Summary: Calcium signalling involves sensor proteins that decode temporal and spatial changes in cellular Ca²⁺ concentration. Calcineurin B‐like proteins (CBLs) represent a unique family of plant calcium sensors that relay signals by interacting with a family of protein kinases, designated as CBL‐interacting protein kinases (CIPKs). In a reverse genetic screen for altered drought tolerance, we identified a loss‐of‐function allele of CIPK23 as exhibiting a drought‐tolerant phenotype. In the cipk23 mutant, reduced transpirational water loss from leaves coincides with enhanced ABA sensitivity of guard cells during opening as well as closing reactions, without noticeable alterations in ABA content in the plant. We identified the calcium sensors CBL1 and CBL9 as CIPK23‐interacting proteins that targeted CIPK23 to the plasma membrane in vivo. Expression analysis of the CIPK23, CBL1 and CBL9 genes suggested that they may function together in diverse tissues, including guard cells and root hairs. In addition, expression of the CIPK23 gene was induced by low‐potassium conditions, implicating a function of this gene product in potassium nutrition. Indeed, cipk23 mutants displayed severe growth impairment on media with low concentrations of potassium. This phenotype correlates with a reduced efficiency of K⁺ uptake into the roots. In support of the conclusion that CBL1 and CBL9 interact with and synergistically serve as upstream regulators of CIPK23, the cbl1 cbl9 double mutant, but not the cbl1 or cbl9 single mutants, exhibit altered phenotypes for stomatal responses and low‐potassium sensitivity. Together with the recent identification of the potassium channel AKT1 as a target of CIPK23, these results imply that plasma membrane‐localized CBL1– and CBL9–CIPK23 complexes simultaneously regulate K⁺ transport processes in roots and in stomatal guard cells.

Nutrient sensing is critical for plant adaptation to the environment. Because of extensive farming and erosion, low content of mineral nutrients such as potassium (K ⁺ ) in soils becomes a limiting factor for plant growth. In response to low-K conditions, plants enhance their capability of K ⁺ uptake through an unknown signaling mechanism. Here we report the identification of a Ca ²⁺ -dependent pathway for low-K response in Arabidopsis . We are not aware of any other example of a molecular pathway for a nutrient response in plants. Earlier genetic analyses revealed three genes encoding two Ca ²⁺ sensors (CBL1 and CBL9) and their target protein kinase (CIPK23) to be critical for plant growth on low-K media and for stomatal regulation, indicating that these calcium signaling components participate in the low-K response and turgor regulation. In this study, we show that the protein kinase CIPK23 interacted with, and phosphorylated, a voltage-gated inward K ⁺ channel (AKT1) required for K ⁺ acquisition in Arabidopsis . In the Xenopus oocyte system, our studies showed that interacting calcium sensors (CBL1 and CBL9) together with target kinase CIPK23, but not either component alone, activated the AKT1 channel in a Ca ²⁺ -dependent manner, connecting the Ca ²⁺ signal to enhanced K ⁺ uptake through activation of a K ⁺ channel. Disruption of both CBL1 and CBL9 or CIPK23 gene in Arabidopsis reduced the AKT1 activity in the mutant roots, confirming that the Ca ²⁺ -CBL-CIPK pathway functions to orchestrate transporting activities in planta according to external K ⁺ availability.

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.

Plants respond to environmental stress by activating “stress genes.” The plant hormone abscisic acid (ABA) plays an important role in stress-responsive gene expression. Although Ca²⁺ serves as a common second messenger in signaling stress and ABA, little is known about the molecular basis of Ca²⁺ action in these pathways. Here, we show that CIPK3, a Ser/Thr protein kinase that associates with a calcineurin B–like calcium sensor, regulates ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis. The expression of the CIPK3 gene itself is responsive to ABA and stress conditions, including cold, high salt, wounding, and drought. Disruption of CIPK3 altered the expression pattern of a number of stress gene markers in response to ABA, cold, and high salt. However, drought-induced gene expression was not altered in the cipk3 mutant plants, suggesting that CIPK3 regulates select pathways in response to abiotic stress and ABA. These results identify CIPK3 as a molecular link between stress- and ABA-induced calcium signal and gene expression in plant cells. Because the cold signaling pathway is largely independent of endogenous ABA production, CIPK3 represents a cross-talk “node” between the ABA-dependent and ABA-independent pathways in stress responses.

Summary: Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium‐binding proteins. In plants, the calcineurin B‐like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs). We report here that the CBL protein CBL10 functions as a crucial regulator of salt tolerance in Arabidopsis. Cbl10 mutant plants exhibited significant growth defects and showed hypersensitive cell death in leaf tissues under high‐salt conditions. Interestingly, the Na⁺ content of the cbl10 mutant, unlike other salt‐sensitive mutants identified thus far, was significantly lower than in the wild type under either normal or high‐salt conditions, suggesting that CBL10 mediates a novel Ca²⁺‐signaling pathway for salt tolerance. Indeed, the CBL10 protein physically interacts with the salt‐tolerance factor CIPK24 (SOS2), and the CBL10–CIPK24 (SOS2) complex is associated with the vacuolar compartments that are responsible for salt storage and detoxification in plant cells. These findings suggest that CBL10 and CIPK24 (SOS2) constitute a novel salt‐tolerance pathway that regulates the sequestration/compartmentalization of Na⁺ in plant cells. Because CIPK24 (SOS2) also interacts with CBL4 (SOS3) and regulates salt export across the plasma membrane, our study identifies CIPK24 (SOS2) as a multi‐functional protein kinase that regulates different aspects of salt tolerance by interacting with distinct CBL calcium sensors.

Potassium (K ⁺ ) is an essential nutrient for plant growth and development. Plants often adapt to low K ⁺ conditions by increasing their K ⁺ uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K ⁺ transporter 1 (AKT1), responsible for K ⁺ uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel. These findings provide evidence that the calcium-sensitive CBL and CIPK families together with 2C-type protein phosphatases form a protein phoshporylation/dephosphorylation network that regulates the AKT1 channel for K ⁺ transport in plants.