Journal of Experimental Botany, Vol. 57, No. 4, pp. 767–774, 2006
doi:10.1093/jxb/erj087 Advance Access publication 22 February, 2006
FOCUS PAPER
Phloem sap proteins: their identities and potential
roles in the interaction between plants and
phloem-feeding insects
Julia Kehr*
Max Planck Institute of Molecular Plant Physiology, Department Willmitzer, Am Mühlenberg 1,
D-14424 Potsdam, Germany
Received 11 July 2005; Accepted 6 December 2005
Abstract
The phloem is a well-known target of sucking and
piercing insects that utilize the transported fluid as
their major nutrient source. In addition to small molecules like sugars and amino acids, phloem sap of
higher land plants contains proteins that can accumulate up to high concentrations. Although the knowledge about the identities of these phloem sap proteins
is increasing, the functions of most of them are still
poorly understood. Since many phloem sap proteins
have predicted roles in wound and defence responses,
they constitute a class of compounds that can potentially influence plant–insect interactions. However,
there are as yet no studies published that have
examined direct effects of phloem sap proteins on
insect feeding or vice versa. This review summarizes
the current knowledge about the identities of phloem
sap proteins, focused on polypeptides with probable
functions in wound and defence reactions, and
their potential impact on plant–insect interactions is
discussed.
Key words: Phloem sap, plant–insect interactions, proteins.
Introduction
The phloem is the major route for the translocation and
distribution of organic metabolites assimilated during
photosynthesis. The sieve elements (SEs) transport a wide
range of compounds like water, minerals, amino acids,
organic acids, sugars, and sugar alcohols (Zimmermann
and Ziegler, 1975).
Research in recent years has shown that the phloem
system is not only responsible for photosynthate allocation,
but has several additional functions. For example, the
phloem is an important mediator of whole-plant communication (Ruiz-Medrano et al., 2001). The transported information molecules include phytohormones (Baker,
2000), and also macromolecules such as proteins (Pearce,
1991) and RNAs (Jorgensen et al., 1998; Jorgensen, 2002).
The occurrence of macromolecules seems surprising, since
mature SEs lack the capability for mRNA and protein
synthesis. However, recent studies have provided accumulating evidence that these macromolecules only not sporadically appear, but a large number of RNAs (Lucas et al.,
2001) and soluble proteins (Hayashi et al., 2000; Walz
et al., 2004) are constantly present in SE exudate. Proteins
can even be regarded as a major component of phloem
sap, given that, for example, cucurbit exudate contains
high concentrations up to 100 mg mlÿ1 (Richardson et al.,
1982). Phloem sap proteins are believed to be imported
through specialized plasmodesmata connecting SEs and the
adjacent companion cells (CCs) where protein synthesis is
taking place (Smith et al., 1987; Clark et al., 1997;
Dannenhoffer et al., 1997).
An increasing number of phloem sap polypeptides have
been identified in recent years. However, identification and
analysis of phloem sap proteins is hampered by the
inaccessibility of the phloem stream to sampling attempts,
and there are only a few plant species from which SE
exudate can easily be obtained in sufficient quantities to
analyse the proteins. Due to these sampling difficulties,
most SE proteins identified originate from Ricinus, cucurbits, and oilseed rape, where relatively large amounts of
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: CC, companion cell; GNA, Galanthus nivalis agglutinin; PP1, phloem protein 1; PP2, phloem protein 2; PI, protease inhibitor; PR,
pathogenesis-related; ROS, reactive oxygen species; SE, sieve element.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
768 Kehr
phloem sap simply exude from incisions (Milburn, 1970).
A few abundant proteins have also been specified in rice,
where phloem-feeding insects allow the collection of small
amounts of phloem sap (Kennedy and Mittler, 1953;
Kawabe et al., 1980). Table 1 lists all known phloem sap
proteins that have predicted functions in signalling, stress
or defence reactions.
Interestingly, most of the identified phloem sap proteins
repeatedly occur in more than one plant species (Table 1).
This indicates a high degree of conservation of the phloem
sap protein composition in higher land plants. A similar
conclusion can be drawn from immunological studies
(Schobert et al., 1998) that showed that several proteins
were found in the phloem sap of many, even completely
unrelated, monocot and dicot species. Since current knowledge about phloem sap proteins is far from being complete
in all species, it can be expected that even more proteins
coincide in several plants, although some species-specific
differences might exist. This reasonable degree of conserved proteins can be explained by the pronounced
Table 1. Compilation of phloem sap proteins that are potentially involved in plant wound and defence reactions
A.th., Arabidopsis thaliana; B.n., Brassica napus; C.m., Cucurbita maxima; C. melo, Cucumis melo; C.s., Cucumis sativus; L.e., Lycopersicon
esculentum; O.s., Oryza sativa; P.v., Phaseolus vulgaris; R.c., Ricinus communis.
Function
Protein
Species
Reference(s)
Reactive oxygen/redox related
Ascorbate peroxidase
Copper homeostasis factor
Dehydroascorbate reductase
Ferredoxin
Glutaredoxin
Glutathione reductase
Glutathione-S-transferase
Iron transport protein
Metallothionein
Monodehydroascorbate reductase
Peroxidase
Peroxiredoxin
Superoxide dismutase, Cu/Zn
Thioredoxin h
Annexin
C2 domain-containing protein
Calmodulin
Kinases
B.n.
A.th., B.n.
B.n., C.m., C.s.
B.n.
B.n., R.c.
C.m.
B.n., O.s.
R.c.
R.c.
B.n.
B.n., C.m., C.s.
B.n.
C.m., C.s.
B.n., C.m., O.s.
B.n., R.c.
B.n.
B.n., R.c.
C.m., O.s.
ACC oxidase
ACC synthase
Allene oxide cyclasea
Allene oxide synthasea
Lipoxygenase
C.m., C.s.
C.m., C.s.
L.e.
L.e.
C.m., C.s.
SAM synthetase
Systemin
Forisome
PP1
PP2
B.n.
L.e.
P.v.
C.m.
B.n., C.m., C.melo, C.s.
Aspartic protease inhibitor
Chymotrypsin inhibitor
Cystatin
C.m.
C.m.
B.n., C.m., C.s., R.c.
Serpin-1
Trypsin inhibitor
Cm lectin 17
C.m.
C.m., C.s.
C.melo, C.s.
Cm lectin 26
Lectin, mannose-binding
CSF-2
Epithiospecifier protein
Myrosinase
Myrosinase-binding protein
SN-1
SWF-1 (peptidase)
SWF-3 (b-glucosidase)
C.s.
C.m.
C.m., C.s.
B.n.
B.n.
B.n.
C.s.
C.m.
C.m.
Giavalisco et al., 2006
Giavalisco et al., 2006; Mira et al., 2001
Walz et al., 2002, 2004
Giavalisco et al., 2006
Szederkenyi et al., 1997
Alosi et al., 1988
Fukuda et al., 2004
Krüger et al., 2002
Barnes et al., 2004
Giavalisco et al., 2006
Walz et al., 2002
Giavalisco et al., 2006
Walz et al., 2002, 2004
Ishiwatari et al., 1995
Barnes et al., 2004
Giavalisco et al., 2006
Barnes et al., 2004; Yoo et al., 2002
Avdiushko et al., 1997; Nakamura et al., 1993;
Yoo et al., 2002
Walz et al., 2004
Walz et al., 2004
Hause et al., 2003
Hause et al., 2003
Avdiushko et al., 1994; Hause et al., 2003;
Walz et al., 2004
Giavalisco et al., 2006
Narvaez-Vasquez et al., 1995
Knoblauch et al., 2001
Clark et al., 1997
Bostwick et al., 1992; Gomez et al., 2005;
Walz et al., 2004
Christeller et al., 1998; Walz et al., 2004
Murray and Christeller, 1995; Walz et al., 2004
Barnes et al., 2004; Haebel and Kehr, 2001;
Schobert et al., 1998; Walz et al., 2004
Walz et al., 2004; Yoo et al., 2000
Murray and Christeller, 1995; Walz et al., 2004
Dinant et al., 2003; Gomez et al., 2005;
Walz et al., 2004
Dinant et al., 2003; Walz et al., 2004
Walz et al., 2004
Haebel and Kehr, 2001; Walz et al., 2004
Giavalisco et al., 2006
Giavalisco et al., 2006
Giavalisco et al., 2006
Walz et al., 2004
Walz et al., 2004
Walz et al., 2004
Calcium-related
Phytohormone- related
SE plugging
Protease inhibitors
Lectins
Others
a
Immunological detection.
