Annals of Botany 79 : 89–100, 1997
The Cellular Pathway of Short-distance Transfer of Photosynthates and Potassium
in the Elongating Stem of Phaseolus vulgaris L.
A Structural Assessment
R O B Y N M. W O O D, C H R I S T I N A E. O F F L E R* and J O H N W. P A T R I CK
Department of Biological Sciences, The UniŠersity of Newcastle, NSW 2308, Australia
Received : 26 May 1995
Accepted : 1 September 1995
The potential cellular pathway of radial transfer of photosynthate and potassium delivered in the phloem to the
elongation zone (apical 0±5–2±5 cm) of internode 2 of Phaseolus Šulgaris L. seedlings was elucidated. This was achieved
using ultrastructural observations of the cell types that constitute the radial pathway and estimates of potential
sucrose and potassium fluxes through the cross-sectional area of interconnecting plasmodesmata and across the
plasma membrane surface areas of selected cell types. The investigation relied on predicting the relative roles of the
mature and developing sieve elements as conduits for the axial delivery of solutes to the elongation zone. In turn, these
predictions led to formulation of two transport models which were subsequently evaluated. It was found that
unloading of sucrose and potassium from the protophloem sieve elements cannot be through the symplast due to the
absence of plasmodesmata. On the other hand, mature metaphloem sieve element-companion cell complexes have the
potential capacity to unload either through the stem symplast or apoplast. The potential symplastic route is proposed
to be via the companion cells to the adjacent large phloem parenchyma cells. Continued radial transfer could occur
either by exchange to the stem apoplast from the large phloem parenchyma cells or continue in the symplast to the
ground tissues. It was further predicted that sucrose utilized for the development of the procambial}small phloem
parenchyma cells could be delivered axially by them and not by the mature sieve elements.
# 1997 Annals of Botany Company
Key words : Phaseolus Šulgaris, apoplast, elongating stem, photosynthates, potassium, transport, symplast.
INTRODUCTION
As a model to examine phloem unloading in growing
tissues, an investigation of the radial transfer of photosynthates and potassium within the elongating zone of
internode 2 of Phaseolus Šulgaris seedlings was undertaken.
Since the bulk of sucrose and 72–82 % of the potassium is
transported in the phloem to the elongation zone (Wood,
Patrick and Offler, 1994), radial transfer of these solutes
occurs from the sieve elements. The predominant sink cells
of the ground tissues of the elongation zone are the pith and
cortex for photosynthates and the pith for potassium
(Wood et al., 1994). The cellular pathways followed by the
radial transfer of sucrose and potassium from the phloem to
these sinks remain to be identified.
In general, maturity of sieve elements would appear to be
a prerequisite for effective long-distance movement of
sucrose (Fisher, 1975 ; Schultz and Gersani, 1990). However,
the elongating stem contains metaphloem sieve element–
companion cell (se–cc) complexes at a range of developmental stages (Wood et al., 1994). In this context, it is
considered that over short distances (mm), axial transfer of
sucrose through differentiating sieve elements may be
sustained at rates comparable to those through mature
elements (Schultz, 1987 ; Schultz and Gersani, 1990).
Therefore, the differentiating sieve elements may contribute
* For correspondence.
0305-7364}97}010089­12 $25.00}0
to the axial delivery of sucrose to the elongation zone. In
contrast, for potassium there is no information available to
predict the extent of axial delivery through developing
phloem cells. Given these circumstances, it was considered
necessary to establish which phloem cells function as
conduits for the axial delivery of solutes to the elongation
zone prior to assessing the cellular pathway of radial
transfer in the elongating stem.
This paper presents a structural assessment of the phloem
cell types comprising the radial pathway for their capacities
to support symplastic and}or apoplastic solute transfer. The
potential for symplastic and apoplastic transfer was assessed
both qualitatively and quantitatively. The qualitative assessment involved an ultrastructural examination of each
cell type. The quantitative assessment was based on an
evaluation of potential sucrose and potassium fluxes through
the total cross-sectional area of plasmodesmata interconnecting the cell types and across their total plasma
membrane surface areas (e.g. see Hayes, Offler and Patrick,
1985 ; Faraday, Quinton and Thomson, 1986). The latter
approach relies on an understanding of the partitioning of
solute delivery through the mature and developing sieve
elements (see above). These predictions lead to the formulation of two transport models to assess the radial
cellular pathway of transfer from the sieve elements to the
surrounding sink cells. The pathway models proposed in
this paper are evaluated physiologically in a subsequent
paper.
bo960307
# 1997 Annals of Botany Company
90
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
F. 1. Electron micrograph showing the spatial arrangement and structural characteristics of the cell types of the proto- and metaphloem in the
elongation zone (apical 0±5–2±5 cm) of internode 2 of Phaseolus Šulgaris L. seedlings. The material was sampled from internodal material excised
on day 4 of elongation (see Fig. 1, Wood et al., 1994). The mature protophloem sieve elements are located towards the epidermis and are
surrounded by phloem parenchyma. Note their lumen and undulating plasma membrane. A group of metaphloem sieve element–companion cell
complexes at varying stages of differentiation (S 1–6) are associated with a few small phloem parenchyma cells and surrounded by large phloem
parenchyma cells. The pre-division procambial}small phloem parenchyma cells (S 1) are densely cytoplasmic and as they commence
differentiation into the sieve element–companion cell complexes they exhibit vacuolation (S 2 and 3). The differentiating sieve elements become
vacuolated, their cytoplasm becomes less dense and peripheral (S 5) and finally is reduced to the parietal form characteristic of mature sieve
elements (S 6). Their walls become thickened and exhibit an electron dense inner portion. The differentiating companion cells retain a large nucleus
and dense cytoplasm which is rich in mitochondria, rough endoplasmic reticulum and free ribosomes (see Fig. 2 B). Bar ¯ 1 µm. lpp, large phloem
parenchyma ; m, mitochondrion ; mcc, metaphloem companion cell ; mse, metaphloem sieve element ; n, nucleus ; pc}spp, procambial}small phloem
parenchyma ; pm, plasma membrane ; pp, phloem parenchyma ; pse, protophloem sieve element ; r, free ribosome ; S1–S6, stages of differentiation
of metaphloem sieve element-companion cell complex ; rer, rough endoplasmic reticulum ; Š, vacuole.
