Members of the Arabidopsis calcineurin B-like Ca 2 ؉ binding protein (AtCBL) family are differentially regulated by stress conditions. One AtCBL plays a role in salt stress; another is implicated in response to other stress signals, including drought, cold, and wounding. In this study, we identified a group of novel protein kinases specifically associated with AtCBL-type Ca 2 ؉ sensors. In addition to a typical protein kinase domain, they all contain a unique C-terminal region that is both required and sufficient for interaction with the AtCBL-type but not calmodulin-type Ca 2 ؉ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca 2 ؉ , suggesting that increases in cellular Ca 2 ؉ concentrations may trigger the formation of AtCBL-kinase complexes in vivo. Unlike most serine/threonine kinases, the AtCBL-interacting kinase efficiently uses Mn 2 ؉ to Mg 2 ؉ as a cofactor and may function as a Mn 2 ؉ binding protein in the cell. These findings link a new type of Ca 2 ؉ sensors to a group of novel protein kinases, providing the molecular basis for a unique Ca 2 ؉ signaling machinery in plant cells. INTRODUCTIONAmong the extracellular signals eliciting changes in Ca 2 ϩ concentration in the cytoplasm of plant cells are plant hormones, light, stress factors, and pathogenic or symbiotic elicitors (Knight et al., 1991(Knight et al., , 1996(Knight et al., , 1997Neuhaus et al., 1993; Trewavas and Knight, 1994; Ehrhardt et al., 1996;McAinsh et al., 1997; Wu et al., 1997). In addition, many intrinsic growth and developmental processes, such as elongation of the root hair and pollen tube, are accompanied by Ca 2 ϩ transients (Franklin-Tong et al., 1996; Felle and Hepler, 1997; Holdaway-Clarke et al., 1997; Wymer et al., 1997). Because different signals often elicit distinct and specific cellular responses, an interesting question is how do cells distinguish between the Ca 2 ϩ signals produced by different stimuli?Studies with both animal and plant cells suggest that a Ca 2 ϩ signal is represented not only by Ca 2 ϩ concentration but also by spatial and temporal information, including Ca 2 ϩ localization and oscillation (Franklin-Tong et al., 1996; Holdaway-Clarke et al., 1997; Dolmetsch et al., 1998;Li et al., 1998). Although such complexity in Ca 2 ϩ parameters may partially explain the specificity of cellular responses triggered by a particular stimulus, the signaling components that "sense" and "interpret" the Ca 2 ϩ signals hold the key to linking the changes in these parameters to specific cellular responses.If Ca 2 ϩ signaling pathways constitute "molecular relays," the first "runner" after Ca 2 ϩ should be a component that serves as the Ca 2 ϩ "sensor" to monitor changes in Ca 2 ϩ parameters. Such sensors often are proteins that bind Ca 2 ϩ and, in so doing, change conformation in a Ca 2 ϩ -dependent manner. Several families of Ca 2 ϩ sensors have been identified in higher plants. Perhaps the best known is the family of calmodulin (CaM) and CaM-related prot...
INTRODUCTIONBimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.
Pollen tubes grow rapidly by very fast rates and reach extended lengths to bring about fertilization during plant reproduction. The pollen tube grows exclusively at its tip. Fundamental for such local, tip-focused growth are the presence of internal gradients and transmembrane fluxes of ions. Consequently, vegetative pollen tube cells are an excellent single cell model system to investigate cell biological processes of vesicle transport, cytoskeleton reorganization and regulation of ion transport. The second messenger Ca(2+) has emerged as a central and crucial modulator that not only regulates but also integrates the coordination each of these processes. In this review we reflect on recent advances in our understanding of the mechanisms of Ca(2+) function in pollen tube growth, focusing on its role in basic cellular processes such as control of cell growth, vesicular transport and intracellular signaling by localized gradients of second messengers. In particular we discuss new insights into the identity and role of Ca(2+) conductive ion channels and present experimental addressable hypotheses about their regulation. This article is part of a Special Issue entitled:12th European Symposium on Calcium.
Fundamental plant functions such as control of the membrane potential, osmo-regulation, and turgor-driven growth and movements are based on the availability to gain high cellular potassium concentrations (1). The absorption of this inorganic osmolyte from the soil by the root therefore represents a pivotal process for plant life. Classical experiments by Epstein et al. in 1963 (2) described K ϩ root uptake as a biphasic process mediated by two uptake mechanisms: high affinity potassium transport with apparent affinities of ϳ20 M and a low affinity transport system with K m values in the millimolar range. During the last decades several molecular components of potassium transport systems have been identified and functionally characterized in plants (3, 4). Mutant analyses, heterologous expression, as well as radiotracer uptake experiments characterized the K ϩ channels AKT1⅐AtKC1 and members of the HAK⅐KT⅐KUP family as major components of the Arabidopsis thaliana root-localized potassium transport system (5-9). In this study we focused on AKT1 and AtKC1, members of the Arabidopsis Shaker-like K ϩ channel family. AKT1 is a voltagedependent inward-rectifying K ϩ channel mediating potassium uptake over a wide range of external potassium concentrations (10 -15). Root cells of the akt1-1 loss-of-function mutant completely lack inward rectifying K ϩ currents (12). As a consequence the growth of akt1-1 seedlings is strongly impaired on low potassium medium (100 M and less) (11,12,15). Rescue of yeast growth on 20 M K ϩ and patch clamp experiments (16, 17) directly demonstrated that plant inward rectifying K ϩ channels are capable of serving as high affinity potassium uptake transporters. AtKC1 shares its expression pattern with . AtKC1 ␣-subunits, however, neither form functional channels in akt1-1 knock-out plants nor in heterologous expression systems. In contrast to root cells of akt1-1 loss of function mutants, root protoplasts of AtKC1 null mutants (atkc1-f) still exhibit inward rectifying potassium currents most likely derived from homomeric AKT1 tetramers (20). Inward K ϩ currents in this atkc1-f mutant were characterized by a more positive activation voltage. These data suggested that the AtKC1 ␣-subunits do not form K ϩ channels per se but modulate the properties of the AKT1⅐AtKC1 heterocomplex (20 -22
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