Physical and chemical interactions between aphids and plants

Physical and chemical interactions between aphids and plants

Journal of Experimental Botany, Vol. 57, No. 4, pp. 729–737, 2006
doi:10.1093/jxb/erj089 Advance Access publication 10 February, 2006
FOCUS PAPER
Physical and chemical interactions between aphids and
plants
Torsten Will and Aart J. E. van Bel*
Plant Cell Biology Research Group, Institute of General Botany, Justus-Liebig-University, Senckenbergstrasse 17,
D-35390 Giessen, Germany
Received 18 August 2005; Accepted 6 December 2005
Abstract
Plants as targets for aphid attack
Aphids feed from sieve tubes deep inside the host
plant. Therefore, aphids must be able to recognize their
host plant(s) and to direct their stylets which must be
long and thin enough to reach and puncture the sieve
tubes at a particular site. Sieve tubes in angiosperms
are longitudinal arrays of sieve element/companion cell
modules which are highly sensitive to disturbance of
any kind. The sieve tubes dispose of elaborate sealing
mechanisms such as protein plugging and callose
sealing which are triggered by a rise in calcium in the
sieve tubes. Aphids seem to have developed a range of
physical and chemical measures to limit the amount
of calcium influx in response to stylet puncturing. Loss
of sieve-element turgor pressure induced by stylet
insertion is minimized by the minute stylet volume.
Turgor-dependent Ca21 influx, possibly mediated by
mechano sensitive Ca21 channels, must therefore be
limited. The components of the sheath and watery
saliva play a pivotal role in establishing the physical
and chemical constraints on the rise of calcium. Most
likely, sheath saliva prevents the influx of calcium from
the apoplast by sealing the stylet puncture site while
watery saliva may prevent plugging and sealing
of sieve plates by potential interaction with SE sap
ingredients.
Xylem and phloem, the transport avenues of the plant
Xylem and phloem are the long-distance transport conduits
in the vascular bundles of angiosperms. Together, they
constitute a water circulation system with high rates of
water intake by roots and water loss from the aerial parts,
respectively (Fig. 1). The transport pipes in vascular bundles are composed of longitudinally arranged modules of
xylem vessel elements or sieve element (SE)/companion
cell (CC) complexes. In both pipe systems, solutes are
transported by mass flow.
Aphids profit from the exuberant supply of food by
insertion of stylets into the sieve tubes. However, aphids are
urged to ‘drink’ xylem sap regularly in order to alleviate the
osmotic effects of ingested phloem sap, the concentration
of which exceeds by far that in xylem vessels (Buchanan
et al., 2000). As the mass flow of xylem sap is brought
about by a difference in hydrostatic potential between roots
and air (Wei et al., 1999), the drinking of xylem sap
requires active sucking by aphids (Spiller et al., 1990) due
to the negative tension (Baker, 1978).
By contrast, mass flow in the phloem is created
by a turgor difference between the sieve tube ends in
the sources and sinks (Münch, 1930). The phloem is
subdivided into three functional zones (van Bel, 1996).
In collection phloem, photoassimilates are accumulated by
the SE/CC-complexes in the minor veins of source leaves
and transported to the sieve tube ends in the release phloem
embedded in sink tissues. Collection phloem and release
phloem are connected by the transport phloem which has
a dual function. In the sieve tubes of the transport phloem,
Key words: Aphids, aphid–plant interaction, phloem, sieve
tubes, sheath saliva, stylet, suppression of plant defence,
watery saliva, wound reaction.
* To whom correspondence should be addressed. E-mail: Aart.v.Bel@bot1.bio.uni-giessen.de
Abbreviations: CC, companion cell; CLSM, confocal laser scanning microscopy; EDTA, ethylenediaminetetraacetate; EGTA, ethylene-glycol-bis(b-aminoethyl-ether) N, N9-tetraacetate; EPG, electrical penetration graph; MEL, mass exclusion limit; paP, parietal structural proteins; PN, protein nets;
PP, structural phloem protein; PPC, phloem parenchyma cell; PPU, pore plasmodesma unit; SE, sieve element; SER, sieve element reticulum; TEM,
transmission electron microscopy; wP, water-soluble proteins.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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730 Will and van Bel
and siRNA (Yoo et al., 2004). These compounds and
several phytohormones (Ruiz-Medrano et al., 2001) are
probably functional in long-distance signalling.
