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.     

Calcineurin B‐like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis

Potassium transporters and channels play crucial roles in K⁺ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K⁺ channel in Arabidopsis roots that is involved in K⁺ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B‐like proteins CBL1/CBL9. The present study showed that another calcineurin B‐like protein (CBL10) may also regulate AKT1 activity. The CBL10‐over‐expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low‐K⁺ conditions. In addition, the K⁺ content of both CBL10‐over‐expressing lines and akt1 mutant plants were significantly reduced compared with wild‐type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two‐hybrid, BiFC and co‐immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1‐mediated inward K⁺ currents. Furthermore, the results from the yeast two‐hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL‐interacting protein kinase‐independent regulatory mechanism of calcineurin B‐like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.     

Insights into the mechanisms of transport and regulation of the arabidopsis high-affinity K+ transporter HAK51

The high-affinity K⁺ transporter HAK5 from Arabidopsis (Arabidopsis thaliana) is essential for K⁺ acquisition and plant growth at low micromolar K⁺ concentrations. Despite its functional relevance in plant nutrition, information about functional domains of HAK5 is scarce. Its activity is enhanced by phosphorylation via the AtCIPK23/AtCBL1-9 complex. Based on the recently published three-dimensionalstructure of the bacterial ortholog KimA from Bacillus subtilis, we have modeled AtHAK5 and, by a mutational approach, identified residues G67, Y70, G71, D72, D201, and E312 as essential for transporter function. According to the structural model, residues D72, D201, and E312 may bind K⁺, whereas residues G67, Y70, and G71 may shape the selective filter for K⁺, which resembles that of K⁺shaker-like channels. In addition, we show that phosphorylation of residue S35 by AtCIPK23 is required for reaching maximal transport activity. Serial deletions of the AtHAK5 C-terminus disclosed the presence of an autoinhibitory domain located between residues 571 and 633 together with an AtCIPK23-dependent activation domain downstream of position 633. Presumably, autoinhibition of AtHAK5 is counteracted by phosphorylation of S35 by AtCIPK23. Our results provide a molecular model for K⁺ transport and describe CIPK-CBL-mediated regulation of plant HAK transporters.

Structure-function analysis of a high-affinity root K⁺ transporter reveals residues involved in transport, regulation by a protein kinase, and autoinhibition.     

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K⁺) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K⁺ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K⁺ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem‐pore outward‐rectifying K⁺ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two‐electrode voltage‐clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K⁺ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K⁺ from the fungus towards the plant .     

Towards an understanding of the molecular basis of plants K+ transport: Characterization of cloned K+ transport cDNAs

Recently, two K⁺-transport cDNAs, KAT1 and AKT1, were cloned in Arabidopsis thaliana. These cDNAs had structural similarities to K⁺ channel genes in animals, and also conferred the ability for growth on micromolar levels of K⁺ when expressed in K⁺ transport-defective yeast mutants. In this study, we examined the possibility that KAT1 encodes the high-affinity K⁺ transport system that has been previously characterized in plant roots, by studying the concentration-dependent kinetics of K⁺ transport for KAT1 expressed in Xenopus oocytes and Saccharomyces cerevisiae. In both organisms, the K⁺ transport system encoded by KAT1 yielded Michaelis-Menten kinetics with a high K_m for K⁺ (35 mM in oocytes, 0.6 mM in yeast cells). Furthermore, Northern analysis indicated that KAT1 is expressed primarily in the Arabidopsis shoot. These results strongly suggest that the system encoded by KAT1 is not a root high-affinity K⁺ transporter.     

Root high-affinity K+ and Cs+ uptake and plant fertility in tomato plants are dependent on the activity of the high-affinity K+ transporter SlHAK5

Root K⁺ acquisition is a key process for plant growth and development, extensively studied in the model plant Arabidopsis thaliana. Because important differences may exist among species, translational research supported by specific studies is needed in crops such as tomato. Here we present a reverse genetics study to demonstrate the role of the SlHAK5 K⁺ transporter in tomato K⁺ nutrition, Cs⁺ accumulation and its fertility. slhak5 KO lines, generated by CRISPR-Cas edition, were characterized in growth experiments, Rb⁺ and Cs⁺ uptake tests and root cells K⁺-induced plasma membrane depolarizations. Pollen viability and its K⁺ accumulation capacity were estimated by using the K⁺-sensitive dye Ion Potassium Green 4. SlHAK5 is the major system for high-affinity root K⁺ uptake required for plant growth at low K⁺, even in the presence of salinity. It also constitutes a pathway for Cs⁺ entry in tomato plants with a strong impact on fruit Cs⁺ accumulation. SlHAK5 also contributes to pollen K⁺ uptake and viability and its absence produces almost seedless fruits. Knowledge gained into SlHAK5 can serve as a model for other crops with fleshy fruits and it can help to generate tools to develop low Cs⁺ or seedless fruits crops.     

ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis in the succulent xerophyte Zygophyllum xanthoxylum

The inward‐rectifying K⁺ channel AKT1 constitutes an important pathway for K⁺ acquisition in plant roots. In glycophytes, excessive accumulation of Na⁺ is accompanied by K⁺ deficiency under salt stress. However, in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits excellent adaptability to adverse environments, K⁺ concentration remains at a relatively constant level despite increased levels of Na⁺ under salinity and drought conditions. In this study, the contribution of ZxAKT1 to maintaining K⁺ and Na⁺ homeostasis in Z. xanthoxylum was investigated. Expression of ZxAKT1 rescued the K⁺‐uptake‐defective phenotype of yeast strain CY162, suppressed the salt‐sensitive phenotype of yeast strain G19, and complemented the low‐K⁺‐sensitive phenotype of Arabidopsis akt1 mutant, indicating that ZxAKT1 functions as an inward‐rectifying K⁺ channel. ZxAKT1 was predominantly expressed in roots, and was induced under high concentrations of either KCl or NaCl. By using RNA interference technique, we found that ZxAKT1‐silenced plants exhibited stunted growth compared to wild‐type Z. xanthoxylum. Further experiments showed that ZxAKT1‐silenced plants exhibited a significant decline in net uptake of K⁺ and Na⁺, resulting in decreased concentrations of K⁺ and Na⁺, as compared to wild‐type Z. xanthoxylum grown under 50 mm NaCl. Compared with wild‐type, the expression levels of genes encoding several transporters/channels related to K⁺/Na⁺ homeostasis, including ZxSKOR, ZxNHX, ZxSOS1 and ZxHKT1;1, were reduced in various tissues of a ZxAKT1‐silenced line. These findings suggest that ZxAKT1 not only plays a crucial role in K⁺ uptake but also functions in modulating Na⁺ uptake and transport systems in Z. xanthoxylum, thereby affecting its normal growth.     

Suppression of Inward-Rectifying K+ Channels KAT1 and AKT2 by Dominant Negative Point Mutations in the KAT1 α-Subunit

The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K⁺ (K⁺ _in ) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K⁺ _in channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K⁺ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not greatly affect the ion selectivity of residual currents for Rb⁺, Na⁺, Li⁺, or Cs⁺. When T256R or G262K were co-expressed with a different K⁺ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs⁺ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers. These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued in vivo by the expression of the dominant negative K ⁺ _in channel mutants described here. Received: 2 July 1998/Revised: 23 October 1998     

Potassium channel AKT1 is involved in the auxin‐mediated root growth inhibition in Arabidopsis response to low K+ stress

The changes in external K⁺ concentration affect plant root growth. However, the molecular mechanism for perceiving a K⁺ signal to modulate root growth remains unknown. It is hypothesized that the K⁺ channel AKT1 is involved in low K⁺ sensing in the Arabidopsis root and subsequent regulation of root growth. Along with the decline of external K⁺ concentration, the primary root growth of wild‐type plants was gradually inhibited. However, the primary root of the akt1 mutant could still grow under low K⁺ (LK) conditions. Application of NAA inhibited akt1 root growth, but promoted wild‐type root growth under LK conditions. By using the ProDR5:GFP and ProPIN1:PIN1‐GFP lines, we found that LK treatment reduced auxin accumulation in wild‐type root tips by degrading PIN1 proteins, which did not occur in the akt1 mutant. The LK‐induced PIN1 degradation may be due to the inhibition of vesicle trafficking of PIN1 proteins. In conclusion, our findings indicate that AKT1 is required for an Arabidopsis response to changes in external K⁺, and subsequent regulation of K⁺‐dependent root growth by modulating PIN1 degradation and auxin redistribution in the root.     

