Lycorine and UV-C stimulate phenolic secondary metabolites production and miRNA expression in Chlamydomonas reinhardtii.

Studying stress pathways on the level of secondary metabolites that are found in very small concentration in the cells is complicated. In the algae, the role of individual metabolites (such as carotenoids, phenolic compounds, organic acids, and vitamins) and miRNAs that participate in plant's defence are very poorly understood during stressful conditions. Therefore, in the present experiment, the model organism Chlamydomonas reinhardtii was exposed to stress conditions (Lyc and UV-C irradiation) to detect these substances, even at very low concentrations. The purpose was to monitored changes at each response level with a future view to identifying their specific roles under different stress factors. In stress-treated cultures, numerous transcriptomic and metabolomic pathways were triggered in C. reinhardtii. Although Lyc significantly decreased the concentration of AA, suggesting that Lyc has a similar function in C. reinhardtii as in plants. The negative effect of UV-C radiation was based on the production of ROS and enhancement of antioxidant responses, resulting in increased levels of polyphenols and simple phenolic compounds. Both treatments did lead to extensive changes in transcript levels and miRNA expression patterns.

A role for glutathione reductase and glutathione in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress.

The role of glutathione reductase (GR; EC 1.6.4.2) in the tolerance of Chlamydomonas reinhardtii P.A. Dangeard to high-intensity light stress (HL, 1400 µmol m-2 s-1 ) was examined. Cells survived under high light (HL) stress, although their growth was inhibited after long-term treatment (9-24 h). GR activity increased 1 h after HL treatment. The contents of total glutathione, reduced glutathione (GSH) and glutathione disulfide (GSSG) increased 1-3 h after HL treatment and then decreased after 24 h, while the GSH:GSSG ratio (glutathione redox potential) decreased after 3-9 h and recovered after 24 h. The transcript abundance of GR, CrGR1 (Cre06.g262100) and CrGR2 (Cre09.g396252) as well as glutathione synthesis-related genes, CrGSH1 (Cre02g077100.t1.1) and CrGSH2 (Cre17.g70800.t1.1), increased with a peak near 1 h after HL treatment. Except for enhanced glutathione synthesis, the GR-mediated glutathione redox machinery is also critical for the tolerance of C. reinhardtii cells to HL stress. Therefore, GR was downregulated or upregulated to investigate the importance of GR in HL tolerance. The CrGR1 knockdown amiRNA line exhibited low GR transcript abundance, GR activity and GSH:GSSG ratio and could not survive under HL conditions. Over-expression of CrGR1 or CrGR2 driven by a HSP70A:RBCS2 fusion promoter resulted in a higher GR transcript abundance, GR activity and GSH:GSSG ratio and led to cell survival when exposed to high-intensity illumination, i.e. 1800 µmol m-2 s-1 . In conclusion, GR-mediated modulation of the glutathione redox potential plays a role in the tolerance of Chlamydomonas cells to photo-oxidative stress.

A penalty on photosynthetic growth in fluctuating light.

Fluctuating light is the norm for photosynthetic organisms, with a wide range of frequencies (0.00001 to 10 Hz) owing to diurnal cycles, cloud cover, canopy shifting and mixing; with broad implications for climate change, agriculture and bioproduct production. Photosynthetic growth in fluctuating light is generally considered to improve with increasing fluctuation frequency. Here we demonstrate that the regulation of photosynthesis imposes a penalty on growth in fluctuating light for frequencies in the range of 0.01 to 0.1 Hz (organisms studied: Synechococcus elongatus and Chlamydomonas reinhardtii). We provide a comprehensive sweep of frequencies and duty cycles. In addition, we develop a 2nd order model that identifies the source of the penalty to be the regulation of the Calvin cycle - present at all frequencies but compensated at high frequencies by slow kinetics of RuBisCO.

Chlamydomonas reinhardtii responding to high light: a role for 2-propenal (acrolein).

