Jens B. Hafke, Alexandra C.U. Furch, Marco U. Reitz and Aart J.E. van Bel*
Plant Cell Biology Research Group, Institute of General Botany, Justus-Liebig University, D–35390 Giessen, Germany
Sieve element (SE) protoplasts were liberated by exposing excisedphloem strands of Vicia faba to cell wall-degrading enzyme mixtures.Two types of SE protoplasts were found: simple protoplasts withforisome inclusions and composite twin protoplasts—twoprotoplasts intermitted by a sieve plate—of which oneprotoplast often includes a forisome. Forisomes are giant proteininclusions of SEs in Fabaceae. Membrane integrity of SE protoplastswas tested by application of CFDA, which was sequestered inthe form of carboxyfluorescein. Further evidence for membraneintactness was provided by swelling of SE protoplasts and forisomedispersion in reaction to abrupt lowering of medium osmolarity.The absence of cell wall remnants as demonstrated by negativeCalcofluor White staining allowed patch-clamp studies. At negativemembrane voltages, the current-voltage relations of the SE protoplastswere dominated by a weak inward-rectifying potassium channelthat was active at physiological membrane voltages of the SEplasma membrane. This channel had electrical properties thatare reminiscent of those of the AKT2/3 channel family, localizedin phloem cells of Arabidopsis (Arabidopsis thaliana). All inall, SE protoplasts promise to be a powerful tool in studyingthe membrane biology of SEs with inherent implications for theunderstanding of long-distance transport and signaling.
Use of fungal enzymes that degrade plant cell walls enablesthe isolation of plant cell protoplasts, which have become aninvaluable tool in plant biology. For example, protoplasts haveyielded considerable insight into plasma membrane-bound ionchannels and carbohydrate carriers in a variety of plant cellsranging from large parenchyma cells to tiny guard cells.
Due to technical barriers, sieve elements (SEs) are missingfrom other cell types that have been protoplasted successfully.A major problem is the tuning of the digesting mixture; theconventional enzyme mixes turn phloem tissues into a mash. Anotherproblem is the unequivocal identification of SE protoplasts.They easily fragment into smaller protoplasts during isolationand, therefore, can hardly be distinguished from those of other,smaller cell types.
Nevertheless, it remains tempting to isolate and identify SEprotoplasts for several reasons. The SE plasma membrane containsnumerous ion channels and carbohydrate carriers that are essentialfor sieve tube functioning (e.g. Patrick et al., 2001; van Bel,2003). Ion channels are not only meaningful for the ion householdof SEs, but also for the regulation of photoassimilate transportrates through sieve tubes (Fromm and Bauer, 1994; Ache et al.,2001; Deeken et al., 2002; van Bel and Hafke, 2005). They alsoplay a central role in long-distance signaling, such as thepropagation of electrical signals via the phloem (Fromm, 1991;Rhodes et al., 1996; Lautner et al., 2005; Furch et al., 2007).
The properties of phloem-associated potassium channels havebeen determined by heterologous expression in Xenopus oocytes(Marten et al., 1999; Bauer et al., 2000; Lacombe et al., 2000;Ache et al., 2001; Deeken et al., 2002; Phillipar et al., 2003).These potassium ion channels have been located in the phloemby in situ hybridization techniques (Marten et al., 1999; Lacombeet al., 2000; Ache et al., 2001). However, the exact cellularlocalization, ion channel densities, ion channel types, anddistribution along the phloem path are unknown. SE protoplastsisolated from the respective phloem sections would provide aunique tool for unequivocal information about these issues.The same applies to calcium channels, which have been postulatedto occur in the SE plasma membrane (Volk and Franceschi, 2000).SE protoplasts from successive phloem sections would also enableto identify, characterize, and quantify carbohydrate carriersin the SE plasma membrane at various sites along the phloemtranslocation pathway. Differential deployment of sugar carriersis likely essential for carbohydrate allocation in intact plants(e.g. Patrick et al., 2001; Kühn, 2003; Hafke et al., 2005).
Isolation of SE protoplasts may also allow the study of membranebiophysics. The mass flow through the pressurized sieve tubesmakes high demands on the physical properties of the SE plasmamembrane. Knowledge of physical properties like elasticity andfluidity and their impact on the activity of transmembrane proteinssuch as mechanosensitive channels is necessary for a betterunderstanding of the pressure regulation of phloem transport.
For isolation of SE protoplasts, we employed Vicia faba phloemsince SEs in this species contain giant calcium-sensitive proteinbodies (forisomes; Knoblauch et al., 2001, 2003) meant to actas tools for SE identification. Here, we present a method forpreparation and identification of functional SE protoplastsof V. faba. The integrity of the SE protoplasts was tested byuse of fluorochromes and osmotic treatments. Furthermore, patch-clampexperiments were carried out to investigate the functionalityof SE protoplasts.