Relief of Arsenate Toxicity by Cd-Stimulated Phytochelatin Synthesis in the Green Alga Chlamydomonas reinhardtii

In most photosynthetic organisms, inorganic arsenic taken up into the cells inhibits photosynthesis and cellular growth. In a green alga, Chlamydomonas reinhardtii, 0.5 mM arsenate inhibited photosynthesis almost completely within 30 min. However, in cells acclimated with a sublethal concentration (0.05 to 0.1 mM) of Cd, the inhibition of photosynthesis at 30 min after the addition of arsenate was relieved by more than 50%. The concentrations of arsenic incorporated into the cells were not significantly different between the Cd-acclimated and the non-acclimated cells. The Cd-acclimated cells accumulated Cd and synthesized phytochelatin (PC) peptides, which are known to play an important role in detoxification of heavy metals in plants. By the addition of an inhibitor of glutathione (an intermediate in the PC biosynthetic pathway) biosynthesis, buthionine sulfoximine, cells lost not only Cd tolerance but also arsenate tolerance. These results suggest that glutathione and/or PCs synthesized in Cd-acclimated cells are involved in mechanisms of arsenate tolerance. The authors contributed equally to this work.     

Carbon Supply and Photoacclimation Cross Talk in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO₂ availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO₂ maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO₂ reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii. This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability.Light sustains virtually all life on Earth through the process of photosynthesis. However, light can be very harmful for oxygenic photosynthetic organisms, as excess absorption can lead to the production of reactive oxygen species. In order to survive and grow, these organisms have developed various photoacclimation mechanisms operating on different time scales that protect the cell from photodamage. In the green alga Chlamydomonas reinhardtii, these mechanisms vary from negative phototaxis and multicomponent nonphotochemical quenching (NPQ) to a number of physiological and biochemical changes (Erickson et al., 2015). C. reinhardtii cells are around 10 μm in diameter, and a large part of their total volume is occupied by a single horseshoe-shaped chloroplast (Sager and Palade, 1957). The photosynthetic machinery responsible for the light reactions is located in thylakoid membranes and contains four major components: PSII, cytochrome b₆f, PSI, and ATP synthase. Both photosystems bind chlorophyll (Chl) and carotenoid (Car) and are composed of a core and several outer antennae pigment-protein complexes, the main function of which is light harvesting and its conversion into chemical energy. The PSII core is composed of D1, D2, CP43, and CP47 pigment-protein complexes and several smaller subunits, the number of which varies between organisms (Shi et al., 2012). The outer antenna contains the light-harvesting complex II (LHCII), which in C. reinhardtii is encoded by nine LHCBM genes, and the minor antennae CP26 and CP29 (Nield et al., 2000; Teramoto et al., 2001; Natali and Croce, 2015). These complexes are assembled together to form PSII-LHCII supercomplexes (Tokutsu et al., 2012; Drop et al., 2014). The PSI core is composed of a PSAA-PSAB heterodimer and a number of smaller subunits (Jensen et al., 2007), and in C. reinhardtii the LHCI antenna consists of nine LHCA proteins (Mozzo et al., 2010) that are associated with the core to form the PSI-LHCI complex (Stauber et al., 2009; Drop et al., 2011).The composition and organization of the thylakoid membrane is light dependent. The gene expression of different LHCs has been reported to be affected by light acclimation (Teramoto et al., 2002; Durnford et al., 2003; Yamano et al., 2008) and to be NAB1 regulated (Mussgnug et al., 2005). It has been observed that long-term high-light exposure of C. reinhardtii cells leads to a 50% decrease of Chl content (Neale and Melis, 1986; Bonente et al., 2012) and to changes in Chl-to-Car ratio (Niyogi et al., 1997a; Baroli et al., 2003; Bonente et al., 2012), suggesting reduction of the antenna size (Neale and Melis, 1986), although, in a more recent report (Bonente et al., 2012), it was concluded that the antenna size is not modulated by light in this alga. Recently, a dependence of the antenna components on the carbon availability also was reported. It was shown that, when cells grown in acetate are shifted from high to low CO₂ concentration, the functional antenna size of PSII decreases and a down-regulation of LHCBM6/8 occurs (Berger et al., 2014).In the short