J. Cell Sci. 4, 155-169 (1969)
Printed in Great Britain
155
DEVELOPMENT AND DISPERSAL OF P-PROTEIN
IN THE PHLOEM OF COLEUS BLUMEI BENTH.
M. W. STEER AND E. H. NEWCOMB
Department of Botany, University of Wisconsin, Madison, Wisconsin, U.S.A.
SUMMARY
The development of P-protein (slime) in the phloem of Coleus stem apices has been studied
electron microscopically using material fixed in glutaraldehyde followed by osmium tetroxide.
In phloem parenchyma cells the earliest-appearing groups of tubular P-protein commonly are
seen in close association with clusters of ' spiny' vesicles similar to those reported in Phaseolus
phloem (Newcomb, 1967). The vesicles break down as the P-protein masses enlarge, and are
assumed to contribute to P-protein formation. Subsequently the groups of tubules are consolidated into a single spindle-shaped body aligned longitudinally in each phloem parenchyma
cell or sieve element. The microtubules observed frequently in the vicinity of the young
P-protein body may play a role in its consolidation or in the longitudinal alignment of its
constituent tubules. Some P-protein bodies acquire a highly organized structure in which the
tubules are arranged hexagonally around lightly staining centres.
Disaggregation of the P-protein body occurs during disintegration of the cytoplasm and
nucleus, and results initially in the presence of swirls of packed fibrils. During disaggregation,
the tubules of the mature P-protein body, which are about 200 A in diameter, are converted
to fibrils about 70 A in diameter in a process apparently with several intermediate stages. In
longitudinal view the fibrils exhibit alternate electron transparent and dense bands that impart
a striated appearance to the mass. During maturation of the sieve element the swirls of fibrillar
masses separate into individual fibrils which become dispersed through the cell lumen.
INTRODUCTION
Several recent papers have considerably expanded our knowledge of the fine structure of slime bodies in a wide range of plants (Lafleche, 1966; Northcote & Wooding,
1966; Tamulevich & Evert, 1966; Wooding, 1967; Cronshaw & Esau, 1967; Northcote & Wooding, 1968). There now seems general agreement that in many cases the
slime body is composed of a mass of tubules approximately 200 A in diameter
(Northcote & Wooding, 1966; Tamulevich & Evert, 1966; Wooding, 1967; Cronshaw
& Esau, 1967). It has been reported that in the maturing sieve elements these tubules
are transformed into a system of fibrils, each having a characteristic alternate light- and
dark-staining band along its length (Northcote & Wooding, 1966; Cronshaw & Esau,
1967; Wooding, 1967). Although their dimensions are somewhat dissimilar, the fibrils
of the above studies probably represent similar stages in the different plants used. The
origin of the slime bodies has not been determined, although Wooding (1967) observed
dense vesicles adjacent to developing tubules.
Work in this laboratory has provided evidence that 'spiny vesicles' (vesicles bearing
small tubular projections) may be involved in the formation of slime bodies in root
apices of Phaseolus vulgaris (Newcomb, 1967). The present paper is part of an overall
156
M. W. Steer and E. H. Newcomb
investigation to confirm this observation and clarify the fine structural changes ocurring
in the developing sieve element. It will describe the formation and breakdown of slime
bodies in the stem apices and leaf primordia of Coleus blumei Benth. Slime body formation has been studied both in phloem parenchyma and in sieve elements undergoing
differentiation. At the level of the light microscope, phloem development in this
material has been followed by Jacobs & Morrow (1958, 1967).
Esau & Cronshaw (1967) have proposed that the slime body components be referred
to as 'P-proteins', and subsequently (Cronshaw & Esau, 1967) have designated the
tubules as 'P-i protein' and the banded or striated fibrils as 'P-2 protein'. In our
opinion the terms 'slime' and 'slime body' are badly in need of replacement, and
' P-protein' as a class designation is brief, convenient, and suggestive. We are therefore
adopting it in this paper. However, the results reported herein as well as the evidence
from other work in our laboratory and from recent publications in the field suggest that
considerable variability of form exists in P-protein both within and among species,
and that different types of P-protein may be interconvertible. We feel, therefore, that
it would introduce confusion if we were to attempt to assign 'P-numbers' to our
components, at least at this time. Much more information is needed on the range in
form and dimensions, the interrelationships, and interconvertibilities of the P-proteins
at different stages in the same plant and in a variety of plant species before the validity
of distinctive P-numbers can be assessed.
