The high-affinity K+ transporter HAK5 from Arabidopsis 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 3D-structure 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 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 proteinprotein 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.
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 Ca 2+ , Mg 2+ , Na + , NO 3 − , 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.
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