term, the main response to high light is the dissipation of energy absorbed in excess heat in a process called qE, or energy-dependent quenching, which is the fastest component of NPQ. In land plants, the main player in this process is the protein PsbS (Li et al., 2002, 2004), while in C. reinhardtii, the process is centered around LHCSR1 and LHCSR3 (Peers et al., 2009; Dinc et al., 2016). LHCSR3, the most studied of the two, is a pigment-protein complex that is expressed within 1 h of high-light exposure (Allorent et al., 2013) in combination with CO₂ limitation (Yamano et al., 2008; Maruyama et al., 2014). The qE onset is triggered by lumen acidification sensed by LHCSR3/1 (Bonente et al., 2011; Liguori et al., 2013; Tokutsu and Minagawa, 2013; Dinc et al., 2016).Cars are well known to be involved in photoprotection. They quench triplet Chl and scavenge singlet oxygen (¹O₂; Frank and Cogdell, 1996). In C. reinhardtii, the antioxidant role of xanthophylls is well illustrated by the mutant npq1 lor1 lacking lutein and zeaxanthin (Niyogi et al., 1997b). This mutant is deficient in qE, but compared with other qE-deficient mutants like npq4 (Peers et al., 2009) and npq5 (Elrad et al., 2002), which are LHCSR3 and LHCBM1 knockouts, respectively, it is extremely light sensitive, due to the absence of quenching of triplet Chl and ¹O₂ by zeaxanthin and lutein.Aquatic oxygenic photosynthetic organisms meet several challenges in CO₂ fixation (Moroney and Ynalvez, 2007). First, the diffusion of CO₂ in water is 10,000 times slower than in air. Second, the CO₂-fixing enzyme Rubisco is not selective for CO₂ and also binds oxygen, resulting in the process of photorespiration. Third, the form of inorganic carbon depends on the pH (i.e. in alkaline pH, it is HCO₃⁻, while in acidic pH, it is CO₂; Beardall, 1981; Gehl et al., 1987). This diminishes even further the availability of CO₂ in the cell. In order to overcome these CO₂ fixation barriers, algae have developed carbon-concentrating mechanisms (CCMs; Moroney and Ynalvez, 2007). The essence of these processes lies in the active pumping of inorganic carbon in the cell via a number of transporters that concentrate it in the pyrenoid, a ball-like structure containing Rubisco, Rubisco activase, and intrapyrenoid thylakoids and surrounded by a starch sheath. In the pyrenoid, HCO₃⁻ is converted to CO₂ by CARBONIC ANHYDRASE3 (CAH3; Blanco-Rivero et al., 2012; Sinetova et al., 2012) and then fixed by Rubisco in the Calvin-Benson-Bassham cycle. CAH3 also is suggested to provide HCO₃⁻ in the proximity of the oxygen-evolving complex, where it may function as a proton carrier, removing H⁺ from water splitting to avoid photoinhibition (Villarejo et al., 2002; Shutova et al., 2008).C. reinhardtii also can grow mixotrophically using alternative organic carbon sources present in its environment. For example, it can take up acetate, which is then incorporated into the citric cycle, producing reducing equivalents and CO₂ (Johnson and Alric, 2012), and into the glyoxylate cycle, producing malate (Lauersen et al., 2016). In the presence of acetate, it has been reported that CO₂ uptake and oxygen evolution were decreased by half under saturating CO₂ and light intensities without affecting PSII efficiency, respiration, and cell growth (Heifetz et al., 2000). In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions (Chapman et al., 2015). Furthermore, acetate can replace PSII-associated HCO₃⁻, reducing ¹O₂ formation and, therefore, acting as a photoprotector during high-light acclimation (Roach et al., 2013).In short, high-light acclimation is a complex, multicomponent process that happens on different time scales. Furthermore, it is embedded in the overall metabolic network and is potentially influenced by different nutrients and metabolic states. A thorough understanding of this process and its regulation is crucial for fundamental research and applications. To determine if different carbon supply conditions trigger different light acclimation strategies and photoprotective responses, we systematically studied C. reinhardtii cells grown in mixotrophic, photoautotrophic, and high-CO₂ photoautotrophic conditions in different light intensities.We show that C. reinhardtii cells use different strategies to acclimate to high light depending on the carbon availability and trophic status. These results underline the strong connection between metabolism and light acclimation responses and reconcile the data from various reports. Furthermore, our study demonstrates how, in a dynamic system such as C. reinhardtii, a single change in growth conditions has large effects at multiple levels.     

