Two calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis

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.     

The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis

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 Ca(2+)-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.     

CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana

The family of calcineurin B-like (CBL) proteins is a unique group of Ca²⁺ sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL-CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL-CIPK involved in cold stress responses. In this study, we analyzed expression of CIPK7 and CBL1 in Arabidopsis during cold treatments. Expression of CIPK7 was induced by cold, and CIPK7 interacted with CBL1 in vitro. Moreover, affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo. Expression of CBL1 was cold inducible, and CBL1 had a role in regulating cold response. By comparing expression patterns of CIPK7 between wild-type and cbl1 mutant plants, we found the induction of CIPK7 by cold stress was influenced by CBL1. This is the first report to demonstrate that CIPK7 may play a role in cold response via its interaction with CBL1.     

The calcium sensor PeCBL1, interacting with PeCIPK24/25 and PeCIPK26, regulates Na+/K+ homeostasis in Populus euphratica

Key message

This paper is the first to directly link two types of ion channel regulation pathway into an emerging and complex CBL–CIPK signal system in wooden plant.

Abstract

In Arabidopsis thaliana, the calcineurin b-like (CBL) 1 gene has been shown to be necessary in response to abiotic stresses. In this study, we identified CBL1 in the woody plant Populus euphratica, designated as PeCBL1. Heterologous expression of PeCBL1 could build the resistance of sensitive phenotypes to low K⁺ stress in the corresponding Arabidopsis cbl1/cbl9 mutant, and display a salt-sensitive phenotype compared with the mutant. Protein interaction analysis showed that PeCBL1 can interact with PeCIPK24, 25 and 26, and form different complexes of PeCBL–PeCIPK. To further investigate the mechanism of PeCBL1, we analyzed the fluxes of K⁺ and Na⁺ in roots of the wild-type Arabidopsis, cbl1/9 mutant, and PeCBL1 transgenic plants under low K⁺ stress and high Na⁺ stress. These analyses revealed that, compared to the cbl1/9 mutant, the PeCBL1 transgenic plant roots exhibited a higher capacity to absorb K⁺ after exposure to low K⁺ stress, and a lower capacity to discharge Na⁺ after exposure to salt stress. The results suggest that CBL1 interacts with CIPK24, CIPK25 and CIPK26 to regulate Na⁺/K⁺ homeostasis in Populus euphratica.     

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

Plant responses to developmental and environmental cues are often mediated by calcium (Ca²⁺) signals that are transmitted by diverse calcium sensors. The calcineurin B-like (CBL) protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases. We report functional analysis of Arabidopsis CBL2 and CBL3, two closely related CBL members that are localized to the vacuolar membrane through the N-terminal tonoplast-targeting sequence. While cbl2 or cbl3 single mutant did not show any phenotypic difference from the wild type, the cbl2 cbl3 double mutant was stunted with leaf tip necrosis, underdeveloped roots, shorter siliques and fewer seeds. These defects were reminiscent of those in the vha-a2 vha-a3 double mutant deficient in vacuolar H⁺-ATPase (V-ATPase). Indeed, the V-ATPase activity was reduced in the cbl2 cbl3 double mutant, connecting tonoplast CBL-type calcium sensors to the regulation of V-ATPase. Furthermore, cbl2 cbl3 double mutant was compromised in ionic tolerance and micronutrient accumulation, consistent with the defect in V-ATPase activity that has been shown to function in ion compartmentalization. Our results suggest that calcium sensors CBL2 and CBL3 serve as molecular links between calcium signaling and V-ATPase, a central regulator of intracellular ion homeostasis.     

Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex

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.     

CBL1, a calcium sensor that differentially regulates salt,drought, and cold responses in Arabidopsis

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.     

Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca2+ signaling complexes in Arabidopsis

Arabidopsis thaliana calcineurin B-like proteins (CBLs) interact specifically with a group of CBL-interacting protein kinases (CIPKs). CBL/CIPK complexes phosphorylate target proteins at the plasma membrane. Here, we report that dual lipid modification is required for CBL1 function and for localization of this calcium sensor at the plasma membrane. First, myristoylation targets CBL1 to the endoplasmic reticulum. Second, S-acylation is crucial for endoplasmic reticulum-to-plasma membrane trafficking via a novel cellular targeting pathway that is insensitive to brefeldin A. We found that a 12-amino acid peptide of CBL1 is sufficient to mediate dual lipid modification and to confer plasma membrane targeting. Moreover, the lipid modification status of the calcium sensor moiety determines the cellular localization of preassembled CBL/CIPK complexes. Our findings demonstrate the importance of S-acylation for regulating the spatial accuracy of Ca2+-decoding proteins and suggest a novel mechanism that enables the functional specificity of calcium sensor/kinase complexes.     

The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis

Calcium plays a pivotal role in plant responses to several stimuli, including pathogens, abiotic stresses, and hormones. However, the molecular mechanisms underlying calcium functions are poorly understood. It is hypothesized that calcium serves as second messenger and, in many cases, requires intracellular protein sensors to transduce the signal further downstream in the pathways. The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plant cells. Here, we report our analysis of the CBL9 member of this gene family. Expression of CBL9 was inducible by multiple stress signals and abscisic acid (ABA) in young seedlings. When CBL9 gene function was disrupted in Arabidopsis thaliana plants, the responses to ABA were drastically altered. The mutant plants became hypersensitive to ABA in the early developmental stages, including seed germination and post-germination seedling growth. In addition, seed germination in the mutant also showed increased sensitivity to inhibition by osmotic stress conditions produced by high concentrations of salt and mannitol. Further analyses indicated that increased stress sensitivity in the mutant may be a result of both ABA hypersensitivity and increased accumulation of ABA under the stress conditions. The cbl9 mutant plants showed enhanced expression of genes involved in ABA signaling, such as ABA-INSENSITIVE 4 and 5. This study has identified a calcium sensor as a common element in the ABA signaling and stress-induced ABA biosynthesis pathways.     

Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress

Many environmental stimuli, including light, biotic and abiotic stress factors, induce changes in cellular Ca(2+) concentrations in plants. Such Ca(2+) signatures are perceived by sensor molecules such as calcineurin B-like (CBL) proteins. AtCBL1, a member of the CBL family which is highly inducible by multiple stress signals, is known to function in the salt stress signal transduction pathway and to positively regulate the plant tolerance to salt. To shed light into the molecular mechanisms of the salt stress response mediated by AtCBL1, a two-dimensional DIGE proteomic approach was applied to identify the differentially expressed proteins in Arabidopsis wild-type and cbl1 null mutant plants in response to salt stress. Seventy-three spots were found altered in expression by least 1.2-fold and 50 proteins were identified by MALDI-TOF/TOF-MS, including some well-known and novel salt-responsive proteins. These proteins function in various processes, such as signal transduction, ROS scavenging, energy production, carbon fixation, metabolism, mRNA processing, protein processing and structural stability. Receptor for activated C kinase 1C (RACK1C, spot 715), a WD40 repeat protein, was up-regulated in the cbl1 null mutant, and two rack1c mutant lines showed decreased tolerance to salt stress, suggesting that RACK1C plays a role in salt stress resistance. In conclusion, our work demonstrated the advantages of the proteomic approach in studies of plant biology and identified candidate proteins in CBL1-mediated salt stress signaling network.     

