Functional modulation of chemical mediators in microbial communities.

Interactions between microorganisms are often mediated by specialized metabolites. Although the structures and biosynthesis of these compounds may have been elucidated, microbes exist within complex microbiomes and chemical signals can thus also be subject to community-dependent modifications. Increasingly powerful chemical and biological tools allow to shed light on this poorly understood aspect of chemical ecology. We provide an overview of loss-of-function and gain-of-function chemical mediator (CM) modifications within microbial multipartner relationships. Although loss-of-function modifications are abundant in the literature, few gain-of-function modifications have been described despite their important role in microbial interactions. Research in this field holds great potential for our understanding of microbial interactions and may also provide novel tools for targeted interference with microbial signaling.

A mutualistic bacterium rescues a green alga from an antagonist.

Photosynthetic protists, known as microalgae, are key contributors to primary production on Earth. Since early in evolution, they coexist with bacteria in nature, and their mode of interaction shapes ecosystems. We have recently shown that the bacterium Pseudomonas protegens acts algicidal on the microalga Chlamydomonas reinhardtii. It secretes a cyclic lipopeptide and a polyyne that deflagellate, blind, and lyse the algae [P. Aiyar et al., Nat. Commun. 8, 1756 (2017) and V. Hotter et al., Proc. Natl. Acad. Sci. U.S.A. 118, e2107695118 (2021)]. Here, we report about the bacterium Mycetocola lacteus, which establishes a mutualistic relationship with C. reinhardtii and acts as a helper. While M. lacteus enhances algal growth, it receives methionine as needed organic sulfur and the vitamins B1, B3, and B5 from the algae. In tripartite cultures with the alga and the antagonistic bacterium P. protegens, M. lacteus aids the algae in surviving the bacterial attack. By combining synthetic natural product chemistry with high-resolution mass spectrometry and an algal Ca2+ reporter line, we found that M. lacteus rescues the alga from the antagonistic bacterium by cleaving the ester bond of the cyclic lipopeptide involved. The resulting linearized seco acid does not trigger a cytosolic Ca2+ homeostasis imbalance that leads to algal deflagellation. Thus, the algae remain motile, can swim away from the antagonistic bacteria and survive the attack. All three involved genera cooccur in nature. Remarkably, related species of Pseudomonas and Mycetocola also act antagonistically against C. reinhardtii or as helper bacteria in tripartite cultures.

A polyyne toxin produced by an antagonistic bacterium blinds and lyses a Chlamydomonad alga.

Algae are key contributors to global carbon fixation and form the basis of many food webs. In nature, their growth is often supported or suppressed by microorganisms. The bacterium Pseudomonas protegens Pf-5 arrests the growth of the green unicellular alga Chlamydomonas reinhardtii, deflagellates the alga by the cyclic lipopeptide orfamide A, and alters its morphology [P. Aiyar et al., Nat. Commun. 8, 1756 (2017)]. Using a combination of Raman microspectroscopy, genome mining, and mutational analysis, we discovered a polyyne toxin, protegencin, which is secreted by P. protegens, penetrates the algal cells, and causes destruction of the carotenoids of their primitive visual system, the eyespot. Together with secreted orfamide A, protegencin thus prevents the phototactic behavior of C. reinhardtii A mutant of P. protegens deficient in protegencin production does not affect growth or eyespot carotenoids of C. reinhardtii Protegencin acts in a direct and destructive way by lysing and killing the algal cells. The toxic effect of protegencin is also observed in an eyeless mutant and with the colony-forming Chlorophyte alga Gonium pectorale These data reveal a two-pronged molecular strategy involving a cyclic lipopeptide and a conjugated tetrayne used by bacteria to attack select Chlamydomonad algae. In conjunction with the bloom-forming activity of several chlorophytes and the presence of the protegencin gene cluster in over 50 different Pseudomonas genomes [A. J. Mullins et al., bioRxiv [Preprint] (2021). https://www.biorxiv.org/content/10.1101/2021.03.05.433886v1 (Accessed 17 April 2021)], these data are highly relevant to ecological interactions between Chlorophyte algae and Pseudomonadales bacteria.

A cyclic lipopeptide produced by an antagonistic bacterium relies on its tail and transient receptor potential-type Ca2+ channels to immobilize a green alga.

