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Phloem Transport: Cellular
Pathways and Molecular
Trafficking
Robert Turgeon1 and Shmuel Wolf 2
1
Department of Plant Biology, Cornell University, Ithaca, New York 14853;
email: [email protected]
2
The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture and the
Otto Warburg Minerva Center for Agricultural Biotechnology, The Hebrew University
of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot
76100, Israel; email: [email protected]
Annu. Rev. Plant Biol. 2009. 60:207–21
Key Words
The Annual Review of Plant Biology is online at
plant.annualreviews.org
apoplast, companion cell, phloem loading, plasmodesmata, sieve
element, symplast
This article’s doi:
10.1146/annurev.arplant.043008.092045
c 2009 by Annual Reviews.
Copyright All rights reserved
1543-5008/09/0602-0207$20.00
Abstract
The phloem transports nutrients, defensive compounds, and informational signals throughout vascular plants. Sampling the complex components of mobile phloem sap is difficult because of the damage incurred
when the pressurized sieve tubes are breached. In this review we discuss
sampling methods, the artifacts that can be introduced by different sampling procedures, the intricate pathways by which materials enter and
exit the phloem, and the major types of compounds transported. Loading and unloading patterns are largely determined by the conductivity
and number of plasmodesmata and the position-dependent function of
solute-specific, plasma membrane transport proteins. Recent evidence
indicates that mobile proteins and RNA are part of the plant’s longdistance communication signaling system. Evidence also exists for the
directed transport and sorting of macromolecules as they pass through
plasmodesmata. A future challenge is to dissect the molecular and cellular aspects of long-distance macromolecular trafficking in the signal
transduction pathways of the whole plant.
207
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Contents
INTRODUCTION . . . . . . . . . . . . . . . . . .
STRUCTURES AND PATHWAYS. . .
SAMPLING THE PHLOEM . . . . . . . .
Stylectomy . . . . . . . . . . . . . . . . . . . . . . . .
Bleeding Plants . . . . . . . . . . . . . . . . . . . .
EDTA-Facilitated Exudation . . . . . . .
Carbon Isotope Labeling . . . . . . . . . . .
SUBSTANCES TRANSPORTED
IN THE PHLOEM . . . . . . . . . . . . . . .
Carbohydrates and Amino Acids . . . .
Secondary Compounds . . . . . . . . . . . . .
Auxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .
RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208
208
209
210
210
211
212
212
212
212
213
213
214
INTRODUCTION
SE: sieve element
CC: companion cell
Size-exclusion limit
(SEL): maximum size
of a molecule that can
pass through
plasmodesmata
passively; often based
on molecular mass but
more properly on its
Stokes radius
Plasmodesmata-pore
unit: a unique
structure that has a
large channel on the
SE side and several
branching channels on
the CC side
Sieve element
reticulum: a
specialized form of
parietal endoplasmic
reticulum in sieve
elements that is often
anastomosing and
stacked
208
The phloem plays an inherently integrative role
in nutrition, development, and defense. Identifying mobile materials in the phloem is difficult because of limitations in available sampling
techniques. Determining how substances enter
and exit the phloem is also difficult because of
the intricacies of the pathways involved. In this
review we consider intercellular routes into and
out of the phloem, sampling methods, and recent information on the major types of compounds transported. Owing to space limitations, readers are directed to recent reviews on
the subjects of virus movement (61), systemic
signaling in plant defense (53, 71), and small
RNA transport (61).
STRUCTURES AND PATHWAYS
The sieve element (SE) forms a functional
complex with its associated companion cell
(CC). This complex is connected to surrounding phloem parenchyma cells and other cell
types by plasmodesmata, primarily via the CC
(Figure 1). The conductivity of plasmodesmata that connect CCs to surrounding cells
ranges greatly, depending on the tissue type
and its stage of development. In some cases
Turgeon
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Wolf
plasmodesmata are numerous and highly permeable and at the other end of the spectrum
they are sparse and seemingly closed to solute
(Figure 1). Differences in channel size may be
difficult to see by electron microscopy unless
the pores are large (99). Permeability can be
calculated from knowledge of the size-exclusion
limit (SEL) (32), but measurements of SEL are
difficult in the small and sequestered cells of the
phloem. Plasmodesmatal frequencies are sometimes used alone as a rough guide to symplastic
continuity, although these numbers can be misleading without information on the SEL (see
below). Plasmodesmata are numerous in the
phloem of plants that load photoassimilate from
the symplast, but they may also be plentiful in
some apoplastic loaders (108, 112).