MATERIALS AND METHODS
Plant material
Plants of French bean, Phaseolus Šulgaris L. cv. Redland
Pioneer, were raised in pots under glasshouse conditions as
described previously (Wood et al., 1994). All studies were
carried out on the apical 0±5–2±5 cm portion of internode 2
during days 2–5 of elongation (Fig. 1, Wood et al., 1994).
Light and transmission electron microscopy
Light microscopy. Tissue slices, 0±01–0±02 cm thick, were
cut transversely from the mid-position of the apical
0±5–2±5 cm elongation zone of internode 2 and fixed for 24 h
in 2±5 % glutaraldehyde and 2±5 % paraformaldehyde
buffered with 20 m sodium phosphate buffer (pH 6±8).
Following removal of fixative, tissue was dehydrated in
acetone through a 10 % step-graded series and infiltrated
and embedded in Spurr’s epoxy resin (Spurr, 1969 and for
details of the procedure, see Wood et al., 1994).
Sections, 3–4 µm thick, were cut with glass knives using
an LKB. (Stockholm, Sweden) Ultramicrotome (Model No.
8800) and stained with toluidine blue in benzoate buffer
(pH 4±4).
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
Transmission electron microscopy. Tissue was sampled
and processed as for light microscopy, but included a 2-h
post-fixation at 4 °C in 2 % osmium tetroxide in 20 m
phosphate buffer.
Sections, 50 nm thick, were cut on a diamond knife using
an Ultracut E Reichert ultramicrotome, collected on
formvar-coated nickel slot grids and stained with uranyl
acetate and lead citrate for 15 min each. The sections were
examined with a Jeol JEM 100 CX electron microscope.
Sucrose and potassium deliŠery rates
Delivery rates of sucrose and potassium were determined
from the data presented by Wood et al. (1994). Two sucrose
delivery rates were estimated depending upon the contribution of stem photosynthesis and the cellular sites of
respiratory loss (see Results and Discussion). These rates
were computed using dry weight gain of the elongation
zone, adjusted to allow for 80 % of dry weight (and
respiratory loss) being delivered to this region as sucrose
(Patrick and Turvey, 1981).
Potassium delivery rates were based on the total amount
of potassium retained by the elongation region and took
into account that 72–82 % of potassium was delivered
through the phloem (Wood et al., 1994).
Potential sucrose and potassium flux estimations
Plasmodesmatal fluxes. Potential sucrose and potassium
fluxes [mol (solute) m−# (plasmodesma) s−"] were computed
assuming that the observed delivery of solutes occurred
exclusively through the total cross-sectional area of plasmodesmata interconnecting contiguous cell types. Total plasmodesmatal cross-sectional area m−" stem length was estimated
for contiguous cell types of the phloem (see justification in
Results and Discussion) using plasmodesmatal frequency
(number µm−# cell wall), the surface area of shared wall
(µm# per m stem length) and mean plasmodesma crosssectional area. Measurements were made from montages of
photomicrographs of cells of the phloem from three stem
portions of four plants fixed on day 4 of elongation (Wood
et al., 1994). At this developmental stage, both immature
(procambial cells) and mature sieve elements were present in
similar numbers (Wood et al., 1994). The position of
plasmodesmata, sectioned longitudinally and obliquely,
were observed during subsequent electron microscopic
examination and recorded on these montages. To obtain
plasmodesmatal density (number µm−" cell wall), the lengths
of cell wall interfaces of all contiguous cell types, within a
particular vascular bundle, were measured on the montages
with a chartometer. The dimensions of longitudinallysectioned plasmodesmata were measured against a photomicrograph of a catalase crystal lattice with a d spacing of
8±75 nm. The density and dimension data were used, with a
section thickness of 50 nm, to compute plasmodesmatal
frequency (number µm−# wall) according to the procedure
described by Robins and Juniper (1980) and verified by
Gunning, Robins and Juniper (1981). Shared wall surface
91
area was computed for 1 m length of stem using the
measured length of cell wall interface. Mean plasmodesma
radii were computed from diameter measures and used to
calculate mean cross-sectional areas per plasmodesma.
The use of total cross-sectional area of plasmodesmata to
compute fluxes for solute transfer allows comparisons to be
made with flux values reported for systems where solute
flow is known to be restricted to the symplast (Kuo, O’Brien
and Canny, 1974 ; Gunning and Hughes, 1976 ; Robards
and Clarkson, 1976). It is appreciated that fluxes computed
on this basis may be an underestimate, as the fine structure
of plasmodesmata and the actual route of solute transfer
through them are not known (for a complete discussion, see
Offler and Patrick, 1993).
Plasma membrane fluxes. Potential fluxes [mol (solute)
m−# (plasma membrane) s−"] were computed for both
sucrose and potassium assuming that the radial transfer of
each solute delivered to the elongation zone was exclusively
through the total plasma membrane surface area of the
specified cell type. Plasma membrane surface areas [m# m−"
(stem)] were estimated for all cell types within the phloem.
Mean cell perimeter, cell length and total cell number were
obtained as described previously (Wood et al., 1994). Total
end wall surface area was computed using cell length to
estimate the number of end-walls per m stem length and
mean perimeter to compute cell radius. Total membrane
surface area was calculated as the sum of (membrane
surface area¬total cell number¬1 m stem) plus (end wall
surface area of a cell in TS¬2¬number of cells in 1 m
length¬number of cells in TS).
RESULTS AND DISCUSSION
Ultrastructural characteristics of internode 2 cells of the
elongation zone
Consideration is given to the ultrastructural features of cells
of internode 2 that are pertinent to plasma membrane and
plasmodesmatal transfer (e.g. Offler and Patrick, 1984 ;
Hayes et al., 1985 ; Warmbrodt, 1985 a, b ; Fahn, 1988).
