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We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant ( Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 m m NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant.

Potassium, the most abundant cation in plant cells, plays essential roles in maintaining the membrane potential, ion homeostasis, in enzyme activation, signal transduction, and many other physiological processes. The molecular mechanism of potassium uptake by plant roots, loading, and transport within plants has been a focus of study during the last decade. Following the isolation of plant potassium channels by yeast ( Saccharomyces cerevisiae) complementation (; ), several proteins capable of transporting potassium have been reported in Arabidopsis, potato ( Solanum tuberosum), barley ( Hordeum vulgare), wheat ( Triticum aestivum), and a few other species. These membrane proteins include inwardly rectifying channels (IRC) and two types of carriers: those in the HKT and HAK/KUP families, respectively (;;; ). Here, we report the characterization of transcripts encoding potassium channel homologs in the Shaker-type subfamily from a halophytic plant, the common ice plant ( Mesembryanthemum crystallinum). We focus on transcript behavior, comparing plants grown under control conditions with plants stressed by high sodium chloride.

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As in animal systems, three families of plant potassium channels are known. They contain a characteristic pore-forming (P) domain conferring ion selectivity, but they differ in the number of transmembrane (TM) and P domains. A nomenclature has become established by which two subfamilies of plant Shaker-type channels became known as members of either the AKT or KAT family. All AKT- and KAT-type channels consist of six TM regions with one P region, but their electrophysiological features vary, as well as the regulation of channel activities (;; for review, see ). In contrast with the outward-rectifying animal Shaker channels (ORC), the functionally characterized KAT1 and AKT1 channels in Arabidopsis and their homologs in other species are IRC (; ).

AKT differ from the KAT type by the presence of carboxy-terminal ankyrin repeat domains, possibly for anchoring to the cytoskeleton. So far, all potassium channels show high specificity for K + over other alkali cations, making unlikely candidates for significant inadvertent sodium intrusion even at high Na + to K + ratios (; ). Mainly expressed in guard cells, KAT1 constitutes a path for potassium influx during stomatal opening.

KAT1 from Arabidopsis and KST1 from potato are activated by extracellular acidification (; ), initiated by increased activity of the plasma membrane H +-ATPase. Although earlier hypotheses, based on physiological observations, had assumed a distinction between channels as low-affinity transporters and carriers as high-affinity transporters, a more complex picture emerges at present. For example, KAT1, when expressed in Arabidopsis guard cells or yeast, mediates K + uptake from media with as low as 10 μ m of external K +. The expression of a second family member, KAT2, has been detected in Arabidopsis leaf mesophyll cells.

At high abundance, AKT1 is predominantly expressed in Arabidopsis roots (; ). Mutant plants with a T-DNA insertion in Akt1 grow poorly on media with potassium concentrations in the micromolar range in comparison with wild type (; ), suggesting that AKT1-type channels can function in the high-affinity range. The expression of other family members, AKT2, has been located most strongly to the leaves , and AKT3 to leaf phloem. The latter seems to be responsible for phloem transport of potassium. Here we report the characterization of three potassium channel transcripts, Mkt1, Mkt2, and Kmt1, from the halophyte common ice plant, which are homologs of the Arabidopsis IRC Akt1, Akt2/3, and Kat1.

We analyzed tissue specificity of these genes at the transcript level, their regulated expression under salt stress, and protein amounts for MKT1. Potassium Channel Transcript Isolation Three members of the AKT/KAT subfamily of inward-rectifying potassium channels were isolated from the common ice plant by a combination of reverse transcription (RT)-PCR amplification with degenerate primers and cDNA library screening (Figs. Partial cDNAs of Mkt1 were obtained by RT-PCR from degenerate primers and 5′-RACE amplifications, and full-length cDNAs were isolated from cDNA libraries. The deduced amino acid sequence of the ice plant Akt1 homolog, Mkt1 (accession no. ), specifies 870 residues (Fig.

Partial cDNAs for Mkt2 and Kmt1 were isolated from total RNA by RT-PCR amplification with degenerate primers ( Mkt2, accession no.; and Kmt1, accession no. All RT-PCR products and cDNAs were sequenced on both strands. Hydropathy plots of MKT1 show the putative membrane topology of this channel, and additional domains are indicated (Fig. The three ice plant sequences show similar hydrophobicity profiles with respect to TM regions.

The membrane-embedded P region between TM5 and TM6 and the voltage-sensing region in TM4 are conserved characteristics of these channels (Fig. The deduced proteins of the three sequences aligned with other AKT/KAT-type proteins as members of the subfamily characterized by six TM domains. The phylogenetic analysis (Fig.

