JXB Advance Access originally published online on July 5, 2007
Journal of Experimental Botany 2007 58(11):2827-2838; doi:10.1093/jxb/erm143
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Ca2+-mediated remote control of reversible sieve tube occlusion in Vicia faba
1Plant Cell Biology Research Group, Institute of General Botany, Justus-Liebig-University, Senckenbergstrasse 17, D-35390 Giessen, Germany
2Department of Plant Biology, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
* To whom correspondence should be addressed. E-mail: aart.v.bel{at}bot1.bio.uni-giessen.de
Received 16 April 2007; Revised 29 May 2007 Accepted 30 May 2007
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Abstract |
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According to an established concept, injury of the phloem triggers local sieve plate occlusion including callose-mediated constriction and, possibly, protein plugging of the sieve pores. Sieve plate occlusion can also be achieved by distant stimuli, depends on the passage of electropotential waves (EPWs), and is reversible in intact plants. The time-course of the wound response was studied in sieve elements of main veins of intact Vicia faba plants using confocal and multiphoton microscopy. Only 15–45 s after burning a leaf tip, forisomes (giant protein bodies specific for legume sieve tubes) suddenly dispersed, as observed at 3–4 cm from the stimulus site. The dispersion was reversible; the forisomes had fully re-contracted 7–15 min after burning. Meanwhile, callose appeared at the sieve pores in response to the heat shock. Callose production reached a maximum after
20 min and was also reversible; callose degraded over the subsequent 1–2 h. The heat induction of both modes of occlusion coincided with the passage of an EPW visualized by electrophysiology or the potential-sensitive dye RH-414. In contrast to burning, cutting of the leaf tip induced neither an EPW nor callose deposition. The data are consistent with a remote-controlled occlusion of sieve plates depending on the longitudinal propagation of an EPW releasing Ca2+ into the sieve element lumen. It is hypothesized that forisome plugs and callose constriction are removed once the cytosolic calcium level has returned to the initial level in those sieve tubes. Key words: Callose, CLSM, forisome, membrane potential, phloem, Vicia faba
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Introduction |
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Sieve elements (SEs), companion cells (CCs), and phloem parenchyma cells (PPCs) are the three phloem elements involved in long-distance transport of photoassimilates in angiosperms (van Bel, 1996; Hafke et al., 2005). While the enucleate SEs form the transport pathway, the CCs are engaged in maintaining their viability. A high density of pore plasmodesma units (PPUs) and tight endoplasmic reticulum (ER) coupling between SEs and CCs underline an intimate symplasmic connection across this boundary (Kempers et al., 1998; Martens et al., 2006). Permanent metabolic and energetic support of the SEs is required since their cellular machinery is largely reduced during ontogeny to make way for mass flow (van Bel and Knoblauch, 2000).
Mature SEs are lacking nucleus, vacuoles, cytoskeleton components, ribosomes, and dictyosomes (van Bel, 2003). They only retain a plasma membrane, a few mitochondria, a set of phloem-specific proteins, and SE plastids, as well as a parietally located smooth ER (Sjolund and Shih, 1983). The cross-walls between the SE modules become transformed into sieve plates, perforated by plasmodesmata (PD) modified into sieve pores with a diameter up to 1 µm (Behnke and Sjolund, 1990; Schulz, 1998; van Bel, 2003).
Translocation in sieve tubes occurs by mass flow, with a high degree of solute exchange with cells surrounding the SEs. The consequences for the rate of mass flow at the sieve pore bottlenecks are unclear (Thompson and Holbrook, 2004; van Bel and Hafke, 2005). However, it is clear that mass flow is impeded by sieve pore occlusion (Knoblauch and van Bel, 1998).
