J. Cell Sd. 3, 71-80 (1968)
71
Printed in Great Britain
RADIOAUTOGRAPHIC AND CHEMICAL
STUDIES OF INCORPORATION INTO
SYCAMORE VASCULAR TISSUE WALLS
F. B. P. WOODING
Department of Biochemistry, University of Cambridge
SUMMARY
Chemical and radioautographic studies on sycamore seedling stems have shown an involvement of the Golgi body in cell-wall polysaccharide synthesis from tritiated glucose. Tritiated
phenylalanine is shown to be incorporated only into lignin after short incubation times. The
patterns of labelling are compared and discussed for the two precursors.
INTRODUCTION
There have been several recent electron-microscopic studies on the development
of the vascular cambium (Bouck & Cronshaw, 1965; Buvat, 1963, 1964; Cronshaw,
1967; Esau & Cheadle, 1965; Srivastava & O'Brien, 1966; Northcote & Wooding,
1966; Wooding & Northcote, 1964; Wooding, 1966). The organelles involved have
been tentatively identified and functions for each suggested. The present report is
of a study using electron-microscope radioautography in parallel with a chemical
analysis of sycamore vascular tissue, in order to define the functions of the organellar
systems involved more closely. Tritiated glucose and phenylalanine were fed to
allow a comparison of wall development including lignification.
METHODS
Young sycamore seedlings, 1 year old, were cut from the root under water and the
cut stem placed in the radioactive solution; 500/ic of either D-[6-3H]glucose or
L-[G-3H]phenylalanine in 0-25 ml of water were used. The shoots were left in the
solutions in sunlight for periods of £, 1 or 2 h and then chased with cold glucose or
phenylalanine (1 mg/ml) for various periods. Samples of the stem to be used for
electron microscopy were sectioned into a glutaraldehyde fixative and the remainder
of the stem was then taken for chemical analysis (see Fig. 1).
Stem segments (1-2 mm) were cut into afixativeconsisting of 4-5 % glutaraldehyde
in phosphate buffer (pH 7-2) containing 2 % sucrose. After 1 h the segments were
washed with buffer and placed for a further hour in 1 % osmium tetroxide solution
buffered with veronal (pH 7-2). The segments were then washed in water, dehydrated
in an ethanol series and embedded in Araldite. Sections were cut on a Porter-Blum
MTa microtome with glass knives, picked up on carbon films on copper grids and
72
F. B. P. Wooding
stained with uranyl acetate (saturated solution in 50% ethanol) followed by lead
hydroxide (Millonig, 1961). After staining the sections on the grids they were covered
with a carbon film and coated with Ilford L4 emulsion by a modification of the
agar block method of Caro & van Tubergen (1962). The grids were then stored at
Stem of seedling after incubation divided into two parts
t
B
A
Fixed for electron microscopy
and radioautography
Washed in water
Extracted 2 x 1 h boiling methanol
2 x 1 h boiling ether
LIPID
FRACTION
Residue divided into three portions
2
Alkaline nitrobenzene oxidation
Hydrolysis with H,SO,
LIGNIN CONSTITUENTS
AMINO ACIDS/SUGAR SEPARATION
Carbohydrate fractionation
Samples refluxed in cold-finger apparatus
0-05 M EDTA pH 6-7 phosphate
buffer 2 h 70°C
PECTIC
SUBSTANCES
H
H
-Residue
Sodium chlorite/acetic
acid 70 °C, 2 h
icetic acic
-Residue
HEMICELLULOSE -*
[3% NaOH,
100 °C, 1 h] x 2
aOH, 10C
-Residue
a-CELLULOSE
Fig i. Analytical scheme.
room temperature for 2-3 months, developed in Kodak Digb (1 min), or Microdol,
washed and fixed with Hypam (Ilford) (diluted 1 :\; 45 sec) and examined in a Philips
EM 100 electron microscope at 80 kV.