) places the ice plant sequences among the Arabidopsis, potato, and grass Shaker-type IRC. Characterization of the ice plant Mkt1 potassium channel transcript. A, DNA and deduced protein sequence of the MKT1 potassium channels from the common ice plant (accession no. Putative functional domains are underlined. They are labeled S1 through S6: TM regions (S4 is also the voltage sensor); P, the pore-forming domain; CNBD, cyclic nucleotide-binding domain; ANKY, ankyrin repeats (underlined); and K HA, a conserved domain rich in hydrophobic and acidic residues is located at the carboxy terminal end (underlined).

A region used for the generation of oligopeptide-directed antibodies is indicated by a dotted line. B, Hydrophobicity plot of MKT1 protein according to; MacVector 6.5, Oxford Molecular Ltd., Oxford). Putative TM domains (I–VI), the pore-forming domain (P), and the K HA domain are indicated. Phylogenetic comparison of ice plant MKT1, MKT2, and KMT1 deduced amino acid sequences with sequences of other plant potassium channel proteins. The phylogenetic tree, including bootstrap values, was constructed using Clustal W AKT1,2,3 and KAT1,2—Arabidopsis; SKT1,2,3 and KST1—potato; MKT1,2 and KMT1—common ice plant (Mkt1, accession no.; Mkt2, accession no.; and Kmt1, accession no. TaAKT1—wheat; ZMK1— Zea mays. KAT2, SKT3, MKT2, and KMT1 are partial sequences.

Table compares the deduced amino acid sequences of the three ice plant genes with their counterparts in Arabidopsis (BestFit in Genetics Computer Group; Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI). The comparable regions of MKT1, MKT2, and KMT1, respectively, share 52% to 62% amino acid sequence identity, and 60% to 64% identity in nucleotide sequence (also based on best fit). The phylogenetic tree of the channels, including a number of sequences from other species (Fig.

), can rely only on few sequences at present, and is complicated by low sequence homology between the AKT and KAT subfamilies. The alignments separate the AKT1 and AKT2/3 subfamilies and place the KAT1 subfamily separate with high bootstrap values. The inclusion of the ice plant sequences places them separate from the other angiosperm orders, most likely reflecting the evolutionary separation of the ice plant (order Caryophyllales).

The inclusion of AKT1 homologs from grasses ( T. Aestivum and Z. Mays, respectively) similarly introduces low bootstrap values separating monocot and dicot AKT1 subfamily members.

Gene Complexity Southern-type hybridizations were performed with the ice plant Mkt1 cDNA. The results of copy number reconstructions (not included), based on an nDNA content of 390 Mb , indicated Mkt1 as one copy of a small gene family with two to three members (Fig. For example, when probed with a full-length Mkt1 coding sequence, genomic DNA digested with XmnI with two restriction sites in the cDNA revealed strong signals at 8.5, 2.3, 2.0, and 1.3 kb.

Weaker bands at 7.3, 6.7, 5.5, 5.0, 2.9, 1.5, 0.9, and 0.8 kb (Fig., lane 2) likely identify a different Mkt1 isoform or isoforms. Expression Patterns RNA was isolated from roots, stems, leaves, flowers, and seed capsules to obtain information on tissue-specific expression by RNA-blot hybridization. The three channel transcripts are expressed at different abundance, and each channel shows distinct tissue-specific patterns (Fig. For Mkt1 and Kmt1, 3′-UTR regions, or sequences close to the 3′ end, were used as probes, and the entire RT-PCR product was used for Mkt2. Mkt1 is mainly expressed in root tissues, similar to its counterpart Akt1 in Arabidopsis.

Mkt2 is expressed in all aerial tissues, including leaves, stems (at low abundance), flowers, and seed capsules. Kmt1 transcripts are most abundant in leaves and seed capsules. No signal was detected in leaves for Mkt1with a 3′-UTR-specific probe, whereas Mkt2 and Kmt1 are absent from roots. Tissue specificity may suggest distinct physiological roles for these potassium channel proteins. Tissue-specific expression of Mkt1, Mkt2, and Kmt1.

3′-untranslated (UTR) regions or sequence-divergent carboxy terminal regions of transcripts were used as probes. Actin was included as a loading standard. Twenty micrograms of total RNA from roots (RT), stems (ST), leaves (LF), flowers (FL), and seed pods (SP) was loaded in each lane. To describe the tissue- or cell-type-specific expression patterns in more detail, in situ hybridizations were performed for Mkt1, using RNA probes corresponding to a fragment downstream of the sixth TM region (amino acid 327–470). Hybridizations with antisense (Fig., A, C, E, and G) and sense (Fig., B, D, F, and H) probes are compared.