Because plasmodesmatal transport is passive, at least for small molecules, as long as a
given molecule is in the cytosolic compartment
its movement between cells is apparently limited only by its size, the conductivity of the
pores, and possibly charge (28). Conversely,
apoplastic transport requires efflux into the cell
wall and subsequent retrieval by another cell.
Such uptake across the plasma membrane can
be passive, as in the puzzling “linear” mechanism of solute uptake (21); transporter driven
(96); or endocytic (93).
The primary communication pathway between the SE and CC is a modified plasmodesmatal complex known as the plasmodesmatapore unit. Green fluorescent protein (GFP)
fusion proteins as large as 67 kDa traffic from
CCs to SEs (103), indicating that many cytosolic proteins can pass between the two cell types.
Within the mature SE, the sieve element
reticulum forms a layer of cisternae that covers almost 100% of the plasma membrane, with
frequent, circular openings (fenestrations) approximately 80 nm in diameter (102). The space
between the plasma membrane and the sieve
element reticulum may sequester and shelter
molecules from the rushing current of sap.
However, the frequency and size of the fenestrations, at least in SEs of cultured tissue
(102), indicates that exchange of unanchored
molecules into the lumen of the SE could
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be rapid. Small, clamp-like structures connect
membranes of the organelles and sieve element
reticulum to each other and to the SE plasma
membrane (27) and apparently act as anchors.
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SAMPLING THE PHLOEM
Phloem sap is notoriously difficult to sample.
SEs are under high hydrostatic pressure and
because they are connected by large sieve plate
pores, sudden pressure release can affect many
cells in a series, rupturing organelles and anchoring molecules, disrupting the sieve element reticulum, altering the configuration of
phloem protein, plugging the pores, and initiating wound reactions (27). It is also possible that
pressure release pulls substances into SEs from
other cell types, especially CCs, leading to contamination of exuding sap. Therefore, demonstrating that a substance is present in phloem
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
sap does not prove that it is mobile in the undamaged translocation stream.
Even mobile molecules may not have
a function. Given the large SEL of the
plasmodesmata-pore units, flux between CCs
Sieve elements
Phloem
parenchyma
Companion cells
PlasmodesmaPlasmodesma=
pore unit
1
Cell wall
Source
Plasmodesma
Sieve
element
reticulum
Endoplasmic
reticulum
3
2
Sieve plate
Figure 1
Primary pathways into and out of the phloem. This
hypothetical scheme is a summary of available
information and may not pertain in all species or in
all tissues. Arrows indicate the direction and
approximate degree of net solute movement. In the
source leaves of many species, solutes are loaded
apoplastically (1) into the companion cells (CC)
from phloem parenchyma (PP) cells via specific
transporters (blue circles). Where such apoplastic
loading occurs, symplastic transport (2) is minimized
by reduction in plasmodesmata (pd) numbers and/or
permeability. Symplastic loading (2) may or may not
involve synthesis of raffinose or stachyose. Both
apoplastic and symplastic loading may occur in a
given species, but each occurs into different CCs.
Flux from CCs to sieve elements (SE) is primarily, if
not exclusively, symplastic through highpermeability plasmodesmata-pore units (ppu).
Solutes leaked from SEs are retrieved by CCs or
other cell types (3). Along the path (conducting)
phloem between sources and sinks, the contents of
the SEs and CCs exchange readily, but the net
direction of flux is out of the phloem to provide
surrounding tissues with nutrients. In path phloem,
CC plasmodesmata (4) have very low permeability,
so efflux is primarily apoplastic. In sink tissues,
solute unloading is commonly symplastic (5), but
may be apoplastic (6), depending on the type of sink
and its stage of development.