Phloem sap collected by sieve tube exudation contains >150 proteins (Schobert et al., 1995). These phloem-specific proteins occur in various aggregation forms and are described to be amorphous, granular, fibrillar, filamentous, tubular, or crystalline (Cronshaw and Sabnis, 1990). In the fully differentiated state, the structural proteins are mostly localized parietally and seem to have the potential for structural changes (Behnke, 1991; Knoblauch and van Bel, 1998). Parietal filamentous networks were reported to transform into longitudinally oriented strands which plug the sieve pores as a response to injury (Evert, 1982). Similar ‘chewing gum-like’ threads were observed in damaged sieve tubes of Vicia faba (Knoblauch and van Bel, 1998). In addition, spindle-like protein bodies which only occur in Fabaceae (Palevitz and Newcomb, 1971) were observed in V. faba sieve tubes. These protein bodies were designated as forisomes (Knoblauch et al., 2003). They disperse instantaneously when triggered by osmotic shock or Ca2+ application, and recondense ‘spontaneously’ into the original state after some time in intact sieve tubes (Knoblauch et al., 2001, 2003). Manipulation of isolated forisomes showed a strongly calcium-dependent dispersion/recontraction.
As a second mode of sieve plate occlusion in response to injury, callose is produced in the form of collars around plasmodesmata and sieve pores (King and Zeevaart, 1974; Zabotin et al., 2002). Callose is a β-1.3-glucan polymer which occurs in different plant tissues such as pollen tubes, plasmodesmata, sieve plates of the phloem, and root hairs (Hong et al., 2001). In various studies, callose deposition was described as a reaction to mechanical or chemical stress (Kudlicka and Brown, 1997; Nakashima, 2003). Like forisome dispersion, callose production is calcium dependent (Thonat et al., 1993).
The phloem is involved in the longitudinal transmission of electropotential waves (also called ‘action potentials’) in response to mechanical and physical stimuli (e.g. heat shocks and cold shocks) (Fromm, 1991; Fromm and Spanswick, 1993; Fromm and Bauer, 1994; Rhodes et al., 1996). These electropotential waves (EPWs) are based on voltage-gated channel activity, which responds to changes in membrane potential (Davies, 2004). During the abrupt depolarization of the plasma membrane, K+ and Cl– efflux are accompanied by Ca2+ influx into the apoplast of leaves and roots (Felle and Zimmermann, 2007). If Ca2+ influx is linked to Ca2+-dependent occlusion mechanisms, distant wounding may induce Ca2+-dependent distant plugging (forisomes) and constriction (callose) of sieve plates in V. faba.
The aim of this study was to investigate if: (i) sieve plate occlusion can be induced by distant stimuli; (ii) the closure is reversible; (iii) it is remotely regulated by an electrical potential wave triggering Ca2+ influx; and (iv) if the occlusion responses depend on the nature of wounding.
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Materials and methods |
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Plant material
Vicia faba cv. Witkiem major plants (Nunhems Zaden BV, Haelen, The Netherlands) and Solanum lycopersicum plants were cultivated in pots in a greenhouse under standard conditions (20–30 °C, 60–70% relative humidity, and a 14/10 h light/dark period). Supplementary lamp light (model SONT Agro 400 W; Philips Eindhoven, The Netherlands) gave an irradiance level of 200–250 µmol m–2 s–1 at the plant apex. Plants were selected for experiments 17–21 d after germination, in the vegetative phase just before flowering.
Preparation of intact plants for microscopy
For in vivo observation of sieve tubes, cortical cell layers were removed down to the phloem from the lower side of the main vein of a mature leaf, still attached to intact V. faba plants. The cell layers were removed by manual paradermal slicing with a fresh razor blade, while avoiding damage to the phloem according to Knoblauch and van Bel (1998). The distance between the observation window and the leaf tip was between 3 cm and 5 cm. The leaf was mounted on a microscope slide with double-sided adhesive tape and the free-lying phloem tissue irrigated with bathing medium (see below). The physiological state of the phloem tissue was checked using a microscope with a water immersion objective, and a drop of fluorochrome mixture was applied to the tissue. The microscope slide carrying the leaf was mounted on the stage of a multiphoton or a conventional confocal laser scanning microscope (CLSM).
