Phosphatases and Differentiation of the Golgi Apparatus

J. Cell Sci. 4, 455-497 (1969) 455 Printed in Great Britain

PHOSPHATASES AND DIFFERENTIATION OF THE GOLGI APPARATUS

MARIANNE DAUWALDER, W. G. WHALEY AND JOYCE E. KEPHART The Cell Research Institute, Tlie University of Texas at Austin, Texas, U.S.A.

SUMMARY Cytochemical techniques for the electron microscopic localization of inosine diphosphatase, thiamine pyrophosphatase, and acid phosphatase have been applied to the developing root tip of Zea mays. Following formaldehyde fixation the Golgi apparatus of most of the cells showed reaction specificity for IDPase and TPPase. Following glutaraldehyde fixation marked localization of IDPase reactivity in the Golgi apparatus was limited to the root cap, the epidermis, and the phloem. A parallelism was apparent between the sequential morphological development of the apparatus for the secretion of a polysaccharide product, the fairly direct incorporation of tritiated glucose into the apparatus to become a component of this product and the development of the enzyme reactivity. Acid phosphatase, generally accepted as a lysosomal marker, was found in association with the Golgi apparatus in only a few cell types near the apex of the root. The localization was usually in a single cisterna at the face of the apparatus toward which the production of secretory vesicles builds up and associated regions of what may be smooth endoplasmic reticulum. Since the cell types involved were limited regions of the cap and epidermis and some initial cells, no functional correlates of the reactivity were apparent. Despite the presence of this lysosomal marker, no structures clearly identifiable as ' lysosomes' were found and the lack of reaction specificity in the vacuoles did not allow them to be so defined.

INTRODUCTION The discriminative localization of enzyme activities may serve as a key to under- standing many of the integrated biochemical processes within the cell. Such intracellular localization is often possible only by cytochemical techniques. Since these techniques are somewhat limited by problems of methodology, the validity of such findings must ultimately be tested by other analytical techniques. The number of instances in which cytochemical results have been supported by biochemical findings is increasing (for example: cytochemically demonstrable mitochondrial ATPase (Lehninger, 1964; Essner, Fogh & Fabrizio, 1965; Roodyn, 1967), certain ATPases of the sarcoplasmic reticulum (Engel & Tice, 1966; Tice & Engel, 1966), microsomal IDPase, GDPase, and UDPase with cytochemical localization in the endoplasmic reticulum (ER) or the Golgi apparatus (Ernster & Jones, 1962; Novikoff, Essner, Goldfischer & Heus, 1962; Novikoff & Heus, 1963; Goldfischer, Essner & Novikoff, 1964), glucose-6-Pase in the ER (Beaufay, Hers, Berthet & de Duve, 1954; Tice & Barrnett, 1962; Terner, Goodman & Spiro, 1965), and lysosomal hydrolases (see dc Reuck & Cameron (eds.), Ciba Symposium on Lysosomes, 1963; de Duve & Wattiaux, 1966; Straus, 1967)). Reid (1967) has reviewed a number of membrane-associated 456 M. Dauwalder, W. G. Whaley and J. E. Kephart enzymic reactivities. The biochemist can characterize the enzyme system or systems of given cell types or of cellular components which can be isolated in reasonably clean preparations. The enzyme cytochemist, often unable to define the enzyme reaction specifically, can study intracellular localization of activity in cells in situ. The cytochemical approach finds particular use in the study of differentiation, where the structural integrity of the cell or tissue system is of major importance and functional diversity is developing. The objective of this study was to determine cytochemically the localization of certain phosphatase activities associated with the Golgi apparatus (Novikoff & Goldfischer, 1961; Goldfischer et al. 1964 and others) in the root tip of Zea mays where morphological sequences of differentiation of the Golgi apparatus for participation in secretory activity have been established. The essentially linear arrangement of cells in this system has allowed close correlation of the cytochemical results with development of functional specialization in three different secretory cell lineages. The enzymes studied were inosine diphosphatase (IDPase), thiamine pyrophosphatase (TPPase), and acid phosphatase (Acid Pase). It will be shown that the Golgi apparatus of most tissue types shows reactivity for IDPase. It will also be shown that the morphological specialization of the Golgi apparatus for participation in the production of a largely carbohydrate secretory product in the root cap, the epidermis, and the developing sieve tube elements is paralleled by localization of a particular IDPase activity within the apparatus. This IDPase activity appears to relate to the differentiation of the apparatus for polysaccharide synthesis. Additionally it will be shown that in this system TPPase activity is a generally specific though not a universal 'marker' for the Golgi apparatus. Notably in regions of the root cap, even though they are characterized by secretory activity, the Golgi apparatus did not show TPPase localization. In a limited number of cell types, reactivity of Acid Pase was found predominantly at the face of the Golgi apparatus most closely associated with secretory vesicles. This observation relates to possible lysosomal activity in plant cells. A report on this work has been published previously in abstract form (Dauwalder, Kephart & Whaley, 1966).

METHODS AND MATERIALS Methods for the cytochemical studies Seeds of a hybrid strain of Zea mays were germinated and grown in moist filter paper at a temperature of approximately 24 °C. After 6 days, the seedlings were removed and central longitudinal slices (usually less than 0-5 mm thick) of the first 3-4 mm (longer than that indicated in Fig. 1 so that all manipulations could be done without damaging the tip) of several root tips were fixed in the following fixatives: 4% formaldehyde in o-i M phosphate buffer, pHy-8; 4% formaldehyde in o-i M cacodylate buffer, pH 7-2; 4% glutaraldehyde in o-i M phosphate buffer, pHy-8; 4% glutaraldehyde in o-i M cacodylate buffer, pH 7-2. The fixatives contained added trace amounts of calcium chloride. Preliminary attempts to use hydroxyadipaldehyde Golgi differentiation and phosp/iatases 457 fixation for these experiments showed modification of the morphology of the Golgi apparatus and other organelles and very limited indications of reaction product. Fixation was at room temperature for at least an hour, and then overnight in a refri- gerator (about 4-5 °C). The samples were then washed in rapidly running water for 3Hh. The reaction mixtures for the IDPase and TPPase were made up according to Novikoff & Goldfischer (1961):

IDP (Sigma Chemical Co.: o-oi M-O-02 M) in distilled water, or TPP (Sigma Chemical Co.: o-oi M-O-03 M) in distilled water i-o ml Distilled water 0-4 ml TrK-maleate buffer, 0-2 M (pH, see below) 2-0 ml 1 % Pb(NO3)2 o-6 ml 0-025 M MnCl2 i-o ml In order to maintain a pH of 7-2 for the final reaction mixture, im-maleate buffer of pH 7-35 was used. Reactions run with the buffer at pH 7-2 or the buffer adjusted to give a final reaction mixture of pH 7-2 gave similar results. Controls were run on all samples by substitution of distilled water for the substrate. Both MnCl2 and MgCl2 were tested as activators. Novikoff et al. (1962) suggested some superiority of MnCl2 at least for the ER and it proved to be slightly better in these experiments; hence it was used in the experiments from which data are reported. All reaction mixture solutions were made up fresh the day the experiment was run and centrifuged prior to use. Incubation was carried out in a water bath at 39 °C for 45 min. In the initial experiments, samples of Helix aspersa ovotestis were run in parallel with the plant material and gave results similar to those reported by Meek & Bradbury (1963). The acid phosphatase medium contained 10 ml 0-05 M acetate buffer, pH 5-0; 13-25 mg Pb(NO3)2, and 30-6 mg sodium /?-glycerophosphate (Sigma Chemical Co.). The mixture was incubated overnight at 37 °C, filtered, and readjusted to a pH of 5-0. Samples were reacted in the mixture for 70 or 90 min at 39 °C. The results were not entirely consistent, but except for a slight variation in the final pH, the procedures were performed in the same way each time. Controls were run in the same mixture minus the substrate. Initial experiments were run on formol-calcium-fixed material, and although evidence of the reaction similar to that to be reported here could be seen, the preservation of the tissue was poor. Following the enzymic incubations, the samples were rinsed in buffer and post-fixed for 4 h in 2% OsO4 made up in the same buffer as the initial fixative, either on ice or at room temperature with comparable results. They were then rinsed again in buffer, dehydrated in graded ethanol, changed into 100% acetone, and embedded in Epon- Araldite plastic stock number I (Mollenhauer, 1964). Sectioning was done with diamond knives on Porter-Blum or LKB microtomes. Sections were observed without post-staining or after post-staining with uranyl acetate and lead citrate, and electron- microscopic investigation was done on RCA EMU3D and 3F microscopes. Both longitudinal and transverse sections of the apical 2 mm including the cap of the 458 M. Dauwalder, W. G. Whaley andj. E. Kephart samples were used to define tissue types and determine the extent of penetration of the reaction mixture. In initial experiments run to determine hydrolysis time, samples for study by light microscopy were treated with ammonium sulphide. Phase-contrast microscopic observation of thick, plastic-embedded sections adjacent to the thin sections for electron microscopy was used as means of verifying the electron-microscopic data. Two or three root tips per experimental run have been checked, and in electron micrographs over 3000 Golgi stacks have been scored for their reactivity. The results of certain preliminary studies, which will not be treated in detail in this paper, are germane to the discussion; a brief description of the techniques is given here. Study of the incorporation of [3H]glucose was carried out with roots with their tips immersed continuously for periods of £, 1 and 4 h, in D-glucose-i-3H (New England Nuclear Corporation, 0-25 mC, 0-0825 mg in 20/tl of dilute salt solution). The samples were washed, fixed with the glutaraldehyde-osmium procedure and processed for electron microscopy as described above. Thick sections (1-2/t) of material freshly embedded in plastic were stained by the PAS method (15-min hydrolysis in 0-5% periodic acid; 2omin in Schiff's reagent; metabisulphite washes— all at room temperature), stripped with Kodak AR-10, and developed with Kodak D-19. For brightfield photography a dark red filter was used. Adjacent thin sections for electron microscopy were coated with Ilford L-4 emulsion and developed with Kodak Microdol-X. Various tritiated amino acids were also used. The silver/PAS reaction was performed on fixed, embedded material according to the procedure of Bryan (1964) using a 15-min hydrolysis in 0-5% periodic acid at room temperature and treatment with the silver reagent for 7^ h at 40 °C.

