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The relationships between damage-induced electropotential waves (EPWs), sieve tube occlusion, and stop of mass flow were investigated in intact Cucurbita maxima plants. After burning leaf tips, EPWs propagating along the phloem of the main vein were recorded by extra- and intracellular microelectrodes. The respective EPW profiles (a steep hyperpolarization/depolarization peak followed by a prolonged hyperpolarization/depolarization) probably reflect merged action and variation potentials. A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring ~10min after burning. Early stop of mass flow, well before completion of callose deposition, pointed to an occlusion mechanism preceding callose deposition. This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids. The early occlusion is probably due to proteins, as indicated by a dramatic drop in soluble sieve element proteins and a simultaneous coagulation of sieve element proteins shortly after the burning stimulus. Mass flow resumed 30–40min after burning, as demonstrated by carboxyfluorescein movement and aphid activities. Stop of mass flow by Ca2+-dependent occlusion mechanisms is attributed to Ca2+ influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca2+ ions.
Sieve elements (SEs) are elongate, enucleate cells lacking most cell components (van Bel, 2003). They are lined by a thin mictoplasmic layer containing a parietally located smooth SE endoplasmic reticulum, SE plastids, a few inactive mitochondria, and clusters of structural proteins (van Bel, 2003). SEs are arranged end-to-end and connected by modified walls perforated by sieve pores (van Bel, 2003). A mixture of metabolites, minerals, phytohormones, soluble phloem-specific proteins (e.g. Giavalisco et al., 2006), and several forms of RNA (e.g. Kehr and Buhtz, 2008) is driven by mass flow through the wide SE lumina.
The mass flow makes plants vulnerable to the consequences of sieve tube damage. Therefore, injury events induce sieve tube occlusion to prevent loss of sieve tube sap (Evert, 1982; Schulz, 1998) and to impede invasion of phytopathogens through the injured site (van Bel, 2003). Sieve tubes may be occluded by callose deposition and by proteins (e.g. Furch et al., 2007).
Callose deposition is a universally observed mode of sieve plate (SP) occlusion (Kudlicka and Brown, 1997; Nakashima et al., 2003). Callose is a β-1,3-glucan polymer which is produced enzymatically and deposited extracellularly around plasmodesmata and sieve pores in the form of collars (Blackman et al., 1998; Zabotin et al., 2002) as a reaction to chemical or mechanical stress (Kudlicka and Brown, 1997; Nakashima et al., 2003; Levy et al., 2007). As for protein plugging, the numerous aggregation forms of phloem-specific proteins (Cronshaw and Sabnis, 1990) promise an immense variation between plant species. However, reports on protein plugging of sieve tubes are scarce. In cucurbits, phloem protein 1 (PP1) produce insoluble aggregates in response to oxidation (Kleinig, 1975; Alosi et al., 1988) and they plug injured sieve tubes (Read and Northcote, 1983). In legumes, spindle-like protein bodies (forisomes) disperse upon wounding and occlude the sieve pores (Knoblauch and van Bel, 1998).
Callose deposition and protein plugging can operate in parallel in a time-shifted fashion in the same species. Burning stimuli elicit distant occlusion in Vicia faba by rapid forisome dispersion and slower callose deposition (Furch et al., 2007, 2009). These occlusion reactions are reversible; forisome dispersion is reversed by the time that callose deposition comes to full development (Furch et al., 2007). It has been speculated therefore that plants may dispose of a universal safety design for sieve tube occlusion, a quick one executed by phloem-specific proteins and a slower one executed by callose (Furch et al., 2007).
As both occlusion mechanisms are Ca2+ dependent (Kauss et al., 1983; Colombani et al., 2004), remote-controlled occlusion was associated with Ca2+ influx during passage of damage-induced electropotential waves (EPWs) (Furch et al., 2007, 2009; Hafke et al., 2009). EPWs communicate sudden and profound physiological changes over long distances (Stankovic et al., 1998; Stahlberg et al., 2006; Grams et al., 2009). They have been recorded in response to stimuli such as wounding, cold, heat, and electrical shocks (Fromm and Spanswick, 1993; Rhodes et al., 1996; Mancuso, 1999; Furch et al., 2007).
