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Published on September 20, 2007; 10.1104/pp.107.105940

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Received July 24, 2007
Accepted September 15, 2007

Isolation of Functional Sieve-Element Protoplasts

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, Senckenbergstrasse 17, D-35390 Giessen, Germany

* Corresponding author; email: Aart.v.Bel{at}bot1.bio.uni-giessen.de.

Sieve-element protoplasts (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, localised in phloem cells of Arabidopsis (Marten et al., 1999; Deeken et al., 2002). 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 signalling.




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