MATERIALS AND METHODS
Stem apices were dissected from greenhouse specimens of Coleus blumei Benth. and
immediately immersed in 3 % glutaraldehyde containing 0-025 M phosphate buffer at
pH 6-8. After fixation in the glutaraldehyde for 3-5 h at room temperature the apices
were washed for 1 h in four changes of 0-025 M phosphate buffer and postfixed in
2 % osmium tetroxide in buffer of the same molarity for 3 h. They were then dehydrated
in an acetone series and embedded in Araldite-Epon (Mollenhauer, 1964). Sections
were cut on a Servall MT-2 Ultramicrotome with a diamond knife. Thick sections
(0-5-1-0/*) were routinely dried on to glass slides and stained with toluidine blue for
examination in the light microscope. Thin sections were collected on 300 x 75 mesh
copper grids, stained with aqueous 2% uranyl acetate followed by lead citrate
(Reynolds, 1963), and viewed in a Hitachi HU-11A microscope at 75 kV with a 30/t
objective aperture.
OBSERVATIONS
Spiny vesicles similar to those first reported from Phaseolus (Newcomb, 1967) are
found in young phloem cells in the stem apices of Coleus. Although the 'spines' on
the vesicles in Coleus are similar in size to those reported from Phaseolus, it has not
been possible to resolve clearly their tubular nature. There is some indication that they
are not as well preserved by fixation in Coleus as in Phaseolus. Clusters of spiny vesicles
are observed frequently in young phloem parenchyma cells of Coleus; however, they
F'-Protein in the phloem of Coleus
157
are smaller and less striking than those encountered in Phaseolus root tips. Since
unequivocal evidence for the presence of spiny vesicles in sieve elements undergoing
differentiation has not yet been obtained, the following description of P-protein formation from spiny vesicles is based on observations made on phloem parenchyma cells.
The description of the subsequent stages is based on observations made on sieve
elements and sieve tubes as well as phloem parenchyma.
In the earliest observed stages of the formation of tubular P-protein (P1-protein of
Cronshaw & Esau, 1967), only a few tubules are present. Frequently these are seen
to be intimately associated with a cluster of spiny vesicles, many of which appear
indistinct as though undergoing dissolution. An example of a cell at a somewhat later
stage is illustrated in Fig. 1, which shows the developing tubules running out from a
localized cluster of disintegrating spiny vesicles. Both longitudinal and transverse
sections of similar cells have shown that the spiny vesicles are interspersed among the
tubules throughout a large part of the cytoplasm. The spiny vesicles in the clusters
appear to break down completely; no trace has been found of a possible stage where
the vesicles persist after losing their spines. These cells also contain an extensive
system of endoplasmic reticulum with associated polysomes.
A darkly staining fibrous material, similar in appearance to that observed in Phaseolus
(Newcomb, 1967), is present in the phloem parenchyma cells and sieve tubes. Larger
accumulations are regularly observed in phloem cells of the stem below the apex and
occasionally in the sieve elements of the leaf primordia. The origin and ultimate fate
of this material in Coleus have not been determined. In some micrographs it can be
seen in association with the spiny vesicles and developing P-protein tubules, suggesting
that it may contribute to formation of the latter.
The tubules of P-protein are 190-220 A in diameter, with an inner electrontransparent core about 50 A across. The wall (70-85 A thick) is not uniformly electrondense in longitudinal section. The staining pattern in transverse sections was examined
be means of the Markham rotational reinforcement method (Markham, Frey & Hills,
1963); a definite radially repeating structure could not be demonstrated.
The aggregation and orientation of the tubules in the sieve elements follows a
consistent sequence of development from association of tubules with spiny vesicles to
the formation of the completed P-protein body. Initially the tubules appear randomly
arranged, but they soon form groups, within each of which they are oriented parallel
to one another (Fig. 1). These groups merge to form a single large P-protein body in
the parenchyma cell or sieve element (Fig. 2). Clusters of spiny vesicles are not found
associated with the larger P-protein bodies in the parenchyma cells, although scattered
individual spiny vesicles are occasionally observed around their edges and amongst
the tubules. In transversely sectioned sieve elements, developing P-protein bodies are
circular to ellipsoidal in outline. Frequently the arrangement of the tubules suggests
that the bodies have a definite twist about their long axes. Microtubules are often seen
running from the plasmalemma into or alongside the developing P-protein body.