The question arises as to whether the ‘safety design’ in sieve tube occlusion is also functioning in plant families without forisomes. Since burning is the most effective stimulus to trigger remote sieve tube occlusion in intact plants, the relationship between remote burning and mass flow was investigated in sieve tubes of intact Cucurbita maxima plants. To this end, intra- and extracellular electrophysiology was used to record the propagation of EPWs, SDS–PAGE separation to determine soluble protein content, aniline blue staining to follow callose deposition, and carboxyfluorescein transport and aphid behaviour to monitor mass flow.
Cucurbita maxima (cv. Gele Reuzen; Enza Zaden, The Nederlands) plants were cultivated in pots in a greenhouse under standard conditions (21°C, 60–70% relative humidity, and a 14/10h light/dark regime). Supplementary lamp light (model SONT Agro 400W; Phillips Eindhoven, The Nederlands) led to an irradiance level of 200–250μmol−2 s−1 at the plant apex. Plants were taken in the vegetative phase just before flowering, 21–28d after germination. For experiments, mature leaves with a size of ~15×15cm were used.
Extracellular voltage measurements were carried out on a vibration-stabilized bench with a Faraday cage. Borosilicate microelectrodes (tip diameter 1–2μm; Hilgenberg GmbH, Malsfeld, Germany), filled with 0.5M KCl in 1% agar in the tip, were pierced blindly into the main vein of a mature leaf of an intact plant, by means of a micromanipulator (model ST 35; Brinkmann Instrumentenbau, Mannheim, Germany). The reference electrode, filled with 0.5M KCl, was inserted into the soil. Electrodes were connected with a high-impedance amplifier (KS-700, World Precision Instruments Inc., New Haven, CT, USA). After the resting potential had settled, the leaf tip was burnt (2–3s) and EPWs were recorded at 4cm and 8cm from the burning site.
For in vivo observation of sieve tubes, cortical cell layers were removed locally down to the phloem from the lower side of the main vein of mature leaves, attached to intact plants. While avoiding damage to the phloem, cell layers were locally removed by manual paradermal slicing with a razor blade to excise an observation window (Knoblauch and van Bel, 1998). The distance between the observation window and the leaf tip was between 3cm and 9cm. The leaf was mounted on a microscope slide with two-sided adhesive tape, fixed onto the stage of a confocal laser scanning microscope (CLSM), and the free-lying phloem tissue was submerged in a phloem physiological buffer. This medium, containing 2mol m−3 KCl, 1mol m−3 CaCl2, 1mol m−3 MgCl2, 50mol m−3 mannitol, and 2.5mol m−3 MES/NaOH buffer, pH 5.7 (Hafke et al., 2005), was also used for dilution of dyes. Integrity of phloem tissue, as indicated by non-swollen SPs, was checked under water immersion objectives.
Intracellular electrophysiology on intact phloem tissue was described in detail previously (Hafke et al., 2005). After submersion of the exposed phloem tissue in the above-mentioned medium for 1h, SEs were impaled by a microelectrode by an micromanipulator (model LN SM-1; Luigs and Neumann, Ratingen, Germany) under microscopic surveillance. After stabilization of the SE potential, the leaf tip was burnt at a distance of 4cm and 8cm from the observation window, and SE membrane potential profile was recorded.
To visualize callose deposition, a 0.005% solution of aniline blue (Merck, Darmstadt, Germany) was employed of which the stock solution (0.5M phosphate buffer, pH 7.4) contained 0.05mg ml−1 aniline blue. The phloem-mobile fluorochrome 5(6)carboxyfluorescein diacetate (CFDA)-mixed isomers (Invitrogen, Karlsruhe, Germany) was used to visualize stop of mass flow in sieve tubes. Stock solutions of CFDA (1mg solubilized in 1ml of dimethylsulphoxide) were diluted (1μl of the stock solution dissolved in 2ml of medium) to a final concentration of ~1μM. Sulphorhodamine 101 (10μM) (Molecular Probes Europe BV, Leiden, The Netherlands) dissolved in medium was employed to visualize protein coagulation.
A CLSM (Leica TCS 4D; Leica Microsystems, Heidelberg, Germany) equipped with a 75mW argon/krypton laser (Omnichrome, Chino, CA, USA) was used to detect CFDA fluorescence by the 488nm line and sulphorhodamine 101 fluorescence by the 564nm line. A multiphoton CLSM (Leica TCS SP2/MP; Leica Microsystems, Bensheim, Germany) equipped with a multiphoton laser was used to detect aniline blue fluorescence. Aniline blue was excited at 800nm and emission was recorded in the spectral window between 450nm and 510nm.
