Cloning and functional characterization of the high-affinity K<Superscript>+</Superscript> transporter HAK1 of pepper

High-affinity K⁺ uptake in plants plays a crucial role in K⁺ nutrition and different systems have been postulated to contribute to the high-affinity K⁺ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K⁺ uptake observed in roots. Pepper plants starved of K⁺ for 3 d showed high-affinity K⁺ uptake (K _m of 6 M K⁺) that was very sensitive to NH and their roots expressed a high-affinity K⁺ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast (Saccharomyces cerevisiae), CaHAK1 mediated high-affinity K⁺ and Rb⁺ uptake with K _m values of 3.3 and 1.9 M, respectively. Rb⁺ uptake was competitively inhibited by micromolar concentrations of NH and Cs⁺, and by millimolar concentrations of Na⁺.     

Recent Advances in Genome-wide Analyses of Plant Potassium Transporter Families

Plants require potassium (K⁺) as a macronutrient to support numerous physiological processes. Understanding how this nutrient is transported, stored, and utilized within plants is crucial for breeding crops with high K⁺ use efficiency. As K⁺ is not metabolized, cross-membrane transport becomes a rate-limiting step for efficient distribution and utilization in plants. Several K⁺ transporter families, such as KUP/HAK/KT and KEA transporters and Shaker-like and TPK channels, play dominant roles in plant K⁺ transport processes. In this review, we provide a comprehensive contemporary overview of our knowledge about these K⁺ transporter families in angiosperms, with a major focus on the genome-wide identification of K⁺ transporter families, subcellular localization, spatial expression, function and regulation. We also expanded the genome-wide search for the K⁺ transporter genes and examined their tissue-specific expression in Camelina sativa, a polyploid oil-seed crop with a potential to adapt to marginal lands for biofuel purposes and contribution to sustainable agriculture. In addition, we present new insights and emphasis on the study of K⁺ transporters in polyploids in an effort to generate crops with high K⁺ Utilization Efficiency (KUE).     

Studies on Arabidopsis athak5, atakt1 double mutants disclose the range of concentrations at which AtHAK5, AtAKT1 and unknown systems mediate K+ uptake

The high‐affinity K⁺ transporter AtHAK5 and the inward‐rectifier K⁺ channel AtAKT1 have been described to contribute to K⁺ uptake in Arabidopsis thaliana. Studies with T‐DNA insertion lines showed that both systems participate in the high‐affinity range of concentrations and only AtAKT1 in the low‐affinity range. However the contribution of other systems could not be excluded with the information and plant material available. The results presented here with a double knock‐out athak5, atakt1 mutant show that AtHAK5 is the only system mediating K⁺ uptake at concentrations below 0.01 mM. In the range between 0.01 and 0.05 mM K⁺ AtHAK5 and AtAKT1 are the only contributors to K⁺ acquisition. At higher K⁺ concentrations, unknown systems come into operation and participate together with AtAKT1 in low‐affinity K⁺ uptake. These systems can supply sufficient K⁺ to promote plant growth even in the absence of AtAKT1 or in the presence of 10 mM K⁺ where AtAKT1 is not essential.     

Molecular biology of K transport across the plant cell membrane: What do we learn from comparison between plant species?

Cloning and characterizations of plant K⁺ transport systems aside from Arabidopsis have been increasing over the past decade, favored by the availability of more and more plant genome sequences. Information now available enables the comparison of some of these systems between species. In this review, we focus on three families of plant K⁺ transport systems that are active at the plasma membrane: the Shaker K⁺ channel family, comprised of voltage-gated channels that dominate the plasma membrane conductance to K⁺ in most environmental conditions, and two families of transporters, the HAK/KUP/KT K⁺ transporter family, which includes some high-affinity transporters, and the HKT K⁺ and/or Na⁺ transporter family, in which K⁺-permeable members seem to be present in monocots only. The three families are briefly described, giving insights into the structure of their members and on functional properties and their roles in Arabidopsis or rice. The structure of the three families is then compared between plant species through phylogenic analyses. Within clusters of ortologues/paralogues, similarities and differences in terms of expression pattern, functional properties and, when known, regulatory interacting partners, are highlighted. The question of the physiological significance of highlighted differences is also addressed.     