Bathing medium and fluorescent probes
A phloem physiological buffer containing 2 mol m–3 KCl, 1 mol m–3 CaCl2, 1 mol m–3 MgCl2, 50 mol m–3 mannitol, and 2.5 mol m–3 MES/NaOH buffer, pH 5.7 was used for the solubilization of dyes.
The voltage-sensitive styryl dye RH-414 [-N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridiniumdibromide] was employed for membrane staining and visualization of the relative changes in membrane potentials. A stock solution was produced by solubilizing 25 mg of RH-414 in 1 ml of dimethylsulphoxide (DMSO). Applied solutions contained 1 µl of the RH-414 stock solution dissolved in 2 ml of bathing medium (final concentration 4.3 µM RH-414). From 100 µl to 500 µl of RH-414 solution were applied depending on the size of the area to be tested.
CMEDA/CMFDA dye mixture was used to highlight the forisome. A 1 mg aliquot of CellTracker Yellow-Green CMEDA (5-chloromethyleosin diacetate) was dissolved in 128 µl of DMSO, and 1 µl of this stock solution was freshly dissolved in 1 ml of bathing medium. A 1 mg aliquot of CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) was dissolved in 215 µl of DMSO, and 1 µl of this stock solution was also diluted with 1 ml of bathing medium. Before application, both stock solutions were mixed in the proportion 1:1 (v/v).
In order to visualize callose, a low concentration (0.005%) of the fluorescent dye aniline blue was used. The stock solution of aniline blue was prepared in 0.1 M phosphate buffer, pH 7.4 (0.05 mg ml–1).
RH-414 and CMEDA/CMFDA were purchased from Molecular Probes (Eugene, OR, USA) and aniline blue was from Merck (Darmstadt, Germany).
Microscopy
A multiphoton CLSM (Leica TCS SP2/MP; Leica Microsystems, Bensheim, Germany) equipped with a multiphoton laser was used to detect the fluorescence signal of aniline blue. A CLSM (Leica TCS 4D; Leica Microsystems, Heidelberg, Germany) equipped with a 75 mW argon/krypton laser (Omnichrome, Chino, CA, USA) was used to detect the fluorescence signals of RH-414 and CMEDA/CMFDA.
The observations were conducted without a cover slip using a special water dipping objective (HCX APO L40x0.80 W U-V-l objective, Leica, Heidelberg, Germany).
RH-414 was excited by the 564 nm line of the 75 mW argon/krypton laser and CMEDA/CMFDA by 488 nm. For observation at the 590 nm and 510 nm wavelength, a long pass filter was used. Aniline blue was excited by 800 nm and the emission was recorded in the spectral window between 450 nm and 510 nm.
Microscopic observation of the events in sieve tubes
After incubation in the bathing medium for at least 1 h, the exposed phloem tissue was stained with RH-414, CMEDA/CMFDA, or aniline blue for 10–20 min. The tissue was washed with bathing medium and distant wounding/damage was imposed either by a heat shock (careful burning of a leaf tip with a match) at a distance of approximately 3–4 cm from the observation window or by cutting the main vein with a razor blade at various distances (3–5 cm) from the observation window. The effects of cutting were observed either side of the incision. During 1 h or 2 h after incubation (aniline blue, RH-414, CMEDA/CMFDA), the time-course of sieve tube occlusion was recorded digitally. The fluorescence intensity was quantified with Leica Confocal Software (version: 2.5 Build 1347). The digital images were treated with Adobe® PhotoShop to optimize brightness, contrast, and colouring, and to overlay the photomicrographs.