The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide (CLiP) orfamide A, which triggers a Ca2+ signal causing rapid deflagellation of the microalga Chlamydomonas reinhardtii. We performed chemical synthesis of orfamide A derivatives and used an aequorin reporter line to measure their Ca2+ responses. Immobilization of algae was studied using a modulator and mutants of transient receptor potential (TRP)-type channels. By investigating targeted synthetic orfamide A derivatives, we found that N-terminal amino acids of the linear part and the terminal fatty acid region are important for the specificity of the Ca2+ -signal causing deflagellation. Molecular editing indicates that at least two distinct Ca2+ -signaling pathways are triggered. One is involved in deflagellation (Thr3 change, fatty acid tail shortened by 4C), whereas the other still causes an increase in cytosolic Ca2+ in the algal cells, but does not cause substantial deflagellation (Leu1 change, fatty acid hydroxylation, fatty acid changes by 2C). Using mutants, we define four TRP-type channels that are involved in orfamide A signaling; only one (ADF1) responds additionally to low pH. These results suggest that the linear part of the CLiP plays one major role in Ca2+ signaling, and that orfamide A uses a network of algal TRP-type channels for deflagellation.

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 C-terminus of a diatom plant-like cryptochrome influences the FAD redox state and binding of interaction partners.

A plant-like cryptochrome of diatom microalgae, CryP, acts as a photoreceptor involved in transcriptional regulation. It contains FAD and 5,10-methenyltetrahydrofolate as chromophores. Here, we demonstrate that the unstructured C-terminal extension (CTE) of CryP has an influence on the redox state of the flavin. In CryP lacking the CTE, the flavin is in the oxidized state (FADox), whereas it is a neutral radical (FADH•) in the full-length protein. When the CTE of CryP is coupled to another diatom cryptochrome that naturally binds FADox, this chimera also binds FADH•. In full-length CryP, FADH• is the most stable redox state and oxidation to FADox is extremely slow, whereas reduction to FADH2 is reversible in the dark in approximately 1 h. We also identified novel interaction partners of this algal CRY and characterized two of them in depth regarding their binding activities. BolA, a putative transcription factor, binds to monomeric and to dimeric CryP via the CTE, independent of the redox state of the flavin. In contrast, an unknown protein, ID42612, which occurs solely in heterokont algae, binds only to CryP dimers. This binding is independent of the CTE and shows slight differences in strength depending on the flavin's redox state.

Total Synthesis and Structure Correction of the Cyclic Lipodepsipeptide Orfamide A.

A total synthesis of the cyclic lipodepsipeptide natural product orfamide A was achieved. By developing a synthesis format using an aminoacid ester building block and SPPS protocol adaptation, a focused library of target compounds was obtained, in high yield and purity. Spectral and LC-HRMS data of all library members with the isolated natural product identified the 5 Leu residue to be d- and the 3'-OH group to be R-configured. The structural correction of orfamide A by chemical synthesis and analysis was confirmed by biological activity comparison in Chlamydomonas reinhardtii, which indicated compound configuration to be important for bioactivity. Acute toxicity was also found against Trypanosoma brucei, the parasite causing African sleeping sickness.

A blue-light photoreceptor mediates the feedback regulation of photosynthesis.

In plants and algae, light serves both as the energy source for photosynthesis and a biological signal that triggers cellular responses via specific sensory photoreceptors. Red light is perceived by bilin-containing phytochromes and blue light by the flavin-containing cryptochromes and/or phototropins (PHOTs), the latter containing two photosensory light, oxygen, or voltage (LOV) domains. Photoperception spans several orders of light intensity, ranging from far below the threshold for photosynthesis to values beyond the capacity of photosynthetic CO2 assimilation. Excess light may cause oxidative damage and cell death, processes prevented by enhanced thermal dissipation via high-energy quenching (qE), a key photoprotective response. Here we show the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green alga Chlamydomonas reinhardtii. We show that PHOT controls qE by inducing the expression of the qE effector protein LHCSR3 (light-harvesting complex stress-related protein 3) in high light intensities. This control requires blue-light perception by LOV domains on PHOT, LHCSR3 induction through PHOT kinase, and light dissipation in photosystem II via LHCSR3. Mutants deficient in the PHOT gene display severely reduced fitness under excessive light conditions, indicating that the sensing, utilization, and dissipation of light is a concerted process that plays a vital role in microalgal acclimation to environments of variable light intensities.

The bacterium Pseudomonas protegens antagonizes the microalga Chlamydomonas reinhardtii using a blend of toxins.