The K+ and NO3− Interaction Mediated by NITRATE TRANSPORTER1.1 Ensures Better Plant Growth under K+-Limiting Conditions

K⁺ and NO₃⁻ are the major forms of potassium and nitrogen that are absorbed by the roots of most terrestrial plants. In this study, we observed that a close relationship between NO₃⁻ and K⁺ in Arabidopsis (Arabidopsis thaliana) is mediated by NITRATE TRANSPORTER1.1 (NRT1.1). The nrt1.1 knockout mutants showed disturbed K⁺ uptake and root-to-shoot allocation, and were characterized by growth arrest under K⁺-limiting conditions. The K⁺ uptake and root-to-shoot allocation of these mutants were partially recovered by expressing NRT1.1 in the root epidermis-cortex and central vasculature using SULFATE TRANSPORTER1;2 and PHOSPHATE1 promoters, respectively. Two-way analysis of variance based on the K⁺ contents in nrt1.1-1/K⁺ transporter1, nrt1.1-1/high-affinity K⁺ transporter5-3, nrt1.1-1/K⁺ uptake permease7, and nrt1.1-1/stelar K⁺ outward rectifier-2 double mutants and the corresponding single mutants and wild-type plants revealed physiological interactions between NRT1.1 and K⁺ channels/transporters located in the root epidermis–cortex and central vasculature. Further study revealed that these K⁺ uptake-related interactions are dependent on an H⁺-consuming mechanism associated with the H⁺/NO₃⁻ symport mediated by NRT1.1. Collectively, these data indicate that patterns of NRT1.1 expression in the root epidermis–cortex and central vasculature are coordinated with K⁺ channels/transporters to improve K⁺ uptake and root-to-shoot allocation, respectively, which in turn ensures better growth under K⁺-limiting conditions.

