Spread the news: systemic dissemination and local impact of Ca2+ signals along the phloem pathway

Journal of Experimental Botany, Apr 2014

We explored the idea of whether electropotential waves (EPWs) primarily act as vehicles for systemic spread of Ca2+ signals. EPW-associated Ca2+ influx may trigger generation and amplification of countless long-distance signals along the phloem pathway given the fact that gating of Ca2+-permeable channels is a universal response to biotic and abiotic challenges. Despite fundamental differences, both action and variation potentials are associated with a sudden Ca2+ influx. Both EPWs probably disperse in the lateral direction, which could be of essential functional significance. A vast set of Ca2+-permeable channels, some of which have been localized, is required for Ca2+-modulated events in sieve elements. There, Ca2+-permeable channels are clustered and create so-called Ca2+ hotspots, which play a pivotal role in sieve element occlusion. Occlusion mechanisms play a central part in the interaction between plants and phytopathogens (e.g. aphids or phytoplasmas) and in transient re-organization of the vascular symplasm. It is argued that Ca2+-triggered systemic signalling occurs in partly overlapping waves. The forefront of EPWs may be accompanied by a burst of free Ca2+ ions and Ca2+-binding proteins in the sieve tube sap, with a far-reaching impact on target cells. Lateral dispersion of EPWs may induce diverse Ca2+ influx and handling patterns (Ca2+ signatures) in various cell types lining the sieve tubes. As a result, a variety of cascades may trigger the fabrication of signals such as phytohormones, proteins, or RNA species released into the sap stream after product-related lag times. Moreover, transient reorganization of the vascular symplasm could modify cascades in disjunct vascular cells.

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Spread the news: systemic dissemination and local impact of Ca2+ signals along the phloem pathway

Journal of Experimental Botany Spread the news: systemic dissemination and local impact of Ca2+ signals along the phloem pathway Aart J. E. van Bel 1 2 Alexandra C. U. Furch 1 Torsten Will 1 Stefanie V. Buxa 1 Rita Musetti 0 Jens B. Hafke 3 0 Department of Agricultural and Environmental Sciences, University of Udine , Via delle Scienze 208, 33100 Udine , Italy 1 Institute of Phytopathology and Applied Zoology, Centre for BioSystems, Land Use and Nutrition, Justus-Liebig-University , Heinrich- Buff-Ring 26-32, D-35392 Giessen , Germany 2 Institute of General Botany, Justus-Liebig University , Senckenbergstrasse 17, D-35390 Giessen , Germany 3 Institute of Plant Physiology, Justus-Liebig University , Senckenbergstrasse 3, D-35390 Giessen , Germany We explored the idea of whether electropotential waves (EPWs) primarily act as vehicles for systemic spread of Ca2+ signals. EPW-associated Ca2+ influx may trigger generation and amplification of countless long-distance signals along the phloem pathway given the fact that gating of Ca2+-permeable channels is a universal response to biotic and abiotic challenges. Despite fundamental differences, both action and variation potentials are associated with a sudden Ca2+ influx. Both EPWs probably disperse in the lateral direction, which could be of essential functional significance. A vast set of Ca2+-permeable channels, some of which have been localized, is required for Ca2+-modulated events in sieve elements. There, Ca2+-permeable channels are clustered and create so-called Ca2+ hotspots, which play a pivotal role in sieve element occlusion. Occlusion mechanisms play a central part in the interaction between plants and phytopathogens (e.g. aphids or phytoplasmas) and in transient re-organization of the vascular symplasm. It is argued that Ca2+-triggered systemic signalling occurs in partly overlapping waves. The forefront of EPWs may be accompanied by a burst of free Ca2+ ions and Ca2+-binding proteins in the sieve tube sap, with a far-reaching impact on target cells. Lateral dispersion of EPWs may induce diverse Ca2+ influx and handling patterns (Ca2+ signatures) in various cell types lining the sieve tubes. As a result, a variety of cascades may trigger the fabrication of signals such as phytohormones, proteins, or RNA species released into the sap stream after product-related lag times. Moreover, transient reorganization of the vascular symplasm could modify cascades in disjunct vascular cells. Calcium hotspots; calcium signatures; eletropotential waves; long-distance signalling; phloem pathway; sieve element cytoskeleton; sieve elements; sieve tube occlusion Introduction In their natural habitat, plants are permanently exposed to countless abiotic and biotic changes imposing a permanent stress. The majority of environmental challenges are communicated via the extracellular microenvironment (i.e. the apoplasmic space) and, from there, via the plasma membrane to the intracellular space. The external stimuli are monitored by a vast battery of sensors which transform the external information into signals triggering adequate cell reactions. One of the initial events in sensing is a Ca2+ influx modulated by Ca2+-permeable channels at the plasma membrane. Since no Ca2+-selective channels have been identified with certainty in the plasma membrane of plant cells thus far (Kudla et  al., 2010), we will refer to these channels as ‘Ca2+-permeable’ (e.g. Sanders et al., 2002). Ca2+ influx elevates the cytosolic Ca2+ level according to stimulus-specific, spatio-temporal, and potentially cell-specific patterns designated Ca2+ signatures © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: 10 s up to 30 min (Stahlberg and Cosgrove, 1997; Stahlberg et al., 2006). The propagation velocities are 5–10 times slower than those of APs (Stahlberg and Cosgrove, 1997) and the amplitude drops along the transmission path (Davies, 2004; Stahlberg et  al., 2005, 2006). As a result, VP amplitudes decrease with increasing distance from the stimulus site and finally extinguish (van Sambeek and Pickard, 1976). The slow repolarization of VPs might result from the shutdown of proton pumps as indicated by pH-dependent fluorochromes and the ineffectiveness of ion channel blockers (Stahlberg and Cosgrove, 1992, 1996). The inhibition of proton pump activity is not fully understood, but may be due to elevated Ca2+ levels (Kinoshita et  al., 1995; Hafke et  al., 2013). Proton pump activity may be equally suppressed by cytosolic Ca2+ in APs, but the reduced pump activity may be hardly detectable due to the lower Ca2+ influx during APs (see ‘Creation of Ca2+ hotspots in sieve elements’). Generation and propagation of VPs