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

ABSTRACT

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.

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 (Webb et al., 1996; Ng and McAinsh, 2003; McAinsh and Pitman, 2009; Kudla et al., 2010). These signatures are decoded and relayed by signalling molecules into a multitude of downstream events.

Ca²⁺ influx is associated with a depolarization that can propagate electrotonically via plasmodesmata towards other cells (e.g. Brinckmann and Lüttge, 1974; Overall and Gunning 1982; van Bel and van Rijen, 1994; Holdaway-Clarke et al., 1996). The depolarization range is mostly limited to a few cells due to the electrical resistance of plasma membranes and plasmodesmata (Overal and Gunning, 1982). However, influx of Ca²⁺ can initiate so-called receptor potentials which trigger action potentials (e.g. Benolken and Jacobson, 1970; Williams and Pickard, 1972b), provided that adjacent cells have become specialized in creating self-amplifying potential waves that can cover appreciable distances. Examples of non-vascular propagation of electropotential waves (EPWs) are the pollen-triggered (Sinyukin and Britikov, 1967) or temperature-triggered effects (Fromm et al., 1995) on ovary respiration. During evolution, conduction of EPWs has been perfected in sieve tubes owing to a reduced resistance of the symplasmic connections in the transverse walls to facilitate mass flow through sieve pores (Fromm and Lautner, 2012; Hafke and van Bel, 2013). The increased pore diameters, the high saline content, and the ability to propagate self-amplifying potential waves made sieve tubes ideal pathways for electrical long-distance signalling (Samejima and Sibaoka, 1983; Fromm, 1991; Wildon et al., 1992; Fromm and Spanswick, 1993; Rhodes et al., 1996; Fromm and Lautner, 2006; Furch et al., 2007).

As mentioned above, Ca²⁺ influx triggers a multidimensional web of intracellular cascades leading to changes in metabolism and/or gene expression, thereby conferring adaptive reactions (Sanders et al., 2002; White and Broadley, 2003; McAinsh and Pittman, 2009; Kudla et al., 2010). Because visibly non-excitable plants also dispose of the capacity for electrical systemic signalling (Fromm and Lautner, 2012; Hafke and van Bel, 2013; Zimmermann and Mithöfer, 2013), the question arises of whether EPWs have universal functions—associated with Ca²⁺ influx—in inter-organ communication. Perhaps electrical long-distance signals in plants primarily serve to distribute Ca²⁺ signals rapidly (e.g. Pyatygin et al., 2008; van Bel et al., 2011a), providing a common platform for several chemical forms of long-distance signalling. In this manner, EPWs may act as vehicles for distribution of Ca²⁺ as the ‘lead currency’ in plant signalling (Kudla et al., 2010). Therefore, we will explore the impact of EPWs on the systemic distribution of Ca²⁺ influx and the inherent responses of diverse cell types along and at the termini of the phloem pathway.

Electropotential waves

Short descriptions and definitions

Diverse types of EPWs propagate along the vascular pathways (Pickard, 1973; Rhodes et al., 1996; Stahlberg and Cosgrove, 1997; Davies, 2006; Davies and Stankovic, 2006; Stahlberg et al., 2006; Furch et al., 2007; Hafke et al., 2009; Zimmermann et al., 2009; Zimmermann and Mithöfer, 2013). Action potentials (APs) are transmitted more rapidly (0.5–10cm s^–1) than variation potentials (0.1–1.0cm s^–1) (VPs) (Stahlberg and Cosgrove, 1997; Fromm and Lautner, 2007), while the propagation velocity of the recently proposed system potentials (SPs; Zimmermann et al., 2009) is 5–6cm min^–1.