The unicellular alga Chlamydomonas reinhardtii and the bacterium Pseudomonas protegens serve as a model to study the interactions between photosynthetic and heterotrophic microorganisms. P. protegens secretes the cyclic lipopeptide orfamide A that interferes with cytosolic Ca2+ homeostasis in C. reinhardtii resulting in deflagellation of the algal cells. Here, we studied the roles of additional secondary metabolites secreted by P. protegens using individual compounds and co-cultivation of algae with bacterial mutants. Rhizoxin S2, pyrrolnitrin, pyoluteorin, 2,4-diacetylphloroglucinol (DAPG) and orfamide A all induce changes in cell morphology and inhibit the growth of C. reinhardtii. Rhizoxin S2 exerts the strongest growth inhibition, and its action depends on the spatial structure of the environment (agar versus liquid culture). Algal motility is unaffected by rhizoxin S2 and is most potently inhibited by orfamide A (IC50  = 4.1 µM). Pyrrolnitrin and pyoluteorin both interfere with algal cytosolic Ca2+ homeostasis and motility whereas high concentrations of DAPG immobilize C. reinhardtii without deflagellation or disturbance of Ca2+ homeostasis. Co-cultivation with a regulatory mutant of bacterial secondary metabolism (ΔgacA) promotes algal growth under spatially structured conditions. Our results reveal how a single soil bacterium uses an arsenal of secreted antialgal compounds with complementary and partially overlapping activities.

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.

A marine Chlamydomonas sp. emerging as an algal model.

The freshwater microalga Chlamydomonas reinhardtii, which lives in wet soil, has served for decades as a model for numerous biological processes, and many tools have been introduced for this organism. Here, we have established a stable nuclear transformation for its marine counterpart, Chlamydomonas sp. SAG25.89, by fusing specific cis-acting elements from its Actin gene with the gene providing hygromycin resistance and using an elaborated electroporation protocol. Like C. reinhardtii, Chlamydomonas sp. has a high GC content, allowing reporter genes and selection markers to be applicable in both organisms. Chlamydomonas sp. grows purely photoautotrophically and requires ammonia as a nitrogen source because its nuclear genome lacks some of the genes required for nitrogen metabolism. Interestingly, it can grow well under both low and very high salinities (up to 50 g · L-1 ) rendering it as a model for osmotolerance. We further show that Chlamydomonas sp. grows well from 15 to 28°C, but halts its growth at 32°C. The genome of Chlamydomonas sp. contains some gene homologs the expression of which is regulated according to the ambient temperatures and/or confer thermal acclimation in C. reinhardtii. Thus, knowledge of temperature acclimation can now be compared to the marine species. Furthermore, Chlamydomonas sp. can serve as a model for studying marine microbial interactions and for comparing mechanisms in freshwater and marine environments. Chlamydomonas sp. was previously shown to be immobilized rapidly by a cyclic lipopeptide secreted from the antagonistic bacterium Pseudomonas protegens PF-5, which deflagellates C. reinhardtii.

Fucoxanthin-Chlorophyll Protein Complexes of the Centric Diatom Cyclotella Meneghiniana Differ in Lhcx1 and Lhcx6_1 Content.

The ecological success of diatoms, key contributors to photosynthesis, is partly based on their ability to perfectly balance efficient light harvesting and photoprotection. Diatoms contain higher numbers of antenna proteins than vascular plants for light harvesting and for photoprotection. These proteins are arranged in fucoxanthin-chlorophyll protein (FCP) complexes. The number of FCP complexes, their subunit composition, and their interactions in the thylakoid membranes remain elusive in different diatoms. We used the recently available genome sequence of the centric diatom Cyclotella cryptica to analyze gene sequences for putative light-harvesting proteins in C. meneghiniana, and to elucidate the FCP complex composition. We analyzed two pools of FCP complexes that were trimeric (FCPa) and nonameric (FCPb). FCPa was composed of four different trimeric subtypes. Two different nonameric FCPb complexes were present. All were distinguished by their polypeptide composition and partly by pigmentation. With use of a milder purification method, two fractions composed of different FCP complexes were isolated. One was enriched in FCPs incorporating the photoprotective subunit Lhcx1, such as the newly identified nonameric FCPb2 and the major trimeric FCPa4 complex, which are predetermined to be involved in energy-dependent nonphotochemical quenching. The other fraction contained mainly FCPs that were devoid of Lhcx1, FCPa3, and FCPb1. Both fractions also included small amounts of trimeric FCPa complexes with the centric diatom-specific Lhcx protein, Lhcx6_1, as subunit. Thus, the antenna organization of centric diatoms, as well as the distribution of different photoprotective Lhcx proteins, differs from that of other diatoms, as well as from plants.

