Interaction of phloem and xylem during phloem loading – functional
symplasmic roles for thin- and thick-walled sieve tubes?
CEJ Botha
Abbreviations: SE = sieve element; ST = sieve tube; ST-CC = sieve tube
companion cell complex; CC = companion cell; TWSE = thick-walled sieve element;
TWST = thick-walled sieve tube; VP = vascular parenchyma.
I Introduction
A. structural considerations in relation to phloem loading:
2
B. structural considerations of the loading pathway
3
I. what is so special about the phloem?
4
C. role of thin- and thick-walled sieve tubes
5
I. the ST-CC complex
6
II is a companion cell really necessary?
7
III. phloem loading – what transport pathways are involved?
7
D visualising phloem loading pathways; answering some questions
8
E Experimental evidence for symplasmic phloem loading
9
I apoplast to symplast uptake of 5,6-CF
II apoplast to symplast transfer – xylem to phloem retrieval
9
10
Concluding remarks
12
Legends to Text Figures
12
References:
1
2
15
I Introduction
A. structural considerations in relation to phloem loading:
The structure of the vascular tissue within the leaf blades of grasses has been
reported in detail in several previous papers (see Botha l, 1982; and references cited; ;
Botha 11992; Cartwright, et al., 1977; Dannenhoffer, et al., 1990; Evert, 1986;; Evert
et al.,1978; Evert and Russin 1993; Evert, et al., 1996a;bt 1996b; Farrar et al. 1992;
Haupt et al., 2001; Kuo and O’Brien 1974; Lush 1976; Matsiliza and Botha 2002;
Russell and Evert 1985).
Phloem loading is achieved via mature metaphloem in small veins, within source
leaves. The process in grasses has been described as being symplasmic, apoplasmic or
mixed-mode apoplasmic-symplasmic (see the review by van Bell 1993 and literature
cited) and is classified according to the presence or absence of plasmodesmata
(frequency) along the loading pathway. If plasmodesmata are present along the
loading pathway, then the assumption has been made that the loading process may be
symplasmic via plasmodesmata between the various cell-cell interfaces involved in
the loading pathway. If plasmodesmata are absent at one or more interfaces along the
loading pathway, and then the more efficient loading process is considered to be
apoplasmic and energy-requiring at the cell-cell interface which either lacks
plasmodesma entirely, or where the frequency is very low (see Table 1).
Given the broad similarity between phloem loading processes between
monocotyledonous and dicotyledonous plants, and that a great deal of attention which
has been focussed on dicotyledonous leaves, this contribution will emphasise
structure-function relationships that influence phloem loading in monocotyledonous
leaves.
2
B. structural considerations of the loading pathway
Whilst dicotyledonous and eudicotyledonous foliage leaves may contain more
than five vein orders, most monocotyledonous leaf blades contain but three, excluding
the marginal vein network and the midrib vein system. Vein structure within a typical
grass leaf can be clearly defined according to vein size, presence or absence of
supporting mechanical tissues, as well as by the arrangement of the vascular tissues
within the veins themselves (Botha and Evert, 1982). Briefly, the large veins contain
large metaxylem vessels and these veins are all associated with a protoxylem lacuna.
Large veins are always associated with hypodermal sclerenchyma fibres, which are
usually associated with the adaxial and abaxial vein faces. The fibres interrupt the
chlorenchymatous bundle sheath, to form girders which directly subtend the vascular
tissue and connect it to the ad- and abaxial epidermis. In contrast, intermediate veins
lack large metaxylem vessels and are usually supported by hypodermal sclerenchyma
associated with one surface only. Small veins are usually entirely surrounded by a
chlorenchymatous bundles sheath, and are thus structurally analogous to the minor
veins in dicotyledonous leaf blades. Like intermediate veins, the xylem in mature
small veins is not associated with a protoxylem lacuna (see Botha and Evert et al.,
1982, Evert et al., 1986 for further explanation). Xylem and phloem elements within
all three size classes of vein are associated with vascular parenchyma cells. The xylem
vessels in particular, are closely coupled with parenchymatic elements via pit
membranes. The intriguing structure of the pit membranes (Fig. 4) and their close
spatial relationship to the plasmamembrane of the contiguous xylem parenchyma
cells, suggests an important physiological role in solute exchange processes. Figs. 3-5
illustrate aspects of the ultrastructure of grass leaf vascular tissue discussed above.
3
I. what is so special about the phloem?
The phloem in grasses is unique, in that two functional types of sieve tube exist