Control of CO(2) Fixation by Cell-free Extracts of Chlamydomonas reinhardtii

Contrary to earlier reports, CO₂ fixation by extracts of Chlamydomonas is inhibited by glutamate and aspartate. These amino acids and some organic acids are shown to be inhibitors of phosphoenolpyruvate carboxylase. Inorganic phosphate is shown to activate CO₂ fixation, but there is a time lag before inorganic phosphate exerts its full activating effect.     

Interactions between Photosynthesis and Respiration in the Green Alga Chlamydomonas reinhardtii (Characterization of Light-Enhanced Dark Respiration)

The rate of respiratory O2 consumption by Chlamydomonas reinhardtii cell suspensions was greater after a period of photosynthesis than in the preceding dark period. This "light-enhanced dark respiration" (LEDR) was a function of both the duration of illumination and the photon fluence rate. Mass spectrometric measurements of gas exchange indicated that the rate of gross respiratory O2 consumption increased during photosynthesis, whereas gross respiratory CO2 production decreased in a photon fluence rate-dependent manner. The rate of postillumination O2 consumption provided a good measure of the O2 consumption rate in the light. LEDR was substantially decreased by the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or glycolaldehyde, suggesting that LEDR was photosynthesis-dependent. The onset of photosynthesis resulted in an increase in the cellular levels of phosphoglycerate, malate, and phosphoenolpyruvate, and a decrease in whole-cell ATP and citrate levels; all of these changes were rapidly reversed upon darkening. These results are consistent with a decrease in the rate of respiratory carbon flow during photosynthesis, whereas the increase in respiratory O2 consumption during photosynthesis may be mediated by the export of photogenerated reductant from the chloroplast. We suggest that photosynthesis interacts with respiration at more than one level, simultaneously decreasing the rate of respiratory carbon flow while increasing the rate of respiratory O2 consumption.     

