Temperature-dependent augmentation of ciliary motility by the TRP2 channel in Chlamydomonas reinhardtii.

Temperature is a critical factor for living organisms. Many microorganisms migrate toward preferable temperatures, and this behavior is called thermotaxis. In this study, the molecular and physiological bases for thermotaxis are examined in Chlamydomonas reinhardtii. A mutant with knockout of a transient receptor potential (TRP) channel, trp2-3, showed defective thermotaxis. The swimming velocity and ciliary beat frequency of wild-type Chlamydomonas increase with temperature; however, this temperature-dependent enhancement of motility was almost absent in the trp2-3 mutant. Wild-type Chlamydomonas showed negative thermotaxis, but mutants deficient in the outer or inner dynein arm showed positive thermotaxis and a defect in temperature-dependent increase in swimming velocity, suggesting involvement of both dynein arms in thermotaxis.

Mechanoresponses mediated by the TRP11 channel in cilia of Chlamydomonas reinhardtii.

Cilia are organelles involved in motility and sensory transduction, but how these two functions coexist has not been elucidated in depth. Here, the involvement of the ciliary transient receptor potential (TRP) channel TRP11 in mechanoresponses is studied in Chlamydomonas reinhardtii using a TRP11-knockout mutant. The mutant has defects in the conversion of the bending mode of the cilium from forward to reverse when tapped with a glass rod, the detachment of cilia when shear is applied, the increase in ciliary beat frequency upon application of mechanical agitation by vortex mixing, and the initiation of gliding while both cilia are attached in opposite directions to a glass surface. These observations indicate that TRP11 can perceive mechanical stimuli with distinct intensities and durations and induce various types of ciliary responses.

Molecular Bulkiness of a Single Amino Acid in the F1 α-Subunit Determines the Robustness of Cyanobacterial ATP Synthase.

Cyanobacteria are promising photosynthetic organisms owing to their ease of genetic manipulation. Among them, Synechococcus elongatus UTEX 2973 exhibits faster growth, higher biomass production efficiency and more robust stress tolerance compared with S. elongatus PCC 7942. This is due to specific genetic differences, including four single-nucleotide polymorphisms (SNPs) in three genes. One of these SNPs alters an amino acid at position 252 of the FoF1 ATP synthase α-subunit from Tyr to Cys (αY252C) in S. elongatus 7942. This change has been shown to significantly affect growth rate and stress tolerance, specifically in S. elongatus. Furthermore, experimental substitutions with several other amino acids have been shown to alter the ATP synthesis rate in the cell. In the present study, we introduced identical amino acid substitutions into Synechocystis sp. PCC 6803 at position 252 to elucidate the amino acid's significance and generality across cyanobacteria. We investigated the resulting impact on growth, intracellular enzyme complex levels, intracellular ATP levels and enzyme activity. The results showed that the αY252C substitution decreased growth rate and high-light tolerance. This indicates that a specific bulkiness of this amino acid's side chain is important for maintaining cell growth. Additionally, a remarkable decrease in the membrane-bound enzyme complex level was observed. However, the αY252C substitution did not affect enzyme activity or intracellular ATP levels. Although the mechanism of growth suppression remains unknown, the amino acid at position 252 is expected to play an important role in enzyme complex formation.

Cells collectively migrate during ammonium chemotaxis in Chlamydomonas reinhardtii.