In root tissue, Mkt1 signals were detected in cells of the epidermis, cortical cells, and in the stele (Fig. Salt stress reduced the absolute signal intensity but did not affect cell specificity (Fig.

Signals were also detected in leaf phloem-related cells and in phloem and xylem parenchyma regions of the leaf with lower intensity signals in mesophyll cells (Fig. As in the roots, the intensity of the signals declined under salt stress conditions (Fig.

We consider the signals in leaves to be caused by a leaf-specific homolog of the root-specific Mkt1. It is unlikely that this form is Mkt2 or Kmt1 because of low sequence identity between Mkt1 and either Mkt2 (60.5%) or Kmt1 (63.7%). Quantitative PCR analysis of root and leaf RNA with the different coding region and 3′-end-specific probes used in RNA-blot and in situ experiments showed that northern hybridization reported the presence of Mkt1 precisely, whereas the in situ data reveal another Mkt-like transcript (data not shown). Transcript Amounts during Salt Stress We were interested in transcript behavior during salt stress. Figure shows results from RNA-blot hybridizations. Mature ice plants were used, which had been exposed to 400 m m NaCl for various time periods.

RNA was probed in those tissues in which a particular transcript was most abundant (see Fig. Mkt1 in root and Mkt2 and Kmt1 in leaf tissue. Mkt1 amounts decreased drastically within 6 h following stress, confirming in situ hybridization data (Fig. ), and suggesting a switch in potassium uptake from Mkt1 to other systems in the roots (Fig. A; see “Discussion”). Root potassium content, based on earlier results (; ), declines long term under salt stress conditions to approximately 60% of the amount found in controls. Potassium content similarly decreased by about 50% in juvenile leaves in plants stressed for 3 d, compared with unstressed plants.

In contrast with whole leaf or root content, the concentration of potassium in the xylem, measured by pressure bomb extrusion of xylem sap in side shoots of the ice plant (Table ), shows no decline under stress conditions. Mkt1, Mkt2, and Kmt1 transcript expression during NaCl stress. A, Northern blots of Mkt1, Mkt2, and Kmt1.

Five- to 6-week-old plants grown in hydroponic tanks were stressed with 400 m m NaCl for the times (hours) indicated. Ten micrograms of total RNA was loaded per lane and actin served as the loading control. B, Transcript levels (concentration based on cDNA amounts generated from cloned transcripts that produced the same signal intensity as transcripts in total RNA) of Mkt1, Mkt2, and Kmt1 in stressed plants detected by semiquantitative RT-PCR. A In micromoles/injection volume (50 μL of undiluted xylem sap in each experiment from three plants; n = 6). Plants, grown hydroponically in 0.25 Hoagland nutrient solution and 6 weeks of age, were stressed for 24 h at 400 m m in the medium. Xylem sap was collected from side-shoot segments by pressure bomb extrusion.

Ions and polyols were determined by HPLC analysis. Suc was absent or below the limit of detection in the samples. Amounts of Mkt2 transcripts in leaves declined to approximately 50% within 6 h, but did not change significantly later during salt stress (Fig. In contrast, expression of Kmt1 showed a different behavior following salt stress. Kmt1 amounts, which were measured in leaf and stem tissue (only leaf shown), increased dramatically but transiently, approaching a peak at 6 h and decreasing rapidly afterward (Fig.

This behavior may indicate an important role of KMT1 in ion homeostasis in leaves during the onset and early periods of salt stress in the ice plant. Very similar patterns were indicated for the three transcripts when RT-PCR was used (Fig. As an average of three repeat experiments using semiquantitative measurements, the decline of root Mkt1 was by a factor of approximately seven, leaf Mkt2 declined to approximately 50% of the prestress value, and the increase of leaf Kmt1 amounted to an approximately 4- to 6-fold transient increase. Protein Expression and Localization Antibodies, which had been generated against an MKT1 oligopeptide sequence in a region that was variable in all alignments (underlined in Fig. ), were used to probe for the presence of MKT1 (Fig. Plasma membrane and tonoplast fractions were isolated by discontinuous Suc gradient centrifugation of membrane vesicles.