The chemical fractionation was as detailed in Fig. 1. Samples of pectin, hemicellulose and a-cellulose were hydrolysed with 72 % sulphuric acid, the hydrolysate
neutralized with barium carbonate, evaporated down and taken up in a known volume
of water for chxomatographic separation of the constituent sugars (pyridine/acetic
acid/water, 8:2:1).
Incorporation into sycamore tissue walls
73
Samples of the original stem were used for alkaline nitrobenzene oxidation in a
stainless steel bomb at 1800 C, which produces syringaldehyde, vanillaldehyde and
/>-hydroxybenzaldehyde from the original lignin material (Stone & Blundell, 1951).
These aldehydes were separated by chromatography of the bomb contents, using
n-butanol/ammonia/water, 4:1:5 (Jeffs & Northcote, 1966). A further sample of the
original material was hydrolysed with 72% sulphuric acid and used for separation
of amino acids and sugars. After neutralization the constituents of the mixture were
separated by electrophoresis at pH 2-1 using acetic acid/formic acid buffer, at 5 kV
for 20 min. At this pH the sugars remain at the origin while the amino acids move
fairly rapidly. The paper strips from each sample separated by chromatography or
electrophoresis were cut into i-cm pieces, placed in 0-5 ml scintillation fluid in
a vial and counted in a Packard scintillation counter. The various constituents in the
samples were identified by use of markers on each paper.
RESULTS
Electron-microscope radioautography
Preliminary experiments with seedlings showed that glucose and phenylalanine
allowed a distinction to be made between the synthesis of cell-wall polysaccharides
and lignin fairly well up to about 3 h after feeding, after which the phenylalanine
label started to appear in the cell walls and also in the general cytoplasm.
Glucose. Experiments without cold chases showed a gradual increase of label into
xylem and phloem walls, but with incubation times between 2 and 4 h little radioactivity was observed in the cytoplasm, except that after 3 h starch grains in the
plastids of all tissues were extensively labelled.
With \ h incubation plus \ h cold chase no label was found in the cytoplasm but
the walls of developing xylem and sieve elements were lightly labelled (Figs. 2, 3).
The only condition found in which labelling was localized over specific organelles
was 1 h of incubation followed by \ h cold chase. Under these conditions, in those
cells which were identified as developing xylem vessels or phloem sieve-tubes/
companion cells, label was localized over the numerous Golgi bodies and their
associated vesicles and also over the developing wall (Figs. 4-7, 19-21, 23). After 2 h
incubation plus \ h cold chase the walls were heavily labelled but no label was found
in the cytoplasm, although numerous Golgi bodies were visible (Figs. 9-11, 17).
When label was incorporated into the wall or seen over the Golgi bodies microtubules were usually present along the xylem cell plasmalemma (Fig. 8). A few starch
grains in ray cells in phloem and xylem were slightly labelled. The callose in the
mature sieve-tube pores was found labelled after all times of incubation, with or
without cold chases (Figs. 22, 26).
The walls of the developing xylem vessels, if lightly labelled, had this label localized
just under the plasmalemma, rather than distributed throughout the wall as in sieve
elements (Figs. 2-4, 7, 8). In both cases the label was found evenly all along the cell
length and never localized at any one place (Figs. 19, 24). At a later stage of development of the sieve tube (stage 3 as defined in Northcote & Wooding, 1966) the only
74
F. B. P. Wooding
part of the wall to be labelled was the callose area under the subtending endoplasmic
reticulum which defines the site of the future sieve pore (Fig. 25).
Phenylalanine. As with glucose, a gradual increase in label was found over the
developing xylem vessel walls, but no label was ever found over cambium or phloem
cell walls. No label has ever been found localized over the cytoplasm of the developing
xylem cells up to 3-4 h of incubation, with or without cold chases. After this period
labelling was found randomly distributed in the cytoplasm of all cells.
In developing xylem the earliest label appears deep in the wall at the middle
lamellar region (Fig. 12). If the developing xylem was spirally thickened (rare in
seedlings of this age) the base of the thickening was labelled initially (Fig. 13).