Electrophysiology
Microelectrodes were fabricated from aluminosilicate microcapillaries with an outer diameter of 1 mm and an internal filament (SM100F-10, Harvard Apparatus Ltd, Edenbridge, Kent, UK) on a vertical electrode puller (GETRA, München, Germany). The tip diameter of these electrodes was 0.5–1 µm. Pulled glass capillaries were back-filled with 500 mol m–3 KCl and clamped in an Ag/AgCl pellet electrode holder (WPI, Sarasota, FL, USA). The microelectrode was connected to the probe of the amplifier (DUO 773 high input impedance differential electrometer, WPI, Sarasota, FL, USA). The Ag/AgCl reference electrode was connected to the bathing medium by a 2% agar bridge (w/v) filled with 500 mol m–3 KCl solution. After incubation of the phloem tissues in standard bathing medium for 1 h, microelectrodes were impaled in the SEs and constantly observed. The microelectrode tip was manoeuvred into the medium close to the phloem tissue by means of an LN SM-1-micromanipulator (Luigs & Neumann, Ratingen, Germany) and carefully impaled through maximally two overlaying cortical cell layers into the SE as previously described (Hafke et al., 2005). All measurements were performed at a room temperature of 23–25 °C. Subsequent to stabilization of the resting potential of the SE, a brief burning stimulus was applied to the leaf tip at an 3 cm distance from the observation window, and the changes in membrane potential due to burning or cutting were recorded.
To correlate electrical signals with the state of the SE forisomes (condensed or dispersed), photographs were taken at different times after applying the burning stimulus. The electrophysiological measurements were monitored under a Leica DM-LB fluorescence microscope (40x HCX APO L40x/0.80 W U-V-I water immersion objective, Leica, Heidelberg, Germany). Micrographs were taken with a digital camera (Canon Power Shot S40) connected to a computer (Canon Digital Camera solutions disk v8.0 software package).
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Results |
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Time-course of the EPW and distant forisome dispersion/recondensation in response to burning the leaf tip of broad bean
Initially, correlations between the EPW along SEs following leaf burning, and forisome behaviour and the depolarization wave were investigated. Following manual removal of the cortical cell layer in the main vein of leaves of intact V. faba plants, at 3–4 cm from the leaf tip, the exposed phloem was immersed in the bathing medium. After incubation, a microelectrode was impaled into a SE at the observation window. Subsequent to insertion of the microelectrode, the forisome initially dispersed (Fig. 1A). After subsequent recondensation of the forisome (Fig. 1B), the experiment was started by burning the leaf tip.
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Electrophysiological measurements demonstrated the passage of an EPW 15–20 s after burning (Fig. 1). The abrupt depolarization was followed by a slow repolarization which extended over several minutes (five replicates in different plants). The behaviour of forisomes in SEs, about 3–4 cm distant from the leaf tip, was documented at the observation window. Forisomes were used to monitor the presumed Ca2+ influx into sieve tubes in response to a remote injury. Forisomes dispersed and plugged the sieve pores simultaneously with depolarization. Seven to 15 min after passage of the depolarization wave, the forisomes recondensed spontaneously and unplugged the sieve plates. The tight coincidence between passage of EPW and forisome behaviour is consistent with Ca2+ influx during depolarization, since Knoblauch et al. (2001) showed a calcium-dependent dispersion and recondensation of forisomes in intact SEs of V. faba.
Time-course of distant callose deposition onto sieve plates after burning the leaf tip of broad bean
The time-course of callose deposition onto the sieve plates and the PPUs in response to burning the leaf tip (Fig. 2) was investigated using multiphoton microscopy (n=12). Ten minutes before the heat shock, aniline blue was applied to the observation window at a concentration of 0.005% (aniline blue toxicity assessed using cytoplasmic streaming in epidermal onion cells was below this concentration; HJ Martens, personal communication). The degree of aniline blue fluorescence was quantified at a range of time intervals as a relative measure for callose deposition and breakdown. In control plants, callose occurred in small, stable amounts at the sieve plate. After burning the leaf tip, aniline blue fluorescence gradually increased both at the sieve plates and at the PPUs (Fig. 2A, B). After having reached a maximum, the fluorescence declined, although more slowly than the rate of increase. Degradation of callose appeared to be more rapid at the PPUs (30–40 min, Fig. 2) than at the sieve pores. One to two hours after its maximum, the fluorescence of the sieve pores had returned to the original level (Fig. 2H).
