Phosphorylation by CIPK23 regulates the high-affinity Mn transporter NRAMP1 in Arabidopsis.

Manganese (Mn) is essential for plants but is toxic when taken up in excess. To maintain Mn homeostasis, the root Mn transporter natural resistance associated macrophage protein 1 (NRAMP1) cycles from the plasma membrane to endosomes upon phosphorylation. To identify the kinase involved, a split-luciferase screening was carried out between NRAMP1 and kinases of the CIPK family and identified CIPK23 as a partner of NRAMP1. The interaction was confirmed by split-mCitrine bimolecular fluorescence complementation and co-immunoprecipitation assays. In vitro phosphorylation assays pinpointed two CIPK23 target residues in NRAMP1, among which serine 20, important for endocytosis. Interestingly, Mn-induced internalization of NRAMP1 was unaffected by cipk23 mutation suggesting a potential redundancy between CIPK23 and other kinase(s). How CIPK23 could regulate NRAMP1 in response to Mn availability is discussed.

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.

Regulation of Root Nutrient Transporters by CIPK23: 'One Kinase to Rule Them All'.

Protein kinases constitute essential regulatory components in the majority of cellular processes in eukaryotic cells. The CBL-INTERACTING PROTEIN KINASE (CIPK) family of plant protein kinases functions in calcium (Ca2+)-related signaling pathways and is therefore involved in the response to a wide variety of signals in plants. By covalently linking phosphate groups to their target proteins, CIPKs regulate the activity of downstream targets, their localization, their stability and their ability to interact with other proteins. In Arabidopsis, the CIPK23 kinase has emerged as a major hub driving root responses to diverse environmental stresses, including drought, salinity and nutrient imbalances, such as potassium, nitrate and iron deficiencies, as well as ammonium, magnesium and non-iron metal toxicities. This review will chiefly report on the prominent roles of CIPK23 in the regulation of plant nutrient transporters and on the underlying molecular mechanisms. We will also discuss the different scenarios explaining how a single promiscuous kinase, such as CIPK23, may convey specific responses to a myriad of signals.

Arabidopsis K+ transporter HAK5-mediated high-affinity root K+ uptake is regulated by protein kinases CIPK1 and CIPK9.

The high-affinity K+ transporter HAK5 is the major contributor to root K+ uptake from dilute solutions in K+-starved Arabidopsis plants. Its functionality is tightly regulated and its activity is enhanced under K+ starvation by the transcriptional induction of the AtHAK5 gene, and by the activation of the transporter via the AtCBL1-AtCIPK23 complex. In the present study, the 26 members of the Arabidopsis CIPK protein kinase family were screened in yeast for their capacity to activate HAK5-mediated K+ uptake. Among them, AtCIPK1 was the most efficient activator of AtHAK5. In addition, AtCIPK9, previously reported to participate in K+ homeostasis, also activated the transporter. In roots, the genes encoding AtCIPK1 and AtCIPK9 were induced by K+ deprivation and atcipk1 and atcipk9 Arabidopsis KO mutants showed a reduced AtHAK5-mediated Rb+ uptake. Activation of AtHAK5 by AtCIPK1 did not occur under hyperosmotic stress conditions, where AtCIPK1 function has been shown to be required to maintain plant growth. Taken together, our data contribute to the identification of the complex regulatory networks that control the high-affinity K+ transporter AtHAK5 and root K+ uptake.

High External K+ Concentrations Impair Pi Nutrition, Induce the Phosphate Starvation Response, and Reduce Arsenic Toxicity in Arabidopsis Plants.

Potassium (K+) and phosphorous (Pi) are two of the most important nutrients required by plants and there is an interest in studying how they are acquired. Most studies have focused on the characterization of the mechanisms involved in K+ and Pi uptake and their distribution within the plants, as well as the regulatory mechanisms involved. Evidence is emerging which points to interactions in the nutrition of different nutrients and to the existence of crosstalk in the signaling cascades regulating their acquisition. However, the interaction between K+ and Pi has been scarcely studied. Here we show that high concentrations of K+ in the external solution inhibit Pi uptake and impair Pi nutrition in Arabidopsis plants, resulting in the induction of phosphate starvation response (PSR) and the upregulation of genes encoding root phosphate uptake systems. The high K+-induced PSR depends on the PHR1 and PHL1 transcription factors that are key pieces of Pi signaling in Arabidopsis. Importantly, high K+ reduces arsenic accumulation in plants and its toxic effects. The results presented may help to design strategies to reduce Pi deficiency as well as the accumulation of arsenic in crops.