Ultrastructure of the phloem cells. The micrograph
presented in Fig. 1 is representative of the spatial arrangement and general ultrastructural features of each cell
type within the phloem in the elongation zone of internode
2. Both proto- and meta-phloem are present within each
vascular bundle (Wood et al., 1994). The protophloem
consists of three cell types, namely sieve elements, phloem
fibres (called pericycle by Doutt, 1932), and phloem
parenchyma ; the metaphloem consists of se–cc complexes,
procambial cells and small and large phloem parenchyma
cells (Wood et al., 1994).
The ultrastructure of the protophloem and mature
metaphloem sieve elements is consistent with that described
for fully functional sieve elements (Fisher, 1975). The
protophloem sieve elements were fully differentiated (cf.
Eleftheriou and Tseko, 1982) and the undulated plasma
membranes remained intact (Fig. 1) during the entire period
of internode elongation. The lumen of some protophloem
sieve elements was almost devoid of stainable material
except for parietally distributed membranous material and
92
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
F. 2. Electron micrographs showing some cell types of the phloem from the elongation zone (apical 0±5–2±5 cm) of internode 2 of Phaseolus
Šulgaris L. seedlings. A, A procambial}small phloem parenchyma cell in the process of division. Note the two nuclei and dense cytoplasm rich
in mitochondria and rough endoplasmic reticulum. Bar ¯ 0±5 µm. B, Part of a metaphloem sieve element–companion cell complex and large
phloem parenchyma cell. The sieve element and companion cell are interconnected by a multiple-branched plasmodesma (indicated by an
arrowhead). The mature sieve element is almost devoid of cytoplasm, but the plasma membrane is evident. The vacuolated companion cell has
dense cytoplasm rich in mitochondria and free ribosomes. The peripheral cytoplasm of the large phloem parenchyma cell is less dense and is
dominated by amyloplasts with starch grains. However, note the two vacuoles and abundant mitochondria. Bar ¯ 0±5 µm. C, Part of a
metaphloem companion cell and procambial}small phloem parenchyma cell. Both cells are densely cytoplasmic with abundant rough endoplasmic
reticulum and free ribosomes. Mitochondria are in evidence in the companion cell, while dictyosomes and small vacuoles are apparent in the
procambial}small phloem parenchyma. Note that the two cells are interconnected by a branched plasmodesma (indicated by an arrowhead). Bar
¯ 0±5 µm. D, Phloem fibre cap cell showing the degenerating cytoplasm with remnant membranous material, mitochondria and amyloplasts. The
plasma membrane is evident and the cell walls are unthickened. Bar ¯ 0±5 µm. a, amyloplast ; d, dictyosome ; dcw, developing cell wall ; lpp, large
phloem parenchyma ; m, mitochondrion ; mcc, metaphloem companion cell ; mm, membranous material ; mse, metaphloem sieve element ; n,
nucleus ; pc}spp, procambial}small phloem parenchyma ; pm, plasma membrane ; r, ribosomes ; rer, rough endoplasmic reticulum ; Š, vacuole.
a few mitochondria and plastids (Fig. 1). However, other
protophloem sieve elements also contained dispersed Pprotein (not shown). The mature metaphloem sieve elements
(developmental stage 6, see Fig. 1, S6) contained a parietal
layer of cytoplasm with a few mitochondria, plastids, Pprotein and smooth endoplasmic reticulum (ER). The latter
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
was usually stacked and closely aligned to the cell wall. The
cytoplasm of the associated companion cells was dense
(Figs 1 and 2 B, C) and contained numerous mitochondria,
a prominent nucleus, rough ER and abundant free ribosomes surrounding one large or many small vacuole(s). The
cell walls of the metaphloem sieve elements were generally
thicker than those of the protophloem and had a characteristic electron-dense inner portion (Fig. 1).
In addition to mature metaphloem se–cc complexes,
differentiating complexes were evident. These were derived
from procambial cells which differentiated into metaphloem
se–cc complexes and small phloem parenchyma. Developmental stages from pre-division procambial cells to
differentiating complexes occurred in each vascular bundle
(Fig. 1). The metaphloem se–cc complexes were associated
in groups of up to ten complexes with none to three mature
complexes (developmental stage 6, see Fig. 1, S6). Those
groups without mature complexes were located closest to
the interfascicular regions of each vascular bundle. Division
and differentiation of these cells appeared to proceed
through previously reported stages of development of such
cells (Evert, Murmanis and Sachs, 1966 ; Cronshaw, 1974 ;
Esau, 1978 ; Esau and Thorsch, 1985). Prior to division to
form se–cc complexes, the procambial cells were densely
cytoplasmic with numerous mitochondria and free ribosomes indicative of highly metabolically-active cells [Fig. 1,
developmental stage 1 (S1)]. In addition, they contained
small vacuoles and a nucleus in which the double nuclear
membrane was prominent. In the developing sieve elements,
the vacuoles became progressively larger (Figs 1, see S2, S3
and 2 A) and the cytoplasm less dense as the cellular content
began to break down (Fig. 1, see S4, S5). The cytoplasm of
these cells (Fig. 2 A, C) contained rough cisternal ER, many
free ribosomes and mitochondria, dictyosomes and a nucleus
in various stages of degradation. The cytoplasm in the
associated companion cell was often more dense (Fig. 1).
Throughout their development (Fig. 1, see S1–S6), the companion cells retained a nucleus, many large, prominent
mitochondria, numerous free ribosomes (Fig. 2 B, C), some
dictyosomes and a varying number of vacuoles. On the basis
of their ultrastructure (Figs 1 and 2 A), the differentiating
se–cc complexes would not be considered by some authors
to be sufficiently mature to conduct (Fisher, 1975).
The small groups of protophloem sieve elements were
surrounded by phloem parenchyma (Wood et al., 1994) and
each group of mature and differentiating metaphloem se–cc
complexes was associated with some small phloem parenchyma cells and surrounded by a single layer of large
phloem parenchyma cells (Fig. 1). The mature se–cc
complexes were generally located on the perimeter of each
group with the companion and small phloem parenchyma
cells contiguous with the surrounding large phloem parenchyma cells. The ultrastructure of the phloem parenchyma
cells of the protophloem and large phloem parenchyma cells
of the metaphloem was indistinguishable (Fig. 1) and was
consistent with a storage function (Fig. 2 B). They contained
either a large central vacuole surrounded by a thin peripheral
cytoplasm or, for some cells, a number of small vacuoles.