The experimental system The first 2-0 mm of the primary root of Zea mays, including the root cap, encom- passes a region in which extensive tissue differentiation takes place, in one direction in the root proper and in a different pattern in the cap (Fig. 1). The initial cells of both the root and the root cap are characterized by relatively few small vesicles associated with the cisternae of the Golgi apparatus. In these cells, for

Fig. 1. The drawing is of a longitudinal section of a root tip of Zea mays. The cross- section is a light micrograph of a i-/t plastic-embedded, PAS-stained section just slightly basipetal to the position indicated. The cross-section was from a typical 'central longitudinal section' and is representative of the tissue thickness used for the enzymic reactions. Fig. 2. Golgi apparatus of cap cells in successive stages of the development of secretory functioning and its apparent cessation. KMnO4 fixation, x 21000. A, Golgi apparatus of a cap initial prior to any conspicuous involvement in secretory vesicle formation; B, Golgi apparatus of a cell toward the periphery of the mid-cap about at the initiation of vesicle formation; c, Golgi apparatus of an outer cap cell showing typical' irregularly ellipsoidal' vesicle formation; D, Golgi apparatus of a free outer cap cell showing the form of the apparatus after major vesicle production has ceased. Cells representing the progressive stages of development between B and c have arbitrarily been termed peripheral mid-cap cells (see text). Golgi differentiation and phosphatases

Xylem element (Fig. 8) Endodermis Pericycle Phloem (Figs. 6, 6Aand 7)

Stele Cortex (Figs. 5 and 5A) Epidermis (Figs. 4 and 4A) Metaxylem element

Apical initials [root proper]

Cap initials (Fig. 2A) Mid cap (Fig. 2B) Outer cap (Fig. 2c) Free outer cap (Fig. 2D) 1 Cap region producing spherical V secretory vesicles (Figs. 3 and 3A) 460 M. Dauwalder, W. G. Wlialey and J. E. Kepliart which no secretory functions have been defined, the Golgi apparatus is assumed to be the least ontogenetically differentiated of those of concern here. In the cap, the central region subjacent to the apex proper is composed of cap initial cells which divide to give rise to additional cap cells. In this region there are none of the large, Golgi apparatus secretory vesicles (Fig. 2 A). Towards the outer cap there is a progressive build-up of secretory-vesicle production, until the outer cells have large numbers of characteristic irregularly ellipsoidal secretory vesicles (Figs. 2B, 2c). The secretory product is predominantly carbohydrate (see below). The cells at the extreme edge of the cap come free from the cap proper and may remain for a time embedded in the secreted layer. In these cells the production of large secretory vesicles ceases (Fig. 2D), and the cells become highly vacuolated. In an area of the cap lateral to the initial cells and near the epidermis the secretory vesicles produced by the Golgi apparatus more closely resemble the spherical secretory vesicles produced by those of the epidermal cells than the ellipsoidal vesicles characteristic of the cap (Figs. 3, 3 A). In the cells of the most apical region of the root proper, termed the 'quiescent zone' (Clowes, 1961), mitotic activity is apparently much lower than in some of the other cell types of the root, as are certain other metabolic activities (Jensen, 1957, 1958). Just basipetal to this region the cells, here termed apical initials, have a higher rate of mitotic activity, but are not yet conspicuously differentiated with respect to tissue type. In these and other cell types during division the Golgi apparatus is active in cell-plate formation. In the cells of the single-layered epidermis large numbers of spherical secretory vesicles are produced by the Golgi apparatus (Figs. 4, 4A). This functional differentiation is first apparent about 10-12 cells from the apex of the root. The cortex, a multilayered tissue between the epidermis and the stele, probably functioning in lateral transport and starch storage, is, in general, not composed of conspicuously differentiated types of cells, nor does the Golgi apparatus produce clearly recognizable secretory vesicles (Figs. 5, 5 A). The innermost part of the cortex is the endodermis. This tissue, with an origin in common with the remainder of the cortex, is in the basipetal regions of these samples, sufficiently differentiated to be considered separately. No apparent structural modification of the Golgi apparatus is seen. Few, if any, starch grains are present. The adjacent internal layer of cells, the pericycle, delineates the stele. The differentiation of two types of vessel elements (the phloem sieve tubes and the large metaxylem elements) can be followed with certainty. The phloem elements which have traditionally been recognized by the early appearance of the sieve plates can in Zea mays be more conveniently delineated in glutaraldehyde-osmium preparations by the presence in their plastids of a crystalline lattice inclusion (Figs. 6, 6A) (Arnott & Dauwalder, in preparation; see also O'Brien & Thimann, 1967). With progressive development of these phloem elements the secretory activity of the Golgi apparatus is markedly enhanced (Fig. 7). Just prior to the extensive vacuolation of these cells to give rise to sieve tube elements, the Golgi apparatus secretory activity ceases and the apparatus resembles those of the vacuolate outer cap except that there are fewer cisternae. The complex development of these cells can be observed in a phloem lineage 10-15 Golgi differentiation and phosphatases 461 long. The cells of the metaxylem elements become much enlarged, both longitudinally and transversely, and highly vacuolate. They can be easily identified at the level of the apical initials. In the stele basipetal to the region in which the youngest phloem cells can be identified, cells of certain files just peripheral to the well-defined metaxylem elements undergo rapid elongation with only a small increase in width. These are presumed to be a type of immature xylem (Fig. 8). The lateral wall thickening characteristic of xylem development is not apparent in any of these primary xylem cells, and no production of secretory vesicles is observed.

RESULTS The results presented here are drawn primarily from the phosphate-buffered glutaraldehyde fixation for the IDPase and Acid Pase and from the phosphate- buffered formaldehyde fixation for TPPase. It is difficult to assess the Golgi apparatus reactivity where the Golgi stacks are apparently not interconnected, are distributed throughout the cytoplasm and show considerable variation in number in different cell types (for example, per section; usually less than 6 are seen in initial cells, 15-30 in secreting epidermal cells, and often more than 50 in secreting cap and phloem cells). To provide a relative comparison of reactivity the data have been expressed as percentage of lead precipitate-labelled organelles. The term 'negative' is not meant to suggest complete absence of the enzyme or complete lack of reactivity, but rather, that for a given set of conditions little, if any, lead precipitate was found in less than 5 % of the organelles. In ultrastructural studies serial sectioning would be needed to yield indications of complete negativity. Under certain conditions of substrate concentration and penetration lead precipitate was found in the nuclei and at sites within the vacuole and along the plasma membrane following all of the reactions studied. In these cases the reactivity was most common in cells near the surface of the reacted sample. Whether this rather generalized lead precipitate is indicative of enzyme activity, or is a result of some other factor in the procedure is not known. Except in the case of some vacuolar labelling such precipitate was not considered in terms of either nature or possible significance. Control samples showed little or no lead precipitate in the Golgi apparatus.

Inosine diphosphatase Following phosphate-buffered glutaraldehyde fixation the cap initial cells showed no lead precipitate associated with the Golgi apparatus at any of the substrate concentrations used (Fig. 9). In peripheral portions of the mid cap, precipitate was found specifically localized in 50% to 70% of the apparatus per cell, both at 0-02 M and 0-0167 M substrate concentrations (Fig. 10). In the outer cap cells with the same substrate concentrations about 90% of the Golgi apparatus per cell showed label, probably indicating enzymic activity in all of the stacks. There was notably more label per Golgi apparatus in the outer cap cells than in those of the peripheral mid cap (Figs. 10—12). As the cells are sloughed off, progressively less indication of IDPase was seen in the Golgi apparatus until little, if any, remained. (The free, 462 M. Dauwalder, W. G. Whaley andj. E. Kephart outermost cells are often lost in processing for electron microscopy, and a complete series has not been obtained.) The more apical cells of the root including the presumptive epidermis did not show any significant amount of lead precipitate at any of the substrate concentrations used. In the secreting epidermis (Fig. 13) about 80% of the Golgi apparatus showed labelling at 0-02 M, 67% at 0-0167 M, and about 25% at o-oi M. Interphase cells and cells in division of the secreting epidermis were consistently highly labelled at both higher substrate concentrations. The epidermis was the only tissue studied that showed appreciable labelling at the o-oi M substrate concentration. The appearance of the lead labelling in the Golgi apparatus of the epidermis coincided with differentiation for secretion.

Table 1. Occurrence and substrate concentration dependence of the IDPase reaction in the Golgi apparatus following phosphate-buffered glutaraldehyde fixation.

Substrate concentration

Cell type o-oi M 0-0167 M

Cap Initials — — — Peripheral mid-cap cells* — + + + + + + Outer cap cells* — + + + + + + + + Root Quiescent zone and apical initials — — — Epidermis prior to secretory activity — — — Epidermis* ++ + + + + + + + Cortex — — — Endodermis — — + + Pericycle — — + Differentiating phloem sieve tubes* — + + + + Phloem companion cells — — + Xylem elements peripheral to metaxylem — — + + Metaxylem elements — — — Central stele — — — • The most consistently highly labelled cell types are in general those for which a well-defined secretory function has been established.

The Golgi apparatus-of the differentiated cortical cells was generally negative at 0-02 M (though in one sample some precipitate was observed). At lower concentrations both the interphase and dividing cells were consistently negative (Fig. 14). The endodermal cells showed lead precipitate in the Golgi apparatus at 0-02 M (Fig. 15), but were negative at lower concentrations. The positive reactivity at 0-02 M occurred with consistency in all cells of the endodermis as viewed in cross-section—an indication of adequate diffusion of the reaction mLxture with respect to demonstration of enzyme activity in the Golgi apparatus. The differentiating elements of the phloem showed a striking reaction at 0-02 M (Figs. 16, I6A), both in the number of Golgi apparatus showing label and in the Golgi differentiation and phosphatases 463 amount of lead per apparatus. A reinvestigation of differentiating phloem showed a very light reaction at 0-0167 M. The exact point in the developing phloem cells at which this activity builds up has not yet been adequately determined, but marked enzymic reaction was found concomitant with demonstrable secretory functioning. In the basipetal cells where the Golgi apparatus is inactive in secretion, the apparatus was negative at 0-02 M. In cross-section the phloem companion cells can be delineated, and at 0-02 M the Golgi apparatus in them showed some low activity as did those of the pericycle and some of the outer stelar cells. At all lower substrate concentrations these tissues were negative. The differentiating elements of the large metaxylem vessels were negative at all concentrations, as were the cells of the central area of the stele. In the immature xylem elements, which are located peripherally to the metaxylem in the stele, a consistent, positive reaction for IDPase in the Golgi apparatus was observed only at 0-02 M (Fig. 17). The reactivity of different cell types at various substrate concentrations is shown in Table 1. The reaction for IDPase following phosphate-buffered formaldehyde fixation is considerably different from the results obtained following glutaraldehyde fixation. Even at low substrate concentrations almost all of the Golgi apparatus of all the tissue types, excepting only the undifferentiated regions of the apex proper and the cap initials, showed a positive reaction. Except for the positive IDPase reaction in the Golgi apparatus of the cap cells (Figs. 18, 19, 19A and 20) which are in general negative for TPPase (Fig. 21,21 A), these results resemble those with TPPase (Table 2) and will not all be illustrated separately.