Stt7-dependent Phosphorylation during State Transitions in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b₆f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7 mutant of Chlamydomonas under state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser⁵³³ in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required for state transitions. Moreover, we also identified phosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent.The primary photochemical reactions of photosynthesis are catalyzed by the pigment-protein complexes photosystem II (PSII)¹ and PSI (PSI), which are linked in series through the plastoquinone pool, the cytochrome b₆f complex, and plastocyanin in the thylakoid membranes. Upon light absorption by the antenna systems of PSII and PSI, charge separations occur across the membrane that lead to the oxidation of water by PSII and electron flow to PSI and ultimately to the reduction of NADP⁺. Because the antenna systems of PSII and PSI have different pigment composition, they are differentially sensitized upon changes in light quality and quantity. However, photosynthetic organisms have the ability to adapt to changes in light. They balance energy input and consumption in the short term through dissipation of excess absorbed light energy into heat through non-photochemical quenching and regulate absorption of excitation energy between PSII and PSI through state transitions (supplemental Fig. 1). This reversible redistribution leads to an overall increase in photosynthetic quantum yield. State transitions occur when preferential excitation of PSII reduces the plastoquinone pool. This leads to the activation of a thylakoid protein kinase as a result of the docking of plastoquinol to the Qo site of the cytochrome b₆f complex (1, 2) and to the phosphorylation of the polypeptides of the light-harvesting complex II (LHCII), a part of which migrates to PSI (state 2) (3–5). The process is reversible as preferential excitation of PSI inactivates the kinase and allows for dephosphorylation of LHCII and its return to PSII (state 1) (3, 6). In the green alga Chlamydomonas reinhardtii, the LHCII protein set consists of Type I (Lhcbm3, Lhcbm4, Lhcbm6, Lhcbm8, and Lhcbm9), Type II (Lhcbm5), Type III (Lhcbm2 and Lhcbm7), and Type IV (Lhcbm1 and Lhcbm10) proteins and of Lhcb7, CP26, and CP29 (7). Because of their nearly identical sequences and sizes, several of these Lhcbm proteins cannot be distinguished by SDS-PAGE. Most of them fractionate into four bands called P11 and P13 (Type I), P16 (Type IV), and P17 (Type III). Whereas P16 is not phosphorylated, phosphorylation events occur on P11, P13, and P17 (7, 8).The association of the mobile part of LHCII to PSI during a transition from state 1 to state 2 requires the PsaH subunit (9) and CP29, which also moves to PSI and is essential for docking LHCII to PSI (10–12). The lateral displacement of LHCII from the PSII-rich grana to the PSI-rich lamellar thylakoid regions results in transfer to PSI of about 80% of the excitation energy absorbed by LHCII in C. reinhardtii (13), a considerably higher amount than in land plants in which only 15–20% of LHCII is mobile (3). In C. reinhardtii, state transitions are associated with a reorganization of the photosynthetic electron transfer chain with a switch from linear to cyclic electron flow during a transition from state 1 to state 2 (14, 15). Thus, cells produce ATP and NADPH in state 1 but only ATP in state 2. It appears that the major function of state transitions in this alga is to adjust the level of ATP and the ATP/NADPH ratio to cellular demands (5).Thylakoid membranes contain appressed grana and nonappressed stromal domains in which PSII and PSI are enriched, respectively. Because LHCII is a major stabilizer of the grana structure (16), the movement of LHCII from PSII to PSI is expected to lead to major rearrangements of these membranes during state transitions. Indeed, based on extensive electron microscope studies, it was proposed that fusion and fission events occur at the interface between the grana and stroma lamellar domains that lead to a remodeling of the membranes (17).Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of Chlamydomonas revealed a total of 19 sites corresponding to 15 genes (18). It was shown that the major changes are clustered at the interface between the PSII core and the associated LHCII proteins during state transitions. Phosphorylation of the PSII core subunits D2 and PsbR and multiple phosphorylations of the minor LHCII antenna subunit CP29 were detected as well as phosphorylation of Lhcbm1, which belongs to the major LHCII complex (18).Although the phosphorylation of LHCII was observed many years ago (6), it is only recently that kinases involved in this process were uncovered. Fleischmann et al. (19) and Kruse et al. (20) used a genetic approach in C. reinhardtii with the aim of dissecting the signal transduction chain of state transitions. Two allelic mutants blocked in state 1 were identified that are affected in the Stt7 gene encoding a thylakoid Ser-Thr protein kinase that is required for LHCII phosphorylation during a transition from state 1 to state 2 (21). This Stt7 kinase is conserved in land plants and has an ortholog, STN7, in Arabidopsis (22).The 754-amino acid Stt7 kinase has a catalytic domain characteristic of Ser-Thr kinases (21). It contains a putative 41-amino acid transit peptide at its N-terminal end, and the protein is localized on the thylakoid membrane. Stt7 is associated with photosynthetic complexes including LHCII, PSI, and the cytochrome b₆f complex (23). Stt7 also contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys residues that are critical for its activity and state transitions (23). Moreover, the level of Stt7 decreases considerably under state 1 conditions, and the kinase acts in catalytic amounts (23). However, it is not yet known whether this kinase directly phosphorylates LHCII or whether it is part of a kinase cascade involved in the signaling pathway of state transitions.In this work, we used a mass spectrometry-based approach (24) to map the in vivo Stt7-dependent protein phosphorylation sites within thylakoid membranes isolated from the green alga C. reinhardtii subjected to state 1 and state 2 conditions. In contrast with the earlier studies via direct MS/MS sequencing of the IMAC-enriched phosphorylated peptides from thylakoid proteins (18, 25), we performed additional LC-MS/MS-based analyses using alternating collision-induced dissociation and electron transfer dissociation of peptide ions. This approach revealed novel phosphorylation sites in LHCII polypeptides, in several other membrane and membrane-associated proteins, and in the thylakoid protein kinases Stt7 and Stl1, suggesting the existence of a thylakoid protein kinase cascade. Relative quantification of phosphorylated peptides labeled with stable isotopes determined the specific Stt7-dependent phosphorylation site in CP29 linker protein under state 2. Moreover, we also identified phosphorylation sites that are redox-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent. This mapping provides new insights into the regulatory network of protein phosphorylation in algal photosynthetic membranes during state transitions.     

Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii

We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.     

Genome-Wide Characterization of Genetic Variation in the Unicellular, Green Alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a model system for studying cilia, photosynthesis, and other core features of eukaryotes, and is also an emerging source of biofuels. Despite its importance to basic and applied biological research, the level and pattern of genetic variation in this haploid green alga has yet to be characterized on a genome-wide scale. To improve understanding of C. reinhardtii's genetic variability, we generated low coverage whole genome resequencing data for nearly all of the available isolates of this species, which were sampled from a number of sites in North America over the past ～70 years. Based on the analysis of more than 62,000 single nucleotide polymorphisms, we identified two groups of isolates that represent geographical subpopulations of the species. We also found that measurements of genetic diversity were highly variable throughout the genome, in part due to technical factors. We studied the level and pattern of linkage disequilibrium (LD), and observed one chromosome that exhibits elevated LD. Furthermore, we detected widespread evidence of recombination across the genome, which implies that outcrossing occurs in natural populations of this species. In summary, our study provides multiple insights into the sequence diversity of C. reinhardtii that will be useful to future studies of natural genetic variation in this organism.     

