The chloroplast as a regulator of Ca2+ signalling


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Many of the attributes associated with multicellular plant life, including a sedentary habit, a decentralized organization, signalling in the absence of a nervous system and a plastic developmental programme, can be attributed to the autotrophism facilitated by the chloroplast. In this issue of New Phytologist, Weinl et al. (pp. 675–686) identify a new role for the chloroplast in Ca2+ signalling, which suggests that the plastid can exert control over signalling events in the cytosol. Weinl et al. report that the 42-kDa Ca2+ receptor protein (CAS) is localized to the chloroplast and that T-DNA knockout of CAS prevents stomatal closure in response to elevated external Ca2+ ([Ca2+]ext) by abolishing oscillations of cytosolic free-Ca2+ ([Ca2+]cyt). Weinl et al. find that cas mutations act upstream of oscillations of [Ca2+]cyt because the stomata of cas mutants close in response to artificially generated oscillations of [Ca2+]cyt.

‘Recent findings suggest that a totally novel pathway that is central to Ca2+ signalling in plants awaits discovery.’

CAS was first identified by a functional screen in which pools of Arabidopsis RNA were introduced into human kidney cells loaded with the Ca2+indicator, FURA2 (Han et al., 2003). External Ca2+ caused moderate increases of [Ca2+]cyt in kidney cells but the expression of Arabidopsis CAS in these cells resulted in large [Ca2+]ext-induced increases of [Ca2+]cyt (Ca2+-induced Ca2+ increase (CICI) (Han et al., 2003)). In Arabidopsis, CAS is expressed in the shoots and is found in guard cells (Han et al., 2003). CAS binds Ca2+ at the N-terminus with low affinity and high capacity (Han et al., 2003). CAS was first proposed to be a plasma membrane receptor that senses [Ca2+]ext (Han et al., 2003). The new data of Weinl et al. suggest that CAS is not a plasma membrane protein and is localized to the chloroplast. Weinl et al. identified an N-terminal chloroplast transit peptide and found that transient expression of CAS : GREEN FLUORESCENT PROTEIN in Nicotiana benthamiana protoplasts results in a chloroplastic localization that is confirmed by subcellular fractionation experiments. These findings are consistent with those of Nomura et al. (2008), who also recently reported a chloroplastic localization for CAS, insensitivity of the stomata of cas mutants to [Ca2+]ext, reduced CICI in cas knockouts and increased stomatal closure in CAS overexpressers.

The data of Weinl et al. and Nomura et al. (2008) suggest that the chloroplast has an essential role in Ca2+ signalling, with CAS as an important intermediary. How does a Ca2+ receptor in the chloroplast function to regulate oscillations of [Ca2+]cyt and CICI? One model is that CAS might sense changes in [Ca2+]cyt following Ca2+ influx across the plasma membrane and act in a feedback loop to regulate [Ca2+]cyt. However, the available data do not support this model. CAS probably senses changes in stromal [Ca2+] ([Ca2+]stroma) because CAS is localized to the thylakoid and the N-terminus Ca2+-binding domain is probably exposed on the stromal side of the membrane (Nomura et al., 2008). Further evidence that CAS does not sense [Ca2+]cyt comes from studies of abscisic acid (ABA) signalling in the guard cell. CAS mutations are without effect on ABA-induced stomatal closure, even though ABA causes oscillations of [Ca2+]cyt that are similar to those caused by [Ca2+]ext (Allen et al., 1999; Staxén et al., 1999; Weinl et al.).