Subsequently the label was found over the entire wall area (Figs. 15, 17). In xylem
vessels in which there was a cross-wall, no label occurred over that part of the wall
which eventually breaks down (Fig. 16). In the protoxylem, formed long before the
seedling was incubated with phenylalanine, heavy labelling has frequently been
found between the spiral thickenings, which are themselves unlabelled (Fig. 14).
Such protoxylem is separated from the labelled developing xylem and cambial
zones by mature xylem with reticulate or continuous wall thickening, which was
completely unlabelled.
Incubations up to 4 h occasionally produced labelling restricted to, and evenly
distributed over, the compact slime body in the developing sieve tube (Fig. 18).
In cambium and other phloem cells the labelling, if any, was at random over the
cytoplasm after such a 4-h incubation; labelling was never found over the walls or
vacuole.
Chemical analysis
The total amount of label taken up by seedlings was determined by sampling the
incubation medium before and after a 2-h experiment (Table 1). The counts removed
by water and ethanol washes were subtracted from this total. Of the total radioactivity
taken up from glucose, 0-7 % was recovered in the polysaccharide and lignin fractions;
l
'5% °f t n e phenylalanine radioactivity was recovered in the lignin constituents
(see Table 2), and 0-5 % in protein. Neither glucose nor phenylalanine gave any
appreciable labelling in the lipid fraction.
The majority of the glucose was incorporated into the wall polysaccharides,
largely into a-cellulose (Table 1). Labelled glucose and xylose were found in the
hemicellulose fraction after 1 h of incubation. The pectin material showed no radioactive
sugars until after 2 h of incubation, when galactose and arabinose were found. The
ratio of the radioactivity in the various sugars in each fraction became fairly constant
(± 10 %) after 1 h. With shorter incubation times radioactivity was found only in
the glucose of the a-cellulose fraction, and to a small extent in the lignin constituents.
Phenylalanine was rapidly incorporated into the lignin, predominantly into substances chromatographically identical with syringaldehyde and vanillaldehyde (Table 2).
The radioactivity in these compounds increased with time and after about 3 h some
trace of radioactivity was also found corresponding to the />-hydroxybenzaldehyde
markers.
Incorporation into sycamore tissue walls
75
Table 1. Radioactivity in analytical fractions
Material fed
(500 nc for each
D-[6-'H] Glucose
L-[G-'H]Phenylalanine
<•)
60
Time of incubation, min
30
Time of cold chase, min
3°
Total count/min/mg dry weight 970
120
3°
30
10870
5060
30
3°
2200
60
30
5600
120
30
25600
0/
/o of counts in each fraction
—
Pectin
8 gal
20 glu
Hemicellulose
a-Cellulose
Lignin
90 glu
5 xyl
10
14 glu
2 xyl
56 glu
17
100
100
100
18
Each value represents a mean of 4 separate determinations with one seedling at each time
interval. The variation on total incorporation and on individual fraction counts was not more
than 7 %. (ara, arabinose; gal, galactose; glu, glucose; xyl, xylose.)
Table
2.
Radioactivity in lignin constituents
D-[6-8H]Glucose
L-[G-'H]Phenylalanine
Material fed
Time of incubation (30 nun
cold chase)
Total count/min/mg dry weight
Syringaldehyde
Vanillaldehyde
^-Hydroxybenzaldehyde
3°
120
60
120
3°
60
120
1000
2500
2200
5600
25600
—
25
100
75
—
—
% of counts in constituents
—
20
80
—
100
—
4
96
—
6
90
2
DISCUSSION
In the stem segment of the sycamore seedling the only part actively growing and
differentiating is the vascular cambium. This study has shown that very few cells
incorporate material. In the xylem most of the developing vessels one or two cells
distant from the cambium incorporate glucose; phenylalanine incorporation continues
to a depth of three or four vessels. In the phloem there are even fewer cells with
incorporation; a single developing sieve tube may be the only phloem cell in an
entire radial longitudinal section to show label in the wall or cytoplasm. Thus over
the periods used in this study the incorporation is largely into phloem or xylem
walls and little label finds its way into cambium, ray or parenchyma cell walls.