have only been observed in intact plants, whilst APs can propagate in isolated organs (Stahlberg et  al., 2006). Therefore, relaxation of the negative hydrostatic pressure in the xylem vessels is the likely source of VP generation (Stahlberg and Cosgrove, 1997). That VPs originate from events in xylem vessels (Fig. 1) was demonstrated by the fact that VPs, in contrast to APs, are able to traverse dead or poisoned areas (Stahlberg et al., 2006). Essential differences and functional similarities between APs and VPs The preceding sections disclose a few essential differences between the APs and VPs (Fig. 1). (i) APs and VPs are of a dissimilar nature (i.e. they are of an electrical or mechanistic origin) and, by implication, the Ca2+-permeable channels responsible for the initial depolarization are either voltagedependent or mechano-sensitive channels, respectively (Fig.  1). (ii) APs are generated in non-vascular or vascular cells, move longitudinally along the sieve tubes, and may disperse in the lateral direction to surrounding vascular cells (Fig.  1). In contrast, VPs are generated by vascular (xylem parenchyma) cells and move laterally across several cell layers to the sieve tubes, so that VPs reflect arrival of successive single depolarizations at the sieve element plasma membrane mimicking electrical propagation along the sieve tubes (Fig. 1; Malone, 1996; Pyatygin et al., 2008; van Bel et al., 2011a). These conclusions call for further exploration of the following questions: (i) which Ca2+-permeable channels reside in the vascular cells and where are they located (Fig. 2); and (ii) how does the symplasmic organization of the phloem strands enable combined longitudinal propagation and lateral dispersion of electrical information (Fig. 3)? Despite their profound differences, EPWs have one essential, functional feature in common. They are all associated with an initial elevation of cytosolic Ca2+ (Trebacz et al., 2006; Davies and Stankovic, 2006; Demidchik and Maathuis, 2007; McAinsh and Pittman, 2009), regardless of the involvement of voltage-dependent, mechano-sensitive, or ligand-activated Ca2+-permeable channels. Hence, the elevation of Ca2+ levels in sieve elements, the involvement of the sieve element cytoskeleton in Ca2+ influx mechanisms, and the impact of Ca2+ influx on sieve element biology are major issues in this frame (Figs. 4–6). Phloem-associated Ca2+-permeable channels and other channels involved in EPW propagation Types of Ca2+-permeable channels localized to the plasma membrane in plants In animals, highly selective Ca2+ channels are responsible for Ca2+ fluxes at the plasma membrane (Tsien et al., 1987; Tsien and Tsien, 1990; McAinsh and Pittman, 2009), whereas non-selective cation channels (NSCCs) or Ca2+-permeable channels seem to enable Ca2+ fluxes in plants to give rise to stimulus-specific Ca2+ signatures (Demidchik and Maathuis, 2007; McAinsh and Pittman, 2009). Ca2+ influx across the plasma membrane can be mediated by the following types of non-specific cation channels (Fig.  2A; Demidchik and Maathuis, 2007; McAinsh and Pittman, 2009): (i) HACCs: hyperpolarization-activated Ca2+-permeable channels, which are gated by an increase in membrane voltage, reactive oxygen species (ROS), and changes in the cytoplasmic Ca2+ level; (ii) DACCs: depolarization-activated Ca2+-permeable channels activated by a decrease in membrane voltage; (iii) MSCs: mechano-sensitive channels the gating of which is modulated by tensile forces exerted on membranes; (iv) CNCGs: cyclic nucleotide-gated channels activated by binding of cyclic nucleotides (e.g. cAMP, cGMP); and (v) GLRs: glutamate receptor-like channels activated by binding of amino acids. Regarding HACCs (in Arabidopsis root cells), the resting value of the membrane potential is more positive than their activation voltage, which, however, can shift to more positive membrane potentials brought about by increased Ca2+ levels (Demidchik et al., 2002; Demidchik and Maathuis, 2007; Miedema et al., 2008). It is likely that DACCs are engaged in cold-induced Ca2+ influx (White, 2009). A  member of the DACCs, named the maxi cation channel, was postulated to be responsible for the creation of complex temperature-dependent Ca2+ signatures (White and Ridout, 1999; White, 2004, 2009). Apart from their gating response to changing tensile forces (Demidchik and Maathuis, 2007), MSCs may act as primary temperature sensors (Minorsky and Spanswick, 1989; Monroy and Dhindsa, 1995; Plieth et  al., 1999), as demonstrated by the gradually increasing activity of MSCs at temperatures dropping below 20 °C (Ding and Pickard, 1993). Putative Ca2+-permeable channels lining the sieve elements Among all possible Ca2+-permeable channels, MSCs have been identified with certainty in the sieve element plasma membrane thus far. Forisome reactions in intact sieve elements (Knoblauch et  al., 2001) and in sieve element protoplasts (Hafke et al., 2007) evidenced Ca2+ influx in response to vigorous turgor changes. MSCs may also be crucial players in the activation of HACCs that may catalyse a long-lasting Ca2+ influx into sieve elements during the prolonged EPW phase after remote burning (Furch et  al., 2009). Recently, GLRs have been discovered in the phloem (Vincill et  al., 2013), but their cellular location is uncertain. Only circumstantial evidence has been obtained for other Ca2+-permeable channels in sieve tubes. Cold-shock induced Ca2+ influx into sieve elements (Thorpe et al., 2010; Hafke et al., 2013) could have been mediated by MSCs or DACCs (Ding and Pickard, 1993; Plieth, 1999; Plieth et al., 1999; White and Ridout, 1999; White, 2009). The wealth of potential ligands associated with VP generation (see ‘Presumptive significance of plasmodesmal connectivity for lateral VP dispersion’) renders the presence of ligandactivated channels on the sieve element plasma membrane highly plausible. Location of the Ca2+-permeable channels in sieve elements Early studies using BODIPY-DHP and antibodies localized voltage-dependent Ca2+-permeable channels to the sieve element (Volk and Franceschi, 2000). A more detailed approach using fluorochrome mixtures (Furch et al., 2009) and reaching a higher spatial resolution visualized Ca2+-permeable channels located in the plasma membrane and the endoplasmic reticulum (ER) stacks of sieve elements (Fig. 2B). Ca2+-permeable channels are unevenly localized to the sieve element plasma membrane. They are mostly aggregated in the vicinity of sieve plates and unilaterally branched plasmodesmata (pore–plasmodesm units; PPUs) towards the companion cells (Furch et al., 2009); hence, at the sieve element side facing the companion cell (Fig. 2B). A false-colour presentation of the ratio between BODIPY-DHP and RH-414 fluorescence confirmed preferential aggregation of Ca2+-permeable channels near sieve plates and PPU orifices (Fig. 2C). Furthermore, distribution of Ca2+-permeable channel clusters closely matched that of sieve element ER distribution in sieve elements (Fig. 2D), as was documented by double-label experiments (Fig. 2E). Cl– channels is entirely lacking, phloem-localized K+ channels of the AKT2/3 type were electrophysiologically characterized and linked to AP depolarization (Marten et al., 1999; Bauer et al., 2000; Lacombe et al., 2000; Deeken et al., 2002). Weak inward rectifying currents matching the features of AKT2/3 channels were recorded in sieve element protoplasts (Hafke et  al., 2007, 2013). The increasing permeability of AKT2/3 channels at more alkaline pH values (Marten et al., 1999) and the extracellular alkalinization during transmission of SPs (Zimmermann et  al., 2009) indicate that AKT2/3 channels are involved in membrane repolarization. As regards proton pumps, immunological approaches localized H+-ATPases to sieve elements and companion cells (Langhans et al., 2001). Symplasmic organization of phloem strands Deployment of other channels and pumps involved in EPW propagation Ultrastructure and plasmodesmal connectivity of sieve elements Although this review does not focus on other channels and pumps involved in EPWs, we will pay some marginal attention to the few facts known. Whilst information on sieve element In sieve elements, cellular substructure is reduced to a plasma membrane envelope lined with a thin margin of gelatinous cytoplasm (mictoplasm) containing a limited number of organelles (e.g. van Bel, 2003). Originally, the mictoplasm was defined as the mixture of cytoplasmic contents with the sieve element fluid (Engleman, 1965). This layer has been re-defined as mictoplasm for practical reasons (van Bel, 2003): Ca2+ concentrations vary greatly in this space during EPWs (Furch et al., 2009). The fact that the mictoplasmic layer is in open contact with the sap stream in the sieve element lumen is the consequence of tonoplast disintegration during sieve element ontogeny (Esau, 1969). Several organelles such as the nucleus, ribosomes, and Golgi apparatus are degraded during sieve element development (Behnke and Sjolund, 1990). The ER that may originate from the cortical ER (Hepler et al., 1990) survives the partial programmed cell death and is aggregated in regular stacks that are often oriented perpendicularly to the plasma membrane (Sjolund and Shih, 1983; Ehlers et al., 2000). The ER stacks are tethered to the plasma membrane and to each other by anchors of unknown nature (Ehlers et al., 2000) to prevent dragging by mass flow and resultant sieve pore occlusion. For the same reason, a special type of plastids of unknown function—considerably smaller than chloroplasts (Behnke and Sjolund, 1990)—are tethered to the plasma membrane (Ehlers et al., 2000). Microscopically visible clusters of phloem-specific structural proteins are located at the margins of the sieve element (Behnke and Sjolund, 1990; Knoblauch and van Bel, 1998) or even in the sieve-tube lumen (Froelich et al., 2011; Knoblauch and Oparka, 2012). In addition, there is a wealth of soluble proteinaceous components in sieve elements (Barnes et  al., 2004; Walz et  al., 2004; Giavalisco et  al., 2006; Aki et  al., 2008; Furch et al., 2010; Dinant and Lucas, 2013). It has been excluded for a long time that a complete cytoskeleton exists in sieve elements (Parthasaraty and Pesacreta, 1980; Thorsch and Esau, 1981; Evert, 1990), although circumstantial structural (Chaffey and Barlow, 2002) and chemical (Kulikova and Puryaseva, 2002; Barnes et  al., 2004; Walz et  al., 2004; Giavalisco et al., 2006; Aki et al., 2008) evidence favoured the opposite view. Recent confocal laser scanning microscopic, immunological, and physiological studies have probably ended the dispute by identification of a complete, parietally located actin network in sieve elements (Hafke et al., 2013). As inferred from dye coupling experiments, the sieve element precursor divides longitudinally and becomes transiently isolated from its neighbouring cells, which has been regarded as an instrument for developmental specialization (van Bel and van Rijen, 1994). The other daughter cell develops into 1–4 companion cells flanking each sieve element (Esau, 1969). Given its limited cellular equipment, a sieve element relies almost completely on its companion cell(s) for its survival, which makes communication between the two of paramount importance (van Bel 2003). Towards the end of the temporary symplasmic seclusion, so-called PPUs (van Bel and Kempers, 1997) arise that have the capacity to traffic a vast spectrum of substances including macromolecules between sieve element and companion cell(s) (Imlau et  al., 1999; Lucas et al., 2001, 2009). Plasmodesmata between companion cells and phloem parenchyma are sparse (Kempers et  al., 1998), which seems to present a symplasmic bottleneck. These plasmodesmata have never been studied in detail, but may be of special nature, since their opening state is related to source–sink relationships (Patrick and Offler, 1996; Hafke et  al., 2005). Moreover, the phloem-specific clostero- and luteoviruses are unable to pass this symplasmic border and, hence, are contained inside the sieve element–companion cell complexes (Stewart et al., 2013). Presumptive significance of plasmodesmal connectivity for lateral AP dispersion The electrical conductivity of the sieve element plasma membrane, the longevity of the sieve elements, and the high electrical conductance of sieve pores make sieve tubes ideal conduits for long-distance electrical signalling (van Bel and Ehlers, 2005). The restriction of longitudinal AP propagation to the sieve tubes indicates a high degree of electrical resistance in the plasmodesmal pathway from sieve elements to other cells (Fig. 3). The scarcity of plasmodesmata between companion cells and phloem parenchyma cells in transport phloem (Kempers et al., 1998), which are closed under sourcelimiting conditions, would fulfil the requirements for electrical insulation (Patrick and Offler, 1996; Hafke et al., 2005). On the other hand, it should be borne in mind that electrical currents are expected to pass plasmodesmata with extremely low molecular exclusion limits or even move along the membranes crossing the cytoplasmic sleeve. Moreover, permanent and full electrical insulation of sieve element–companion cell complexes is unlikely, as inferred from symplasmic unloading of excess photoassimilates under sink-limiting conditions (Patrick and Offler, 