Plant Cell, Vol. 13, 1221-1230, May 2001, Copyright © 2001, American Society of Plant Physiologists
Reversible Calcium-Regulated Stopcocks in Legume Sieve
Tubes
Michael Knoblaucha,
Winfried S. Petersb,
Katrin Ehlersa, and
Aart J. E. van
Bela
a Institut für Allgemeine Botanik und
Pflanzenphysiologie, Justus-Liebig-Universität, Senckenbergstrasse 17-21, D-35390 Giessen,
Germany
b AK Kinematische Zellforschung, Biozentrum der Johann Wolfgang
Goethe-Universität, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany
Correspondence to:
Michael Knoblauch, michael.knoblauch{at}bot1.bio.uni-giessen.de (E-mail), 49-641-99-35119 (fax)
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ABSTRACT |
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Sieve tubes of legumes (Fabaceae)
contain characteristic P-protein crystalloids with controversial function. We studied their behavior
by conventional light, electron, and confocal laser scanning microscopy. In situ, crystalloids are
able to undergo rapid (<1 sec) and reversible conversions from the condensed resting state into a
dispersed state, in which they occlude the sieve tubes. Crystalloid dispersal is triggered by plasma
membrane leakage induced by mechanical injury or permeabilizing substances. Similarly, abrupt turgor
changes imposed by osmotic shock cause crystalloid dispersal. Because chelators generally prevent the
response, divalent cations appear to be the decisive factor in crystalloid expansion. Cycling between
dispersal and condensation can be induced in opened cells by repetitive exchange of bathing media
containing either Ca2+ or chelators. Sr2+ and Ba2+, but not
Mg2+, are equally active. In conclusion, the fabacean P-protein crystalloids represent a
novel class of mechanically active proteinaceous structures, which provide an efficient mechanism with
which to control sieve tube conductivity.
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INTRODUCTION |
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In higher
plants, long-distance photoassimilate transport takes place through sieve tubes, which are
longitudinal arrays of sieve elements (SEs; Behnke and Sjolund 1990 
; Schulz 1998 ). SEs develop by a unique differentiation, during which they abandon their nucleus,
ribosomes, dictyosomes, and tonoplast (van Bel and Knoblauch 2000 ). Plasmodesmata in the
cross walls between SEs develop into large pores, thus turning these walls into sieve plates (SPs),
the characteristic histological feature of the elongate adult SE (Esau and Thorsch 1984 ).
According to Munch 1930 theory, transport through SEs occurs as mass flow driven by
pressure gradients. This notion has long been in dispute due to the observation of structural barriers
(proteinaceous plugs on the SP pores) in several electron microscopy studies (Weatherley and Johnson 1968 ; Robidoux et al. 1973 ; Johnson et al. 1976 ). However,
alternative hypotheses proved even more controversial, and so pressure flow has remained the most
plausible mechanism for phloem transport (Evert 1982 ).
The pressure flow theory has
gained substantial experimental support recently from the visualization of operating SEs in vivo by
confocal laser scanning microscopy (CLSM; Knoblauch and van Bel 1998 ) and from
structural studies based on the development of appropriate preparation techniques (Ehlers et al. 2000 ). As a result, it became indisputable that unimpeded mass flow occurs at velocities in the
range of 1 cm min-1 in SEs of the model system broad bean (Vicia faba).
Proteinaceous SP plugs occur within seconds after wounding by laser light or mechanical damage (Knoblauch and van Bel 1998 ) or as artifacts caused by inadequate preparation techniques for
electron microscopy (Ehlers et al. 2000 ). Such barriers aggregate from parietal P-proteins
and the contents of phloem-specific SE-plastids, which in the functional state are located near the
longitudinal walls of the SEs (Ehlers et al. 2000 ). Thus, at least some of the confusingly
multifarious structures previously described (Cronshaw and Sabnis 1990 ; Sabnis and Sabnis 1995 ) actually represent the same material at different states of a cellular
wound response.