Freudenberg (1965) has suggested that in the synthesis of lignin, soluble precursors
are formed in the cambium region which pass to the xylem cell wall after it is fully
secondarily thickened. Lignification then occurs by polymerization of these precursors,
76
F. B. P. Wooding
commencing at the level of the middle lamella. Other workers have suggested that
the lignification is a continuous process in a single xylem cell, deposition of the lignin
starting soon after secondary thickening of the wall commences. Evidence for this
comes largely from electron microscopy of permanganate-fixed, lead-stained sections,
where the very densely stained areas are said to indicate lignin (Wooding & Northcote,
1964). The present work shows that label from phenylalanine is incorporated into
xylem vessels at a very early stage in the development of their secondary wall. Since
on chemical extraction of the tissue only lignin constituents are found to be labelled
by phenylalanine, this demonstrates that lignin synthesis occurs in cells contiguous
with the cambium and at a very early stage of secondary wall formation.
No cytoplasmic labelling has been observed when the seedlings are fed phenylalanine. This would indicate that the precursors of the lignin are soluble as Freudenberg
suggests. Thus the site of the synthesis of the more immediate precursors of lignin
from phenylalanine cannot be distinguished by the radioautographic methods used
in this study. Only those xylem cells which still had recognizable fine structure showed
label on their walls, which would suggest that the synthesis of lignin was dependent
upon the cell possessing a functional cytoplasm.
One exception to the above is found in the protoxylem. Here the wall remaining
between the spirally thickened vessels is labelled but the thickenings themselves are
unlabelled. This effect is consistently found, and there is no question of the protoxylem
cells developing during the incubation. However, there are many ray and parenchyma
cells among the protoxylem vessels and it is possible that these living cells are involved
in lignification of the primary wall (which is not usually lignified) between the spiral
thickenings.
The radioactivities of the syringaldehyde and vanillaldehyde increase in parallel
and there is no indication of a precursor relationship between the two.
Apart from their incorporation into the xylem walls, after 3 or 4 h the label from
phenylalanine begins to appear at random in the cytoplasm of all cells, with no
association with any organelle. There is one exception to this, in a sieve element at
an early stage of development when slime bodies are being formed (stage 2 of Northcote
& Wooding, 1966). In such a cell, label is found over only the slime body. The label
is evenly distributed, with no indication of any directional growth. This shows that
the sieve-tube metabolism is geared at this stage solely to the production of slime-body
material. Since phenylalanine labelling is usually random in a cell the label is most
likely to occur in protein. Buvat (1963) has claimed that slime bodies contain RNA
in Cucurbita but they are generally considered to be largely protein, and the labelling
shown in this study would confirm this.
The earlier suggestions (Wooding & Northcote, 1964; Northcote & Wooding,
1966) that Golgi bodies are involved in synthesis of polysaccharide wall material in
both xylem and phloem is borne out by this study. After 1 h incubation plus A h cold
chase the label can be clearly seen over the Golgi bodies or vesicles with a small
amount in the cell walls, while after 2 h the Golgi bodies are unlabelled and the wall
heavily labelled. On two occasions a trace of cytoplasmic label was seen after half an
hour of incubation; this again was associated with a Golgi body and/or vesicles which
Incorporation into sycamore tissue walls
could be derived from a Golgi body. Apart from this no cytoplasmic organelle was
significantly labelled after any of the various periods of incubation tried. Chemical
analysis shows that the radioactive materials isolated after 1 or 2 h of incubation
with glucose were lignin, hemicellulose and a-cellulose, in order of increasing label
(Table 1). Since phenylalanine is a much better precursor of lignin than glucose
and yet never produces a labelled Golgi body, the radioactive material in the latter
is unlikely to be a lignin forerunner.
