Isolation of AtSUC2 promoter-GFP-marked companion cells for patch-clamp studies and expression profiling.
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.
Ivashikina
Natalya
N
Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany.
Deeken
Rosalia
R
Ache
Peter
P
Kranz
Erhard
E
Pommerrenig
Benjamin
B
Sauer
Norbert
N
Hedrich
Rainer
R
D017360:Arabidopsis / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D017931:DNA Primers; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000502:physiology; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011523:Protoplasts / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D005819:Genetic Markers; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; C078729:sucrose transport protein, plant; D049452:Green Fluorescent Proteins; D011188:Potassium