However, despite recent progress, the biological role of most structural
phloem-specific proteins (P-proteins) remains elusive. For example, sieve tubes of the legumes
(Fabaceae) contain characteristic protein crystalloids (Lawton 1978a ; Behnke 1991 ), whose function is entirely obscure. On the basis of cytological studies, some
authors have suggested that these crystalline P-proteins undergo a transition from a crystalloid to a
dispersed state during SE differentiation (Wergin and Newcomb 1970 ; Palevitz and Newcomb 1971 ). Others have argued that the apparent dispersal in the adult state is
an artifact attributable to turgor loss during tissue preparation for electron microscopy (Fisher 1975 ; Lawton 1978b ). Here, we demonstrate that these crystalloids
represent a novel type of mechanically active intracellular structure: they reversibly plug sieve
tubes by performing rapid Ca2+-controlled cycles of dispersal and reassembly in response to
turgor changes.
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RESULTS |
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Crystalloids Visible in Intact
SEs
Protein crystalloids were studied in living SEs in the midrib of fully grown broad bean
(V. faba) leaves. After phloem tissue had been made accessible to light microscopy by a
shallow surface cut, a few crystalloids usually were visible. The number of crystalloids seemed to
increase with time; in some cases, crystalloids could not be detected before 10 to 15 min after
preparation. Crystalloids were usually seen first in SEs a few cell layers below the cut surface;
in the uppermost layer, they regularly appeared some minutes later.
As a rule, each SE contained
one elongate crystalloid. Rarely, two crystalloids were observed in the same SE. Crystalloids were
mostly located close to the SP on the downstream end of the SE. Our attempts to isolate individual
crystalloids failed because crystalloids vanished within a few seconds when the surrounding cell wall
was damaged mechanically. Thus, it seemed that crystalloids dispersed and assembled spontaneously
within the cells. Unfortunately, conventional light microscopy did not provide further insights into
the mechanisms of these phenomena.
Variable Ultrastructure of Crystalloids
To
gain information about the nature of spontaneous crystalloid assembly and dispersal, we determined
which of the ultrastructural appearances of P-protein crystalloids described previously (cf. Sabnis and Sabnis 1995 ) were present in virtually intact SEs (cf. Ehlers et al. 2000 ). In transmission electron micrographs, crystalloids from SEs of adult leaves appeared as
elongate electron-dense bodies, up to 30 µm long and 2 to 6 µm wide (Fig 1A). Higher magnification showed that crystalloids consisted of coaligned fibrils
with a superimposed, regularly striped pattern perpendicular to fibril orientation (Fig 1B). Occasionally, crystalloids were found in which the dense ultrastructure seen
in their center became less ordered along their edges (Fig 1C and Fig 1D). In some cases, crystalloids consisted entirely of irregularly
interwoven fibrils (Fig 1F). These flakelike structures extended over
the diameter of the sieve tube and usually were found in contact with a SP (Fig 1E).
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Figure 1.
Ultrastructural Appearance of P-Protein Crystalloids in SEs of
Adult Broad Bean Leaves.
(A) Compact crystalline P-protein body (CP) within a SE. Note
undamaged SE-plastids (PL) and masses of parietal P-protein (PP; interspersed with membrane
cisternae of the endoplasmic reticulum) located close to the longitudinal SE wall, indicating that the
cell remained intact during preparation. c, chloroplast; CC, companion cell. Bar = 3
µm.
(B) Compact crystalline P-protein body as in (A) at higher magnification.
Note the regular striped pattern perpendicular to the longitudinal fibrillar structure. Bar = 0.5
µm.
(C) Crystalline P-protein body (asterisk) located near a SP, showing the
structural transition from the dense, ordered state at its center to a more fluffy appearance along
its edges. Bar = 5 µm.
(D) Detail of (C) at higher magnification. Bar = 0.3
µm.
(E) Crystalline P-protein body (asterisk) with dispersed appearance. The
fibrillar mass extends over almost the entire diameter of the SE, next to a SP. Bar = 5
µm.
(F) Detail of (E) at higher magnification. Bar = 0.2
µm.
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Our ultrastructural findings were consistent with the idea that in
living SEs, P-protein crystalloids could dissolve into filamentous masses or assemble from that
material. However, due to the static nature of electron micrographs, alternative interpretations could
not be excluded.
Plasma Membrane Leakage Induces Crystalloid Dispersal
To
further examine the hypothesis that P-protein crystalloids transform reversibly into unordered
fibrillar masses, we searched for a vital dye specific for P-protein crystalloids. We tested numerous
fluorescent dyes commonly used in plant cell biology, but only one member of the dimethylfluorescein
diacetate family, CDMFDA (for 5[6]-carboxy-4',5'-dimethylfluorescein diacetate)
transiently contrasted the crystalloids. When the dye was applied and washed out after 5 min, it
allowed the observation of crystalloids by CLSM for a period of a few minutes.