Extracted a-cellulose is labelled at an early stage, whereas hemicellulose becomes
detectably labelled only after an hour's incubation, coincidentally with the appearance
of the label over the Golgi bodies. The synthesis of the a-cellulose with its oriented
microfibrils is considered more likely to involve incorporation of glucose at a site
at or just outside the plasmalemma. The present work clearly shows that label from
glucose occurs initially at this site.
The particulate preparations synthesizing cellulose so far isolated (Ordin & Hall,
1967; Barber, Elbein & Hassid, 1964) produce a-cellulose, rather than high-molecularweight precursors which would subsequently be incorporated into a-cellulose chains.
On the other hand the hemicellulose provides amorphous packing material around the
oriented a-cellulose fibrils, and need not be synthesized at the cell boundary, as the
mechanism of exopinocytosis exists to transfer the high-molecular-weight precursors
to the wall. The nucleoside diphospho-sugar precursors at either a-cellulose or the
hemicelluloses would almost certainly be removed from the tissue during specimen
preparation. Thus the Golgi label seems most likely to originate from high-molecularweight precursors of hemicellulose, synthesis of a-cellulose from soluble precursors
occurring at or very close to the plasmalemma.
Northcote & Pickett-Heaps (1966) demonstrated an incorporation of tritiated
glucose into a pectic material in wheat root tips. Pectin does not become labelled in
the sycamore seedlings until after at least 2 h of incubation, which would rule out the
possibility of the Golgi label arising from this material in sycamore stem, whereas
the pectic material in wheat root tips is indisputably associated with the Golgi body.
The sycamore stem hemicellulose production demonstrated in this paper shows
that the same organelle is capable of a radical change in its metabolic product
depending upon the differentiation of the cell.
Thornber & Northcote (1961) have shown that in sycamore xylem the main
hemicellulose constituents are glucose and xylose, which are the only two sugars
found to be labelled in this study. In a-cellulose glucose is the predominant sugar,
and as would be expected only this sugar is labelled in this fraction.
The incorporation into pectic substances occurred only after 2 h of incubation,
and the sugars labelled were galactose and arabinose. Northcote & Pickett-Heaps
(1966) demonstrated considerable glucose-to-galactose conversion in wheat root
caps, but the label in arabinose is unexpected, since glucose labelled at position 6
would lose its label on conversion to arabinose (via galactose). Thus this label must
be derived from glucose which has undergone considerable metabolic transformation
and is not produced by the most direct route of arabinose synthesis from glucose.
The localization of the wall label near the plasmalemma from glucose and at
77
78
F. B. P. Wooding
middle-lamellar level from phenylalanine confirms what has been suggested by
Freudenberg (1965) and others. The label from glucose is predominantly in the
a-cellulose and in xylem the wall thickening is in layers, thus label should appear
localized near the plasmalemma. Since the hemicellulose is also labelled, some of the
label on the wall will arise from these polysaccharides. The hemicellulose is amorphous
material and is probably laid down throughout the wall rather than solely in the
surface layer which is being synthesized. This could account for the occasional
departure from the labelling of the plasmalemma side of the developing xylem wall.
In the sieve tube the glucose label is not so clearly restricted toward the plasmalemma
side of the wall. The impression is of synthesis throughout the wall, as found by
Setterfield & Bayley (1958)111 coleoptile epidermal cell walls.
Since labelled Golgi vesicles are found in both xylem and phloem it is considered
that radioactive hemicellulose is formed in both cases, although from the work of
Thornber & Northcote (1961, 1962) the amount of hemicellulose is far lower in
phloem as compared with xylem, both in proportion to the amount of a-cellulose
and also in total amount.
The even labelling found along the xylem (and phloem) walls from either phenylalanine or glucose shows that incorporation of materials was simultaneous at all
points along the wall. No indication was seen that secondary thickening starts at the
centre of a xylem vessel and works toward the tip as suggested by Wardrop & Harada
(1965). Although label is frequently seen over xylem cell wall and adjacent longitudinal
microtubules, such sections can be interpreted as showing a very thin layer of
recently deposited, labelled wall material, on top of or under which the microtubules
are visible. It is not thought that there is any labelling of the microtubules.