Conceivably they may be involved in the longitudinal alignment of the P-protein body
in the sieve element.
The completed tubular P-protein body is characteristically spindle-shaped in longi-
158
M. W. Steer and E. H. Newcomb
tudinal section (Fig. 3). In transverse section many of the tubules appear to be arranged
in hexagonal patterns around small, central, lightly staining structures. Although this
arrangement is observed in earlier stages where the tubules are not all parallel to the
long axis of the cell, greater regularity is found in later stages where most of the tubules
are parallel as illustrated in Fig. 5. Occasionally fine lines are seen radiating from the
tubules (Fig. 5, inset). The possibility that these are interconnecting strands between
the tubules has been mentioned by Cronshaw & Esau (1967).
Several authors (Northcote & Wooding, 1966; Cronshaw & Esau, 1967; Wooding,
1967) have recorded the conversion of similar P-protein tubules to striated or banded
fibrils (P2-protein of Cronshaw & Esau, 1967). Some further details of this conversion
have come to light in our observations on Coleus. Accompanying this conversion is the
breakdown of the P-protein body.
The early stages of the disaggregation of the large spindle-shaped P-protein body
occur before extensive disintegration of the cytoplasm and nucleus has taken place.
As seen in transverse section the outermost portions of the P-protein body spread
outward in a centrifugal manner, so that commonly the cytoplasm is almost filled with
large swirls of P-protein. An early stage in this process is shown in Fig. 4. The cytoplasm disintegrates leaving the lumen of the sieve tube largely filled with individual
P-protein fibrils and fibrillar aggregates (Figs. 6,7). The latter eventually break down to
form uniformly dispersed fibrils.
Alteration of structure in~the tubules occurs as the P-protein body disintegrates.
Initially, as noted previously, the diameter of the tubules is 190-220 A and that of the
central core 50 A. However, the diameter of the tubules at the earliest recorded stages
of P-protein body disaggregation is 170 A and that of the central core is only 30 A.
This is illustrated in Fig. 4, where a mass of fibrils has separated from the main body
of tubules. At later stages still smaller tubules, with a diameter of about 100 A (central
core 25 A), as well as fibrils about 70 A in diameter, can be found together in the same
sieve element. These fibrils sometimes appear to have a more lightly staining core, an
observation which will be discussed later.
Initially, the fibrils to which the tubules give rise are in aggregates. Transverse
sections of these aggregates reveal that the fibrils are packed into rows, each row being
staggered with respect to the next so that the fibrils are separated from one another by
a 10—20 A electron-transparent space (Fig. 6). In longitudinal section each fibril shows
a characteristic structure, consisting of alternating light and dark bands, each approximately 60 A in width. The bands on neighbouring fibrils are coincident, imparting a
transversely striped appearance to the aggregates (Fig. 4). Some of these aggregates
also have a longitudinal, darkly staining band giving rise to a pattern of squares (Fig. 7).
This is believed to result from sectioning the rows of fibrils at right angles. As stated
above, these aggregates disperse to form the individual banded fibrils.
DISCUSSION
The original suggestion that spiny vesicles in Phaseolus root tips contribute to
formation of the P-protein tubules (Newcomb, 1967) is further supported by the present
F'-Protein in the phloem of Coleus
159
study of Coleus stem apices. Groups of tubules in the earliest stages of formation are
seen lying at the edges of clusters of apparently disintegrating spiny vesicles; as the
tubular groups enlarge and consolidate, the vesicles disappear. It seems reasonable to
assume, therefore, that spiny vesicles contribute some or all of the material required
for tubule synthesis, at least in some of the differentiating cells of the procambial
strand. Most of this material is presumably proteinaceous, since protein is the major
component of P-protein bodies (e.g. see Cronshavv & Esau, 1967). A mechanism must
exist for transferring the protein from its site of synthesis on the ribosomes to the spiny
vesicles. Some observations that appear relevant to this problem are that in Phaseolus
the spiny vesicles are closely associated physically with both endoplasmic reticulum
and dictyosomes (Newcomb, 1967), and in Coleus a conspicuous group of cisternae of
endoplasmic reticulum is commonly observed lying near the spiny vesicles and developing tubules. Jamieson & Palade (1967 a, b) have reported that in certain animal systems
proteins produced by the ribosomes are transferred to the endoplasmic reticulum and
thence to the dictyosomes, and are subsequently released in vesicles. It may be suggested that the P-protein is transferred from the endoplasmic reticulum to the dictyosomes similarly, and is then incorporated into the structure of the spiny vesicles. On
the other hand, it is possible that the spiny vesicles arise directly from the endoplasmic
reticulum. The P-protein may be localized in the spines, since they constitute the
unique feature of these vesicles. However, since the entire vesicle structure appears
to break down during formation of the P-protein tubules, the fate of the remainder of
the vesicle is also in question.
