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The Plant Cell, Vol. 13, 989, May 2001, www.plantcell.org © 2001 American Society of Plant Physiologists
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A Calcium-Regulated Gatekeeper in Phloem Sieve Tubes
The phloem is a highly specialized long
distance transport tissue in vascular
plants. Despite detailed knowledge of
the development and ultrastructure of
phloem tissue in a wide variety of plant
species, surprisingly little is known about
the mechanisms and regulation of
phloem transport. The main function of
the phloem is transport of the immediate products of photosynthesis (e.g.,
sugars) from “source” tissue (actively
photosynthesizing leaves) to “sink” tissue (immature leaves, growing root
tips, and developing flowers, fruit, and
seed). It is sometimes overlooked that a
wide variety of other material is also
transported through the phloem, including proteins, amino acids, solutes, viruses, and various signaling molecules.
PHLOEM STRUCTURE
AND FUNCTION
All students of plant physiology are familiar with the mass flow concept of
phloem transport. According to this
concept, the transport of materials
through phloem sieve tubes is passive,
nonselective, and driven entirely by
pressure gradients that are maintained
by active loading of photosynthate in
source tissue and unloading of materials in sink tissue.
Phloem tissue is well designed for
long distance transport. The flow of
materials takes place through sieve
tubes, which are made up of long,
tube-like, enucleate sieve elements arranged end to end and connected by
specialized end walls known as sieve
plates. The sieve plates contain large
(up to 1 to 2 ␮m) pores that allow for
passage of materials between sieve elements. Typically, each sieve element
in angiosperms is accompanied by one
or more companion cells, which interact intimately with the sieve element
and play a crucial role in regulating
phloem loading and unloading and in
the turnover of sieve element proteins
and other components (reviewed by
Oparka and Turgeon, 1999). Sieve elements and companion cells are derived
from unequal longitudinal division of a
single “fusiform mother cell.” The cell
that becomes the sieve element undergoes a highly regulated partial autolysis, not unlike that of programmed cell
death, in which the central vacuole
breaks down and the nucleus, ribosomes, cytoskeleton, and Golgi bodies
are degraded. The result is a large,
nearly empty cell that is well suited for
conductance of a wide range of molecules. However, specific components
and organelles are retained that may
have functions in transport and/or cell–
cell interactions within the sieve tubes
(van Bel and Knoblauch, 2000).
P-PROTEINS AND
PHLOEM TRANSPORT
Mature sieve elements retain a modified endoplasmic reticulum (ER), mitochondria, numerous types of proteins,
and specialized sieve element plastids.
Most of these components are arranged along the lateral walls of the
sieve element and collectively constitute a system known as the parietal
layer (Figure 1). A wide variety of sieve
element proteins and plastids have been
observed that can be distinguished by
genus or family.
Behnke (1991a, 1991b) presented electron micrographs and descriptions of
plastids and crystalloid proteins from the
sieve elements of many different species. Behnke (1991a) distinguished two
types of sieve element plastids: S-type
plastids, which contain only starch inclusions, and P-type plastids, which
contain mainly protein inclusions. Behnke
(1991b) referred to “nondispersive” versus “dispersive” protein bodies, also
called P-proteins or structural sieve element proteins. These terms arose because of changes that are observed
during sieve element ontogeny. Early in
the maturation process, many protein
bodies are often observed, some of
which disperse as the sieve element
matures and some that remain unchanged in mature sieve elements.
There are many shapes of nondispersive fibrous and crystalloid protein bodies, also called P-proteins, which may
be quite large and are often observed in
the lumen of sieve elements (Figure 1).
Electron micrographic images sometimes show masses of fibrous or amorphous protein located directly in front of
or within the pores of sieve plates and
that appear to block transport through
the pores. To date, the large crystalloid
P-protein bodies that occur exclusively
in Fabaceae were classified as being
nondispersive (Behnke, 1991b).
It has long been thought that some
sieve element proteins may function to
block the pores of injured sieve tubes
to prevent the loss of assimilate. Sieve
elements appear to be extremely sensitive to injury, making it difficult to isolate and fix tissues for observation
without wounding them, possibly leading to an overabundance of micrographic images with blocked pores.
Some researchers have considered
the possibility that sieve element proteins might regularly block mass flow
through sieve tubes; thus, an additional
transport mechanism might be required
to assist materials through sieve tube
pores. One possibility put forth was
electro-osmosis (see Spanner, 1979)
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Figure 1. Schematic Representation of the Histology of the Sieve Element/Companion CellComplex in Vicia faba.