Fig. 1. Circulatory water flow through the plant. Water enters the
transport system for water mass flow in the roots by uptake from the soil
and is transported via the xylem. The driving force is mainly a negative
pressure or suction imposed by the evaporation of water from the leaves.
Part of the apoplasmic water is retrieved into the phloem system
associated with the loading of osmotically active photoassimilates. A
difference in turgor pressure between phloem loading and unloading
sites drives a mass flow through the phloem. In the sinks, amounts
of apoplasmic water which are not invested in sink growth flow back to
the xylem.
photoassimilates are retained for the supply of terminal
sinks and released to support, growth and maintenance of
the axial sinks along the pathway (Minchin and Thorpe,
1987; Hafke et al., 2005).
The collection of osmotically active photoassimilates
by the source ends of the sieve tubes results in a high local
turgor pressure, whereas the local turgor pressure is low at
the sink ends due to the release of photoassimilates. Hence,
mass flow in the phloem originates from a pressure
difference (Patrick, 1997). The high concentration of
osmotic substances causes a high sieve-tube turgor, which
allows almost passive food ingestion by aphids (Prado and
Tjallingii, 1994). Mass flow velocities in the phloem have
reported in the order of up to 50–100 cm h1 (Canny,
1975). This implies that an aphid’s stylet ‘sees’ a volume
of 56.6–113.2 nl h1 from a SE with a diameter of 12 lm.
Sieve tube sap contains a multitude of substances,
several of which are key nutrients of aphids. The phloem
sap is rich in carbohydrates and amino acids, but also
contains a wealth of minerals (Ziegler, 1975). Apart from
these compounds, the sieve tubes also translocate macromolecules such as soluble proteins which have largely
unknown functions (van Bel, 2003). Recently discovered
macromolecules in the phloem sap encompass several
classes of RNA-molecules such as, for example, miRNA
SE/CC-complexes as the sieve tube modules
Sieve elements (SEs) and companion cells (CCs) of
angiosperms originate from an unequal longitudinal division of a cambial precursor cell (Esau, 1969). During
differentiation of the SE, most of the organelles degenerate.
This process leaves a nearly empty long-stretched cell that
contains only an intact plasma membrane, P-plastids, a sieve
element endoplasmic reticulum (SER, Sjolund and Shih,
1983), a few mitochondria, and an extensive set of phloemspecific proteins (reviewed by Hayashi et al., 2000). Most
of these phloem-specific proteins have low molecular
weights (Schobert et al., 1995, 1998; Sjolund, 1997,
Hayashi et al., 2000). The CCs contain high numbers of
mitochondria, chloroplasts, vacuoles, a huge nucleus, and
a cytoplasm with a high optical density. The inferred high
metabolic activity of CCs is presumed to support the
physiological achievements of SEs.
SEs and CCs are connected via special plasmodesmata,
so called pore-plasmodesma units (PPUs) with branches
at the CC-side and only one single corridor at the SE-side.
PPUs possess MELs (up to 40 kDa) larger than most other
plasmodesmata (Balanchandran et al., 1997; Kempers and
van Bel, 1997; Imlau et al., 1999). Plasmodesmata are
absent at other SE-interfaces (Kempers et al., 1998).
Standard plasmodesmata occur at the interface between
CCs and phloem parenchyma cells (PPCs). As a third
plasmodesma-type of the SE/CCs, meristemic plasmodesmata are transformed into the sieve pores that traverse the
transverse wall which becomes modified into a sieve plate
(Evert, 1990).
Callose sealing of the sieve pores as a phloem defence
mechanism
As sessile organisms, plants must cope with a broad variety
of physical and chemical, abiotic and biotic stresses. One
particular stress is injury which is inflicted by abiotic (wind,
fire) or biotic (stinging, browsing, chewing) agents. SEs are
particularly sensitive to injury; they immediately react to
damage by a reorganization ranging up to a full collapse
(Knoblauch and van Bel, 1998). As a first response to
injury, sieve pores become plugged. It is obvious that these
plugs impede mass flow and prevent the nutrition flow for
piercing–sucking insects. Aphids must therefore suppress
the mechanisms triggering sieve plate plugging. In order to
understand the challenge an aphid is confronted with, the
plugging mechanisms must be looked at in more detail.