The hexagonal arrangement of the tubules in the P-protein bodies may be a peculiarity of the Coleus phloem since it has not been reported from other plants. Also the
electron-dense 'centres' around which many of the tubules are arranged have not been
reported previously. These centres appear to be an integral part of the P-protein body
since they are observed consistently in the later stages of its formation. It is not possible
to determine whether the centres persist when the P-protein body disaggregates since
they would not be distinguishable from the fibrils in transverse sections of the fibrillar
aggregates.
The conversion of tubules to fibrils has been investigated in Acer by Northcote &
Wooding (1966). Fibrils 90-100 A in diameter were observed apparently fraying out
directly from tubules 180-240 A in diameter. They suggested that fibrils are formed
initially, and that these then associate to form tubules during P-protein body development. These tubules apparently disaggregate to form the original fibrils when the
sieve element matures. A subsequent study by Wooding (1967) on the same material
has provided further support for this process of disaggregation.
In Coleus there does not appear to be a stage at which the tubules are converted
suddenly to fibrils; rather, there is a gradation of intermediate forms which are recognizable in thin sections as tubules of progressively smaller diameter. Initially, as the
diameter of the tubule decreases from 200 A, the thickness of the wall is only slightly
reduced. Later the wall thickness is markedly reduced, since tubules 100-130 A in
diameter can be found alongside fibrils with a diameter of 70 A (Fig. 6). We have been
unable to observe the precise manner of formation of fibrils from tubules. The thick-
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M. W. Steer and E. H. Newcomb
ness of the tubule wall before P-protein body disaggregation occurs is similar to the
diameter of the fibrils formed subsequently (70 A), so that the simple disaggregation
process envisaged by Northcote and Wooding would be quite feasible. However, this
leaves unexplained the observation of tubules of intermediate dimensions in Coleus.
Cronshaw & Esau (1967) have suggested that in sieve elements of Nicotiana the
tubules may be converted to fibrils as a result of changes induced in the cytoplasm
by the vacuolar contents following breakdown of the tonoplast. Such a mechanism
does not appear to occur in Coleus since the tonoplast is still intact when the first fibrils
are formed. A possibly related difference between the two species is that in Coleus,
tubules are not observed to persist in mature sieve tubes, as reported for Nicotiana.
In Acer (Northcote & Wooding, 1966; Wooding, 1967) the alternating lightly and
darkly staining bands of the fibrils impart a striated appearance to the fibrillar aggregates. A similar staining pattern has also been observed in the fibrils of Nicotiana and
their aggregates by Cronshaw & Esau (1967). It should be emphasized that in Coleus
the fibrillar aggregates are derived directly from the P-protein body and that subsequently they disaggregate to give free, individual fibrils. In the aggregates many of
the fibrils appear to have a more lightly staining core in transverse section, suggesting
a tubular structure. However, this may be due to an uneven staining pattern, as can be
seen in longitudinal sections of the fibrils.
It appears that there is considerable variation in the form of P-protein even in the
relatively few_species of plants so far examined in detail. The sequence followed in its
development in Coleus seems to resemble the general pattern of development previously
reported for Nicotiana and Acer. However, Phaseolus, in which a large crystalline
P-protein body is produced (Lafleche, 1966), and Cucurbita, in which several darkly
staining spherical bodies are formed in each sieve element (Esau & Cheadle, 1965;
Evert, Murmanis & Sachs, 1966), are two examples of species having apparently quite
different patterns of development. Thus the pathway of P-protein differentiation seems
to be variable among different species of plants, suggesting that a detailed study of this
in a wide range of materials will be required before any general conclusions can be
reached. Of particular interest is the form that the P-proteins take in the mature
and presumably actively transporting sieve tubes. It is interesting to note, in light of
the current view that they may participate in the translocation process, that in the
three species (Acer, Nicotiana and Coleus) for which sufficiently detailed observations
are available the P-proteins of the mature sieve tubes are quite similar in appearance.
This work was supported in part by Grant GB-6161 from the National Science Foundation.