Specific
transporter
Path
4
6
Sink
5
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Laser-assisted
microdissection: a
laser is used in
conjunction with
conventional
compound microscopy
to manually or
robotically harvest
specific tissues or cells
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and SEs appears to be indiscriminate for many
compounds. These freely exchanged materials
are presumably metabolized in sink tissues. Furthermore, where secondary phloem, derived
from the vascular cambium, is present, it may be
possible for the degraded contents of autolyzing
SEs to enter the sap as sieve pores mature.
The uses of parasitic plants and various dissection methods, including laser-assisted microdissection (55), are discussed below in the
context of the compounds they are used to
identify. The more commonly employed sampling approaches are discussed in this section.
To date, no method provides a complete and
artifact-free picture of the contents of moving
phloem sap.
Stylectomy
Severed stylets of phloem-feeding insects can
be used to measure sieve-tube solute composition, solute concentration, turgor pressure, and
membrane potential (33, 38, 56). Aphids are
most commonly employed, but scales, mealybugs (33), and planthoppers (36) may also be
used.
Although this technique has been instrumental in phloem research, it has limitations.
Stylectomy is technically challenging and restricted to certain plant-insect combinations.
Also, exudate quantity is low, and many plants
rapidly seal the phloem. It must also be kept in
mind that insect-host interactions are biological, as well as mechanical. The very fact that
sap exudes suggests that the insect has counteracted sealing defenses. Kennedy & Mittler
(63) cautioned that stylet exudate might differ
somewhat from sieve-tube sap in an aphid-free
plant. Since then researchers have learned that
aphids exude saliva, apparently to inhibit plugging (120), and thus they potentially alter sap
chemistry (56). Aphid feeding also induces callose deposition (12) and localized changes in
gene expression (79). Aphids may also alter sap
composition to enhance their diet (106).
Although the effects listed above are gross
changes resulting from heavy infestations, one
wonders if individual aphids induce more sub210
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tle, local changes in the chemical composition
of sap. In this regard note that the amino acid
compositions of the sap from stylets of different
aphid species on the same host plant have been
reported to differ (94).
Bleeding Plants
Some plants, especially trees (39, 127), produce
exudate from severed phloem. The cucurbits
(23), Ricinus communis (76), and legumes (101)
exude copiously. Yucca and palms can provide
hundreds of liters of exudate (113).
Sap authenticity is often inferred from its expected molecular profile: high concentration of
transport carbohydrate, high molar ratio of sugars to amino acids, and little, if any, monosaccharide. However, this reasoning can be circular
because it presupposes the results the experiment is designed to obtain. Furthermore, certain phloem contents may not differ much from
those of other cell types (Reference 121 and
references therein), limiting their usefulness as
markers. In practice, the absence of monosaccharides is usually considered the best indicator
of phloem sap purity. However, monosaccharides are largely confined to vacuoles, at least
in mesophyll cells (80), which means that contamination could originate from the cytosol of
nonphloem cells and not include hexose.
Physiological correlates can also help identify the exudate source. Sap exudes most profusely from the source side of wounds in Eucalyptus (84), and a source-to-sink gradient also
exists in the sugar content of bleeding sap.
Some authors have noted diel variation in the
sugar concentration of sap (e.g., 77). However, source-to-sink and diel patterns of carbohydrate storage in surrounding, nonphloem
cells could also exist. Isotopic labeling can help
prove that exudate contains translocated materials (e.g., 100), but this type of analysis does
not prove that the exudate is free of unlabeled
contaminants.