Within the cytoplasm, there was a nucleus, cisternal and}or
stacks of rough ER, amyloplasts containing one or more
93
starch grains, some free ribosomes and mitochondria with
well-developed cristae. The ultrastructure of the small
phloem parenchyma cells of the metaphloem was similar to
that of the procambial cells described previously (Fig. 2 C).
Outward of the phloem parenchyma of the protophloem lie
fibre cap cells (Wood et al., 1994). The plasma membrane of
these cells remained intact and close to the unthickened cell
wall (Fig. 2 D). In contrast, the tonoplast was not evident
during any stage of elongation and, while organelles were
still evident, the cytoplasm had undergone substantial
degeneration (Fig. 2 D). It is apparent from the above
descriptions that the phloem parenchyma of the protophloem could not be confidently distinguished from the
large phloem parenchyma of the metaphloem nor were the
procambial and small phloem parenchyma cells readily
identified. Thus, for the purposes of the quantitative
assessment the protophloem phloem parenchyma have been
treated as large phloem parenchyma and the procambial
and small phloem parenchyma cells are referred to subsequently as procambial}small phloem parenchyma (pc}spp).
Symplastic continuity of the radial pathway. The protophloem sieve elements were symplastically interconnected to
one another by single or double, simple or branched
plasmodesmata, but were symplastically isolated from the
other cell types (Table 1). The metaphloem sieve elements
also were symplastically joined to one another but
symplastically isolated from contiguous cells except via
their associated companion cells (Table 1). The plasmodesmata interconnecting the metaphloem sieve elements
and companion cells were multiple-branched (Fig. 2 B). The
branches were typically on the side of the companion cell
within an extended cell wall (see Hayes et al., 1985). The
companion cells were connected with both the procambial}
small (Fig. 2 C) and large phloem parenchyma cells by
simple and bifurcating plasmodesmata situated within a
thickened wall (Table 1).
A symplastic discontinuity existed between the phloem
parenchyma cells of the protophloem and adjacent phloem
fibre cap cells. In contrast, the phloem parenchyma cells
associated with the metaphloem se–cc complexes were
symplastically interconnected to the cells of both the
interfascicular region and the vascular cambium (data not
shown). Inward from the phloem, plasmodesmata were
present between the cells of the vascular cambium, xylem
parenchyma and pith, and outwardly, between the cells of
the cortex and epidermis (data not shown). Generally these
plasmodesmata occurred singly at relatively low densities.
On the basis of the distribution of plasmodesmata
described above, it can be concluded that symplastic
unloading from the sieve elements is only possible from the
metaphloem se–cc complexes. Subsequent symplastic transfer towards the pith may be possible through the
procambial}small and large phloem parenchyma cells.
Transfer could occur either in series or parallel through
these phloem cells to the vascular cambium and interfascicular region. Movement beyond the phloem and
through the cells of the pith and interfascicular parenchyma
may be limited by the relatively low numbers of plasmodesmatal connections. Similarly, symplastic transfer to the
cortical and epidermal cells may be restricted by the absence
94
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
T     1. Structural characteristics, diameters and frequencies of plasmodesmata interconnecting the contiguous cell types
within the phloem of the apical 0±5–2±5 cm of internode 2 of Phaseolus vulgaris L. seedlings during elongation growth
Plasmodesmatal characteristics
Contiguous
cell types*
Protophloem
pse-pse
pse-pc}spp
pse-lpp
Metaphloem
mse-mse
mse-mcc
mse-pc}spp
mcc-pc}spp
mcc-lpp
Phloem parenchyma
pc}spp
pc}spp-lpp
lpp-lpp
Plasmodesmatal frequency (¬10()
Type
Internal
diameter
(10−( cm)
Simple, branched
n.d.†
n.d.
32³2
—
—
0±47
—
—
0±28
—
—
Branched
Multiply-branched
n.d.
Simple, bifurcating
Simple, bifurcating
—
35³2
—
31³2
31³3
0±86
0±81
—
0±43
0±59
0±88
2±04
—
0±32
1±25
Simple
Simple
Simple, bifurcating
30³3
33³2
35³1
1±67
1±57
3±74
7±03
10±35
70±00
(µm−# wall) (cm−" stem)
* pse, protophloem sieve element ; mse, metaphloem sieve element ; mcc, metaphloem companion cell ; pc}spp, procambial}small phloem
parenchyma ; lpp, large phloem parenchyma.
† n.d., plasmodesmata not detected.
of plasmodesmata between the phloem parenchyma and
fibre cap cells of the protophloem. Transfer through the
fibre cap also may be restricted by the apparent breakdown
of the cytoplasmic content of these cells (Fig. 2 A). This
means that a symplastic route would involve by-passing
these cells by moving through the interfascicular parenchyma.
Cross-sectional area of plasmodesmata. The total crosssectional areas of plasmodesmata interconnecting phloem
cell types were determined from plasmodesmatal dimensions
and densities and the length of contact wall within the
elongation zone of internode 2. The internal diameters of
the plasmodesmata (Table 1) were similar to the equivalent
plasmodesmata in mature stems of P. Šulgaris (Hayes et al.,
1985) and of Ricinus (van Bel and Kempers, 1991). This
indicates that plasmodesmatal size did not alter with stem
development. The frequencies of plasmodesmata (Table 1)
were expressed on a wall surface area basis (µm−# wall), to
allow for comparison with other published values ; and per
centimetre length of stem (cm−" stem). Plasmodesmatal
frequencies (µm−# wall) were well within the range of 0±1–
10±0 µm−# wall (Robards and Lucas, 1990) and were
comparable with those reported in the mature bean stem
(Hayes et al., 1985). The latter observation suggests that the
plasmodesmata must be continually laid down during stem
development of the conducting cells. The exception to the
above generalization is the plasmodesmata interconnecting
the large phloem parenchyma. Their frequency in the
elongating stem is seven-fold higher than that of the mature
stem (0±57 µm−# and 3±7 µm−#, respectively). These comparisons infer that, during expansion growth, plasmodesmatal numbers are reduced in frequency between the
large phloem parenchyma. Such a change in frequency
during development has been demonstrated in developing
tomato fruit (Johnson, Hall and Ho, 1988), in leaves of
tobacco (Ding et al., 1988) and in the floral nectary gland of
Hibiscus (Robards and Lucas, 1990).