The sensing, by CAS, of changes in [Ca2+]stroma could potentially affect the release or uptake of Ca2+ by the chloroplast. It is possible that the plastids act either as Ca2+ stores that release Ca2+ into the cytosol or as a Ca2+ buffer that removes Ca2+ from the cytosol following stimulation. The plastids could have a similar role to mitochondria in Ca2+ signalling. In mammals, mitochondria act as Ca2+buffers that take up Ca2+ from the cytosol following release from the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR). The tight coupling between ER/SR release and mitochondrial uptake has profound effects on localized [Ca2+]cyt dynamics, and mitochondria also contain a pool of releasable Ca2+ (Hetherington & Brownlee, 2004). If plastids, like mitochondria, act as Ca2+ buffers that also have a pool of releasable Ca2+, this might explain how CAS sensing of the [Ca2+]stroma could feed back to affect oscillations of [Ca2+]cyt. There is evidence that plastids are capable of both Ca2+ uptake and release (Johnson et al., 2006), although the data from different systems conflict as to whether chloroplastic uptake of Ca2+ has consequences for [Ca2+]cyt (Miller & Sanders, 1987; Sai & Johnson, 2002; Johnson et al., 2006). Chloroplasts take up Ca2+ in the light (Miller & Sanders, 1987; Xiong et al., 2006), and CASTOR and POLLUX are required for nodulation (NOD) factor-induced Ca2+ oscillations in root hairs of Lotus japonicus and are predicted to encode plastid-localized ion channels of unknown selectivity (Imaizumi-Anraku et al., 2005). Similarly, the pea PPF1 protein localizes to the chloroplast, delays flowering when expressed in Arabidopsis and is capable of carrying Ca2+ currents (Wang et al., 2003).

In addition to plastid regulation of [Ca2+]cyt, there are dark-induced increases in [Ca2+]stroma that can persist with a circadian rhythm in constant dark (Sai & Johnson, 2002). Circadian oscillations of [Ca2+]stroma appear to be independent of [Ca2+]cyt because in constant dark there are usually no oscillations of [Ca2+]cyt (Johnson et al., 1995). Sai & Johnson (2002) proposed that the thylakoid is a dark-dischargeable Ca2+ store that releases into the stroma. The thylakoid is suggested to be filled with Ca2+ from the cytosol via the stroma as a result of the action of a Ca2+/H+ antiporter acting in the light (Ettinger et al., 1999). Lengthening the light period appears to increase the amount of Ca2+ stored in the thylakoid because dark-induced discharge is increased with longer periods of light (Sai & Johnson, 2002).

It is not known if CAS affects the daily dark-induced increase in [Ca2+]stroma but CAS antisense reduces the amplitude of daily oscillations of [Ca2+]cyt in light/dark cycles (Tang et al., 2007). This appears to be related to the role of CAS in CICI because increases in [Ca2+]ext increase the amplitude of daily oscillations of [Ca2+]cyt (Tang et al., 2007). These findings led to a model being proposed in which the daily oscillations of [Ca2+]cyt are a consequence of similar oscillations in [Ca2+]ext caused by rhythmic water fluxes in response to daily stomatal movements. In this model, CAS was proposed to sense the rhythms of [Ca2+]ext to drive daily oscillations of [Ca2+]cyt through an inositol(1,4,5)trisphosphate-mediated pathway (Tang et al., 2007). However, recent data suggest that this may not be the case. Circadian oscillations of [Ca2+]cyt are driven by cyclic ADP ribose and are insensitive to U73182, an inhibitor of inositol(1,4,5)trisphosphate production (Dodd et al., 2007). Furthermore, circadian [Ca2+]cyt oscillations and stomatal movements are not functionally linked because the circadian rhythms of stomatal opening and [Ca2+]cyt run with different periods in both the timing of cab1-1 and zeitlupe-1 circadian mutants (Dodd et al., 2004; Xu et al., 2007). The localization of CAS to the chloroplast, and the evidence that rhythmic changes in [Ca2+]ext caused by stomatal movements are not likely to drive circadian [Ca2+]cyt oscillations, suggests that the effects of CAS should be reconsidered as evidence for the chloroplast modulating daily [Ca2+]cyt oscillations, with CAS acting in an unknown pathway.

The localization of CAS to the chloroplast by Weinl et al. and Nomura et al. (2008) identifies a new aspect of Ca2+ signalling. There are essential roles for the chloroplast in the sensing of [Ca2+]ext by stomata, timing of flowering, NOD factor-induced oscillations of [Ca2+] and daily oscillations of [Ca2+]cyt. Forming a model of how CAS affects [Ca2+]cyt is difficult as so little is known about Ca2+ fluxes associated with plastids and because the biological action of CAS is unknown. The findings reported in this issue by Weinl et al., those of Nomura et al. (2008) and the work of those in the Pei laboratory, who first identified CAS along with its role in regulating CICI, oscillations of [Ca2+]cyt and Ca2+-induced stomatal closure (Han et al., 2003; Tang et al., 2007), suggest that a totally novel pathway that is central to Ca2+ signalling in plants awaits discovery.