Cucurbits are often used in exudate studies. However, there is reason to question the
origin and nature of cucurbit exudate. First,
the sugar concentration is sometimes less than
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20 mM (31, 89), though in some cases it is
closer to 100 mM (77). Cucurbit sap has been
suggested to be naturally dilute, but from histochemical analysis (87), plasmolysis studies
(110), observations of surging (24), and measurement of sugars in microdissected minor
veins (48) it is clear that this is not so. Such studies are consistent with aphid stylectomy measurements of 839 mM sugar in the sap of Alonsoa
(Scrophulariaceae) (115), which appears to use
the same phloem loading mechanism as cucurbits (109, 115). It could be argued that cucurbit exudate is highly concentrated as it bleeds
but is immediately diluted by water from the
xylem. This is unlikely given that the water column is under tension (101). Furthermore, dilution does not account for the unexpectedly
low concentration of stachyose compared with
monosaccharides and amino acids in such sap
(31, 89), in direct conflict with radiolabeling experiments (97, 117).
Perhaps the low osmotic potential and unusual composition of bleeding sap in cucurbits
is associated with the presence of an extrafascicular system of anastomosing phloem strands
around the periphery of the bundles and in the
cortex (23, 24, 30). The sieve pores of extrafascicular, but not fascicular, SEs are open in severed
tissue (24), perhaps explaining why exudate is so
copious. Eschrich and coworkers (29) reported
that exuding sap originates from vascular bundles and from extrafascicular phloem in the cortex. Therefore, cucurbit sap may be a mixture
of the contents of the fascicular and extrafascicular systems. If the extrafascicular system is
not part of the long-distance transport network,
some of the material in cucurbit exudate could
be stationary in unsevered tissue.
Unfortunately, little is known about the
role of extrafascicular phloem in cucurbits.
Radiolabel is present in extrafascicular phloem
following 14 CO2 fixation in the lamina (117),
suggesting that it is part of the long-distance
transport system, but this radiolabel may simply
represent sugars exchanging between the two
systems. Another possible role for extrafascicular phloem is that of a biosynthetic reservoir
(17).
EDTA-Facilitated Exudation
King & Zeevaart (65) demonstrated that EDTA
facilitates exudation of 14 C from cut petioles or
shoots after 14 CO2 fixation in the lamina. Their
labeling procedure assured that mobile photoassimilates were being sampled. However, the
EDTA technique is also used to identify unlabeled compounds, and in this case there is a danger of sampling nonmobile materials. This issue
is the same one discussed above for bleeding
sap, but it is potentially more serious because
EDTA softens the tissue and induces leakage of
ions and metabolites, including carbohydrates
(54). Darkening leaves during exudation (65)
prevents movement of the EDTA into the lamina in the transpiration stream, where it can
cause more extensive damage, but the effects on
the submerged portion of the petiole cannot be
avoided. At higher EDTA concentrations, the
proportion of exuded hexose is elevated, consistent with additional leakage (45).
There is also a risk that exuded compounds
may degrade during the collection period.
Hexoses in EDTA exudate are sometimes ascribed to invertase activity. This may or may not
be the case, but should not simply be assumed.
Problematically, in some studies hexoses are included in the calculation of exuded sucrose, assuming that they arose by hydrolysis. This is a
suspect procedure considering that hexoses may
have leaked instead from the vacuolar compartment of compromised, nonphloem cells.
Discrepancies in metabolite profiles of sap
from EDTA-facilitated exudation and aphid
stylets have been noted. Fructans and loliose are
present in EDTA exudate, but not aphid stylet
sap, of Lolium perenne (1), and gamma amino butyric acid is present in EDTA exudate, but not
aphid sap, of Medicago sativa (19). In both studies, monosaccharide levels were much higher
in the EDTA exudate. Similarly, metabolite
studies on Lucerne (41) and oat and barley
(118) yielded significant differences when the
two methods were compared. When using the
EDTA-facilitated exudation technique, identification of novel or unexpected compounds in
sap should be corroborated by other methods.
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The method also does not lend itself to measuring transport rates, because the source/sink
balance is drastically altered and the amount
exuded is generally much less than that transported in intact plants (65).