Assessed capacity for radial symplastic and apoplastic
transfer
The structural capacity for symplastic unloading from the
metaphloem sieve elements and subsequent radial transfer
to the surrounding phloem cells was assessed quantitatively
based on estimates of the potential plasmodesmatal fluxes
of sucrose and potassium. These were computed on the
assumption that the observed rates of solute accumulation
by the elongation zone (Wood et al., 1994) resulted from
solute passage through the total cross-sectional area of
plasmodesmata interconnecting the various phloem cell
types.
The absence of plasmodesmata from the protophloem
sieve elements and low plasmodesmatal frequencies interconnecting the phloem parenchyma cells with the adjacent
ground tissues suggested the possibility of apoplastic steps
at these interfaces. To examine this possibility, the capacity
of the various types of phloem cells to support solute
transfer to the stem apoplast was assessed using estimates of
potential solute fluxes across their plasma membranes.
However, before the radial transfer pathway could be
considered it was necessary to estimate the relative proportion of solute delivered in each conducting cell type.
Two models for radial transfer (Fig. 3) were considered.
Model 1 was constructed on the assumption that the axial
delivery of solutes occurred exclusively through the mature
proto- and meta-phloem sieve elements (Fisher, 1975).
Model 2 was proposed in recognition of the fact that
developing sieve elements and small phloem parenchyma
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
Model 1
pse
mse
4
lpp
3
4
mcc
pc-spp
1
1
A
2
3
2
2
B
Model 2
pse
mse
lpp
4
6
5
3
pc-spp
mcc
1
2
1
A
3
2
2
B
C
F. 3. Diagrammatic representation of the possible axial and radial
pathways of sucrose and potassium transfer through the elongation
zone (apical 0±5–2±5 cm) of internode 2 of Phaseolus Šulgaris L.
seedlings. Model 1 is based on the proposal that the solutes are
delivered axially via (A) the protophloem sieve elements and (B) the
metaphloem se–cc complexes. Model 2 is based on the proposal that
the solutes are delivered axially via (A) the protophloem sieve elements,
(B) the metaphloem se–cc complexes, and (C) the procambial}small
phloem parenchyma cells. lpp, large phloem parenchyma ; mcc,
metaphloem companion cell ; mse, metaphloem sieve element ; pc}spp,
procambial}small phloem parenchyma ; pse, protophloem sieve element. Potential symplastic –*Y, and apoplastic –DY routes.
(Numbers are referred to in text).
cells, in addition to mature sieve elements, may be involved
in axial delivery of solutes to the elongation zone (Schultz
and Gersani, 1990). The former cells are arranged in axial
continuity with mature sieve elements located immediately
beneath the elongation zone. It was further assumed that
sucrose delivered to this region is retained by the differentiating metaphloem se–cc complexes for their growth.
Axial rates of deliŠery of sucrose and potassium. For
models 1 and 2, partitioning of the total solute delivery
between the conducting cell types was based on the
assumption that their hydraulic conductivities were comparable. Under these conditions, solute flow rate through
each cell type would be a function of their relative crosssectional areas. In the case of the mature sieve elements,
their hydraulic conductivities would be determined by the
number and radii of the sieve pores located in their
95
transverse walls. It is probable that these sieve pore
characteristics do not differ markedly between proto- and
meta-phloem sieve elements. For the second model, the
hydraulic conductivity of differentiating sieve elements
would be less than that of mature elements but is unknown.
However, if transfer occurred through the differentiating
elements this would involve distances of a millimetre or less.
Hence, the hydraulic conductivities of these cells could well
approximate that of mature sieve elements in the elongation
zone. At this stage of development, the sieve pores have not
formed in the differentiating sieve elements and the
interconnections are still in the form of plasmodesmata.
Hence, based on their ultrastructure there is probably very
little difference between the small phloem parenchyma and
the procambial cells in this regard and so they can be treated
equally in terms of the axial passage of solutes through
them.
The quantity of the total imported sucrose depends on the
contribution of stem photosynthesis (Wood et al., 1994) to
the carbon budget of the elongating zone. Two scenarios
were considered for model 2. The first was that stem
photosynthesis re-fixed all respired carbon dioxide so that
the total carbon gains by the elongation zone resulted
exclusively from import through the phloem thus providing
maximum estimates of the sucrose rates of radial transfer
(Table 2). The second scenario assumed that stem photosynthesis drew exclusively on carbon dioxide supply from
the ambient atmosphere and that all the respiratory losses
could be ascribed to the metabolic activity of the conducting
cells (i.e. pse, mse–cc and pcc}spp). This second scenario,
therefore, provides a minimal estimate for the total rate of
sugar unloading from the conducting cells of the phloem
(Table 2). Applying the above assumption, the respiratory
rates of the conducting cells were estimated to be 4300 µl g−"
(f.wt) h−". This estimate was derived from the observed stem
respiratory rate of 215 µl g−" (f.wt) h−" (Wood et al., 1994)
and a relative stem volume occupied by the conducting cells
of 5 % (Wood, unpubl. res.). Our estimate exceeds measured
respiratory rates of excised phloem strips by some two-fold
(Bieleski, 1966). At least part of this difference could be
accounted for the higher respiratory activity of differentiating tissues (e.g. see Ho, Grange and Picken, 1989). Thus,
these considerations provide some qualified justification for
the proposed scenario that the bulk of the stem respiratory
activity could be confined to the conducting tissues. In
addition, for model 2, we assumed that all carbon that
entered the elongation zone through the procambium}small
phloem parenchyma cell path was retained by these cells
and utilised in their respiratory and growth activities. The
latter assumption finds support in the observation that
procambial}small phloem parenchyma cells have been
shown to be strong sinks for photoassimilates (Schultz and
Gersani, 1990).