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The time-course of callose production/degradation was qualitatively similar between the plants, but there was variation in the time scale. Maximum build-up was achieved between 15 min and 20 min, and full breakdown between 1.5 h and 2 h after burning (n=7). A minor increase in fluorescence was detected at PDs of the PPCs of V. faba 10–15 min after burning the leaf tip.
Simultaneous light microscopy showed forisome reactions (Fig. 3A–H) similar to those observed before (Fig. 1). The time-course of forisome dispersion/recondensation did not coincide with that of callose production/degradation (Fig. 3I). Forisome dispersion occurred in a matter of seconds; recondensation was completed by the time that the repolarization had been concluded. In contrast, callose deposition had largely reached a maximum after the forisomes had recondensed (Fig. 3I). Thus, forisome plugging is much quicker than callose constriction.
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Wounding reactions in response to cutting the main vein of broad bean leaves
It has previously been shown that both mechanical wounding and heat injury result in a slow electrical signal of a long duration along the phloem pathway of Mimosa (Fromm, 1991). Since cutting and burning produced similar reactions in Mimosa, sieve plate occlusion in response to distant cutting was investigated here.
Similar to heat shock (Fig. 2), forisomes dispersed about 30 s after cutting the main vein, when observed at about 3 cm from the cutting site (n=8; results not shown). In contrast, following 15 min of preincubation with aniline blue, there was no evidence of increased callose production at the observation window (n=5; results not shown). The amount of callose observed was comparable with the small, stable amounts seen in control leaves. Callose induction by cutting sieve plates and sieve pores was restricted to an area of about 0.5 cm from the site of cutting (results not shown).
Sieve plate occlusion after a heat shock or cutting the main vein of tomato leaves
Given the contradictory effects of burning and cutting on callose deposition, experiments were carried out with leaves of intact tomato plants (Solanum lycopersicum) identical to those carried out with broad bean leaves. As the sieve tubes of Solanaceae do not contain forisomes, only the callose production could be recorded. Callose synthesis and breakdown in both tomato and broad bean sieve tubes react in a similar way to burning (Figs 2, 4). After burning the leaf tip of an intact tomato plant, callose fluorescence reached a maximum between 20 min and 25 min at the observation window at approximately 3 cm from the leaf tip (Fig. 4B, C). Full breakdown of callose was detected between 2 h and 3 h (n=4).
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After cutting the main vein, however, callose formation was limited to sieve plates and PPUs in an area about 0.5 cm from the site of injury after about 5 min (n=3; results not shown). No callose deposition on the sieve plates was detected at the observation window, 3–4 cm from the site of cutting. Thus, the different rates of callose deposition in response to either burning or cutting may be a general phenomenon. The contrasting reaction may be related to a difference in the triggering mechanisms of EPWs induced by cutting and burning. To elucidate this further, generation of EPWs along the sieve tubes was investigated in response to burning or cutting.
Visualization of changes in membrane potential with RH-414 after burning the leaf tips of broad bean and tomato
Using a non-invasive approach, propagation of EPWs was investigated by application of the voltage-sensitive styryl fluorochrome RH-414 (Schild et al., 1995). In this method, passage of an EPW after a distant stimulus could be identified by an increase of fluorescence followed by a decrease (Schild et al., 1995).
After 15 min of RH-414 incubation of the tissue at the observation window, the leaf tip of broad bean was burned for 3 s and the degree of fluorescence was recorded at the observation window 3 cm from the tip. In keeping with the short time lapse between heat stimulus and SE depolarization (Fig. 1), SEs of V. faba react almost instantaneously, with an increase of RH-414 fluorescence accompanied by forisome dispersion (Fig. 5; n=12).