Modulation of K+ translocation by AKT1 and AtHAK5 in Arabidopsis plants.

Root cells take up K+ from the soil solution, and a fraction of the absorbed K+ is translocated to the shoot after being loaded into xylem vessels. K+ uptake and translocation are spatially separated processes. K+ uptake occurs in the cortex and epidermis whereas K+ translocation starts at the stele. Both uptake and translocation processes are expected to be linked, but the connection between them is not well characterized. Here, we studied K+ uptake and translocation using Rb+ as a tracer in wild-type Arabidopsis thaliana and in T-DNA insertion mutants in the K+ uptake or translocation systems. The relative amount of translocated Rb+ to the shoot was positively correlated with net Rb+ uptake rates, and the akt1 athak5 T-DNA mutant plants were more efficient in their allocation of Rb+ to shoots. Moreover, a mutation of SKOR and a reduced plant transpiration prevented the full upregulation of AtHAK5 gene expression and Rb+ uptake in K+ -starved plants. Lastly, Rb+ was found to be retrieved from root xylem vessels, with AKT1 playing a significant role in K+ -sufficient plants. Overall, our results suggest that K+ uptake and translocation are tightly coordinated via signals that regulate the expression of K+ transport systems.

The combination of K+ deficiency with other environmental stresses: What is the outcome?

Potassium (K+ ) is a macronutrient known for its high mobility and positive charge, which allows efficient and fast control of the electrical balance and osmotic potential in plant cells. Such features allow K+ to remarkably contribute to plant stress adaptation. Some agricultural lands are deficient in K+ , imposing a stress that reduces crop yield and makes fertilization a common practice. However, individual stress conditions in the field are rare, and crops usually face a combination of different stresses. As plant response to a stress combination cannot always be deduced from individual stress action, it is necessary to gain insights into the specific mechanisms that connect K+ homeostasis with other stress effects to improve plant performance in the context of climate change. Surprisingly, plant responses to environmental stresses under a K+ -limiting scenario are poorly understood. In the present review, we summarize current knowledge and find substantial gaps regarding specific outcomes of K+ deficiency in addition to other environmental stresses. In this regard, combined nutrient deficiencies of K+ and other macronutrients are covered in the first part of the review and interactions arising from K+ deficiency with salinity, drought and biotic factors in the second part. Information available so far suggests a prominent role of potassium and nitrate transport systems and their regulatory proteins in the response of plants to several stress combinations. Thus, such molecular pathways, which are located at the crossroad between K+ homeostasis and environmental stresses, could be considered biotechnological targets in future studies.

Tolerance to Stress Combination in Tomato Plants: New Insights in the Protective Role of Melatonin.

Abiotic stresses such as drought, heat or salinity are major causes of yield loss worldwide. Recent studies have revealed that the acclimation of plants to a combination of different environmental stresses is unique and therefore cannot be directly deduced from studying the response of plants to each of the different stresses applied individually. The efficient detoxification of reactive oxygen species (ROS) is thought to play a key role in enhancing the tolerance of plants to abiotic stresses. Here, we report on the role of melatonin in the protection of the photosynthetic apparatus through the increase in ROS detoxification in tomato plants grown under the combination of salinity and heat, two of the most common abiotic stresses known to act jointly. Plants treated with exogenous melatonin showed a different modulation in the expression on some antioxidant-related genes and their related enzymes. More specifically, ascorbate peroxidase, glutathione reductase, glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase (APX, GR, GPX and Ph-GPX, resepctively) showed an antagonistic regulation as compared to plants that did not receive melatonin. This translated into a better antioxidant capacity and to a lesser ROS accumulation under stress combination. The performance of the photosynthesis parameters and the photosystems was also increased in plants treated with exogenous melatonin under the combination of salinity and heat. In accordance with these findings, tomato plants treated with melatonin were found to grow better under stress combination that the non-treated ones. Our study highlights the important role that exogenous melatonin plays in the acclimation of plants to a combination of two different abiotic stresses, and how this compound can specifically regulate oxidative stress-related genes and enzymes to increase plant tolerance.