Carbon Isotope Labeling
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In a common and useful isotope labeling approach, leaves are exposed to 14 CO2 and isotopically labeled substances are detected downstream. 11 C can also be employed, but its short
half-life precludes its use in most analytical procedures. 14 CO2 labeling was instrumental in
early studies that identified transported photoassimilates (9). Labeled compounds can be
metabolized in transit, leading to the possible misidentification of metabolic products as
transport compounds, but over short periods
this effect is minimal (117).
Isotopically labeled compounds, such as hormones, can be applied to one region of the
plant and detected downstream (e.g., 13). This
approach allows researchers to determine if a
compound of interest is phloem-mobile, but
it does not guarantee that the same endogenous compound is mobile because it may not
be in a compartment that makes it available for
transport.
SUBSTANCES TRANSPORTED
IN THE PHLOEM
Carbohydrates and Amino Acids
All plants transport sucrose, whereas some also
transport raffinose and stachyose and/or sugar
alcohols, sometimes in greater quantity than sucrose (70, 108). These compounds enter the
phloem primarily in mature leaves but also
exchange with surrounding tissues along the
transport route (5).
Numerous plasmodesmata are present in the
minor veins of mature leaves in some species
(37), providing a potential symplastic pathway
for sucrose to enter the phloem passively, down
its concentration gradient, as long as there is
enough sucrose in the mesophyll to obviate the
212
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need for a concentrating step in the phloem
(111). In some other species, such as the cucurbits, sucrose diffuses into the minor vein CCs
and is converted there to raffinose and stachyose
(74, 108), a polymer trapping mechanism that
concentrates the sugars. A third loading mechanism, which is apparently the most common,
is through the apoplast. Sucrose effluxes into
the apoplast and is then actively loaded into the
minor vein phloem by cotransport with protons
(70). Some plants apparently load by more than
one mechanism, using different CC types in the
same veins (108).
Less is known about sugar alcohol loading.
Sorbitol and mannitol transporters have been
localized in the minor vein phloem (Reference
60 and references therein). However, because
transporters are also used for retrieval, their
presence in the phloem does not prove that
loading is apoplastic (108). Sugar alcohol concentrations in the cytosol of Plantago and celery mesophyll cells are much lower than those
in aphid stylet exudate (80), providing strong
evidence for active, apoplastic loading. However, similar measurements in Prunus produced
equivocal results (80).
Organic N may enter the phloem by several routes (88). Inorganic N may be carried
into mature leaves in the xylem, converted to
organic form, and subsequently exported. Organic N may also enter leaves in the xylem
to be metabolized, used in protein synthesis,
or transferred to the phloem. Comparisons of
amino acid concentrations in the mesophyll and
phloem (122) and the effects of downregulating
an amino acid transporter (66) suggest an active
loading mechanism in plants that load sucrose
from the apoplast (83). In symplastic loading
species, active apoplastic loading may not be
required or even possible, because amino acids
are smaller than sucrose and should have free
access to the phloem via plasmodesmata.
Secondary Compounds
Many secondary compounds are toxic and are
excluded from symplastic compartments, including the phloem. However, the phloem
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does transport several types of secondary
compounds.
Glucosinolates are sulfur-containing glucosides activated by myrosinase (46). Glucosinolates and myrosinase are present in different
cellular or intracellular compartments and are
mixed by herbivory, releasing toxic products
(46). In Arabidopsis, glucosinolate is found in S
cells, external to the vascular bundles. Glucosinolates are present in aphid stylet exudate (68),
but it is still not known where or how they enter the phloem. Glucosinolate transporters have
been characterized (15), and glucosinolate enters the phloem when applied to leaves (16), but
transporter localization does not definitively
prove that loading of endogenous material is
apoplastic (see above).
Iridoid glycosides are terpene-derived compounds that protect against nonadapted insect
herbivores, fungi, and bacteria (14). Antirrhinoside (44, 115) and catalpol (112) are translocated in Asarina and Catalpa, respectively. Voitsekhovskaja and coworkers (115) measured high
concentrations of antirrhinoside (786 mM) in
aphid stylet exudate from Asarina. One wonders
if such high levels in exudate are due to release
of antirrhinoside into the phloem in response
to aphid attack.