Using CLSM on
CDMFDA-stained sieve tubes, we were able to verify that the apparent disappearance of crystalloids
actually was a transformation of the dense elongate crystalloid structure into a less dense roundish
plug that extended over the width of the SE (Fig 2A and Fig 2B). The reaction could be triggered reliably (four repetitions) by micropipette
insertion into the SEs. The transformation obviously entailed a change in refractive index that
rendered the dispersed plug invisible in the conventional light microscope.
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Figure 2.
Dynamics of Crystalline P-Protein Bodies Observed in CDMFDA-Stained Phloem
Tissue of Broad Bean (V. faba) and Runner Bean (Phaseolus vulgaris) by
CLSM.
(A) and (B) Crystalloid P-protein body in broad bean before (A) and
after (B) insertion of a micropipettete (not visible; tip diameter of 2 µm).
Micropipettete insertion triggers the transformation of the dense, elongate crystalloid into a
roundish sieve tube plug.
(C) to (E) Dispersal of a crystalloid P-protein body in
a broad bean SE in response to retraction of an inserted GEF (tip diameter of 0.1 µm). (C)
In the undisturbed SE, an elongate crystalline P-protein body is visible. (D) GEF (arrow)
insertion has no effect. (E) GEF (arrow) retraction triggers plug formation.
(F)
Crystalline P-protein body in a sieve tube in runner bean. Crystalloid dispersal had been induced by
micropipettete insertion (as in [A] and [B]). Although the main body of the crystalloid
forms a sieve tube plug as in broad bean (cf. [B] and [E]), the two tail-like
protrusions (arrowheads), which are characteristic for crystalline P-protein bodies in runner bean,
remain unchanged.
Asterisks, crystalline P-protein bodies; CC, companion
cell.
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The reaction to
micropipette insertion depended on the pipette tip size. With tip diameters >2 µm, crystalloids
always dispersed within 1 to 3 sec (>30 experiments). Decreasing tip diameter slowed the
response; 1-µm tips triggered plug formation within an average of 4 sec (n = 8).
Upon insertion of galinstan-expansion-femtosyringes (GEFs; tip diameter of 0.1 µm), a pipette
type developed for leak-free microinjection into high-turgor cells such as SEs (Knoblauch et al. 1999 ), crystalloids remained undisturbed (in six out of seven experiments; Fig 2C and Fig 2D). However, retraction of the GEFs
always caused instantaneous crystalloid dispersal (Fig 2E). Thus,
membrane leakage, and not the penetration as such, triggered the response. This interpretation was
corroborated by the reaction to the application of Triton X-100 and digitonin, substances that induce
unspecific membrane permeability. Solutions of 1% (v/v) Triton X-100 and 1 mM digitonin induced
dispersal of the crystalloids within 20 to 30 sec (two repetitions of each experiment; data not
shown).
Crystalloid Dispersal Is Triggered by Turgor Changes
Crystalloid
dispersal in response to membrane leakage might be triggered by turgor changes in the leaky cell. We
tested this possibility by challenging SEs osmotically according to a variety of experimental
protocols. Changes in extracellular osmolarity in steps <400 mosmol (equivalent to a change in
osmotic pressure of 1 MPa) were ineffective, whereas changes in steps >600 mosmol (1.5 MPa) triggered
crystalloid dispersal; intermediate steps yielded mixed results. These observations were
independent not only of the osmolyte used (KCl, CaCl2, MgCl2, and sucrose were
tested) but also of the osmolarity prevailing before the step. External osmolarity could be increased
slowly from 100 to 700 mosmol in three steps of 200 mosmol each at 5-min intervals without any
response of the crystalloids. Further increase from 700 to 1300 mosmol in one step triggered
crystalloid dispersal in the same way that a single step from 100 to 700 mosmol did. SEs actually
could be plasmolyzed with crystalloids remaining intact if extracellular osmolarity was increased
stepwise. If the plasma membrane of plasmolyzed cells was damaged with micropipettes, then
crystalloids dispersed within a few seconds (in both of two experiments).