Strong injuries such as burning trigger both APs and VPs, resulting in composite signals (Houwink, 1935; van Sambeek and Pickard, 1976; Roblin, 1985; Roblin and Bonnemain, 1985; Stankovic et al., 1997, 1998; Hlavackova et al., 2006), when recorded in the close vicinity of the burning site (Davies and Stankovic, 2006; Furch et al., 2007, 2009; Hafke et al., 2009). With increasing distance, the signals drift apart due to the different propagation velocities (Davies et al., 1991; Stankovic et al., 1998; Davies and Stankovic, 2006; Furch et al., 2010), which implies that EPW profiles can differ between various locations along the propagation pathway.

A proper assessment of EPW profiles requires some further awareness of the caveats in recording. Overlapping APs and VPs giving rise to ‘mixed’ EPWs often impede proper signal analysis of the AP and VP component. Furthermore, EPW types are distinguished on the basis of kinetic parameters such as amplitude, duration, and profile (e.g. Stahlberg et al., 2006). However, kinetic features alone (Stankovic et al., 1997; Dziubinska et al., 2001) might be a doubtful criterion for a distinction between APs and VPs, because VPs often mimick AP kinetics (Stahlberg et al., 2006; Furch et al., 2008; Fromm et al., 2013). For instance, cutting often causes VPs that have a close similarity to APs, while crushing results in long-lasting VPs (van Sambeek and Pickard, 1976).

Action potentials

APs are mostly associated with non-invasive stimuli (Stahlberg et al., 2006; Trebacz et al., 2006). Examples of AP-induced events propagating along sieve tubes are the fast thigmonastic movements in touch-sensitive plants (Sibaoka, 1969, 1991), the touch-induced closure of the Venus flytrap (Sibaoka, 1966; Hodick and Sievers, 1989), the movements of the waterwheel plant (Iijima and Sibaoka, 1981), and the complex bending of sundew leaf tentacles (Williams and Pickard, 1972a, b, 1974; Williams and Spanswick, 1976). APs are also induced by dipping leaves in ice-cold water (Fromm, 1991; Fromm and Bauer, 1994) as well as by drought-mimicking treatments of roots (Fromm and Fei, 1998).

APs are characterized by spike-like changes of the membrane potential (Stahlberg and Cosgrove, 1997). A period of >1min between the onset of the depolarization and completion of the repolarization usually identifies an EPW as an AP (Pickard, 1973; Fromm, 1991). As in neurons, APs seem to be all-or-nothing events (Fromm and Spanswick, 1993; Pyatygin et al., 2008). Below a critical membrane potential threshold, APs or self-amplifying depolarizations start their propagation with defined amplitudes and velocities independent of the stimulus strength (Fromm, 1991; Zawadzki et al., 1991; Wildon et al., 1992; Fromm and Spanswick, 1993; Davies and Stankovic, 2006; Fromm and Lautner, 2007).

AP kinetics depend on the integrated activities of voltage-dependent ion channels in the plasma membrane (Lunevsky et al., 1983; Okihara et al., 1991; Zawadzki et al., 1991; Fromm and Bauer, 1994; Wayne, 1994; Davies, 2004). The ionic exchanges during APs (Trebacz et al., 2006) seem to bear a strong resemblance throughout the entire plant kingdom (Iijima and Sibaoka, 1981, 1982, 1985; Hodick and Sievers, 1988; Fromm and Spanswick, 1993; Fromm and Bauer, 1994; Trebacz et al., 1994; Opritov et al., 2002; Krol et al., 2003, 2004; Fisahn et al., 2004; Felle and Zimmermann, 2007) and in some algal taxa (Lunevsky et al., 1983; Okihara, 1991; Homann and Thiel, 1994; Thiel et al., 1997).

The ion channels involved differ from those engaged in stimulus conduction along nerve cells. An initial depolarization is associated with a transient increase in Ca²⁺ concentration mediated by stimulus-triggered gating of Ca²⁺-permeable channels in the plasma membrane (Fig. 1). The elevated Ca²⁺ level brings about Cl^– efflux through Ca²⁺-dependent anion channels also located in the plasma membrane (Lunevsky et al., 1983; Tsutsui et al., 1986; Okihara et al., 1991; Homann and Thiel, 1994). Voltage-dependent K⁺ channels are gated just before the steady-state potential for Cl^– ions is reached, which causes K⁺ efflux to repolarize the membrane potential (Homann and Thiel, 1994; Thiel et al., 1997). Since full repolarization is limited by the Nernst potential of K⁺, re-establishment of the resting potential is achieved by auxiliary input of proton pumps (Kishimoto et al., 1985).

