Strategies to identify transport systems in plants.

Since the first molecular structures of plant transporters were discovered over a decade ago, considerable advances have been made in the study of plant membrane transport, but we still do not understand transport regulation. The genes encoding the transport systems in the various cell membranes are still to be identified, as are the physiological roles of most transport systems. A wide variety of complementary strategies are now available to study transport systems in plants, including forward and reverse genetics, proteomics, and in silico exploitation of the huge amount of information contained in the completely known genomic sequence of Arabidopsis.

Guard cell inward K+ channel activity in arabidopsis involves expression of the twin channel subunits KAT1 and KAT2.

Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K(+) channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K(+) influx that enables stomatal opening. We report that another Arabidopsis K(+) channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K(+) channels in guard cells. Coexpression in Xenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery.

Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co-expressed in insect cells.

KAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker-like plant K+ channels. They were biochemically characterized after expression in insect cells using recombinant baculoviruses. The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one, L-alpha-lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion-exchange and size exclusion (AKT1) techniques. The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co-expressed in insect cells. The use of the two-hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides. Because of these negative results, the hypothesis that plant K+-channel subunits are able to co-assemble without any discrimination, previously put forward based on co-expression in Xenopus oocytes of various K+-channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches. Co-localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.

A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis.

RNA gel blot and reverse transcription-polymerase chain reaction experiments were used to identify a single K(+) channel gene in Arabidopsis as expressed throughout the plant. Use of the beta-glucuronidase reporter gene revealed expression of this gene, AKT2/AKT3, in both source and sink phloem tissues. The AKT2/AKT3 gene corresponds to two previously identified cDNAs, AKT2 (reconstructed at its 5' end) and AKT3, the open reading frame of the latter being shorter at its 5' end than that of the former. Rapid amplification of cDNA ends with polymerase chain reaction and site-directed mutagenesis was performed to identify the initiation codon for AKT2 translation. All of the data are consistent with the hypothesis that the encoded polypeptide corresponds to the longest open reading frame previously identified (AKT2). Electrophysiological characterization (macroscopic and single-channel currents) of AKT2 in both Xenopus oocytes and COS cells revealed a unique gating mode and sensitivity to pH (weak inward rectification, inhibition, and increased rectification upon internal or external acidification), suggesting that AKT2 has enough functional plasticity to perform different functions in phloem tissue of source and sink organs. The plant stress hormone abscisic acid was shown to increase the amount of AKT2 transcript, suggesting a role for the AKT2 in the plant response to drought.

pH control of the plant outwardly-rectifying potassium channel SKOR.

SKOR, an Arabidopsis depolarisation-activated K+-selective channel, was expressed in Xenopus oocytes, and external and internal pH effects were analysed. Internal pH was manipulated by injections of alkaline or acidic solutions or by acid load from acetate-containing medium. An internal pH decrease from 7.4 to 7.2 induced a strong (ca. 80%) voltage-independent decrease of the macroscopic SKOR current, the macroscopic gating parameters and the single channel conductance remained unchanged. An external acidification from 7.4 to 6.4 had similar effects. It is proposed that pH changes regulate the number of channels available for activation. Sensitivity of SKOR activity to pH in the physiological range suggests that internal and external pH play a role in the regulation of K+ secretion into the xylem sap.

Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap.

SKOR, a K+ channel identified in Arabidopsis, displays the typical hydrophobic core of the Shaker channel superfamily, a cyclic nucleotide-binding domain, and an ankyrin domain. Expression in Xenopus oocytes identified SKOR as the first member of the Shaker family in plants to be endowed with outwardly rectifying properties. SKOR expression is localized in root stelar tissues. A knockout mutant shows both lower shoot K+ content and lower xylem sap K+ concentration, indicating that SKOR is involved in K+ release into the xylem sap toward the shoots. SKOR expression is strongly inhibited by the stress phytohormone abscisic acid, supporting the hypothesis that control of K+ translocation toward the shoots is part of the plant response to water stress.

Tetramerization of the AKT1 plant potassium channel involves its C-terminal cytoplasmic domain.

