## 1 Introduction  
Chloroplasts are key organelles for plant autotrophism, where many essential metabolic processes take place. In addition to photosynthesis, plastids perform functions such as fatty acid biosynthesis, nitrite and sulphate reduction and amino acid biosynthesis. To ensure proper function and regulation, chloroplast metabolism is tightly coordinated with the requirements of the surrounding cell. Accordingly, environmental and developmental signals have to be transduced into the organelle. Calcium is a ubiquitous secondary messenger of eukaryotic organisms involved in the regulation of multiple cellular processes [1,2]. In chloroplasts, calcium has been shown to be involved in the regulation of processes such as photosynthesis, CO\({}_{2}\) fixation, protein transport and protein phosphorylation. As recently reviewed, about twenty proteins are so far known to be potentially involved in the chloroplast calcium network, however, only a few have been shown to bind calcium in a direct manner [3]. The photosystem II protein PsbO has been reported as a weak calcium-binding protein [4,5] and calcium was suggested to influence its proton-dependent activation [6]. Ferredoxin, the electron donor of photosystem I, is also able to bind calcium with high capacity in its reduced state [7]. The thylakoid localized calcium sensing protein (CAS) is one of the most extensively studied calcium-binding proteins in plastids. It is involved in stomatal closure [8, 9, 10, 11] and photoacclimation [12] and was one of three thylakoid targets of calcium-dependent phosphorylation identified in a recent study [13]. The same study also showed Var1 and PsaN to be phosphorylated in a calcium-dependent manner.  
Present in all eukaryotes, calmodulins (CaMs) are one of the best studied families of calcium-binding proteins. They are comprised of four EF-hand structural motifs that are able to bind calcium with high affinity [14] and bind to their targets in a calcium-dependent manner. Several chloroplast proteins have been shown to behave as CaM-binding proteins, including the photosystem I component PsaN [15], the chaperonine CPN10 [16], as well as the AAA\({}^{+}\) proteins CIP111 and AFG1L1 [17,18]. However, in chloroplasts, no CaM has been so far identified. Indeed, typical EF-hand containing calcium-binding proteins seems to be very rare in chloroplasts. In the outer envelope, a member of the calcium-dependent mitochondrial carrier family SAMTL has been identified [19,20] that possesses a single EF-hand domain and was shown to specifically bind calcium _in vitro_[21]. Also, the (p)ppGpp synthase-degradase CRSH has an EF-hand domain and requires calcium for its activity [22]. The latter is the only stromal EF-hand protein identified so far.  
In the present work, we investigated the presence of additional calcium-binding proteins in the stroma. To that end, proteins were separated by 2D SDS-PAGE and tested for the ability to bind radiolabeled calcium by calcium overlay assays. CP12 was identified as a potential candidate for calcium-binding and this property could be confirmed _in vitro_ using recombinant protein. Together, our data suggest that CP12 represents a novel calcium-binding protein in chloroplasts.