Increased tolerance to low K+, and to cationic stress of Arabidopsis plants by expressing the F130S mutant version of the K+ transporter AtHAK5.

Potassium (K+) selectivity of high-affinity K+ uptake systems is crucial for plant growth under low K+ and in the presence of inhibitors of K+ uptake that are toxic to plants such as Na+ or Cs+. Here, we express a mutated version of the Arabidopsis AtHAK5 high-affinity K+ transporter consisting on a change of phenylalanine 130 to serine (F130S) in athak5 akt1 double mutant plants. F130S-expressing plants show better growth, increased K+ uptake from low external concentrations and higher K+ contents when grown at low K+ (10 µM) and when grown at low K+ in the presence of Na+ (15 mM) or Cs+ (1 µM). In addition, these plants accumulate less Na+ and Cs+, resulting in lower Na+/K+ and Cs+/K+ ratios, which are important determinants of plant tolerance to salt stress and to Cs+-polluted soils. Structure analysis of AtHAK5 suggest that the F130 residue approaches the intracellular gate of the K+ tunnel of AtHAK5, affecting somehow its ionic selectivity. Modification of transport systems has a large potential to face challenges of future agriculture such as sustainable production under abiotic stress conditions imposed by climate change.

Relevance of the SlCIPK23 kinase in Na+ uptake and root morphology in K+-starved tomato plants.

The beneficial effects of Na+ as a substitute for K+ have been well-documented at the physiological level. However, the transport systems and regulatory mechanisms that allow Na+ acquisition under K+ deficiency remain poorly understood in the majority of land plants. In tomato, SlCIPK23 kinase was involved in Na+ accumulation in K+-starved plants, in addition to activating the LKT1 K+ channel and the K+ transporter SlHAK5. We used the central role of SlCIPK23 in K+ and Na+ acquisition to study which molecular entities mediate Na+ uptake with knockout tomato mutants and expression in heterologous systems. Two main pathways for Na+ uptake were deduced in tomato plants: an NH4+-sensitive pathway dependent on SlCIPK23, and a second one sensitive to Ba2+, Ca2+, La3+, and Li+. The addition of Na+ (10 mM) to lkt1, slhak5, or slcipk23 mutant KO lines produced interesting changes in root morphology. In particular, the roots of slcipk23 plants were longer and lighter than those of the WT under K+-deficient conditions and this effect was reversed by the addition of 10 mM Na+. These results provide a stimulating perspective for the study of the beneficial effects of Na+ in crops.

Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 uncovers CIPK-CBL-target network rewiring in land plants.

Transport of K+ to the xylem is a key process in the mineral nutrition of the shoots. Although CIPK-CBL complexes have been widely shown to regulate K+ uptake transport systems, no information is available about the xylem ones. Here, we studied the physiological roles of the voltage-gated K+ channel SlSKOR and its regulation by the SlCIPK23-SlCBL1/9 complexes in tomato plants. We phenotyped gene-edited slskor and slcipk23 tomato knockout mutants and carried out two-electrode voltage-clamp (TEVC) and BiFC assays in Xenopus oocytes as key approaches. SlSKOR was preferentially expressed in the root stele and was important not only for K+ transport to shoots but also, indirectly, for that of Ca2+ , Mg2+ , Na+ , NO3 - , and Cl- . Surprisingly, the SlCIPK23-SlCBL1/9 complexes turned out to be negative regulators of SlSKOR. Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 was observed in Xenopus oocytes and tomato plants. Regulation of SKOR-like channels by CIPK23-CBL1 complexes was also present in Medicago, grapevine, and lettuce but not in Arabidopsis and saltwater cress. Our results provide a molecular framework for coordinating root K+ uptake and its translocation to the shoot by SlCIPK23-SlCBL1/9 in tomato plants. Moreover, they evidenced that CIPK-CBL-target networks have evolved differently in land plants.

Non-autonomous stomatal control by pavement cell turgor via the K+ channel subunit AtKC1.

Stomata optimize land plants' photosynthetic requirements and limit water vapor loss. So far, all of the molecular and electrical components identified as regulating stomatal aperture are produced, and operate, directly within the guard cells. However, a completely autonomous function of guard cells is inconsistent with anatomical and biophysical observations hinting at mechanical contributions of epidermal origins. Here, potassium (K+) assays, membrane potential measurements, microindentation, and plasmolysis experiments provide evidence that disruption of the Arabidopsis thaliana K+ channel subunit gene AtKC1 reduces pavement cell turgor, due to decreased K+ accumulation, without affecting guard cell turgor. This results in an impaired back pressure of pavement cells onto guard cells, leading to larger stomatal apertures. Poorly rectifying membrane conductances to K+ were consistently observed in pavement cells. This plasmalemma property is likely to play an essential role in K+ shuttling within the epidermis. Functional complementation reveals that restoration of the wild-type stomatal functioning requires the expression of the transgenic AtKC1 at least in the pavement cells and trichomes. Altogether, the data suggest that AtKC1 activity contributes to the building of the back pressure that pavement cells exert onto guard cells by tuning K+ distribution throughout the leaf epidermis.