The dispersal response
was transient; spontaneous crystalloid reassembly usually occurred 4 to 6 min after the dispersal
event. However, reassembly could not be induced by bringing external osmolarity back to the initial
value immediately after the disappearance of the crystalloid. Surprisingly, decreases of extracellular
osmolarity worked exactly as increases did. The reaction to a 600-mosmol step down was
indistinguishable from the response to a corresponding step up.
The main results of our
osmolarity experiments are exemplified by a typical test shown in Fig 3; similar tests with modified treatments were performed on >20 crystalloids
with consistent results.
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Figure 3.
Reversible Dispersal of a Crystalline P-Protein Body in a Broad Bean SE in
Response to Osmotic Shock.
The time course of extracellular osmolarity is given, with
photographs of the SE taken at the times indicated by arrows. The crystalloid did not react to
osmolarity steps of 350 mosmol (corresponding to a change in osmotic pressure of almost 0.9 MPa;
upper row). Steps of 750 mosmol (corresponding to a change in osmotic pressure of  1.9 MPa)
induced dispersal of the crystalline P-protein body regardless of the direction of the step (lower
row). Note that the crystalloid spontaneously reassembled at 5 min after osmotic shock–induced
dispersal. In this experiment, external osmolarity was adjusted with KCl. Identical results were
obtained using CaCl2, MgCl2, and sucrose.
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Calcium Acts as Dispersing Agent
As an alternative
to changes in turgor, membrane leakage might also provoke crystalloid dispersal because of increased
membrane fluxes of signaling substances. Coincidentally, we had noted that more crystalloids seemed to
be visible immediately after tissue preparation if the bathing medium contained a chelator. Therefore,
we studied the influence of extracellular Ca2+ on crystalloid dispersal.
The
incubation medium in all experiments described above contained 10 mM CaCl2;
repetitions of these experiments with Ca2+-free standard medium always yielded identical
results. However, when endogenous Ca2+ that might have originated from the cell wall was
intercepted by EDTA or EGTA (
1 mM) in the bathing solution, none of the treatments that in
standard medium had triggered crystalloid dispersal (micropipette insertion, >80 repetitions;
application of Triton X-100, two repetitions; changes in extracellular osmolarity, >20
repetitions) showed any effect. When holes up to 150 µm long were torn into SE walls with
micropipettes in the presence of 10 mM EDTA, crystalloids remained unaffected even when agitated with
micropipette tips (Fig 4). In these experiments, the addition of excess
Ca2+ (12 mM) to chelator-containing media always caused rapid crystalloid dispersal. When
bathing solutions containing 10 mM of either Ca2+ or EDTA were exchanged, crystalloids in
opened SEs instantaneously dispersed or assembled, respectively (Fig 5; this experiment was repeated on 16 crystalloids with identical results).
In individual crystalloids, such reaction cycles could be repeated at least 15 times. Crystalloid
dispersal and assembly could be initiated in severed cells even after they had been stored for several
hours (in all of five tests). As a corollary of the results obtained with damaged SEs, a direct
involvement of the membrane potential in triggering crystalloid dispersal could be ruled
out.
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Figure 4.
Mechanical Robustness of a
Crystalline P-Protein Body in a Severed Broad Bean SE in the Presence of a Chelator (10 mM
EDTA).
(A) A crystalline P-protein body (asterisk) is seen located next to a SP in a
kinked sieve tube; two SEs are visible. A micropipette tip touches a longitudinal SE wall
(circle).
(B) The micropipette is inserted into the SE. The crystalloid can be pushed
around and rotated along its longitudinal axis in this situation.
(C) After retraction of
the micropipette, crystalloid dispersal is induced by exchanging 10 mM EDTA for 10 mM CaCl2
in the external medium.
A video showing the complete experiment is available (supplemental
material under file name crystalloids movie 1.mov).
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Figure 5.
Rapid Dispersal and Reassembly of a Crystalloid P-Protein Body
in a Severed Broad Bean SE.
(A) A crystalline P-protein body (asterisk) is seen in a SE
that has been ripped open in the presence of 10 mM EDTA.
(B) The crystalloid disperses
instantaneously when EDTA is exchanged for 10 mM CaCl2 in the external
medium.
(C) Instantaneous crystalloid reassembly is induced by exchanging
CaCl2 for EDTA again.
A video showing the complete experiment is available
(supplemental material under file name crystalloids movie 2.mov).