After incubation in medium for at least 1h, the exposed phloem tissue was stained with aniline blue or sulphorhodamine 101 for 15–20min. Subsequently, the tissue was washed with the medium and distant damage was inflicted by a burning stimulus (careful burning by a match for 2s) at the major vein near the tip at a distance of 9cm from the observation window. Every 5–10min, events in sieve tubes after diverse remote stimuli were documented in real-time during 1–2h after the stimulus. Fluorescence was quantified with ImageJ 1.38 (Wayne Rosband, National Institute of Health, USA).
CFDA was applied to a loading window, 3.5cm upstream from the observation window. CFDA permeates the plasma membrane in the non-fluorescent acetate form and is cleaved by cytosolic enzymes producing membrane-impermeant fluorescent carboxyfluorescein which is transported by mass flow inside SEs. After the carboxyfluorescein had reached the observation window situated at 9cm from the tip, the leaf tip was burnt.
Observations were performed without a coverslip by means of a water immersion objective (HCX APO L40×0.80 W U-V-l, Leica, Heidelberg, Germany). Digital image processing was executed using Adobe® PhotoShop 8.0 and Adobe® Illustrator 11.0 (Adobe Systems Inc., USA) to optimize brightness, contrast, and colouring.
Macrosiphum euphoribiae was reared on 20- to 28-d-old plants of C. maxima in a controlled-environment room at 25°C and a 17/7h light/darkness regime in Perspex cages with large gauze-covered windows (cf. Will et al., 2009).
Aphid activities were recorded by the electrical penetration graph (EPG) technique (Tjallingii, 1978, 1985; Will et al., 2007). Aphids (M. euphorbiae) were placed on the main vein of a mature leaf of C. maxima, at various distances from the leaf tip. After the aphids started phloem sap ingestion, aphid activities in response to EPW passage triggered by burning the leaf tip were analysed according to Prado and Tjallingii (1994). For recording and analysis of EPGs, the software package PROBE 3.5 (EPG Systems, Wageningen, The Netherlands) was adopted. Mann–Whitney rank sum tests were done using SigmaStat 3.0 software (SPSS Inc., Chicago, IL, USA) and SigmaPlot 8.0 software (SPSS Inc.).
Phloem sap was collected from the stump of cut petioles by glass pipettes and transferred to 4-fold concentrated reducing sample buffer (Roti-Load 1; Carl-Roth, Karlsruhe, Germany) in the proportion of 1:3.
One-dimensional SDS–PAGE of phloem sap was carried out according to Laemmli (1970) by using a 4% stacking gel and a 12% separation gel in a MiniProtean 3 Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA) with Precision Plus Protein Standard–All Blue (Bio-Rad) as a protein size marker. The gels were stained by Coomassie blue (Roti-Blue; Carl-Roth, Karlsruhe, Germany), scanned with the Gel Doc XR documentation system (Bio-Rad), and proteins in the respective lanes were quantified by means of Quantity One 1-D Analysis Software (Bio-Rad). Statistical significance was analysed by the Holm–Sidak method using SigmaStat 3.0 software (SPSS Inc.)
Microelectrodes were inserted blindly into the apoplast of the main vein. Following stabilization of the apoplastic electrical potential, the leaf tip was burnt. EPWs were recorded extracellulary at 4cm (Fig. 1B) and 8cm (Fig. 1C) from the burning site (Fig. 1A).
At both recording points, the passage of EPWs was observed. A minute short hyperpolarization of –20mV to –10mV directly after burning was observed ahead of EPWs in all extracellularly conducted experiments. Since they reflect the sudden, transient turgor disbalance of the system, these hyperpolarizations are most probably irrelevant for the present studies.
At 4cm, a fast and short hyperpolarization (66mV) 27s after burning was followed by a fast short repolarization for 18s (Fig. 1B). Subsequently, a second hyperpolarization commenced (65mV) which was followed by a prolonged repolarization including a plateau phase of 48min and an undulating profile, before finally reaching the resting potential.
At 8cm, the first hyperpolarization (46mV) arose after 72s (Fig. 1C). The second phase of hyperpolarization (46mV) started after 99s and was much shorter (19.5min duration) than the prolonged one at 4cm distance (Fig. 1C). The variability in strength and duration of the EPWs at both recording sites is summarized in Table 1.