Potassium (K) is an essential element for plant growth and development and contributes to determining the yield and quality of crops in agriculture production (Wang and Wu, 2013). However, the concentrations of soluble K⁺ in most soils are relatively low, which often limits plant growth (Maathuis, 2009). Although crop production can be increased by applying large amounts of potassic fertilizers to agricultural fields, only approximately one-half of the applied fertilizers is available to plants; the remainder accumulates as residues in soils, consequently leading to environmental contamination (Meena et al., 2016). Therefore, there is a pressing need to gain a more complete understanding of the molecular mechanisms underlying K⁺ transport and regulation in order to enhance the K⁺ utilization efficiency of plants. Accordingly, in the past few decades, researchers have focused on identifying K⁺ channels and transporters in plants, as well as the mechanisms underlying their regulation.In Arabidopsis (Arabidopsis thaliana), 71 K⁺ channels and transporters have been identified and categorized into three channel (Shaker, Tandem-Pore K⁺, and K_ir-like) and three transporter (K⁺ uptake permeases [KT/HAK/KUP], High-affinity K⁺ transporters [HKT], and cation/proton antiporter [CPA]) families (Wang and Wu, 2010). Among these, the shaker inward K⁺ channel K⁺ TRANSPORTER1 (AKT1) and the KT/HAK/KUP K⁺ transporter HIGH-AFFINITY K⁺ TRANSPORTER5 (HAK5) have been characterized as the two major components that contribute to K⁺ uptake in roots, although they have been found to operate at different K⁺ levels (Pyo et al., 2010; Wang and Wu, 2013). AKT1 functions in plant K⁺ uptake over a wide range of K⁺ concentrations, whereas HAK5 shows high-affinity K⁺ transport activity (Gierth et al., 2005). Following its uptake into root epidermal cells, K⁺ is distributed to different plant organs or tissues. The Arabidopsis shaker-like outward-rectifying K⁺ channel STELAR K⁺ OUTWARD RECTIFIER (SKOR), the expression of which was first identified in stelar tissues, has been shown to facilitate K⁺ secretion into xylem sap, which is a critical step in long-distance K⁺ transport from roots to shoots (Gaymard et al., 1998). Recently, K⁺ UPTAKE PERMEASE7 (KUP7), a member of the KT/HAK/KUP family, was functionally characterized as a K⁺ transporter participating in both root K⁺ uptake and root-to-shoot K⁺ allocation, particularly under K⁺-limiting conditions (Han et al., 2016). However, the uptake affinity for K⁺ has been found to be considerably lower in KUP7 than in HAK5 (Wang and Wu, 2017).In addition to the aforementioned K⁺ channels and transporters, other mineral elements, including Na⁺, Ca²⁺, and N, are known to have pronounced effects on K⁺ nutrition in plants. Given that N is the nutrient that is required in the greatest quantity by most plants and is the most widely used fertilizer nutrient in crop production, the relationships between N and K have long been investigated (Fageria and Baligar, 2005; Wang and Wu, 2013; Meng et al., 2016; Shin, 2017). Since the 1960s, physiological studies have revealed a close relationship between NO₃⁻ and K⁺ with regard to uptake and translocation (Zioni et al., 1971; Blevins et al., 1978; Barneix and Breteler, 1985; Drechsler et al., 2015). However, the coordination between these two nutrients in plant transport pathways remains to be extensively studied at the molecular level. We hypothesized that transporters involved in the transference of NO₃⁻ across cell membranes may play a role in controlling K⁺ nutrition in plants. Recently, NITRATE TRANSPORTER1.5 (NRT1.5), a member of the nitrate transporter1/peptide transporter family (NPF), initially identified as a pH-dependent bidirectional NO₃⁻ transporter (Lin et al., 2008), was shown to be involved in the control of K⁺ allocation in plants (Drechsler et al., 2015; Li et al., 2017; Du et al., 2019). Nevertheless, it was subsequently established that this function was merely associated with its role as a proton-coupled H⁺/K⁺ antiporter for K⁺ loading into the xylem (Li et al., 2017; Du et al., 2019), which is not associated with the transport of NO₃⁻. In this study, we showed that the loss of another nitrate transporter1 member, NRT1.1/NPF6.3, in nrt1.1 mutants led to the development of a more pronounced K⁺-deficiency phenotype under conditions of low-K⁺ stress. Further physiological and genetic evidence revealed that both the uptake and root-to-shoot allocation of K⁺ in plants require NRT1.1. However, NRT1.1 acts as a coordinator rather than a K⁺ channel/transporter in K⁺ uptake and root-to-shoot allocation, which could depend on its NO₃⁻-related transport activity. Our findings highlight the significance of nutrients and nutrient interactions in ensuring plant growth, and indicate that the modification of NRT1.1 homolog activity in crops using biological engineering techniques might be a promising approach that could simultaneously contribute to enhancing the utilization efficiencies of K and N fertilizers in agricultural production.     

The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis

Caesium (Cs⁺) is a potentially toxic mineral element that isreleased into the environment and taken up by plants. AlthoughCs⁺ is chemically similar to potassium (K⁺), and much is knownabout K⁺ transport mechanisms, it is not clear through whichK⁺ transport mechanisms Cs⁺ is taken up by plant roots. In thisstudy, the role of AtHAK5 in high affinity K⁺ and Cs⁺ uptakewas characterized. It is demonstrated that AtHAK5 is localizedto the plasma membrane under conditions of K⁺ deprivation, whenit is expressed. Growth analysis showed that AtHAK5 plays arole during severe K⁺ deprivation. Under K⁺-deficient conditionsin the presence of Cs⁺, Arabidopsis seedlings lacking AtHAK5had increased inhibition of root growth and lower Cs⁺ accumulation,and significantly higher leaf chlorophyll concentrations thanwild type. These data indicate that, in addition to transportingK⁺ in planta, AtHAK5 also transports Cs⁺. Further experimentsshowed that AtHAK5 mediated Cs⁺ uptake into yeast cells andthat, although the K⁺ deficiency-induced expression of AtHAK5was inhibited by low concentrations of NH     