Pyrrolizidine alkaloids (PAs) are defensive
compounds (49) and are found in the phloem
(123). In Senicio, synthesis of the backbone
structure of PA is restricted to roots (78).
How PAs are loaded into the phloem and
transported from roots to shoots against the
typical direction of flow is unknown (78).
Complex compartmentation and the translocation of intermediates between cell types are
common in alkaloid synthesis (126). Many of
these pathways operate close to, if not within,
the phloem, perhaps taking advantage of the
carbohydrate supply. In opium poppy, alkaloids
synthesis is associated with the phloem and the
compounds accumulate in laticifers. Whether
this synthesis occurs in phloem parenchyma
cells and laticifers themselves (119) or in SEs
(10) is a matter of contention.
Cardenolides are defensive steroid compounds. On the basis of aphid feeding patterns,
Botha and colleagues (11) suggested that cardenolides are transported in the internal phloem.
These compounds are found in aphid honeydew (20) and Cuscuta growing on Digitalis (91).
Auxin
Indole-3-acetic acid (IAA) is transported cellto-cell (35) and also in the phloem. Exogenous
radioactive IAA migrates at 3–5 cm h−1 (18),
5–10 times faster than cell-to-cell transport
(35). Also, endogenous auxin (Aux) is present
in phloem sap of Ricinus communis (6) and in
sap collected from Pisum sativum leaves using
the EDTA-facilitated exudation protocol (59).
Interestingly, inhibition of polar Aux transport
from shoot apex to roots does not reduce the
Aux effect on lateral root development, suggesting that phloem-based transport of IAA is also
involved in controlling root architecture (18).
Indole-3-acetic acid
(IAA): a native form
of the plant hormone
auxin; involved in
regulation of spatial
and temporal aspects
of plant growth and
development
Proteins
Proteins are present in phloem sap (Reference
107 and references therein). Several hundred
proteins have been identified in the sap of cucurbits, Ricinus, and oilseed rape (Reference 98
and references therein). Protein concentration
in Ricinus sap is 0.1 to 2 mg ml−1 (47) and up to
100 mg ml−1 in cucurbit exudate (89).
Because mature SEs cannot synthesize protein, the most likely origin of phloem-sap proteins is the immature SE or the CC. Considering that GFP-fusion proteins greater than
67 kDa traffic from CCs to SEs in transgenic
plants (103), it is reasonable to propose that
many proteins in sieve tube sap have nonspecifically diffused (leaked) out of the CCs and into
the SEs.
Conversely, some proteins in phloem sap are
larger than 100 kDa in size (7, 34, 98, 116), and
protein profiles of phloem sap are significantly
different from those of surrounding cells (e.g.,
40, 116). These findings suggest facilitated and
regulated protein movement into SEs. Indeed,
proteins derived from pumpkin sap have the
capacity to alter the SEL of plasmodesmata
that interconnect mesophyll cells (7), though
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evidence on plasmodesmata-pore unit regulation is lacking.
Additional evidence suggests that protein
trafficking into and out of the SE is regulated.
Although the P-proteins of cucurbits, PP1 and
PP2, traffic in both directions between CCs and
SEs (42, 69), serpin, a defense-related protein
smaller than PP1 (43 kDa versus 88 kDa), appears unable to move from SE to CC (69). Protein profiles in SE sap are site specific. For example, new soluble proteins in wheat phloem
sap are added and other proteins are removed
along the transport pathway (34), more proteins are present in sap collected from large
than from small pumpkin fruits (62), and differences are seen in comparisons of proteins in
sap collected from different organs of melon
plants (D. Malter & S. Wolf, unpublished data).
In addition, allene oxide cyclase, a key enzyme
in the jasmonate pathway, but not its mRNA, is
present in SE plastids in tomato,which suggests
synthesis in the CC and directed export to SE
plastids (50).