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The Ca2+ effect in these experiments could be mimicked reliably by equimolar
concentrations of the divalent cations Sr2+ (in all of eight crystalloids tested;
Fig 6) and Ba2+ (six crystalloids; data not shown).
However, Mg2+ was ineffective (13 crystalloids tested; Fig 6), as were monovalent cations (K+ and Na+) and
Cl- (eight crystalloids tested; data not shown).
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Figure 6.
Induction of Crystalline P-Protein
Body Dispersal by Ca2+ and Sr2+ but Not by Mg2+ in Broad Bean
SEs.
(A) Crystalloids (asterisks) in SEs severed in the presence of 10 mM
EDTA.
(B) Effect of exchanging EDTA for 10 mM chloride salt of the divalent cation
indicated to the left of each row. Crystalloid dispersal is induced by Ca2+ and
Sr2+ but not by Mg2+.
(C) Crystalloid reassembly by exchanging
divalent cation salts for 10 mM EDTA.
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Reversible
Crystalloid Dispersal Is Characteristic of Fabaceae
Elongate crystalline P-protein bodies are
typical of the Fabaceae (Behnke 1991 ). To establish the general validity of our findings
in broad bean, we examined protein crystalloids of runner bean (Phaseolus vulgaris) and
Lupinus polyphyllos. Despite subtle morphological differences between the crystalloids of the
different species (such as the crystalloid "tails" in runner bean seen in Fig 2F; cf. Lawton 1978a ), sieve tube plug formation was induced by
micropipette insertion (three repetitions in each species), as described above for broad bean. In all
species, spontaneous crystalloid assembly occurred a few minutes after preparation of the
leaves.
More or less globular P-protein bodies, which remain nondispersive during SE ontogeny
and ultrastructurally resemble the fabacean P-protein crystalloids, exist in numerous angiosperm
families (Behnke 1991 ). We tested the reaction to wounding in Urtica dioica
(Urticaceae; two repetitions) and Rubus fruticosus (Rosaceae; three repetitions).
P-protein bodies in these species were truly nondispersive, that is, they failed to react even when
SEs were severely damaged mechanically in the presence of free Ca2+ (data not
shown).
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DISCUSSION |
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Structural Dynamics of P-Protein
Crystalloids
Crystalline P-proteins from legume SEs were first described more than a century
ago (Baccarini 1892 ) and have been studied ever since. However, on the basis of
descriptive studies alone, the structural state of these protein bodies in transporting SEs could not
be established unequivocally (reviewed in Cronshaw and Sabnis 1990 ; Sabnis and Sabnis 1995 ). In our plant material, we were able not only to reproduce all major
ultrastructural features described previously, including crystalloid, fibrillar, and intermediate
states (Fig 1), but also to observe directly the transformation of
crystalloids into filamentous plugs in intact SEs (Fig 2). Moreover, we
established the reversibility of the structural transformation in living (Fig 3) and dead (Fig 4 Fig 5 Fig 6) cells. Our results provide conclusive
evidence for the material identity of crystalloid and filamentous structures in fabacean SEs. Their
remarkable structural dynamics distinguish crystalline P-proteins of the legumes from the
nondispersive phloem-specific protein bodies of other families (such as the Urticaceae and Rosaceae
tested here).
Control of Crystalloid Conformation by Ca2+
Because
extracellular chelators prevent crystalloid dispersal in vivo, an influx of divalent cations into the
SE seems a prerequisite for the response. Cytoplasmic Ca2+, typically kept to submicromolar
levels in plant cells (Gilroy et al. 1993 ), is a well-established link of various signal
transduction chains (Poovaiah and Reddy 1993 ; Bush 1995 ). In sieve tubes,
the concentration of free Ca2+ has been reported to be 20- to 100-fold higher than it is in
typical plant cells (Brauer et al. 1998 ). Nevertheless, the electrochemical
Ca2+ gradient is directed into the SE cytoplasm, where the concentration of free
Ca2+ may be controlled by sequestration in the so-called SE reticulum (Arsanto 1986 ), by specific Ca2+ binding proteins (McEuen et al. 1981 ), and probably
by Ca2+-ATPases, which seem to be ubiquitous in plant plasma membranes (Askerlund and Sommarin 1996 ). Thus, changes in free Ca2+ have the potential to function as signals
in SEs. It must be expected, though, that phloem-specific Ca2+-regulated proteins respond
to relatively high Ca2+ concentrations in the micromolar or low millimolar range (Brauer et al. 1998 ).
