The UV-A Receptor CRY-DASH1 Up- and Downregulates Proteins Involved in Different Plastidial Pathways.

Algae encode up to five different types of cryptochrome photoreceptors. So far, relatively little is known about the biological functions of the DASH (Drosophila, Arabidopsis, Synechocystis and Homo)-type cryptochromes. The green alga Chlamydomonas reinhardtii encodes two of them. CRY-DASH1 also called DCRY1 has its maximal absorption peak in the UV-A range. It is localized in the chloroplast and plays an important role in balancing the photosynthetic machinery. Here, we performed a comparative analysis of chloroplast proteins from wild type and a knockout mutant of CRY-DASH1 named cry-dash1mut, using label-free quantitative proteomics as well as immunoblotting. Our results show upregulation of enzymes involved in specific pathways in the mutant including key enzymes of chlorophyll and carotenoid biosynthesis consistent with increased levels of photosynthetic pigments in cry-dash1mut. There is also an increase in certain redox as well as photosystem I and II proteins, including D1. Strikingly, CRY-DASH1 is coregulated in a D1 deletion mutant, where its amount is increased. In contrast, key proteins of the central carbon metabolism, including glycolysis/gluconeogenesis, dark fermentation and the oxidative pentose phosphate pathway are downregulated in cry-dash1mut. Similarly, enzymes of histidine biosynthesis are downregulated in cry-dash1mut leading to a reduction in the amount of free histidine. Yet, transcripts encoding for several of these proteins are at a similar level in the wild type and cry-dash1mut or even opposite. We show that CRY-DASH1 can bind to RNA, taking the psbA RNA encoding D1 as target. These data suggest that CRY-DASH1 regulates plastidial metabolic pathways at the posttranscriptional level.

Evolution of circadian clocks along the green lineage.

Circadian clocks govern temporal programs in the green lineage (Chloroplastida) as they do in other photosynthetic pro- and eukaryotes, bacteria, fungi, animals, and humans. Their physiological properties, including entrainment, phase responses, and temperature compensation, are well conserved. The involvement of transcriptional/translational feedback loops in the oscillatory machinery and reversible phosphorylation events are also maintained. Circadian clocks control a large variety of output rhythms in green algae and terrestrial plants, adjusting their metabolism and behavior to the day-night cycle. The angiosperm Arabidopsis (Arabidopsis thaliana) represents a well-studied circadian clock model. Several molecular components of its oscillatory machinery are conserved in other Chloroplastida, but their functions may differ. Conserved clock components include at least one member of the CIRCADIAN CLOCK ASSOCIATED1/REVEILLE and one of the PSEUDO RESPONSE REGULATOR family. The Arabidopsis evening complex members EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHMO are found in the moss Physcomitrium patens and in the liverwort Marchantia polymorpha. In the flagellate chlorophyte alga Chlamydomonas reinhardtii, only homologs of ELF4 and LUX (named RHYTHM OF CHLOROPLAST ROC75) are present. Temporal ROC75 expression in C. reinhardtii is opposite to that of the angiosperm LUX, suggesting different clock mechanisms. In the picoalga Ostreococcus tauri, both ELF genes are missing, suggesting that it has a progenitor circadian "green" clock. Clock-relevant photoreceptors and thermosensors vary within the green lineage, except for the CRYPTOCHROMEs, whose variety and functions may differ. More genetically tractable models of Chloroplastida are needed to draw final conclusions about the gradual evolution of circadian clocks within the green lineage.

The World of Algae Reveals a Broad Variety of Cryptochrome Properties and Functions.

Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together with the cyanobacteria they contribute to about half of global carbon fixation. As primary producers, they are at the basis of many food webs and they are involved in biogeochemical processes. Algae are evolutionarily distinct and are derived either by primary (e.g., green and red algae) or secondary endosymbiosis (e.g., diatoms, dinoflagellates, and brown algae). Light is a key abiotic factor needed to maintain the fitness of algae as it delivers energy for photosynthesis, regulates algal cell- and life cycles, and entrains their biological clocks. However, excess light can also be harmful, especially in the ultraviolet range. Among the variety of receptors perceiving light information, the cryptochromes originally evolved as UV-A and blue-light receptors and have been found in all studied algal genomes so far. Yet, the classification, biophysical properties, wavelength range of absorbance, and biological functions of cryptochromes are remarkably diverse among algal species, especially when compared to cryptochromes from land plants or animals.

DASH cryptochrome 1, a UV-A receptor, balances the photosynthetic machinery of Chlamydomonas reinhardtii.

Drosophila, Arabidopsis, Synechocystis, Homo (DASH) cryptochromes belong to the cryptochrome/photolyase family and can act as DNA repair enzymes. In bacteria and fungi, they also can play regulatory roles, but in plants their biological functions remain elusive. Here, we characterize CRY-DASH1 from the green alga Chlamydomonas reinhardtii. We perform biochemical and in vitro photochemical analysis. For functional characterization, a knock-out mutant of cry-dash1 is used. CRY-DASH1 protein is localized in the chloroplast and accumulates at midday. Although the photoautotrophic growth of the mutant is significantly reduced compared to the wild-type (WT), the mutant has increased levels of photosynthetic pigments and a higher maximum photochemical efficiency of photosystem II (PS II). Hyper-stacking of thylakoid membranes occurs together with an increase in proteins of the PS II reaction center, D1 and its antenna CP43, but not of their transcripts. CRY-DASH1 binds fully reduced flavin adenine dinucleotide and the antenna 5,10-methenyltetrahydrofolate, leading to an absorption peak in the UV-A range. Supplementation of white light with UV-A increases photoautotrophic growth of the WT but not of the cry-dash1 mutant. These results suggest a balancing function of CRY-DASH1 in the photosynthetic machinery and point to its role as a photoreceptor for the UV-A range separated from the absorption of photosynthetic pigments.