Vascular expression driven by the promoter of a gene encoding a high-affinity potassium transporter HAK5 from <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

The construction of expression cassettes harboring tissue-specific promoters is a viable alternative to limit transgene expression to specific organs and cell types. In this study, we have functionally characterized the promoter of a Eucalyptus grandis gene encoding a putative high-affinity HAK5-like potassium (K⁺) transporter (designated EgHAK5) showing root-specific expression. The ability of the EgHAK 5′-flanking region (~1.3 kb) to drive root-specific expression of a reporter gene (β-glucuronidase; GUS) was examined using transgenic tobacco plants. Histochemical analysis revealed enhanced GUS staining in the vasculature of leaves, hypocotyls and roots, which was also confirmed in histological cross-sections. Moreover, the relative expression of GUS in the roots of the generated transgenic lines was increased in response to K⁺ starvation. Overall, our results indicate that, in a heterologous system, the EgHAK5 promoter shows expression in vascular tissues, mainly within the phloem, and is up-regulated upon potassium deprivation.     

Molecular mechanisms of potassium and sodium uptake in plants

Potassium (K⁺) is an essential nutrient and the most abundant cation in plants, whereas the closely related ion sodium (Na⁺) is toxic to most plants at high millimolar concentrations. K⁺ deficiency and Na⁺ toxicity are both major constraints to crop production worldwide. K⁺ counteracts Na⁺ stress, while Na⁺, in turn, can to a certain degree alleviate K⁺ deficiency. Elucidation of the molecular mechanisms of K⁺ and Na⁺ transport is pivotal to the understanding – and eventually engineering – of plant K⁺ nutrition and Na⁺ sensitivity. Here we provide an overview on plant K⁺ transporters with particular emphasis on root K⁺ and Na⁺ uptake. Plant K⁺-permeable cation transporters comprise seven families: Shaker-type K⁺ channels, `two-pore' K⁺ channels, cyclic-nucleotide-gated channels, putative K⁺/H⁺ antiporters, KUP/HAK/KT transporters, HKT transporters, and LCT1. Candidate genes for Na⁺ transport are the KUP/HAK/KTs, HKTs, CNGCs, and LCT1. Expression in heterologous systems, localization in plants, and genetic disruption in plants will provide insight into the roles of transporter genes in K⁺ nutrition and Na⁺ toxicity.     

A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5

A chimeric CaHAK1–LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K⁺ uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K⁺ uptake shown by K⁺-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K⁺ uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K⁺ uptake was not correlated with the root K⁺ content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K⁺ starvation or growth in the presence of NH₄ ⁺, but which do not decrease the K⁺ content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K⁺ starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K⁺ transporter.     

Kinetics of high-affinity K+ uptake in plants, derived from K+-induced changes in current-voltage relationships

To investigate coupled, charge-translocating transport, it is imperative that the specific transporter current-voltage (IV ) relationship of the transporter is separated from the overall membrane IV relationship. We report here a case study in which the currents mediated by the K⁺-H⁺ symporter, responsible for high-affinity K⁺ uptake in Arabidopsis thaliana (L.) Heynh. cv. Columbia roots, are analyzed with an enzyme kinetic reaction scheme. The model explicitly incorporates changes in membrane voltage and external substrate, and enables the derivation of the underlying symport IV relationships from the experimentally obtained difference IV data. Data obtained for high-affinity K⁺ transport in A. thaliana root protoplasts were best described by a 1:1 coupled K⁺-H⁺ symport-mediated current with a parallel, outward non-linear K⁺ pathway. Furthermore, the large predictive value of the model was used to describe symport behaviour as a function of the external K⁺ concentration and the cytoplasmic K⁺ concentration. Symport activity is a complex function of the external K⁺ concentration, with first-order saturating kinetics in the micromolar range and a strong activity reduction when external K⁺ is in the millimolar range and the membrane depolarises. High cytoplasmic K⁺ levels inhibit symport activity. These responses are suggested to be part of the feedback mechanisms to maintain cellular K⁺ homeostasis. The general suitability of the model for analysis of carrier-mediated transport is discussed. Received: 23 November 1996 / Accepted: 22 April 1997     