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The increase of RH-414 fluorescence is especially high in the vicinity of the sieve plates (Fig. 5, arrowheads) and in the SE plasma membranes adjacent to the CC (Fig. 5). The forisome recondensed 5–10 min after burning, and the fluorescence decreased.
In tomato, a similar transient increase in fluorescence was observed in the SE plasma membrane at 3 cm from the burned leaf tip (n=5). A sudden increase of the RH-414 fluorescence shortly (30 s) after burning was followed by a slow extinction of the fluorescence. In both species, a steady increase of fluorescence of PPC plasma membranes was observed.
Visualization of changes in membrane potential with RH-414 after cutting the main vein of broad bean and tomato leaves
The same experimental procedure as described above was used to identify propagation of EPWs after cutting the main vein of V. faba (n=15) and S. lycopersicum (n=5). After an incubation time of 15–20 min, the main vein was cut at a distance of 3 cm from the observation window. An increase of RH-414 fluorescence was never observed. Moreover, electrophysiological measurements show negligible membrane depolarizations (results not shown) at about 3 cm from the site of cutting in both leaves. The absence of callose deposition thus concurs with the absence of an EPW along V. faba and S. lycopersicum sieve tubes. It is intriguing that the forisome disperses in response to cutting (Fig. 6) despite the apparent absence of an EPW.
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Time-course of protein plugging and callose constriction of sieve plates
Sieve plate constriction by callose as a response to damage has been established for a long time (King and Zeevaart, 1974). Sieve plate plugging by proteins may act in a complementary fashion (Cronshaw and Sabnis, 1990; Knoblauch and van Bel, 1998). Electron microscopy (EM) studies of wounded phloem in pea roots confirm both the process of sieve plate plugging by phloem proteins and callose deposition as well as the removal of both substances after restoration of the wounded sieve tubes, which were re-established as functional phloem 2 d after wounding (Schulz, 1988).
None of the parameters involved in sieve plate occlusion has so far been quantified. Experimental artefacts caused by tissue excision can complicate interpretation. Consequently, sieve plate occlusion was studied by remote induction of injury signals in intact plants. The dependence of sieve plate occlusion on EPWs was investigated, focusing on a presumed concurrent influx of calcium. The approach used here further enabled the quantification of the response time of occlusion events, the time dependence of build-up and breakdown of occluding substances, and the degree of occlusion. Finally, sieve plate occlusion frequently turned out to be a reversible event in intact plants.
At a distance of 3 cm, callose deposits around the sieve pores and PPUs reach their maximum size at about 10–15 min (Fig. 2A, B) in response to burning the leaf tip of V. faba. Callose degradation occurs in a matter of hours (1.5–2 h) for sieve plates but only requires 30–40 min for PPUs, where the deposits are thinner (Fig. 2E). Similar results for callose synthesis and breakdown in the sieve tubes were obtained with S. lycopersicum, although in this species the breakdown velocity was lower (Fig. 4). Potential for communication between SEs and CCs is recovered long before long-distance transport is re-established. Rapid reactivation of CC–SE interaction is essential since the metabolic functioning of SEs is dependent on the activities of the CC (van Bel, 2003) and so must be restored quickly. For example, crucial components for metabolic functioning (i.e. ATP; van Bel, 1996) and proteins are delivered via the PPUs into the SEs.