All plant channels identified so far show high conservation throughout the polypeptide sequence except in the ankyrin domain which is present only in those closely related to AKT1. In this study, the architecture of the AKT1 protein has been investigated. AKT1 polypeptides expressed in the baculovirus/Sf9 cells system were found to assemble into tetramers as observed with animal Shaker-like potassium channel subunits. The AKT1 C-terminal intracytoplasmic region (downstream from the transmembrane domain) alone formed tetrameric structures when expressed in Sf9 cells, revealing a tetramerization process different from that of Shaker channels. Tests of subfragments from this sequence in the two-hybrid system detected two kinds of interaction. The first, involving two identical segments (amino acids 371-516), would form a contact between subunits, probably via their putative cyclic nucleotide-binding domains. The second interaction was found between the last 81 amino acids of the protein and a region lying between the channel hydrophobic core and the putative cyclic nucleotide-binding domain. As the interacting regions are highly conserved in all known plant potassium channels, the structural organization of AKT1 is likely to extend to these channels. The significance of this model with respect to animal cyclic nucleotide-gated channels is also discussed.

The baculovirus/insect cell system as an alternative to Xenopus oocytes. First characterization of the AKT1 K+ channel from Arabidopsis thaliana.

Two plant (Arabidopsis thaliana) K+ transport systems, KAT1 and AKT1, have been expressed in insect cells (Sf9 cell line) using recombinant baculoviruses. Microscopic observation after immunogold staining revealed that the expressed AKT1 and KAT1 polypeptides were mainly associated with internal membranes, but that a minute fraction was targeted to the cell membrane. KAT1 was known, from earlier electrophysiological characterization in Xenopus oocytes, to be an inwardly rectifying voltage-gated channel highly selective for K+, while similar experiments had failed to characterize AKT1. Insect cells expressing KAT1 displayed an exogenous inwardly rectifying K+ conductance reminiscent of that described previously in Xenopus oocytes expressing KAT1. Under similar conditions, cells expressing AKT1 showed a disturbed cell membrane electrical stability that precluded electrophysiological analysis. Use of a baculovirus transfer vector designed so as to decrease the expression level allowed the first electrophysiological characterization of AKT1. The baculovirus system can thus be used as an alternative method when expression in Xenopus oocytes is unsuccessful for electrophysiological characterization of the ion channel of interest. The plant AKT1 protein has been shown in this way to be an inwardly rectifying voltage-gated channel highly selective for K+ ions and sensitive to cGMP.

Characterization of a ferritin mRNA from Arabidopsis thaliana accumulated in response to iron through an oxidative pathway independent of abscisic acid.

A ferritin cDNA, AtFer1, from seedlings of Arabidopsis thaliana has been characterized. The deduced amino acid sequence of the AtFer1 protein indicates that A. thaliana ferritin shares the same characteristics as the plant ferritin already characterized from the Leguminosae and Graminacea families: (i) it contains an additional sequence in its N-terminal part composed of two domains: a transit peptide responsible for plastid targeting and an extension peptide; (ii) amino acids that form the ferroxidase centre of H-type animal ferritin, as well as Glu residues characteristic of L-type animal ferritin, are conserved in AtFer1; (iii) the C-terminal part of the A. thaliana ferritin subunit defining the E-helix is divergent from its animal counterpart, and confirms that 4-fold-symmetry axis channels are hydrophilic in plant ferritin. Southern blot experiments indicate that AtFer1 is likely to be encoded by a unique gene in the A. thaliana genome, although a search in the NCBI dbEST database indicates that other ferritin genes, divergent from AtFer1, may exist. Iron loading of A. thaliana plantlets increased ferritin mRNA and protein abundance. In contrast to maize, the transcript abundance of a gene responding to abscisic acid (RAB18) did not increase in response to iron loading treatment, and A. thaliana ferritin mRNA abundance is not accumulated in response to a treatment with exogenous abscisic acid, at least in the culture system used in this study. In addition, iron-induced increases in ferritin mRNA abundance were the same as wild-type plants in abi1 and abi2 mutants of A. thaliana, both affected in the abscisic acid response in vegetative tissues. Increased AtFer1 transcript abundance in response to iron is inhibited by the antioxidant N-acetylcysteine. These results indicate that an oxidative pathway, independent of abscisic acid, could be responsible for the iron induction of ferritin synthesis in A. thaliana.