High light causes photosystem II to generate singlet oxygen (1 O2 ), a reactive oxygen species (ROS) that can react with membrane lipids, releasing reactive electrophile species (RES), such as acrolein. To investigate how RES may contribute to light stress responses, Chlamydomonas reinhardtii was high light-treated in photoautotrophic and mixotrophic conditions and also in an oxygen-enriched atmosphere to elevate ROS production. The responses were compared to exogenous acrolein. Non-photochemical quenching (NPQ) was higher in photoautotrophic cells, as a consequence of a more de-epoxidized state of the xanthophyll cycle pool and more LHCSR3 protein, showing that photosynthesis was under more pressure than in mixotrophic cells. Photoautotrophic cells had lowered α-tocopherol and ß-carotene contents and a higher level of protein carbonylation, indicators of elevated 1 O2 production. Levels of glutathione, glutathione peroxidase (GPX5) and glutathione-S-transferase (GST1), important antioxidants against RES, were also increased in photoautotrophic cells. In parallel to the wild-type, the LHCSR3-deficient npq4 mutant was high light-treated, which in photoautotrophic conditions exhibited particular sensitivity under elevated oxygen, the treatment that induced the highest RES levels, including acrolein. The npq4 mutant had more GPX5 and GST1 alongside higher levels of carbonylated protein and a more oxidized glutathione redox state. In wild-type cells glutathione contents doubled after 4 h treatment, either with high light under elevated oxygen or with a non-critical dose (600 ppm) of acrolein. Exogenous acrolein also increased GST1 levels, but not GPX5. Overall, RES-associated oxidative damage and glutathione metabolism are prominently associated with light stress and potentially in signaling responses of C. reinhardtii.

Sensitivity of the green algae Chlamydomonas reinhardtii to gamma radiation: Photosynthetic performance and ROS formation.

The aquatic environment is continuously exposed to ionizing radiation from both natural and anthropogenic sources, making the characterization of ecological and health risks associated with radiation of large importance. Microalgae represent the main source of biomass production in the aquatic ecosystem, thus becoming a highly relevant biological model to assess the impacts of gamma radiation. However, little information is available on the effects of gamma radiation on microalgal species, making environmental radioprotection of this group of species challenging. In this context, the present study aimed to improve the understanding of the effects and toxic mechanisms of gamma radiation in the unicellular green algae Chlamydomonas reinhardtii focusing on the activity of the photosynthetic apparatus and ROS formation. Algal cells were exposed to gamma radiation (0.49-1677mGy/h) for 6h and chlorophyll fluorescence parameters obtained by PAM fluorometry, while two fluorescent probes carboxy-H2DFFDA and DHR 123 were used for the quantification of ROS. The alterations seen in functional parameters of C. reinhardtii PSII after 6h of exposure to gamma radiation showed modifications of PSII energy transfer associated with electron transport and energy dissipation pathways, especially at the higher dose rates used. Results also showed that gamma radiation induced ROS in a dose-dependent manner under both light and dark conditions. The observed decrease in photosynthetic efficiency seems to be connected to the formation of ROS and can potentially lead to oxidative stress and cellular damage in chloroplasts. To our knowledge, this is the first report on changes in several chlorophyll fluorescence parameters associated with photosynthetic performance and ROS formation in microalgae after exposure to gamma radiation.

Enhanced Ascorbate Regeneration Via Dehydroascorbate Reductase Confers Tolerance to Photo-Oxidative Stress in Chlamydomonas reinhardtii.