Thiamine pyrophosphatase Following phosphate-buffered formaldehyde fixation, there is a consistent reactivity of the Golgi apparatus for TPPase in all tissue types at all substrate concentrations (Figs. 22-30) except in cells of a limited area at the very apex of the root, perhaps equivalent to the quiescent zone and including the epidermal initials, and the major portion of the cap. This reactivity was observed in most, if not all, of the Golgi apparatus per section and was seen in at least several cisternae per apparatus. By comparison with the consistent distinctive labelling shown in other cell types, the cap initials have been classified as negative to low in activity as have the mid-cap cells. Only at 0-03 M substrate concentration could reactivity be determined in these cells, but it was limited in extent and not consistently found. In the cap initials the Golgi apparatus occasionally showed what appeared to be cisternal precipitate; in large areas of the mid cap no reaction product was found in the Golgi apparatus. A small number of Golgi apparatus could be found in this area and in the cap adjacent to the epidermis which exhibited lead precipitate not in the cisternae generally but in a few of the small vesicles associated with the distal cisternae (see Acid Pase for definition), or at the juncture between the cisterna and the expanding incipient secretory vesicle (not illustrated). Areas of the outer cap seem to show some Golgi apparatus reaction for TPPase at the highest substrate concentration; however, in this region there is an extremely 464 M. Dauwalder, W. G. Whaley and J. E. Kephart high precipitation of lead throughout the ground cytoplasm not characteristic of the other tissues, which makes delineation of the Golgi apparatus reaction questionable. At lower substrate concentrations the Golgi apparatus of the outer cap cells were negative or of very low activity (Table 2). No localization of IDPase or TPPase was found in the endoplasmic reticulum of any of the cell types. As will be discussed, following glutaraldehyde fixation the Golgi apparatus-TPPase reaction is generally not observed. The diffuse reaction over the nucleus was found, but the cytoplasm was negative except at the highest concentration used (0-03 M). In the latter case the Golgi apparatus reaction was found only in the outermost cap cells and in some of the epidermal cells.

Table 2. Pliosphatases in the Golgi apparatus by tissue type

IDPase IDPase Acid Functional (gluta- (formal- phos- Tissue state* raldehyde) dehyde) TPPase phatase Cap Initials Cell division — + -to± ± Peripheral mid-cap Differentiating for + + + + + - to ± + + secretion (near epidermis) Outer cap Secretory + + + + + + -to ? Root Quiescent zone and — — - -to± apical initials Epidermis Secretory + + + + + + + + + + + (near apex) Cortex (accumulation of — + + + + + + — storage material)! Endodermis + + + + + + + — Phloem elements Secretory + + + + + + + Peripheral xylem Differentiating for + + + + + + + elements secretion (?) Metaxylem ele- Prior to differentiation — + + + + + + ments for conduction • Recognized1 functional state to which the enzyme activity may relate. f Activities which do not appear to involve the Golgi apparatus directly.

Acid plwsphatase The results of the Acid Pase reaction were somewhat more variable and the samples showed a somewhat higher generalized scattering of lead than in the IDPase and TPPase reactions. Because both the literature and the experience in this laboratory indicate that 'over reactions' frequently obscure specific enzyme distribution, particular efforts were made to limit the reaction to give specific organelle localization. In most cases following phosphate-buffered glutaraldehyde fixation the Golgi apparatus Acid Pase reactivity, when found, was only in or associated with the distal cisternae. (For discussion of the Golgi apparatus, the form and orientation of which is highly variable with respect to organism and cell type, we have adopted the purely morpho- Golgi differentiation and phosphatases 465 logical terms 'distal' and 'proximal' (Grasse, 1957) to refer only to the two opposite faces of the Golgi stack where they can be determined. In this system the organelles do not show a consistent positional relationship to other structures. In secreting cells the cisternae concerned with the final elaboration of the secretory vesicles or granules are here arbitrarily termed distal (for discussion see Whaley, 1966).) The Golgi apparatus-associated-Acid Pase reaction was more limited with respect to tissue type than either the IDPase or TPPase reactions. The reaction was characteristically found in areas of the cap lateral to the initial region and near the epidermis (Figs. 31A-F, 32A-F) and in some mid-cap cells, but not in outer cap cells (Fig. 33). A few of the cap initials showed a low response. It was found consistently in secretory epidermal cells (Fig. 34), but only in the more apical ones. The reaction was rarely found in other cells of the root although it was seen occasionally in lateral initial cells adjacent to the quiescent zone (Table 2). A similar pattern of reactivity was found after phosphate-buffered formaldehyde fixation and after formol-calcium fixation, although the tissue preservation was not good enough to delineate the specific relationship of the lead precipitate to the Golgi apparatus in the latter case. In a few experiments fine granular precipitate appeared in the nuclear envelope and endoplasmic reticulum and occasionally in the proximal cisternae of the Golgi apparatus. Where this reactivity pattern was observed in epidermal cells in which the proximal and distal faces are clearly distinguishable the reaction associated with the distal cisternae was not found. There was also a fine scattering of lead throughout the cytoplasm and in both mitochondria and plastids. Factors in technique may be responsible for this distributional variation.

[3H]glucose incorporation and PAS staining The radioautographic data presented are based on visual estimation of grain den- sities and consideration of the relative sizes of the cell types studied. Only limited grain counts were made. The incorporation of glucose by the nuclei and nucleoli will not be discussed. Following 30 min in a solution containing D-glucose-i-3H, the labelling of the cap initial cells and of the cells of the apical region of the root was quite low. The youngest mid-cap cells did not show a much greater labelling of the cytoplasm in general, but they did have heavy accumulations of label over the starch grains. In the central mid-cap region where the cells are conspicuously larger, the grains per cell were more numerous but, per unit area of cytoplasm, there was not a marked increase in labelling. The starch grains were still heavily labelled. In mid-cap cells further displaced from the initials, there was somewhat less labelling of starch grains. Toward the periphery of the mid-cap and in the outer cap cells there was a marked increase in the general cytoplasmic labelling. The starch grains in the outer cap cells did not show labelling nor did the outer cap cells show evidence of a particularly high localization of label at their surfaces (Fig. 35). Electron microscopic radioautography of the mid-cap region showed in addition to the heavy labelling over the plastids that few, if any, grains were found associated with the Golgi apparatus. In the outer cap a high percentage of the cytoplasmic labelling 30 Cell Sci. 4 466 M. Dauwalder, W. G. Whaley and J. E. Kephart was over the secretory vesicles of the Golgi apparatus, and a few of the grains in the secreted material just outside the plasma membrane (Fig. 36). By the standard method the secreted material is highly PAS-positive, and with the PAS/silver method the secreted material, material in the secretion vesicles, and some material in the Golgi apparatus is reactive (Fig. 37). The epidermal cells which had become functional in secretion were heavily labelled. The onset of this activity can be readily distinguished in light microscopy preparations. In addition to heavy cytoplasmic labelling there is a dense band of label along the outer surface of these cells, i.e. the root surface (Fig. 35). Electron microscopic analysis has shown a situation comparable to that in outer cap cells, i.e. a high predominance of the cytoplasmic label localized over the secretory vesicles of the Golgi apparatus. Here, however, there is heavy labelling of surface material immediately adjacent to the plasma membrane (Fig. 38). Again by standard methods the secreted material is PAS-positive, and the PAS/silver method shows reactivity additionally in the secretion vesicles with very little reactivity in the Golgi apparatus. The cortical and stelar cells showed, in general, a higher grain density per unit cytoplasm (excluding the plastids) than did the cap initials and the most apical cells of the root, but were distinctly less highly labelled than are the outer cap and epidermal cells. Even the most apical cortical and metaxylem cells showed labelling of starch in the plastids (see Fig. 35). The other stelar cells did not show much starch labelling. The only cell type showing a particularly high general cytoplasmic glucose incorporation in the stele was the developing phloem. The labelling was not quite so high as that seen in the outer cap and epidermal cells, but was significantly higher than that in the other cell types (Fig. 39). In general the cell walls were distinctively outlined by the silver grains. Cell plates showed, however, higher grain density than was seen in the general wall pattern and much higher incorporation than the cytoplasmic matrix of dividing cells. A particularly interesting incorporation pattern is seen in dividing epidermal cells. With the 30-min incubation time, in cells fixed in telophase and late anaphase there is a heavy labelling of the cell plate while the dense band of labelled material at the root surface is lacking (Fig. 40A, 40B). In these cells the secretory vesicles going into the plate are morphologically similar to those normally secreted at the cell surface (Fig. 41). After 1 h in pHJglucose the tissue patterning was not markedly different, except that the outer root cap cells showed a heavy band of label at the cell periphery and the entire thickness of the layer of material coating the epidermis was labelled. After 4 h, labelling differences among the tissues were less distinctive. The secretory materials of the cap and epidermis were highly labelled, and there was a heavy scattering of label in the extracellular slime surrounding the cap. The starch grains of the enlarged middle cap cells and outer cap cells were not labelled. The most conspicuous tissue difference was the low amount of incorporation in the very apex of the root, even lower than that of the adjacent cap initials. Golgi differentiation and phosphatases 467

