Functional Sieve Element Protoplasts

Abstract

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.

Aphid watery saliva counteracts sieve-tube occlusion: a universal phenomenon?

“…Induction of sieve-plate occlusion by electropotential waves (Fromm and Bauer, 1994;Furch et al, 2007) and turgor shocks (Knoblauch et al, 2001;Hafke et al, 2007) triggers a release of Ca 2+ into the SE lumen which then induces occlusion. Furch and colleagues demonstrated that sieve-tube occlusion is reversible probably through active removal of Ca 2+ ions.…”

Global Characterization of Cell-Specific Gene Expression through Fluorescence-Activated Sorting of Nuclei

“…The third explanation is based on the knowledge that the formation of mature phloem sieve elements and companion cells from meristematic tissue involves a controlled process of programmed cell death, resulting in anucleate sieve elements, which are linked to companion cells by complex plasmodesmatal connections. Despite a recent report (Hafke et al, 2007), the production of transcriptionally functional protoplasts from anucleate tubes is self-inconsistent, and would be problematic for companion cells. Furthermore, the process of protoplast sorting discriminates in favor of protoplasts that are mechanically resilient, whereas that of nuclear sorting automatically excludes anucleate cells or cells in mitosis.…”

Plant- and stimulus-specific variations in remote-controlled sieve-tube occlusion

“…Callose deposition was restricted to a distance of about 0.5 cm from the site of cutting and results from local wound effects. In the vicinity of cutting loss of turgor pressure may cause activation of mechano-sensitive Ca 2+ -channels 21 , but reduced Ca 2+ -influx into the SE lumen is not sufficient to induce callose production further away from the cut. In conclusion, forisome dispersion may have a lower Ca 2+ threshold than callose synthesis.…”