For model 1 (Fig. 3), in accordance with the changing
number of the two types of mature sieve elements during
elongation (Wood et al., 1994), the predicted rates of axial
delivery of sucrose and potassium through the protophloem
sieve elements halved, while those for the metaphloem se–cc
complexes doubled (Tables 2 and 3). Thus, model 1 predicts
that the protophloem sieve elements play a decreasing role
96
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
T     2. ObserŠed and predicted rates of axial deliŠery of sucrose to the apical 0±5–2±5 cm of internode 2 of Phaseolus
vulgaris L. seedlings during 4 d of elongation growth (i.e. days 2–5 ; see Wood et al., 1994)
Rate of sucrose delivery (10−* mol m−"
stem s−")
days 2–3
days 3–4
days 4–5
Refixation of respired imported carbon with no net photosynthetic gain†
Estimated total
7±96
8±63
Predicted by model 1§
pse*
4±35
3±49
mse–cc
3±58
5±13
Predicted by model 2s
pse
1±86
1±71
mse–cc
1±53
2±52
pc}spp
4±56
4±40
All respiratory losses from conducting cells (i.e. Mse–cc and Pc/spp)‡
Estimated total
5±75
5±32
Predicted by model 2s
pse
1±35
1±06
mse–cc
1±11
1±56
pc}spp
3±30
2±71
8±88
2±17
6±71
1±41
4±34
3±13
5±64
0±89
2±75
1±99
* pse, protophloem sieve element ; mse–cc, metaphloem sieve element-companion cell complex ; pc}spp, procambial}small phloem
parenchyma.
† Total sucrose delivery was determined from the dry weight gain and dry weight loss as a balance between net photosynthesis and respiration
(Table 2, Wood et al., 1994) and adjusted to allow for 80 % being delivered as sucrose to the elongation zone.
‡ Total sucrose delivery was determined for dry weight gain (Table 2, Wood et al., 1994) and adjusted to allow for 80 % being delivered as
sucrose to the elongation zone.
Rates of delivery for Models 1 and 2 are assumed to be proportional to the relative cross-sectional area occupied by each proposed cell type.
§ Based on the assumption that sucrose is delivered axially in the mature sieve elements of the proto- and meta-phloem (Fig. 3, model 1).
s Based on the assumption that sucrose is delivered axially in the protophloem sieve elements, metaphloem se–cc complexes and
procambial}small phloem parenchyma cells (Fig. 3, model 2).
T     3. ObserŠed and predicted rates of axial deliŠery of potassium to the apical 0±5–2±5 cm of internode 2 of Phaseolus
vulgaris L. seedlings during 4 d of elongation growth (i.e. days 2–5 ; see Wood et al., 1994)
Rate of potassium delivery (10−* mol m−" stem s−")
days 2–3
days 3–4
days 4–5
2±32
2±98
3±23
1±27
1±05
1±20
1±76
0±80
2±44
0±54
0±45
0±13
0±59
0±87
1±52
0±51
1±58
1±14
Observed†
Predicted by model 1‡
pse*
mse–cc
Predicted by model 2§
pse
mse–cc
pc}spp
* pse, protophloem sieve element ; mse–cc, metaphloem sieve element-companion cell complex ; pc}spp, procambial}small phloem
parenchyma.
† Total potassium delivery was determined as the total amount of potassium retained by the elongation zone.
Rates of delivery are assumed to be proportional to the relative cross-sectional area occupied by each cell type :
‡ Based on the assumption that potassium is delivered axially in the mature sieve elements of the proto- and metaphloem.
§ Based on the assumption that potassium is delivered axially in the protophloem sieve elements, metaphloem se–cc complexes and
procambial}small phloem parenchyma.
in delivery of the two solutes during elongation. In contrast,
the role of the metaphloem se–cc complexes increased
significantly as elongation progressed (Tables 2 and 3).
For model 2 (Fig. 3), the rates of axial delivery through
the protophloem sieve elements for sucrose, estimated from
dry weight gain and with and without dry weight loss as a
balance between net photosynthesis and respiration (Table
2), and for potassium (Table 3), were predicted to decrease
slightly from 23 to 16 % of the total delivery rate. A
concomitant decrease in the axial delivery rates through the
procambial}small phloem parenchyma cells is also predicted
resulting from a decline in their cross-sectional area as the
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
97
T     4. Estimates of sucrose and potassium fluxes through the total cross-sectional area of plasmodesmata interconnecting
contiguous cell types of the metaphloem in the apical 0±5–2±5 cm of internode 2 of Phaseolus vulgaris L. seedlings on day 4
of elongation growth
Estimated flux† (mol m−# s−")
Cell types*
Mse
Mcc
Pc}spp
Lpp
mcc
pc}spp
lpp
2±9¬10−$ (1)‡
not applicable
not applicable
1±8¬10−# (2)
4±7¬10−$ (3)
8±3¬10−%
5±6¬10−% (4)
5±3¬10−&
mcc
pc}spp
lpp
1±0¬10−$ (1)
not applicable
not applicable
6±5¬10−$ (2)
1±7¬10−$ (3)
2±9¬10−%
2±0¬10−%
3±0¬10−)
Sucrose
Potassium
* mse, metaphloem sieve element ; mcc, metaphloem companion cell ; pc}spp, procambial}small phloem parenchyma ; lpp, large phloem
parenchyma.
† Estimated from the average for days 3–4 and 4–5 predicted rates of axial delivery through the metaphloem se–cc complexes of 5±92¬10−*
mol (sucrose) m−" (stem) s−" for sucrose and 2±11¬10−* mol (potassium) m−" (stem) s−" (for more details, see Tables 2 and 3 and accompanying
text). Radial fluxes were estimated assuming transfer was exclusively through the total cross-sectional area of plasmodesmata interconnecting the
test cell types.
‡ Numbers refer to the symplastic route number shown in Fig. 3.
