of cotton embryos: A quantitative study with image analysis. Ceil Biol. Int. Reports 13, 47-64. WOHLRAB, F. a GOSSRAU, R. (1991) Katalytische Enzymhistochemie. Grundlagen und Methoden f~r die Etektronenmikroskopie. Jena: Fischer (in press).