The mechanisms governing chemotaxis in Chlamydomonas reinhardtii are largely unknown compared to those regulating phototaxis despite equal importance on the migratory response in the ciliated microalga. To study chemotaxis, we made a simple modification to a conventional Petri dish assay. Using the assay, a novel mechanism governing Chlamydomonas ammonium chemotaxis was revealed. First, we found that light exposure enhances the chemotactic response of wild-type Chlamydomonas strains, yet phototaxis-incompetent mutant strains, eye3-2 and ptx1, exhibit normal chemotaxis. This suggests that Chlamydomonas transduces the light signal pathway in chemotaxis differently from that in phototaxis. Second, we found that Chlamydomonas collectively migrate during chemotaxis but not phototaxis. Collective migration during chemotaxis is not clearly observed when the assay is conducted in the dark. Third, the Chlamydomonas strain CC-124 carrying agg1-, the AGGREGATE1 gene (AGG1) null mutation, exhibited a more robust collective migratory response than strains carrying the wild-type AGG1 gene. The expression of a recombinant AGG1 protein in the CC-124 strain suppressed this collective migration during chemotaxis. Altogether, these findings suggest a unique mechanism; ammonium chemotaxis in Chlamydomonas is mainly driven by collective cell migration. Furthermore, it is proposed that collective migration is enhanced by light and suppressed by the AGG1 protein.

Changes in intracellular energetic and metabolite states due to increased galactolipid levels in Synechococcus elongatus PCC 7942.

The lipid composition of thylakoid membranes is conserved from cyanobacteria to green plants. However, the biosynthetic pathways of galactolipids, the major components of thylakoid membranes, are known to differ substantially between cyanobacteria and green plants. We previously reported on a transformant of the unicellular rod-shaped cyanobacterium Synechococcus elongatus PCC 7942, namely SeGPT, in which the synthesis pathways of the galactolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol are completely replaced by those of green plants. SeGPT exhibited increased galactolipid content and could grow photoautotrophically, but its growth rate was slower than that of wild-type S. elongatus PCC 7942. In the present study, we investigated pleiotropic effects that occur in SeGPT and determined how its increased lipid content affects cell proliferation. Microscopic observations revealed that cell division and thylakoid membrane development are impaired in SeGPT. Furthermore, physiological analyses indicated that the bioenergetic state of SeGPT is altered toward energy storage, as indicated by increased levels of intracellular ATP and glycogen. We hereby report that we have identified a new promising candidate as a platform for material production by modifying the lipid synthesis system in this way.

Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.

Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins.

Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔµH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔµH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔµH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔµH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants.

Observation of Photobehavior in Chlamydomonas reinhardtii.

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii's photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii.

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Györgyi demonstrated in the mid-20th century, in vitro reactivation of demembranated cells has been a traditional and potent way to examine cell motility. The fundamental advantage of this experimental method is that the composition of the reactivation solution may be easily changed. For example, a high-Ca2+ concentration environment that occurs only temporarily due to membrane excitation in vivo can be replicated in the lab. Eukaryotic cilia (a.k.a. flagella) are elaborate motility machinery whose regulatory mechanisms are still to be clarified. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism in the research field of cilia. The reactivation experiments using demembranated cell models of C. reinhardtii and their derivatives, such as demembranated axonemes of isolated cilia, have significantly contributed to understanding the molecular mechanisms of ciliary motility. Those experiments clarified that ATP energizes ciliary motility and that various cellular signals, including Ca2+, cAMP, and reactive oxygen species, modulate ciliary movements. The precise method for demembranation of C. reinhardtii cells and reactivation of the cell models is described here.

Verification of the Relationship between Redox Regulation of Thioredoxin Target Proteins and Their Proximity to Thylakoid Membranes.

Thioredoxin (Trx) is a key protein of the redox regulation system in chloroplasts, where it modulates various enzyme activities. Upon light irradiation, Trx reduces the disulfide bonds of Trx target proteins (thereby turning on their activities) using reducing equivalents obtained from the photosynthetic electron transport chain. This reduction process involves a differential response, i.e., some Trx target proteins in the stroma respond slowly to the change in redox condition caused by light/dark changes, while the ATP synthase γ subunit (CF1-γ) located on the surface of thylakoid membrane responds with high sensitivity. The factors that determine this difference in redox kinetics are not yet known, although here, we hypothesize that it is due to each protein's localization in the chloroplast, i.e., the reducing equivalents generated under light conditions can be transferred more efficiently to the proteins on thylakoid membrane than to stromal proteins. To explore this possibility, we anchored SBPase, one of the stromal Trx target proteins, to the thylakoid membrane in Arabidopsis thaliana. Analyses of the redox behaviors of the anchored and unanchored proteins showed no significant difference in their reduction kinetics, implying that protein sensitivity to redox regulation is determined by other factors.