The role of ascorbate (AsA) recycling via dehydroascorbate reductase (DHAR) in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress was examined. The activity of DHAR and the abundance of the CrDHAR1 (Cre10.g456750) transcript increased after moderate light (ML; 750 µmol m-2 s-1) or high light (HL; 1,800 µmol m-2 s-1) illumination, accompanied by dehydroascorbate (DHA) accumulation, decreased AsA redox state, photo-inhibition, lipid peroxidation, H2O2 overaccumulation, growth inhibition and cell death. It suggests that DHAR and AsA recycling is limiting under high-intensity light stress. The CrDHAR1 gene was cloned and its recombinant CrDHAR1 protein was a monomer (25 kDa) detected by Western blot that exhibits an enzymatic activity of 965 µmol min-1 mg-1 protein. CrDHAR1 was overexpressed driven by a HSP70A:RBCS2 fusion promoter or down-regulated by artificial microRNA (amiRNA) to examine whether DHAR-mediated AsA recycling is critical for the tolerance of C. reinahartii cells to photo-oxidative stress. The overexpression of CrDHAR1 increased DHAR protein abundance and enzyme activity, AsA pool size, AsA:DHA ratio and the tolerance to ML-, HL-, methyl viologen- or H2O2-induced oxidative stress. The CrDHAR1-knockdown amiRNA lines that have lower DHAR expression and AsA recycling ability were sensitive to high-intensity illumination and oxidative stress. The glutathione pool size, glutathione:oxidized glutathione ratio and glutathione reductase and ascorbate peroxidase activities were increased in CrDHAR1-overexpressing cells and showed a further increase after high-intensity illumination but decreased in wild-type cells after light stress. The present results suggest that increasing AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity in C. reinhardtii against photo-oxidative stress.

Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii.

Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity.

Acclimation of Chlamydomonas reinhardtii to ultraviolet radiation and its impact on chemical toxicity.

The toxicity of chemical pollutants can be modulated under stressful environmental conditions, such as increased temperature, salinity or ultraviolet radiation (UVR), due to the interaction of effects during simultaneous stressor exposure. However, organisms may acclimate to such conditions by activation of physiological and biochemical defence mechanisms. In sequential exposures, organisms acclimated to environmental stressors may display an increased sensitivity or co-tolerance towards chemical pollutants. It has been suggested that co-tolerance might be expected for similarly acting stressors due to common defence mechanisms. To test this for combinations of UVR and chemical stressors, we first acclimatized the model green alga Chlamydomonas reinhardtii to UVR and subsequently compared the sensitivity of UVR pre-exposed and control algae towards chemicals. Selected chemicals all act on photosynthesis and thus share a common physiological target, but display distinct toxicity mechanisms. Results showed that UVR pre-exposure for four days partially inhibited algal growth and photosynthesis, but also increased algal tolerance to higher UVR levels, confirming UVR acclimation. HPLC analysis of algal pigments indicated that UVR acclimation might in part be explained by the protective function of lutein while the contribution of UVR absorbing compounds was less clear. Challenge exposure to chemicals in the absence of UVR showed that acclimated algae were co-tolerant to the photosensitizer rose bengal, but not to the herbicides paraquat and diuron, suggesting that the fast physiological and biochemical defence mechanisms that conferred tolerance of algae towards higher UVR levels were related to singlet oxygen defence. The presented study suggests that knowledge of the molecular toxicity mechanisms of chemicals, rather than their general physiological target, is needed in order to predict co-tolerance between environmental and chemical stressors.

Multiple stressor effects in Chlamydomonas reinhardtii--toward understanding mechanisms of interaction between effects of ultraviolet radiation and chemical pollutants.

The effects of chemical pollutants and environmental stressors, such as ultraviolet radiation (UVR), can interact when organisms are simultaneously exposed, resulting in higher (synergistic) or lower (antagonistic) multiple stressor effects than expected based on the effects of single stressors. Current understanding of interactive effects is limited due to a lack of mechanism-based multiple stressor studies. It has been hypothesized that effect interactions may generally occur if chemical and non-chemical stressors cause similar physiological effects in the organism. To test this hypothesis, we exposed the model green alga Chlamydomonas reinhardtii to combinations of UVR and single chemicals displaying modes of action (MOA) similar or dissimilar to the impact of UVR on photosynthesis. Stressor interactions were analyzed based on the independent action model. Effect interactions were found to depend on the MOA of the chemicals, and also on their concentrations, the exposure time and the measured endpoint. Indeed, only chemicals assumed to cause effects on photosynthesis similar to UVR showed interactions with UVR on photosynthetic yield: synergistic in case of Cd(II) and paraquat and antagonistic in case of diuron. No interaction on photosynthesis was observed for S-metolachlor, which acts dissimilarly to UVR. However, combined effects of S-metolachlor and UVR on algal reproduction were synergistic, highlighting the importance of considering additional MOA of UVR. Possible mechanisms of stressor effect interactions are discussed.