Variation potentials

VPs are mostly triggered by damaging treatments such as burning (Houwink, 1935; van Sambeek and Pickard, 1976; Roblin, 1985; Wildon et al., 1992; Stankovic et al., 1998; Mancuso, 1999; Furch et al., 2007, 2010; Hafke et al., 2009), hot water (van Sambeek and Pickard, 1976), vigorous cutting (Mancuso, 1999), strong crushing (van Sambeek and Pickard, 1976), and injury by chewing insects (Alarcon and Malone, 1994).

VPs do not obey the all-or-nothing law: the signals are positively correlated with the stimulus strength and last for from 10 s up to 30min (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 voltage-dependent 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 ligand-activated 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).

Deployment of other channels and pumps involved in EPW propagation

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 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

Ultrastructure and plasmodesmal connectivity of sieve elements

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 source-limiting 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 waves and plasmodesmal opening is unnecessary or even counter-productive.

Creation of Ca²⁺ hotspots in sieve elements

Formation and necessity of Ca²⁺ hotspots

In legumes, Ca²⁺ concentration changes in sieve elements were monitored in vivo by innate bioindicators. Their sieve elements contain so-called forisomes (Knoblauch et al., 2001), giant multimodular, striated protein bodies up to 100 μm in length (Schwan et al., 2009; Peters et al., 2010; Tuteja et al., 2010). The frequently forked forisome tips may reside in the mictoplasm (Furch et al., 2009). It is unclear, up to date if the modular ‘forisomette’ structure (Schwan et al., 2009; Tuteja et al., 2010) and the forked tips are fixation or manipulation artefacts. Upon Ca²⁺ elevation in sieve elements, forisomes disperse and reach up to six times their contracted volume (Knoblauch et al., 2003, 2012; Peters et al., 2006), so that the sieve element is blocked and mass flow stops (Thorpe et al., 2010; Knoblauch et al., 2012). After removal of Ca²⁺, probably by Ca²⁺ pumps (Kudla et al., 2010; Huda et al., 2013), forisomes recontract to their original size (Knoblauch et al., 2003; Furch et al., 2007, 2009).

During passage of a sufficiently effective EPW (Fig. 4A), forisomes disperse instantaneously and recontract after 5–20min (Fig. 4B–E), which is indicative of sudden Ca²⁺ influx and gradual Ca²⁺ removal (Furch et al., 2007, 2009). The reversible forisome reaction has been observed in vivo and in vitro (Knoblauch et al., 2001, 2003; Furch et al., 2007, 2009). Forisomes may block sieve pores in response to damage to prevent loss of the precious sieve tube sap and invasion by phytopathogens (van Bel, 2003).

The fact that Ca²⁺ channels act as relay stations between EPWs and Ca²⁺ influx into sieve elements has been demonstrated by Ca²⁺ channel blockers (Fig 4F–I). Membrane-impermeant La³⁺ and Gd³⁺ ions as well as the membrane-permeant blockers nifedipin and verapamil prevented the usual forisome dispersion in response to distant burning (Furch et al., 2009). Inhibitory effects of nifedipin and verapamil seem to confirm that Ca²⁺ channels are located on ER membranes (see Fig. 2D, E).