Structure of the bifunctional cryptochrome aCRY from Chlamydomonas reinhardtii.

Photolyases and cryptochromes form an almost ubiquitous family of blue light photoreceptors involved in the repair and maintenance of DNA integrity or regulatory control. We found that one cryptochrome from the green alga Chlamydomonas reinhardtii (CraCRY) is capable of both, control of transcript levels and the sexual cycle of the alga in a positive (germination) and negative manner (mating ability), as well as catalyzing the repair of UV-DNA lesions. Its 1.6 Å crystal structure shows besides the FAD chromophore an aromatic tetrad that is indispensable in animal-like type I cryptochromes for light-driven change of their signaling-active redox state and formation of a stable radical pair. Given CraCRY's catalytic activity as (6-4) photolyase in vivo and in vitro, we present the first co-crystal structure of a cryptochrome with duplex DNA comprising a (6-4) pyrimidine-pyrimidone lesion. This 2.9 Å structure reveals a distinct conformation for the catalytic histidine His1, H357, that challenges previous models of a single-photon driven (6-4) photolyase mechanism.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Time-Resolved Infrared and Visible Spectroscopy on Cryptochrome aCRY: Basis for Red Light Reception.

Cryptochromes function as flavin-binding photoreceptors in bacteria, fungi, algae, land plants, and insects. The discovery of an animal-like cryptochrome in the green alga Chlamydomonas reinhardtii has expanded the spectral range of sensitivity of these receptors from ultraviolet A/blue light to almost the complete visible spectrum. The broadened light response has been explained by the presence of the flavin neutral radical as a chromophore in the dark. Concomitant with photoconversion of the flavin, an unusually long-lived tyrosyl radical with a red-shifted ultraviolet-visible spectrum is formed, which is essential for the function of the receptor. In this study, the microenvironment of this key residue, tyrosine 373, was scrutinized using time-resolved Fourier transform infrared spectroscopy on several variants of animal-like cryptochrome and density functional theory for band assignment. The reduced tyrosine takes on distinct hydrogen bond scenarios depending on the presence of the C-terminal extension and of a neighboring cysteine. Upon radical formation, all variants showed a signal at 1400 cm-1, which we assigned to the ν7'a marker band of the CO stretching mode. The exceptionally strong downshift of this band cannot be attributed to a loss of hydrogen bonding only. Time-resolved ultraviolet-visible spectroscopy on W322F, a mutant of the neighboring tryptophan residue, revealed a decrease of the tyrosyl radical lifetime by almost two orders of magnitude, along with a shift of the absorbance maximum from 416 to 398 nm. These findings strongly support the concept of a π-π stacking as an apolar interaction between Y373 and W322 to be responsible for the characteristics of the tyrosyl radical. This concept of radical stabilization has been unknown to cryptochromes so far but might be highly relevant for other homologs with a tetrad of tryptophans and tyrosines as electron donors.

A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii.

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non-toxic algae, including those of many green algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in 2-day-old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild-type zygospores contain knob-like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild-type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption in zygospore development. In agreement with the role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild-type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions.

A Musashi Splice Variant and Its Interaction Partners Influence Temperature Acclimation in Chlamydomonas.

Microalgae contribute significantly to carbon fixation on Earth. Global warming influences their physiology and growth rates. To understand algal short-term acclimation and adaptation to changes in ambient temperature, it is essential to identify and characterize the molecular components that sense small temperature changes as well as the downstream signaling networks and physiological responses. Here, we used the green biflagellate alga Chlamydomonas reinhardtii as a model system in which to study responses to temperature. We report that an RNA recognition motif (RRM)-containing RNA-binding protein, Musashi, occurs in 25 putative splice variants. These variants bear one, two, and three RRM domains or even lack RRM domains. The most abundant Musashi variant, 12, with a molecular mass of 60 kD, interacts with two clock-relevant members of RNA metabolism, the subunit C3 of the RNA-binding protein CHLAMY1 and the 5'-3' exoribonuclease XRN1. These proteins are able to integrate temperature information by up- or down-regulation of their protein levels in cells grown at low (18°C) or high (28°C) temperature. We further show that the 60-kD Musashi variants with three RRM domains can bind to (UG)7 repeat-containing RNAs and are up-regulated in cells grown at a higher temperature during early night. Intriguingly, the 60-kD Musashi variant 12, as well as C3 and XRN1, confer thermal acclimation to C. reinhardtii, as shown with mutant lines. Our data suggest that these three proteins of the RNA metabolism machinery are key members of the thermal signaling network in C. reinhardtii.