Thioredoxin-dependent redox regulation of chloroplastic phosphoglycerate kinase from Chlamydomonas reinhardtii.

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (-335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys(227) and Cys(361). Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.

Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii.

During oxygenic photosynthesis, metabolic reactions of CO2 fixation require more ATP than is supplied by the linear electron flow operating from photosystem II to photosystem I (PSI). Different mechanisms, such as cyclic electron flow (CEF) around PSI, have been proposed to participate in reequilibrating the ATP/NADPH balance. To determine the contribution of CEF to microalgal biomass productivity, here, we studied photosynthesis and growth performances of a knockout Chlamydomonas reinhardtii mutant (pgrl1) deficient in PROTON GRADIENT REGULATION LIKE1 (PGRL1)-mediated CEF. Steady state biomass productivity of the pgrl1 mutant, measured in photobioreactors operated as turbidostats, was similar to its wild-type progenitor under a wide range of illumination and CO2 concentrations. Several changes were observed in pgrl1, including higher sensitivity of photosynthesis to mitochondrial inhibitors, increased light-dependent O2 uptake, and increased amounts of flavodiiron (FLV) proteins. We conclude that a combination of mitochondrial cooperation and oxygen photoreduction downstream of PSI (Mehler reactions) supplies extra ATP for photosynthesis in the pgrl1 mutant, resulting in normal biomass productivity under steady state conditions. The lower biomass productivity observed in the pgrl1 mutant in fluctuating light is attributed to an inability of compensation mechanisms to respond to a rapid increase in ATP demand.

Transcriptional regulation of the stress-responsive light harvesting complex genes in Chlamydomonas reinhardtii.

Dissipating excess energy of light is critical for photosynthetic organisms to keep the photosynthetic apparatus functional and less harmful under stressful environmental conditions. In the green alga Chlamydomonas reinhardtii, efficient energy dissipation is achieved by a process called non-photochemical quenching (NPQ), in which a distinct member of light harvesting complex, LHCSR, is known to play a key role. Although it has been known that two very closely related genes (LHCSR3.1 and LHCSR3.2) encoding LHCSR3 protein and another paralogous gene LHCSR1 are present in the C. reinhardtii genome, it is unclear how these isoforms are differentiated in terms of transcriptional regulation and functionalization. Here, we show that transcripts of both of the isoforms, LHCSR3.1 and LHCSR3.2, are accumulated under high light stress. Reexamination of the genomic sequence and gene models along with survey of sequence motifs suggested that these two isoforms shared an almost identical but still distinct promoter sequence and a completely identical polypeptide sequence, with more divergent 3'-untranscribed regions. Transcriptional induction under high light condition of both isoforms was suppressed by treatment with a photosystem II inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a calmodulin inhibitor W7. Despite a similar response to high light, the inhibitory effects of DCMU and W7 to the LHCSR1 transcript accumulation were limited compared to LHCSR3 genes. These results suggest that the transcription of LHCSR paralogs in C. reinhardtii are regulated by light signal and differentially modulated via photosynthetic electron transfer and calmodulin-mediated calcium signaling pathway(s).

Down-regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii.

In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H2 O2 in the 10 µm range. This concentration range seems to be necessary to activate H2 O2 -dependent signalling pathways stimulating the expression of H2 O2 responsive genes, such as the heat shock protein HSP22C. Catalase knock-down mutants had lost the transient accumulation of H2 O2 , suggesting that a decrease in catalase activity was the key element for establishing a transient H2 O2 burst. Catalase was inactivated by a one-electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H2 O2 , inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast-directed protection enzymes.

The chloroplast calcium sensor CAS is required for photoacclimation in Chlamydomonas reinhardtii.

