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.

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.

No abstract

No abstract

No abstract

No abstract

MATERIALS AND METHODSCell Wall Isolation Procedure II. Excised roots were put in 1 mM CaCl2, 1% v/v Triton X-100. This solution was periodically renewed during 3 weeks, which was sufficient for the lysis of the cellular content to take place as seen by electronic microscopy (3).The surfactant was then eliminated by washing with 1 mm CaCI2 for 10 days. The whole preparation was done at 5 C. The resulting cell walls retained the original shape and dimensions they had in the roots.Experimental Treatments. Just before the experiments, the cell walls were repeatedly washed with 10 mM HC1 until no further displacement of cations was observed. This treatment allowed an easier control of the ionic composition of incubation media during the experiments, especially at low concentrations. The pH and the ionic concentrations of the different incubation solutions changed following the introduction of cell walls. The solutions were renewed until their ionic characteristics remained unchanged.A 12-h incubation in the last bath insured that equilibrium was reached. The cell walls were thereafter blotted on filter paper and the fresh and dry weights of the samples were measured. The experiments were done at room temperature. Assays. The ions were extracted from the dry samples with 100 mM HC1. The concentrations of K+, Ca2+, and Mg2+ in the extracts and in the incubation solutions were determined by flame spectrophotometry. Proton displacements were measured by titrations of the medium. Assays for uronic acids were made by colorimetric method (1). All contents are expressed on a dry matter weight basis.Correcting Procedure for the Contaminating Medium. The assays of the acid extract gave the sum of the ions which were fixed by the cell walls and of the ions present in the contaminating interstitial medium. The quantity of the latter was estimated by: (fresh weight of the sample -dry weight) x (concentration in the medium) and substracted from the total. This procedure avoided rinsing, which was shown to be at variance with the equilibrium measurement requirements (see under "Discussion"). (Mi')= (exp _e*T This relation is useful to convey the concept, but, for practical use, the exponential will be written as r, the so-called Donnan ratio, so the electric potential (A) no longer needs to be explained. U) r2(1) (Me) For the sake of simplicity, yi has been given the value 1. Association. If zi fixed charges bind one cation i (see under "Discussion"), the mass action law is Comparison of these theoretical values with the experimental values is achieved by converting them into contents. This can be made by using an estimate of the volume v of the polyelectrolytic phase. Hence, the parameters which should be estimated before using the model are RT, v, the jiU and the Ki values (see under "Results"). RESULTSJustification of the Measurements. The validity of the correcting procedure for the contaminating interstitial medium was checked by equilibrating Horse bean cell wails (isolate II) with an aqueous 1 mm CaCl2 solution a...

No abstract