Cellular and Physiological Implications

The ability to coordinate the Ca2+-signalling network, i.e. to achieve specific spatiotemporal characteristics of the signal, is of great importance for an agonist-stimulated signal to progress properly. Altering the abundance of different Ca2+-signalling components may consequently skew the calcium signal and hence affect different sets of downstream targets. These targets may range from signal transmitters to signal effectors (Sanders et al. 2002). The signal transmitters, such as transcription factors and CaM-like proteins, may undergo conformational changes in response to Ca2+-fluxes, which alter their binding ability to target molecules. In contrast, the signal effectors, such as various kinases and phosphatases, may directly trigger a covalent modification of their target molecules.

A major breakthrough in the visualization of calcium signals has been the development of calcium sensitive probes (Knight et al. 1991; Miyawaki et al. 1997). The most widely used probes are aequorins and cameleons. Whereas the aequorins are useful when measuring calcium changes in whole tissues or a cell population, the cameleons can be used to measure Ca2+-oscillations on a single-cell level. The cameleon construct consists of a recombinant protein based on a CaM flanked by two fluorescent proteins (Fig. 7A), which can be transformed into any organism and targeted to different organelles. When the calcium levels increase the CaM backbone undergoes a conformational change which brings the two fluorescent proteins in close proximity to each other (Fig. 7A; Miyawaki et al. 1997). By choosing fluorescent protein-parts with different excitation and emission wavelengths, the energy from one of the fluorescent protein-parts may be transferred to the other, referred to as fluorescent resonance energy transmission (FRET).

Combining cameleons with reverse genetic and molecular approaches has begun to increase our understanding for how different components affect the cellular calcium status in plants (Allen et al. 1999, 2000). This approach was used to dissect how a vacuolar H+-ATPase affects Ca2+-signalling in guard cells (Allen et al. 2000). By introducing cameleons into a line with a mutated vacuolar H+-ATPase (det3), Allen et al. (2000) showed that the spatiotemporal calcium patterns generated by different stimuli changed in the mutant line compared to wild-type, and resulted in altered physiological responses of the guard cell (Fig. 7B,C; Allen et al. 2000). This report provided the first genetic evidence that distinct stimulus-specific calcium oscillations may facilitate specific physiological responses in plants.

The only component in the plant ER calcium network that has been investigated so far using genetic approaches is the Ca2+-pump ECA1 (Wu et al. 2002). Disruption of the ECA1 gene did not result in any visible phenotype when grown on standard Murashige Skoog growth medium (Wu et al. 2002). However, when grown on either low calcium (0.2 mM) or high manganese (0.5 mM) levels, seedlings grew very poorly (Fig. 8A). This may be due to an inhibition of cell expansion or cell division (Wu et al. 2002), similar to what has been reported for the yeast pmr1 mutant, lacking a Golgi Ca2+/Mn2+-pump (Durr et al. 1998). In addition, the pmr1 mutant showed defects in protein sorting and glycosylation processes, indicating a link between calcium and internal ER processes. Perturbing the cellular calcium homeostasis in plants by deletion of ECA1 related Ca2+-pumps may also affect intracellular organization (Adamikova et al. 2004). Disruption of an ECA1 homolog in the fungus Ustilago maydis resulted in randomization of the microtubule network due to a sustained elevation of cytosolic calcium (Adamikova et al. 2004).

Overexpression of CRT also results in a conditional growth phenotype (Persson et al. 2001). Arabidopsis seedlings expressing a maize Crt1a/b homolog showed higher resistant to low levels of calcium in the growth medium (Fig. 8B). The increase in CRT expression further mediated a higher Ca2+-holding potential of the ER in vitro (Persson et al. 2001). In addition, Arabidopsis plants expressing GFP fused to the high capacity Ca2+-binding C domain of Crt showed an overall increase in cellular calcium (Wyatt et al. 2002).

Fig. 7 Schematic model of the Ca2+-indicator cameleon and its utilization in measuring defective Ca2+-oscillations in the det3 mutant. A The cameleon molecule, and its activation, consisting of a calmodulin (CaM) backbone fused to two fluorescent proteins. Modified from Miyawaki et al. (1997). B Application of external calcium evokes differences in cytosolic calcium responses in guard cells from wild-type and det3 mutants. Intracellular Ca2+-oscillations were induced applying 10 mM external calcium. Whereas the wild-type exhibited oscillatory Ca2+-signals, Ca2+-oscillations were abolished in the det3 mutant. C The increase in external calcium caused stomatal closure in wild-type but not in the det3 mutant. Reproduced from Allen et al. (2000) with permission. YFP: Yellow fluorescent protein, CFP: Cyan fluorescent protein, FRET: Fluorescent Resonance Energy Transfer

Fig. 7 Schematic model of the Ca2+-indicator cameleon and its utilization in measuring defective Ca2+-oscillations in the det3 mutant. A The cameleon molecule, and its activation, consisting of a calmodulin (CaM) backbone fused to two fluorescent proteins. Modified from Miyawaki et al. (1997). B Application of external calcium evokes differences in cytosolic calcium responses in guard cells from wild-type and det3 mutants. Intracellular Ca2+-oscillations were induced applying 10 mM external calcium. Whereas the wild-type exhibited oscillatory Ca2+-signals, Ca2+-oscillations were abolished in the det3 mutant. C The increase in external calcium caused stomatal closure in wild-type but not in the det3 mutant. Reproduced from Allen et al. (2000) with permission. YFP: Yellow fluorescent protein, CFP: Cyan fluorescent protein, FRET: Fluorescent Resonance Energy Transfer

