Molecular and functional characterization of a novel low-affinity cation transporter ( LCT 1 ) in higher plants

The transport of cations across membranes in higher plants plays an essential role in many physiological processes including mineral nutrition, cell expansion, and the transduction of environmental signals. In higher plants the coordinated expression of transport mechanisms is essential for specialized cellular processes and for adaptation to variable environmental conditions. To understand the molecular basis of cation transport in plant roots, a Triticum aestivum cDNA library was used to complement a yeast mutant deficient in potassium (K1) uptake. Two genes were cloned that complemented the mutant: HKT1 and a novel cDNA described in this report encoding a cation transporter, LCT1 (lowaffinity cation transporter). Analysis of the secondary structure of LCT1 suggests that the protein contains 8–10 transmembrane helices and a hydrophilic amino terminus containing sequences enriched in Pro, Ser, Thr, and Glu (PEST). The transporter activity was assayed using radioactive isotopes in yeast cells expressing the cDNA. LCT1 mediated low-affinity uptake of the cations Rb1 and Na1, and possibly allowed Ca21 but not Zn21 uptake. LCT1 is expressed in low abundance in wheat roots and leaves. The precise functional role of this cation transporter is not known, although the competitive inhibition of cation uptake by Ca21 has parallels to whole plant and molecular studies that have shown the important role of Ca21 in reducing Na1 uptake and ameliorating Na1 toxicity. The structure of this higher plant ion transport protein is unique and contains PEST sequences. Most plants are rooted and are therefore stationary organisms that must adapt to harsh and highly variable conditions including changes in the mineral concentrations and water content of soils (1). Acquisition of essential mineral nutrients from soils may become difficult for plants when the soil solution contains very low concentrations of available macro and micro nutrients or contains high concentrations of toxic metal ions such as Na1 or Al31. To ensure a supply of minerals from soils under a wide range of environmental conditions, multiple membrane transport mechanisms are present in the plasma and vacuole membranes of higher plants (2). The cation K1 is important in plant growth because of its vital role in cell expansion, osmoregulation, and cellular homeostasis. In higher plants recent molecular evidence (3–5) supports the model (6, 7) that plant roots contain both highand low-affinity K1 transporters that ensure an adequate supply of this cation across a wide range of soil K1 concentrations. In saline soils, when K1 availability is low, the uptake and transport of Na1 across membranes may also become important for plant growth because Na1 can partially replace K1 in some species (8). However, because the uptake of excessive amounts of Na1 is toxic to nonhalophytic plants, the compartmentation of Na1 into the vacuole is vital. Physiological mechanisms for transport of Na1 into the plant vacuole have been well described, whereas the mechanistic bases for plasma membrane Na1 uptake are only beginning to be understood. Although the precise mechanisms of plasma membrane Na1 uptake are poorly understood, Na1 tracer flux experiments have shown that Na1 uptake pathways are sensitive to inhibition by Ca21, where the presence of this divalent cation reduces one or more components of Na1 uptake (9). More than one low-affinity pathway for Na1 and K1 uptake has been suggested to exist in plants. The presence of multiple loci linked to salt tolerance and Na1 exclusion supports the idea of multiple pathways for Na1 uptake (10). The emerging molecular evidence supports the classical tracer flux data (11, 12), showing that higher plants have multiple K1 and Na1 uptake mechanisms in the plasma membrane for acquisition. Recent studies have further shown that the high-affinity K1 uptake transporter HKT1 (3) has both a high-affinity Na1–K1 coupled uptake component and a low-affinity Na1-selective uptake component at toxic extracellular Na1 concentrations that also block high-affinity K1 uptake via HKT1 (13, 14). Inward rectifying K1 channels are the only low-affinity pathway for K1 uptake in higher plants that have been identified (4, 15–17). However, tracer flux studies have indicated that multiple low-affinity K1 uptake transporters may exist in plants (12). The molecular structure as well as the mode of transport of other low-affinity Na1 and K1 uptake pathways remain unknown. The cloning of plant membrane transporters by yeast complementation has provided a potent tool that has greatly enhanced our understanding of the physiological roles and specific functional and structural characteristics of ion transporters in higher plants (18). Sucrose, potassium, ammonium, sulfate, copper, amino acid, and iron transporters have all been cloned and characterized in yeast mutants deficient in the uptake of these ions. The yeast system has been highly successful for cloning plant transporters because the complementation strategy relies on functional similarities between yeast and plant physiological mechanisms rather than structural similarities in genes. In principle, this strategy can identify transporters that have a specific substrate, such as K1, but also others that are more promiscuous, such as general transporters for a variety of cations. The application of the yeast complementation system has allowed us to identify a novel cation transporter in higher plants that could function as a component of the multiple low-affinity Na1 and K1 uptake pathways in roots. In the present study we describe the structure and function of a novel plant membrane transporter (low-affinity cation transporter, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9411079-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: LCT1, low-affinity cation transporter; RT-PCR, reverse transcription–PCR; PEST, sequences rich in Pro, Ser, Thr, and Glu residues. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF015523). †To whom reprint requests should be addressed. e-mail: dschachtman@botany.adelaide. edu.au.