The plant-specific calcium binding protein CAS (calcium sensor) has been localized in chloroplast thylakoid membranes of vascular plants and green algae. To elucidate the function of CAS in Chlamydomonas reinhardtii, we generated and analyzed eight independent CAS knockdown C. reinhardtii lines (cas-kd). Upon transfer to high-light (HL) growth conditions, cas-kd lines were unable to properly induce the expression of LHCSR3 protein that is crucial for nonphotochemical quenching. Prolonged exposure to HL revealed a severe light sensitivity of cas-kd lines and caused diminished activity and recovery of photosystem II (PSII). Remarkably, the induction of LHCSR3, the growth of cas-kd lines under HL, and the performance of PSII were fully rescued by increasing the calcium concentration in the growth media. Moreover, perturbing cellular Ca(2+) homeostasis by application of the calmodulin antagonist W7 or the G-protein activator mastoparan impaired the induction of LHCSR3 expression in a concentration-dependent manner. Our findings demonstrate that CAS and Ca(2+) are critically involved in the regulation of the HL response and particularly in the control of LHCSR3 expression.

bop5 Mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric waveforms.

I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability.

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas.

The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii. NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.

Does single-dose cell resistance to the radio-mimetic zeocin correlate with a zeocin-induced adaptive response in Chlamydomonas reinhardtii strains?

This study aimed to test whether a correlation exists between single-dose resistance to zeocin and the ability to develop a zeocin-induced adaptive response (AR) in Chlamydomonas reinhardtii strains. Three genotypes were used: wild type (WT) strain 137C and two strains (H-3 and AK-9-9), which are highly resistant to radiation based on survival studies. Based on a micro-colony assay, the strains could be arranged according to their single-dose resistance to zeocin as follows: AK-9-9 > H-3 > 137C. However, zeocin induced a similar level of DSB in strains AK-9-9, H-3 and 137C. The radio- and zeocin-resistant strains AK-9-9 and H-3 showed higher DSB rejoining capacity than the WT strain 137C, suggesting that DSB rejoining can at least partly account for different cell survival. Both WT and radio-resistant strains develop zeocin-induced AR involving increased DSB rejoining. The radio- and zeocin-resistant strains AK-9-9 and H-3 again showed higher DSB rejoining capacity than the WT strain 137C. The higher resistance of strains H-3 and AK-9-9 did not abrogate their ability to adapt, albeit with a smaller magnitude as compared to the WT strain. The obtained results characterize new radio-resistant C. reinhardtii strains, which enrich the collection of resistant C. reinhardtii strains.

Chlamydomonas reinhardtii, a model system for functional validation of abiotic stress responsive genes.

Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress specific. Although many of these have been cloned, their functional significance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for different abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further confirmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be efficiently validated for diverse abiotic stresses using Chlamydomonas system.

Growth condition-dependent sensitivity, photodamage and stress response of Chlamydomonas reinhardtii exposed to high light conditions.

Different substrate conditions, such as varying CO(2) concentrations or the presence of acetate, strongly influence the efficiency of photosynthesis in Chlamydomonas reinhardtii. Altered photosynthetic efficiencies affect the susceptibility of algae to the deleterious effects of high light stress, such as the production of reactive oxygen species (ROS) and PSII photodamage. In this study, we investigated the effect of high light on C. reinhardtii grown under photomixotrophy, i.e. in the presence of acetate, as well as under photoautotrophic growth conditions with either low or high CO(2) concentrations. Different parameters such as growth rate, chlorophyll bleaching, singlet oxygen generation, PSII photodamage and the total genomic stress response were analyzed. Although showing a similar degree of PSII photodamage, a much stronger singlet oxygen-specific response and a broader general stress response was observed in acetate and high CO(2)-supplemented cells compared with CO(2)-limited cells. These different photooxidative stress responses were correlated with the individual cellular PSII content and probably directly influenced the ROS production during exposure to high light. In addition, growth of high CO(2)-supplemented cells was more susceptible to high light stress compared with cells grown under CO(2) limitation. The growth of acetate-supplemented cultures, on the other hand, was less affected by high light treatment than cultures grown under high CO(2) concentrations, despite the similar cellular stress. This suggests that the production of ATP by mitochondrial acetate respiration protects the cells from the deleterious effects of high light stress, presumably by providing energy for an effective defense.

Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.

Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+ -binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility.