Reyes Ródenas

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

The protein kinase CIPK23 activates high-affinity K+ uptake in roots and is essential for growth in K+-limiting conditions. 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 Ca2+ 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.

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.

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 Ki 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 Vmax 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.

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.

High Ca2+ 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.

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.

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.

DELLAs Contribute to Set the Growth and Mineral Composition of Arabidopsis thaliana Plants Grown Under Conditions of Potassium Deprivation

DELLAs proteins play a major role in the modulation of plant responses to fluctuations in environmental conditions. In this work, we examined to what extent Arabidopsis thaliana plants lacking DELLAs activity (5xdella mutant) or carrying an altered function allele of one of the DELLAs coding genes (gai-1 mutant) display differential responses, in terms of growth and shoot elemental composition, relative to WT plants when deprived of potassium (K). Studies with plants grown in hydroponic media unveiled that the shoot mineral composition of gai-1 constitutively differs from that of WT and 5xdella plants. Tolerance to K-deprivation, as estimated by the relative decline of biomass accumulation, followed the order gai-1 > WT > 5xdella. In turn, the degree of responsiveness of the shoot composition to the stress condition followed the order 5xdella > WT > gai-1, suggesting a correspondence between the degree of injury and changes in the elemental composition. Internal efficiency of K-utilization was maximized in WT relative to 5xdella plants. Interestingly, the acquisition of K was severely impaired in gai-1 plants well supplied, or deprived of, K. Complementary studies indicated that influx and root-to-shoot transport of Rubidium, a K-analogue, were reduced in those plants. Furthermore, evidence obtained supports the view that the effect of altered DELLAs derives, at least partially, from controlling the accumulation of transcripts coding for the AtHAK5 transporter. These results, together with the observation that K-deprivation promotes the accumulation of a DELLA protein (RGA) fused to GFP in root cells, suggest a pivotal role of DELLAs in key plant responses to K-deprivation.

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

ABSTRACT 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.