Phloem sap protein–insect interactions
structural similarity of SEs in all angiosperms (van Bel,
1999) that is probably caused by the high functional
specialization of SEs.
769
The following sections will therefore focus on the known
classes of defence-related phloem sap proteins summarized
in Table 1 and will discuss how they could influence plant–
insect interactions.
Potential influences of phloem sap proteins on
plant–insect interactions
ROS, calcium and phytohormones
The transported metabolites make SEs an attractive target
for insects that are specialized to feed exclusively on
phloem sap, like, for example, whiteflies or aphids.
In contrast to herbivores, phloem-feeding insects establish a sustained interaction with SEs. They release saliva
that inhibits plant stress responses and prevents closure of
pierced SEs by callose or polymerized proteins (Miles,
1999). This allows the insects to ingest large amounts of
phloem sap to obtain enough nutrients for their survival.
Due to this feeding behaviour, phloem-sucking insects are
directly exposed not only to nutrients but to all components
of the transport fluid, including proteins. Interestingly,
a high proportion of the phloem sap proteins so far
identified is predicted to be involved in stress and defence
reactions, although their exact physiological functions
remain to be established. To date, there is no study showing
either an impact of phloem sap proteins on insects or of
insect feeding on phloem sap protein composition or
activities. However, due to the direct contact of phloemfeeding insects to SE contents, an influence of phloem sap
proteins on insects is easily conceivable.
At the whole plant level, phloem-feeding insects induce
local responses, including the activation of salicylic- and
jasmonic acid-dependent pathways (Moran and Thompson,
2001), although they cause only limited tissue damage to
the plant when targeting SEs. In addition, genes involved
in oxidative stress, calcium-dependent signalling, and
pathogenesis-related responses [such as lipoxygenase,
chitinases, peroxidases and other pathogenesis-related (PR)
proteins] are key components of the plant defence response
(Moran et al., 2002).
In addition to these local reactions, a recent publication
demonstrated a systemic effect of aphid infestation on gene
expression in isolated phloem tissue (containing sieve
elements, companion cells, sclerenchyma, and parenchyma
cells) of celery (Divol et al., 2005). Here, genes typically
responding to non-chewing insects were not altered. Instead, genes involved in cell wall modification, water
transport, vitamin biosynthesis, photosynthesis, and carbon
and nitrogen assimilation were induced. This indicates that
the phloem response might be quite different from the
whole plant reaction.
However, the observed changes in phloem tissue
gene expression do not allow conclusions about alterations
of phloem sap protein composition, because the location of
protein synthesis in CCs might differ from their site of
accumulation in SEs.
As mentioned above, oxidative stress is one of the first
general reactions to the injury caused by phloem-sucking
insects penetrating the tissue. In addition, it has previously
been noticed that aphid salivary secretions can themselves
alter plant oxidative conditions (Jiang and Miles, 1993;
Walling, 2000). It was also proposed that the saliva and the
injury caused by aphid feeding induce not only a local but
also a systemic production of reactive oxygen species
(ROS) in the phloem (Moran et al., 2002; Zhu-Salzman
et al., 2004; Divol et al., 2005). Accordingly, several genes
associated with oxidative stress are up-regulated by aphid
infestation at the whole plant level in A. thaliana (Moran
et al., 2002), as well as systemically in phloem tissue
isolated from celery (Divol et al., 2005). In soybean, insect
feeding has also been shown to trigger the generation of
ROS. Here, a direct anti-insect activity of ROS has been
proposed (Bi and Felton, 1995). ROS are obviously not
excluded from SEs, since ROS scavenging enzymes are
a protein class generally contained in phloem sap and their
activities are up-regulated by stresses such as drought
(Walz et al., 2002). Glutathione-S-transferases and the
metal ion scavengers, metallothioneins, and the copper
homeostasis factor can detoxify radicals (Marrs, 1996) and
the expression of the corresponding genes was significantly
up-regulated by aphid feeding in systemic phloem tissue
(Divol et al., 2005). Phloem tissue-specific regulation of
the redox status in response to insect attack is substantiated by the observation that even one week of aphid infestation of A. thaliana did not result in necrosis or other
symptoms indicative of a breakdown in the regulation
of oxidation (Moran et al., 2002).