DISCUSSION Fixatives, substrate concentrations, and buffers To interpret the results presented and assess their comparability with results reported elsewhere in the literature, the dependence of the final amount and patterning of reaction product on the fixative and buffer used must be considered. Lead precipitate indicating IDPase or TPPase activity is dependent both on the aldehyde used (Sabatini, Bensch & Barrnett, 1963; Goldfischer et al. 1964) and the length of the fixation period. In general, TPPase activity is not found following glutaraldehyde fixation. However, this fixation dependency could be modified by varying the level of substrate used in the reaction mixtures. For example, some reactivity was found in the Golgi apparatus following glutaraldehyde fixation with the 0-03 M TPP substrate but not with lower concentrations. Poux (1967) has reported TPPase activity in the Golgi apparatus of the cucumber root epidermis (protoderm) following 45 min fixation in glutaraldehyde using a final substrate concentration of 0-004 M (equivalent to adding 0-02 M substrate in making up the reaction mixture). It is not yet possible to determine the extent to which positive indications of TPPase following glutaraldehyde (Tice & Barrnett, 1963; Osinchak, 1964, 1966; Lane, 1968) are tissue or species specific or are due to procedural variations. In the system studied here and with the procedures used, reaction product indicating IDPase was generally present in the Golgi apparatus after formaldehyde fixation, whereas after glutaraldehyde fixation the distribution of reaction product appears quite specific with respect to both tissue and developmental stage. The IDPase reactivity was obtained after overnight fixation in glutaraldehyde, whereas in many cases 60- 90 min fixation markedly reduces the amount of demonstrable IDPase reactivity (Goldfischer et al. 1964). Extreme sensitivity to very short-term fixation has been demonstrated in some systems (Carasso, Favard & Goldfischer, 1964). The presence of the IDPase reaction was, with a few exceptions, correlated in the system with an observable morphological development. Additionally, the tissue dependence (epidermis > cap > phloem) of this localization on concentration of substrate seems to be correlated with a similar rank order of the reactive tissue types in apparent incorporation of [3H]glucose. The possibility that these patterns in the cytochemical localization of the reaction product may represent some form of fixation 'artifact' must be allowed, but their reproducibility plus the observable morphological and physiological correlates suggest a clear biological basis for them even if they are 'artifacts'. This situation has recently been argued by Novikoff (1967) and Moses & Rosenthal (1967). Whether such fixation-dependent differences in cytochemical patterns are actually indicative of different enzymes remains to be determined by more direct chemical methods. The suggestion that differential fixation by formaldehyde and glutaraldehyde may delineate tissue specific ATPases has been made by Torack (1965). Similarly, in the case of the IDPase reaction studied here, it would appear that the differential fixation has perhaps allowed the demonstration of a specific nucleoside diphosphatase, either one which is differentially sensitive to the two aldehydes or one of particularly 30-2 468 M. Dauwalder, W. G. Whaley andj. E. Kephart high reactivity. With 45 min glutaraldehyde fixation Poux (1967) reported reactivity to additional nucleoside phosphate substrates in the Golgi apparatus of the cucumber root epidermis, but the substrate giving the most dense reaction precipitate was IDP. The extent to which effects of fixative and of substrate concentration are interrelated is difficult to assess. The most consistent IDPase reaction following glutaraldehyde was found in three tissues showing secretory differentiation of the Golgi apparatus. At the highest IDP substrate concentration some reactivity was found in a few additional tissue types for which no clear functional correlates have been established. Whether this reactivity is similar to that of the secretory tissues or to the more generalized one following formaldehyde fixation is not known. A group of related enzymes each with a particular reactivity for the substrate and with varying fixation ssnsitivity could be involved. The fixative buffer is also a factor in the final appearance of reaction product. In this study the cacodylate-buffered fixations generally showed a higher scattering of lead throughout the cytoplasm. While essentially the same Golgi apparatus activity could be observed, the amount of reaction product was much lower at a given substrate concentration than that in the phosphate-buffered samples. The most consistent results were obtained with phosphate buffer. The fixatives and buffers used here did not give differential cytochemical results with respect to Acid Pase.

The acid phosphatase reaction and the question of plant lysosomes Concern for the presence of acid phosphatase in the Golgi apparatus and its possible relationship to lysosomes was brought into focus in 1958-59 by three papers: one dealing with biochemical identification of an Acid Pase in association with isolated Golgi membranes (Kuff & Dalton, 1959), another with studies of lysosomes as hydrolytic cellular components containing Acid Pase (de Duve, 1959), and the third, a suggestion of an histochemical approach which it was thought might clarify possible in vivo relations of the Golgi apparatus and the lysosomes (Novikoff, 1959). The concept of the lysosome as a cellular component containing a number of hydrolases distinguished by an acid pH optimum and the role of the lysosome in cellular functioning have undergone considerable refinement (de Duve & Wattiaux, 1966), but the transfer of acid phosphatase, which is a principal lysosomal marker, from its probable site of formation in association with the ribosomes to the functional lysosome remains poorly understood. The most frequent suggestions concern 'direct' transfer from the endoplasmic reticulum or transfer 'via' the Golgi apparatus (see Novikoff, Essner & Quintana, 1964; Novikoff, Roheim & Quintana, 1966), and though not unequivocal proof the findings by Novikoff et al. (1964) and Friend & Farquhar (1967) of Acid Pase activity in small Golgi-derived vesicles support the latter contention. The particular functional significance of this enzymic activity in the limited number of instances where it can be defined in the Golgi apparatus per se awaits elucidation. Acid Pase reactivity is not consistently found in Golgi apparatus of cell types characterized by lysosomes nor is it clear whether Acid Pase activity when defined in or associated with the apparatus necessarily indicates that this activity is or will be 'lysosomal'. Golgi differentiation and phosphatases 469 Whether Acid Pase activity is a consistent feature of the functioning of the Golgi apparatus demonstrable cytochemically only in certain phases of activity or whether its presence indicates a specific differentiation of the apparatus for a particular function is not clear. It would appear, however, that the enzyme reaction is correlated in many cases with some function which is predominantly expressed at the distal face of the apparatus. In those tissues where the reaction was commonly observed in the Golgi apparatus, it was limited to the distalmost cisterna or what was possibly associated endoplasmic reticulum (Novikoff's GERL, see below) (Fig. 31 E). A similar reaction, extending over more cisternae but with an obvious predominance at the distal face, has been observed by Pickett-Heaps (1967a) in epidermal cells of wheat roots, and a fairly clear-cut gradient across the apparatus has been observed in TSH-stimulated thyroid cells by Seljelid (1965) and in Euglena by Sommer & Blum (1965). Restriction of Acid Pase reactivity to what is here termed the distal face has been reported by Smith & Farquhar (1966) in rat anterior pituitary cells producing mammotrophic hormone, Friend & Farquhar (1967) in rat vas deferens associated with 'coated' vesicle formation, Frank & Christensen (1968) in guinea pig testis interstitial cells possibly related to autophagy, and Osinchak (1964) in rat hypothalamic neurosecretory cells (although the author calls it proximal, using the term in reference to the nucleus). Novikoff and his associates (Novikoff et al. 1966; Novikoff & Biempica, 1966; Holtz- man, Novikoff & Villaverde, 1967) have found Acid Pase reactivity in what they defined as GERL, a region of the smooth endoplasmic reticulum associated with the distal cisternae of the Golgi apparatus, in liver cells and neurons of spinal and cranial ganglia. Acid Pase has been reported both in the distal cisternae of the Golgi apparatus and in the equivalent of GERL by Hugon & Borgers (1967) in absorbing cells of the duodenal mucosa of the mouse and by Lane (1968) in thoracic ganglionic neurons of a grasshopper. In additional instances involving several types of cells, the reactivity has been found to be limited to one or a few cisternae at one face of the apparatus, but whether these represent the distal face pattern is not clear (Moe, Rostgaard & Behnke, 1965; Osinchak, 1966; Wetzel, Spicer & Horn, 1967). In the latter paper (Wetzel et al. 1967) certain of the illustrations suggest the distal-face reaction. Additionally, their finding of Acid Pase in the Golgi apparatus of early and some late heterophilic leucocytes and not in intermediate stage cells correlates with phosphatases in the granules being elaborated and supports the concept of particular enzyme specialization of the apparatus concurrent with cellular differentiation. In a majority of cell types studied the Golgi apparatus appears to be negative for Acid Pase (Goldfischer et al. 1964; Miller & Palade, 1964) as was true in our experiments. In several cases an Acid Pase reaction can be demonstrated in the apparatus only after the cells have been subjected to abnormal conditions. Here again, the reaction seems to be limited to a single, probably distal, cisterna or shows a gradient (Bertolini & Hassan, 1967; perhaps also Lane & Novikoff, 1965; Holtzman & Novi- koff, 1965). Following absorption of horseradish peroxidase Friend & Farquhar (1967) noted an increase in reactivity of the Golgi apparatus of the rat vas deferens which did not seem to show a clear-cut gradient. In some cases a more general reactivity of 47° M. Dauwalder, W. G. Whaley and J. E. Kephart the cisternae across the apparatus has been reported (Brandes, Buetow, Bertini & Mal- koff, 1964; Jurand, 1965; Poux, 1963a; Seljelid, 1967). The difficulties in precise localization of Acid Pase activity by use of the Gomori technique are widely recognized (see especially Novikoff's papers; Holt & Hicks, 1961a, b). The observations of Seeman & Palade (1967) on Acid Pase localization in rabbit eosinophils have re-emphasized that the intactness of structure, the relative hydration of the matrix, and the possibility of inhibitors must also be considered. However, despite numerous variables, the distal-face patterns are the most consistent in the many experimental materials, both plant and animal, in which Golgi apparatus localization is reasonably clear. While this reactivity appears in some cases possibly to be related to the production of lysosomes, no morphologically distinguishable lysosomes of either primary or secondary types (de Duve & Wattiaux, 1966) are apparent in the root cells studied. (See, however, discussion of vacuolar labelling.) The absence of definable lysosomes in cells showing the distal-face reaction suggests that this Acid Pase is not necessarily incorporated into vesicles identifiable as primary lysosomes. In the present experiment the cell types showing the distal-face reaction were varied, and no common denominator was identified which would permit relating this to a functional activity. Acid Pase activity has been found to be a characteristic of the endoplasmic reticulum of certain Protista (Goldfischer, Carasso & Favard, 1963) and that of certain cell types of higher organisms in abnormal conditions including injury (Holtzman & Novikoff, 1965; Lane & Novikoff, 1965). The possibility that this labelling pattern may be related to a different pattern of lysosomal activity is discussed by some of the investigators named. Acid Pase was not found characteristically in the endoplasmic reticulum of any of the cells in this system except in a limited number of samples where variations in the technique seemed to be responsible. The question of whether plant cells contain lysosomes as do many animal cells is one for which no clear answer is yet available. That no such bodies have been unques- tionably identified in this experimental system does not imply their nonexistence. Poux (1963 a, b, 1965) has described Acid Pase reactivity in aleurone grains of hydrated and germinating plant seed where a considerable breakdown of storage material is occurring. With a combination of cytochemical and isolation techniques, Yatsu & Jacks (1968) have shown that in the cotyledons of cottonseed (and peanut seed, T. Jacks, personal communication) most of the cytochemical reaction for Acid Pase is associated with the aleurone grains. By biochemical assay, they found that the aleurone grain fraction contained 75% of the total protein, 77% of the Acid Pase activity, and 100% of the acid proteinase activity of unfractionated homogenates. They have concluded that with respect to these two enzyme activities and a probable role in intracellular digestive processes aleurone grains resemble animal cell lysosomes. (There are several papers ascribing lysosomal equivalence to the 'sphero- somes' of plant cells. The term 'spherosome' has, however, been variously applied to structures of differing morphology, composition, and development. Most commonly the term has been used to identify 'lipid droplets' or lipid-containing structures. In maize, in both seeds and very young seedlings there are a number of different mem- Golgi differentiation and phosphatases 471 brane-bound storage bodies some of which clearly contain lipid. As do the aleurone grains some of these bodies may also contain hydrolytic enzymes. It would seem that discussion of these structures as lysosomes should await further information. In the root tips studied here these storage bodies are no longer present, and no reaction product was localized in the non-membrane-bound 'lipid droplets' of the cells by any of the procedures used.) Acid phosphatase activity has also been observed in plant cell vacuoles during germination (Poux, 19636), and the general reactivity of vacuoles with cytochemical techniques should be considered. In the present experiments vacuolar labelling was common not only after the reaction for Acid Pase but also after that for IDPase and TPPase independent of fixative or buffer. Routinely, deposition of lead along the membrane or at limited sites along the membrane of some vacuoles within cells of all tissue types was found, especially near the surface of the reacted sample. No apparent reproducible pattern was observed. When seen with the light microscope this reactivity was suggestive of lysosomes as defined cytologically. Poux (1967) has also described plant cell vacuolar precipitate with a number of substrates. The apparent lack of specificity of the vacuolar labelling makes it impossible to interpret this Acid Pase reactivity as a reliable indicator of lysosomal activity. (A range of substrate specificity has been observed in lysosomes of invertebrates (Lane, 1968 and Discussion). That this reactivity can reproducibly be defined in the same membrane-bound, electron- dense structures showing Acid Pase activity makes their equation to lysosomes reasonably certain.) Furthermore, analysis of vacuolating cells of the outer cap, xylem and phloem did not, following the reaction for Acid Pase, show any increase in the precipitate associated with the vacuoles. This would suggest that even were this a reliable localization of Acid Pase, the progress of vacuolation cannot, by this technique, in general be directly correlated with the build-up of that enzyme. Despite the fact that the cytochemically demonstrable vacuolar precipitate may not be a specific enzymic reaction, it could be that plant vacuoles have certain 'lysosomal' activities. De Duve has repeatedly emphasized (see de Duve & Wattiaux, 1966) that vacuoles of various types in animal cells come to contain lysosomal enzymes. Matile (1968) has found acid hydrolases in cell fractions obtained from maize root tips which he inter- prets to be isolated vacuoles or derived from vacuoles. Very little is known about the functional activities of plant vacuoles, but there is increasing evidence from other studies in this laboratory that vacuoles may vary considerably in differentiation and function from one plant cell type to another. In another instance in this study there was evidence suggesting that a clearer interpretation of vacuolar localization might be possible. In the developing phloem cells, both after the phosphate-buffered formaldehyde IDPase and TPPase reactions and after the phosphate-buffered glutaraldehyde IDPase reaction (and discernibly, though perhaps to a slightly lesser extent, in the same reactions after cacodylate buffering), the reactivity of the vacuoles seemed particularly high even when those of adjacent cells were low to negative. In several cases a gradual morphological sequence was followed to the point of formation of a large vacuole, presumably by fusion of smaller vacuoles. As seen in section, these large vacuoles were almost filled with precipitate. This 472 M. Dauwalder, W. G. Whaley and J. E. Kephart sequence is most striking in light microscopy. The observation suggests the progressive development of a particular metabolic function in the vacuole, and that perhaps further study by enzyme cytochemistry could be used to study vacuole differentiation and the possible lysosomal-like activity of plant vacuoles. The question regarding the possible origins of the enzyme or enzymes in the vacuole remains unanswered. In other plant cell structures such as the aleurone grains, the association of acid phosphatase with stored materials presents a picture that is somewhat comparable to that recorded for the polymorphonuclear leucocyte granules by Bainton & Farquhar (1966), for macrophages by North (1966), and for the eosinophil granules by Seeman & Palade (1967). Lytic enzymes immediately associated with stored materials would thus seem to be a common denominator for both plants and animals. In total, the evidence suggests the presence of lysosomal activity as defined in terms of acid phosphatase in several types of plant cells, but it also suggests that in the cells of growing root tips cytologically distinguishable lysosomes do not exist.

