Previous studies on guard cells of the model plant Arabidopsis have shown that the guard cell inward rectifier is composed of different K+-channel α-subunits belonging to the plant Shaker channel family with A. thaliana inward rectifying potassium channel 1 (KAT1) representing the dominant transcript (Szyroki et al., 2001). In contrast, the outward rectifier is assembled from just one channel subunit A. thaliana guard cell outward rectifying K+ channel (GORK) (Ache et al., 2000; Hosy et al., 2003). To examine the molecular basis of channel-mediated K+ transport in poplar, we searched the EST database from P. tremula × P. tremuloides (Sterky et al., 1998), for sequence homologies, to K+ transporters known. Thereby, we isolated DNA fragments with relevant homologies to Arabidopsis guard cell potassium channels (Szyroki et al., 2001). Following complete sequencing of these fragments, we identified distinct orthologues to guard-cell-expressed K+ channel of the KAT1-type, A. thaliana potassium channel (K+ transporter AKT)2/3 and GORK (for review, see Very and Sentenac, 2002). Based on ESTs and degenerated primers against conserved K+-channel domains (Ache et al., 2001), we cloned the corresponding full-length cDNAs and named the KAT1 orthologue KPT1 (GenBank Accession number AJ344623; Langer et al., 2002). KPT1 exhibited all structural features of members of the ‘green’Shaker channel family: six transmembrane domains and an amphiphilic linker – containing the K+ selectivity filter – between transmembrane segments 5 and 6 (Hedrich and Becker, 1994), and an ankyrin-like domain at the C-terminus (cf. Pratelli et al., 2002). Based on amino acid alignments with known plant K+ channels, KPT1 showed highest homologies to guard cell K+-uptake channels of the KAT1-type from Arabidopsis (Figure 4), potato, maize and grapevine (Mueller-Roeber et al., 1995; Philippar et al., 2003; Pratelli et al., 2002). So far, KAT1-type channel with ankyrin-like domains have been identified in Populus and Vitis vinifera only. In a previous study on the molecular mechanism of potassium-dependent wood growth, we identified P. tremula potassium channel (PTK)2 as an AKT2/3 orthologue and P. tremula outward rectifying potassium channel (PTORK) as a member of the A. thaliana stelar K+ outward rectifier (SKOR)/GORK family (Langer et al., 2002). KPT1, sharing closest homologies to the KAT1-like guard cell K+ channels, was found in guard-cell-enriched epidermis peels (Figure 5a). In roots and shoots, almost no KPT1-specific mRNA could be detected by quantitative real-time PCR. In the ‘guard cell’ fractions, PTK2 and PTORK co-localised with KPT1 (Figure 5b). Close inspection of the KPT1, PTK2, as well as the PTORK promoter, identified guard cell localisation elements (cf. Langer, 2003; Mueller-Roeber et al., 1995; Plesch et al., 2001). This indicates that PTK2 and PTORK, which have been functionally expressed in Xenopus oocytes and characterised as K+-selective channels before (Langer et al., 2002), mediate K+ transport in poplar guard cells just as their counterparts in Arabidopsis.
Figure 4. The KPT1 gene encodes a Shaker-like K+ channel.
(a) Schematic presentation of the predicted domains of KPT1. The ion channel consists of a transport domain (ion trans) with six linked transmembrane domains and a conserved pore region, a cyclic nucleotide-binding site (cNBD) and additionally an ankyrin domain.
(b) Alignment of KPT1 (AJ344623), SIRK (AF359521), KAT1 (NM_123993), KAT2 (NM_117939), KZM1 (AJ421640) and KST1 (X79779) S4, pore and ankyrin. Identical amino acids are labelled in white with black background; similar amino acids are shown in black on a grey background. Alignments were generated with clustalw on EBI server (http://www.ebi.ac.uk/clustalw/) and represented on Boxshade Server (http://www.ch.embnet.org/software/BOX_form.html).
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Figure 5. Expression pattern of KPT1 analysed by quantitative RT-PCR.
Quantification of KPT1 transcripts calculated by external standards relative to actin.
(a) High amounts of KPT1 transcripts were detected in guard-cell-enriched fractions only. Total RNAs isolated from sink (si) and source leaves (so), guard cells (gc), petioles (pet), xylem (xy), phloem (ph) and roots (ro) were analysed with primers specific for poplar potassium transporters.
(b) KPT1, PTORK and PTK2 expression in guard-cell-enriched epidermal fractions.
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