Jens B. Hafke

Sieve Element Ca2+ Channels as Relay Stations between Remote Stimuli and Sieve Tube Occlusion in Vicia faba

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 Ca2+ channels and cytosolic Ca2+ elevation as a link between EPWs and forisome dispersion. Ca2+ channel antagonists affect the initial phase of the EPW as well as the prolonged plateau phase. Resting levels of sieve tube Ca2+ of ∼50 nM were independently estimated using Ca2+-selective electrodes and a Ca2+-sensitive dye. Transient changes in cytosolic Ca2+ were observed in phloem tissue in response to remote stimuli and showed profiles similar to those of EPWs. The measured elevation of Ca2+ in sieve tubes was below the threshold necessary for forisome dispersion. Therefore, forisomes need to be associated with Ca2+ release sites. We found an association between forisomes and endoplasmic reticulum (ER) at sieve plates and pore-plasmodesma units where high-affinity binding of a fluorescent Ca2+ channel blocker mapped an increased density of Ca2+ channels. In conclusion, propagation of EPWs in response to remote stimuli is linked to forisome dispersion through transiently high levels of parietal Ca2+, release of which depends on both plasma membrane and ER Ca2+ channels.

Thermodynamic Battle for Photosynthate Acquisition between Sieve Tubes and Adjoining Parenchyma in Transport Phloem

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.

Remote-controlled stop of phloem mass flow by biphasic occlusion in Cucurbita maxima

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.

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

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.

Functional Sieve Element Protoplasts

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.

Rapid cooling triggers forisome dispersion just before phloem transport stops

Phloem transport stops transiently within dicot stems that are cooled rapidly, but the cause remains unknown. Now it is known that (1) rapid cooling depolarizes cell membranes giving a transient increase in cytoplasmic Ca(2+), and (2) a rise of free calcium triggers dispersion of forisomes, which then occlude sieve elements (SEs) of fabacean plants. Therefore, we compared the effects of rapid chilling on SE electrophysiology, phloem transport and forisomes in Vicia faba. Forisomes dispersed after rapid cooling with a delay that was longer for slower cooling rates. Phloem transport stopped about 20 s after forisome dispersion, and then transport resumed and forisomes re-condensed within similar time frames. Transport interruption and forisome dispersion showed parallel behaviour--a cooling rate-dependent response, transience and desensitization. Chilling induced both a fast and a slow depolarization of SE membranes, the electrical signature suggesting strongly that the cause of forisome dispersion was the transient promotion of SE free calcium. This apparent block of SEs by dispersed forisomes may be assisted by other Ca(2+)-dependent sealing proteins that are present in all dicots.

(Questions)n on phloem biology. 1. Electropotential waves, Ca2+ fluxes and cellular cascades along the propagation pathway

This review explores the relationships between electrical long-distance signalling, Ca(2+) influx coincident with propagation of electropotential waves, and cellular responses to Ca(2+) influx including the consequences for sieve-tube conductivity and mass flow. Ca(2+) influx is inherent to electropotential waves and appears to constitute the key link between rapid physical signals and resultant chemical cascades in sieve tubes and adjacent cells. Members of several channel groups are likely involved the regulation of Ca(2+) levels in sieve elements. Among them are hyperpolarization-activated, depolarization-activated, and mechanosensitive Ca(2+) channels located in the plasma membrane and Ca(2+) dependent Ca(2+) channels that reside in ER-membranes of sieve elements. These channels collectively determine intracellular Ca(2+) levels in sieve elements and their neighbour cells. The latter cells react to Ca(2+) elevation by inducing diverse functional responses dependent on the cell type. If the Ca(2+) concentration in sieve elements surpasses a threshold level, dual sieve-plate occlusion by proteins and callose deposition is triggered. Occlusion is reversed when Ca(2+) levels subside. Electrical messages may regulate the degree of sieve plate hydraulic conductivity in intact plants by partial sieve-plate occlusion that has a major impact on volume flow through sieve tubes. Furthermore, complete but temporary occlusion of sieve tubes may modify mass flow patterns in intact plants.

Physiochemical Determinants of Phloem Transport

Publisher Summary This chapter identifies several questions as to the physiochemical determinants of phloem transport. There is a general quantitative unawareness with regard to physiochemical parameters. Moreover, one must be thoughtful of potentially large differences between plant species due to disparate structural/functional conditions. The steepness of the hydraulic gradient depends on the physiological activities along the whole sieve tube stretch. Thus, diversity in modes of phloem loading, release/retrieval along the transport pathway, and in modes of phloem unloading, may have a strong impact on generation of the hydraulic pressure gradient. This recognition requires detailed studies on deployment and functioning of proteins involved in shuttling osmotic equivalents and water through the sieve element/companion cell (SE/CC) plasma membrane of the successive phloem zones and the structural frame of the SE/CCs in various plant groups. Furthermore, the reciprocal feedback regulation of sources and sinks and the compensatory and buffering activities of transport phloem must be investigated on the cell-biological level.

Forisome dispersion in Vicia faba is triggered by Ca2+ hotspots created by concerted action of diverse Ca2+ channels in sieve element

Remote-controlled Ca2+ 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 Ca2+ influx into SEs. The cooperative recruitment of Ca2+ channels results in a graded responses of forisome responses culminating in full sieve-tube occlusion. Several lines of evidence are integrated into a model that links the mode and strength of the EPWs with forisome dispersion, mediated by transiently enhanced levels of local Ca2+ release dependent on both plasma membrane and ER Ca2+ channels.

Interaction of xylem and phloem during exudation and wound occlusion in Cucurbita maxima

Collection of cucurbit exudates from cut petioles has been a powerful tool for gaining knowledge on phloem sap composition without full notion of the complex exudation mechanism. Only few publications explicitly mentioned that exudates were collected from the basal side of the cut, which exudes more copiously than the apical side. This is surprising since only exudation from the apical side is supposedly driven by phloem pressure gradients. Composition of carbohydrates and pH values at both wounding sides are equal, whereas protein concentration is higher at the basal side. Apparently, exudation is far more complex than just the delivery of phloem sap. Xylem involvement is indicated by lower protein concentrations after elimination of root pressure. Moreover, dye was sucked into xylem vessels owing to relaxation of negative pressure after cutting. The lateral water efflux from the vessels increases turgor of surrounding cells including sieve elements. Simultaneously, detached parietal proteins (PP1/PP2) induce occlusion of sieve plates and cover wound surface. If root pressure is strong enough, pure xylem sap can be collected after removal of the occlusion plug at the wound surface. The present findings provide a mechanism of sap exudation in Cucurbita maxima, in which the contribution of xylem water is integrated.