In addition to ROS, tissue damage is normally accompanied by an elevation in cytosolic calcium. In undisturbed
SEs, calcium is low and an increase upon wounding
is thought to initiate a long-distance signalling cascade
(Fromm and Bauer, 1994; Knoblauch et al., 2001; van Bel
and Gaupels, 2004). It was proposed that elevations of
phloem Ca2+ levels also participate in the regulation of
phloem enzymes (Eschrich and Heyser, 1975). The occurrence of calcium-binding proteins in phloem sap was
recognized early (McEuen et al., 1981), but some time
elapsed before the first of them could be identified. For
example, enzymes and activity of calcium-activated protein
kinases that act as major mediators in Ca2+ signalling occur
in phloem sap. In addition, annexins and calmodulins were
found in phloem samples from different species (Table 1).
There is evidence that calcium and calcium-binding
770 Kehr
proteins are involved in the regulation of plant wound
responses (Bergey and Ryan, 1999; Leon et al., 2001) and
reports actually suggest a relationship between calmodulin gene expression and ROS generation (Harding et al.,
1997). Moreover, calmodulin expression is increased by
systemin (Bergey and Ryan, 1999), a peptide hormone found
in Solanaceae, that has also been detected inside SEs
(Narvaez-Vasquez et al., 1995).
In addition, phloem sap contains enzymes involved in
the synthesis of phytohormones, namely ethylene and the
jasmonic acid (Table 1). Formation of these phytohormones
within SEs may lead to an amplification of locally induced
signals that can then trigger a systemic response, as has
been proposed for jasmonic acid (Hause et al., 2003).
Occlusion of SEs
It is well known that, upon mechanical damage, plants have
different mechanisms to plug affected SEs to avoid the loss
of organic nutrients. This can be regarded as a quick and
straightforward response to the damage caused by insects.
Calcium is an important mediator for plugging SEs and
calcium antagonists such as EDTA have long been known
to prevent this sieve tube occlusion (King and Zeevaart,
1974).
Different mechanisms that are all based on the action of
phloem sap proteins seem to be involved in SE occlusion
responses. Legumes contain unique crystalloid proteins, the
so-called forisomes, that can undergo rapid and reversible
conversions from the condensed resting state into a dispersed state, in which they close SEs. Crystalloid dispersal
is mediated by calcium levels that increase following
plasma membrane leakage, rapid turgor loss, or mechanical
injury (Knoblauch et al., 2001, 2003).
Another well-known class of SE proteins involved in the
plugging of sieve pores are PP1 and PP2 that were first
described in cucurbit phloem sap (Read and Northcote,
1983a), but seem to occur in all dicots. Under oxidative
conditions, PP1 monomers and PP2 dimers are covalently
cross-linked via disulphide bonds, forming high molecular
weight polymers that close the sieve pores (Read and
Northcote, 1983a, b). This response is normally accompanied by the synthesis of the b-1,3-glucan callose by callose
synthase that accumulates on sieve plates after different
stress treatments (McNairn and Currier, 1968) to prevent
assimilate loss from cut SEs (Sjölund, 1997). While the
closure by proteins is a rapid and reversible mechanism,
callose is responsible for long-term plugging of sieve plates
that is almost irreversible (van Bel, 2003).
As a response to herbivore attack, phloem sap is
squeezed from SEs and accumulates at wounded sites.
This phloem sap will prevent further herbivory and reduces
the risk of infection of wounds with opportunistic pathogens like fungi (Christeller et al., 1998). The formation of
phloem filaments by PP1 and PP2 as well as the closure by
callose constitute a potent physical barrier against further
invasion.
By contrast, phloem-sucking insects can locate and
access SEs avoiding the normal plant wound response.
Components of aphid saliva injected immediately after
phloem puncture inhibit the normal callose deposition and
P-protein gelation and thus enable sap uptake without
phloem sealing. After feeding sites are established, the
stylets of phloem-piercing insects can stay in continuous
contact with plant cells for h up to weeks (Walling, 2000).