TPPase and IDPase reactions following formaldehyde fixation TPPase has been generally recorded by Novikoff et al. (1962), Allen (1963 a), Goldfischer et al. (1964), and others as a consistent marker of the Golgi apparatus. In the current study most of the cell types showed a distinct, consistent TPPase reactivity of the Golgi cisternae, usually apparent in all the Golgi apparatus per section. Such was not found in the most apical cells of the root or in cells of certain regions of the cap. The lack of reactivity in the apical cells for which low over-all rates of metabolic activity have been suggested (Jensen, 1957, 1958; Clowes, 1961) could imply enzyme levels below that demonstrable by these methods. The general lack of reactivity for TPPase in the Golgi apparatus of those cells in the cap which have a highly developed functional activity and IDPase reactivity is more significant. It must be supposed that TPPase activity is either not characteristic of these secretory cell Golgi apparatus, or that if it is present it somehow differs with respect to the reaction used or the fixation from that of other cells. Note of the absence of TPPase activity in some plant cells was made by Novikoff & Goldfischer (1961). Whatever is responsible for the negative results, caution is indicated in considering TPPase to be a ' universal' Golgi apparatus marker. Little if any TPPase labelling of the endoplasmic reticulum was observed in any of the cell types. Such labelling has been reported for a limited number of animal cells (Novikoff et al. 1962; Goldfischer et al. 1964). The distribution of lead precipitate is quite similar for both IDPase and TPPase reactions following formaldehyde fixation. This raises the obvious question of whether two enzymes are actually involved. In the cap, however, IDPase activity can be demonstrated in the Golgi apparatus of cells of the mid-cap and outer cap in which there is no significant TPPase activity. The possible effects of differential fixation cannot be ruled out, but evidence from other laboratories (Allen, 19636) as well as the distribution pattern reported here indicates reaction specificity which distinguishes between the nucleoside diphosphatases and thiamine pyrophosphatase. Either Golgi differentiation and phosphatases 473 TPPase or nucleoside diphosphatase reactivity is a common characteristic of the Golgi apparatus in the plant cell types studied here as is the case with animal cell types (Novikoff et al. 1962). In the material studied here, after glutaraldehyde fixation a particular nucleoside diphosphatase activity has been shown to be localized in the Golgi apparatus of three different cell types concurrent with the development of secretory functioning.

The IDPase reaction following phosphate-buffered glutaraldehyde fixation: differentiated activity of the Golgi apparatus The tissue reactivity for IDPase has both morphological and other correlates which give rise to an hypothesis about its functional significance. The tissues in which a high reactivity for IDPase is consistently found in the Golgi apparatus—the outer root cap, the epidermis, and the phloem—are characterized by specific developmental modification of the apparatus for secretion of a product with a high polysaccharide content. This is indicated by the fact that the material secreted is strongly PAS- positive and that the secretory vesicles of the cap and epidermis show high specificity with the use of the silver/PAS method with electron microscopy (see also, Pickett- Heaps, 19676) and supported by the pattern of uptake of [3H]glucose (see also, Northcote & Pickett-Heaps, 1966). None of the standard cytochemical tests for protein—for example, mercuric bromphenol blue (Mazia, Brewer & Alfert, 1953), low pH fast green—gives a positive reaction, and such protein labels as [3H]proline, [3H]leucine, [3H]histidine, and [3H]tyrosine, though readily incorporated into the cells, do not appear in the secretory product. Whether protein is actually absent, or a small percentage is masked, has not been determined. The evidence indicates that IDPase in the Golgi apparatus appears just prior to, or concurrently with, the morphological modification indicative of secretory vesicle production. When the morphological evidence indicates maximum secretory activity, indications of IDPase in the cisternae are also at a maximum. The appearance of the precipitate in association with the membranes of the cisternae and along the membranes of the separated vesicles in contrast to its absence from the non-membrane- bound surface aggregations of secreted product suggests, in so far as morphological evidence can suggest, that the enzyme is membrane-associated. The plasma membranes of these secreting cells show lead precipitate accumulations after the reaction, but too little is known about the specificity of the reactions and the matter of surface membrane labelling is too complex to permit the attractive conclusion that IDPase has been transferred with incorporation of vesicle membranes into the plasma membrane. In the vacuolated outermost cap cells in which Golgi apparatus have reverted to a compact form, a reduced amount of IDPase, probably residual, is found. A similar pattern is seen in the phloem where the Golgi apparatus are negative for IDPase after the apparent cessation of secretory activity. The IDPase reaction can be considered as a nucleoside diphosphatase reaction with specificity for IDP, UDP, and GDP substrates, and only occasionally responding with ADP or CDP substrates (Goldfischer et al. 1964). It is possible that UDP-type reactions concerned with the synthesis of a polysaccharide component of the secretory 474 M. Dauwalder, W. G. Wlialey and J. E. Kephart material (Northcote, 1964; Leloir, 1964; Nordin & Kirkwood, 1965; Northcote & Pickett-Heaps, 1966) are somehow, either indirectly or directly, demonstrated by the IDPase reaction. This reactivity could, therefore, reflect enzymic differentiation within the Golgi apparatus for polysaccharide synthesis. Support for this hypothesis was obtained from experiments in this laboratory on the incorporation of [3H] glucose which confirm that during the secretory phase the epidermis, the outer regions of the cap, and the phloem are markedly more active in incorporation than are the other tissues studied (except those involved in starch synthesis). Radioautographic observations suggest movement of the glucose label via the Golgi apparatus vesicles into the mass of secretory product which accumulates outside the protoplast. The time relationships of the appearance of label in the cells and in the secretory product of these tissues as well as the degree to which the secretory products are labelled correlate with the enzyme substrate concentration dependence data. Morphological modification for secretory activity, glucose incorporation and IDPase activity seem to be parallel events in the Golgi apparatus in the developmental sequences of three different tissue types even though the time of onset of differentiation and the particular morphological characteristics of the apparatus differ. The concept that the Golgi apparatus is involved in the secretion of and probably the synthesis of polysaccharides in both plant and animal materials has now been adequately supported by several investigators (Northcote & Pickett-Heaps, 1966; Neutra & Leblond, 1966a, b; Deck, Hay & Revel, 1966; Berlin, 1967; Schmalbeck & Rohr, 1967; Barland, Smith & Hamerman, 1968; Wooding, 1968). Northcote & Pickett-Heaps (1966; see also, later Pickett-Heaps papers), from correlated radioautographic and chemical analyses of a similar plant system, have shown that glucose taken in by the root cap cells during secretory functioning moves rapidly into the Golgi apparatus to be incorporated into polysaccharide (probably pectic) material of the secretory vesicles. Localization of the radioautographic labelling precisely at the Golgi apparatus is difficult in cells with generally distributed Golgi apparatus, but a study of many micrographs leaves little doubt about this localization. Neutra & Leblond (1966a) in their study of the specifically positioned Golgi complex in secreting goblet cells of the intestine have proposed a comparable pathway for the incorporation of glucose fairly directly into the Golgi apparatus there to become part of the strongly PAS-positive polysaccharide-protein product. IDPase reactivity has also been reported in the Golgi apparatus of these cells (Otero-Vilardeb6, Lane & Godman, 1964). Barlandeia/. (1968) have demonstrated that [3H]glucosamine incorporated into cultured human synovial cells is rapidly localized over the Golgi apparatus and have presented biochemical evidence that the glucosamine is a relatively specific precursor of the anionic polysaccharide, hyaluronic acid, secreted by these cells into the medium. In all these systems, the very rapid labelling of the Golgi apparatus without evidence of transport of materials from other cellular organelles suggests synthesis in the apparatus per se. Northcote & Pickett-Heaps (1966) have noted three possibly separate pathways of incorporated [3H]glucose: (1) into the secretory material via the Golgi apparatus, Golgi differentiation and phosphatases 475 (2) into the starch of the plastids, and (3) into the cellulose of the wall. The tissue pattern of active incorporation of glucose into starch is of interest here. In the preliminary labelling experiments in this laboratory, those tissues which show the most active labelling of starch—the cortex, the metaxylem elements, the cap initials and the inner portions of the mid-cap—do not show the Golgi apparatus IDPase reaction. Additionally, starch grains have not been demonstrated morphologically or by the PAS reaction in the plastids of either the epidermis or phloem, and although starch grains are conspicuous in the outer portions of the cap in these cells, they are not labelled. This labelling pattern in the cap and an apparent decrease in the total volume of starch from the inner mid-cap cells outward suggests that the starch grains in the outer regions of the cap are preformed storage products, possibly being degraded. However, it could be that variations in size of the metabolic glucose pool or rate factors affecting the incorporated glucose determine the apparent lack of incorporation, as has been suggested by Northcote & Pickett-Heaps (1966). Radioautographic techniques alone are, in this case, insufficient to delineate between sequential events and metabolic variations (see Perry, 1964). The evidence suggests that in this system there is a predominance of one glucose pathway in cells actively engaged in synthesis of starch in the plastids, and predominance of another path in cells actively engaged in synthesis of polysaccharide for secretion. (This study was concerned only with processes of secretion which involve the Golgi apparatus.) In other types of cells where processes of storage and secretion may occur at the same time, a comparable contrast in the pathways of utilization of monosaccharides apparently holds. Neutra & Leblond (19666; see also, Coimbra & Leblond, 1966) from a comparison of the incorporation of [3H]glucose and [3H]galactose in a variety of animal cell types have postulated that whereas glycogen is synthesized outside the Golgi apparatus, the carbohydrate moiety of 'glycoproteins' or 'mucopolysaccharides' destined for secretion is synthesized in the Golgi region. It is perhaps indicative of the known diversity of the Golgi apparatus that this pattern is not clearly indicated for some of the other types of tissues. The IDPase labelling of the Golgi apparatus of the endodermis and the peripheral xylem elements has not been correlated with a detectable secretory functioning. In both these cases IDPase is detectable only at the highest substrate concentration. In neither case is there a notably high cellular incorporation of [3H]glucose. The formation of the cell plate in dividing plant cells involves incorporation of Golgi apparatus-produced vesicles into the plate region. The plate is PAS-positive and shows a higher labelling following the introduction of [3HJglucose just prior to or during cytokinesis than does the remainder of the cell. Evidence of IDPase is, however, lacking except in the case of dividing epidermal cells. In the epidermal cells the vesicles going into the plate have distinctive form and staining characteristics which are comparable to those secreted outside the cell. These results may indicate that the IDPase activity recorded here is associated only with synthesis of some particular polysaccharide component which is formed in relative abundance in the secretory activity of the cap, phloem, and epidermal cells, and in the latter is also moved into the cell plate. 476 M. Dauwalder, W. G. Whaley andj. E. Kephart Attempts to correlate cytochemical and radioautographic results in such a system as the one studied can at best lead to only tentative conclusions. Where such a range of developmental stages is involved—including various phases in mitosis, initiation of tissue differentiation, development of storage materials, and specific differentiation for secretion—there are, of course, wide differences in some of the metabolic activities in the various cells and tissue types. Despite the limitations in the techniques and concern with different metabolic states, however, the data clearly suggest correlation of three developments in the differentiation of the Golgi apparatus—morphological modification, IDPase activity, and synthesis of a polysaccharide secretory product. The authors wish to acknowledge encouragement and critical opinion of Professor Alex B. Novikoff. Thanks are due to Mrs P. Behrens and Mrs J. Zeagler for expert technical assistance and to Thomas P. Leffingwell for electron microscopic radioautographs. This work was supported in part by National Science Foundation Grant no. GB 7218 to Dr W. G. Whaley and by a Faith Foundation Grant to Dr Paul A. Weiss.