1996), to fill axial storage compartments along the phloem pathway rapidly. ‘Electrical leakiness’ (Fig. 3A) is indicated by small depolarizations of phloem parenchyma cells coincident with the passage of EPWs (Rhodes et  al., 1996). All in all, there is a good chance that the electrical insulation of sieve tubes is incomplete. Voltage-dependent Ca2+ channels in sieve elements would be the initiators of longitudinal AP propagation that is diverted by electrotonic leakage bringing about Ca2+ influx into vascular parenchyma cells. The concept of functional current leakage is supported by events in the excitable plant Mimosa pudica, in which long distances are covered by APs owing to an insulating sclerenchyma sheath around the sieve element–companion cell complexes (Fleurat-Lessard and Roblin, 1982). This shield is interrupted in the pulvini, where numerous plasmodesmata provide ample symplasmic access to flexor parenchyma cells (Fleurat-Lessard and Bonnemain, 1978) with inherent facilitation of current leakage. The flexor cells react to Ca2+ influx by instantaneous loss of osmotic substances giving rise to leaf and leaflet bending (Fleurat-Lessard and Bonnemain, 1978). These phenomena may exemplify less prominent events in non-excitable plants with lower rates of current leakage and less eye-catching reactions by the flanking parenchyma cells. Altogether, it appears that incomplete insulation of sieve tubes is not a defect, but highly functional in lateral dispersion of Ca2+ waves and Ca2+-mediated information. Presumptive significance of plasmodesmal connectivity for lateral VP dispersion While the lateral events accompanying APs allow a straightforward assessment, there is more room for speculation regarding VPs. Disturbance of the hydraulic equilibrium in xylem vessels leads to water intake by the adjacent parenchyma cells which causes membrane depolarization due to increased turgor (Malone and Stankovic, 1991; Stahlberg and Cosgrove, 1992, 1997; Mancuso, 1999; Davies, 2006). Therefore, receptor potentials are probably triggered here by mechano-sensitive Ca2+-permeable channels (probably MSCs), but the mode of subsequent lateral electrical transmission to sieve tubes is a matter of debate. As a first possibility (Fig.  3B), pressure-induced receptor potentials activate voltage-dependent Ca2+-permeable channels (perhaps DACCs) that generate EPW propagation towards the sieve tubes. The second and, at first sight, most likely option (Fig. 3C) is that the turgor of all vascular cells including the sieve tubes rises by intake of water after vessel damage, as argued for the mechanisms of cucurbit phloem exudation (Zimmermann et al., 2013). According to this scenario, VP propagation results from the action of mechano-sensitive channels which perceive local turgor changes in each vascular cell. This concept explains the attenuation of VPs with distance, provided that the relaxation in the vessels is increasingly dampened further away from the site of damage. Nevertheless, a few essential problems remain with this concept. Why is the VP generation not almost equally rapid along the vascular pathway, because pressure loss must propagate very quickly. In other words, why does it take so much longer for the VP to be expressed far away from the site of wounding, and why is the reaction to crushing so much more vigorous than to cutting, although the number of vessels damaged is approximately identical? Therefore, it has been postulated as a third alternative (Fig.  3D) that MSC-mediated Ca2+ influx triggers cascades that produce chemical signals (Ricca, 1916; van Sambeek and Pickard, 1976; van Sambeek et al., 1976; Boari and Malone, 1993; Malone, 1996; Stahlberg and Cosgrove, 1997; Mancuso, 1999; Pyatygin et  al., 2008). Oligosaccharides as well as the peptide systemin in solanacean species (Narvaez-Vasquez and Ryan, 2004) are potential messengers triggering VPs (Thain et al., 1995; Moyen and Johannes, 1996) after docking to ligand-activated Ca2+-permeable channels (Fig.  3D). The period to accumulate sufficient second messengers which may be correlated with the degree of relaxation would explain the increasing lag time between wounding and VP generation along the pathway. In view of the co-occurrence of diverse Ca2+-permeable channels in plasma membranes (Kudla et  al., 2010), combinations of the above scenarios are likely to occur. Irrespective of the mode of lateral EPW transmission, open plasmodesmata are compulsory (Fig.  3B, D; van Bel et  al., 2011a), unless information is transferred by lateral pressure An elevated mictoplasmic Ca2+ level may boost its own concentration by Ca2+-stimulated Ca2+ efflux from ER stacks in analogy to Ca2+-induced Ca2+ release at the tonoplast (CICRs, or calcium-induced calcium release channels; Bewell et al., 1999; Sanders et al., 2002). Similarly, Ca2+ would trigger presumptive Ca2+-dependent Ca2+ channels on the ER membranes (Fig. 4F; Furch et al., 2009; Hafke et al., 2009). Ca2+ recruitment from internal stores is an established event during cold shocks (Knight et  al., 1996; Gong et  al., 1998; White and Broadley, 2003). Further evidence (Furch et  al., 2009; Thorpe et al., 2010; van Bel, 2011a) also points to the ER as an important Ca2+ store which seems a major reason why ER stacks have been retained during sieve element evolution (Sjolund and Shih, 1983; van Bel, 2003). All in all, Ca2+ hotspots are probably created where high densities of Ca2+-permeable channels in the plasma membrane and an abundance of ER stacks meet (Fig. 4J; Hafke et al., 2009). In line with putative Ca2+ accumulation at these sites, the reactivity of forisomes increases when their tips are located in the vicinity of Ca2+ hotspots in sieve elements (Furch et  al., 2009). Further functional support for Ca2+ hotspots is provided by the fact that the forisome tips, being positioned between the ER stacks, are the only forisome parts that disperse as a reaction to weaker stimuli (Fig.  4J). The frequently perpendicular orientation of the ER stacks (Ehlers et al., 2000) facilitates insertion of the tips into a space (Furch et al., 2009), where Ca2+ levels may reach the threshold value needed for forisome dispersion. The interstices of the ER offer an undisturbed microenvironment for creation of Ca2+ hotspots (Furch et al., 2009; Hafke et al., 2009). Correlation between the Ca2+ concentration in hotspots and forisome responses As argued above, forisomes can be regarded as innate indicators for the Ca2+ thresholds and the approximate Ca2+ concentration in hotspots. APs seem to generate low-concentrated hotspots, since APs seldom lead to forisome responses (Fig. 4J) or, if they do so, lead to a slight