The function of SE-plugging may be less obvious than it
appears. Sealing of sieve plates is often seen as a means
against pressure-sustained leakage of the precious phloem
sap. However, a driving force for phloem mass flow ceases
Plant–aphid interactions
upon removal of the source, when leaves or twigs are torn
off by wind or herbivores. Therefore, maintenance of the
pressure conditions in the plant or a defence against
phytopathogens are more likely functions of sieve plate
plugging. The lectin character of the structural phloem
protein PP2 in Cucurbita (Read and Northcote, 1983),
which takes part in forming sieve plate plugs, also suggests
that sealing acts a barrier against bacteria, viruses, and
fungi. This attributes protein plugs to another function, but
the leakage argument may still hold for tree-stem browsing
by herbivores or beetles.
Plugging of sieve plates may consist of successive steps
which are related to the degree of injury. A long-described
reaction is the production of callose, a 1,3-b-glucan with
a few 1,6 branches (Aspinall and Kessler, 1957) synthesized by a membrane-spanning 1,3-b-glucan synthase
(Kauss, 1987) (or callose synthase (EC 2.4.1.34)). Callose
production is often observed around plasmodesmata induced by wounding (Stone and Clarke, 1992) or by
microbial attack (Kauss, 1996; Donofrio and Delaney,
2001). Callose is produced in the cell wall outside the
plasma membrane, i.e. the so-called callose plugs in sieve
plates are callose collars that ‘strangle’ the sieve pores.
Callose can be degraded by endo-1,3-b-glucanase (or
callase (EC 3.2.1.6)). It appears that it takes some time
(several minutes) before such extensive callose structures
are being synthesized or broken down. Therefore, more
rapid plugging mechanisms are suspected to be responsible
for a ready (a few seconds) response to injury whereas
callose serves as an evolutionarily rudimental longer-term
mechanism.
Protein plugging of the sieve pores as a phloem
defence mechanism
As a wealth of different protein structures has been found
in TEM pictures of SEs (Evert, 1990), proteins are the
most likely candidates for fast plugging events. There is
a variety of protein forms in the sieve tubes. Presumably
most of the phloem-specific proteins are in a soluble form
in the phloem sap, a few are present as insoluble deposits
along the SE plasma membrane of dicotyledons, while
again others are dispersed as a fine meshwork throughout
the lumina of the sieve elements (Ehlers et al., 2000). Apart
from proteins free-lying in the sieve tube lumen, others do
occur in a more compact configuration. Spindle-like protein bodies (or forisomes), up to a size of 100 lm in length,
have been found in the sieve tubes of legumes. Furthermore, proteinaceous bodies are enclosed in so-called SE
plastids in many higher plant families. SE plastids occur in
SEs of all angiospermes and are of a smaller size (<1 lm)
than plastids in other cell types.
Thick clots of proteins on the sieve plates in conventionally prepared TEM samples (Ehlers et al., 2000) gave
the impression that proteins are dispersed throughout the
731
SE lumen and, carried by mass flow, move to the sieve
plates in case of damage. This suspicion has been confirmed by CLSM studies in which SEs of Vicia faba were
damaged. In response to injury inflicted by a microcapillary
tip, thick protein deposits readily appeared on the sieve
plates and mass flow stopped immediately (Knoblauch and
van Bel, 1998). The protein plugs may be composed of
dispersed forisomes and detached parietal proteins. Moreover, the protein meshwork in the lumen may also
contribute to the plugging reaction (Ehlers et al., 2000).
Forisomes, sieve tube stopcocks in legumes
A unique feature of fabacean SEs is the presence of
forisomes. Forisomes have been designated as crystalline
protein bodies in the past (Zee, 1968). However, recent investigations showed that they hardly have light-polarizing
properties (WS Peters et al., unpublished results). Forisomes
mostly possess a spindle-like appearance with or without
tails (Lawton, 1978a, b). They disperse in response to damage or turgor disturbance (Knoblauch et al., 2001) and seem
to re-contract spontaneously in vivo (Knoblauch et al.,
2001), but not in vitro (Knoblauch et al., 2003).