Values were compared with the reported range of 1±5¬10−% mol m−# (plasmodesmata) s−" (Kuo et al., 1974) to 8±4¬10−$ mol m−#
(plasmodesmata) s−" (derived from data presented by Gunning and Hughes, 1976).
se–cc complexes mature. In contrast, the rate of delivery by
the metaphloem se–cc complexes was predicted to more
than double, from 19 to 49 % of the total solute delivered,
during the same period (Tables 2 and 3). This is consistent
with the metaphloem sieve elements maturing.
Potential sucrose and potassium fluxes through the radial
pathway. For model 1, apart from one exception, the
estimated sucrose fluxes through the plasmodesmata connecting the cell types of the metaphloem (Fig. 3 and Table
4) all fell within the range reported for exclusive symplastic
transfer (Kuo et al., 1974 ; Gunning and Hughes, 1976). The
exception was the flux rate between the companion and
procambial}small phloem parenchyma cells (Symplastic
Route No. 2 in model 1 of Fig. 3). This was 2±2 times higher
than the highest reported value. This suggests a possible
symplastic bottle-neck at this point. However, based on
maximal plasmodesmatal fluxes, some 50 % of the sucrose
could be transferred in this direction. If symplastic unloading
does occur from the metaphloem sieve elements, the flux
data presented in Table 4 suggest that the preferential route
would be a circuitous one through the companion cells to
the large phloem parenchyma cells and onwards to the
procambial}small phloem parenchyma cells (Symplastic
Route Nos 1, 3 and 4 in model 1 of Fig. 3).
In the case of the estimated plasmodesmatal fluxes for
potassium (Fig. 3 and Table 4), similar conclusions can be
made as those for sucrose. That is, unloading from the
metaphloem sieve elements could be entirely symplastic
with subsequent flows preferentially from the metaphloem
companion cells to large phloem parenchyma and ultimately
procambial}small phloem parenchyma cells.
For model 1, potential plasma membrane fluxes were
estimated for sucrose and potassium for the period days 3–4
of elongation (Fig. 3 and Membrane Transfer Steps Nos
1–4). No values have been reported for the direct measure-
ment of sucrose fluxes across the plasma membranes of sieve
elements. However, an estimated value of 16¬10−) mol
(sucrose) m−# (plasma membrane) s−" has been given for
fluxes through the se–cc complex membranes in the minor
vein of Beta Šulgaris L. (Fondy and Geiger, 1977). Since the
estimated potential membrane fluxes of sucrose during
elongation were four to six times higher (Table 5) than those
reported (Fondy and Geiger, 1977 ; Luttge and
Higinbotham, 1979), it can be concluded that exclusive
membrane transfer across the membranes of the sieve
elements is unlikely. Thus, the possibility that the cells of the
metaphloem may be acting as a functional unit to provide
sufficient membrane surface area for an apoplastic step was
considered.
Based on the sucrose flux data provided in Table 5, the
only cell type that has sufficient plasma membrane surface
area to support sucrose exchange to the apoplast at the rates
of radial transfer is the large phloem parenchyma (see Fig.
3, Membrane Transfer Step No. 4 for model 1). Significantly,
the extent of symplastic coupling between these cells and the
metaphloem sieve elements and their associated companion
cells is also sufficient to support the radial fluxes of sucrose
(Table 4). Similar conclusions can be drawn for potassium
transfer (Tables 4 and 5). Therefore, the large phloem
parenchyma cells have the potential to function as a
significant cellular site for membrane exchange of phloemimported solutes to the stem apoplast. Inclusion of
membrane exchange across the symplastic complex of
metaphloem sieve element–companion cells–large phloem
parenchyma cells–procambial}small phloem parenchyma
cells clearly has spare transport capacity (Table 5). However,
the sucrose fluxes, but not those of potassium, predicted for
exchange across the plasma membranes of the protophloem
sieve elements by model 1 exceed the maximal values by
three to four times. This inconsistency may be accounted for
98
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
T     5. Model 1 predictions of potential plasma membrane fluxes of sucrose and potassium for each cell type of the phloem
following axial deliŠery to the apical 0±5–2±5 cm of internode 2 of Phaseolus vulgaris L. seedlings during days 3–4 of elongation
growth
Phloem cell
type*
Transfer
step
(number†)
Membrane surface
area‡ ¬10−$
(m# m−" stem)
pse
mse–cc
lpp
pc}spp
mse–cc–lpp–pc}spp
1
2
4
3
2a­3a­4a
8±19
9±13
62±96
19±91
Potential membrane flux§ ¬10−)
(mol m−# plasma membrane s−")
Sucrose
Potassium
42±7
56±2
8±1
25±8
0±6
14±7
19±3
2±8
8±9
* pse, protophloem sieve element ; mse–cc, metaphloem sieve element-companion cell complex ; pc}spp, procambial}small phloem
parenchyma ; lpp, large phloem parenchyma.
† Numbers refer to apoplastic route number shown for model 1 in Fig. 3.
‡ Data collected from transverse sections of one quarter of internodes harvested from two replicate plants for each day of elongation—refer
to Fig. 1, Wood et al., 1994.
§ Assumes that axial transfer of sucrose and potassium at the observed rates of delivery was proportional to the cross-sectional area of each
type of sieve element (Tables 2 and 3 and accompanying text). Estimated fluxes were exclusively across the total plasma membrane of each test
cell type.
Values were compared with maximal fluxes for facilitated transfer of sugars of 1–10¬10−) mol m−# s−# (Luttge and Higinbotham, 1979) and
16¬10−) mol m−# s−" (Fondy and Geiger, 1977) and of potassium of 1–10¬10−) mol m−# s−" (Robards and Clarkson, 1976).
T     6. Model 2 predictions of potential plasma membrane
fluxes for sucrose and potassium for the Šarious cell types
within the phloem following axial deliŠery to the apical
0±5–2±5 cm of internode 2 of Phaseolus vulgaris L. seedlings
during days 3–4 of elongation growth
Potential membrane flux‡
(¬10−) mol m−# plasma membrane s−")
Phloem cell
type*
pse
mse–cc
pc}spp
lpp
Transfer
step
(number†)
Sucrose
Potassium
Scenario 1§
Scenario 2§
1
2
3
4
5
6
21±0
27±6
22±1
4±0
7±0
7±5
12±9
17±0
13±6
2±5
4±3
8±4
7±2
9±7
6±2
2±4
1±7
!1
* pse, protophloem sieve element ; mse–cc, metaphloem sieve
element–companion cell complex ; pc}spp, procambial}small phloem
parenchyma ; lpp, large phloem parenchyma.