However, a recent study revealed that massive deposits of
callose are caused by infestation with phloem-feeding
aphids, with callose associated with sieve plates and the
pore-plasmodesmata between CCs and their associated
SEs. Even leaves that had been colonized by aphids, but
from which the aphids were removed, showed extensive
wound callose deposits, which persisted for up to 48 h after
the removal of aphid colonies, suggesting that the damage
caused by aphid feeding is a long-term, non-transient event
in non-resistant plants (Botha and Matsiliza, 2004).
Anti-nutritive proteins
If insects manage to overcome the first levels of plant
defence, phloem sap still provides an option to inflict
damage on them by supplying toxic or inhibitory compounds, including proteins. Plants can accumulate phloem
sap proteins up to high concentrations. Since phloem sap
accumulates at sites wounded by herbivores and also
phloem-sucking insects take up large amounts of phloem
sap, proteins with anti-insect properties constitute a passable
route for directed plant defence responses. The first
important question in this regard is if and how insects can
take up and digest phloem sap proteins.
Uptake and digestion of phloem sap proteins
by insects
Insects with different feeding behaviours can be expected to
get into contact with phloem sap by different mechanisms.
Herbivores will undoubtedly ingest a portion of phloem sap
together with the tissue consumed, while phloem-piercing
insects can take up large quantities of phloem sap. Aphids
normally process many times their body weight of phloem
sap in order to assimilate sufficient quantities of amino
acids, which occur in low concentrations in phloem, and
defecate large amounts of sugars as honeydew, because the
carbohydrate content of phloem exceeds their ability to use
it (Law et al., 1992). Therefore, toxic proteins transported
in SEs could make sucking problematic for some phloemfeeding insects. Previous studies indicate that insects take
up proteins from phloem sap together with all the other
transport sap components without any obvious restrictions.
Phloem sap protein–insect interactions
Accordingly, phloem exudate collected from excised aphid
stylets contains a range of different phloem proteins (Fisher
et al., 1992; Ishiwatari et al., 1995; Barnes et al., 2004) and
additional macromolecules like RNAs (Sasaki et al., 1998).
Also the phloem-feeding whiteflies ingested leaf proteins
from their diet when either a 35S-labelled mixture or a single
fluorescently labelled protein was applied. 35S label was
recovered either as labelled amino acids in honeydew or as
labelled proteins and amino acids in the whiteflies’ bodies
(Salvucci et al., 1998). In a study of the aphid Acyrthosiphon pisum, proteins supplied with an artificial diet were
recovered in intact form in honeydew (Rahbe et al., 1995),
which provides direct evidence for protein ingestion by
living aphids.
For insects feeding on plant tissues by chewing, proteolysis of dietary proteins is essential for survival. The
widespread production of protease inhibitors (PIs) by plants
in response to insect attack (Ryan, 1990) reflects the
importance of this step.
However, in recent years the question whether piercingsucking insects directly feeding on phloem sap are also
dependent on protein digestion has been a matter of debate.
Earlier studies suggested that phloem-sucking insects from
the superorder Hemiptera do not contain significant levels
of protease activity (Terra, 1990; Rahbe et al., 1995) and it
was also shown that aphid salivary secretions contain no
proteases (Cherqui and Tjallingii, 2000). Previously, the
fact that lectins fed in artificial diets can be recovered intact
in honeydew was interpreted as evidence against protein
digestion (Rahbe et al., 1995). However, lectins are
proteins that are very difficult to digest, even for herbivorous lepidopteran insects (Gatehouse et al., 1994). By
contrast, more recent experiments indicate that the occurrence of proteases allowing digestive proteolysis might
indeed be a more general feature, not only in herbivores
but also in sap-sucking insects, and may be important for
their proper nutrition (Salvucci et al., 1998; Foissac et al.,
2002; Habibi et al., 2002; Cristofoletti et al., 2003).