As far as the knowledge on ontogeny of the SE/CCcomplex in the Fabaceae goes, forisome structure remains
unchanged after its formation, which probably takes place
during SE ontogeny. There is no sign of turnover, which
may be different for other phloem-specific proteins. In
cucurbits, mRNAs encoding for the most abundant SE
proteins PP1 and PP2 (both involved in sieve plate protein
plugging) were only found in the CCs, not in the SEs
(Bostwick et al., 1992; Clark et al., 1997; Dannenhoffer
et al., 1997). Turnover requires the transport of PP1 intact
through the PPUs. Several phloem-specific proteins have
been discovered (Balanchandran et al., 1997) which
enlarge the MEL of the PPUs. Therefore, several soluble
phloem-specific proteins may possess chaperone-like properties (Schobert et al., 1995).
Role of calcium in sieve plate plugging by forisomes
and sealing by callose
Dispersion of forisomes has been linked to binding of Ca2+
ions. Both in vivo and in vitro (isolated forisomes),
forisomes disperse in response to Ca2+ supply and contract
upon addition of the calcium chelator ethylenediaminetetraacetate (EDTA) (Knoblauch et al., 2001, 2003). The
forisome reaction to damage has been ascribed to abundant
Ca2+ influx into the SE lumen through the wound
(Knoblauch et al., 2001). That forisomes react to drastic
turgor changes as well has been related to mechanosensitive Ca2+ channels or membrane-bound mechanoreceptors closely linked with a calcium channel (Knoblauch
et al., 2001). The spontaneous re-contraction in intact
SEs may be due to the presence of calcium pumps lowering
the luminal Ca2+ concentration.
732 Will and van Bel
The sensitivity of the forisome reaction suggests a
mechanism which is highly reactive to minor changes
in intracellular calcium concentration, which requires
a low intracellular basic calcium concentration. Indeed,
the free Ca2+ concentration in SEs is excessively low
(50–70 nM) as measured in drops of sieve tube sap from
cut aphid stylets using Ca2+-selective electrodes (JB
Hafke et al., unpublished results). In parenchymatous
cells, cytosolic concentrations of free Ca2+ lie in the
range between 100 and 200 nM (Evans et al., 1991;
Bush, 1995).
One must realize that forisomes only occur in a minor
part of the angiosperms, which raises question concerning
plugging in other species. There may be several proteinmediated plugging mechanisms which may even operate
in parallel (Fig. 2).
With the exception of protein kinases (Yoo et al., 2002;
Kumar and Jayabashkaran, 2004), any evidence in favour
of Ca2+-binding of phloem-specific proteins is lacking.
Nevertheless, it is tempting to speculate that proteinclogging reactions in SEs are generally calcium-dependent
in view of the low Ca2+ concentration in the phloem sap.
Interestingly, callose deposition has also been claimed to be
Ca2+-sensitive for a long time. The classic experiment in
which phloem exudate can be collected from excised stems
is carried out by addition of EDTA or ethylene-glycol-bis(b-aminoethyl-ether) N, N9-tetraactetate (EGTA) to the
medium (King and Zeevaart, 1974) on the premise that
callose synthesis is Ca2+-dependent. By implication, both
plugging and sealing of the sieve pores depend on the
enhancement of free Ca2+ in SEs.
Possible sources for Ca2+ in the case of the Ca2+ influx
into the SE are the sieve element reticulum (SER), as well
as the apoplast. There are indications for both given the
presence of Ca2+ channels in the SE plasma membrane
(Volk and Franceschi, 2000) as well as the presence of
Ca2+-ATPase in the SER membrane (Arsanto, 1986).
Aphids on the search for nutrient sources in plants
As has been set out before, aphids navigate their stylets
through the plant tissues in order to detect the SEs
containing rich food supplies. The stylet consists of two
mandibular (enclosing a food channel and a saliva channel)
and two maxillary (each containing a neuronal canal) parts.
In making its way to the SEs, the stylet was postulated
to advance between the primary and secondary cell wall
layers (Tjallingii and Hogen Esch, 1993). However, parenchyma cells in plant axes tend to have large radial intercellular spaces up to 1 lm (van der Schoot and van Bel,
1989) which would greatly facilitate the progress of aphid
stylets. Progression of the stylet is visible as B-wave
forms in electrical penetration graph (EPG) measurements
(Tjallingii, 1995).