Potassium Substitution by Sodium in Plants

Soil salinity is an ever-increasing constraint to crop productivity worldwide especially in countries with irrigated agriculture. In contrast to all the soil reclamation strategies to decrease salt concentrations in root zone, the use of sodium (Na⁺) in plant nutrition may be an interesting tactic. The roles of potassium (K⁺) and Na⁺ in plant nutrition suggest that K⁺ is the only monovalent cation which is essential for most higher plants and is involved in three important functions, i.e., enzyme activation, charge balance and osmoregulation. Plants need a small amount but high concentration of K⁺ for specific functions in the cytoplasm and a major portion (～90%) of it is localized in vacuoles, where it acts as an osmoticum. Maintenance of osmotic potential in vacuoles, a nonspecific function of K⁺, can be achieved by other cations such as Na⁺. For decades an ample amount of work has been done on the substitution of K⁺ by Na⁺ in plant nutrition. In this regard, Na⁺ has the potential to replace K⁺ for some of its functions. In some plants, supplementation of Na⁺ in reduced amounts can eliminate K⁺ deficiency symptoms under limited K⁺ supply. Thus, the question of K⁺ substitution by Na⁺ in plant physiology is not only of academic interest but has considerable practical implications in relation to fertilizer management and plant growth in salt-affected environments. In this review, we discuss the possibilities of K⁺ substitution by Na⁺ under specific soil and environmental conditions.     

Complexity of potassium acquisition: How much flows through channels?

The involvement of potassium (K⁺)-selective, Shaker-type channels, particularly AKT1, in primary K⁺ acquisition in roots of higher plants has long been of interest, particularly in the context of low-affinity K⁺ uptake, at high K⁺ concentrations, as well as uptake from low-K⁺ media under ammonium (NH₄⁺) stress. We recently demonstrated that K⁺ channels cannot mediate K⁺ acquisition in roots of intact barley (Hordeum vulgare L.) seedlings at low (22.5 µM) external K⁺ concentrations ([K⁺]_ext) and in the presence of high (10 mM) external NH₄⁺, while the model species Arabidopsis thaliana L. utilizes channels under comparable conditions. However, when external NH₄⁺ was suddenly withdrawn, a thermodynamic shift to passive (channel-mediated) K⁺ influx was observed in barley and both species demonstrated immediate and dramatic stimulations in K⁺ influx, illustrating a hitherto unexplored magnitude and rapidity of K⁺-uptake capacity and plasticity. Here, we expand on our previous work by offering further characterization of channel-mediated K⁺ fluxes in intact barley, with particular focus on anion effects, root respiration and pharmacological sensitivity and highlight key additions to the current model of K⁺ acquisition.     

Overexpression of a novel soybean gene modulating Na+ and K+ transport enhances salt tolerance in transgenic tobacco plants

Salt is an important factor affecting the growth and development of soybean in saline soil. In this study, a novel soybean gene encoding a transporter (GmHKT1) was identified and its function analyzed using transgenic plants. GmHKT1 encoded a protein of 419 amino acids, with a potential molecular mass of 47.06 kDa and a predicted pI value of 8.59. Comparison of the genomic and cDNA sequences of GmHKT1 identified no intron. The deduced amino acid sequence of GmHKT1 showed 38–49% identity with other plant HKT‐like sequences. RT‐PCR analysis showed that the expression of GmHKT1 was upregulated by salt stress (150 mM NaCl) in roots and leaves but not in stems. Overexpression of GmHKT1 significantly enhanced the tolerance of transgenic tobacco plants to salt stress, compared with non‐transgenic plants. To investigate the role of GmHKT1 in K⁺ and Na⁺ transport, we compared K⁺ and Na⁺ accumulation in roots and shoots of wild‐type and transgenic tobacco plants. The results suggested that GmHKT1 is a transporter that affected K⁺ and Na⁺ transport in roots and shoots, and regulated Na⁺/K⁺ homeostasis in these organs. Our findings suggest that GmHKT1 plays an important role in response to salt stress and would be useful in engineering crop plants for enhanced tolerance to salt stress.     

Leaf cell membrane stability‐based mechanisms of zinc nutrition in mitigating salinity stress in rice

