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• Low concentrations of CO₂ cause stomatal opening, whereas [CO₂] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO₂-induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+-binding proteins that function in the stomatal CO₂ response remain unknown. • Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. • We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO₂, light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO₂-induced stomatal closure and, unexpectedly, also in low CO₂-induced stomatal opening. Furthermore, K⁺-uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO₂-regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. • Our findings describe combinatorial cpk mutants that function in CO₂ control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.

Summary In plants, potassium (K+) homeostasis is tightly regulated and established against a concentration gradient to the environment. Despite the identification of Ca2+‐regulated kinases as modulators of K+ channels, the immediate signaling and adaptation mechanisms of plants to low‐K+ conditions are only partially understood. To assess the occurrence and role of Ca2+ signals in Arabidopsis thaliana roots, we employed ratiometric analyses of Ca2+ dynamics in plants expressing the Ca2+ reporter YC3.6 in combination with patch‐clamp analyses of root cells and two‐electrode voltage clamp (TEVC) analyses in Xenopus laevis oocytes. K+ deficiency triggers two successive and distinct Ca2+ signals in roots exhibiting spatial and temporal specificity. A transient primary Ca2+ signature arose within 1 min in the postmeristematic stelar tissue of the elongation zone, while a secondary Ca2+ response occurred after several hours as sustained Ca2+ elevation in defined tissues of the elongation and root hair differentiation zones. Patch‐clamp and TEVC analyses revealed Ca2+ dependence of the activation of the K+ channel AKT1 by the CBL1–CIPK23 Ca2+ sensor‐kinase complex. Together, these findings identify a critical role of cell group‐specific Ca2+ signaling in low K+ responses and indicate an essential and direct role of Ca2+ signals for AKT1 K+ channel activation in roots.

Potassium (K + ) channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K + channels remain poorly understood. Here, we show that the calcium (Ca 2+ ) sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM) targeting of the K + channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2 , cbl4 and cipk6 mutants share similar developmental and delayed flowering phenotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4- and Ca 2+ -dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca 2+ sensor modulates K + channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.

Fluorescence complementation (FC) techniques are expedient for analyzing bimolecular protein–protein interactions. Here we aimed to develop a method for visualization of ternary protein complexes using dual-color trimolecular fluorescence complementation (TriFC). Dual-color TriFC combines protein fragments of mCherry and mVenus, in which a scaffold protein is bilaterally fused to C-terminal fragments of both fluorescent proteins and combined with potential interacting proteins fused to an N-terminal fluorescent protein fragment. For efficient visual verification of ternary complex formation, TriFC was combined with a cytoplasm to plasma membrane translocation assay. Modular vector sets were designed which are fully compatible with previously reported bimolecular fluorescence complementation (BiFC) vectors. As a proof-of-principle, the ternary complex formation of the PP2B protein phosphatase Calcineurin-A/Calcineurin-B with Calmodulin-2 was investigated in transiently transformed Nicotiana benthamiana leaf epidermal cells. The results indicate a Calcineurin-B-induced interaction of Calmodulin-2 with Calcineurin-A. TriFC and the translocation of TriFC complexes provide a novel tool to investigate ternary complex formations with the simplicity of a BiFC approach. The robustness of FC applications and the opportunity to quantify fluorescence complementation render this assay suitable for a broad range of interaction analyses.

The collective function of calcineurin B-like (CBL) calcium ion (Ca2+ ) sensors and CBL-interacting protein kinases (CIPKs) in decoding plasma-membrane-initiated Ca2+ signals to convey developmental and adaptive responses to fluctuating nitrate availability remained to be determined. Here, we generated a cbl-quintuple mutant in Arabidopsis thaliana devoid of these Ca2+ sensors at the plasma membrane and performed comparative phenotyping, nitrate flux determination, phosphoproteome analyses, and studies of membrane domain protein distribution in response to low and high nitrate availability. We observed that CBL proteins exert multifaceted regulation of primary and lateral root growth and nitrate fluxes. Accordingly, we found that loss of plasma membrane Ca2+ sensor function simultaneously affected protein phosphorylation of numerous membrane proteins, including several nitrate transporters, proton pumps, and aquaporins, as well as their distribution within plasma membrane microdomains, and identified a specific phosphorylation and domain distribution pattern during distinct phases of low and high nitrate responses. Collectively, these analyses reveal a central and coordinative function of CBL-CIPK-mediated signaling in conveying plant adaptation to fluctuating nitrate availability and identify a crucial role of Ca2+ signaling in regulating the composition and dynamics of plasma membrane microdomains.

Calcium (Ca2+) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca2+- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca2+-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 angstrom resolution, revealed an unusual calmodulin-fold of the Ca2+-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca2+-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca2+-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.