In the phloem the callose labelling is of interest. The callose in the mature sieve
plate labels very rapidly as has been reported previously (Northcote & Wooding, 1966).
Callose, a 1-3/? glucan is extremely resistant to extraction and might well be present
in the a-cellulose fraction which really represents the residue from the other extractions
(see Table 1). Thus some of the labelled glucose demonstrated after half an hour
could be callose rather than a-cellulose, since the walls of the developing tissues
have little label after this time. Insufficient radioactive material was isolated to allow a
distinction to be made. If the sieve tubes are still functioning as conduits the tritiated
glucose would permeate the sieve-tube system very rapidly during the incubation, as
well as passing up the stem via the xylem.
In the young sieve tube the pattern of labelling depends upon the stage of development reached. At first, when active wall deposition is occurring, the callose around
developing sieve pore and companion-cell pore is labelled, as is the wall around. But
at a later stage label is incorporated only into the callose areas. This indicates either
that the callose synthesis continues after wall synthesis is complete, or that the callose
is produced in both developing and mature phloem only when the stem is killed in
the fixative and is not present in vivo. The latter is considered less likely.
Incorporation into sycamore tissue walk
79
REFERENCES
BARBER, G. A., ELBEIN, A. D. & HASSID, W. Z. (1964). Synthesis of cellulose by enzyme systems
from higher plants. J. biol. Chem. 339, 4056-4161.
BOUCK, G. B. & CRONSHAW, J. (1965). The fine structure of differentiating sieve tube elements.
J. Cell Biol. as, 79-96.
BUVAT, R. (1963). Leg infrastructures et la difterenciation des cellules cribl6es de Cucurbita
pepo L. Portug. Acta biol. Serie A 7, 249-299.
BUVAT, R. (1964). Comportement des membranes plasmiques lors de la differentiation des
parois laterales des vaisseaux de Cucurbita pepo L. C. r. hebd. Static. Acad. Set., Parti 358,
L. C. & TUBERGEN, R. P. VAN (1962). High resolution autoradiography. J. Cell Biol.
15, 173-188.
CRONSHAW, J. (1967). Tracheid differentiation in tobacco pith cultures. Planta 7a, 78-90.
ESSAU, K. & CHEADLE, V. I. (1965). Cytologic studies on phloem. Univ. Calif. Publs Bot.
36, 253-344FREUDENBERG, K. (1965). Lignin, its constitution and formation from £-hydroxycinnamyl
alcohols. Science, N. Y. 148, 595-600.
JEFFS, R. A. & NORTHCOTE, D. H. (1966). Experimental induction of vascular tissue in an
undifferentiated plant callus. Biochem. J. 101, 146-152.
MILLONIG, C. (1961). Modified procedure for lead staining of thin sections. J. biophys. biochem.
Cytol. 11, 736-739NORTHCOTE, 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.
NORTHCOTE, D. H. & WOODING, F. B. P. (1966). Development of sieve tubes in Acer pseudoplatanus. Proc. R. Soc. B 163, 524-537.
ORDIN, L. & HALL, M. A. (1967). Studies on cellulose synthesis by a cell free oat coleoptile
enzyme system. Inactivation by airborne oxidants. PI. Physiol., Lancaster 42, 205-212.
SETTERFIELD, G. & BAYLEY, S. T. (1958). Deposition of wall material in thickened primary
walls of elongating plant cells. Expl Cell Res. 14, 622-625.
SRIVASTAVA, L. M. & O'BRIEN, T. P. (1966). On the ultrastructure of cambium and its vascular
derivatives. II. Secondary phloem of Pinus strobus. L. Protoplasma 61, 277—293.
STONE, J. E. & BLUNDELL, M. J. (1951). Rapid micromethod for alkaline nitrobenzene
oxidation of lignin and determination of aldehydes. Analyt. Chem. 21, 771-774.
THORNBER, J. P. & NORTHCOTE, D. H. (1961). Changes in the chemical composition of a cambial
cell during its differentiation into xylem and phloem tissue in trees. Parts 1 and 2. Biochem.
J. 81, 449-464THORNBER, J. P. & NORTHCOTE, D. H. (1962). Changes in the chemical composition of a
cambial cell during its differentiation into xylem and phloem tissue in trees. Part 3. Biochem.
J. 8a, 340-346.
WARDROP, A. B. & HARADA, H. (1965). The formation and structure of the cell wall in fibres
and tracheids. J. exp. Bot. 16, 356-364.
WOODING, F. B. P. (1966). The development of the sieve elements of Pinus pinea. Planta 69,
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WOODING, F. B. P. & NORTHCOTE, D. H. (1964). The development of the secondary wall of the
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CARO,
{Received 25 July 1967)
8o
F. B. P. Wooding
ABBREVIATIONS ON PLATES
c cambium
cc companion cell
dip developing sieve plate
dst developing sieve tube
8 Golgi body
P plastdd
s spiral thickening
sp sieve plate
st sieve tube
V
vacuole
X
xylem vessel
w cell wall
All figures are of radial longitudinal sections of sycamore stem.
Figs. 2, 3. Radioautographs from stems fed [6-*H]glucose for J h, followed by a cold
chase of glucose for \ h. The micrographs show developing xylem vessels with
labelling restricted to the boundary of the cell wall and the cytoplasm. None of the
cytoplasmic organelles is labelled. Fig. 2, x 25000; Fig. 3, x 16500.
Fig. 4. Radioautograph from stem fed [6-'H]glucose for 1 h followed by a cold
chase of glucose for i h. The micrograph shows a developing xylem vessel with
labelling at the boundary between wall and cytoplasm and also label over the cytoplasm
confined to the Golgi bodies, x 15600.
Figs. 5, 6. Radioautographs from stems fed [6-3H]glucose for 1 h followed by a cold
chase of glucose for \ h. Two micrographs of developing xylem vessels demonstrating
the restriction of cytoplasmic labelling to the Golgi cisternae and associated vesicles.
Fig. 5, x 23000; Fig. 6, x 40000.
Journal of Cell Science, Vol. 3, No. 1
F. B. P. WOODING
(Facing p. 80)
Fig. 7. Radioautograph from stem fed [6-3H]glucose for 1 h followed by a cold
chase of glucose for % h. Micrograph of a developing xylem vessel with Golgi bodies
in the cytoplasm. The only labelling in the cytoplasm is found over these organelles.
x 14800.
Fig. 8. Radioautograph from stem fed [6-3H]glucose for 1 h followed by a cold chase
of glucose for ^ h. Micrograph of a developing xylem vessel showing the even distribution of labelling over both the wall and microtubules just under the plasmalemma.
x 16800.
Fig. 9. Radioautograph from stem fed [6-3H]glucose for 2 h followed by a cold chase
of glucose for £ h. The micrograph shows a developing xylem vessel with greatly
thickened wall. No label can be seen over the Golgi bodies or over the cytoplasm;
only the wall is labelled, x 15000.
Fig. 10. Radioautograph from stem fed [6-3H]glucose for 2 h followed by a cold
chase of glucose for £ h. The micrograph shows a developing xylem vessel with
spiral thickenings. There is no label over the cytoplasm, only the top layer of the
thickenings toward the cytoplasm is labelled, x 7700.
Fig. 11. Radioautograph from stem fed [6-3H]glucose for 2 h followed by a cold chase
of glucose for i h. The micrograph shows the heavily labelled walls of mature xylem
vessels. The cytoplasm is of a xylem ray cell, x 3600.
Fig. 12. Radioautograph from a stem fed [G- 3 H]phenylalanine for i h followed by
a cold chase of phenylalanine for £ h. The radioautograph was developed in Microdol,
not D19b, to allow closer localization. The developing xylem vessel shows incorporation
only in the region of the middle lamella, x 14800.