Ca2+ controlled the conformational state of crystalloids
in severely damaged cells (Fig 4 Fig 5 Fig 6). It seems inconceivable that a cellular signaling machinery should be
functional under these conditions. Therefore, it is plausible that Ca2+ acts directly on
P-protein crystalloids. Under the low Ca2+ conditions prevailing in the undisturbed SE,
crystalloids probably exist in the condensed resting state. Presumably, they transform into plugs in
direct response to surges in intracellular Ca2+ caused by Ca2+ influx from
extracellular sources (Fig 7). If this idea is correct, then the
reversible conformational changes would actually be controlled by those cellular activities that
establish and regulate the inwardly directed electrochemical Ca2+ gradient (e.g.,
Ca2+-ATPase action). Generally, the action of mechanically active proteins is often
controlled but not usually driven by Ca2+. To our knowledge, only spasmonems, the
contractile organelles of certain ciliates, show similar behavior. Spasmonemes are regulated by the
free Ca2+ concentration as controlled by storage and release from intracellular stores
(Amos et al. 1976 ; Mahadevan and Matsudaira 2000 ). It remains to be
determined whether spasmonemes provide a valid model for the regulation of P-protein crystalloids in
sieve tubes.
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Figure 7.
Hypothetical Regulation of the Conformational State of Crystalline P-Protein
Bodies in the Fabaceae.
The conformation of the crystalline P-protein is controlled directly by
intracellular Ca2+. High Ca2+ levels lead to dispersal of the protein body;
in the absence of Ca2+, the protein transforms into a dense "crystalloid"
state. Intracellular Ca2+ is regulated by events in the plasma membrane (PM). Low
Ca2+ concentrations in the cells probably are maintained by Ca2+ pumps, which
establish a steep electrochemical Ca2+ gradient directed into the cells. Therefore,
membrane leakage as well as Ca2+ channel activity result in Ca2+ influx, leading
to dispersal of the crystalline P-protein bodies. Water fluxes over the membrane provoked by changes
in external osmolarity modify turgor pressure (P). Changes in turgor may cause increases in
intracellular Ca2+ by the activation of Ca2+ channels; alternatively,
osmotic shock might induce membrane leakage. The resulting dispersal of crystalline P-protein bodies
is fully reversible, because normal intracellular Ca2+ levels are restored by the pumps
when Ca2+ channel activity ceases or membrane leaks are repaired.
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P-protein crystalloids are responsive only to specific divalent cations, as
demonstrated by the lack of response to Mg2+ (Fig 6). Interestingly, crystalloids respond to Sr2+ and Ba2+, which, just like
Ca2+, are excluded efficiently from the sieve tubes. On the other hand, Mg2+ is
taken up and transported (Ziegler 1975 ). Thus, crystalline P-proteins seem adapted to
detect perturbations of membrane function by responding to increasing intracellular levels of ions,
which normally are kept to low concentrations in the sieve tube sap.
Stimuli
Triggering Crystalloid Dispersal: Membrane Permeability and Turgor
Increased nonspecific
membrane permeability, that is, leakage induced by micropipette insertion or chemical effectors,
triggered crystalloid dispersal if Ca2+ was present in the extracellular medium. This is
explained readily by the Ca2+ dependence of crystalloid conformation, because extracellular
Ca2+ enters leaky SEs following its electrochemical gradient (Fig 7).
Turgor-dependent crystalloid dispersal as induced by sudden steps in
external osmolarity also depends on extracellular Ca2+. However, it remains unclear whether
Ca2+ enters the cell via nonspecific pathways (membrane leakage induced by osmotic shock)
or whether the rapid change in turgor is a signal that leads to the opening of Ca2+
channels in the plasma membrane (Fig 7). The latter explanation would
appear to make sense in a wider context. Cytoplasmic Ca2+ has been implicated as a signal
in turgor regulation in a range of taxa (Okazaki and Tazawa 1990 ). In characean algae,
turgor steps in either direction trigger action potentials if they exceed a threshold of 0.2 to 0.5
MPa (Zimmermann and Beckers 1978 ). Cytoplasmic Ca2+ rises sharply during these
action potentials (Kikuyama and Tazawa 1983 ; Thiel et al. 1997 ). In
higher plants, similar potential waves are transmitted along sieve tubes (Eschrich et al. 1988 ; Fromm 1991 ; Rhodes et al. 1996 ), where they seem to raise
levels of cytoplasmic Ca2+ (Fromm and Spanswick 1993 ). It seems a reasonable
working hypothesis that exogenously induced turgor changes trigger membrane potential waves in SEs
that are accompanied by intracellular Ca2+ surges. Increased intracellular Ca2+
then could cause reversible sieve tube occlusion by controlling P-protein crystalloid conformation
(Fig 7).