Microelectrodes were inserted under microscopic surveillance into a submersed SE of the main vein. Following stabilization of the resting membrane potential, the leaf tip was burnt and EPWs were recorded intracellularly at 4cm (Fig. 1D) and 8cm (Fig. 1E) from the burning site.
At 4cm (Fig. 1D), a transient depolarization after 9s (142mV) was followed by a second depolarization (106mV) 24s after burning, with a prolonged repolarization (52mV) including small transients (60mV) over 13.5min.
At 8cm (Fig. 1E), a fast depolarization (87mV) developed 30s after burning with a duration of 30s. Subsequently, a second depolarization (79mV) occurred after 78s and was followed by a sustained repolarization of 16min.
Stop of mass flow in sieve tubes of C. maxima induced by leaf tip burning (at a distance of 9cm from the observation window) was observed using CFDA, as recorded using CLSM (Fig. 2).
CFDA was applied onto the loading window located at 3.5cm from the observation window (Fig. 2A) and transported downstream in the form of carboxyfluorescein through the sieve tubes. After carboxyfluorescein fluorescence had reached a saturation level at the observation window, the leaf tip was burnt (Fig. 2B). Non-recovery of carboxyfluorescein photobleaching (Fig. 2C, J) indicated that the SPs were fully occluded ~1min after burning. The rationale is that fluorescence will not recover unless the SPs are unblocked and mass flow resumes. Between 45min and 90min after burning (Fig. 2D–F, J) the fluorescence increased again, indicating that mass flow had recovered. A second decrease of fluorescence took place between 100min and 120min (Fig. 2G–I, J).
The time course of callose deposition onto SPs in response to burning the leaf tip (Fig. 3) was investigated by means of multiphoton microscopy. Ten minutes before the burning stimulus at 9cm distance, a non-toxic concentration of 0.005% aniline blue was applied onto the observation window (Furch et al., 2007). In control plants, the amounts of callose at the SPs were stable and low after the same preparation procedure without stimulus (results not shown). After burning the leaf tip, aniline blue fluorescence gradually rose, with a maximum at 10min (Fig. 3A–D, I), after which callose appeared to be degraded. After the original low level of fluorescence had been reached (45–50min; Fig. 3E–F, I), callose deposition recommenced, but increased at a slower rate than before (60–105min; Fig. 3G, H). The second wave of callose deposition may explain the second inhibition phase of mass flow as found with carboxyfluorescein (Fig. 2G–I).
The activities of aphids (M. euphorbiae), feeding on the main vein at distances of 4.5cm (Fig. 4A–C) and 7.5cm (Fig. 4A, D–G) from the leaf tip, were monitored by EPG profiles before, during, and after the passage of burning-induced EPWs. Prior to the burning stimulus, the aphids show an E2 profile (Fig. 4B, D) representing ingestion of SE sap accompanied by rhythmic secretion of small amounts of watery saliva that is resorbed together with phloem sap (Tjallingii and Hogen Esch, 1993). The first depolarizations shortly after burning did not influence aphid activities and may therefore reflect a general turgor imbalance of the system in response to burning.
At 4.5cm distance from the burning site, aphids switched their activities from E2 to E1 behaviour [representing secretion of watery saliva into the SE lumen (Prado and Tjallingii, 1994)] 10s after leaf tip burning. They stayed for ~25min in the E1 phase, showed a mixed E1/E2 profile for ~2.5min, and subsequently returned to ingestion characterized by E2 activities (Fig. 4B). Aphids located at 7.5cm from the leaf tip changed their behaviour from E2 to E1 after 7.5min. At this site, the E1 and mixed E1/E2 profiles lasted for a much shorter time (E1, 66s; mixed E1/E2, 27.5s) than those at 4.5cm before the aphids switched back to the E2 behaviour (Fig. 4D). A box–whisker plot (Fig. 4H) displays the time until aphids reacted to leaf tip burning by switching from E2 to E1 behaviour. With increasing distance from the burning site, the time until aphids changed their behaviour increased in a non-linear fashion (Fig. 4H), indicating a declining EPW propagation velocity along the phloem. Statistical comparisons by Mann–Whitney rank sum test of data groups are presented as a table inset in Fig. 4H. P-values <0.05 indicate significant differences between two groups.
