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Stomata are pores on the surface of plants which regulate the uptake of carbon dioxide for photosynthesis and the loss of water vapor by transpiration (Hetherington & Woodward, 2003). The aperture of the pore is controlled by the state of turgor of the two guard cells. Many environmental signals (e.g. humidity, light and CO2) and endogenous plant hormones such as abscisic acid (ABA) and auxin modulate stomatal aperture (Assmann & Wang, 2001; Hetherington, 2001; Schroeder et al., 2001; Outlaw, 2003; Vavasseur & Raghavendra, 2005; Roelfsema & Hedrich, 2005). A plethora of studies have established that guard cell signaling is controlled by a network of components influencing turgor, gene expression, cytoskeletal dynamics, protein trafficking and ion channel activity (Hetherington & Woodward, 2003). An intricate signaling network interconnecting second messengers such as cyclic ADP ribose (cADPR), inositol-3-phosphate (IP3) and sphingosine-1-phosphate (SP1P) with alterations in the concentrations of plant hormones such as ABA and with the regulation of ion channels has been shown to control guard cell aperture in response to environmental cues. Cell physiological investigations have revealed that an increase in cytoplasmic Ca2+ concentration ([Ca2+]cyt) represents a key component involved in coupling a range of extracellular signals such as CO2, ABA, CaCl2 and H2O2 to changes in stomatal aperture.
The function of Ca2+ as a secondary messenger is not restricted to guard cells. Temporally and spatially defined modulations of cellular Ca2+ concentration represent primary events in response to many signals and also regulate developmental programs and various physiological processes (Hetherington & Brownlee, 2004). Such signals include plant hormones, light, stress factors, and pathogenic or symbiotic elicitors (Trewavas & Knight, 1994; Ehrhardt et al., 1996; Knight et al., 1996, 1997; Neuhaus et al., 1997; Sanders et al., 1999, 2002; Harper & Harmon, 2005). In addition, physiological processes such as guard cell regulation, root hair elongation and pollen tube growth are accompanied by distinct spatio-temporal changes in calcium concentration (Evans et al., 2001; Sanders et al., 2002).
Recent studies suggest that a Ca2+ signal is not only represented by the Ca2+ concentration but also by spatial and temporal information, including Ca2+ localization and oscillation (Allen et al., 2000; Allen et al., 2001; Young et al., 2006). Critical for the spatially distinct generation of cellular calcium transients are the stores from which this messenger ion is released. Calcium signals are generated through the opening of channels that either mediate Ca2+ entry from outside the cell or mediate release from intracellular stores. The vacuole and the endoplasmatic reticulum represent major intracellular Ca2+ sinks with endogenous Ca2+ concentrations in the millimolar range, while the resting concentration of Ca2+ in the cytoplasm has been estimated to be approx. 200 µM (Sanders et al., 1999). In addition, plant nuclei represent a potential calcium store and stimulus-induced dynamic changes in nuclear calcium concentration that appear to be independent of cytoplasmic calcium signaling have been reported (Pauly et al., 2000). Although chloroplasts have been shown to contain high concentrations of Ca2+ (between 4 and 23 mM; Portis & Heldt, 1976) their potential role in cellular Ca2+ homeostasis and signaling has remained largely unexplored (Johnson et al., 2006). Light stimulates uptake of Ca2+ into the chloroplast but the Ca2+ concentrations in the stroma do not change significantly during illumination (Kreimer et al., 1988). However, circadian chloroplast Ca2+ oscillations were observed after transfer of plants to constant darkness (Johnson et al., 1995). Such dark-induced increases in stroma Ca2+ concentrations precede the generation of elevations of [Ca2+]cyt in tobacco (Nicotiana plumbaginifolia) leaves (Sai & Johnson, 2002).
In addition, several recent studies point to an unexpected functional interconnection of the chloroplast-localized Ca2+ pool with cytoplasmic signaling events. Characterization of the chloroplast membrane protein Pisum post-floral-specific gene 1 (PPF1) revealed that it represents a putative calcium ion carrier that affects the flowering time of transgenic Arabidopsis thaliana by modulating Ca2+ storage capacities in chloroplasts (Wang et al., 2003; Li et al., 2004). Interaction of Lotus japonicus with mycorrhiza-forming fungi critically involves the function of the two loci CASTOR and POLLUX (Ehrhardt et al., 1996). Both proteins are indispensable for microbial admission to plant cells and for the occurrence of intracellular calcium spiking in response to symbiotic stimulation (Imaizumi-Anraku et al., 2005). Surprisingly, positional cloning of both genes revealed that they encode chloroplast-localized proteins with potential ion channel function, thereby assigning a novel function to chloroplasts in generating cytoplasmic Ca2+ signatures (Imaizumi-Anraku et al., 2005).
Here we describe the functional analysis of the plant-specific protein Ca2+-sensing receptor (CAS). This 42-kDa Ca2+-binding protein was originally described as a plasma membrane-localized extracellular Ca2+-sensing protein exhibiting low-affinity/high-capacity Ca2+ binding through an N-terminal domain of the protein (Han et al., 2003). Functional characterization of CAS antisense lines indicated a function of the protein in regulating stomatal responses to elevation of extracellular Ca2+ concentrations and indicated that CAS mediates Ca2+-induced [Ca2+]cyt increases.
In contrast to the proposed plasma membrane localization, comprehensive proteomics-based studies identified the CAS protein in A. thaliana as well as in the green alga Chlamydomonas reinhardtii in the chloroplast thylakoid membrane (Friso et al., 2004; Allmer et al., 2006). In this study we verified the chloroplast localization of CAS by subcellular fractionation analyses and investigation of green fluorescent protein (GFP) fusion proteins in protoplasts and leaf guard cells. Analysis of two independent T-DNA induced loss-of-function alleles uncovered a crucial function of CAS in regulating stomatal closure in response to elevations of extracellular Ca2+. This mutant phenotype coincided with impaired increases in the concentration of cytosolic Ca2+ normally induced in response to this stimulus. Consequently, our study reveals a crucial function of chloroplasts and in particular of the thylakoid-localized CAS protein in regulating guard cell responses and uncovers a critical contribution of these organelles to the generation and fine-tuning of cytoplasmic calcium signals.