Pharmacological and gene regulation properties point to the SlHAK5 K+ transporter as a system for high-affinity Cs+ uptake in tomato plants.

Potassium (K+ ) and cesium (Cs+ ) are chemically similar but while K+ is an essential nutrient, Cs+ can be toxic for living organisms, plants included. Two different situations could lead to problems derived from the presence of Cs+ in agricultural systems: (1) presence of Cs+ at high concentrations that could produce toxic effects on plants, (2) presence of micromolar concentrations of radiocesium, which can be accumulated in the plant and affect animal and human health through the food chain. While K+ uptake has been well described in tomato plants, information on molecular mechanisms involved in Cs+ accumulation in this species is absent. Here, we show that in tomato plants, high concentrations of Cs+ produce deficiency of K+ but do not induce high-affinity K+ uptake or the gene encoding the high-affinity K+ transporter SlHAK5. At these concentrations, Cs+ uptake takes place through a Ca2+ -sensitive pathway, probably a non-selective cation channel. At micromolar concentrations, Cs+ is accumulated by a high-affinity uptake system upregulated in K+ -starved plants. This high-affinity Cs+ uptake shares features with high-affinity K+ uptake. It is sensitive to NH4+ and insensitive to Ba2+ and Ca2+ and its presence parallels the pattern of SlHAK5 expression. Moreover, blockers of reactive oxygen species and ethylene action repress SlHAK5 and negatively regulate both high-affinity K+ and Cs+ uptake. Thus, we propose that SlHAK5 contributes to Cs+ uptake from micromolar concentrations in tomato plants and can constitute a pathway for radiocesium transfer from contaminated areas to the food chain.

How DELLAs contribute to control potassium uptake under conditions of potassium scarcity? Hypotheses and uncertainties.

Maintenance of the inward transport of potassium (K) by roots is a critical step to ensure K-nutrition for all plant tissues. When plants are grown at low external K concentrations a strong enhancement of the activity of the AtHAK5 transporter takes place. In a recent work, we observed that the gai-1 mutant of Arabidopsis thaliana, which bears an altered function version of a DELLA regulatory protein, displays reduced accumulation of AtHAK5 transcripts and reduced uptake of Rubidium, an analog for K. In this Addendum we discuss some hypotheses and uncertainties regarding how DELLAs could contribute to the control of K uptake under those conditions. We advance the idea that, following K-restriction, there is a zone and tissue specific regulation of DELLAs by gibberellins through a pathway that likely involves ethylene. According to this model in the epidermis of non-apical zones, DELLAs repress transcription factors that promote AtHAK5 accumulation.

NO3- , PO43- and SO42- deprivation reduced LKT1-mediated low-affinity K+ uptake and SKOR-mediated K+ translocation in tomato and Arabidopsis plants.

Regulation of essential macronutrients acquisition by plants in response to their availability is a key process for plant adaptation to changing environments. Here we show in tomato and Arabidopsis plants that when they are subjected to NO3- , PO43- and SO42- deprivation, low-affinity K+ uptake and K+ translocation to the shoot are reduced. In parallel, these nutritional deficiencies produce reductions in the messenger levels of the genes encoding the main systems for low-affinity K+ uptake and K+ translocation, i.e. AKT1 and SKOR in Arabidopsis and LKT1 and the tomato homolog of SKOR, SlSKOR in tomato, respectively. The results suggest that the shortage of one nutrient produces a general downregulation of the acquisition of other nutrients. In the case of K+ nutrient, one of the mechanisms for such a response resides in the transcriptional repression of the genes encoding the systems for K+ uptake and translocation.

Uneven HAK/KUP/KT Protein Diversity Among Angiosperms: Species Distribution and Perspectives.

