Download phosphatases and differentiation of the golgi apparatus

J. Cell Sci. 4, 455-497 (1969)
Printed in Great Britain
455
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 understanding 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 refrigerator (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
i-o ml
TPP (Sigma Chemical Co.: o-oi M-O-03 M) in distilled water
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 EponAraldite 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 electronmicroscopic 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, encompasses 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 crosssection 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. KMnO 4 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)
V
1
Cap region producing spherical
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 phosphatebuffered 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
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
0-0167
M
—
—
—
—
+ + +
+ + + +
—
+ + +
+ + + +
—
—
++
—
—
—
—
—
—
—
—
—
—
+ + +
—
—
—
+
—
—
—
—
—
—
+ + + +
—
+ +
+
+ + +
+
+ +
—
—
• 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
Tissue
Functional
state*
IDPase
IDPase
(formal(glutaraldehyde) dehyde)
TPPase
Acid
phosphatase
Cap
Initials
Cell division
Peripheral mid-cap Differentiating for
secretion
Outer cap
Secretory
Root
Quiescent zone and
apical initials
Epidermis
Secretory
Cortex
(accumulation of
storage material)!
—
++
+++
+++
+++
—
—
+++
+ ++
—
+
+++
-to±
±
- to ±
++
(near epidermis)
-to ?
-
-to±
+++
++
(near apex)
+++
—
+
Endodermis
+++
—
+ ++
++
Phloem elements Secretory
+ +
+++
+
Peripheral xylem Differentiating for
+ ++
+++
secretion (?)
elements
Prior to differentiation —
Metaxylem ele+++
+++
for conduction
ments
• 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 densities 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 6090 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; Holtzman, 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 & Novikoff, 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 & Malkoff, 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 unquestionably 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 'spherosomes' 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, electrondense 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 interprets 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 PASpositive 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, PickettHeaps, 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-membranebound 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.
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(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 completely 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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M. Dauwalder, W. G. Whaley and J. E. Kephart
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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M. Dauwalder, W. G. Whaley and J. E. Kephart
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
A
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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. [ 3 H]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. [ 3 H]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. [ 3 H]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 darklystained Golgi apparatus secretory vesicles into the plate. KMnO 4 fixation, x 4000.
Golgi differentiation and phosphatases
Cell Sci. 4