† Numbers refer to apoplastic route number shown for model 2 in
Fig. 3.
‡ Assumes that axial rates of delivery of solutes are proportional to
the cross- sectional area of the protophloem sieve elements, metaphloem
se–cc complexes and the cells of procambial}small phloem parenchyma.
Estimated fluxes were exclusively across the total plasma membrane of
each test cell type.
§ Scenario 1 : refixation of all respired carbon to give a maximal rate
of radial transfer. Scenario 2 : all respiratory losses from the stem
incurred by the conducting cells to give a minimal rate of radial
transfer.
Values were compared with maximal fluxes for facilitated membrane
transfer of sugars of 1–10¬10−) mol m−# s−" (Luttge and Higinbotham,
1979) and 16¬10−) mol m−# s−" (Fondy and Geiger, 1977) and of
potassium of 1–10¬10−) mol m−# s−" (Robards and Clarkson, 1976).
by taking into account the potential transport capacity of
the procambial}small phloem parenchyma cells as outlined
for model 2 discussed below.
The plasmodesmatal fluxes were not re-calculated to
conform with the proposed solute flows in model 2 as there
was sufficient plasmodesmatal cross-sectional area to support the higher rates of delivery from the procambial}small
phloem parenchyma cells to the large phloem parenchyma
cells predicted for model 1 (see Table 4). Under conditions
where stem photosynthesis re-fixed all respired carbon
dioxide so that the radial transfer rates were maximal (Table
2 and see Scenario 1), the computed membrane fluxes of
sucrose demonstrate that only the large phloem parenchyma
cells have sufficient plasma membrane surface area to
support the predicted radial rates of sucrose transfer
contributed by import through the metaphloem sieve
elements and procambial}small phloem parenchyma cells
(Table 6 and see Fig. 3, Membrane Transfer Steps Nos 4–6).
However, this model does not account for the sucrose flux
across the protophloem sieve elements (Table 6 and see Fig.
3, Membrane Transfer Step No. 1). The alternative scenario
is that the bulk of carbon is respired by the conducting cells
and results in a minimal radial flow (Table 2 and the earlier
discussion to support the validity of this model). If these
circumstances applied, the computed membrane fluxes of
sucrose indicate that the protophloem sieve elements and
procambial}small phloem parenchyma cells have sufficient
combined plasma membrane surface area to support radial
sucrose transfer from these cells (Table 6). The metaphloem
se–cc complexes may just have sufficient plasma membrane
surface area, and in particular, if the excess sucrose is
transferred to the large phloem parenchyma for exchange to
the stem apoplast (Table 6 and see Fig. 3). The membrane
flux data for potassium (Table 6) indicate that model 2 can
account adequately for the radial flows of potassium at
Wood et al.—Photosynthate and Potassium Transfer in Elongating Bean Stems
predicted rates from the various cells types. Overall, these
considerations lead to the conclusion that model 2 provides
the most satisfactory description of the radial solute flows
with the proviso that the bulk of the stem respiratory
activity is restricted to the conducting tissues.
CONCLUSIONS
A structural assessment of the possible pathways of sieve
element unloading and subsequent radial transfer of sucrose
and potassium within the elongating internode 2 of
Phaseolus Šulgaris L. seedlings has been undertaken. The
composition of the phloem was found to be more complex
than that described in other studies of sink regions (but see
Eschrich, 1983). In the elongating stem, there were two
different types of mature sieve elements and a large number
of differentiating metaphloem se–cc complexes and
procambial}small phloem parenchyma cells. In the case of
the mature protophloem sieve elements, the absence of
plasmodesmatal
interconnections
rendered
them
symplastically isolated from their contiguous cells and
hence restricted unloading to a membrane transfer step. In
contrast, identification of the potential pathway of
unloading from the mature metaphloem sieve elements
proved more difficult. The quantitative assessment of the
possible cellular pathways of radial transfer from these
conducting elements to the phloem parenchyma cells
suggested that (a) the protophloem sieve elements, metaphloem se–cc complexes and procambial}small phloem
parenchyma cells, are collectively responsible for the delivery
of photosynthates to the elongation region, and (b) the bulk
of sucrose is respired or utilised for growth within the cells
of the phloem and therefore not unloaded.
Based on this quantitative assessment and the ultrastructural evidence, it has been concluded that unloading of
sucrose and potassium from the mature sieve elements
occurs ; (a) across the plasma membrane of the protophloem
sieve elements, and (b) either through the symplast or
apoplast of the metaphloem. If unloading from the
metaphloem se–cc complexes occurs via the symplast then
transfer would be direct to the cells of the large phloem
parenchyma. Furthermore, it has been predicted that
potassium follows the same axial and radial pathways as
proposed for sucrose.
The absence of plasmodesmata within the fibre cap cells
and between them and the adjacent large phloem parenchyma of the protophloem suggested that, if subsequent
radial transfer occurs through the symplast from the phloem
to the cortical and epidermal tissues then transfer would be
through the interfascicular parenchyma cells. Symplastic
transfer to the pith can potentially occur via the cells of the
vascular cambium. Alternatively, an entirely apoplastic
route may occur to all cells of the ground tissues.
The pathway of sieve element unloading and subsequent
radial transfer of sucrose and potassium described by model
2 forms the basis of the next paper (Wood et al., unpubl.
res.). Two main issues need to be resolved. First, whether
the plasmodesmata interconnecting the metaphloem se–cc
complexes with the large phloem parenchyma cells and
between the cells within the ground tissues are indeed
99
functioning. Second, the proportion of the unloaded solutes,
if any, that are reloaded back into the sieve elements.
A C K N O W L E D G E M E N TS
We thank Mrs Stella Savory for her technical assistance.
R. M. W. was supported by a Commonwealth Postgraduate
Research Scholarship. The investigation was assisted by
funds from The University of Newcastle Research Management Committee.
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