Journal of Cell Science, Vol. 3, No. 1
F. B. P. WOODING
Fig. 13. Radioautograph from a stem fed [G-*H]phenylalanine for £h followed by
a cold chase of phenylalanine for i h. The radioautograph was developed in Microdol,
not Dio,b, to allow closer localization. The micrograph shows part of a spirally
thickened xylem vessel with incorporation restricted to the base of the thickenings,
x 6200.
Fig. 14. Radioautograph from a stem fed [G-3H]phenylalanine for 2 h followed by
a cold chase of phenylalanine for $ h. Micrograph of a mature xylem vessel. Label can
be seen over the wall area between the spiral thickenings which are unlabelled.
X7600.
Fig. 15. Radioautograph from a stem fed [G-3H]phenylalanine for 2 h followed by
a cold chase of phenylalanine for £ h. A micrograph showing xylem, cambium, and
phloem, with labelling restricted to the developing xylem vessel, x 2700.
Fig. 16. Radioautograph from a stem fed [G-'H]phenylalanine for 2 h followed by
a cold chase of phenylalanine for i h. Micrograph of a developing xylem vessel,
demonstrating that label is restricted to those areas of wall which will persist at
maturity. The cross-wall (between arrows) destined to break down, is unlabelled.
x 5100.
Fig. 17. Radioautograph from a stem fed [G-3H]phenylalanine for £ h followed by
a cold chase of phenylalanine for £ h. Micrograph showing a xylem vessel near the
completion of secondary wall deposition. Note the restriction of labelling to the wall
areas only, x 7 600.
Fig. 18. Radioautograph from a stem fed [G-'H]phenylalanine for 4 h followed by
a cold chase of phenylalanine for 2 h. The micrograph shows a developing sieve element
with a characteristic slime body, which is evenly labelled, x 8900.
Journal of Cell Science, Vol. 3, No. 1
F. B. P. WOODING
Fig. 19. Radioautograph from a stem fed [6-3H]glucose for 1 h followed by a cold
chase of glucose for £ h. Micrograph of phloem showing that a developing sieve tube
is the only cell with appreciably labelled walls. Note the even labelling along the cell
length. Label over the cytoplasm is restricted to the Golgi bodies and associated
vesicles (see the higher power view in Fig. 20). x 2900.
Figs. 20, 21. Radioautographs from stems fed [6-'H]glucose for 1 h followed by
a cold chase of glucose for ^ h. Micrographs demonstrating the restriction of labelling
to the Golgi bodies, their associated vesicles, and the wall. Fig. 20, x 22000;
Fig. 21, x 23000.
Fig. 22. Radioautograph from a stem fed [6-3H]glucose for 1 h followed by a cold
chase of glucose for £ h. Micrograph showing a phloem area with labelling restricted to
the callose on a mature sieve plate. No label is seen over the walls of the sieve tube or
companion cell, x 8000.
Journal of Cell Science, Vol. 3, No. 1
F. B. P. WOODING
Fig. 23. Radioautograph from a stem fed [6-'H]glucose for 1 h followed by a cold
chase of glucose for £ h. Micrograph of an area from a developing sieve tube. Labelling
is restricted to the Golgi bodies and the wall, x 30000.
Figs. 24, 25. Radioautograph from stems fed [6-3H]glucose for 2 h followed by a cold
chase of glucose for £ h. Micrographs of developing sieve tubes. Fig. 24 is at stage 2 of
of development; Fig. 25 is at stage 3 (Northcote & Wooding, 1966). In Fig. 24 label
occurs over the walls and the sieve plate, in Fig. 25 over only the callose areas on the
sieve plate. Fig. 24, X4500; Fig. 25, x 8250.
Fig. 26. Radioautograph from a stem fed [6-3H]glucose for 2 h followed by a cold
chase of glucose for £ h. Micrograph of a mature sieve tube; only the callose on the
sieve plates (arrows) is labelled. X4000.
Journal of Cell Science, Vol. 3, No. 1
F. B. P. WOODING