Physiological Significance of Reversible Plug
Formation
The appearance of the dispersed fibrillar masses by CLSM (Fig 2) and transmission electron microscopy (Fig 1)
leaves little doubt that crystalline P-proteins drastically increase the hydraulic resistance of the
sieve tubes when they disperse (cf. Weatherley and Johnson 1968 ; Weatherley 1972 ; Ehlers et al. 2000 ). Thus, SEs potentially control the rate of phloem flux by
regulating intracellular Ca2+. Ca2+ antagonists such as EDTA have long been
known to prevent sieve tube occlusion (King and Zeevaart 1974 ; Fellows et al. 1978 ). The effect is commonly ascribed to the inhibition of Ca2+-dependent
stimulation of callose synthesis (Sabnis and Sabnis 1995 ). However, the moderate
Ca2+-mediated increase of callose formation on SPs detectable after hours of treatment
(King and Zeevaart 1974 ) does not seem to provide a sufficient explanation for the
dramatic stimulation of phloem exudation by chelating agents (Fellows et al. 1978 ;
Urquhart and Joy 1981 ; Schulz 1998 ). Our results demonstrate that an
additional Ca2+-dependent mechanism operates in the Fabaceae. The process of
Ca2+-triggered crystalloid dispersal and plug formation is much more rapid than sieve tube
occlusion by callose synthesis, and it is reversible within seconds. These features render crystalloid
dispersal an ideal first line of defense against loss of sieve tube contents, particularly in cases of
localized and transient disturbances, such as attacks of phloem-feeding aphids. Fabaceae are
notoriously poorly suited for phloem exudate studies using severed aphid stylets (Fisher and Frame 1984 ). The reason may be that they have evolved a unique type of cellular stopcock to control
the hydraulic conductivity of their sieve tubes.
Concluding Remarks—A Wider
Perspective
We have demonstrated that crystalline P-proteins of the Fabaceae occlude SEs by a
rapid and reversible conformational switch in response to wounding, changes in turgor, and
disturbances of membrane integrity. Available evidence suggests that the control of crystalline
P-protein conformation is tightly linked with the regulation of intracellular Ca2+.
Previously, several lines of indirect evidence suggested a possible relation between intracellular
Ca2+-regulation and the functioning of phloem-specific proteins other than the fabacean
P-protein crystalloids (Kleinig et al. 1971 ; McEuen et al. 1981 ;
Arsanto 1986 ). It now seems reasonable to generalize our findings and hypothesize that
intracellular Ca2+ controls interconversions between different structural states of
P-proteins and thereby regulates the flow through the
phloem.
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METHODS |
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Plant Material
Vicia faba cv
Witkiem major (Nunhems Zaden BV, Haarlem, The Netherlands) was grown individually in pots in a
greenhouse at 20°C, with 60 to 70% relative humidity, and a 14/10-hr light/dark period (daylight
plus additional lamp light [model SONT Agro 400 W; Phillips, Eindhoven, The Netherlands], with a
minimum irradiance of 250 mol m-2 sec-1 at plant level). Plants were
used for experiments 17 to 21 days after germination. Lupinus polyphyllos, Phaseolus
vulgaris, Urtica dioica, and Rubus fruticosus were collected from the Botanical
Garden of Giessen University (Giessen, Germany).
Conventional Light
Microscopy
Vascular tissue for microscopical in vivo observation was prepared from detached
leaves by a shallow pericline cut as described previously (Knoblauch and van Bel 1998 ).