REFERENCES ALLEN, J. M. (1963a). The properties of Golgi-associated nucleoside diphosphatase and thiamine pyrophosphatase. I. Cytochemical analysis. X Histocliein. Cytochem. 11, 529-541. ALLEN, J. M. (19636). The properties of Golgi-associated nucleoside diphosphatase and thiamine pyrophosphatase. II. Electrophoretic separation and identification. J. Histochem. Cytocliem. 11, 542-552. BAINTON, D. F. & FARQUHAR, M. G. (1966). Origin of granules in polymorphonuclear leuko- cytes. J. Cell Biol. 28, 277-301. BARLAND, P., SMITH, C. & HAMERMAN, D. (1968). Localization of hyaluronic acid in synovial cells by radioautography. J. Cell Biol. 37, 13-26. BEAUFAY, H., HERS, H.G., BERTHET, J. & DUVE, C.DE(I954). Lesysteme hexosephosphatasique. V. Influence de divers agents sur l'activiteet la stability de la glucose-6-phosphatase. Bull. Soc. Chim. biol. 36, 1539-1568. BERLIN, J. D. (1967). The localization of acid mucopolysaccharides in the Golgi complex of intestinal goblet cells. J. Cell Biol. 32, 760-766. BERTOLINI, B. & HASSAN, G. (1967). Acid phosphatase associated with the Golgi apparatus in human liver cells. J. Cell Biol. 32, 216-219. BRANDES, D., BUETOW, D. E., BERTINI, F. & MALKOFF, D. B. (1964). Role of lysosomes in cellular lytic processes. I. Effect of carbon starvation in Euglena gracilis. Expl molec. Path. 3, 583-609- BRYAN, J. H. D. (1964). A silver-Schiff reaction for cytological studies: light microscope observations. Q. Jl microsc. Sci. 105, 363-366. CARASSO, N., FAVARD, P. & GOLDFISCHER, S. (1964). Localisation, a l'dchelle des ultrastructures, d'activites de phosphatases en rapport avec les processus digestifs chez un Cilie (Campanella timbellaria). J. Microscopie 3, 297-322. CLOWES, F. A. L. (1961). Apical Mcristems. Oxford: Blackwell. COIMBRA, A. & Leblond, C. P. (1966). Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-H3. J. Cell Biol. 30, 151-175. DAUWALDER, M., KEPHART, J. E. & WHALEY, W. G. (1966). Phosphatases and the Golgi apparatus in differentiating cells. J. Cell Biol. 31, 25A-26A. DECK, J. D., HAY, E. D. & REVEL, J.-P. (1966). Fine structure and origin of the tunic of Perophora viridis. J. Morph. 120, 267-280. DUVE, C. DE (1959). Lysosomes, a new group of cytoplasmic particles. In Subcellular Particles (ed. T. Hayashi), pp. 128-159. New York: Ronald Press. DUVE, C. DE & WATTIAUX, R. (1966). Functions of lysosomes. A. Rev. Physiol. 28, 435-492. ENGEL, A. G. & TlCE, L. W. (1966). Cytochemistry of phosphatases of the sarcoplasmic reticulum. I. Biochemical studies. J. Cell Biol. 31, 473-487. Golgi differentiation and phosphatases 477 ERNSTER, L. & JONES, L. C. (1962). A study of the nucleoside tri- and diphosphate activities of rat liver microsomes. J. Cell Biol. 15, 563-578. ESSNER, E., FOGH, J. & FABRIZIO, P. (1965). Localization of mitochondrial adenosine triphosphatase activity in cultured human cells. J. Histochem. Cytochem. 13, 647-656. FRANK, A. L. & CHRISTENSEN, A. K. (1968). Localization of acid phosphatase in lipofuscin granules and possible autophagic vacuoles in interstitial cells of the guinea pig testis. J. Cell Biol. 36, 1-13. FRIEND, D. S. & FARQUHAR, M. G. (1967). Functions of coated vesicles during protein absorption in the rat vas deferens. J. Cell Biol. 35, 357-376. GOLDFISCHER, S., CARASSO, N. & FAVARD, P. (1963). The demonstration of acid phosphatase activity by electron microscopy in the ergastoplasm of the ciliate Campanella umbellaria L. J. Microscopie 2, 621-628. GOLDFISCHER, S., ESSNER, E.&NOVIKOFF, A. B. (1964). The localization of phosphatase activities at the level of ultrastructure. J. Histochem. Cytochem. 12, 72-95. GRASSE, P.-P. (1957). Ultrastructure, polarite et reproduction de l'appareil de Golgi. C. r. hebd. Sianc. Acad. Sci., Paris 245, 1278-1281. HOLT, S. J. & HICKS, R. M. (1961a). Studies on formalin fixation for electron microscopy and cytochemical staining purposes. J. biophys. biochem. Cytol. n, 31-45. HOLT, S. J. & HICKS, R. M. (19616). The localization of acid phosphatase in rat liver cells as revealed by combined cytochemical staining and electron microscopy. J. biophys. biochem. Cytol. 11, 47-66. HOLTZMAN, E. & NOVIKOFF, A. B. (1965). Lysosomes in the rat sciatic nerve following crush. J. Cell Biol. 27, 651-670. HOLTZMAN, E., NOVIKOFF, A. B. & VILLAVEKDE, H. (1967). Lysosomes and GERL in normal and chromatolytic neurones of the rat ganglion nodosum. J. Cell Biol. 33, 419-435. HUCON, J. & BORCERS, M. (1967). Fine structural localization of lysosomal enzymes in the absorbing cells of the duodenal mucosa of the mouse. J. Cell Biol. 33, 212-218. JENSEN, W. A. (1957). The incorporation of C14-adenine and Cu-phenylalanine by developing root-tip cells. Proc. natn. Acad. Sci. U.S.A. 43, 1038-1046. JENSEN, W. A. (1958). The nucleic acid and protein content of root tip cells of Vicia faba and Alliurn cepa. Expl Cell Res. 14, 575-583. JURAND, A. (1965). UltrastnJctural aspects of early development of the fore-limb buds in the chick and the mouse. Proc. R. Soc. B 162, 387-405. KUFF, E. L. & DALTON, A. J. (1959). Biochemical studies of isolated Golgi membranes. In Subcellular Particles (ed. T. Hayashi), pp. 114-127. New York: Ronald Press. LANE, N. J. (1968). Distribution of phosphatases in the Golgi region and associated structures of the thoracic ganglionic neurons in the grasshopper, Melanoplus differ entialis. J. Cell Biol. 37, 89-104. LANE, N. J. & NOVIKOFF, A. B. (1965). Effects of arginine deprivation, ultraviolet radiation, and X-radiation on cultured KB cells. A cytochemical and ultrastructural study. J. Cell Biol. 27, 603-620. LEHNINCER, A. L. (1964). The Mitochondrion. New York: Benjamin. LELOIR, L. F. (1964). Nucleoside diphosphate sugars and saccharide synthesis. Biochem. J. 91, 1-8. MATILE, PH. (1968). Lysosomes of root tip cells in corn seedlings. Planta 79, 181-196. MAZIA, D., BREWER, P. A. & ALFKRT, M. (1953). The cytochemical staining and measurement of protein with mercuric bromphenol blue. Biol. Bull. mar. biol. Lab., Woods Hole 104, 57-67. MEEK, G. A. & BRADBURY, S. (1963). Localization of thiamine pyrophosphatase activity in the Golgi apparatus of a mollusc, Helix aspersa. jf. Cell Biol. 18, 73-85. MILLER, F. & PALADE, G. E. (1964). Lytic activities in renal protein absorption droplets. An electron microscopical cytochemical study. J. Cell Biol. 23, 519-552. MOE, H., ROSTGAARD, J. & BEHNKE, O. (1965). On the morphology and origin of virgin lysosomes in the intestinal epithelium of the rat. J. Ultrastruct. Res. 12, 396-403. MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 1 u-i 14. MOSES, H. L. & ROSENTHAL, A. S. (1967). On the significance of lead-catalysed hydrolysis of nucleoside phosphates in histochemical systems. J. Histochem. Cytochem. 15, 354-355. 478 M. Dauwalder, W. G. Whaley and J. E. Kephart NEUTRA, M. & LEBLOND, C. P. (1966a). Synthesis of the carbohydrate of mucus in the Golgi complex as shown by electron microscope radioautography of goblet cells from rats injected with glucose-H*. J. Cell Biol. 30, 119-136. NEUTRA, M. & LEBLOND, C. P. (19666). Radioautographic comparison of the uptake of galactose-H3 and glucose-H' in the Golgi region of various cells secreting glycoproteins or mucopolysaccharides. J. Cell Biol. 30, 137-150. NORDIN, J. H. & KIRKWOOD, S. (1965). Biochemical aspects of plant polysaccharides. A. Rev. PI. Physiol. 