wiggling of the forisome tails or a partial dispersion of the tips. Forisome dispersion coincident with prolonged EPW profiles indicates strong accumulation of Ca2+ at sieve element hotspots in response to VPs (Fig. 4J). Violent stimuli (burning, crushing) trigger APs and VPs in parallel that will collaborate in generating Ca2+ influx, the more so as Ca2+ potentiates it own hotspot concentration via Ca2+ liberation from ER stacks (Fig. 4J; Hafke et al., 2009). Ca2+ hotspots could also be meaningful for callose synthesis since Ca2+ concentrations required for this reaction greatly exceed those in the sieve tube sap (Hafke et  al., 2009; Furch et al., 2009), at least in vitro (Colombani et al., 2004). Involvement of the sieve element cytoskeleton in EPW propagation It has been known for a long time that cold shocks induce transient blockage of sieve tubes (Pickard and Minchin, 1990), which has been related to Ca2+ channel reactivity (Thorpe et  al., 2010). Cold shocks [>0.5  °C s–1 (Thorpe et  al., 2010) or 4.2  °C in less than a second (Hafke et  al., 2013)] induced sieve element depolarization followed by forisome dispersion (Fig.  5A). The depolarization was strongly reduced by the Ca2+ channel blocker La3+, and forisome dispersion also failed to occur. The apparent cold-triggered Ca2+ influx was originally ascribed to gating of mechano-sensitive Ca2+-permeable channels (Fig. 5A; Thorpe et al., 2010) due to a change of the tensile force exerted on the plasma membrane. Involvement of the cytoskeleton, however, was not excluded given the resemblance between cold-induced Ca2+ influx into the mictoplasm and other cell types (Knight et al., 1996; Plieth et al., 1999; White, 2009). The latter has become more plausible after the recent discovery of a complete, dense actin network in sieve elements (Fig. 5E; Hafke et al., 2013). The actin disruptor latrunculin A (Lat A) has similar inhibitory effects on the cold-induced events (depolarization and forisome dispersion) in the presence or absence of La3+ (Fig. 5B; Hafke et al., 2013). Their equal impact indicates that LatA and La3+ target the same Ca2+ influx mechanism that is linked in some way to actin action. All in all, the presumptive interaction between Ca2+permeable channels and actin (Hafke et  al., 2013) predicts that the cytoskeleton plays a pivotal role in EPW propagation. Interaction between Ca2+ channels and the cytoskeleton in sieve elements is further supported by an intimate connection between the plasma membrane and the actin meshwork as indicated by dense anti-actin immunochemical labelling of the face of the plasma membrane (Fig. 5C; Hafke et al., 2013). Forisomes probably must be kept in position for optimal sensing of Ca2+ changes in hotspots, although no compelling evidence for anchoring has been obtained thus far. The virtual absence of actin on dispersed forisomes (Fig. 5D; Hafke et al., 2013) seems to exclude that forisomes are linked to actin, unless actin filaments are torn apart during the fixation procedure due to forisome swelling. Other modes of linkage could be provided by protein filaments of unknown nature that anchor sieve element organelles to the plasma membrane (Fig. 5F; Ehlers et al., 2000) or tubulin. As expected, tubulin occurs in sieve elements (JBH, unpublished results) based on preliminary experiments using the tubulin disruptor oryzalin. Actin and tubulin may be coupled to different Ca2+ channels, since the activity of depolarization-activated (Mazars et al., 1997; Thion et al., 1998) and mechano-sensitive (Wang et al., 2004; Zhang et al., 2007) Ca2+-permeable channels was modulated by microtubules and microfilaments, respectively, in other cell types. The interaction between cytoskeleton elements and Ca2+ channels and the inherent cytoskeleton involvement in shaping Ca2+ signatures and triggering intracellular signal cascades (Mazars et  al., 1997; Trewavas and Malho, 1997; Drøbak et  al., 2004; Davies and Stankovic, 2006) may be of paramount significance for EPW propagation. The question now arises as to how actin and Ca2+-permeable channels are linked (Fig.  5F). In general, cytoskeleton disruptors that destabilize either F-actin (Liu and Luan, 1998; Wang et  al., 2004; Zhang et al., 2007) or microtubules (Thion et al., 1998) affect the action of ion channels. Protein complexes designated as ‘transducons’, which consist of an aggregate of receptors, Ca2+-permeable channels, bound calmodulin, protein kinases, and phosphatases, have been invoked to explain the intimate interaction between Ca2+ and the cytoskeleton (Trewavas and Malho, 1997) via members of the NETWORKED superfamily (Deeks et al., 2012). Transducons have been proposed to be tethered by integrins (Trewavas and Malho, 1997; Knepper et al., 2011) to the plasma membrane and cell wall. Ca2+-induced sieve element occlusion mechanisms: a safety design? Full sieve element occlusion achieved by forisomes had been a matter of debate (Peters et al., 2006) until in vitro experiments demonstrated that the swelling capacity was more than sufficient (Knoblauch et al., 2012). In intact V. faba plants, forisomes dispersed within seconds after EPW passage induced by burning and recontracted after 10–20 min (Fig.  6A–C; Furch et al., 2007, 2009). Forisome dispersion turned out to be quicker than callose production (Furch et al., 2007, 2009). By the time that a forisome had recontracted, probably due to active Ca2+ removal (e.g. Huda et al., 2013), callose build-up reached its maximum followed by a slower degradation up to 3 h (Fig. 6D; Furch et al., 2007, 2008, 2010). Both modes of occlusion are under the control of Ca2+ ions (Fig. 6E, F), the difference being that protein-mediated occlusion may have a lower Ca2+ threshold (50  μM; Furch et  al., 2009; Hafke et  al., 2009) than callose synthesis. In vitro callose synthesis required a concentration of 8 mM Ca2+ (Colombani et  al., 2004). Alternatively, the time lag of maximal callose deposition under the control of the Cal7 gene (Barratt et al., 2011; Xie et al., 2011) is due to the relative slowness of the complex de novo callose synthesis (Chen and Kim, 2009) with a Vmax of 45.5 nmol min–1 mg–1 (Li and Brown, 1993). A dual sieve plate occlusion mechanism was also found in Cucurbita maxima (Furch et al., 2010). Rapid, apparent coagulation of the phloem proteins, PP1 and PP2, several centimetres away from the site of burning preceded callose deposition (Furch et al., 2010). As shown for various species, callose deposition reaches its maximum after 10–30 min and is gradually degraded thereafter (Furch et al., 2008). Commensurate with the amount of callose deposited, PPUs reopen before the sieve pores do (Furch et al., 2007, 