Nobuyuki Uozumi

Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2‐1 and sas2‐2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2‐1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2‐1 mutation strongly decreased Na+ concentration in the phloem sap. It led to Na+ overaccumulation in every aerial organ (except the stem), but to Na+ underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na+ recirculation from shoots to roots, probably by mediating Na+ loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na+ from the shoot and playing a crucial role in plant tolerance to salt.

AtKUP1: An Arabidopsis Gene Encoding High-Affinity Potassium Transport Activity

Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants. We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database. The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively. Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E. coli triple mutant. In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent Km of ~22 μM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo. A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells. RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation. It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types.

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild‐type in short‐term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+‐hypersensitive in long‐term growth assays. Thus wild‐type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.

Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase

The Arabidopsis thaliana K+ channel KAT1 has been suggested to have a key role in mediating the aperture of stomata pores on the surface of plant leaves. Although the activity of KAT1 is thought to be regulated by phosphorylation, the endogenous pathway and the primary target site for this modification remained unknown. In the present study, we have demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for the Arabidopsis calcium-independent ABA (abscisic acid)-activated protein kinase SnRK2.6 (Snf1-related protein kinase 2.6). This was confirmed by LC-MS/MS (liquid chromatography tandem MS) analysis, which showed that Thr306 and Thr308 of KAT1 were modified by phosphorylation. The role of these specific residues was examined by single point mutations and measurement of KAT1 channel activities in Xenopus oocyte and yeast systems. Modification of Thr308 had minimal effect on KAT1 activity. On the other hand, modification of Thr306 reduced the K+ transport uptake activity of KAT1 in both systems, indicating that Thr306 is responsible for the functional regulation of KAT1. These results suggest that negative regulation of KAT1 activity, required for stomatal closure, probably occurs by phosphorylation of KAT1 Thr306 by the stress-activated endogenous SnRK2.6 protein kinase.

Phytosiderophore Efflux Transporters Are Crucial for Iron Acquisition in Graminaceous Plants

Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released 14C-labeled deoxymugineic acid but not 14C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.

Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants

Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K+ channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na+-coupled K+ transport. Arabidopsis AtHKT1, however, transports only Na+ in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKT1/HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K+ selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K+ permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K+ permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K+ channel selectivity filter GYG motif. The importance of such filter glycines for K+ selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2. Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na+ transporters in plants and that Na+/K+ symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K+ channels.

KtrAB and KtrCD: Two K+ Uptake Systems in Bacillus subtilis and Their Role in Adaptation to Hypertonicity

Recently, a new type of K+ transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T. Nakamura, R. Yuda, T. Unemoto, and E. P. Bakker, J. Bacteriol. 180:3491-3494, 1998). The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane. The genome sequence of Bacillus subtilis contains two genes for each of these subunits: yuaA (ktrA) and ykqB (ktrC) encode homologues to the V. alginolyticus KtrA protein, and yubG (ktrB) and ykrM (ktrD) encode homologues to the V. alginolyticus KtrB protein. We constructed gene disruption mutations in each of the four B. subtilis ktr genes and used this isogenic set of mutants for K+ uptake experiments. Preliminary K+ transport assays revealed that the KtrAB system has a moderate affinity with a Km value of approximately 1 mM for K+, while KtrCD has a low affinity with a Km value of approximately 10 mM for this ion. A strain defective in both KtrAB and KtrCD exhibited only a residual K+ uptake activity, demonstrating that KtrAB and KtrCD systems are the major K+ transporters of B. subtilis. Northern blot analyses revealed that ktrA and ktrB are cotranscribed as an operon, whereas ktrC and ktrD, which occupy different locations on the B. subtilis chromosome, are expressed as single transcriptional units. The amount of K+ in the environment or the salinity of the growth medium did not influence the amounts of the various ktr transcripts. A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective. In the ktrAB strain, the osmotically mediated growth defect was associated with a rapid loss of K+ ions from the cells. Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically induced proHJ, opuA, and gsiB genes was not impaired, demonstrating that a high cytoplasmic K+ concentration is not essential for the transcriptional activation of these genes at high osmolarity. Taken together, our data suggest that K+ uptake via KtrAB and KtrCD is an important facet in the cellular defense of B. subtilis against both suddenly imposed and prolonged osmotic stress.

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters

The Arabidopsis thaliana AtHKT1 protein, a Na+/K+ transporter, is capable of mediating inward Na+ currents in Xenopus laevis oocytes and K+ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K+ transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K+ channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na+ currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135–142 and 377–384, face the cytosol, whereas the region of residues 55–62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

Potassium channels in plant cells.

Potassium (K+) is the most abundant inorganic cation in plant cells. Unlike animals, plants lack sodium/potassium exchangers. Instead, plant cells have developed unique transport systems for K+ accumulation and release. An essential role in potassium uptake and efflux is played by potassium channels. Since the first molecular characterization of K+ channels from Arabidopsis thaliana in 1992, a large number of studies on plant potassium channels have been conducted. Potassium channels are considered to be one of the best characterized class of membrane proteins in plants. Nevertheless, knowledge on plant potassium channels is still incomplete. This minireview focuses on recent developments in the research of potassium transport in plants with a strong focus on voltage‐gated potassium channels.

Pollen Tubes Lacking a Pair of K + Transporters Fail to Target Ovules in Arabidopsis

How pollen tubes respond to female cues and precisely deliver sperm cells to the ovule is largely unknown. This article shows that two members of a cation transporter family are required in pollen tube navigation and in shifting polar tip growth. Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guidance signals from female cells are being identified; however, how pollen responds to those cues is largely unknown. Here, we show that two predicted cation/proton exchangers (CHX) in Arabidopsis thaliana, CHX21 and CHX23, are essential for pollen tube guidance. Male fertility was unchanged in single chx21 or chx23 mutants. However, fertility was impaired in chx21 chx23 double mutant pollen. Wild-type pistils pollinated with a limited number of single and double mutant pollen producing 62% fewer seeds than those pollinated with chx23 single mutant pollen, indicating that chx21 chx23 pollen is severely compromised. Double mutant pollen grains germinated and grew tubes down the transmitting tract, but the tubes failed to turn toward ovules. Furthermore, chx21 chx23 pollen tubes failed to enter the micropyle of excised ovules. Green fluorescent protein–tagged CHX23 driven by its native promoter was localized to the endoplasmic reticulum of pollen tubes. CHX23 mediated K+ transport, as CHX23 expression in Escherichia coli increased K+ uptake and growth in a pH-dependent manner. We propose that by modifying localized cation balance and pH, these transporters could affect steps in signal reception and/or transduction that are critical to shifting the axis of polarity and directing pollen growth toward the ovule.