16, 393-414. NORTH, R. J. (1966). The localization by electron microscopy of acid phosphatase activity in guinea pig macrophages. J. Ultrastruct. Res. 16, 96—108. NORTHCOTE, D. H. (1964). Polysaccharides. A. Rev. Biochem. 33, 51-74. NORTHCOTE, D. H. & PICKETT-HEAPS, J. D. (1966). A function of the Golgi apparatus in polysaccharide synthesis and transport in the root-cap cells of wheat. Biochem. J'. 98, 159-167. NOVIKOFF, A. B. (1959). Approaches to the in vivo function of subcellular particles. In Siibcellu- lar Particles (ed. T. Hayashi), pp. 1-22. New York: Ronald Press. NOVIKOFF, A. B. (1967). Enzyme localizations with Wachstein-Meisel procedures: real or artifact. J. Histochem. Cytochem. 15, 353-354. NOVIKOFF, A. B. & BIEMPICA, L. (1966). Cytochemical and electron microscopic examination of Morris 5123 and Reuber H-35 hepatomas after several years of transplantation. In Biological and Biochemical Evaluation of Malignancy in Experimental Hepatomas. Garni Monograph. 1, 65-87. NOVIKOFF, A. B., ESSNER, E., GOLDFISCHER, S. & HEUS, M. (1962). Nucleosidephosphatase activities of cytomembranes. In The Interpretation of Ultrastructure, vol. 1 (ed. R. J. C. Harris) (Symp. int. Soc. Cell Biol.), pp. 149-192. New York and London: Academic Press. NOVIKOFF, A. B., ESSNER, E. & QUINTANA, N. (1964). Golgi apparatus and lysosomes. Fedn. Proc. Fedn Am. Socs exp. Biol. 23, 1010-1022. NOVIKOFF, A. B. & GOLDFISCHER, S. (1961). Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc. natn. Acad. Sci. U.S.A. 47, 802-810. NOVIKOFF, A. B. & HEUS, M. (1963). A microsomal nucleoside diphosphatase. J. biol. Chem. 238, 710-716. NOVIKOFF, A. B., ROHEIM, P. S. & QUINTANA, N. (1966). Changes in rat liver cells induced by orotic acid feeding. Lab. Invest. 15, 27-49. O'BRIEN, T. P. & THIMANN, K. V. (1967). Observations on the fine structure of the oat coleoptile. III. Correlated light and electron microscopy of the vascular tissues. Protoplasma 63. 443-478. OSINCHAK, J. (1964). Electron microscopic localization of acid phosphatase and thiamine pyrophosphatase activity inhypothalamicneurosecretorycellsof the rat. 7. Cell Biol. 21, 35-47. OSINCHAK, J. (1966). Ultrastxuctural localization of some phosphatases in the prothoracic gland of the insect Leucophaea maderae. Z. Zellforsch. mikrosk. Anat. 72, 236-248. OTERO-VILARDEB6, L. R., LANE, N. & GODMAN, G. C. (1964). Localization of phosphatase activities in colonic goblet arid absorptive cells. J. Cell Biol. 21, 486-490. PERRY, R. P. (1964). Quantitative autoradiography. In Methods in Cell Physiology, vol. 1 (ed. D. M. Presscott), pp. 305-326. New York: Academic Press. PiCKETT-Heaps, J. D. (1967a). Further observations on the Golgi apparatus and its functions in cells of the wheat seedling. J. Ultrastruct. Res. 18, 287-303. PICKETT-HEAPS, J. D. (19676). Preliminary attempts at ultrastmctural polysaccharide localization in root tip cells. J. Histochem. Cytochem. 15, 442-455. Poux, N. (1963 a). Localisation de la phosphatase acide dan les cellules meristematiques de Bl£ (Triticum vulgare Vill.). J. Microscopie 2, 485-490. Poux, N. (19636). Localisation des phosphates et de la phosphatase acide dans les cellules des embryons de Ble (Triticum vulgare Vill.) lore de la germination. J. Microscopie 2, 557-568. Poux, N. (1965). Localisation de l'activite phosphatasique acide et des phosphates dans les grains d'aleurone. I. Grains d'aleurone renfermant a la fois globoides et cristalloides. J. Microscopie 4, 771-782. Poux, N. (1967). Localisation d'activites enzymatiques dans les cellules du meristeme radicu- laire de Cucumis sativus L. I.—Activites phosphatasiques neutres dans les cellules du proto- derme. J. Microscopie 6, 1043-1058. Golgi differentiation and phosphatases 479 REID, E. (1967). Membrane systems. In Enzyme Cytology (ed. D. B. Roodyn), pp. 321-406. London: Academic Press. ROODYN, D. B. (1967). The mitochondrion. In Enzyme Cytology (ed. D. B. Roodyn), pp. 103- 180. London: Academic Press. SABATINI, D. D., BENSCH, K. & BARRNETT, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58. SCHMALBECK, J. & ROHR, H. (1967). Die Mukopolysaccharid-Synthese in ihrer Beziehung zur Eiweiss-Synthese in der Brunnerschen Druse der Maus. (Elektronmikroskopische-Auto- radiographische Untersuchung mit 3H-Glukose). Z. Zellforsch. mikrosk. Anat. 80, 329-344. SEEMAN, P. M. & PALADE, G. E. (1967). Acid phosphatase localization in rabbit eosinophils. y. Cell Biol. 34, 745-756. SELJELID, R. (1965). Electron microscopic localization of acid phosphatase in rat thyroid follicle cells after stimulation with thyrotropic hormone. J. Histochem. Cytochem. 13, 687-690. SELJELID, R. (1967). Endocytosis in thyroid follicle cells. IV. On the acid phosphatase activity in thyroid follicle cells, with special reference to the quantitative aspects. J. Ultra- stnict. Res. 18, 237-256. SMITH, R. E. & FARQUHAR, N. G. (1966). Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland. J. Cell Biol. 31, 319—347. SOMMER, J. R. & BLUM, J. J. (1965). Cytochemical localization of acid phosphatases in Euglena gracilis. J. Cell Biol. 24, 235-251. STRAUS, W. (1967). Lysosomes, phagosomes and related particles. In Enzyme Cytology (ed. D. B. Roodyn), pp. 239—319. London: Academic Press. TERNER, J. Y., GOODMAN, R. M., & SPIRO, D. (1965). Glucose-6-phosphatase in the salivary glands of Sciara coprophila: A histochemical and biochemical study. J. Histochem. Cytochem. 13, 168-181. TICE, L. W. & BARRNETT, R. J. (1962). The fine structural localization of glucose-6-phosphatase in rat liver. J. Histochem. Cytochem. 10, 754-762. TICE, L. W. & BARRNETT, R. J. (1963). The fine structural localization of some testicular phosphatases. Anat. Rec. 147, 43-63. TICE, L. W. & ENGEL, A. G. (1966). Cytochemistry of phosphatases of the sarcoplasmic reticulum. II. In situ localization of the MG-dependent enzyme. J. Cell Biol. 31, 489—499. TORACK, R. M. (1965). Adenosine triphosphatase activity in rat brain following differential fixation with formaldehyde, glutaraldehyde, and hydroxyadipaldehyde. J. Histochem. Cytochem. 13, 191-205. WETZEL, B. K., SPICER, S. S. & HORN, R. G. (1967). Fine structural localization of acid and alkaline phosphatases in cells of rabbit blood and bone marrow. J. Histochem. Cytochem. 15, 311-334- WHALEY, W. G. (1966). Proposals concerning replication of the Golgi apparatus. In Funktionelle und morphologische Organisation der Zelle III; Probleme der biologischen Reduplikation. 3rd wissenschaftliche Konferenz der Gesellschaft Deutscher Naturforscher und Arzte (ed. P. Sitte), pp. 340-371. Berlin: Springer. WOODING, F. B. P. (1968). Radioautographic and chemical studies of incorporation into syca- more vascular tissue walls. J. Cell Sci. 3, 71-80. YATSU, L. Y. & JACKS, T. J. (1968). Association of lysosomal activity with aleurone grains in plant seeds. Archs Biochem. Biophys. 124, 466-471. (Received 5 June 1968) 480 M. Dauwalder, W. G. Whaley and J. E. Kephart