REFERENCES
J. & ESAU, K. (1967). Tubular and fibrillar components of mature and differentiating sieve elements. J. Cell Biol. 34, 801-815.
ESAU, K. & CHEADLE, V. I. (1965). Cytologic studies on phloem. Univ. Calif. Publ. Bot. 36, 253344ESAU, K. & CRONSHAW, J. (1967). Tubular components in cells of healthy and tobacco-mosaic
virus infected Nicotiana. Virology 33, 26—32.
EVERT, R. F., MURMANIS, L. & SACHS, I. B. (1966). Another view of the ultrastructure of
Cucurbita phloem. Ann. Bot. N.S. 30, 563-585.
CRONSHAW,
P-Protein in the phloem of Coleus
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JACOBS, W. P. & MORROW, I. B. (1958). Quantitative relations between stages of leaf development and differentiation of sieve-tubes. Science, N.Y. 128, 1084-1085.
JACOBS, W. P. & MORROW, I. B. (1967). A quantitative study of sieve-tube differentiation in
vegetative shoot apices of Coleus. Am. J. Bot. 54, 425-431.
JAMIESON, J. D. & PALADE, G. E. (1967a). Intracellular transport of secretory proteins in the
pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex..?. Cell Biol.
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JAMIESON, J. D. & PALADE, G. E. (19676). Intracellular transport of secretory proteins in the
pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J. Cell
Biol. 34, 597-6i5LAFLECHE, D. (1966). Ultrastructure et cytochimie des inclusions flagellees des cellules criblees
de Phaseolus vulgaris. J. Microscopie 5, 493-510.
MARKHAM, R., FREY, S. & HILLS, G. T. (1963). Methods for the enhancement of image detail
and accentuation of structure in electron microscopy. Virology 20, 88-102.
MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy.
Stain Technol. 39, 111-114.
NEWCOMB, E. H. (1967). A spiny vesicle in slime-producing cells of the bean root. J. Cell Biol.
35, C17-C22.
NORTHCOTE, D. H. & WOODING, F. B. P. (1966). Development of sieve tubes in Acer pseudoplatanus. Proc. R. Soc. B 163, 524-537.
NORTHCOTE, D. H. & WOODING, F. B. P. (1968). The structure and function of phloem tissue.
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REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron
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TAMULEVICH, S. R. & EVERT, R. F. (1966). Aspects of sieve element ultrastructure in Primula
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(Received 20 March 1968)
Cell Sci. 4
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M. W. Steer and E. H. Newcomb
Fig. i. A glancing longitudinal section of a differentiating phloem parenchyma cell in
which P-protein tubules are apparently arising from clusters of disintegrating spiny
vesicles. The circled cluster of vesicles is shown at higher magnification in the inset.
Other spiny vesicles are also discernible at the edges of the tubular mass. A sieve
tube (st) is present in the lower left, x 32000; inset, x 80000.
P-Protein in the phloem of Coleus
st
163
164
M. W. Steer and E. H. Newcomb
Fig. 2. Longitudinal section of a differentiating phloem parenchyma cell showing
a stage in the aggregation of groups of tubules into a P-protein body, x 16000.
Fig. 3. Longitudinal section of a fully developed P-protein body in a sieve element.
Most of the tubules lie parallel to one another and approximately parallel to the long
axis of the cell, x 18000.
P-Protein in the phloem of Cokus
.*• - ^ j j ^
166
M. W. Steer and E. H. Newcomb
Fig. 4. Oblique section through a P-protein body in an early stage of disaggregation.
Many of the fibrillar aggregates in the lower part of the figure exhibit a characteristic
striated pattern in longitudinal section, x 45 000.
P-Protein in the phloem of Coleus
•
^
#
•
168
M. W. Steer and E. H. Newcomb
Fig. 5. Transverse section of a sieve element containing a completed P-protein body.
The regular hexagonal arrangement of the tubules around electron-dense centres is
evident. The inset shows this at higher magnification. Fine lines can be seen radiating
from some of the tubules, x 30000; inset, x 80000.
Fig. 6. Transverse section of fibrillar aggregates formed at later stages of P-protein
body disaggregation. A region can be seen where the rows of fibrils are staggered
with respect to one another, x 100000.
Fig. 7. A relatively uncommon square pattern seen in longitudinal sections of fibrillar
aggregates. It is possible that this pattern results from sectioning an aggregate normal
to the rows of fibrils, x 100000.
P-Protein in the phloem of Coleus