The presence of MKT1 protein was detected in the plasma membrane in roots (Fig. A), consistent with the information obtained for AKT1 , which is expressed in the root epidermis and functions as the potassium uptake machinery in roots from the soil. Like the transcript, MKT1 protein amount decreases under salt stress (Fig. Plant Material Seedlings of the common ice plant ( Mesembryanthemum crystallinum) were transferred to aerated hydroponic tanks about 2 weeks after germination. Plants were grown in 0.5× Hoagland nutrition solution with the amount of iron doubled and 3 m m potassium. For salt stress treatments, the plants were watered with one-half-strength Hoagland solution including 400 m m NaCl at the age of 4 to 5 weeks for the time periods indicated.

Unstressed control plants were grown in parallel and harvested at the same time. Nucleic Acid Isolation and Hybridizations For RNA isolations, each 4 g of frozen, ground tissues was suspended in 16 mL of extraction medium containing 100 m m Tris-HCl (pH 8.0), 150 m m NaCl, 10 m m EDTA, 2 m m aurin tricarboxylic acid, 1% (w/v) SDS, and 1% (w/v) sarcosyl. After adding an equal volume of phenol, the extracts were incubated on ice for 1 h with shaking. The aqueous phases, collected by centrifugation at 5,000 g for 10 min at 4°C, was incubated with an equal volume of isopropyl alcohol overnight at −20°C. Total precipitated nucleic acids were collected by centrifugation at 6,000 g for 20 min at 4°C and dissolved in diethyl pyrocarbonate-treated water. RNA was precipitated by incubation with an equal volume of 4 m LiCl twice at 4°C for 4 h and collected by centrifugation. Concentrations of nucleic acids were measured at 260 nm.

Genomic DNA was isolated from 2- to 3-week-old plants as described. Poly(A +) mRNA was selected from total RNA by using the PolyATract mRNA Isolation System IV (Promega, Madison, WI). For Southern-blot analyses, 10 μg of genomic DNA was completely digested with different restriction enzymes and DNA fragments were separated on 0.7% (w/v) agarose gels in 0.5× TBE buffer.

For RNA blot analysis, 10 to 20 μg of total RNA was separated on 1% (w/v) agarose gel containing 5.5% (w/v) formaldehyde. Gel treatment, transfer of nucleic acid, and the hybridizations were performed according to the instruction manual of membranes (Stratagene, La Jolla, CA). All blot hybridizations were conducted with Duralon-UVTM membranes (Stratagene) and probed with 32P-labeled DNA fragments (ICN Biomedicals, Inc., Irvine, CA).

Semiquantitative RT-PCR One to 5 μg of total RNA or 50 to 500 ng of poly(A +)-RNA was used for reverse transcription. RNA and 50 ng of oligo(dT) 12-18 in a volume of 12 μL were incubated at 70°C for 10 min. After quenching on ice, 4 μL of 5× first-strand buffer, 2 μL of 0.1 m dithiothreitol, and 1 μL of 10 m m dNTP mix were added and tubes were incubated at 42°C for 2 min. One microliter of Supercript II (Life Technologies, Inc., Rockville, MD) was added and the incubation was continued for 50 min.

The reaction was stopped by heating (70°C, 15 min) and 1 μL of ribonuclease A (2 units) was added to remove RNA complementary to the cDNA. Amplification reactions were carried out with 1 μL of the first-strand cDNA, 1× PCR buffer, 1.5 m m MgCl2, 0.2 m m dNTPs, 0.4 m m primers, and 2.5 units Taq DNA polymerase in a volume of 50 μL (Life Technologies, Inc.). After denaturation (94°C, 3 min), samples were subjected to 32 cycles of 1.5 min at 94°C, 1.5 min (55°C or temperature gradient), and 2 min at 72°C. PCR products were separated on 0.8% (w/v) agarose gels (0.5× TBE). In Situ Hybridizations Tissues from the root tip and from the second leaf pair of the plants were fixed in formaldehyde, dehydrated, and embedded as described.

The tissues were embedded in Paraplast Plus (Fisher Scientific, Pittsburgh) and 10-μm sections were mounted on poly-L-Lys-coated slides. Sense and antisense RNA transcripts labeled with digoxigenin-UTP (Boehringer, Mannheim, Germany) were synthesized by T3 and T7 RNA polymerase from linearized pBluescript harboring the cDNA from position 980 to 1,410 in the MKT1 cDNA. Transcripts were hydrolyzed to an average length of 200 nucleotides by alkaline treatment. In situ hybridizations were performed as described. Signal detection was done with antidigoxigenin alkaline phosphatase-conjugated Fab fragments (Boehringer) and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as a substrate.