Specimens were covered with standard medium (10 mM KCl, 10 mM CaCl2, and 5 mM NaCl,
buffered at pH 7.5 with 50 mM Hepes; in experiments in which extracellular osmolarity was varied,
quarter-strength standard medium was used) or calcium-free medium (10 mM disodium-salt of EGTA or EDTA
instead of CaCl2). Leaves were fixed on microscope slides with adhesive tape so that
crystalloids in sieve elements (SEs) could be observed with a microscope (Leica DMLB equipped with an
HCX APO L40X/0.80 W U-V-I water immersion objective; Leica, Wetzlar, Germany) in the bright-field
mode. Attached was a video camera (model TK-C1360; JVC, Tokyo, Japan) connected to a Miro Video
Capture Card (Pinnacle Systems, Braunschweig, Germany) to store digital images on a desktop computer.
Bathing solutions were changed with laboratory pipettes and a glass capillary that was connected to a
suction pump. When necessary, cells and P-protein crystalloids were agitated by micropipettes with
different tip diameters mounted on four-axis micromanipulators (model MMW-204; Narishige, Tokyo,
Japan). Pipettes were produced from borosilicate glass capillaries (Clark Electromedical Instruments,
Reading, UK) by using a microelectrode puller (model P-200; Sutter Instruments, Novato,
CA).
The effect of nonspecific membrane leakage was tested by adding either 1% (v/v) Triton
X-100 or 1 mM digitonin to the standard medium. For osmolarity tests, standard medium or calcium-free
medium was used; osmolarities were adjusted by adding KCl, CaCl2, MgCl2, or
sucrose. To test the ion specificity of the dispersal response, standard medium was used in which
CaCl2 had been exchanged for the equivalent concentrations of MgCl2,
BaCl2, or SrCl2. The effects of monovalent ions were tested in calcium-free
medium in which the contents of NaCl and KCl were modified to adjust the concentration of
Na+, K+, or Cl-, respectively, to either 0 or 10
mM.
Confocal Laser Scanning Microscopy
Confocal laser scanning microscopy was
performed using a Leica (Heidelberg, Germany) TCS 4D microscope as described in detail previously
(Knoblauch and van Bel 1998 ). To specifically contrast crystalloids, we used
5[6]-carboxy-4',5'-dimethylfluorescein diacetate (CDMFDA; Molecular Probes, Eugene,
OR). A droplet of 0.02
(w/v) CDMFDA in a solution containing 10 mM KCl, 10 mM
CaCl2, and 5 mM NaCl was applied for 5 min and washed out; crystalloids then were
clearly visible for a few minutes. Excitation light (488 nm) was produced with a 75-mW argon/krypton
laser (Omnichrome, Chino, CA). Emitted light passed through a fluorescein band pass
filter.
Electron Microscopy
To keep the SEs intact, vascular tissue was laid
open in whole leaves, as described previously (Ehlers et al. 2000 ), and a solution of 3%
(v/v) paraformaldehyde, 4% (v/v) glutaraldehyde, 50 mM sodium cacodylate buffer, and 2 mM
CaCl2, pH 7.2, was applied to the exposed tissue. The solution was replaced every 30 min
for 6 hr. Pieces (15 x 5 mm) were excised from the midrib and transferred to fresh solution.
After 3 hr at room temperature, tissue samples were washed and incubated overnight in 50 mM sodium
cacodylate buffer with 2 mM CaCl2, pH 7.2, and 1% (w/v) OsO4 at 4°C. After
contrasting in 0.5% (w/v) aqueous uranyl acetate for 2 hr at 4°C, samples were dehydrated in a
graded ethanol series and propylene oxide and were transferred to Spurr's epoxy resin. Ultrathin
sections were cut with a diamond knife on a Reichert Om U2 ultramicrotome (Leica, Bensheim, Germany)
and collected on Formvar-coated single-slot copper grids. After poststaining with 2% aqueous
uranylacetate and Reynolds' lead citrate, sections were examined at 80 kV with an electron
microscope (model EM 300; Phillips, Eindhoven, The Netherlands).
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FOOTNOTES |
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Online version contains Web-only
data.
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ACKNOWLEDGMENTS |
|---|
Helpful suggestions and comments given during the course of
this study by Hubert Felle and Gerhard Thiel are gratefully acknowledged. We thank Jürgen
Bereiter-Hahn, Julian Hibberd, and Gerhard Thiel for critical discussion of earlier versions of the
manuscript.
Received December 21, 2000; accepted March 20, 2001.
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TOP
ABSTRACT
INTRODUCTION