• Excess salt affects about 955 million ha of arable land worldwide, and 49% of agricultural land is Zn‐deficient. Soil salinity and zinc deficiency can intensify plant abiotic stress. The mechanisms by which Zn can mitigate salinity effects on plant functions are not well understood.
• We conducted an experiment to determine how Zn and salinity effects on rice plant retention of Zn, K⁺ and the salt ion Na⁺ affect chlorophyll formation, leaf cell membrane stability and grain yield. We examined the mechanisms of Zn nutrition in mitigating salinity stress by examining plant physiology and nutrition. We used native Zn‐deficient soils (control), four salinity (EC ) and Zn treatments – Zn 10 mg·kg^?1 (Zn₁₀), EC 5 dS ·m^?1 (EC ₅), Zn₁₀+EC ₅ and Zn₁₅+EC ₅, a coarse rice (KS ‐282) and a fine rice (Basmati‐515) in the study.
• Our results showed that Zn alone (Zn₁₀) significantly increased rice tolerance to salinity stress by promoting Zn/K⁺ retention, inhibiting plant Na⁺ uptake and enhancing leaf cell membrane stability and chlorophyll formation in both rice cultivars in native alkaline, Zn‐deficient soils (P  <  0.05). Further, under the salinity treatment (EC ₅), Zn inputs (10–15 mg·kg^?1) could also significantly promote rice plant Zn/K⁺ retention and reduce plant Na⁺ uptake, and thus increased leaf cell membrane stability and grain yield. Coarse rice was more salinity‐tolerant than fine rice, having significantly higher Zn/K⁺ nutrient retention.
• The mechanistic basis of Zn nutrition in mitigating salinity impacts was through promoting plant Zn/K⁺ uptake and inhibiting plant Na⁺ uptake, which could result in increased plant physiological vigour, leaf cell membrane stability and rice productivity.

     

Expression of the AKT1-type K+ channel gene from Puccinellia tenuiflora, PutAKT1, enhances salt tolerance in Arabidopsis

Potassium channels are important for many physiological functions in plants, one of which is to regulate plant adaptation to stress conditions. In this study, a K⁺ channel PutAKT1 cDNA was isolated from the salt-tolerant plant Puccinellia tenuiflora. A phylogenetic analysis showed that PutAKT1 belongs to the AKT1-subfamily in the Shaker K⁺ channel family. PutAKT1 was localized in the plasma membrane and it was preferentially expressed in the roots. The expression of PutAKT1 was induced by K⁺-starvation stress in the roots and was not down-regulated by the presence of excess Na⁺. Arabidopsis plants over-expressing PutAKT1 showed enhanced salt tolerance compared to wild-type plants as shown by their shoot phenotype and dry weight. Expression of PutAKT1 increased the K⁺ content of Arabidopsis under normal, K⁺-starvation, and NaCl-stress conditions. Arabidopsis expressing PutAKT1 also showed a decrease in Na⁺ accumulation both in the shoot and in the root. These results suggest that PutAKT1 is involved in mediating K⁺ uptake (i) both in low- and in high-affinity K⁺ uptake range, and (ii) unlike its homologs in rice, even under salt-stress condition.     

Coordination of AtHKT1;1 and AtSOS1 facilitates Na+ and K+ homeostasis in Arabidopsis thaliana under salt stress

Reducing Na⁺ accumulation and maintaining K⁺ stability in plant is one of the key strategies for improving salt tolerance. AtHKT1;1 and AtSOS1 are not only the salt tolerance determinants themselves, but also mediate K⁺ uptake and transport indirectly. To assess the contribution of AtHKT1;1 and AtSOS1 to Na⁺ homeostasis and K⁺ nutrition in plant, net Na⁺ and K⁺ uptake rate, Na⁺ and K⁺ distributions in Arabidopsis thaliana wild type (WT), hkt1;1 mutant (athkt1;1) and sos1 mutant (atsos1) were investigated. Results showed that under 2.5 mM K⁺ plus 25 or 100 mM NaCl, athkt1;1 shoot concurrently accumulated more Na⁺ and less K⁺ than did WT shoot, suggesting that AtHKT1;1 was critical for controlling Na⁺ and K⁺ distribution in plant; while atsos1 root accumulated more Na⁺ and absorbed lower K⁺ than did WT root, implying that AtSOS1 was determiner of Na⁺ excretion and K⁺ acquisition. Under 0.01 mM K⁺, athkt1;1 absorbed lower Na⁺ than did WT with 100 mM NaCl, suggesting that AtHKT1;1 is involved in Na⁺ uptake in roots; while atsos1 shoot accumulated less Na⁺ than did WT shoot no matter with 25 or 100 mM NaCl, implying that AtSOS1 played a key role in controlling long-distance Na⁺ transport from root to shoot. We present a model in which coordination of AtHKT1;1 and AtSOS1 facilitates Na⁺ and K⁺ homeostasis in A. thaliana under salt stress: under the normal K⁺, the major function of AtHKT1;1 is Na⁺ unloading and AtSOS1 is mainly involved in Na⁺ exclusion, whereas under the low K⁺, AtHKT1;1 may play a dominant role in Na+ uptake and AtSOS1 may be mainly involved in Na⁺ loading into the xylem.