[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Circadian clocks can be entrained by light-dark or temperature cycles. In the green alga Chlamydomonas reinhardtii, 12h changes in temperature between 18°C and 28°C synchronize its clock. Both subunits of the circadian RNA-binding protein CHLAMY1, named C1 and C3, are able to integrate temperature information. C1 gets hyper-phosphorylated in cells grown at 18°C and the level of C3 is up-regulated at this temperature. In the long period mutant per1, where temperature entrainment is disturbed, the temperature-dependent regulation of C1 and C3 is altered. Up-regulation of C3 at the low temperature is mediated predominantly by an E-box element situated in its promoter region. This cis-acting element is also relevant for circadian expression of c3 as well as of its up-regulation in cells, where C1 is overexpressed. Among the few identified factors interacting with the E-box region, C3 is also present, suggesting that it feedbacks on its own transcription.
[Show abstract][Hide abstract] ABSTRACT: 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 degrees C and 28 degrees C) were in the physiological range of C. reinhardtii. While C1 is hyperphosphorylated at low temperature, the C3 expression level is up-regulated at 18 degrees 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 degrees 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 up-regulation 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.
[Show abstract][Hide abstract] ABSTRACT: The research of this thesis focused on the circadian RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii that consists of two subunits, C1 and C3. CHLAMY1 binds specifically to (UG)≥7-repeat sequences situated in the 3'-UTRs of several mRNAs such as nitrite reductase 1 (nii1).
The role of the C1 subunit within the circadian system was characterized by silencing its gene by an RNAi. The expression level of C1 was silenced down to 25 - 85%. To show the influence of CHLAMY1 on circadian output, NII activities were measured. In wild-type, NII activity peaks around the beginning of subjective day. In the transgenic strain arrhythmicity was observed. Circadian phototaxis was chosen as a second output rhythm. In this case, arrhythmicity was observed immediately or in the first three days under constant conditions in transgenic lines. In addition to these results, a co-regulation between C1 and C3 subunits was found. These data indicate a central role of the C1 subunit in the circadian system of C. reinhardtii.
It was also analyzed if the two subunits play a role in temperature integration. C1 was found to be hyper-phosphorylated at 18°C and hypo-phosphorylated at 28°C. The C3 expression level was found to be up-regulated at 18°C, which was shown to occur at the transcriptional level. The clock-relevant CASEIN KINASE1 (CK1) and Ser-/Thr-PROTEIN PHOSPHATASEs (PPs) were found to mediate the temperature dependent regulation of C1 and C3. The expression of CK1 was itself temperature controlled and increased at 28°C. In the long period clock mutant per1 temperature integration of both C1 and C3 was shown to be altered: a low phosphorylation level of C1 and a high expression level of C3 was observed at different temperatures. Altogether, the data suggest that a temperature controlled functional network of clock-relevant proteins exists in C. reinhardtii including C1, C3, CK1, PPs and PER1.
[Show abstract][Hide abstract] ABSTRACT: Chlamydomonas reinhardtii Dangeard generates photoreceptor currents (PCs) upon light excitation. These currents play a key role in the signal transduction chain for photomotility responses. We have previously found that inhibition of PCs by tryptone occurs only in gametes that display chemotaxis toward this agent, and is not observed in chemotactically insensitive vegetative cells. Here we show that the sensitivity to tryptone is characteristic of gametes of both mating types, and examine the influence of gamete mating on PCs and their sensitivity to tryptone. The amplitude of PCs increases after cell fusion, but the sensitivity of these currents to tryptone decreases upon flagellar adhesion and/or an increase in the intracellular cAMP concentration. Net chemotaxis toward tryptone is reduced in young zygotes compared to gametes. We conclude that gamete mating leads to rapid inactivation of a gamete-specific chemosensory system.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.