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.

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.

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.

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.

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.

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.

SlCIPK9 regulates pollen tube elongation in tomato plants via a K+-independent mechanism.

Potassium (K+) is an essential macronutrient which contributes to osmotic- and turgor-related processes in plants. Calcineurin-B like Interacting Protein Kinases (CIPKs) play crucial roles in plants under low-K+ supply since they activate root K+ uptake transport systems such as AKT1 and AtHAK5. In Arabidopsis, AtCIPK9 is important for low-K+ tolerance since atcipk9 plants exhibited poor growth and leaf chlorosis when K+ was scarce. Part of these phenotypes could be ascribed to the activation of AtHAK5 by AtCIPK9. It has been reported that important differences exist between Arabidopsis and other plant species such as tomato with respect to the regulation of K+ uptake systems. Thus, our aim was to evaluate the contribution of SlCIPK9, the homologous protein of AtCIPK9 in tomato, to K+ nutrition. Unexpectedly, phenotyping experiments carried out with slcipk9 loss-of-function mutants revealed that SlCIPK9 did not play a clear role in tomato K+ homeostasis. By contrast, it was found that SlCIPK9 contributed to pollen tube elongation, but not to pollen germination, via a K+-independent mechanism. Therefore, our results highlight the remarkable differences that exist in Ca2+ signaling pathways between plant species and encourage the realization of more comparative studies as the one presented here.

Polyurethane Composites Recycling with Styrene-Acrylonitrile and Calcium Carbonate Recovery.

The glycolysis process of flexible polyurethane foams containing styrene-acrylonitrile and calcium carbonate as fillers was explored in detail. The use of DABCO as a catalyst allowed us to reduce the catalyst concentration and the polyurethane-to-glycol mass ratio to 0.1% and 1:1, respectively. The glycolysis process allowed us to obtain a high-purity polyol (99%), which can totally replace raw polyols in the synthesis of new flexible polyurethane foams, maintaining the standard mechanical properties of the original one and modifying the ratio of isocyanates employed to correct the closed cell structure caused by the impurities present in the recovered polyol. This isocyanate mixture was also optimized, resulting in a ratio of 30 and 70% of the isocyanates TDI80 and TDI65, respectively. Additionally, the fillers incorporated in the glycolyzed foams were recovered. Both recovered fillers, styrene-acrylonitrile and calcium carbonate, were fully characterized, showing a quality very similar to that of commercial compounds. Finally, the replacement of commercial fillers by the recovered ones in the synthesis of new polyurethane foams was studied, demonstrating the feasibility of using them in the synthesis of new foams without significantly altering their properties.

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.

Glycolysis of Polyurethanes Composites Containing Nanosilica.

Rigid polyurethane (RPU) foams have been successfully glycolyzed by using diethylene glycol (DEG) and crude glycerol (CG) as transesterification agents. However, DEG did not allow to achieve a split-phase process, obtaining a product with low polyol purity (61.7 wt %). On contrary, CG allowed to achieve a split-phase glycolysis improving the recovered polyol purity (76.5%). This is an important novelty since, up to now, RPUs were glycolyzed in single-phase processes giving products of low polyol concentration, which reduced the further applications. Moreover, the nanosilica used as filler of the glycolyzed foams was recovered completely pure. The recovered polyol successfully replaced up to 60% of the raw polyol in the synthesis of RPU foams and including the recovered nanosilica in the same concentration than in glycolyzed foam. Thus, the feasibility of the chemical recycling of this type of polyurethane composites has been demonstrated. Additionally, PU foams were synthesized employing fresh nanosilica to evaluate whether the recovered nanosilica has any influence on the RPU foam properties. These foams were characterized structurally, mechanically and thermally with the aim of proving that they met the specifications of commercial foams. Finally, the feasibility of recovering the of CG by vacuum distillation has been demonstrated.