The Importance of the C-Terminal Cys Pair of Phosphoribulokinase in Phototrophs in Thioredoxin-Dependent Regulation.

Phosphoribulokinase (PRK), one of the enzymes in the Calvin-Benson cycle, is a well-known target of thioredoxin (Trx), which regulates various enzyme activities by the reduction of disulfide bonds in a light-dependent manner. PRK has two Cys pairs conserved in the N-terminal and C-terminal regions, and the N-terminal one near the active site is thought to be responsible for the regulation. The flexible clamp loop located between the N-terminal two Cys residues has been deemed significant to Trx-mediated regulation. However, cyanobacterial PRK is also subject to Trx-dependent activation despite the lack of this clamp loop. We, therefore, compared Trx-mediated regulation of PRK from the cyanobacterium Anabaena sp. PCC 7120 (A.7120_PRK) and that from the land plant Arabidopsis thaliana (AtPRK). Interestingly, peptide mapping and site-directed mutagenesis analysis showed that Trx was more effective in changing the redox states of the C-terminal Cys pair in both A.7120_PRK and AtPRK. In addition, the effect of redox state change of the C-terminal Cys pair on PRK activity was different between A.7120_PRK and AtPRK. Trx-mediated redox regulation of the C-terminal Cys pair was also important for complex dissociation/formation with CP12 and glyceraldehyde 3-phosphate dehydrogenase. Furthermore, in vivo analysis of the redox states of PRK showed that only one disulfide bond is reduced in response to light. Based on the enzyme activity assay and the complex formation analysis, we concluded that Trx-mediated regulation of the C-terminal Cys pair of PRK is important for activity regulation in cyanobacteria and complex dissociation/formation in both organisms.

The mammalian-type thioredoxin reductase 1 confers a high-light tolerance to the green alga Chlamydomonas reinhardtii.

Reactive oxygen species (ROS) can both act as a poison causing cell death and important signaling molecules among various organisms. Photosynthetic organisms inevitably produce ROS, making the appropriate elimination of ROS an essential strategy for survival. Interestingly, the unicellular green alga Chlamydomonas reinhardtii expresses a mammalian form of thioredoxin reductase, TR1, which functions as a ROS scavenger in animal cells. To investigate the properties of TR1 in C. reinhardtii, we generated TR1 knockout strains using CRISPR/Cas9-based genome editing. We found a reduced tolerance to high-light and ROS stresses in the TR1 knockout strains compared to the parental strain. In addition, the regulation of phototactic orientation, known to be regulated by ROS, was affected in the knockout strains. These results suggest that TR1 contributes to a ROS-scavenging pathway in C. reinhardtii.

Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana.

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.

The four-celled Volvocales green alga Tetrabaena socialis exhibits weak photobehavior and high-photoprotection ability.

Photo-induced behavioral responses (photobehaviors) are crucial to the survival of motile phototrophic organisms in changing light conditions. Volvocine green algae are excellent model organisms for studying the regulatory mechanisms of photobehavior. We recently reported that unicellular Chlamydomonas reinhardtii and multicellular Volvox rousseletii exhibit similar photobehaviors, such as phototactic and photoshock responses, via different ciliary regulations. To clarify how the regulatory systems have changed during the evolution of multicellularity, we investigated the photobehaviors of four-celled Tetrabaena socialis. Surprisingly, unlike C. reinhardtii and V. rousseletii, T. socialis did not exhibit immediate photobehaviors after light illumination. Electrophysiological analysis revealed that the T. socialis eyespot does not function as a photoreceptor. Instead, T. socialis exhibited slow accumulation toward the light source in a photosynthesis-dependent manner. Our assessment of photosynthetic activities showed that T. socialis chloroplasts possess higher photoprotection abilities against strong light than C. reinhardtii. These data suggest that C. reinhardtii and T. socialis employ different strategies to avoid high-light stress (moving away rapidly and gaining photoprotection, respectively) despite their close phylogenetic relationship.

