Consequences of state transitions on the structural and functional organization of Photosystem I in the green alga Chlamydomonas reinhardtii

State transitions represent a photoacclimation process that regulates the light‐driven photosynthetic reactions in response to changes in light quality/quantity. It balances the excitation between photosystem I (PSI) and II (PSII) by shuttling LHCII, the main light‐harvesting complex of green algae and plants, between them. This process is particularly important in Chlamydomonas reinhardtii in which it is suggested to induce a large reorganization in the thylakoid membrane. Phosphorylation has been shown to be necessary for state transitions and the LHCII kinase has been identified. However, the consequences of state transitions on the structural organization and the functionality of the photosystems have not yet been elucidated. This situation is mainly because the purification of the supercomplexes has proved to be particularly difficult, thus preventing structural and functional studies. Here, we have purified and analysed PSI and PSII supercomplexes of C. reinhardtii in states 1 and 2, and have studied them using biochemical, spectroscopic and structural methods. It is shown that PSI in state 2 is able to bind two LHCII trimers that contain all four LHCII types, and one monomer, most likely CP29, in addition to its nine Lhcas. This structure is the largest PSI complex ever observed, having an antenna size of 340 Chls/P700. Moreover, all PSI‐bound Lhcs are efficient in transferring energy to PSI. A projection map at 20 Å resolution reveals the structural organization of the complex. Surprisingly, only LHCII type I, II and IV are phosphorylated when associated with PSI, while LHCII type III and CP29 are not, but CP29 is phosphorylated when associated with PSII in state2.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.     

A novel gene with antisalt and anticadmium stress activities from a halotolerant marine green alga Chlamydomonas sp. W80

A novel gene with antistress activities against both salt (NaCl) and cadmium stresses was isolated from the cDNA library of halotolerant green alga Chlamydomonas sp. strain W80 by a functional expression screening with Escherichia coli. The C-terminal region of this protein is responsible for the antistress activity, because N-terminal truncated clone of this gene retains the antistress activity, and the C-terminal truncated clone loses the activity. In the C-terminal region, there is a histidine and aspartic acid-rich domain (HD-rich domain).     

Study of photosystem 2 heterogeneity in the sulfur-deficient green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

A set of chlorophyll fluorescence methods, including PEA (Plant Efficiency Analyser), PAM (Pulse Amplitude Modulated fluorometer), and picosecond fluorometer, was employed to study PS 2 heterogeneity in sulfur deprived green algae Chlamydomonas reinhardtii. The regression method and JIP test were applied to analyze chlorophyll fluorescence kinetics. The fractions of PS 2 characterized by the energetic disconnection, smaller antenna size, elevated constant rate of primary photochemistry, and inability to maintain ΔpH-dependent energy dissipation increased essentially already after 12 h of incubation in sulfur depleted medium. The amount of PS 2 centers with reduced Q_A (closed state), Q_B-non-reducing centers with impaired water splitting function, and centers coupled to the plastoquinone pool with the slow cycle rate increased dramatically after 24 h period of deprivation. The mechanisms of PS 2 inactivation under sulfur deprivation are discussed.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

In vitro and in vivo investigation of chlorophyll binding sites involved in non‐photochemical quenching in Chlamydomonas reinhardtii

Non‐photochemical quenching (NPQ) of the light energy absorbed is one of the main photoprotective mechanisms evolved by oxygenic photosynthetic organisms to avoid photodamage, at a cost of reduced photosynthetic efficiency. Tuning of NPQ has been reported as a promising biotechnological strategy to increase productivity in both higher plants and unicellular microalgae. Engineering of NPQ induction requires the comprehension of its molecular mechanism(s), strongly debated in the last three decades with several different models proposed. In this work, the molecular details of NPQ induction was investigated at intramolecular level by in vitro and in vitro site‐specific mutagenesis on chlorophyll binding sites of the Light‐Harvesting Complex Stress‐Related 3 (LHCSR3) protein, the pigment binding complexes identified as the quencher during NPQ induction in the model organism for green algae Chlamydomonas reinhardtii. The results obtained demonstrate a correlation between the quenching activity of LHCSR3 variants in vitro and the NPQ phenotypes observed in vivo. In particular, multiple quenching sites in LHCSR3 cooperatively dissipating the excitation energy were revealed with a peculiar role of Chl 613, a chromophore located a close distance to carotenoid binding site L1.     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P₇₀₀ recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.     