Fig. 3. A portion of a cell from the peripheral region of the cap near the apical epidermal cells characterized by epidermal-type secretory vesicles. KMnO4 fixation, x 4000. A, Golgi apparatus at higher magnification, x 16000. Fig. 4. Epidermal cells in which the Golgi apparatus produce large numbers of spherical secretory vesicles. KMnO4 fixation, x 4000. A, Golgi apparatus at higher magnification sectioned approximately perpendicular and parallel to the plane of the cisternae. x 20000. Fig. 5. Cells of the cortex without conspicuous specialization of the Golgi apparatus. Some apparent small vesicles are associated with the cisternae. KMnO4 fixation, x 4000. A, Golgi apparatus at higher magnification, x 16000. Golgi differentiation and phosphatases 4I1

Cell Sci. 4 482 M. Dauwalder, W. C. Whaley and J. E. Kephart

Fig. 6. A portion of a phloem cell showing dark inclusions in the plastids. The Golgi apparatus are involved in secretory vesicle production. Glutaraldehyde—osmium preparation, x 11000. A, Part of a plastid inclusion showing the ordered substructure, x 85000. Fig. 7. Cells of a developing phloem file showing the Golgi apparatus specialization. The numerous, small, darkly stained, spherical secretory vesicles contribute to the build-up of the secondary wall. No such specialization is seen in the Golgi apparatus of the adjacent stelar cells. The ' crystalline' inclusions in the plastids are not preserved with this fixation. KMnO4 fixation, x 4000. Fig. 8. Portion of cell of a xylem element located peripheral to the metaxvlem files. KMnO4 fixation, x 4000. Golgi differentiation and phosphatases

3'- 484 M. Dauwalder, W. G. Whaley and J. E. Kephart

Figs. 9-12. G (Glutaraldehyde fixation)-IDPase reaction (00167 M substrate). Fig. 9. A portion of a cap initial cell. No reaction product is seen in the Golgi apparatus. Some vacuolar (i>) reactivity is seen in the upper right corner, x 18000. Fig. 10. A portion of a cell from the peripheral region of the mid-cap. Reaction product is seen in the cisternae of the Golgi apparatus and in the forming vesicles, x 25 500. Fig. 11. A portion of an outer cap cell showing reaction product in all of the Golgi apparatus. Some vacuolar (v) precipitate is also seen, x 11 000. Fig. 12. A Golgi apparatus from an outer cap cell at higher magnification showing enhanced secretory vesicle production and increased enzymic reactivity when compared with regions of the mid-cap, x 32800. Golgi differentiation and phosphatases 485 486 M. Dauwalder, W. G. Whaley and J. E. Kephart

Fig. 13. G-IDPase reaction (0-0167 M substrate). A portion cf an actively secreting epidermal cell showing reaction product in the cisternae of the Golgi apparatus cut both parallel and transversely to the long axis of the cisternae. Although not shown here, reaction product is localized along the membranes of the forming secretory vesicles as in Fig. 12. x 28600. Fig. 14. G-IDPase reaction (00167 M substrate). A portion of a cortical cell in anaphase (ch, chromosome). Reaction product is not characteristically found in the Golgi apparatus (arrows) in this or later stages of cell division in other than epidermal cells. Some vacuolar (v) precipitate can be seen, x 13000. Fig. 15. G-IDPase reaction (0-0201 M substrate). A portion of an endodermal cell showing reaction product in the Golgi apparatus. The dense material seen in a few areas in the endoplasmic reticulum and nuclear envelope (tie) can be shown at higher magnification to be a sporadic fixation artifact and not to be lead reaction product (see, for example, the cell membrane shown in Fig. 34). x 11 000. Fig. 16. G-IDPase reaction (0-0201 M substrate). A portion of a phloem file (left) and adjacent stelar cells (right). Reaction product is seen in almost all of the Golgi apparatus (arrows) of the phloem cells while the apparatus (arrows) is almost com- pletely negative in the adjacent cells. The phloem cells also show reactivity along the vacuolar (v) membrane, x 6600. A, Golgi apparatus at higher magnification showing the localization of reaction product within the cisternae and in association with the forming secretory vesicles, x 23400. Fig. 17. G-IDPase reaction (00201 M substrate). Regions of a cell from a xyleni element peripheral to the large metaxylem elements showing localization of reaction product in the Golgi apparatus, x 11000. Golgi differentiation and phosphatases

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Fig. 18. F (Formaldehyde fixation)-1 DPase reaction (00201 M substrate). A portion of an outer cap cell showing reaction product in the Golgi cisternae and at the surfaces of the secretory vesicles (arrows). As in Fig. 15, the apparent density in the endoplasmic reticulum is not due to lead reaction product. At this substrate concentration the precipitate in the Golgi apparatus is quite heavy and for fine localization the tissue is probably 'over-reacted'. It is shown, however, for contrast with the TPPase reaction following which the Golgi apparatus remain negative even at higher substrate concentrations (see Fig. 21 A), X 3800. Fig. 19. F-IDPase reaction (0-0201 M substrate). A region showing the reactivity of the Golgi apparatus (arrows) in the epidermis (left) and adjacent cap cells (right). X3100. A, Golgi apparatus from the peripheral region of the mid cap showing localization of reaction product, x 11 800. Fig. 20. F-IDPase reaction (0-0201 M substrate). A phase-contrast micrograph of the outer region of the cap taken from a thick plastic section near the area shown in Fig. 18. The small, scattered Golgi apparatus are made visible by the lead reaction product, x 1000. Fig. 21. F-TPPase reaction (00296 M substrate). A phase-contrast micrograph of the peripheral mid- and outer regions of the cap taken from a thick plastic section. No reactivity is seen in the Golgi apparatus, x 1000. A, Golgi apparatus from a thin section from the peripheral mid-cap region. Scattered background precipitate is visible in the electron micrograph but the Golgi apparatus are negative, x 18200. Golgi differentiation and phosphatases

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Fig. 22. F-TPPase reaction (0-0286 M substrate). Portions of secretory epidermal cells. Except for the nuclei, reaction product is limited to the Golgi apparatus (arrows), x 4000. Fig. 23. F-TPPase reaction (00296 M substrate). A phase-contrast micrograph of a thick plastic section of a group of epidermal cells showing the Golgi apparatus reactivity, x 1000. Fig. 24. F-TPPase reaction (0-0266 M substrate). A Golgi apparatus from a secretory epidermal cell showing localization of reaction product, x 40800. Fig. 25. F-TPPase reaction (0-0296 M substrate). A portion of a phloem cell and adjacent stelar cells. Reactivity is seen in the Golgi apparatus (arrows) and in the small vacuoles (v) found in early phloem development. Reactive Golgi apparatus can also be seen in the adjacent stelar cells, x 6000. Fig. 26. F-IDPase reaction (00201 M substrate). A phase-contrast micrograph of a thick plastic section showing a developing phloem file. In the least differentiated phloem cell shown (bottom) the reactivity is mostly confined to the Golgi apparatus. In the cells showing increasing differentiation, the vacuolar labelling becomes more evident. Little vacuolar labelling is seen in the adjacent cells, x 1000. differentiation and phosphatases

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26 492 M. Dauwalder, W. G. Whaley and J. E. Kepliart

Fig. 27. F-TPPase reaction (00296 M substrate). A phase-contrast micrograph from a thick plastic section of metaxylem elements showing Golgi apparatus reactivity, x 1000. Fig. 28. F-TPPase reaction (00296 M substrate). Metaxylem elements showing the electron microscopic localization of reactivity in the Golgi apparatus (arrows), x 5000. Fig. 29. F-TPPase reaction (00296 M substrate). A phase-contrast micrograph of a thick plastic section of cortical cells showing the Golgi apparatus reactivity, x 1000. Fig. 30. F-TPPase reaction (0-0296 M substrate). A cortical cell showing the electron microscopic localization of reactivity in the Golgi apparatus (arrows), x 5500. A, Golgi apparatus from a similarly reacted sample (00266 M substrate) at higher magnification, x 43000. Golgi differentiation and phosphatases 453 494 M. Dauwalder, W. G. Wlialey and J. E. Kephart

Figs. 31, 32. G-Acid Pase reaction (70 min incubation). Serial sections through two Golgi apparatus from the same cell in the region of the peripheral cap adjacent to the epidermis. The two different section planes through the apparatus show the distal-face pattern of reactivity. The pattern seen in Fig. 31 E (arrow) is particularly suggestive that the localization is also in a limited region of smooth endoplasmic reticulum. x 28500. Fig. 33. G-Acid Pase reaction (90 min incubation). Portions of two outer cap cells. Lead precipitate is not found in the Golgi apparatus or stacks of vesicles (arrows) but is limited to the vacuoles (v). x 4600. Fig. 34. G-Acid Pase reaction (70 min incubation). Portion of a secreting epidermal cell showing the Golgi apparatus distal-face-associated reaction. The greyish droplets of material along the cell surface are a fixation artifact, x 33000. Golgi differentiation and phosphatases 495 496 M. Dauwalder, W. G. Wlialey and J. E. Kephart

Fig. 35- [°H]Glucose 30 min, radioautographic exposure 3 days. In the inner regions of the mid-cap the predominant incorporation has been into the starch grains. Incorporation into starch decreases to negligible levels in the outer mid-cap and outer cap regions. Relatively heavy ' cytoplasmic' (non-plastid) incorporation is seen in the outer cap cells. The epidermal cells show a dense band of grains at the secretory surface. Some incorporation into starch is seen in the cortex, x 250. Fig. 36. [3H]Glucose 30 min, radioautographic exposure 99 days. Electron-microscopic radioautograph of a portion of an outer cap cell showing localization of grains over the secretory vesicles of the Golgi apparatus (arrows). Little of the incorporated glucose is seen in the secreted material at this time interval, x 10400. Fig- 37- Glutaraldehyde fixation, plastic embedding, silver/PAS reaction. A portion of an outer cap cell similar to that in Fig. 36 showing staining of the material outside the protoplast and in the Golgi apparatus (arrows) and secretory vesicles, x 8300. Fig. 38. [3H]Glucose3omin, radioautographic exposure 65 days. Electron-microscopic radioautograph of a portion of an epidermal cell showing localization of grains predominantly over the secretory vesicles of the Golgi apparatus (arrows) and at the cell surface, x 7000. Fig. 39. pHJGlucose 30 min, radioautographic exposure 3 days. A region of the stele is shown. The developing phloem file is identifiable at this magnification by heavy labelling of the walls, x 250. Fig. 40. [3H]Glucose 30 min, radioautographic exposure 3 days. Bright-field (A) and phase-contrast micrograph (B) of the same epidermal cells one of which is in late telophase. Compared to the adjacent interphase cells, in the cell in telophase little material has been secreted at the cell surface toward the outside of the root and the forming cell plate is heavily labelled (arrows), x 1000. Fig. 41. An epidermal cell in late telophase showing incorporation of typical darkly- stained Golgi apparatus secretory vesicles into the plate. KMnO4 fixation, x 4000. Golgi differentiation and phosphatases

Cell Sci. 4