in mature vascular bundles (see Fig. 5). The first type is termed early metaphloem and
fits the classical description of phloem sieve tube members, in that the STM are
associated with companion cells (Evert et al., 1977, 1985; 1996a,b and references
cited; Botha and Cross 1997 and references cited; Haupt et al., 2001). These sieve
tubes have thin walls and are thus commonly referred to as thin-walled (metaphloem)
sieve elements. The second recognizable type is the last to differentiate in the leaf
blade bundles, and is usually spatially closely associated with the vessels. These lateformed sieve elements characteristically have thick walls and lack recognizable
companion cells. In all but two instances, (Cartwright, et al., 1977; Kuo and O’Brien,
1974) they are reportedly not lignified. Thick-walled sieve tubes were first reported in
wheat by Kuo and O’Brien (1974) and later found to occur in all Gramineae (see
Walsh 1974, Miyake and Maeda 1976, Cartwright et al., 1977; Evert, et al., 1977;
Evert 1986 and references cited al., 1978;Colbert and Evert 1982 1985; Botha and
Evert 1988; Botha 1992; Botha and van Bel, 1992; Evert and Russin 1993; Evert et
al., 1996a; Botha and Cross 1997, and Haupt et al., 2001 and literature cited) and also
occur in other monocotyledons, such as the Commelinaceae (van Bel et al., 1988).
Electron microscopy studies have shown that with the apparent exception of Zea
mays, (see Evert, 1986) thick-walled sieve are generally poorly connected with the
surrounding vascular parenchyma, including the early metaphloem (thin-walled) sieve
tube-companion cell complexes of the thin-walled phloem. (Botha, 1992; Botha and
van Bel 1992; Evert 1986; Evert et al., 1996a,b)In other words, they could be
symplasmically isolated, or symplasmically poorly connected. Sieve element isolation
from the surrounding vascular parenchyma was inferred from electrophysiological
4
studies (Farrar et al., 1992; Botha and Cross, 1997). This included thin-walled SE’s
and vascular parenchyma. The TWSE in many grasses such as Zea Evert 1986) and
as illustrated here by Eragrostis plana (Fig 5) abut large vascular parenchyma (VP)
cells, with which elements of the phloem may be well-connected (Table 1). Numerous
connections of thick-walled sieve tubes to the VP suggest a strong potential
symplasmic pathway between the VP and the TWST in Z. mays at least. In other
grasses, plasmodesmal frequencies may be low across this interface, (Table 1)
suggesting the existence of a less significant symplasmic pathway. Interestingly, there
is no evidence for the TWST being involved in long distance transport (Evert 1986).
C. role of thin- and thick-walled sieve tubes
The proportion of thick- to thin-walled SE’s changes with the vein order. In the
small longitudinal veins, the number of ST to TWST is much lower (between 1 and 3
ST:1 TWST) in the smaller veins, than is commonly observed in the large
longitudinal veins (usually 1 to 2 TWST, up to 5-10 ST; Colbert and Evert 1982;
Russell and Evert 1984; Dannenhoffer et al., 1990). The smallest veins usually
contain one ST and one TWST, but Dannenhoffer et al., (1990) report that the
smallest of the small veins in barley may contain only one TWST and no thin-walled
sieve tube.
Cartwright et al., (1977) and later Fritz et al., (1983) demonstrated that that
phloem loading is probably executed by the thin-walled sieve tubes exclusively as
was demonstrated by microautoradiography. Additional evidence supporting loading
via the ST comes from plasmolytic studies (Evert et al., 1978).which demonstrated
that osmotic potentials were much higher in thin-walled than in thick-walled SE’s.
Recently, Matsiliza and Botha (2002) provided the first direct evidence that the aphid,
5
Sitobion yakini selectively feeds on ST and furthermore, that the aphid had a distinct
preference for those in the small longitudinal veins of barley. The aphids were more
strongly attracted to the ST than to the TWST – presumably due to the nature of the
content of these sieve tubes.
All longitudinal leaf blade veins in the grasses are connected by numerous small
transverse (also termed lateral or cross) veins. Good supportive evidence comes from
a number of sources which show that they are not involved in assimilate uptake, but
rather, in lateral transfer of assimilate. Firstly,
14
C-labelled photosynthate was shown
to be accumulated in the small longitudinal veins of Panicum (Lush 1976) and that the
transverse veins in leaves of Panicum were heavily labelled with 14C some time after
the main pulse of 14C had passed out of the leaves. A subsequent paper by Fritz et al.,
(1983) showed conclusively that the thin-walled ST of the small and intermediate
longitudinal veins in maize were the channels through which
14
C-labelled