A large scale structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii. I. Generation of 3433 non-redundant expressed sequence tags.

To understand genetic information carried in a unicellular green alga, Chlamydomonas reinhardtii, normalized and size-selected cDNA libraries were constructed from cells at photoautotrophic growth, and a total of 11,571 5'-end sequence tags were established. These sequences were grouped into 3433 independent EST species. Similarity search against the public non-redundant protein database indicated that 817 groups showed significant similarity to registered sequences, of which 140 were of previously identified C. reinhardtii genes, but the remaining 2616 species were novel sequences. The coverage of full-length protein coding regions was estimated to be over 60%. These cDNA clones and EST sequence information will provide a powerful source for the future genome-wide functional analysis of uncharacterized genes.     

Ultrastructural,cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

Summary— The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.     

Ultrastructure and properties of the sexual agglutinins of the biflagellate green alga Chlamydomonas moewusii

Summary The mt^– agglutinins of the interfertile species Chlamydomonas moewusii and Chlamydomonas eugametos are very similar fibrous molecules. The mt^– agglutinin of C. moewusii has the same Stokes radius (39 nm) and sedimentation coefficient (9.3 S) as its counterpart in C. eugametos; its length (336 nm) and its ultrastructure, including the position of four kinks are also the same as in C. eugametos. The sugar compositions of both agglutinins are very similar, and they react equally well with the monoclonal antibody Mab 66.3 raised against the mt^– agglutinin of C. eugametos. Finally, they are equally thermoresistant, with half-lives at 100 °C of 50 min (C. moewusii) and 57 min (C. eugametos). The mt⁺ agglutinins of both species are different. Both are fibrous molecules with a terminal head, but the fibrous part of the molecule in C. moewusii is shorter (210 nm compared to 276 nm). The mt⁺ agglutinin of C. moewusii is also significantly more sensitive to heating with a half-life of 6 min at 40 °C compared to the 20 min shown by the mt⁺ agglutinin of C. eugametos. Their sugar compositions are, however, very similar, and they react equally well with Mab 66.3. The mt⁺ agglutinin of C. moewusii is sensitive to denaturing reagents and proteolytic attack, whereas the mt^– agglutinin is highly resistant. It is proposed that the globular head of the mt⁺ agglutinin acts as its recognition domain and interacts with a carbohydrate ligand on the mt^– agglutinin.     

Temperature-dependent photoinhibition and recovery of photosynthesis in the green alga Chlamydomonas reinhardtii acclimated to 12 and 27°C

Photoinhibition of photosynthesis and subsequent recovery were studied in cultures of the unicellular green alga Chlamydomonas reinhardtii L. (wt strain 137 c mating type +) acclimated at high (27°C) and low (12°C) temperature, Photoinhibition was assayed by fluorescence kinetics (77K) and oxygen evolution measurements under growth temperature conditions Inhibition of 50% was obtained by exposing cultures acclimated at high temperature to a photosynthetic photon flux density (PPFD) of 1 600 μmol m⁻² S⁻¹ at. 27°C. and cultures acclimated at low temperature to a PPFD of 900 μmol m⁻² s⁻¹ at 12°C When the photoinhibitory conditions were shifted it was revealed that algae acclimated at low temperature had acquired an increased resistance to photoinhibition at both 12 and 27°C. Furthermore, acclimation at low temperature increased the capacity to recover from 50% photoinhibition at both 12 and 27°C Studies of photoinhibition in the presence of the protein synthesis inhibitor, chloramphenicol, revealed that in response to acclimation at low temperature during growth the algae became more dependent on protein synthesis to avoid photoinhibition. It is suggested that acclimation at low temperature rendered C. reinhardtii an increased resistance to photoinhibition by. increasing the rate of turnover of photodamaged proteins in photosystem II (PS II). However, we cannot exclude the possibility that the increased resistance to photoinhibition of C. reinhardtii acclimated at low temperature also involves modifications of the mechanism of photoinhibition.     

