In plants, as expected by analogy to well studied animal systems, the ER is emerging as an important Ca2+-source for intracellular signalling. Several important signalling components have been identified and characterized (Table 1; Sanders et al. 2002). However, approaches to reveal how these components are functionally orchestrated will be necessary to understand calcium related functions within the organelle, as well as cytosolic signalling events. Comparable studies in animal systems have revealed the ER, although a physical continuum, as a diverse and heterogenous organelle (Papp et al. 2003).

The construction and distribution of Arabidopsis insertion lines have provided the plant scientific community with a powerful tool to explore functional implications of genes of interest. The first gene involved in ER Ca2+-signalling to be investigated using this approach was the ECA1 Ca2+-

ATPase (Wu et al. 2002). Disruption of the ECA1 gene resulted in reduced growth on medium with lower calcium contents. A similar growth defect was evident when an antisense construct of Crt was introduced into Ara-bidopsis plants (Persson et al. 2001). Combining molecular and genetic tools may therefore be a fruitful approach to assess the effects of altered ER calcium on plant growth. Crossing individual insertion lines may further reveal the extent of redundancy and functional overlap among different genes. Subsequent introduction of cameleons would then allow for a direct connection between the disruption/overexpression of genes and changes in calcium signatures.

Several additional approaches may be utilized to uncover the dynamics of the plant ER calcium network: 1) Comparable distribution of components using either fluorescently-labelled proteins or immuno-techniques, 2) Introduction of compatible fluorescently-labelled components for FRET analyses, 3) Large-scale microarray data mining to expose coregulatory networks of genes, 4) Assessment of protein-protein interactions through various pro-teomic efforts. These techniques should provide spatio-temporal information about how different components work in concert with each other under various conditions.

The emerging view in animal cells is that different organelles may sense and respond to the status of other organelles. This view has not been explored for Ca2+-signalling and holding compartments in plants. The cross-talk in animal systems between, for example, the ER, PM and mitochondria has generated information to how changes in Ca2+-homeostasis may affect the cellular status, e.g. apoptosis, protein synthesis and secretion. Given that the vacuole is the main Ca2+-holding source in plant, a disruption of the calcium equilibrium between this organelle and the ER may provide insight into how the cellular distribution of calcium is facilitated.

Whilst most of the suggested approaches here refer to a macroscopic view of the ER and cellular Ca2+-signalling, it is equally important to uncover structural information of the different enzymes. The structure of the P domain of Crt was solved using NMR and revealed a extended hairpin with three anti-parallel beta-sheets (Schrag et al. 2001). This information provided useful clues to how Crt interacts with other Ca2+-regulated chaperones, such as ERp57 (Frickel et al. 2002). The structure of SERCA1a has similarly provided important information for functional aspects of Ca2+-pumps (Toyoshima et al. 2004). These data may also reveal evolutionary relationships which may not be evident solely from sequence homology.

In this chapter we have tried to high-light the versatile aspects of calcium and its impact on a variety of ER processes. The tight coordination of cy-tosolic and organellar calcium contents emphasizes the importance of the ion in different compartments. The Ca2+-fluxes should therefore simultaneously be viewed as a regulatory switch for cytosolic as well as internal organellar processes.

Acknowledgements We would like to thank Drs Marianne Sommarin, Wendy F. Boss and Thorsten Hamann for valuable suggestions. SP was a recipient of a Carl Tryggers fellowship (CTS 03:258).

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