photosynthate trafficked. It is important to note that lack of evidence for involvement
in active phloem loading does not preclude a role in temporary storage, or in deviating
photosynthate streams, — possibly coordinating functioning of the different
longitudinal vein orders through an as yet, unknown mechanism (Lush 1976). It
seems, however (based upon available evidence including the example represented in
Fig 2) that the cross-veins function as transfer conduits between the small,
intermediate and large veins in the leaf blade, which we know to be involved in
uptake, transfer, and transport and export of assimilate respectively.
I. the ST-CC complex
TWST lack CC (even though this is incorrectly reported to the contrary in Haupt et
al., (2001), for Hordeum vulgare (see Table 1). This is clearly illustrated in the
6
transaction of an intermediate vascular bundle of Eragrostis curvula a common veld
grass in southern Africa (Fig. 5). The bundle lacks a protoxylem lacuna, and contains
two late-formed thick-walled sieve tubes (cells shown with solid dots, Fig. 5) in close
spatial proximity to the metaxylem. A column of four thin-walled metaphloem sieve
tubes occurs centrally, surrounded by contiguous companion cells and phloem
parenchyma cells.
II is a companion cell really necessary?
The companion cell-less TWST, poses questions relative to the role that we know the
companion cell plays in the phloem of dicotyledonous and eudicotyledonous plants,
and, by inference what we suspect the CC’s role is in monocot phloem. Companion
cells are recognised as having an important role related directly to the functionality of
the phloem – indeed; Oparka and Turgeon (1999) have likened the companion cells to
the phloem’s ‘traffic control centers'.
Lacking companion cells means that two
physiologically and functionally important questions need to be asked — if traffic
control is absent (lack of CC associated with the TWST), then are these TWST nonfunctional, or is the ‘control function’ simply taken over by the adjacent large
vascular parenchyma cells, which then function as pseudo companion-cells? Or
indeed, is a CC really necessary?
III. phloem loading – what transport pathways are involved?
Based upon plasmodesmal frequencies and other evidence in barley (Botha and Cross,
1997) suggests that the CC-ST complex is commonly isolated (with low
plasmodesmal frequencies evident between VP and the CC-ST complex) in grasses.
Lack of plasmodesmal or pore-plasmodesmal connections to the TWST (with the
possible exceptions of Zea mays, and Hordeum vulgare (as in Haupt et al 2001, Table
7
1 for H vulgare black hull-less)) suggests that TWST are isolated from adjacent
parenchymatous cells. If transport in the TWST does indeed occur, then the loading
process is suggested to be under apoplasmic control.
D visualising phloem loading pathways; answering some questions
Many of the grasses are known to host a range of systemic viruses, which appear in
the phloem. This must mean that the phloem is not as symplasmically isolated as
indicated by plasmodesmal frequency studies. Virus transmission and movement must
involve functional plasmodesma along the transport route. This brings into question
frequency — how many plasmodesma are sufficient or indeed required to support
symplasmic phloem loading processes?
To answer the questions asked here relating to the role of the apoplast in loading;
plasmodesmal continuity; potential symplasmic loading and unloading pathways; the
role of cross veins in phloem loading; connectivity and functionality of TWST and ST
with contiguous parenchymatic elements, requires the use of exogenously-applied
molecules (preferably fluorescent) to track symplasmic passage from the mesophyll to
the sieve tubes, if indeed such a pathway is present and can be demonstrated.
The xenobiotic 5,6 – carboxyfluorescine (5,6-CF) has proved to be a useful tool
in plant physiology. Applied in the diacetate form, 5,6-CF does not fluoresce, is nonpolar polar and is readily taken up by plant cells, usually across cell walls, and
membranes of damaged cells. Once contained within physiologically intact systems, it
is cleaved, and the polar free 5,6-CF fluoresces. It is reasonably membrane
impermeant, but is known to accumulate in some cell vacuoles. Appearance in
contiguous cells is thought to be confined to plasmodesmal trafficking. Grignon et al.,
(1989) suggest that 6(5) CF to be a stable, non-permeant, and probably innocuous
8
molecule, which obeyed source-sink relationships, and in their studies, it remained
confined to the phloem for up to four days. A word of caution may be necessary
however; as the pKa’s suggest that a small percentage (less than 0.1%) may remain
undissociated, and could thus make a not insignificant contribution to the movement
of this probe (Wright and Oparka, 1996) in longer-term experiments.