The protein kinase SlCIPK23 boosts K+ and Na+ uptake in tomato plants.

Regulation of root transport systems is essential under fluctuating nutrient supply. In the case of potassium (K+ ), HAK/KUP/KT K+ transporters and voltage-gated K+ channels ensure root K+ uptake in a wide range of K+ concentrations. In Arabidopsis, the CIPK23/CBL1-9 complex regulates both transporter- and channel-mediated root K+ uptake. However, research about K+ homeostasis in crops is in demand due to species-specific mechanisms. In the present manuscript, we studied the contribution of the voltage-gated K+ channel LKT1 and the protein kinase SlCIPK23 to K+ uptake in tomato plants by analysing gene-edited knockout tomato mutant lines, together with two-electrode voltage-clamp experiments in Xenopus oocytes and protein-protein interaction analyses. It is shown that LKT1 is a crucial player in tomato K+ nutrition by contributing approximately 50% to root K+ uptake under K+ -sufficient conditions. Moreover, SlCIPK23 was responsible for approximately 100% of LKT1 and approximately 40% of the SlHAK5 K+ transporter activity in planta. Mg+2 and Na+ compensated for K+ deficit in tomato roots to a large extent, and the accumulation of Na+ was strongly dependent on SlCIPK23 function. The role of CIPK23 in Na+ accumulation in tomato roots was not conserved in Arabidopsis, which expands the current set of CIPK23-like protein functions in plants.

Involvement of salicylic acid in the response to potassium deficiency revealed by metabolomics.

Potassium (K) deficiency has consequences not only on cellular ion balance and transmembrane potential but also on metabolism. In fact, several enzymes are K-dependent including enzymes in catabolism, causing an alteration in glycolysis and respiration. In addition, K deficiency is associated with the induction of specific pathways and accumulation of metabolic biomarkers, such as putrescine. However, such drastic changes are usually observed when K deficiency is established. Here, we carried out a kinetic analysis with metabolomics to elucidate early metabolic events when nutrient conditions change from K-sufficiency to K-deficiency in Arabidopsis rosettes from both wild type and mutants affected in both K absorption and low-K signalling (hak5 akt1 cipk23). Our results show that mutants have a metabolomics pattern similar to K-deficient wild-type, showing a constitutive metabolic response to low K. In addition, shifting to low K conditions induces (i) changes in sugar metabolism and (ii) an accumulation of salicylic acid metabolites before the appearance of biomarkers of K deficiency (putrescine and aconitate), and such an accumulation is more pronounced in mutants. Our results suggest that early events in the response to low K conditions involve salicylic acid metabolism.

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.

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.

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.

Functional characterization and physiological roles of the single Shaker outward K+ channel in Medicago truncatula.

The model legume Medicago truncatula possesses a single outward Shaker K+ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K+ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K+ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch-clamp experiments on root hair protoplasts, besides the Shaker-type slowly activating outwardly rectifying K+ conductance encoded by MtGORK, a second K+ -permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula. A knock-out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K+ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod-factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.

Doing 'business as usual' comes with a cost: evaluating energy cost of maintaining plant intracellular K+ homeostasis under saline conditions.

Salinization of agricultural lands is a major threat to agriculture. Many different factors affect and determine plant salt tolerance. Nonetheless, there is a consensus on the relevance of maintaining an optimal cytosolic potassium : sodium ion (K+  : Na+ ) ratio for salinity tolerance in plants. This ratio depends on the operation of plasma membrane and tonoplast transporters. In the present review we focus on some aspects related to the energetic cost of maintaining that K+  : Na+ ratio. One of the factors that affect the cost of the first step of K+ acquisition - root K+ uptake through High Affinity K+ transporter and Arabidopsis K+ transport system 1 transport systems - is the value of the plasma membrane potential of root cells, a parameter that may differ amongst plant species. In addition to its role in nutrition, cytosolic K+ also is important for signalling, and K+ efflux through gated outward-rectifying K+ and nonselective cation channels can be regarded as a switch to redirect energy towards defence reactions. In maintaining cytosolic K+ , the great buffer capacity of the vacuole should be considered. The possible role of high-affinity K+ transporters (HKT)2s in mediating K+ uptake under saline conditions and the importance of cycling of K+ throughout the plant also are discussed.

Genome-wide identification and expression analysis of two component system genes in Cicer arietinum.

The two-component system (TCS) plays an important role in signal transduction pathways, cytokinin signaling and stress resistance of prokaryotes and eukaryotes. It is comprised of three types of proteins in plants; histidine kinases (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide with special economic value in semi-arid tropics. Availability of complete genome sequence of chickpea presents a valuable resource for comparative analysis among angiosperms. In current study, Arabidopsis thaliana and Oryza sativa were used as reference plant species for comparative genomics analysis with C. arietinum. A genome-wide computational survey enabled us to identify putative members of TCS protein family including 18HKs, 26 RRs (7 type-A, 7 type-B, 2 type C and 10 pseudo) and 7 HPs (5 true and 2pseudo) genes in chickpea. The predicted TCS genes displayed family specific intron/exon organization and were randomly distributed across all the eight chromosomes. Comparative phylogenetic and evolutionary analysis suggested a variable conservation of TCS genes in relation to mono/dicot model plants and segmental duplication was the principal route of expansion for this family in chickpea. The promoter regions of TCS genes exhibited several abiotic stress-related cis-elements indicating their involvement in abiotic stress response. The expression analysis of TCS genes demonstrated stress (drought, heat, osmotic and salt) specific differential expression. Current study provides insight into TCS genes in C. arietinum, which will be helpful for further functional analysis of these genes in response to different abiotic stresses.