H(2) and CO(2) Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60

Using manometric and enzymic techniques, H₂ and CO₂ evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.     

The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation

The cell wall–lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the Tn₅ transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10⁶ mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10³ Hyg^R transformants per 10⁶ recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation ofCh. reinhardtii with heterologous genes are discussed.     

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga,Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

The Cytotoxic Effects of Camptothecin and Mastoparan on the Unicellular Green Alga Chlamydomonas reinhardtii

We have recently reported that protease inhibitors affecting the activity of the proteasome cause necrotic cell death in Chlamydomonas reinhardtii instead of inducing apoptosis as shown for some mammalian cell lines. Therefore, we have studied other well‐known inducers of apoptosis in mammalian cells for their effects on C. reinhardtii cells. Mastoparan caused rapid cell death without a prominent lag‐phase under all growth conditions, whereas the cytotoxic effect of the topoisomerase I inhibitor camptothecin exclusively occurred during the cell‐division phase. Essentially no differences between wall‐deficient and wild‐type cells were observed with respect to dose‐response and time‐course of camptothecin and mastoparan. In cultures of the wall‐deficient strain, cell death was accompanied by swelling and subsequent disruption of the cells, established markers of necrosis. In case of the wild‐type strain, camptothecin and mastoparan caused accumulation of apparently intact, but dead cells instead of cell debris due to the presence of the wall. Both in cultures of the wall‐deficient and the wild‐type strains, cell death was accompanied by an increase of the protein concentration in the culture medium indicating a lytic process like necrosis. Taking together, we have severe doubts on the existence of an apoptotic program in case of C. reinhardtii.     

Adaptation of Chlamydomonas reinhardtii High-CO(2)-Requiring Mutants to Limiting CO(2)

Photosynthetic characteristics of four high-CO₂-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO₂ concentrations. The four mutants represent two loci involved in the CO₂-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO₂ concentration, indicating that the genetic lesion in each is expressed even at elevated CO₂ concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO₂ characteristic of the induction of the CO₂-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO₂-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO₂-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.     

Blue Light-Induced Lethality of a Cell Wall-Deficient Mutant of the Unicellular Green Alga Chlamydomonas reinhardtii

When synchronized cultures of a cell wall-deficient Chlamydomonasreinhardtii mutant strain were grown under heterotrophic conditionsand subsequently transferred to the light, a considerable decreaseof the cell number was observed during transition to the celldivision phase. Lethality of the wall-deficient cells was inducedby blue light, but not by red or far-red light, and could notbe prevented by addition of the photosystem II inhibitor DCMU.The light-induced lethality was found to be restricted to wall-deficientcells which were agitated by bubbling with filtered air or nitrogenor vigorously shaken during the transition to the cell divisionphase. Therefore, a (blue) light-induced sensitivity to anymechanical stress seems to be the cause for cell death. In heterotrophicallygrowing cultures of the Chlamydomonas wild-type, illuminationwith blue or white light did not cause a decrease of the cellnumber but only a delay of cell divisions. The latter effectwas also observed in case of the wall-deficient mutant. Bothblue light effects are observed during the transition to thecell division phase and can be induced during the same periodof the cell cycle. Furthermore, the (blue) light-induced lethalityof wall-deficient cells was found to be prevented when the transitionto the cell division phase was inhibited by addition of antibiotics.Therefore, we assume that there is a connection between theblue light-induced sensitivity to mechanical stress and theblue light-induced delay of cell divisions. (Received September 3, 1993; Accepted November 12, 1993)     

Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii

The “Phot” protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 μs with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.     

Hydrogen Production by CO2 Deprived Photoautotrophic Chlamydomonas reinhardtii Cultures

Biochemistry (Moscow) - Light-dependent hydrogen production by microalgae attracts attention of researchers because of the potential practical application. It is generally recognized that...     

nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii

Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.