ROC75 is an Attenuator for the Circadian Clock that Controls LHCSR3 Expression.

Strong light intensity leads to harmful overexcitation of the photosystems in green algae. In Chlamydomonas reinhardtii, LHCSR3 is required for the rapid protective response known as energy-dependent quenching (qE). Because the majority of photoacclimation analysis has been conducted under controlled laboratory conditions, physiological responses to natural environmental changes such as light/dark cycles have not been examined in detail. Regarding fitness in higher plants and microalgae, light-dark cycles represent a major Zeitgeber for synchronizing the circadian clock to multiple physiological responses, yet there is little consensus with respect to the clock response to high-intensity light in photosynthetic organisms. In a previous study, 105 circadian rhythm insertional mutants were isolated as rhythm of chloroplast (roc) mutants. Here, we report our characterization of the roc75 mutant, which exhibited a significantly higher qE value and LHCSR3 protein accumulation when grown under red light. We performed transcript analysis of ROC75 in the pcry (plant-cryptochrome) and phot mutants and found that only the former accumulated lower levels of ROC75 mRNA, suggesting that the blue light photoreceptor pCRY positively regulates ROC75. However, the degradation of pCRY by high-light exposure contributes to prevent over-accumulation of ROC75, which in turn facilitates the PHOT mediated main activation pathway for LHCSR3. Furthermore, LHCSR3 mRNA exhibited a circadian rhythm, though its basal expression level in the roc75 mutant was higher than that in WT. We therefore conclude that ROC75 acts as an attenuator of the circadian clock to control LHCSR3 expression with blue and red light as stimuli for attenuation.

An Animal-Like Cryptochrome Controls the Chlamydomonas Sexual Cycle.

Cryptochromes are known as flavin-binding blue light receptors in bacteria, fungi, plants, and insects. The animal-like cryptochrome (aCRY) of the green alga Chlamydomonas reinhardtii has extended our view on cryptochromes, because it responds also to other wavelengths of the visible spectrum, including red light. Here, we have investigated if aCRY is involved in the regulation of the sexual life cycle of C. reinhardtii, which is controlled by blue and red light at the steps of gametogenesis along with its restoration and germination. We show that aCRY is differentially expressed not only during the life cycle but also within the cell as part of the soluble and/or membrane-associated protein fraction. Moreover, localization of aCRY within the algal cell body varies between vegetative cells and the different cell types of gametogenesis. aCRY is significantly (early day) or to a small extent (late night) enriched in the nucleus in vegetative cells. In pregametes, gametes and dark-inactivated gametes, aCRY is localized over the cell body. aCRY plays an important role in the sexual life cycle of C. reinhardtii: It controls the germination of the alga, under which the zygote undergoes meiosis, in a positive manner, similar to the regulation by the blue light receptors phototropin and plant cryptochrome (pCRY). However, aCRY acts in combination with pCRY as a negative regulator for mating ability as well as for mating maintenance, opposite to the function of phototropin in these processes.

A Plant Cryptochrome Controls Key Features of the Chlamydomonas Circadian Clock and Its Life Cycle.

Cryptochromes are flavin-binding proteins that act as blue light receptors in bacteria, fungi, plants, and insects and are components of the circadian oscillator in mammals. Animal and plant cryptochromes are evolutionarily divergent, although the unicellular alga Chlamydomonas reinhardtii (Chlamydomonas throughout) has both an animal-like cryptochrome and a plant cryptochrome (pCRY; formerly designated CPH1). Here, we show that the pCRY protein accumulates at night as part of a complex. Functional characterization of pCRY was performed based on an insertional mutant that expresses only 11% of the wild-type pCRY level. The pcry mutant is defective for central properties of the circadian clock. In the mutant, the period is lengthened significantly, ultimately resulting in arrhythmicity, while blue light-based phase shifts show large deviations from what is observed in wild-type cells. We also show that pCRY is involved in gametogenesis in Chlamydomonas pCRY is down-regulated in pregametes and gametes, and in the pcry mutant, there is altered transcript accumulation under blue light of the strictly light-dependent, gamete-specific gene GAS28 pCRY acts as a negative regulator for the induction of mating ability in the light and for the loss of mating ability in the dark. Moreover, pCRY is necessary for light-dependent germination, during which the zygote undergoes meiosis that gives rise to four vegetative cells. In sum, our data demonstrate that pCRY is a key blue light receptor in Chlamydomonas that is involved in both circadian timing and life cycle progression.