HAK/KUP/KT K(+) transporters have been widely associated with K(+) transport across membranes in bacteria, fungi, and plants. Indeed some members of the plant HAK/KUP/KT family contribute to root K(+) uptake, notably at low external concentrations. Besides such role in acquisition, several studies carried out in Arabidopsis have shown that other members are also involved in developmental processes. With the publication of new plant genomes, a growing interest on plant species other than Arabidopsis has become evident. In order to understand HAK/KUP/KT diversity in these new plant genomes, we discuss the evolutionary trends of 913 HAK/KUP/KT sequences identified in 46 genomes revealing five major groups with an uneven distribution among angiosperms, notably between dicotyledonous and monocotyledonous species. This information evidenced the richness of crop genomes in HAK/KUP/KT transporters and supports their study for unraveling novel physiological roles of such transporters in plants.

The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

High Ca(2+) reverts the repression of high-affinity K(+) uptake produced by Na(+) in Solanum lycopersycum L. (var. microtom) plants.

Potassium (K(+)) is an essential nutrient for plants which is acquired by plant roots through the operation of specific transport systems. Abiotic stress conditions such as salinity impair K(+) nutrition because, in addition to other effects, high salt concentrations in the solution bathing the roots inhibit K(+) uptake systems. This detrimental effect of salinity is exacerbated when external K(+) is very low and the only system capable of mediating K(+) uptake is one with high-affinity for K(+), as that mediated by transporters of the HAK5 type. Increasing external Ca(2+) has been shown to improve K(+) nutrition under salinity and, although the specific mechanisms for this beneficial effect are largely unknown, they are beginning to be understood. The genes encoding the HAK5 transporters are induced by K(+) starvation and repressed by long-term exposure to high Na(+). This occurs in parallel with the hyperpolarization and depolarization of root cell membrane potential. In the present study it is shown in tomato plants that the presence of high Ca(2+) during the K(+) starvation period that leads to LeHAK5 induction, counteracts the repression exerted by high Na(+). High Ca(2+) reduces the Na(+)-induced plasma membrane depolarization of root cells, resorting one of the putative first steps in the low-K(+) signal cascade. This allows proper LeHAK5 expression and functional high-affinity K(+) uptake at the roots. Thus, the maintenance of HAK5-mediated K(+) nutrition under salinity by high Ca(2+) can be regarded as a specific beneficial effect of Ca(2+) contributing to salt tolerance in plants.

The F130S point mutation in the Arabidopsis high-affinity K(+) transporter AtHAK5 increases K(+) over Na(+) and Cs(+) selectivity and confers Na(+) and Cs(+) tolerance to yeast under heterologous expression.

Potassium (K(+)) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K(+) acquisition by plant roots at low external K(+) concentrations. Certain abiotic stress conditions such as salinity or Cs(+)-polluted soils may jeopardize plant K(+) nutrition because HAK5-mediated K(+) transport is inhibited by Na(+) and Cs(+). Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na(+) tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs(+) tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K(+) and reduces the K i values for Na(+) and Cs(+), suggesting that the F130 residue may contribute to the structure of the pore region involved in K(+) binding. In addition, this mutation increases the V max for K(+). All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter.

A low K+ signal is required for functional high-affinity K+ uptake through HAK5 transporters.

The high-affinity K(+) transporter HAK5 is a key system for root K(+) uptake and, under very low external K(+), the only one capable of supplying K(+) to the plant. Functional HAK5-mediated K(+) uptake should be tightly regulated for plant adaptation to different environmental conditions. Thus, it has been described that the gene encoding the transporter is transcriptionally regulated, being highly induced under K(+) limitation. Here we show that environmental conditions, such as the lack of K(+), NO(3)(-) or P, that induced a hyperpolarization of the plasma membrane of root cells, induce HAK5 transcription. However, only the deprivation of K(+) produces functional HAK5-mediated K(+) uptake in the root. These results suggest on the one hand the existence of a posttranscriptional regulation of HAK5 elicited by the low K(+) signal and on the other that HAK5 may be involved in yet-unknown functions related to NO(3)(-) and P deficiencies. These results have been obtained here with Solanum lycopersicum (cv. Micro-Tom) as well as Arabidopsis thaliana plants, suggesting that the posttranscriptional regulation of high-affinity HAK transporters take place in all plant species.