E Experimental evidence for symplasmic phloem loading
I apoplast to symplast uptake of 5,6-CF
Uptake through abraded epidermal cells occurred rapidly and as expected, 5,6-CFDA
was incorporated rapidly and transferred to adjacent cells (presumably) undamaged
cells, where fluorescence (from 5,6-CF) was seen within 10 min in the cytosol of
mesophyll cells in maize, wheat and barley leaves (Fig. 1). With time, 5,6-CF spreads
and accumulated within the cytosol principally, in to a larger group of mesophyll
cells, and is taken up into vascular bundles (Fig. 2). After several hours, intense
fluorescence may be detected up to 6 cm away from the point of application. More
importantly, cross veins seem to be implicated in the transport process (Fig. 2. No
evidence of outward leakage from the bundle sheath (BS, Fig. 2) suggests that local
unloading does not occur via the BS occurs after uptake. The intensity of 5,6-CF
associated with the cross veins was surprising. Double staining with aniline blue to
colocalize callose associated with plasmodesma and or pore plasmodesma between
the companion cells and the sieve tube members, as well as confocal microscopy,
(data not shown) confirmed that the fluorescence was associated with the sieve tubes.
Frequency data presented in Table 1 indicates low plasmodesmal frequencies between
VP and the CC-SE complexes, as well as between VP and the TWST, in all but Z.
mays. The 5,6-CF experiments illustrated here, illustrate some symplasmic uptake.
9
However, the slow appearance and movement after uptake – movement was limited,
after 2h, to about 3-6 cm from the point of application of 5,6-CFDA. Slow movement
supports the low frequency data – there cannot be many functional plasmodesma in
this putative symplasmically-controlled pathway.
II apoplast to symplast transfer – xylem to phloem retrieval
Notwithstanding the above findings, it is not possible to ignore the possibility that
there is a potential for some of the applied 5,6-CFDA to have been translocated and in
an undissociated state, crossing walls and plasmalemma and thus entering the CC-SE
complex, or even directly within sieve tubes themselves, effectively ‘delivering’ 5,6CFDA before dissociation took place. Experimentally, this would not be difficult to
follow. The cell location within which acetate was cleaved, could be visualised by
making use of uptake via the xylem, in effect, a transpiration-driven system could be
used, much like Fritz et al’s (1983) protocol in which
14
C- sucrose was applied, to
deliver 5,6-CFDA to possible cleavage sites. A retrieval pathway exists from the
xylem, which, as mentioned, was first demonstrated by Fritz et al., (1983). This,
together with information gained from previous experiments using Prussian blue
localization, (Botha and Evert, 1986) the assumption was that 5,6-CFDA should be
able to cross from the apoplast (xylem) to the symplast (xylem parenchyma) with
consummate ease.
The question was however, where would 5,6-CF appear first? Would it be in the
xylem vessels, or in the xylem parenchyma? If 5,6-CFDA dissociated in the xylem
vessels, would the resultant 5,6-CF be able to cross the pit membrane?
The hypothesis was simple: If no fluorescence was visible in either the xylem vessels
or the xylem parenchyma, or if fluorescence was not detected in any cells within the
10
retrieval pathway other than the phloem-associated ones, (VP, CC-SE complex, or
TWST) this would imply that undissociated 5,6-CFDA was likely to have been
transported and could have loaded apoplasmically. However, if 5,6-CFDA dissociated
in the xylem parenchyma first, and if 5,6-CF subsequently accumulated in the
phloem, (including the CC-SE complex, or TWST) then the loading pathway could
only be symplasmic.
Fig. 3 shows a small loading vein in a mature wheat leaf. This vein contains two
vascular parenchyma cells associated with two tracheary elements and beneath these,
are two sieve tubes. No companion cell or phloem parenchyma cells are visible in this
vein, which makes it difficult to determine if both phloem sieve tubes are thin-walled
or not — usually at least one is thick-walled. Of immediate interest is the structure of
the common walls between the xylem parenchyma cells and their concomitant
tracheary elements (box, Fig. 3, and shown in detail in Fig. 4). The pit membrane (W,
Fig. 4) is clearly a very leaky wall structure, composed of a fenestrated net-like
structure, effectively forming an open meshwork on the tracheary element side. This
structure is delimited by plasmamembrane on the vascular parenchyma cell side,
which forms a physiological barrier between the xylem apoplast from the symplast of
the parenchyma cell.
Uptake experiments (Figs 6 A,B) demonstrated conclusively that 5,6-CF appeared