Four sieve elements (SE) connected by sieve plates (SP) are shown with their adjacent companion cells (CC). Only the companion cells contain nuclei (N), vacuoles (V), and chloroplasts
(C); mitochondria (M) and endoplasmic reticulum (ER) are also indicated. Typical features of
the sieve elements in this species are the sieve element plastids (Pl), the parietal P-proteins
(PP), and the P-protein crystalloids (PC). One crystalloid is shown in the dense state, in which
it permits the sieve element contents to flow past it. The other is depicted in the dispersed
state (DPC), in which it blocks the tube. The direction of flow is downwards, as indicated.
involving either a potassium ion or a
proton pump connected to the sieve
plate to maintain sufficient pressure to
allow the flow of assimilates across the
plate. Other theories invoked an active
role for sieve element proteins. Evert
(1982) reviewed various theories for
phloem transport and concluded that
the pressure flow mechanism remained
the most likely; he also stated that it
was difficult to conceive an active role
for sieve element proteins, or P-proteins, in phloem transport. For one
thing, the amount and structure of
sieve element proteins are highly variable among different plant families.
Many monocots and some dicots appear to lack crystalloid P-proteins entirely. Also, it has been shown that
some “nondispersive” crystalloid P-pro-
tein can disperse and move very rapidly
to plug sieve pores after injury. Evert
concluded that the most likely function
of P-protein is to seal the sieve plate
pores of injured sieve elements as a
rapid first line of defense against the
loss of assimilates.
It is possible to obtain electron micrographs from well-preserved and largely
uninjured sieve tubes using gentle tissue preparation techniques. Evert (1982)
showed an electron micrograph of Cucurbita maxima sieve tubes with completely unoccluded pores. Ehlers et al.
(2000) presented a detailed analysis of
sieve elements in Vicia faba (broad
bean) and Lycopersicon esculentum (tomato). Well-preserved sieve elements
clearly showed open, unoccluded sieve
pores. Deposits of stacked ER cisternae, plastids, and proteins were observed along the parietal walls and
along the walls of sieve plates (particularly in V. faba), but gaps that were free
of materials were maintained in front of
the sieve pores. These authors also
showed that, after injury, the sieve plate
pores became occluded by dispersed
P-proteins, but the sieve element organelles remained intact and in place along
the parietal walls. Numerous clamp-like
structures were observed that appeared
to anchor the plastids, mitochondria,
and ER cisternae in place along the
plasma membrane of the parietal wall.
The clamps appear to be specific to organelles of the sieve elements and
likely function to prevent the components of sieve elements from being carried along through the sieve tubes by
the mass flow turbulence.
P-PROTEIN IS AN ACTIVE
PARTICIPANT IN THE REGULATION
OF PHLOEM TRANSPORT
Knoblauch and van Bel (1998) used
confocal laser scanning microscopy to
visualize fluorescent dyes moving in
sieve tubes of V. faba, which provided
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definitive evidence of unimpeded mass
flow in intact plants. This study also reported that P-type plastids in V. faba
actually exploded upon injury of sieve
elements, releasing their protein contents,
which, together with the dispersed
crystalloid protein, rapidly occluded the
sieve plate pores. In this issue of The
Plant Cell, Knoblauch et al. (pages
1221–1230) extend their previous work
with confocal microscopy to show that
crystalloid P-proteins of V. faba rapidly
disperse and occlude sieve plate pores
after injury or osmotic shock. Furthermore, they make the striking observation that this process is rapidly
reversible and controlled by calcium
fluxes. It is shown that protein from the
large crystalloid protein bodies in V.
faba sieve elements dispersed to plug
the sieve plate pores after injury from
micropipette injection or osmotic shock
induced by various osmolytes. Addition
of the chelating agent EDTA completely
prevented crystalloid P-protein dispersal, and repeated exchanges of
Ca2⫹- and EDTA-containing media induced the alternate dispersal and reassembly, respectively, of crystalloid
P-proteins in injured sieve tubes. This
work represents an important advance
in our knowledge of phloem transport.