The second behavioural component is the secretion of
gel saliva (Spiller et al., 1985; Kimmins, 1986; Tjallingii
and Hogen Esch, 1993) recognized as C-wave forms
in EPG (Tjallingii, 1995). The regular alternation of B- and
C-patterns indicates that gel saliva is producing a sheath
through which the stylet can move forwards and backwards. Most likely, the sheath is needed for stabilization of
the stylet (Pollard, 1973) and better movement through the
cell walls (Smith, 1985), but further functions are discussed. Gelling or sheath saliva contains a mixture of
proteins such as pectinase (Ma et al., 1990), cellulase and
phenoloxidase (Miles, 1985) as well as peroxidase (Miles
and Peng, 1989) which renders credence to a function of
sheath saliva in cell wall breakdown. The functional
importance of sheath saliva proteins has hardly been
discussed, since stylets seem to progress too fast to allow
adequate functioning of the enzymes (Tjallingii and
Hogen Esch, 1993; Miles, 1999).
On their way to the phloem, aphids puncture and readily
seal almost every cell along the stylet pathway. Apparently,
the short punctures serve aphids to probe the cell content, which may give information on the direction to be
Fig. 2. Several protein-mediated sealing reactions in response to sieve-element wounding. The reactions are presented in a hypothetical order of
increased complexity. (A) Sealing by water-soluble proteins (wP) may occur in grasses which mostly lack structural phloem-specific proteins
(Eleftheriou, 1990). (B) The next step may be combined sealing by water-soluble proteins and proteins from a fine meshwork (PN) throughout the sieve
element lumen (Ehlers et al., 2000). (C) The previous sealing devices may be used in combination with parietal structural proteins (paP) and possibly
P-plastids (PP) (Knoblauch and van Bel, 1998). (D) The sealing equipment may be completed by forisomes which occur in legumes (Knoblauch
et al., 2001).
Plant–aphid interactions
followed. The stylet mostly follows a radial route in the
cortex tissue and starts searching laterally in the vicinity of
the phloem (Tjallingii and Hogen Esch, 1993). Puncturing a suitable SE coincides with an E1-wave form,
followed by an E2 wave form once phloem sap is being
collected (Prado and Tjallingii, 1994).
How do aphids overcome plant defence?
As pointed out before, aphids are confronted with a number
of constraints which determine the degree of access to the
food supplies. There must be a number of plant factors
responsible for the specificity of the aphid/plant interaction.
One of the selection criteria must be the length and diameter
of the stylet: SEs must be in reach of the stylet. Thus, the
distance between the outer surface of the plant and the
phloem is an important selection criterion (S Bauer and
E Komor, unpublished results). Furthermore, the diameter
of the stylet tip should be far smaller than the SE diameter, since impalement is otherwise physically impossible.
The degree to which an aphid species can cope with the
chemical defence of the plant is probably another selection
criterion. However, virtually nothing is known about
deterrents or poisons which should withhold aphids from
penetration. A trypsin inhibitor (Murray and Christeller,
1995) and an aspartic acid protease inhibitor (Christeller
et al., 1998) collected from the phloem of Cucurbita may
play a role as protease inhibitors in the defence against
insect herbivores (Christeller et al., 1998; Dannenhoffer
et al., 2001). However, it is a matter of debate if aphids
are able to digest proteins. Only if aphids are able to do so,
proteinase inhibitors would be meaningful for aphid
control.
It is of paramount importance for aphids to prevent
wound-induced plugging of the sieve plates upon stylet
insertion, so that their nutrition supply is not interrupted.
As plugging by phloem proteins and sealing by callose
is probably related to calcium influx, it is in the interest of
aphids to prevent calcium binding to protein sealants. This
can be achieved by either reduction of calcium influx into
SEs or by sequestering of calcium ions inside SEs. There
are indications that aphids do both.
Physical devices
One wonders why a glass capillary with a tip diameter of
1 lm causes immediate plugging of the sieve plates
(Knoblauch and van Bel, 1998), whereas penetration of
a stylet with the same tip diameter hardly affects sieve plate
plugging. Both may cause nearly the same degree of injury.
The difference between them seems to be that the wound
inflicted by stylet penetration is immediately sealed by
sheath saliva (Miles, 1987) so that influx of cell wall
Ca2+ is prevented (Fig. 3). Furthermore, insertion of both
‘tips’ may cause a local stretching of the cell wall before
733
penetration and thus a direct induction of stretch-activated
calcium channels. However, turgor pressure relaxation
induced by microcapillary insertion is much larger, since
the volume of the capillary into which SE turgor pressure is released is several factors larger than the stylet
volume with corresponding rates of compression potential
(Table 1; Fig. 3).