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.

Characterization of the grapevine Shaker K+ channel VvK3.1 supports its function in massive potassium fluxes necessary for berry potassium loading and pulvinus-actuated leaf movements.

In grapevine, climate changes lead to increased berry potassium (K+ ) contents that result in must with low acidity. Consequently, wines are becoming 'flat' to the taste, with poor organoleptic properties and low potential aging, resulting in significant economic loss. Precise investigation into the molecular determinants controlling berry K+ accumulation during its development are only now emerging. Here, we report functional characterization by electrophysiology of a new grapevine Shaker-type K+ channel, VvK3.1. The analysis of VvK3.1 expression patterns was performed by qPCR and in situ hybridization. We found that VvK3.1 belongs to the AKT2 channel phylogenetic branch and is a weakly rectifying channel, mediating both inward and outward K+ currents. We showed that VvK3.1 is highly expressed in the phloem and in a unique structure located at the two ends of the petiole, identified as a pulvinus. From the onset of fruit ripening, all data support the role of the VvK3.1 channel in the massive K+ fluxes from the phloem cell cytosol to the berry apoplast during berry K+ loading. Moreover, the high amount of VvK3.1 transcripts detected in the pulvinus strongly suggests a role for this Shaker in the swelling and shrinking of motor cells involved in paraheliotropic leaf movements.

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.

Using Tomato Recombinant Lines to Improve Plant Tolerance to Stress Combination Through a More Efficient Nitrogen Metabolism.

The development of plant varieties with a better nitrogen use efficiency (NUE) is a means for modern agriculture to decrease environmental pollution due to an excess of nitrate and to maintain a sufficient net income. However, the optimum environmental conditions for agriculture will tend to be more adverse in the coming years, with increases in temperatures, water scarcity, and salinity being the most important productivity constrains for plants. NUE is inherently a complex trait, as each step, including N uptake, translocation, assimilation, and remobilization, is governed by multiple interacting genetic and environmental factors. In this study, two recombinant inbred lines (RIL-66 and RIL-76) from a cross between Solanum lycopersicum and Solanum pimpinellifoilum with different degree of tolerance to the combination of salinity and heat were subjected to a physiological, ionomic, amino acid profile, and gene expression study to better understand how nitrogen metabolism is affected in tolerant plants as compared to sensitive ones. The ionomics results showed a different profile between the two RILs, with K+ and Mg2+ being significantly lower in RIL-66 (low tolerant) as compared to RIL-76 (high tolerant) under salinity and heat combination. No differences were shown between the two RILs in N total content; however, N-NO3 - was significantly higher in RIL-66, whereas N-Norg was lower as compared to the other genotype, which could be correlated with its tolerance to the combination of salinity and heat. Total proteins and total amino acid concentration were significantly higher in RIL-76 as compared to the sensitive recombinant line under these conditions. Glutamate, but more importantly glutamine, was also highly synthesized and accumulated in RIL-76 under the combination of salinity and heat, which was in agreement with the upregulation of the nitrogen metabolism related transcripts studied (SlNR, SlNiR, SlGDH, SlGLT1, SlNRT1.2, SlAMT1, and SlAMT2). This study emphasized the importance of studying abiotic stress in combination and how recombinant material with different degrees of tolerance can be highly important for the improvement of nitrogen use efficiency in horticultural plants through the targeting of N-related markers.

Coping With Water Shortage: An Update on the Role of K+, Cl-, and Water Membrane Transport Mechanisms on Drought Resistance.

Drought is now recognized as the abiotic stress that causes most problems in agriculture, mainly due to the strong water demand from intensive culture and the effects of climate change, especially in arid/semi-arid areas. When plants suffer from water deficit (WD), a plethora of negative physiological alterations such as cell turgor loss, reduction of CO2 net assimilation rate, oxidative stress damage, and nutritional imbalances, among others, can lead to a decrease in the yield production and loss of commercial quality. Nutritional imbalances in plants grown under drought stress occur by decreasing water uptake and leaf transpiration, combined by alteration of nutrient uptake and long-distance transport processes. Plants try to counteract these effects by activating drought resistance mechanisms. Correct accumulation of salts and water constitutes an important portion of these mechanisms, in particular of those related to the cell osmotic adjustment and function of stomata. In recent years, molecular insights into the regulation of K+, Cl-, and water transport under drought have been gained. Therefore, this article brings an update on this topic. Moreover, agronomical practices that ameliorate drought symptoms of crops by improving nutrient homeostasis will also be presented.