In V. faba, forisomes dispersed 20–30 s after burning and had re-condensed shortly before callose deposition reached its maximum (Fig. 3). The current data suggest that V. faba possesses a two-step occlusion mechanism: a rapid one is responsible for provisional plugging until the second, slower one, has come to full development. The apparent absence of such a double security system provides a functional explanation for the longer presence of callose deposits (up to 3 h) in S. lycopersicum in comparison with those in V. faba (2 h) (compare Figs 2 and 4). However, the interspecific differences in callose deposition may be purely coincidental, as S. lycopersicum sieve tubes may also possess other protein occlusion mechanisms (Ehlers et al., 2000). Aggregates of proteins observed in EM photomicrographs of numerous species (Cronshaw and Sabnis, 1990) indicate a general role for phloem proteins in sieve plate occlusion. Moreover, parietal proteins are part of the giant protein plugs in V. faba sieve tubes (Knoblauch and van Bel, 1998). It remains to be investigated in detail if and how parietal SE proteins act in sieve plate occlusion.
Electric potential waves as triggers for sieve plate occlusion
Burning triggers a longitudinal propagation wave of an electric potential along the phloem (Fig. 1) as previously observed for tomato (Wildon et al., 1989; Stankovic and Davis, 1996) and sunflower (Stankovic et al., 1998). Also touch, cutting, and electrical stimulation induce phloem-related potential waves (Fromm, 1991; Fromm and Spanswick, 1993). The passage of the depolarization almost coincides with forisome dispersion (Fig. 5C), whereas sieve plate constriction by callose is a delayed reaction (Fig. 3). A relationship between burning and remote sieve plate occlusion has also been found for Cucurbita and Hordeum leaves at 9 cm and 12 cm, respectively, from the site of burning (T Will, unpublished results).
The passage of depolarization is also concomitant with an increase in RH-414 fluorescence (Fig. 5). The observed correlation between disturbance of the electric balance and SE reactions provides support for the indirect involvement of ion channels in sieve plate occlusion.
Calcium dependence of sieve plate occlusion
Dispersion and re-condensation of forisomes has been described as being exclusively Ca2+ dependent (Knoblauch et al., 2001, 2003). Furthermore, calcium is known to induce callose formation; the Ca2+-chelator EDTA is employed to prevent sieve plate constriction by callose (King and Zeevaart, 1974). Thus, it can be suggested that EPW passage triggers include the release of Ca2+ into the SE lumina, with the resultant elevated Ca2+ concentration there responsible for triggering both modes of occlusion.
Calcium import into the SE via the plasma membrane generates intracellular signal cascades (Trevawas, 1999; Bootman et al., 2001), since calcium plays a regulatory role in a vast range of cellular processes by acting as a second messenger (White and Broadly, 2003). For instance, calcium was associated with adaptation to heat and cold shocks in plant cells (White and Broadly, 2003).
Cytosolic Ca2+ concentrations range between 100 nM and 200 nM in most plant cells (Mithöfer and Mazars, 2002; Logan and Knight, 2003). Ca2+ concentrations were expected to be of the same order for phloem sap, as the content of sieve tubes is to be regarded as a highly aqueous cytosolic fluid. Preliminary observations (JB Hafke, unpublished data) seem to indicate that the Ca2+ concentration in stylet exudates of sieve tube sap is extraordinarily low (50 nM), which would explain the vigorous forisome reaction to EPWs. On the basis of in vitro experiments with isolated forisomes, Knoblauch and Peters (2004) predicted the dispersion threshold to be in the micromolar range. Possibly, the Ca2+ concentration suddenly rises up to the micromolar range in the vicinity of the SE plasma membrane. In contrast, the optimal reaction mixture for in vitro callose synthesis contained 8 mM Ca2+ (Colombani et al., 2004). However, the latter values may not be valid for the in vivo situation. In view of the apparently conflicting results, further research is required to assess in vivo cytosolic Ca2+ concentrations in SEs.