Low concentrations of CO 2 cause stomatal opening, whereas [CO 2 ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca 2+ and protein phosphorylation in CO 2 ‐induced stomatal closing. Calcium‐dependent protein kinases (CPKs) and calcineurin‐B‐like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca 2+ into specific phosphorylation events. However, Ca 2+ ‐binding proteins that function in the stomatal CO 2 response remain unknown. Time‐resolved stomatal conductance measurements using intact plants, and guard cell patch‐clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO 2 , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO 2 ‐induced stomatal closure and, unexpectedly, also in low CO 2 ‐induced stomatal opening. Furthermore, K + ‐uptake‐channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light‐mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO 2 ‐regulated stomatal movement kinetics were not clearly affected in plasma membrane‐targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO 2 control of stomatal movements and support the results of classical studies showing a role for Ca 2+ in this response.

* In plants, potassium (K super(+)) homeostasis is tightly regulated and established against a concentration gradient to the environment. Despite the identification of Ca super(2+)-regulated kinases as modulators of K super(+) channels, the immediate signaling and adaptation mechanisms of plants to low-K super(+) conditions are only partially understood. * To assess the occurrence and role of Ca super(2+) signals in Arabidopsis thaliana roots, we employed ratiometric analyses of Ca super(2+) dynamics in plants expressing the Ca super(2+) reporter YC3.6 in combination with patch-clamp analyses of root cells and two-electrode voltage clamp (TEVC) analyses in Xenopus laevis oocytes. * K super(+) deficiency triggers two successive and distinct Ca super(2+) signals in roots exhibiting spatial and temporal specificity. A transient primary Ca super(2+) signature arose within 1 min in the postmeristematic stelar tissue of the elongation zone, while a secondary Ca super(2+) response occurred after several hours as sustained Ca super(2+) elevation in defined tissues of the elongation and root hair differentiation zones. Patch-clamp and TEVC analyses revealed Ca super(2+) dependence of the activation of the K super(+) channel AKT1 by the CBL1-CIPK23 Ca super(2+) sensor-kinase complex. * Together, these findings identify a critical role of cell group-specific Ca super(2+) signaling in low K super(+) responses and indicate an essential and direct role of Ca super(2+) signals for AKT1 K super(+) channel activation in roots.

In plants, potassium (K+) homeostasis is tightly regulated and established against a concentration gradient to the environment. Despite the identification of Ca2+-regulated kinases as modulators of K+ channels, the immediate signaling and adaptation mechanisms of plants to low-K+ conditions are only partially understood. To assess the occurrence and role of Ca2+ signals in Arabidopsis thaliana roots, we employed ratiometric analyses of Ca2+ dynamics in plants expressing the Ca2+ reporter YC3.6 in combination with patch-clamp analyses of root cells and two-electrode voltage clamp (TEVC) analyses in Xenopus laevis oocytes. K+ deficiency triggers two successive and distinct Ca2+ signals in roots exhibiting spatial and temporal specificity. A transient primary Ca2+ signature arose within 1 min in the post-meristematic stelar tissue of the elongation zone, while a secondary Ca2+ response occurred after several hours as sustained Ca2+ elevation in defined tissues of the elongation and root hair differentiation zones. Patch-clamp and TEVC analyses revealed Ca2+ dependence of the activation of the K+ channel AKT1 by the CBL1–CIPK23 Ca2+ sensor-kinase complex. Together, these findings identify a critical role of cell group-specific Ca2+ signaling in low K+ responses and indicate an essential and direct role of Ca2+ signals for AKT1 K+ channel activation in roots.

In plants, potassium (K + ) homeostasis is tightly regulated and established against a concentration gradient to the environment. Despite the identification of Ca 2+ ‐regulated kinases as modulators of K + channels, the immediate signaling and adaptation mechanisms of plants to low‐K + conditions are only partially understood. To assess the occurrence and role of Ca 2+ signals in Arabidopsis thaliana roots, we employed ratiometric analyses of Ca 2+ dynamics in plants expressing the Ca 2+ reporter YC 3.6 in combination with patch‐clamp analyses of root cells and two‐electrode voltage clamp (TEVC) analyses in Xenopus laevis oocytes. K + deficiency triggers two successive and distinct Ca 2+ signals in roots exhibiting spatial and temporal specificity. A transient primary Ca 2+ signature arose within 1 min in the postmeristematic stelar tissue of the elongation zone, while a secondary Ca 2+ response occurred after several hours as sustained Ca 2+ elevation in defined tissues of the elongation and root hair differentiation zones. Patch‐clamp and TEVC analyses revealed Ca 2+ dependence of the activation of the K + channel AKT 1 by the CBL 1– CIPK 23 Ca 2+ sensor‐kinase complex. Together, these findings identify a critical role of cell group‐specific Ca 2+ signaling in low K + responses and indicate an essential and direct role of Ca 2+ signals for AKT 1 K + channel activation in roots.