first in the xylem parenchyma and moved from the xylem parenchyma cells adjacent
to the large metaxylem, transferred radially via the mestome and bundle sheath cells,
and re-entered the phloem via phloem parenchyma. Clearly, some plasmodesma must
have been present along the whole pathway; else this retrieval pattern would not have
occurred. None of the experiments showed no evidence of 5,6-CF localization in
TWST.
11
Concluding remarks
Phloem loading in grasses (into the CC-SE complex) appears to have a minor
symplasmic component, which seems to be confined to the thin-walled sieve tubes.
The slow rate of uptake (measured in hours) suggests that it is not a major transport
route, but perhaps a relic of some inefficient ancestral symplasmic loading system,
exploited today by viruses.
One question remains -- what about the role of the thick-walled sieve tubes?
Clearly they do not seem to transport 5,6-CF (even in Z mays, in which the TWST are
well connected to VP) and one would have assumed that TWST have direct roles in
symplasmically-mediated transport. They remain an enigma. Scanning electron
micrograph images of fossil leaf fragments of a C4 species some 7 to 5 million years
old, (Thomasson et al., 1986) just like modern-day chloridoid grasses, suggests that
thick-walled sieve tube occurred within the phloem. So, structurally, as well as
functionally, phloem loading in grasses is complicated – with thin- and thick-walled
sieve tubes, with only the thin-walled associated with companion cells, and the thickwalled sieve tubes more closely allied spatially to the xylem. Much remains
unanswered.
Legends to Text Figures
Fig. 1. Shows uptake of 5,6-CF and symplasmic transport into mesophyll cells in
Hordeum vulgare leaf and the distribution of fluorescence, 10 min after application to
a lightly-abraded region of the leaf tissue (unlabelled arrow points to abraded cell)
5,6-CF had spread to a large number of cells.
Fig. 2. Shows distribution of 5,6-CF in a small (left) and intermediate (right)
vascular bundle in Zea mays leaf blade tissue. Bright fluorescence in cross vein is
12
associated with the single thin-walled sieve tube interconnecting the small with the
intermediate longitudinal bundle.
Figs. 3.-4 Electron micrographs showing aspects of the ultrastructure of a
small vein in a wheat leaf. Fig 3 shows a small vascular bundle in wheat, which
contains two vascular parenchyma cells (VP) associated with two tracheary elements
(T), below which are two sieve tubes (S). Note that no companion cell is visible in
this micrograph. In Fig. 4, the highly fenestrated wall area between the vascular
parenchyma cell (VP), and a tracheary element (TE) below is shown in detail. The
wall structure is highly porous and possibly ‘holy’ in the pit region, which will allow
transfer of water and solutes from the vascular parenchyma cell to the tracheary
element and vice versa. This is the route available for retrieval of solute from the
xylem.
Fig. 5. Shows an intermediate vascular bundle in the Eragrostis plana, C4 a C4
grass.
This micrograph shows that the phloem is composed of two thick-walled, (solid
dots) and four functional thin-walled sieve tubes. Thick-walled sieve tubes are
spatially close to xylem. The thin-walled sieve tubes (S) are associated with
companion cells (CC) as well as vascular parenchyma (VP) cells. The thick-walled
sieve tubes (solid circles) are associated with vascular parenchyma only. Note the
very large lateral vascular parenchyma cells situated either side of the central phloem
core in this bundle. High plasmodesmal frequencies between these parenchyma cells
and the mestome sheath – bundle sheath- mesophyll interface, suggest that this may
be an important symplasmic loading pathway.
Figs 6 Shows retrieval of 5,6-CF from the xylem after 120 (A, left) and 180 (B,
right) min uptake of 5,6-CFDA from the cut end of a severed source leaf of Zea mays.
13
Sections cut into silicone oil and viewed with a narrow-band filter set specific for 5,6CF and lignin-free. Fluorescence is very intense in the XVP, as well as in BS and
vascular parenchyma cells, but is absent from the xylem, suggesting that dissociation
of 5,6-CFDA occurred within the xylem parenchyma. One VP and one SE contain
intense label. After 180 min (B, right) the fluorescence intensity is greatest in the
phloem, with VP and SE show intense label. Mestome sheath and the hypodermal
sclerenchyma girder cells contain label as well.
14
References:
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15
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16
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17
Table 1: Summary of plasmodesmal frequencies between vascular
parenchyma cells and thin and thick-walled sieve tubes.
Plant spp
Key Interface
BS/MS
VP
CC
ST
TWST
Zm 1