In Vicia faba, Ca²⁺ concentrations in sieve tube sap collected via cut aphid stylets and in sieve element mictoplasm were in the range between 50 nmol and 100 nmol (Furch et al., 2009). These concentrations were similar to those measured in the cytoplasm of other cells (Malho et al., 1998; Trewavas, 1999), but were different from values (in the micromolar to millimolar range) reported for sieve tube sap collected in other studies (Fromm and Brauer, 1994; Brauer et al., 1998). Concentrations in the low nanomolar range indicate a classic role for Ca²⁺ as a signalling ion (White and Broadley, 2003; Kudla et al., 2010) in sieve elements (Furch et al., 2009). Remote burning triggered prolonged EPWs along sieve tubes (Furch et al., 2007, 2009; Hafke et al., 2009) that coincided with a rise to 200–500 nmol Ca²⁺ in the mictoplasm and dispersion of the forisomes (Furch et al., 2009). However, these Ca²⁺ concentrations fail to meet by a long way the 50 μmol threshold needed for in vitro forisome dispersion (Knoblauch et al., 2001, 2005; Furch et al., 2009). This discrepancy suggests special conditions in the sieve elements needed for forisome dispersion in vivo (Hafke et al., 2009).

Ca²⁺ concentrations exceeding a few hundred nanomoles might only exist temporarily at the cytoplasmic mouth of the Ca²⁺ channels (Trewavas, 1999). Such high local Ca²⁺ concentrations have been detected in animal cells (Llinas et al., 1992, 1995), but have not been documented as yet for plant cells. Fluorescent reporters (i.e. Oregon Green-488-BAPTA-1; Furch et al., 2009) used for Ca²⁺ level determination probably dissipate steep Ca²⁺ microgradients (Bolsover and Silver, 1991; Malho et al., 1998; Demuro and Parker, 2006). Thus, the absence of forisome reactions in the presence of fluorescent Ca²⁺ buffers can be interpreted as indirect evidence in favour of high concentrated Ca²⁺ microdomains (Ca²⁺ hotspots) in sieve elements (Furch et al., 2009; Hafke et al., 2009).

A prerequisite for establishing Ca²⁺ hotspots is the local aggregation of Ca²⁺-permeable channels (White and Broadley, 2003). Punctuate patterns (Fig. 4B) indicate clusters of Ca²⁺-permeable channels in the vicinity of sieve plates and around PPUs (Fig. 2B, C; Furch et al., 2009). Voltage-dependent Ca²⁺-permeable channels were also localized to ER membranes in sieve elements (Fig. 2E; Furch et al., 2009), so that voltage changes in the plasma membrane (Furch et al., 2007, 2009) conducted by protein anchors between the plasma membrane and ER (Ehlers et al., 2000) might activate voltage-dependent channels at the ER stacks (Klüsener et al., 1995; Klüsener and Weiler, 1999; McAinsh and Pittman, 2009).

Ca²⁺ release from ER cisternae (Fig. 4J) implies that these are intracellular Ca²⁺ sequestration compartments (Buchen et al., 1983; Sjolund and Shih, 1983). It is uncertain to what extent second messengers such as the inositol phosphates IP₃ (inositol trisphosphate; Gilroy et al., 1990) and IP₆ (myo-inositol hexakisphosphate; Lemtiri-Chlieh et al., 2003), or cADPR (cyclic ADP-ribose; Lecki et al., 1998) play a role in Ca²⁺ release from the ER stacks in sieve elements. Inhibition of APs by the phospholipase C inhibitor neomycin—– phospholipase C is involved in the formation of IP₃—in a variety of plant species (Krol et al., 2003, 2004) hints at the engagement of second messengers.

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 10–20min (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 3h (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 8mM 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 V_max 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 10–30min 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 long-term 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.

• Which Ca²⁺-permeable channels are involved in the propagation of EPWs and the processing of electrical information in vascular cells?

• Is there an impact of a temporary symplasmic organization on the production of signalling substances?

• 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?

• Do the Ca²⁺ signatures and the resultant cascades depend on the receptor or target cell types; that is, are there cell-specific responses to identical Ca²⁺ signatures?

• What is the relationship between Ca²⁺ influx and the production of various mobile signals along the pathway such as phytohormones, reactive oxygen species, lipidic substances, proteins, and RNA species?

References