It is not known how protein trafficking into
and out of SEs is regulated, but viral protein
movement may provide clues. For example,
virus infection enables the movement of GFP
from the mesophyll to the sieve tube (85), and
the 3a movement protein of Cucumber mosaic
virus fused to GFP can traffic out of the SE-CC
complex in tissue where there is no movement
of solute through the same plasmodesmata (57).
Perhaps viruses exploit an endogenous mechanism for protein trafficking across apparently
closed symplastic boundaries.
Control over the import and export of proteins from the SEs presumably involves protein
modifications inside the sieve tube. The finding
that numerous pumpkin phloem-sap proteins
can be phosphorylated and/or glycosylated suggests that these proteins are subjected to posttranslational modifications required for their
interaction with a chaperone (protein-protein
interaction) to enable their trafficking out of the
sieve tube (105). A unique motif that facilitates
trafficking through plasmodesmata was characterized in a chaperone isolated from Cucurbita
maxima phloem sap (2).
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Perhaps the most pronounced alteration in
plant development is the transition from vegetative to floral growth, which is governed
by environmental and internal signals. Longdistance trafficking of the protein product of
FLOWERING LOCUS T (FT) is associated
with the promotion of flowering in Arabidopsis,
pumpkin, and rice (22, 72, 104), which provides
strong evidence that FT is the long-sought
florigen. The current challenge is to inhibit specific phloem trafficking of FT and explore the
influence of this inhibition on flowering.
It is logical to assume that long-distance
protein trafficking is directed by mass flow
from source to sink organs. However, rootward
movement of C. maxima PP16-1 apparently depends on its interaction with specific phloem
proteins (2, 3). Note, however, that these pioneering studies were based on microinsertion of
biotinylated pumpkin protein into a rice sieve
tube via cut stylets. Detection of the tracer proteins in the various plant organs might have
been due to this ultrasensitive experimental
system.
RNA
Almost 40 years ago, traces of RNA were found
in phloem sap, but were considered to be contaminants from neighboring cells (67, 125).
More recently, transcripts encoding proteins
related to metal homeostasis, stress response,
protein degradation or turnover, and phloem
structure and metabolism have been identified
and characterized in phloem-enriched tissues
(86, 114). The laser-assisted microdissection
technique has been used to collect clusters of
phloem cells that include several cell types for
a cDNA library (4) and for microarray experiments (81). However, the technique has not yet
yielded samples from single cell types.
RNA analysis has been conducted on sap
from aphid stylectomy (25, 95), but these
studies have provided only limited genomic
information. Therefore, sap collection from
bleeding plants is still the primary option for
high-throughput analysis of phloem-sap transcripts (26, 82, 92). Functional classification
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(based on BLAST descriptions) of almost 1000
transcripts identified in phloem sap from cut
melon stems showed that the transcripts fall
into the following categories: cellular response
to hormones and stress (40%); signal transduction, transcriptional control and nucleic
acid binding (15%); and defense-associated responses (10%). Interestingly, only 2% of transcripts are related to metabolism (82).
In an alternative approach, a CC-specific library was constructed from Arabidopsis cells selected on the basis of GFP expression driven
by the AtSUC2 promoter (58). Partial sequencing of 2000 clones revealed that 29% of the
genes are associated with redox regulation and
16% with metabolism. The pronounced differences between the profiles of transcripts identified in phloem sap (82) and CCs (58), albeit from different species, suggests that sap
RNA is not simply contamination from CCs.
Moreover, the inability to detect CC-specific
gfp RNA in wild-type scions on transgenic
stocks (51, 85) suggests that entry of endogenous mRNA molecules into the sieve tube is
sequence specific.
Is it true that the macromolecules present
in the sieve tube, including mRNA molecules,
are in the process of trafficking long distances?
Support for this notion has been provided
mainly by experiments in which species-specific
mRNA molecules pass through unions in heterografts (Reference 82 and references therein)
or from host to parasite (90). The latter discovery raises the fascinating possibility of interspecies communication.