Cloning and functional expression in <Emphasis Type="Italic">Saccharomyces cereviae</Emphasis> of a K<Superscript>+</Superscript> transporter,AlHAK, from the graminaceous halophyte, <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

High-affinity K⁺ transporters play an important role in K⁺ absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WΔ3, AlHAK performed high-affinity K⁺ uptake with a K_m value of 8 μM, and the growth of transformants was dramatically inhibited by 150 mM Rb⁺ and 150 mM Cs⁺ but less affected by 300 mM Na⁺. AlHAK may thus improve the capacity of plants to maintain a high cytosolic K⁺/Na⁺ ratio at high salinity.     

AtNHX5 and AtNHX6 Control Cellular K+ and pH Homeostasis in Arabidopsis: Three Conserved Acidic Residues Are Essential for K+ Transport

AtNHX5 and AtNHX6, the endosomal Na⁺,K⁺/H⁺ antiporters in Arabidopsis, play an important role in plant growth and development. However, their function in K⁺ and pH homeostasis remains unclear. In this report, we characterized the function of AtNHX5 and AtNHX6 in K⁺ and H⁺ homeostasis in Arabidopsis. Using a yeast expression system, we found that AtNHX5 and AtNHX6 recovered tolerance to high K⁺ or salt. We further found that AtNHX5 and AtNHX6 functioned at high K⁺ at acidic pH while AtCHXs at low K⁺ under alkaline conditions. In addition, we showed that the nhx5 nhx6 double mutant contained less K⁺ and was sensitive to low K⁺ treatment. Overexpression of AtNHX5 or AtNHX6 gene in nhx5 nhx6 recovered root growth to the wild-type level. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for K⁺ homeostasis and plant growth. nhx5 nhx6 had a reduced vacuolar and cellular pH as measured with the fluorescent pH indicator BCECF or semimicroelectrode. We further show that AtNHX5 and AtNHX6 are localized to Golgi and TGN. Taken together, AtNHX5 and AtNHX6 play an important role in K⁺ and pH homeostasis in Arabidopsis. Three conserved acidic residues are essential for K⁺ transport.     

Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high‐affinity range of concentrations

The relative contribution of the high‐affinity K⁺ transporter AtHAK5 and the inward rectifier K⁺ channel AtAKT1 to K⁺ uptake in the high‐affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col‐0). The results obtained with wild‐type lines, with T‐DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the ‐sensitive and the Ba²⁺‐sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high‐affinity range of Rb⁺ concentrations. Using Rb⁺ as a K⁺ analogue, it was shown that AtHAK5 mediates absorption at lower Rb⁺ concentrations than AtAKT1 and depletes external Rb⁺ to values around 1 μM. Factors such as the presence of K⁺ or during plant growth determine the relative contribution of each system. The presence of in the growth solution inhibits the induction of AtHAK5 by K⁺ starvation. In K⁺‐starved plants grown without , both systems are operative, but when is present in the growth solution, AtAKT1 is probably the only system mediating Rb⁺ absorption, and the capacity of the roots to deplete Rb⁺ is reduced.     

Alternating Hemiplegia of Childhood mutations have a differential effect on Na+,K+-ATPase activity and ouabain binding

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na⁺,K⁺-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na⁺,K⁺-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K⁺/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na⁺,K⁺-ATPase. Functional impairment of Na⁺,K⁺-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.