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

Abstract

These authors contributed equally to this work. SummaryK channels control K homeostasis and the membrane potential in the sieve element/companion cell complexes. K channels from Arabidopsis phloem cells expressing green¯uorescent protein (GFP) under the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RT-PCR. Single green¯uorescent 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-speci®c marker genes SUC2 and AHA3, formed the basis for a cell-speci®c cDNA library and expressed sequence tag (EST) collection. Although we used primers for all members of the Shaker K channel family, we identi®ed 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 identi®ed distinct K channel types. Time-dependent inward rectifying K currents were activated upon hyperpolarization and were characterized by a pronounced Ca 2 -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 ¯uxes required for phloem action and potassium cycling.

Increased Functional Diversity of Plant K+ Channels by Preferential Heteromerization of the Shaker-like Subunits AKT2 and KAT2

“…In protoplasts of companion cells from main leaf veins, AKT2 is expressed essentially along with KAT1, the twin of KAT2. Instantaneous current of AKT2-type was not detected in these cells, but the time-dependent inward current (KAT1 type) recorded displayed a voltage-dependent Ca 2ϩ block attributable to AKT2 subunits (47). The absence of instantaneous current might be explained by the weakness of the leak current in AKT2/KAT1 heteromers, as shown in Baizabal-Aguirre et al (35).…”

“…The two genes are co-expressed in the phloem vasculature. AKT2 is expressed in phloem throughout the whole plant (44,46,47), and KAT2 is expressed in the leaf minor vein phloem (36). AKT2 and KAT2 transcripts are also co-localized in guard cells (41).…”

Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels

“…Based on quantitative real-time PCR, leaf phloem cells express AKT2 and KAT1 Shaker genes at much higher levels than KAT2 (23). Thus, expression of the kat2 domneg construct under control of KAT2 promoter may mainly affect K in channel activity in guard cells, leaving phloem K in channels largely unaffected.…”

A Novel Calcium Binding Site in the Slow Vacuolar Cation Channel TPC1 Senses Luminal Calcium Levels

“…First-strand cDNA was prepared using the M-MLV-RT kit (Promega) and diluted for qPCR 20-fold in water. Quantification and calculation of actin and marker transcripts were performed by real-time PCR as described previously (Ivashikina et al, 2003) using the ABsolute SYBR Capillary Mix (ABgene). The following primers were used: ACT2/8fwd (59-GGTGATGGTGTGTCT-39), ACT2/ 8rev (59-ACTGAGCACAATGTTAC-39), TPC1fwd (59-CTGTGTATCTA-CTGCTC-39), TPC1rev 59-ACGAAATATGTAATGCTC-39), VHP2;1+2fwd (59-TTTAGTGCG TATATGGAC-39),VHP2;1+2rev (59-CTTCTTACTTCG-TTGACA-39), VHP1;1fwd (59-CAGTATATTCGTTAGCTT-39), VHP1;1rev (59-TGATAACTTTAGGGGTC-39), VHA-a2fwd (59-TGTTCCGATGTATC-AGA-39), and VHA-a2rev (59-CAAATGGGAATTTTACCT-39).…”