Tissue-specific expression of Arabidopsis AKT1 gene is consistent with a role in K+ nutrition.

AKT1, a putative inwardly directed K+ channel of Arabidopsis, restores long-term potassium uptake in a yeast mutant defective in K+ absorption. In this paper, the expression pattern of the gene encoding AKT1 is described. Northern blots indicate that AKT1 transcripts are preferentially accumulated in Arabidopsis roots. Owing to the difficulties in producing large quantities of Arabidopsis roots under hydroponic conditions, experiments were undertaken with Brassica napus, a related species. Potassium starvation experiments on B. napus plants show that changes in the K+ status of the organs do not modify AKT1 mRNA accumulation. Western blot analysis of B. napus proteins confirms the presence of AKT1 at the root plasma membrane. Tissue-specific expression directed by the Arabidopsis AKT1 gene promoter was investigated by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing an AKT1-GUS gene fusion. As determined by fluorimetric and histochemical tests, the AKT1 promoter directs preferential expression in the peripheral cell layers of root mature regions. The discrete activity found in leaves relates to leaf primordia and to small groups of cells, hydathodes, found on toothed margins of the Arabidopsis leaf lamina. These data are discussed with regard to a possible role of AKT1 in K+ nutrition of plants.

Functional expression of the plant K+ channel KAT1 in insect cells.

Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1, and KST1 have recently been identified on the molecular level. Among them, we focussed on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as -60 to -80mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-traditional modification and assembly of a green inward rectifier.

Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents.

The open reading frame from the Arabidopsis thaliana KAT1 cDNA was cloned in a transcription plasmid between the 3' and 5' untranslated regions of a beta-globin cDNA from Xenopus oocyte. The polyadenylated transcripts resulting from in vitro transcription gave rise to high levels of expression of KAT1 channel when injected in Xenopus oocytes. Upon hyperpolarization, a slow activating current could be recorded, inwardly- or outwardly-directed, depending on K+ external concentration. Predictions of the voltage-gated channel theory were shown to fit the data well. The equivalent gating charge and the half-activation potential ranged around 2 and -145 mV, respectively. KAT1 gating characteristics did not depend on K+ external concentration nor on external pH in the 5.0-7.5 range. KAT1 conductance was, however, increased (40%) when external pH was decreased from 6.5 to 5.0. The apparent affinity constant of KAT1 for K+ lay in the range 15-30 mM, at external pH 7.4. As for many K+ channels of animal cells, external caesium caused a voltage-dependent blockage of inward (but not outward) KAT1 current, whereas tetraethylammonium caused a voltage-independent block of both inward and outward KAT1 currents. In conclusion, high levels of expression made it possible to carry out the first quantitative analysis of KAT1 macroscopic currents. KAT1 channel was shown to display features similar to those of as yet uncloned inward-rectifying voltage-gated channels described in both plant cells (namely guard cells) and animal cells.

Level of expression in Xenopus oocytes affects some characteristics of a plant inward-rectifying voltage-gated K+ channel.

The plant K+ channel KAT1 shows some similarity to animal voltage-gated channels of the Shaker superfamily. Contrary to these animal counterparts, this plant channel is inwardly rectifying, being gated upon hyperpolarization. Different levels of expression of KAT1 in Xenopus oocytes could be obtained by increasing the amount of injected cRNA. The resulting KAT1 gating and sensitivity to external caesium were significantly changed. Similar findings have been published regarding animal voltage-gated channels. The present data show that plant channels may also undergo modification of their activity upon modification of their level of expression.

A test for screening monoclonal antibodies to membrane proteins based on their ability to inhibit protein reconstitution into vesicles.

The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.

Cloning and expression in yeast of a plant potassium ion transport system.

A membrane polypeptide involved in K+ transport in a higher plant was cloned by complementation of a yeast mutant defective in K+ uptake with a complementary DNA library from Arabidopsis thaliana. A 2.65-kilobase complementary DNA conferred ability to grow on media with K+ concentration in the micromolar range and to absorb K+ (or 86Rb+) at rates similar to those in wild-type yeast. The predicted amino acid sequence (838 amino acids) has three domains: a channel-forming region homologous to animal K+ channels, a cyclic nucleotide-binding site, and an ankyrin-like region.