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins.

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein-based or yellow fluorescent protein-based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes "ROBINc" and "ROBINy." ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.

Characterization of Chlamydomonas reinhardtii Mutants That Exhibit Strong Positive Phototaxis.

The most motile phototrophic organisms exhibit photo-induced behavioral responses (photobehavior) to inhabit better light conditions for photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study photobehavior. Several years ago, we found that C. reinhardtii cells reverse their phototactic signs (i.e., positive and negative phototaxis) depending on the amount of reactive oxygen species (ROS) accumulated in the cell. However, its molecular mechanism is unclear. In this study, we isolated seven mutants showing positive phototaxis, even after the induction of negative phototaxis (ap1~7: always positive) to understand the ROS-dependent regulatory mechanism for the phototactic sign. We found no common feature in the mutants regarding their growth, high-light tolerance, and photosynthetic phenotypes. Interestingly, five of them grew faster than the wild type. These data suggest that the ROS-dependent regulation of the phototactic sign is not a single pathway and is affected by various cellular factors. Additionally, the isolation and analyses of mutants with defects in phototactic-sign regulation may provide clues for their application to the efficient cultivation of algae.

Redox regulation of NADP-malate dehydrogenase is vital for land plants under fluctuating light environment.

Many enzymes involved in photosynthesis possess highly conserved cysteine residues that serve as redox switches in chloroplasts. These redox switches function to activate or deactivate enzymes during light-dark transitions and have the function of fine-tuning their activities according to the intensity of light. Accordingly, many studies on chloroplast redox regulation have been conducted under the hypothesis that "fine regulation of the activities of these enzymes is crucial for efficient photosynthesis." However, the impact of the regulatory system on plant metabolism is still unclear. To test this hypothesis, we here studied the impact of the ablation of a redox switch in chloroplast NADP-malate dehydrogenase (MDH). By genome editing, we generated a mutant plant whose MDH lacks one of its redox switches and is active even in dark conditions. Although NADPH consumption by MDH in the dark is expected to be harmful to plant growth, the mutant line did not show any phenotypic differences under standard long-day conditions. In contrast, the mutant line showed severe growth retardation under short-day or fluctuating light conditions. These results indicate that thiol-switch redox regulation of MDH activity is crucial for maintaining NADPH homeostasis in chloroplasts under these conditions.

Thioredoxin pathway in Anabaena sp. PCC 7120: activity of NADPH-thioredoxin reductase C.

To understand the physiological role of NADPH-thioredoxin reductase C (NTRC) in cyanobacteria, we investigated an NTRC-deficient mutant strain of Anabaena sp., PCC 7120, cultivated under different regimes of nitrogen supplementation and light exposure. The deletion of ntrC did not induce a change in the cell structure and metabolic pathways. However, time-dependent changes in the abundance of specific proteins and metabolites were observed. A decrease in chlorophyll a was correlated with a decrease in chlorophyll a biosynthesis enzymes and photosystem I subunits. The deletion of ntrC led to a deregulation of nitrogen metabolism, including the NtcA accumulation and heterocyst-specific proteins while nitrate ions were available in the culture medium. Interestingly, this deletion resulted in a redox imbalance, indicated by higher peroxide levels, higher catalase activity and the induction of chaperones such as MsrA. Surprisingly, the antioxidant protein 2-CysPrx was downregulated. The deficiency in ntrC also resulted in the accumulation of metabolites such as 6-phosphogluconate, ADP and ATP. Higher levels of NADP+ and NADPH partly correlated with higher G6PDH activity. Rather than impacting protein expression levels, NTRC appears to be involved in the direct regulation of enzymes, especially during the dark-to-light transition period.

A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light.

Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.