Fig. 8 Conditional growth phenotypes exhibited in ECA1 knock-out plants and seedlings over expressing CRT. A Deletion of the ECA1 gene results in delayed growth on medium containing reduced levels of calcium. Seedlings (5-day-old) transferred from MS medium to medium containing reduced calcium and grown for 10 days. B Correlation of CRT expression and seedlings ability to maintain growth on calcium-depleted medium. Seedlings (19 day-old) transferred from MS medium to medium containing reduced calcium (10 mM EGTA) and grown for 9 days. Reproduced from Wu et al. (2002) and Persson et al. (2001) with permission

Fig. 8 Conditional growth phenotypes exhibited in ECA1 knock-out plants and seedlings over expressing CRT. A Deletion of the ECA1 gene results in delayed growth on medium containing reduced levels of calcium. Seedlings (5-day-old) transferred from MS medium to medium containing reduced calcium and grown for 10 days. B Correlation of CRT expression and seedlings ability to maintain growth on calcium-depleted medium. Seedlings (19 day-old) transferred from MS medium to medium containing reduced calcium (10 mM EGTA) and grown for 9 days. Reproduced from Wu et al. (2002) and Persson et al. (2001) with permission

The unique and redundant functions of different calcium stores in plant cells are not fully understood. However, calcium signals originating from the ER are expected to affect localized and global events, e.g. regulation of ER-generated calcium signatures and transcriptional events. The components decoding these calcium signals include CDPKs, CaM, as well as many of the other approximately 400 proteins in plants that have defined calcium binding EF-hands (Reddy and Reddy 2004). Of the 34 identified CDPKs in Arabidop-sis (Harper and Harmon 2004), only AtCPK2 has been shown to localize to the cytosolic surface of the ER (Lu and Hrabak 2002). There is experimental evidence that this association is dependent on myristoylation at the N terminus of AtCPK2. While the in vivo target substrates of CPK2 have yet to be determined, CDPKs may phosphorylate the ER calcium pump ACA2 (Hwang et al. 2000). In this case, phosphorylation is predicted to slow the rate of calcium efflux back into the ER, thereby possibly increasing the duration and magnitude of a calcium signal in the micro-environment around the ER. Since CDPKs are thought to be multifunctional kinases (Harper et al. 2004), many ER surface proteins are expected to be regulated by calcium signals decoded by isoform CPK2.

While investigations of how altered levels of Ca2+-signalling components affect physiological responses have been initiated in plants, potential crosstalk between different Ca2+-containing organelles are still largely unexplored. The ER calcium status in animal cells is tightly linked to Ca2+-channels at the PM (Randriamampita and Tsien 1993; Fasolato et al. 1993; Putney et al. 1999). Consequently, depletion of the ER calcium stores activates the PM Ca2+-channels and allows for refilling of the ER stores. This feedback process is referred to as capacitative calcium entry (Putney et al. 2001). The presently favoured model for capacitative calcium entry is a direct communication between PM Ca2+-channels and the cADPR activated ryanodine receptors in the ER (Putney et al. 2001). The tight monitoring of the ER calcium status further strengthens the notion that the calcium ion is essential for proper maintenance of a variety of ER functions.

The ER calcium fluxes in animal cells also appear to influence the calcium status of the mitochondria (Rizzuto et al. 1993). This may be facilitated via the "hot-spot" hypothesis, in which IP3-activated receptors in the ER are enriched at ER/mitochondria interfaces (Rizzuto et al. 2004). When calcium is released through the receptors, mitochondrial Ca2+-uniporters facilitate uptake of calcium into the mitochondria. The increase in mitochondrial calcium may activate ATP production, but could also alter the organelle structure and trigger a release of apoptosis-activating substrates (Rizzuto et al. 2004).

Potential organellar cross-talk in the plant cell may, however, offer clues to phenotypic behaviours generated by genetic and molecular procedures. As discussed above, lower expression of either CRT or ECA1 results in reduced growth on medium containing low levels of calcium (Fig. 8; Persson et al. 2001; Wu et al. 2002). These phenotypes were largely attributed to an overall decrease in ER calcium. However, overexpression of the vacuolar Ca2+/H+-antiport, CAX1, in tobacco displayed severe symptoms of calcium deficiency and contained a two-fold increase in cell calcium (Hirschi 1999). The phe-notype could be reversed by adding exogenous calcium. Thus, a decrease in expression of at least two ER calcium network components, and an increase in expression of a vacuolar calcium network component, causes similar phenotypes. The explanation for this deceptive contradiction may lie in the distribution of calcium between the two organelles. If we assume that the protein level of a specific calcium handling protein is directly related to its activity, then the decrease in both Crt and ECA1 should result in lower ER calcium levels. Similarly, increasing the CAX1 expression may increase the vacuolar calcium efflux efficiency and therefore reduce the levels of accessible calcium for other organelles, e.g. the ER. Introducing cytosolic- and ER-targeted cameleons in crossed combinations of Crt, ECA1 and CAX1 plants should certainly present useful tools for visualization of potential cross-talk between the vacuole and the ER.

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