Zmb,7




Tt 2




Hvm3




Hvd4




Hvbh5,b




Ep6




Pm6




Bu6

VP

CC

ST

TWST
***


***
Zmb
?


****
Tt
**



Hvm
****



Hvd
****



Hvbh
****



Ep
**



Pm
**



Bu
****

CC

ST

TWST

**
VP
Zm
CC
Zm

**
Tt


Hvm


**
Hvd

Hvbh


**

**
Ep



Pm



Bu



Zm
b

Key:
a = Bundle sheath or vascular parenchyma, concomitant with vascular parenchyma
b = sink leaf tissue.
*****
****
***
*



?
= very high
= plentiful
= high
= low
= scarce
= rare
= absent/not structurally possible
= not recorded
Zm= Zea mays ; Tt = Themeda triandra; (small loading bundle); Hv = Hordeum vulgare cv. Morex
(small); Hvd = Hordeum vulgare cv. Dyan; Hvbh = Hordeum vulgare cv. Black hullless; Ep = Eragrostis
plana; Pm = Panicum maximum; BU = Bromus unioloides 1 = Evert et al 1997; 2 = Botha and Evert
18
1988; 3 = Evert et al., 1996; 4 = Botha and Cross, 1997; 5 = Haupt et al, 2001; 6 = Botha, 1992; 7 =
Evert and Russin, 1993.
19