Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. To further understand the molecular organization of the eyespot apparatus and the phototactic movement that is controlled by light and the circadian clock, a detailed understanding of all components of the eyespot apparatus is needed. We developed a procedure to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the identification of 202 different proteins with at least two different peptides (984 in total). These data provide new insights into structural components of the eyespot apparatus, photoreceptors, retina(l)-related proteins, members of putative signaling pathways for phototaxis and chemotaxis, and metabolic pathways within an algal visual system. In addition, we have performed a functional analysis of one of the identified putative components of the phototactic signaling pathway, casein kinase 1 (CK1). CK1 is also present in the flagella and thus is a promising candidate for controlling behavioral responses to light. We demonstrate that silencing CK1 by RNA interference reduces its level in both flagella and eyespot. In addition, we show that silencing of CK1 results in severe disturbances in hatching, flagellum formation, and circadian control of phototaxis.

The RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits. One (named C1) contains three lysine homology motifs and the other (named C3) has three RNA recognition motifs. CHLAMY1 binds specifically to uridine-guanine-repeat sequences and its circadian-binding activity is controlled at the posttranslational level, presumably by time-dependent formation of protein complexes consisting of C1 and C3 or C1 alone. Here we have characterized the role of the two subunits within the circadian system by measurements of a circadian rhythm of phototaxis in strains where C1 or C3 are either up-or down-regulated. Further, we have measured the rhythm of nitrite reductase activity in strains with reduced levels of C1 or C3. In case of changes in the C3 level (both increases and decreases), the acrophase of the phototaxis rhythm and of the nitrite reductase rhythm (C3 decrease) was shifted by several hours from subjective day (maximum in wild-type cells) back towards the night. In contrast, both silencing and overexpression of C1 resulted in disturbed circadian rhythms and arrhythmicity. Interestingly, the expression of C1 is interconnected with that of C3. Our data suggest that CHLAMY1 is involved in the control of the phase angle and period of the circadian clock in C. reinhardtii.

The circadian RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits named C1 and C3. Changes in the C1 level cause arrhythmicity of the phototaxis rhythm, while alterations in the level of C3 lead to acrophase shifts. Thus, CHLAMY1 is involved in maintaining period and phase of the circadian clock. Here, we analyzed the roles of the two subunits in the integration of temperature information, the basis for other key properties of circadian clocks, including entrainment by temperature cycles and temperature compensation. Applied temperatures (18°C and 28°C) were in the physiological range of C. reinhardtii. While C1 is hyperphosphorylated at low temperature, the C3 expression level is upregulated at 18°C. An inhibitor experiment showed that this up-regulation occurs at the transcriptional level. Promoter analysis studies along with single promoter element mutations revealed that individual replacement of two DREB1A-boxes lowered the amplitude of c3 up-regulation at 18°C, while replacement of an E-box abolished it completely. Replacement of the E-box also caused arrhythmicity of circadian-controlled c3 expression. Thus, the E-box has a dual function for temperature-dependent upregulation of c3 as well as for its circadian expression. We also found that the temperature-dependent regulation of C1 and C3 as well as temperature entrainment are altered in the clock mutant per1, indicating that a temperature-controlled network of C1, C3, and PER1 exists.

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Although the number of reconstructed metabolic networks is steadily growing, experimental data integration into these networks is still challenging. Based on elementary flux mode analysis, we combine sequence information with metabolic pathway analysis and include, as a novel aspect, circadian regulation. While minimizing the need of assumptions, we are able to predict changes in the metabolic state and can hypothesise on the physiological role of circadian control in nitrogen metabolism of the green alga Chlamydomonas reinhardtii.