The volume of a 400 lm stylet of Aphis fabae with
a closed valve inside the food channel is, inclusive of
the saliva channel, calculated to be 95.03 lm3, whilst the
volume of a microcapillary tip of 5 cm is approximately
1.3210 lm3. Loss of pressure will be negligible in the case
of stylet insertion since the volume of the hardly compressible stylet fluid is 419 times smaller than that of a single
SE. Hence, stimulation of mechanosensitive Ca2+ channels
will be much stronger in the case of microcapillary insertion.
All in all, aphids seem to impose appreciable physical
constraints on calcium influx.
Chemical devices
The question arises whether aphids also possess chemical
devices to avert the injury-triggered reactions of SEs
(Tjallingii and Hogen Esch, 1993). There are some preliminary indications that aphid saliva is able to bind calcium
which would provide a second tool against sieve plate
plugging (T Will and AJE van Bel, unpublished results).
An alternative explanation is provided by the ‘redox
hypothesis’ which assumes that phenolic polymers and
phenol–protein conjugates are responsible for sieve plate
plugging. These compounds would be produced in response to a disturbance of the redox potential following
stylet insertion (Miles and Oertli, 1993). According to this
view, saliva compounds stabilize the redox potential of the
SE sap or interfere with coagulation reactions. The phloemspecific proteins are potential candidates for cross-linking
by phenolics which may be suppressed by salivary phenol
oxidases (Miles and Oertli, 1993).
Similarly, cross-linking reactions may be triggered by
oxygen invasion during stylet impalement. Oxygen concentration in the phloem is generally low (van Dongen
et al., 2003) and a sudden rise of oxygen concentration in
SEs may result in protein coagulation as has been demonstrated for cucurbit phloem sap (Alosi et al., 1988).
Therefore, binding of reactive oxygen species may also be
among the functions of salivary compounds.
As outlined above, watery saliva may have a key
function in stabilizing phloem-specific proteins. That
watery saliva is being excreted into SEs during the E1
phase, which lasts up to a few minutes (Tjallingii, 1994) at
the beginning of every SE puncture, speaks for a crucial
role of saliva compounds in events coincident with stylet
insertion. Moreover, short shots of watery saliva are
continuously secreted during nutrient ingestion (Tjallingii,
1978, 1988). It is surmized that these short shots of watery
saliva keep the food channel open by interaction with SE
734 Will and van Bel
Fig. 3. Physical reduction of the plant defence mechanism in response to stylet puncture. Differential consequences of the impalement of stylets and
microcapillaries with a tip diameter of 1 lm into sieve elements, although both punctures inflict the same wound size. However, the stylet puncture is
readily sealed by sheath saliva, which strongly reduces the influx of calcium. Furthermore, turgor pressure release into the microcapillary exceeds by far
that in the stylet, because the compressible volume in the microcapillary is several million times higher than that in the valved stylet. As a result, the
turgor drop in the sieve tubes will be much higher in the case of microcapillary impalement with commensurate consequences for calcium influx via
mechanosensitive calcium channels. The sealing by sheath saliva and the minute volume thus impose considerable physical constraints on calcium
influx. These physical factors reduce sealing of the sieve plates, as a rise in calcium concentration is probably responsible for sealing of the sieve tubes.
Table 1. Calculation of the potential compression of stylet and microcapillary content
To simplify the calculation, the bulk modulus (K) of elasticity for water was chosen (23109 Pa). The pressure inside the sieve element (PSE) was set at
13106 Pa (10 bar). (Physical effects like viscosity of SE sap and the friction of the fluids on the corresponding surfaces like glass or chitin were
disregarded.) Data for the stylet were taken from Tjallingii and Hogen Esch (1993). Data for the capillary were taken from package of WPI capillaries.
SE data were produced in this laboratory.
Length (h)
Radius (r)
Volume (V) (V=p3r23h)
SE ðPaÞ
Compression (DV) of microcapillary and stylet volume (V) DV = V PKðPaÞ
proteins in the nutrient flow (Tjallingii and Cherqui, 1999).
Some support for this suspicion is provided by events in
microcauterized aphid stylets. Shortly after stylet excision
(e.g. about 10 min in Vicia faba; Fisher and Frame, 1984),
proteins in the phloem exudate coagulate and phloem
bleeding stops, probably due to the absence of watery
saliva (Tjallingii and Cherqui, 1999).