The sieve plate wall originates from cambial cross-walls of SE precursors, which are perforated by PDs which transform into sieve pores during development (Evert, 1990). Occlusion of sieve pores may represent cellular isolation in response to damage, a mechanism inherited from ancestral PDs during their evolution to SEs (van Bel, 2006). Indeed, PD closure shows features strongly reminiscent of sieve plate occlusion. Abrupt turgor shocks have been shown to be responsible for PD closure in tobacco leaf hairs (Oparka and Prior, 1992). Moreover, PD closure has often been related to Ca2+-induced production of callose in the walls around the PD neck region (Botha et al., 2000; Iglesias and Meins, 2000; Roberts and Oparka, 2003). As callose synthesis seems to be universally Ca2+ dependent, PD closure and sieve pore constriction are most probably related mechanisms. This raises the question of whether PDs can also be plugged by proteins.
Putative involvement of calcium channels in Ca2+ release into the sieve tube lumina
Electrical propagation along the plasma membrane of giant algal cells has been shown to be a process in which K+, Cl–, and Ca2+ channels are involved (Kikuyama and Tazawa, 1983). Similarly, the depolarization wave along sieve tubes has been postulated to be dependent on a sudden Ca2+ influx, K+ efflux, and Cl– efflux (Davies, 1987). In general, at least three Ca2+ channel types [voltage-gated (Pineros and Tester, 1997), ligand-gated (Zimmermann et al., 1997), and stretch-activated (Taylor et al., 1996)] have been identified in plant membranes. One Ca2+ channel type has been visualized in sieve tubes (Volk and Franceschi, 2000).
Ca2+ channels may be involved in the propagation of action potentials (APs) and variation potentials (VPs). APs are triggered by treatments such as electrical, cold, and turgor shocks (Davies, 2006); VPs are induced by wounding (Stankovic et al., 1998; Davies, 2006). The current view on turgor-dependent generation of APs suggests that turgor loss induces a weak membrane depolarization by Ca2+ influx followed by an activation of voltage-gated Ca2+ channels when depolarization exceeds a certain threshold (Felle and Zimmermann, 2007). The rapidly increasing cytosolic Ca2+ concentration then activates Cl– channels, which in turn results in the gating of K+ channels (Felle and Zimmermann, 2007). The role of Ca2+ channels in VPs is less well defined (Davies, 2006; Fromm and Lautner, 2007). A distant burning stimulus evokes an AP followed by a VP (Stankovic et al., 1998; Davies, 2006). It is also suspected that burning induces an AP as well as a VP (the sharp depolarization in Fig. 1 and the longer lasting shoulder in Fig. 1). Cutting would only induce a VP (Dziubinska, 2003) which is apparently very small in the present system.
In contrast to burning (Fig. 1), cutting only triggers a negligible depolarization wave as monitored by the voltage-sensitive dye RH-414 (Fig. 6) and recorded by electrophysiological measurements. It is plausible that, in the case of cutting, only a minute amount of calcium ions is released into the SE lumen which may be insufficient to induce callose production. Forisome dispersion may have a much lower Ca2+ threshold than callose synthesis. In the case of burning, the depolarization period is longer (Fig. 1), which is suggestive of a higher rate of Ca2+ influx. Experiments with the membrane-permeant Ca2+ marker Oregon Green indeed show an increased Ca2+ influx into the SE lumen over a period of several minutes in response to burning (ACU Furch et al., unpublished results).
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Acknowledgements |
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We thank Helle Martens and Michael Hansen (University of Copenhagen) for helpful guidance in using the multiphoton microscope, and Christian Gerken for expert assistance in preparing the figures. We also thank Dr Jeremy Pritchard for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft in the frame of Schwerpunktprogramm 1108.
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Abbreviations |
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AP, action potential; CC, companion cell; CLSM, confocal laser scanning microscope; CMEDA, 5-chloromethyleosin diacetate; CMFDA, 5-chloromethylfluorescein diacetate; DMSO, dimethylsulphoxide; EM, electron microscopy; EPW, electropotential wave; ER, endoplasmic reticulum; PD, plasmodesmata; PPC, phloem parenchyma cell; PPU, pore plasmodesma unit; SE, sieve element; VP, variation potential.
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References |
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