Protease inhibitors and lectins
One class of well-known defence proteins, the alreadymentioned protease inhibitors, are widespread in phloem
sap of different plant species (Table 1). Squash phloem
exudate has been shown to contain high amounts of trypsin,
chymotrypsin, serine, and aspartic protease inhibitors and
cysteine protease inhibitors have also been detected in rape
and Ricinus phloem sap (Table 1). PIs are proteins that
tightly bind proteolytic enzymes and thereby inhibit their
activity. Herbivores ingesting a diet high in PIs are thought
to experience metabolic deficiencies, including the lack of
essential amino acids (Ryan, 1990). There is no question
that protease inhibitors from plants can inhibit the digestive proteases of herbivorous insects and that they may,
771
in certain instances, suppress growth, development, and
survival when they are fed to these insects (Murdock and
Shade, 2002). This led to the introduction of genes
encoding protease inhibitors into plants by transgenic approaches in order to increase resistance towards chewing
insects (Hilder et al., 1987; Gatehouse et al., 1997). The
widespread distribution of PIs in vulnerable tissue and
their wound- and herbivore inducibility in diverse plant
families, together with the results from transgenic plants,
demonstrate the important role of PIs against herbivores
(Constable, 1999). The high abundance of PIs in phloem
sap in concert with the growing evidence that also phloemfeeders contain digestive proteases (see above) suggests an
important defensive role of the broad range of phloem sap
PIs against herbivores as well as phloem feeders.
In addition to PIs, another group of defence proteins
show a widespread occurrence in phloem sap, the lectins
(Table 1). Lectins are proteins that reversibly bind to
specific mono- or oligosaccharides. Chitin-binding lectins
from the Cucurbitaceae are a small group of lectins that
were first identified in cucurbit phloem sap (Read and
Northcote, 1983b). Arabidopsis also contains homologous
phloem-expressed PP2-like lectins (Dinant et al., 2003). In
addition to PP2-like proteins, the phloem sap of different
plants contains additional lectins (Table 1). Many lectins
are toxic to both insects and also vertebrates, although only
a few are known to be herbivore- or wound-induced
(Chrispeels and Raikhel, 1991). In insects, dietary lectins
are thought to bind to insect midgut tissue and disrupt
feeding and digestion and thus interfere with growth and
development (Murdock and Shade, 2002). Feeding experiments showed lectin effects on chewing (Murdock et al.,
1990) and sucking (Powell et al., 1993) insects.
The anti-insect properties of lectins led to their biotechnological exploitation. The most intensely studied antiinsect lectin, snowdrop lectin (Galanthus nivalis agglutinin:
GNA), showed a high toxicity against phloem-sucking
insects, not only in tests with artificial diets but also in
experiments with transgenic plants (Hilder et al., 1995;
Down et al., 1996; Gatehouse et al., 1996), indicating that
these toxins are translocated within the phloem sap.
Accordingly, Shi et al. (1994) detected GNA lectin in the
honeydew produced by aphids feeding on transgenic
tobacco plants.
Other defence-related proteins
Several components of the myrosinase system have been
detected in phloem exudate from Brassica (Table 1). The
myrosinase system is able to produce cyanates and nitriles
(Bones and Rossiter, 1996) from glucosinolates that are
also transported inside the phloem (Chen et al., 2001).
These toxic hydrolysis products are induced by wounding,
micro-organisms, and insects and could lead to a deterrence
of herbivorous as well as phloem-feeding insects.
772 Kehr
Phloem sap contains additional proteins known to be
induced by wounding (CSF-2, SN-1) or insect feeding
(SLW-1, SLW-3), but whose modes of action are unknown.
The SLW proteins are specifically induced by whitefly
feeding. While SLW-1 transcription is regulated by jasmonic acid and ethylene, two phytohormones that can
potentially be synthesized in SEs (see above), SLW-3 does
not respond to any known wound signal, indicating a new
signalling pathway for activation (Walling, 2000).
Conclusions
The recent identification of a growing number of proteins
from phloem sap of different plant species now allows first
insights into the potential functions of these polypeptides.
Functional classification maps them to diverse categories
but, interestingly, a number of them are functionally related
to defence responses and therefore influences on plant–
insect interactions are conceivable.
The most likely functions are connected to instant wound
signalling, plugging of SEs to avoid nutrient loss, and, since
there is evidence that herbivores as well as phloem feeders
are able to take up and digest phloem sap proteins, the
dispersal of directly acting anti-insect polypeptides.
Future studies analysing the direct effects of insect
infestation on local and systemic changes of phloem sap
protein composition and activity will elucidate their exact
involvement in plant defence against herbivores and
phloem-sucking insects. This knowledge will be useful to
develop novel biotechnological strategies to enhance the
resistance of crop plants against phloem-feeding insects.
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