As a weapon against callose, the other sealing device
of SEs, the presence of the callose-hydrolysing enzyme 1,
3-b-glucanase in watery saliva (postulated by Dorschner,
1990) may assist in removal of sieve-plate callose. On the
other hand, it is difficult to envisage how glucanase released
Sieve element
Microcapillary
Stylet (food- and
saliva-channel)
352 lm
6 lm
3.983104 lm3
5
290
1.3210
6.63106
400
0.275
95.03
0.05
cm
lm
lm3
lm3
lm
lm
lm3
lm3
into the SE may assist in hydrolysis of callose in the SE
apoplast.
Other potential roles of watery saliva proteins
transported in the sieve tubes
Prevention of sieve-plate plugging near the side of puncture
is thus regarded as a principal function of watery saliva.
Additional functions such as collective suppression of
the plant defence or communication between individuals
may also be taken into consideration. An absolute prerequisite for systemic functioning is long-distance transport of saliva proteins.
Plant–aphid interactions
Long-distance transport of phloem-specific proteins has
been reported before (Fisher et al., 1992; Golecki et al.,
1998, 1999). Even structural P proteins, for example, PP1
and PP2, can cross intergeneric graft borders of cucurbit
species (Golecki et al., 1998, 1999). The filamentous
protein PP1 of Cucurbita maxima can undergo conformational changes and is transported in an 88 kDa globular
form (Leineweber et al., 2000) which can easily pass sieve
pores. Sieve pores have a diameter between 0.5 and 1.3 lm
in Vicia faba (Resch, 1954) and around 1 lm in Cucurbita
(Sjolund, 1997). Although sieve plates are partially covered
with protein deposits (Ehlers et al., 2000), the corridors
may be large enough to allow passage of larger proteins.
Even suspension particles of carbon black with a MEL of
375 kDa are able to pass sieve pores with a diameter of
0.4 lm in Heracleum sphondylium (Barclay and Fensom,
1973). However, 40 kDa dextrans had some problems in
passing sieve pores, probably due to the coarse preparation
procedures (Kempers and van Bel, 1997) triggering
massive protein deposition on the sieve plates.
Most likely, protein components of aphid saliva reported
to range from 30–240 kDa (Schizaphis graminum; Cherqui
and Tjallingii, 2000), 51–300 kDa (Schizaphis graminum,
Baumann and Baumann, 1995), and 6–200 kDa (spotted
alfalfa aphid; Madhusudhan and Miles, 1998), and 120–
300 kDa (Acyrtosiphon pisum; Madhusudhan and Miles,
1998) can move freely through the phloem system. Forrest
and Noordink (1971) found that radiolabelled saliva of
aphids fed on a 32P-labelled diet was distributed throughout
the apple plants and ingested by non-labelled aphids.
Do aphids profit from the long-distance translocation
of saliva proteins? Ubiquitous presence of watery saliva
proteins may lower the risk of failing punctures and may
be used by social aphids such as Aphis fabae to give other
individuals, nymphs in particular, a better chance of nutrient acquisition (Prado and Tjallingii, 1997). The disadvantage of a reduced food supply in dense aphid
colonies would be compensated by securing the flow of
nutrients by elimination of plant defence mechanisms. The
distinct presence of proteins such as polyphenoloxidase
and peroxidase in watery saliva (Miles, 1990; Peng and
Miles, 1991; Urbanska et al., 1998) may lead to a severe
suppression of plant defence. In addition, the conversion of
toxic phenolics (chinones) into feeding stimulants may
attract aphids to the colony (Peng and Miles, 1988). Watery
saliva proteins may also be employed as a means of communication between aphids sitting at different plant parts
(Forrest and Noordink, 1971).
Conclusions
(i) Sieve-tubes dispose of elaborate defence mechanisms
against wounding such as protein clogging and callose
sealing, most of which are calcium dependent. (ii) Aphids
735
most likely reduce the wound effects inflicted by stylet
insertion into SEs by preventing a rise in the sieve-tube
calcium concentration by physical and chemical means.
The sheath saliva plays a key role in the elimination of
calcium influx; watery saliva may be involved in SE
calcium binding. (iii) Other potential sealing triggers
such as reactive oxygen species may also be detoxified by
watery saliva proteins. (iv) The contents of watery saliva
may have additional functions such as a collective suppression of the plant defence and the communication between
aphid individuals.
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