The previous results (Fig. 3, ,4)4) suggest ready occlusion of SPs at the time that callose deposition has hardly started. In analogy to the events in V. faba (Furch et al., 2007, 2009), proteins may be engaged in sieve tube occlusion. Therefore, the response of SE proteins to burning the leaf tip/major vein at a distance of 9 cm was observed using sulphorhodamine 101 (Fig. 5) and separation by 1-D SDS-PAGE (Fig. 6). Twenty minutes prior to burning, 10μM sulphorhodamine 101 (cf. Peters et al., 2010), which preferentially associates with membranes, was applied onto the observation window. From 5min after burning onwards, a cloud of fluorescence was observed at the SPs (Fig. 5B, C) indicative of protein clogging. It was technically impossible to further shorten the period between burning and observation.
SE proteins of untreated (Fig. 6A, lane 2; B, grey curve) and treated (collection of phloem sap 5min after burning; Fig. 6A, lane 3; B, black curve) plants were separated by 1-D SDS–PAGE, Coomassie stained (Fig. 6A), and quantified (Fig. 6B). Quantities of the three major proteins of phloem sap samples—PP1 (95kDa), phloem protein 2 (PP2)-dimer (48kDa), and PP2-monomer (24kDa)—differ considerably (Fig. 6A, B). In treated samples, the quantities of PP1 and PP2-dimer are strongly reduced (P <0.001), which indicates a disappearance of the water-soluble forms. PP2-monomer protein bands are equally stained under both conditions (Fig. 6A, B).
As in other plants (e.g. Wildon et al., 1989; Stankovic and Davies, 1996; Stankovic et al., 1998; Furch et al., 2007, 2008, 2009), burning triggers EPWs along the phloem of cucurbits (Fig. 1). The intracellular EPW profiles—a steep transient of depolarization, followed by a long-lasting repolarization (Fig. 1)—hint at the merging of a rapid action potential and a slower variation potential (Stankovic et al., 1998; Hafke et al., 2009). A similar assessment was made for EPWs induced by burning leaf tips of other species (Stankovic et al., 1998; Hlavackova et al., 2006; Hafke et al., 2009). In keeping with this interpretation, the first depolarization peak and the onset of the second depolarization wave drift apart with increasing distance from the burning site (Fig. 1, Table 1).
The decline of the putative action potential depolarization between the points of recording (Fig. 1) seems to contradict the definition of an action potential (Zawadzki et al., 1991; Dziubinska, 2003). In contrast to the prevailing opinion, however, we agree with Stahlberg et al. (2006) that action potentials can fade along the sieve tubes, in particular when, as in Cucurbita, symplasmic continuity with surrounding phloem parenchyma cells is high (Kempers et al., 1998). Leakage of electrical current also explains the occurrence of reduced EPWs in phloem cells adjacent to the sieve tubes in tomato leaf veins (Rhodes et al., 1996). In species specialized in the transmission of action potentials such as Mimosa pudica, sieve tubes are electrically isolated to prevent current efflux to adjoining cells (Fleurat-Lessard and Roblin, 1982).
It should be noted that analysis of different kinetic components is complex; for example, apoplasmic recordings (Fig. 1B,C) express the overlapping reactions of different cell types. The high variability of both kinetic phases here in terms of duration and strength is in agreement with the variability of electrical signals recorded by microelectrodes blindly pierced into the main vein of tomato leaves (Herde et al., 1998).
The initial depolarization of an action potential is thought to involve activation of plasma membrane-localized voltage-sensitive Ca2+ channels in the plasma membrane (e.g. Felle and Zimmermann, 2007). Depolarization of the plasma membrane may be communicated to SE endoplasmic reticulum-located voltage-sensitive Ca2+ channels leading to supplementary Ca2+ influx into the SE mictoplasm (Hafke et al., 2009). Action potentials do not seem to mediate substantial Ca2+ influx (Hafke et al., 2009). In contrast, synergistic Ca2+ channel activation in plasma membrane and SE endoplasmic reticulum is proposed to result in massive Ca2+ influx in sieve tubes during variation potentials (Hafke et al., 2009). Variation potentials depend upon direct activation of mechano-sensitive Ca2+ channels or indirect activation of ligand-activated Ca2+ channels via signalling cascades (Stahlberg and Cosgrove, 1997; Stahlberg et al., 2006).
