SummaryThe 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 proteinprotein 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 calciummediated 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.

The plant hormone abscisic acid (ABA) mediates seed dormancy, controls seedling development and triggers tolerance to abiotic stresses, including drought. Core ABA signaling components consist of a recently identified group of ABA receptor proteins of the PYRABACTIN RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family that act as negative regulators of members of the PROTEIN PHOSPHATASE 2C (PP2C) family. Inhibition of PP2C activity enables activation of SNF1-RELATED KINASE 2 (SnRK2) protein kinases, which target downstream components, including transcription factors, ion channels and NADPH oxidases. These and other components form a complex ABA signaling network. Here, an in depth analysis of the evolution of components in this ABA signaling network shows that (i) PYR/RCAR ABA receptor and ABF-type transcription factor families arose during land colonization of plants and are not found in algae and other species, (ii) ABA biosynthesis enzymes have evolved to plant- and fungal-specific forms, leading to different ABA synthesis pathways, (iii) existing stress signaling components, including PP2C phosphatases and SnRK kinases, were adapted for novel roles in this plant-specific network to respond to water limitation. In addition, evolutionarily conserved secondary structures in the PYR/RCAR ABA receptor family are visualized.

). † These authors contributed equally to this work. SummaryCalcium 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.

Drought stress triggers an increase in the level of the plant hormone abscisic acid (ABA), which initiates a signaling cascade to close stomata and reduce water loss. Recent studies have revealed that guard cells control cytosolic ABA concentration through the concerted actions of biosynthesis, catabolism as well as transport across membranes. Substantial progress has been made at understanding the molecular mechanisms of how the ABA signaling core module PYR/PYL/RCAR-PP2C-SnRK2 controls the activity of anion channels and thereby stomatal aperture. In this review, we focus on our current mechanistic understanding of ABA signaling in guard cells including the role of the second messenger Ca2+ as well as crosstalk with biotic stress responses.

SUMMARYDuring 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.