A Novel Role for Arabidopsis CBL1 in Affecting Plant Responses to Glucose and Gibberellin during Germination and Seedling Development

Glucose and phytohormones such as abscisic acid (ABA), ethylene, and gibberellin (GA) coordinately regulate germination and seedling development. However, there is still inadequate evidence to link their molecular roles in affecting plant responses. Calcium acts as a second messenger in a diverse range of signal transduction pathways. As calcium sensors unique to plants, calcineurin B-like (CBL) proteins are well known to modulate abiotic stress responses. In this study, it was found that CBL1 was induced by glucose in Arabidopsis. Loss-of-function mutant cbl1 exhibited hypersensitivity to glucose and paclobutrazol, a GA biosynthetic inhibitor. Several sugar-responsive and GA biosynthetic gene expressions were altered in the cbl1 mutant. CBL1 protein physically interacted with AKINβ1, the regulatory β subunit of the SnRK1 complex which has a central role in sugar signaling. Our results indicate a novel role for CBL1 in modulating responses to glucose and GA signals.     

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.     

A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis

Potassium is an essential mineral element for plant growth and development. Although it is known that plants absorb and transport K+ through membrane transporters, it remains unclear how these transporters are regulated. Here we show that the protein kinase CIPK23, encoded by the LKS1 gene, regulates K+ uptake under low-K+ conditions. Lesion of LKS1 significantly reduced K+ uptake and caused leaf chlorosis and growth inhibition, whereas overexpression of LKS1 significantly enhanced K+ uptake and tolerance to low K+. We demonstrate that CIPK23 directly phosphorylates the K+ transporter AKT1 and further find that CIPK23 is activated by the binding of two calcineurin B-like proteins, CBL1 and CBL9. We propose a model in which the CBL1/9-CIPK23 pathway ensures activation of AKT1 and enhanced K+ uptake under low-K+ conditions.     

Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network

Plant development and reproduction depend on a precise recognition of environmental conditions and the integration of this information with endogenous metabolic and developmental cues. Calcium ions have been firmly established as ubiquitous second messengers functioning in these processes. Calcium signal deciphering and signal-response coupling often involve calcium-binding proteins as responders or relays in this information flow. Here we review the calcineurin B-like protein (CBL) calcium sensor/CBL-interacting protein kinase (CIPK) network as a newly emerging signaling system mediating a complex array of environmental stimuli. We focus particularly on the mechanisms generating signaling specificity. Moreover, we emphasize the functional implications that are emerging from the analyses of CBL and CIPK loss-of-function mutants.     

The CBL–CIPK network mediates different signaling pathways in plants

The calcineurin B-like protein–CBL-interacting protein kinase (CBL–CIPK) signaling pathway in plants is a Ca²⁺-related pathway that responds strongly to both abiotic and biotic environmental stimuli. The CBL–CIPK system shows variety, specificity, and complexity in response to different stresses, and the CBL–CIPK signaling pathway is regulated by complex mechanisms in plant cells. As a plant-specific Ca²⁺ sensor relaying pathway, the CBL–CIPK pathway has some crosstalk with other signaling pathways. In addition, research has shown that there is crosstalk between the CBL–CIPK pathway and the low-K⁺ response pathway, the ABA signaling pathway, the nitrate sensing and signaling pathway, and others. In this paper, we summarize and review research discoveries on the CBL–CIPK network. We focus on the different modification and regulation mechanisms (phosphorylation and dephosphorylation, dual lipid modification) of the CBL–CIPK network, the expression patterns and functions of CBL–CIPK network genes, the responses of this network to abiotic stresses, and its crosstalk with other signaling pathways. We also discuss the technical research methods used to analyze the CBL–CIPK network and some of its newly discovered functions in plants.     

Calcineurin B‐like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis

Potassium transporters and channels play crucial roles in K⁺ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K⁺ channel in Arabidopsis roots that is involved in K⁺ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B‐like proteins CBL1/CBL9. The present study showed that another calcineurin B‐like protein (CBL10) may also regulate AKT1 activity. The CBL10‐over‐expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low‐K⁺ conditions. In addition, the K⁺ content of both CBL10‐over‐expressing lines and akt1 mutant plants were significantly reduced compared with wild‐type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two‐hybrid, BiFC and co‐immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1‐mediated inward K⁺ currents. Furthermore, the results from the yeast two‐hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL‐interacting protein kinase‐independent regulatory mechanism of calcineurin B‐like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.