N-myristoylation and S-acylation are common modifications of Ca2+ -regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel.

N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+ -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

The BIG protein distinguishes the process of CO2 -induced stomatal closure from the inhibition of stomatal opening by CO2.

We conducted an infrared thermal imaging-based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO2 . This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO2 insensitive 1 (cis1). BIG mutants are compromised in elevated CO2 -induced stomatal closure and bicarbonate activation of S-type anion channel currents. In contrast with the wild-type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO2 . However, like the wild-type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO2 . BIG mutants also display wild-type stomatal aperture responses to the closure-inducing stimulus abscisic acid (ABA). Our results indicate that BIG is a signaling component involved in the elevated CO2 -mediated control of stomatal development. In the control of stomatal aperture by CO2 , BIG is only required in elevated CO2 -induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO2 -mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO2 -mediated responses.

Phosphorylation of calcineurin B-like (CBL) calcium sensor proteins by their CBL-interacting protein kinases (CIPKs) is required for full activity of CBL-CIPK complexes toward their target proteins.

Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K(+) channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins.

FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²âº dynamics.

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.

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.

CBL-mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores.

During adaptation and developmental processes cells respond through nonlinear calcium-decoding signaling cascades, the principal components of which have been identified. However, the molecular mechanisms generating specificity of cellular responses remain poorly understood. Calcineurin B-like (CBL) proteins contribute to decoding calcium signals by specifically interacting with a group of CBL-interacting protein kinases (CIPKs). Here, we report the subcellular localization of all 10 CBL proteins from Arabidopsis and provide a cellular localization matrix of a plant calcium signaling network. Our findings suggest that individual CBL proteins decode calcium signals not only at the plasma membrane and the tonoplast, but also in the cytoplasm and nucleus. We found that distinct targeting signals located in the N-terminal domain of CBL proteins determine the spatially discrete localization of CBL/CIPK complexes by COPII-independent targeting pathways. Our findings establish the CBL/CIPK signaling network as a calcium decoding system that enables the simultaneous specific information processing of calcium signals emanating from different intra- and extracellular stores, and thereby provides a mechanism underlying the specificity of cellular responses.

A plastid protein crucial for Ca2+-regulated stomatal responses.

* Guard cell movements are regulated by environmental cues including, for example, elevations in extracellular Ca(2+) concentration. Here, the subcellular localization and physiological function of the Ca(2+)-sensing receptor (CAS) protein was investigated. * CAS protein localization was ascertained by microscopic analyses of green fluorescent protein (GFP) fusion proteins and biochemical fractionation assays. Comparative guard cell movement investigations were performed in wild-type and cas loss-of-function mutant lines of Arabidopsis thaliana. Cytoplasmic Ca(2+) dynamics were addressed in plants expressing the yellow cameleon reporter protein YC3.6. * This study identified CAS as a chloroplast-localized protein that is crucial for proper stomatal regulation in response to elevations of external Ca(2+). CAS fulfils this role through modulation of the cytoplasmic Ca(2+) concentration. * This work reveals a novel role of the chloroplast in cellular Ca(2+) signal transduction.

CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi.

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.