In V. faba, Ca2+ influx into SEs, which probably corresponds to the intensity of the stimulus, is decisive for the successive occlusion by forisomes and callose (Furch et al., 2007). As in Vicia, EPWs in Cucurbita represent merged potential waves, so that the proportional contribution of action potential (if any) and variation potential to Ca2+ influx and, hence, their individual effects on sieve tube occlusion cannot possibly be distinguished.
Bleached carboxyfluorescein fluorescence failed to recover within the first 1–2min after burning. That the dye was not replaced and thus failed to reach the site of observation indicated cessation of mass flow by blockage of the pathway. About 40min later, fluorescence re-emerged and phloem transport appeared to have resumed (Fig. 3D).
In an independent approach, aphids were used as biosensors for sieve tube occlusion, because they start secreting massive amounts of watery saliva in response to SE occlusion (Will et al., 2007, 2009). This saliva aims to prevent Ca2+-induced sieve tube occlusion by supplying Ca2+-binding proteins (Will et al., 2007). While feeding on SEs, aphids switch from phloem sap ingestion (E2) to secretion of watery saliva (E1) in response to the stop of mass flow triggered by leaf tip burning (Will et al., 2007, 2009). The fact that aphids resume E2 behaviour much faster further away from the burning site (Fig. 4) is consistent with the idea that variation potentials strongly dissipate along the propagation pathway (Davies, 2006). Accordingly, aphid saliva would be able to bind more rapidly to the lower amount of Ca2+ ions flowing into SEs further away from the leaf tip.
Like carboxyfluorescein (Fig. 2), aphids provide compelling physiological evidence in favour of a stop of mass flow due to occlusion in response to remote damage. However, the time delay between stop of mass flow and callose deposition is difficult to interpret. Aphids feeding on the main vein showed an abrupt change in behaviour (Fig. 4) well after the time point when mass flow had stopped (Fig. 7). It may take some time before sieve tube occlusion is perceived by aphids as a loss of pressure (Will et al., 2008). If the aphid response indeed presents a delayed reaction to sieve tube occlusion, aphid behaviour could reflect stoppage of mass flow long before callose deposition is being completed (Fig. 7). Similarly, carboxyfluorescein movement stops (Fig. 2) well before callose deposition is completed (Fig. 3). That stoppage of mass flow does not need maximal deposition of callose could explain this time-incongruence, but the following alternative may be attractive.
In V. faba, sieve tube occlusion by callose was preceded by fast forisome dispersion (Furch et al., 2007) triggered during the initial action potential-like phase. Such a safety design may also operate in a modified form in cucurbits: an immediate protein reaction precedes the slower callose deposition (Fig. 5). Candidates for sieve tube occlusion are PP1 and PP2 (Fig. 6), two major proteins from cucurbit exudates (Read and Northcote, 1983) which easily coagulate in the presence of oxygen (Alosi et al., 1988; Golecki et al., 1998). Moreover, PP1 and PP2-dimers are covalently cross-linked by disulphide bonds, forming high molecular weight polymers (Read and Northcote, 1983). These filaments were released from the plasma membrane and the SE endoplasmic reticulum and washed towards the SP in the case of wounding (Smith et al., 1987). The loss of the water-soluble forms of PP1 and PP2 (Fig. 6) in response to burning indicates that part of these proteins have become water insoluble due to polymerization. This conformational transition is in accordance with the visual appearance of protein plugs (Fig. 5). Most probably, therefore, the protein plugs observed represent polymerized PP1 and PP2. Given the Ca2+ dependence of the occlusion mechanisms in V. faba (Furch et al., 2009; Hafke et al., 2009), potential Ca2+-binding sites on PP1 (McEuen et al., 1981; Arsanto, 1986) make this compound a prime candidate for proteinaceous occlusion prior to callose occlusion in cucurbits.
We acknowledge the guidance of Alexander Schulz and Helle Martens (University of Copenhagen, Denmark) in using multiphoton microscopy. Furthermore, we are grateful to Steffanie V. Buxa, Aline Koch, and Sarah R. Kornemann (Justus-Liebig-University Gießen, Germany) for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft in the frame of the Schwerpunktprogramm 1108 (BE1925/8-2, 8-3, 15-1).