Not all phloem sap mRNA molecules traffic systemically: Only 6 of 43 randomly selected
melon phloem sap transcripts were identified in
scions of heterografts (82). Interestingly, three
of the six transcripts encoded Aux/IAA and
small auxin up RNA (SAUR) proteins. To date
it is unclear how many transcripts are phloem
mobile, how stationary mRNA is anchored, and
most importantly, what biological roles most
transcripts play in enucleated SEs or their target sink tissues.
Although it has been suggested that mRNA
molecules can operate as long-distance signals
(73), evidence to date is limited. Transmission
of a phenotype across a graft union in conjunction with mRNA movement was first demonstrated with the mouse ears (Me) mutation, where
wild-type scions developed octapinnate compound leaves characteristic of Me mutant plants
(64). These results were corroborated in one
study (52), but only in low-light growth conditions, and were not corroborated in a second study under any light conditions (75). Using another system, Haywood and coworkers
(52) demonstrated that transport of gibberellic
acid-insensitive ( gai) transcripts in heterografts
altered leaflet morphology in wild-type scions,
consistent with impaired GA signaling (52). In
a third system, trafficking of BEL1-like transcription factor mRNA in grafted plants resulted in enhanced potato tuber formation (8).
The results of these studies are highly intriguing. Given the importance of the concept of
long-distance RNA signaling and the possibility of sequence-specific RNA sorting (52), these
experiments need to be corroborated and extended. In particular, the relevance of systemic
RNA signaling to natural environmental responses needs to be established.
Although phloem sap contains no detectable
RNAse activity (25, 95), the current perception,
based on virus movement studies and the identification of RNA binding proteins in phloem
sap (43, 124), is that mRNA molecules traffic long distances as ribonucleoprotein (RNP)
complexes and not as naked molecules. Because
transcripts cannot be translated in the SEs, such
movement must require a chaperoning mechanism to load them into the SEs and unload them
in their target sinks.
The generality of the concept that mRNA
molecules act as signals to coordinate developmental processes at the whole-plant level is still
under debate. More information is needed on
the sites of synthesis of these molecules and the
sequence motifs (or chaperoning mechanisms)
required for transcripts to enter, exit, and traffic
long distances in sieve tubes. Other important
questions relate to the synthesis and possible
movement and function of the respective proteins in target tissue. A special challenge will
www.annualreviews.org • Trafficking in the Phloem
Heterograft: a
vegetative tissue
(upper stem and shoot
apex) of a plant from
one species that is
transplanted on a
tissue (lower stem and
roots) of a plant from
another species
Ribonucleoprotein
(RNP) complex: a
complex that is formed
between RNA and
protein
215
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be to develop experimental systems in which
phenotypic changes can be evaluated following
specific inhibition of the long-distance movement of each transcript.
SUMMARY POINTS
1. Pathways into and out of the phloem are determined primarily by the distribution and
permeability of plasmodesmata and the location, activity, and specificity of transmembrane transporters.
2. Because of its elevated hydrostatic pressure, the phloem is difficult to sample without
displacing its contents and possibly mobilizing normally anchored materials and pulling
substances into the sieve elements (SEs) from surrounding cells.
Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org
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3. Several methods have been developed to sample the phloem but all, including insect
stylectomy, can introduce artifacts.
4. Carbohydrates are the primary osmotic agents in phloem sap. The mechanisms of sugar
and sugar alcohol loading in leaves are species specific and still under investigation.
5. The functions of most proteins in phloem sap are unknown, as are the mechanisms that
confer specificity to their intercellular distribution and mobility.
6. Functions have been attributed to a few RNA molecules in phloem sap, but this area of
research is in its infancy.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of this
review.
ACKNOWLEDGMENTS
Research in the authors’ laboratories is currently supported by the U.S.-Israel Binational Agricultural Research and Development Fund (BARD: IS-3884–06). R.T.’s research is also funded by
the National Science Foundation (IOB-0444119) and the U.S. Department of Agriculture (2005–
35318-16231). S.W.’s research is also supported by the Israel Science Foundation (ISF 380/06).
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