Alonso Rodríguez-Navarro

The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter.

The high-affinity K+ uptake system of plants plays a crucial role in nutrition and has been the subject of extensive kinetic studies. However, major components of this system remain to be identified. We isolated a cDNA from barley roots, HvHAK1, whose translated sequence shows homology to the Escherichia coli Kup and Schwanniomyces occidentalis HAK1 K+ transporters. HvHAK1 conferred high-affinity K+ uptake to a K(+)-uptake-deficient yeast mutant exhibiting the hallmark characteristics of the high-affinity K+ uptake described for barley roots. HvHAK1 also mediated low-affinity Na+ uptake. Another cDNA (HvHAK2) encoding a polypeptide 42% identical to HvHAK1 was also isolated. Analysis of several genomes of Triticeae indicates that HvHAK1 belongs to a multigene family. Translated sequences from bacterial DNAs and Arabidopsis, rice, and possibly human cDNAs show homology to the Kup-HAK1-HvHAK1 family of K+ transporters.

A novel P‐type ATPase from yeast involved in sodium transport

The gene ENA1 was cloned by its ability to complement the Li+ sensitivity of a low Li+‐efflux strain. The nucleotide sequence of the cloned DNA fragment showed that there are two almost identical genes in tandem, and predicts that they encode P‐ATPases. Disruption of both genes originated a strain defective in Na+ and Li+ effluxes, and sensitive to Na+, to Li+ and to alkaline pH. By transformation with ENA1 the defective effluxes and tolerances were repaired.

Ion homeostasis during salt stress in plants

Recent progress has been made in the characterization of cation transporters that maintain ion homeostasis during salt stress in plants. Sodium-proton antiporters at the vacuolar (NHX1) and plasma membrane (SOS1) have been identified in Arabidopsis. SOS1 is regulated by the calcium-activated protein kinase complex SOS2-SOS3. In yeast, a transcription repressor, Sko1, mediates regulation of the sodium-pump ENA1 gene by the Hog1 MAP kinase. The recent visualization at the atomic level of the inhibitory site of sodium in the known target Hal2 has helped identify the interactions determining Na(+) toxicity.

Inventory and Functional Characterization of the HAK Potassium Transporters of Rice

Plants take up large amounts of K+ from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K+from the soil solution to its final destination is mediated by channels and transporters. To better understand K+ movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativacv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1–17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K+ influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K+ influx in yeast and in roots of K+-starved rice seedlings demonstrated the involvement of HAK transporters in root K+ uptake. We discuss that all HAK transporters may mediate K+ transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast.

ECTOPIC POTASSIUM UPTAKE IN TRK1 TRK2 MUTANTS OF SACCHAROMYCES CEREVISIAE CORRELATES WITH A HIGHLY HYPERPOLARIZED MEMBRANE POTENTIAL

Null trk1 trk2 mutants ofSaccharomyces cerevisiae exhibit a low-affinity uptake of K+ and Rb+. We show that this low-affinity Rb+ uptake is mediated by several independent transporters, and that trk1Δ cells and especially trk1Δ trk2Δcells are highly hyperpolarized. Differences in the membrane potentials were assessed for sensitivity to hygromycin B and by flow cytometric analyses of cellular DiOC6(3) fluorescence. On the basis of the latter analyses, it is proposed that Trk1p and Trk2p are involved in the control of the membrane potential, preventing excessive hyperpolarizations. K+ starvation and nitrogen starvation hyperpolarize both TRK1 TRK2 and trk1Δ trk2Δ cells, thus suggesting that other proteins, in addition to Trk1p and Trk2p, participate in the control of the membrane potential. The HAK1 K+ transporter fromSchwanniomyces occidentalis suppresses the K+-defective transport of trk1Δ trk2Δ cells but not the high hyperpolarization, and the HKT1 K+transporter from wheat suppresses both defects, in the presence of Na+. We discuss the mechanism involved in the control of the membrane potential by Trk1p and Trk2p and the causal relationship between the high membrane potential (negative inside) of trk1Δ trk2Δ cells and its ectopic transport of alkali cations.

Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae

SummaryThe ENA2 gene encoding a P-type ATPase involved in Na+ and Li+ effluxes in Saccharomyces cerevisiae has been isolated. The putative protein encoded by ENA2 differs only in thirteen amino acids from the protein encoded by ENA1/PMR2. However, ENA2 has a very low level of expression and for this reason did not confer significant Li+ tolerance on a Li+ sensitive strain. ENA1 and ENA2 are the first two units of a tandem array of four highly homologous genes with probably homologous functions.

HKT1 mediates sodium uniport in roots. Pitfalls in the expression of HKT1 in yeast.

The function of HKT1 in roots is controversial. We tackled this controversy by studying Na+ uptake in barley (Hordeum vulgare) roots, cloning the HvHKT1 gene, and expressing the HvHKT1 cDNA in yeast (Saccharomyces cerevisiae) cells. High-affinity Na+ uptake was not detected in plants growing at high K+ but appeared soon after exposing the plants to a K+-free medium. It was a uniport, insensitive to external K+ at the beginning of K+ starvation and inhibitable by K+ several hours later. The expression of HvHKT1 in yeast was Na+ (or K+) uniport, Na+-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed. The Na+ uniport function was insensitive to external K+ and mimicked the Na+ uptake carried out by the roots at the beginning of K+ starvation. The K+ uniport function only took place in yeast cells that were completely K+ starved and disappeared when internal K+ increased, which makes it unlikely that HvHKT1 mediates K+ uptake in roots. Mutation of the first in-frame AUG codon of HvHKT1 to CUC changed the uniport function into symport. The expression of the symport from either mutants or constructs keeping the first in-frame AUG took place only in K+-starved cells, while the uniport was expressed in all conditions. We discuss here that the symport occurs only in heterologous expression. It is most likely related to the K+ inhibitable Na+ uptake process of roots that heterologous systems fail to reproduce.

Diversity in expression patterns and functional properties in the rice HKT transporter family.

Plant growth under low K+ availability or salt stress requires tight control of K+ and Na+ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K+ Transporters), permeable either to K+ and Na+ or to Na+ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na+ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na+ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na+-K+ symport, Na+ uniport, or inhibited states, depending on external Na+ and K+ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K+ and Na+ accumulation in monocots.

Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae

Two Neurospora crassa genes, trk‐1 and hak‐1, encode K+ transporters that show sequence similarities to the TRK transporters described in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and to the HAK transporters described in Schwanniomyces occidentalis and barley. The N. crassa TRK1 and HAK1 transporters expressed by the corresponding cDNAs in a trk1Δ trk2Δ mutant of S. cerevisiae exhibited a high affinity for Rb+ and K+. Northern blot analysis and comparison of the kinetic characteristics of the two transporters in the trk1Δ trk2Δ mutant with the kinetic characteristics of K+ uptake in N. crassa cells allowed TRK1 to be identified as the dominant K+ transporter and HAK1 as a transporter that is only expressed when the cells are K+ starved. The HAK1 transporter showed a high concentrative capacity and is identified as the K+–H+ symporter described in N. crassa, whereas TRK1 might be a K+ uniporter. Although the co‐existence of K+ transporters of the TRK and HAK types in the same species had not been reported formerly, we discuss whether this co‐existence may be the normal situation in soil fungi.

A potassium transport mutant of Saccharomyces cerevisiae

A mutant in Saccharomyces cerevisiae required one hundred times more K+ than wild type for the same half maximal growth rate. Mutant cells and wild type cells grown at millimolar K+ did not show significant differences in Rb+ transport. In the mutant, a rapid K+ loss induced by azide or incubation (4 h) in K+-free medium decreased the Rb+ transport Km by one half; in the wild type, those treatments decreased the Rb+Km twenty and one hundred times, respectively. Mutant and wild type did not show significant differences in Na+ transport and in the Na+ inhibition of Rb+ transport, either in normal-K+ cells or in K+-starved cells. The results suggest that either two systems or one system with two interacting sites mediate K+ transport in S. cerevisiae.