2008, 2010). Its occurrence in systematically distant families suggests that dual occlusion is widespread and functions to safeguard sieve tube contents. In this safety design, protein occlusion guarantees quick sieve plate sealing, which bridges the time until callose deposition is completed (van Bel et al., 2011a). Although evidence in favour of a dual occlusion mechanisms is growing, numerous questions have to be addressed, in particular concerning the diversity of occlusion mechanisms. (i) It is unclear if the Ca2+ thresholds for protein reactivity and callose synthesis are different (Fig.  6E, F). There might be a vast spectrum of Ca2+ thresholds needed for diverse occlusion mechanisms (Furch et  al., 2007, 2008, 2009, 2010), which in particular pertains to VPs which are positively related to the stimulus strength (Stahlberg and Cosgrove, 1997; Stahlberg et  al., 2006). (ii) Most probably, not every protein clogging event in sieve tubes is Ca2+ dependent. Forisomes comprise SEO proteins (Pélissier et al., 2008), a widespread family among dicotyledons (Rüping et al., 2010; Anstead et  al., 2012; Ernst et  al., 2012; Jekat et  al., 2012). SEO proteins are claimed to be Ca2+ binding in general (Ernst et al., 2012), although firm direct evidence seems to be lacking. Furthermore, PPs in cucurbit sieve tubes (Cronshaw and Sabnis, 1990; Dinant et al., 2003) do not belong to the SEO family (Ernst et al., 2012) and may react to (reactive) oxygen (species) or interact due to oxidation (Alosi et al., 1988). (iii) Since structural phloem-specific proteins are virtually absent in grasses (Eleftheriou, 1990), protein occlusion seems less important there (van Bel, 2003). However, emergence of protein plugs in gramineous sieve tubes indicates the presence of soluble proteins that are able to coagulate in response to injury (Will et  al., 2009). (iv) The capacity to remove Ca2+ from sieve elements may be decisive for the reversibility of occlusion and achieved by a battery of Ca2+ efflux facilitators (Kudla et al., 2010; Huda et al., 2013). Their activities bear strongly on the mechanisms of Ca2+ homeostasis in sieve elements. Ca2+ efflux facilitators such as Ca2+ ATPases at the plasma membrane and the endomembranes, as well as Ca2+ exchangers (McAinsh and Pittman, 2009; Kudla et al., 2010) could modulate Ca2+ signatures and are responsible for cytoplasmic Ca2+ homeostasis. It has been speculated that soluble Ca2+-binding proteins fine-tune and shape Ca2+ transients during signalling (McAinsh and Pittman, 2009). Given the wealth of soluble proteins in sieve tube sap (e.g. Nakamura et al., 1993; Lin et al., 2009; Gaupels et al., 2012; Dinant and Lucas, 2013), this type of Ca2+ sequestration may be of paramount importance for Ca2+ buffering in the sieve element. Ca2+-binding proteins associated with the cytoskeleton could also act as modulators of Ca2+ signatures (Malho et al., 1998). Relationships between Ca2+-mediated sieve element occlusion and pathogenic attacks Apart from the involvement of Ca2+-permeable channels in the long-distance signalling of pathogenic infections and the implementation of defence mechanisms (e.g. Lecourieux et al., 2006; Cheval et al., 2013), Ca2+ channels are also locally and directly involved in putting up anti-pathogenic barriers. As reported below, penetration of aphid stylets and the presence of phytoplasmas elicit sieve tube occlusion mechanisms related to Ca2+ influx. Aphid infestation Sieve elements in Lupinus albus and V. faba occlude instantaneously by virtue of forisome dispersion in response to micropipette impalement (tip diameter 1 μm) due to Ca2+ influx (van Bel and van Rijen, 1994; Knoblauch and van Bel, 1998). After impalement, cell wall Ca2+ will diffuse into the sieve element via the wound edges created by the micropipette (Fig.  7A; Will and van Bel, 2006). Concomitantly, sieve element turgor is dissipated by the large micropipette volume, which may affect the gating of mechano-sensitive Ca2+-permeable channels (Fig. 7A). Remarkably, aphid stylets which have a similar tip diameter do not cause forisome dispersion (Walker and Medina-Ortega, 2012). Pressure loss into the stylet is minimal due to the minute volume and sealing of the wound edges by gel saliva (Miles, 1999; Tjallingii, 2006; Will et  al., 2013) so that passive Ca2+ influx and activation of Ca2+-permeable channels are constrained (Fig. 7A; Will and van Bel, 2006). When sieve element occlusion is triggered by remote burning, feeding aphids react within a few seconds (Will et al., 2007; Furch et al., 2010). Several aphid species switch from ingestion to secretion of watery saliva probably to counteract sieve tube occlusion by Ca2+ binding (Fig. 7B–D; Will et al., 2007, 2009). In vitro studies using forisomes (Will et al., 2007), biochemical techniques (Will et  al., 2007), and proteomics (Carolan et  al., 2009; Rao et  al., 2013)  confirmed that watery saliva contains Ca2+-binding proteins. In vitro, dispersion of Ca2+-treated forisomes was reversed by addition of the Ca2+ chelator EDTA or watery saliva concentrate from the aphid species Megoura viciae (Fig. 7E–I; Will et al., 2007). As a supplementary function, Ca2+-binding proteins may interfere with Ca2+-mediated defence and signalling mechanisms (Will and van Bel, 2008). Up-regulation of genes encoding calmodulin, calmodulin-like proteins, calcium-dependent protein kinases, and calcium-binding reticulin in response to infestation (Coppola et  al., 2013) suggests a major role for Ca2+ in plant defence against aphids. The fact that Ca2+-binding proteins have been identified in the phloem-feeding green rice leafhopper (Hattori et  al., 2012) indicates that comparable strategies for suppression of plant defence may exist in diverse hemipteran families. Thus far, however, evidence in favour of in vivo suppression of Ca2+-induced sieve element occlusion by aphid saliva is lacking. On the contrary, forisome reversibility after leaf tip burning was found to be similar in distant sieve tubes with or without aphid stylet penetration in intact broadbean plants (Medina-Ortega and Walker, 2013). Infection by phytoplasms Phytoplasmas are frequently transmitted to plants by phloem-feeding leafhoppers and distributed via the sieve tubes (Christensen et  al., 2005; McLean and Hogenhout, 2013) which become occluded in response to the infection (Braun and Sinclair, 1978; Kartte and Seemüller, 1991; Musetti and Favali, 1999). Phytoplasma-infected sieve tubes in V.  faba contain consistently dispersed forisomes (Fig. 7J–M) hinting at Ca2+ levels appreciably higher than in healthy sieve tubes (Musetti et al., 2013). The Ca2+ concentration is indeed higher in infected sieve