According to an established concept, injury of the phloem triggers local sieve plate occlusion including callose-mediated constriction and, possibly, protein plugging of the sieve pores. Sieve plate occlusion can also be achieved by distant stimuli, depends on the passage of electropotential waves (EPWs), and is reversible in intact plants. The time-course of the wound response was studied in sieve elements of main veins of intact Vicia faba plants using confocal and multiphoton microscopy. Only 15-45 s after burning a leaf tip, forisomes (giant protein bodies specific for legume sieve tubes) suddenly dispersed, as observed at 3-4 cm from the stimulus site. The dispersion was reversible; the forisomes had fully re-contracted 7-15 min after burning. Meanwhile, callose appeared at the sieve pores in response to the heat shock. Callose production reached a maximum after approximately 20 min and was also reversible; callose degraded over the subsequent 1-2 h. The heat induction of both modes of occlusion coincided with the passage of an EPW visualized by electrophysiology or the potential-sensitive dye RH-414. In contrast to burning, cutting of the leaf tip induced neither an EPW nor callose deposition. The data are consistent with a remote-controlled occlusion of sieve plates depending on the longitudinal propagation of an EPW releasing Ca(2+) into the sieve element lumen. It is hypothesized that forisome plugs and callose constriction are removed once the cytosolic calcium level has returned to the initial level in those sieve tubes.

Damage induces remote occlusion of sieve tubes in Vicia faba by forisome dispersion, triggered during the passage of an electropotential wave (EPW). This study addresses the role of Ca 2+ channels and cytosolic Ca 2+ elevation as a link between EPWs and forisome dispersion. Ca 2+ channel antagonists affect the initial phase of the EPW as well as the prolonged plateau phase. Resting levels of sieve tube Ca 2+ of ;50 nM were independently estimated using Ca 2+ -selective electrodes and a Ca 2+ -sensitive dye. Transient changes in cytosolic Ca 2+ were observed in phloem tissue in response to remote stimuli and showed profiles similar to those of EPWs. The measured elevation of Ca 2+ in sieve tubes was below the threshold necessary for forisome dispersion. Therefore, forisomes need to be associated with Ca 2+ release sites. We found an association between forisomes and endoplasmic reticulum (ER) at sieve plates and pore-plasmodesma units where highaffinity binding of a fluorescent Ca 2+ channel blocker mapped an increased density of Ca 2+ channels. In conclusion, propagation of EPWs in response to remote stimuli is linked to forisome dispersion through transiently high levels of parietal Ca 2+ , release of which depends on both plasma membrane and ER Ca 2+ channels.

We explored the idea of whether electropotential waves (EPWs) primarily act as vehicles for systemic spread of Ca(2+) signals. EPW-associated Ca(2+) influx may trigger generation and amplification of countless long-distance signals along the phloem pathway given the fact that gating of Ca(2+)-permeable channels is a universal response to biotic and abiotic challenges. Despite fundamental differences, both action and variation potentials are associated with a sudden Ca(2+) influx. Both EPWs probably disperse in the lateral direction, which could be of essential functional significance. A vast set of Ca(2+)-permeable channels, some of which have been localized, is required for Ca(2+)-modulated events in sieve elements. There, Ca(2+)-permeable channels are clustered and create so-called Ca(2+) 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 Ca(2+)-triggered systemic signalling occurs in partly overlapping waves. The forefront of EPWs may be accompanied by a burst of free Ca(2+) ions and Ca(2+)-binding proteins in the sieve tube sap, with a far-reaching impact on target cells. Lateral dispersion of EPWs may induce diverse Ca(2+) influx and handling patterns (Ca(2+) 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.

SummaryElectrophysiological studies using the patch-clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoe¨daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current-voltage relations of the tonoplast were dominated by a strongly inward-rectifying anion-selective channel that was active at cytoplasmic-side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage-dependent gating of a 3-pS malate-selective ion channel, which showed a half-maximal open probability at À43 mV. Also, the timeaveraged unitary currents following a step to a negative voltage corresponded to the time-dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion-selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent K m of 2.5 mM malate 2À activity), a selectivity sequence of fumarate 2À > malate 2À > Cl À > maleate 2-% citrate 3-, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 mM niflumate reversibly blocked the activity of the 3-pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 lm À2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3-pS, inward-rectifying, anion-selective channel represents the principal pathway for malate influx into the vacuole.

In transport phloem, photoassimilates escaping from the sieve tubes are released into the apoplasmic space between sieve element (SE)/companion cell (CC) complexes (SE/CCs) and phloem parenchyma cells (PPCs). For uptake respective retrieval, PPCs and SE/CCs make use of plasma membrane translocators energized by the proton motive force (PMF). Their mutual competitiveness, which essentially determines the amount of photoassimilates translocated through the sieve tubes, therefore depends on the respective PMFs. We measured the components of the PMF, membrane potential and ΔpH, of SE/CCs and PPCs in transport phloem. Membrane potentials of SE/CCs and PPCs in tissue slices as well as in intact plants fell into two categories. In the first group including apoplasmically phloem-loading species (e.g. Vicia, Solanum), the membrane potentials of the SEs are more negative than those of the PPCs. In the second group including symplasmically phloem-loading species (e.g. Cucurbita, Ocimum), membrane potentials of SEs are equal to or slightly more positive than those of PPCs. Pure sieve tube sap collected from cut aphid stylets was measured with H+-selective microelectrodes. Under our experimental conditions, pH of the sieve tube saps was around 7.5, which is comparable to the pH of cytoplasmic compartments in parenchymatous cells. In conclusion, only the membrane potential appears to be relevant for the PMF-determined competition between SE/CCs and PPCs. The findings may imply that the axial sinks along the pathway withdraw more photoassimilates from the sieve tubes in symplasmically loading species than in apoplasmically loading species.

The relationships between damage-induced electropotential waves (EPWs), sieve tube occlusion, and stop of mass flow were investigated in intact Cucurbita maxima plants. After burning leaf tips, EPWs propagating along the phloem of the main vein were recorded by extra- and intracellular microelectrodes. The respective EPW profiles (a steep hyperpolarization/depolarization peak followed by a prolonged hyperpolarization/depolarization) probably reflect merged action and variation potentials. A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring ∼10 min after burning. Early stop of mass flow, well before completion of callose deposition, pointed to an occlusion mechanism preceding callose deposition. This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids. The early occlusion is probably due to proteins, as indicated by a dramatic drop in soluble sieve element proteins and a simultaneous coagulation of sieve element proteins shortly after the burning stimulus. Mass flow resumed 30–40 min after burning, as demonstrated by carboxyfluorescein movement and aphid activities. Stop of mass flow by Ca2+-dependent occlusion mechanisms is attributed to Ca2+ influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca2+ ions.

Remote-controlled Ca(2+) influx, elicited by electropotential waves, triggers local signaling cascades in sieve elements and companion cells along the phloem of Vicia faba plants. The stimulus strength seems to be communicated by the rate and duration of Ca(2+) influx into sieve elements (SEs). The cooperative recruitment of Ca(2+) channels results in a graded response of forisome culminating in full sieve-tube occlusion. Several lines of evidence are integrated into a model that links the mode and strength of the electropotential waves (EPWs) with forisome dispersion, mediated by transiently enhanced levels of local Ca(2+) release dependent on both plasma membrane and ER Ca(2+) channels.

Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts-two protoplasts intermitted by a sieve plate-of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling.