Isolation of AtSUC2 promoter-GFP-marked companion cells for patch-clamp studies and expression profiling

Abstract

K+ channels control K+ homeostasis and the membrane potential in the sieve element/companion cell complexes. K+ channels from Arabidopsis phloem cells expressing green fluorescent protein (GFP) under the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RT-PCR. Single green fluorescent protoplasts were selected after being isolated enzymatically from vascular strands of rosette leaves. Companion cell protoplasts, which could be recognized by their nucleus, vacuole and chloroplasts, and by their expression of the phloem-specific marker genes SUC2 and AHA3, formed the basis for a cell-specific cDNA library and expressed sequence tag (EST) collection. Although we used primers for all members of the Shaker K+ channel family, we identified only AKT2, KAT1 and KCO6 transcripts. In addition, we also detected transcripts for AtPP2CA, a protein phosphatase, that interacts with AKT2/3. In line with the presence of the K+ channel transcripts, patch-clamp experiments identified distinct K+ channel types. Time-dependent inward rectifying K+ currents were activated upon hyperpolarization and were characterized by a pronounced Ca2+-sensitivity and inhibition by protons. Whole-cell inward currents were carried by single K+-selective channels with a unitary conductance of approximately 4 pS. Outward rectifying K+ channels (approximately 19 pS), with sigmoidal activation kinetics, were elicited upon depolarization. These two dominant phloem K+ channel types provide a versatile mechanism to mediate K+ fluxes required for phloem action and potassium cycling.