Data from the current study, together
with that of Knoblauch and van Bel (1998),
suggest that P-protein originating from
all sources within V. faba sieve elements (e.g., P-plastids, parietal proteins, and larger crystalloid P-proteins)
takes part in the occlusion of sieve plate
pores after injury to cells. Repeated observations now suggest that crystalloid
proteins in V. faba are much more
sensitive to perturbation than are the
P-type plastids. Unlike the reversibility of
crystalloid protein dispersal, explosion
and dispersal of the P-type plastids appears to be irreversible and presages the
death of a sieve element (M. Knoblauch
and A.J.E. van Bel, personal communication). Dispersal of parietal proteins after
injury has also been observed and was
found to be more sensitive to perturba-
tion than was the explosion of P-type
plastids (Knoblauch and van Bel, 1998).
In addition to the “structural” sieve
element proteins discussed here, a
wide variety of soluble proteins have
been reported from phloem exudates
of many plant species. Hayashi et al.
(2000) reported that more than 100
polypeptides could be detected in
phloem exudates from a variety of species, including wheat and rice. Unlike
the protein composition of other plant
tissues, low-molecular-weight proteins
appeared to be the dominant proteins
in the phloem. In addition, thioredoxin
h, glutaredoxin, and glutathione reductase have been found in reasonably
high concentrations in phloem exudates, suggesting a redox mechanism
for the regulation of sieve tube function.
Interestingly, some of the principal sieve
element proteins from Oryza sativa, C.
maxima, and Ricinus communis can interact with plasmodesmata to increase
their size exclusion limit. Two C. maxima proteins, CmPP16 and CmPP36,
also appear to mediate the transport of
RNAs (Xoconostle-Cázares et al., 1999,
2000). It is likely that the primary function of many of these proteins is the
regulation of macromolecular trafficking via the unilaterally branched plasmodesmata located at the junction of
sieve elements and companion cells (van
Bel and Knoblauch, 2000). As shown
by Knoblauch et al. in the present work,
other phloem-specific proteins appear
to function in the regulation of conductance through the sieve plate pores.
A GENERAL PHENOMENON?
Large crystalloid P-proteins are a particular characteristic of the Fabaceae
(legumes). Knoblauch et al. examined
sieve elements of Urtica dioica (Urticaceae) and Rubus fruticosus (Rosaceae) in response to wounding and
found that the P-protein bodies present
in these species failed to disperse and
occlude sieve pores, even when severely damaged in the presence of free
Ca2⫹. P-type plastids also appear to
have somewhat limited distribution. Proteinaceous P-type plastids were observed in just 64 of 382 dicot families
studied by Behnke (1991a); the majority
of families contained starch-filled S-type
plastids.
Calcium is an important component
of many signal transduction pathways,
and calcium regulation has been implicated in phloem function. In the current
study, Knoblauch et al. show that an
influx of calcium into legume sieve elements stimulates the rapid and reversible dispersal of crystalloid P-protein
to occlude sieve plate pores. It will be
important to determine if this phenomenon is limited strictly to the Fabaceae
or if other families have similar mechanisms, perhaps involving the parietal
and/or other P-proteins or plastids. The
concentration of free calcium in sieve
tubes of R. communis has been found
to be significantly higher than that in
surrounding tissue (Brauer et al., 1998),
and calcium-dependent protein kinases
have been detected in rice phloem sap
(Nakamura et al., 1993). Volk and
Franceschi (2000) showed evidence of
a calcium channel in the sieve element
plasma membrane of tobacco and of
the aquatic plant Pistia stratiotes using
immunolabeling with antibodies to a
calcium channel protein. These authors
proposed that calcium channels become activated during wounding or
pathogen attack, facilitating calcium influx into phloem tissues. McEuen et al.
(1981) detected a calcium binding protein distinct from calmodulin in phloem
exudates of C. maxima and speculated
that it might be associated with P-protein function. Do any of these species
have a calcium-regulated reversible
mechanism for sealing sieve elements?
The grasses may represent an interesting case for the investigation of sieve
tube sealing after injury; they appear to
lack structural sieve element proteins
(i.e., crystalloid or fibrous P-proteins
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The Plant Cell
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and parietal proteins), and sealing of injured sieve elements might be achieved
only by the contents of exploded
plastids and/or the slower process of
callose formation.
Ehlers, K., Knoblauch, M., and van Bel,
A.J.E. (2000). Ultrastructural features of
well-preserved and injured sieve elements:
Minute clamps keep the phloem transport
conduits free for mass flow. Protoplasma
214, 80–92.
Nancy A. Eckardt
News and Reviews Editor
Evert, R. (1982). Sieve-tube structure in relation to function. BioScience 32, 789–795.
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