The chloroplast calcium sensor protein CAS affects phototactic behaviour in Chlamydomonas reinhardtii (Chlorophyceae) at low light intensities

Here we showed enrichment of the orthologue of the plant specific Ca2+ binding protein CAS in Chlamydomonas reinhardtii (CrCAS) in both the thylakoids and the eyespot. Ca2+ binding to recombinant CrCAS was analysed for the first time in detail. Binding of Ca-45(2+) (2.9 mu M) occurred in the presence of an excess of Mg2+ (5 mM) and was not affected by variation in pH from 6.8 to 8.0. Competition experiments with the Ca2+ antagonists La3+ (50 mu M) and unlabelled Ca2+ (5 mM) indicated that CrCAS probably acts as a low-affinity/high-capacity Ca2+ binding protein. Fe2+ (5 mu M) had no effect on Ca-45(2+) binding. To elucidate a possible role of CrCAS in the phototactic signalling cascade, knockdown and CrCAS overexpressing lines were generated. All 17 knockdown lines quickly reverted, making a detailed analysis impossible. In contrast, overexpressing lines were stable for at least 6 to 9 mo. Four of these lines with overexpression levels between 169% and 270% were analysed in detail for their phototactic behaviour. At low photon fluence rates, all showed an initial positive phototactic orientation. After a few minutes, however, they switched to an orientation away from the light source. The threshold fluence rate for this switch was determined and negatively correlated with the CrCAS overexpression level. In contrast, the negative phototactic behaviour of the lines at higher light intensities was unaffected, and empty vector control lines responded like the wild-type, pointing to the specificity of this effect. These results indicate that C. reinhardtii CAS, in addition to its known roles in acclimation of photosynthesis to high light, is also involved in adaptation responses to low light at an intermediate time scale and also affects processes outside the chloroplast.