tubes (Fig. 7N, O). Moreover, the sieve tubes are sealed with thick deposits of callose (Fig. 7P, Q; Musetti et al., 2013). Undoubtedly, phytoplasmas induce Ca2+ influx with inherent consequences for forisome dispersion and callose synthesis. Thus far, it is unclear if sieve element occlusion is part of the plant’s strategy against phytoplasma spread or if phytoplasmas induce and explore symplasmic isolation for undisturbed multiplication. Physiological and genetic impact of EPWs A diversity of physiological and genetic remote responses to EPWs have been reported, many of which are likely to be due to Ca2+ influx (Kudla et al., 2010). EPWs induce the expression of the proteinase inhibitor gene (pin2; Wildon et  al., 1992; Pena-Cortes et  al., 1995; Stankovic and Davies, 1997) and other genes (Davies, 2004) in distant parts of tomato plants. EPW propagation and gene expression are linked by the fact that Ca2+ influx and a consequent, transient increase in cytosolic Ca2+ is required for pin2 gene expression (Fisahn et al., 2004). Interestingly, the level of IP3, a second messenger potentially responsible for Ca2+ liberation from ER stacks (Gilroy et al., 1990; Krol et al., 2003, 2004), abruptly increases after EPW passage (Davies, 2004). Furthermore, touch-triggered EPWs evoke an arsenal of transcriptional downstream responses pertinent to 2.5% of the genes (Braam, 2005) including the enhanced expression of Ca2+-binding proteins (Lee et  al., 2005). These data strongly suggests a relationship between EPWs, Ca2+ influx, and remote effects on gene expression. A range of physiological responses to EPWs have been documented (Retivin et al., 1997; Fromm and Lautner, 2012). A  Ca2+-controlled shutdown of proton pumps (Kinoshita et  al., 1995; Hafke et  al., 2013) during and after passage of a VP triggered by heating led to a transient decrease in the cytosolic pH from 7.0 to 6.4 and a concomitant increase of the apoplasmic pH from 4.5 to 5.2 (Grams et al., 2009). The lowered cytosolic pH, in turn, may bring about depressed CO2 uptake rates and reduced photosynthetic quantum yields (Koziolek et  al., 2004; Lautner et  al., 2005; Grams et  al., 2009). In keeping with these observations, occasional sudden changes in osmotic potential of sieve tubes (Knoblauch et al., 2001) or pressure waves may cause VPs involved in the restoration of the source–sink balance. Remarkably, APs triggered by sudden flooding of drought-stressed roots produced the opposite effect: they induced an increase in stomatal conductance and photosynthesis (Grams et al., 2007) in the absence of appreciable pH changes. Responses of distant cells to EPWs may depend on two major factors: the Ca2+ signatures in question and the equipment of the recipient cells. Ca2+ signatures (McAinsh and Pittman, 2009) will depend on the Ca2+-permeable channels involved, their cellular location, their Ca2+-mediated interaction, the available Ca2+ binding components, and the final Ca2+ compartmentation. There is a wealth of data on Ca2+ receptors and downstream signalling cascades (e.g. Dodd et al., 2010), and it seems that Ca2+ influx is of crucial importance for initiation of numerous cascades (Sanders et  al., 2002; Kudla et al., 2010). The diverse Ca2+ reactivity of various cell types has not yet been studied in detail, but there are obvious differences. Differential Ca2+ responsiveness of diverse cell types along the phloem pathway is exemplified by: sieve plate occlusion in sieve elements (Furch et  al., 2007, 2009), production of NO in companion cells (Gaupels et al., 2008), systemin production in phloem parenchyma cells (Narvaez-Vazquez and Ryan, 2004), and massive water release in pulvinar flexor cells (Fleurat-Lessard and Bonnemain, 1978). It will be fascinating to explore further the impact of EPWs on gene expression, metabolism, and physiology of distant conductive elements and adjoining cells. Speculations on whole-plant effects of EPW-modulated Ca2+ waves Symplasmic organization of phloem strands changes in response to Ca2+ fluxes Ca2+ influx during APs mostly is insufficient to induce forisome dispersion and sieve element occlusion (Fig. 4J). After passage of EPWs of sufficient strength, namely VPs or, in particular, a combination of VPs and APs (Fig. 4J), Ca2+ levels exceed an activation threshold. The resultant occlusion of the intercellular corridors may impose a transient symplasmic reorganization of the sieve tube tracks (Fig.  8). Apart from the proven occlusion of sieve plates and PPUs (Figs. 4,6), elevated Ca2+ levels in the adjoining parenchyma cells may induce the deposition of callose collars around their plasmodesmata, as demonstrated for several tissues (e.g. Tucker, 1990; Kauss and Jeblick, 1991; Radford et al., 1998; Holdaway-Clarke et  al., 2000; Sivaguru et  al., 2000, 2005; Michard et  al., 2011). Formation of callose deposits blocking photoassimilate loading by sieve tubes in response to APs (Fromm et al., 2013) is in agreement with this concept. Without the usual interaction with their neighbours, occlusion of symplasmic contacts would render vascular cells temporarily autonomous units (Figs. 8,9). Under these conditions, vascular cells may be able to switch to other cascades implementing more discrete metabolic or genetic programmes. The effects of (transient) plasmodesmal closure on cell autonomy were demonstrated for the differentiation of the stomatal apparatus (Palevitz and Hepler, 1985), the divergent development of the sieve element and companion cell (van Bel and van Rijen, 1994), the formation of symplasmic domains (Ehlers et al., 1999), the synchronization of metabolic activity (Ehlers and Kollmann, 2000), and the explosive elongation of cotton hair cells (Ruan et  al., 2001). As soon as the lifelines (PPUs) between companion cells and sieve elements have been restored, Ca2+-induced products can be released into the sieve elements and translocated to target cells when the sieve pores become re-opened (van Bel et al., 2011a). Ca2+-triggered systemic signalling occurs in partly overlapping waves Lateral transfer of EPWs, either focused in the pulvini (Fleurat-Lessard and Bonnemain, 1978) or distributed along the entire pathway (Rhodes et  al., 1996), may reflect a fundamental difference between EPWs in animals and plants. Instead of the minor ion displacements occurring in animals, gating of ion channels causes massive ion displacement in plants (Pyatygin et  al., 2008). Apart from the regulation of Ca2+ influx, ion displacement in plants may strongly contribute to ion homeostasis (e.g. Mummert and Gradmann, 1991; Trebacz et al., 1994; Zimmermann and Felle, 2009). Dissemination of electrical signalling implies that both cells along the phloem pathway and those at the termini of the phloem track are targets for EPWs. The multitude of potential combinations of Ca2+ influx and its differential effects on diverse cell types potentiate the complexity of the responses and provide an endless wealth of possibilities (Kudla et  al., 2010; Dempsey and Klessig, 2012) exemplified by the ‘myriad plant responses’ to herbivores (Walling, 2000). We explore the possibility—as advanced before in a less elaborate way (van Bel and Ehlers, 2005)—that phloem-borne signalling passes through partly overlapping waves which are distinct in time scale, site of origin, and nature (Fig.  9). At the forefront of EPWs, Ca2+ ions are released into sieve elements which may readily attach to constitutive Ca2+-binding proteins in the sieve tube sap such as Ca2+-dependent protein kinases (Nakamura et  al., 1993; Yoo et  al., 2002; Gaupels et al., 2012). Thus, the first wave of signals (time scale: seconds to minutes to arrive in target cells) may include free Ca2+ ions accompanied by Ca2+-activated or Ca2+-binding proteins. As a result, Ca2+ signatures induce proactive responses to imminent changes. The signatures will depend on the stimulus; that is, disparate signatures are obtained from diverse Ca2+permeable channels which are functionally linked with different cytoskeleton components (Mazars et al., 1997; Thion et al., 1998; Wang et al., 2004; Zhang et al., 2007). A second wave of signals (time scale: minutes to hours) may comprise compounds from the vascular parenchyma that are readily manufactured under the control of Ca2+ influx. If the EPW is accompanied by symplasmic reorganization, the longer residence time could make the stagnant contents of sieve elements into reaction vessels for Ca2+ binding to constitutive sieve element proteins, and vascular cells may follow alternative signalling cascades as argued above. Thus, without sieve element occlusion, the compounds released into the sieve tubes for further translocation may differ from those released after relief of symplasmic re-organization (Fig.  9). During this second stage, various parallel cascades may be initiated by Ca2+ influx. For instance, calmodulin-like and calmodulin (McCormack and Braam, 2003; Lee et al., 2005; McCormack et  al., 2005), as well as other specific Ca2+binding proteins (White and Broadley, 2003; Kudla et  al., 2010) are attached to the cytoskeleton (Malho et al., 1998). In this way, information conferred by Ca2+ signatures is decoded and transformed into protein–protein interactions, resulting in Ca2+-dependent phosphorylation cascades like transcriptional responses that lead to downstream reactions (Luan et al., 2002; Sanders et al., 2002; Kudla et al., 2010). Whether Ca2+ is directly related to the synthesis of jasmonic acid (Fisahn et al., 2004) and/or salicylic acid is uncertain, but there seems little doubt that Ca2+ ions are engaged in the action of jasmonic acid (Munemasa et al., 2011) and salicylic acid (Du et al., 2009; Boursiac et al., 2010). In addition, cytosolic Ca2+ elevation is linked to downstream nitric oxide production, as shown for companion cells (Gaupels et al., 2008) via the intervention of calmodulin-(like) proteins (Ma et al., 2008). The third wave (time scale: hours) would encompass longterm implementation of Ca2+ effects exemplified by the production of various types of RNA (Kehr and Buhtz, 2013), proteins (Lin et  al., 2009; Dinant and Lucas, 2013), and even lipidic substances (Guelette et al., 2012) present in the sieve tube sap. For the impact of Ca2+ signals on the production of macromolecules, the reader is referred to an excellent review (Kudla et al., 2010), but a few examples are given here. Ca2+ signals are converted into transcriptional responses for a fair number of genes (Lee et al., 2005; Kaplan et al., 2006) which may comprise ~3% of the protein-coding genes in Arabidopsis (Kudla et al., 2010). Many of these expression responses depend on Ca2+ regulation of the transcription factors (e.g. Finkler et  al., 2007). As an interesting note in the present context, one of these transcription factors interacts with the promoter of AtEDS1, a regulator of salicylic acid synthesis (Du et al., 2009). Macromolecules produced in the vascular cells and released into the sieve tube sap via PPUs (Lucas et al., 2001; Chen and Kim, 2006; Lough and Lucas, 2006; Ding and Itaya, 2007; Lin et  al., 2009) might find their way to target cells by molecular tagging (zip codes) so that compounds required for local and remote use can be distinguished (van Bel et  al., 2011b). In this way, macromolecules are recognized to remain within the sieve element into which they had been released or move either to companion cells along the pathway (Fisher et al., 1992; Golecki et al., 1999) or to sink cells. Interactions on the interface between ER stacks and the sieve element cytoskeleton may play a crucial part in the distribution of macromolecules inside the sieve element and delivery of macromolecules into the sieve tube sap. Presumably, some of the macromolecules are back-trafficked into companion cells by the aid of non-cell autonomous agents (Schulz, 1999; Itaya et al., 2000; Lucas et al., 2009). This ‘molecular hopping’ (van Bel et al., 2011b) may provide a complex basis for amplification or attenuation of systemic signals. Macromolecules enter sink cells via permanently widened plasmodesmata (Fisher and Cash-Clarke, 2000), each of which may demand specific entrance codes (Foster et al., 2002). Concluding remarks This review is a plea for further research on the link between EPWs and chemical systemic signalling. It appears to be worth investigating if and to what extent EPWs provide a common basis for the rapid distribution of Ca2+ signals. A  limited number of studies demonstrate the immense and remote effects of EPWs on the genetics and physiology of plants. There may be a few prime targets for investigation. (i) Which Ca2+-permeable channels are involved in the propagation of EPWs and the processing of electrical information in vascular cells? (ii) Is there an impact of a temporary symplasmic organiza tion on the production of signalling substances? (iii) Do the Ca2+ signatures and the resultant cascades depend on the nature of electrical signalling; for example, is there a difference between Ca2+ signatures induced by VPs or APs? 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Aart J. E. van Bel, Alexandra C. U. Furch, Torsten Will, Stefanie V. Buxa, Rita Musetti, Jens B. Hafke. Spread the news: systemic dissemination and local impact of Ca2+ signals along the phloem pathway, Journal of Experimental Botany, 2014, 1761-1787, DOI: 10.1093/jxb/ert425