Studies of excitation energy transfer within the green alga Chlamydomonas reinhardtii and its mutants at 77 K

The 77 K picosecond fluorescence of intact Chlamydomonas reinhardtii exhibits a 680-nm band (F680) that can be identified with light-harvesting chlorophyll. Analysis of the time and spectral dependence of F680 reveal a forward transfer rate of 1/(15 ps) from this 680-nm species to photosystem II. The possibility of transfer through LHC I, the light-harvesting complex closely associated with photosystem I with a transfer time of 60 to 100 ps, is indicated by analysis of similar data in the 700–720 nm region. Simple kinetic models that account for the time dependence of the emissions F707, F703 and F715 are proposed.Based in part on a thesis submitted in partial fulfillment of the requirements for the Ph.D. Degree, University of Rochester (SL).     

Temperature- and pH-dependent accumulation of heat-shock proteins in the acidophilic green alga Chlamydomonas acidophila

Chlamydomonas acidophila, a unicellular green alga, is a dominant phytoplankton species in acidic water bodies, facing severe environmental conditions such as low pH and high heavy metal concentrations. We examined the pH-, and temperature-dependent accumulation of heat-shock proteins in this alga to determine whether heat-shock proteins play a role in adaptation to their environment. Our results show increased heat-shock proteins accumulation at suboptimal pHs, which were not connected with any change in intracellular pH. In comparison to the mesophilic Chlamydomonas reinhardtii, the acidophilic species exhibited significantly higher accumulations of heat-shock proteins under control conditions, indicating an environmental adaptation of increased basal levels of heat-shock proteins. The results suggest that heat-shock proteins might play a role in the adaptation of C. acidophila, and possibly other acidophilic algae, to their extreme environment.     

Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas

Defects in complex I (NADH:ubiquinone oxidoreductase (EC 1.6.5.3)) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which may hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (i) respiratory complex-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (ii) transformation of the mitochondrial genome is feasible, and (iii) Chlamydomonas complex I is similar to that of humans. To illustrate this proposal, we introduced a Leu157Pro substitution into the Chlamydomonas ND4 subunit of complex I in two recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defects when present in the heteroplasmic state in a patient with chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent assembly of whole complex I (950 kDa) and the NADH dehydrogenase activity of the peripheral arm of the complex is mildly affected. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect binding of ubiquinone to the membrane domain. The in vitro defects correlate with a decrease in dark respiration and growth rate in vivo.     

Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii

Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii . The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii .     

Short Communication: Analysis of effective light in different photobioreactors: its influence on growth,photosynthetic activity and glycerol production by the freshwater green alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii synthesizes glycerol as an osmoregulatory metabolite when exposed to high saline concentrations (200 mM NaCl). Response to osmotic stress can be used for biotechnological production of this compound. When synthesis of a substance is linked to photosynthetic capacity and consequently to effective light, the production on a large scale makes an efficient utilization of light necessary. In the present work a model for evaluation of effective light has been tested.     

Homologous and heterologous protein import into mitochondria isolated from the green alga Chlamydomonas reinhardtii

We have established a homologous system for studying mitochondrial protein import in Chlamydomonas reinhardtii, using C. reinhardtii precursor proteins and mitochondria isolated from C. reinhardtii. The precursors of the F₁ ATP synthase subunit and the Rieske FeS protein were imported into mitochondria with high efficiency, while the F₁ subunit precursor was imported with much lower efficiency. The import of heterologous precursor proteins from higher plants was also less efficient. The precursor of the C. reinhardtii PsaF chloroplast protein was converted into a protease-protected form upon incubation with mitochondria. In vitro processing studies revealed that in contrast to the situation in higher plants, the processing of the precursors was catalysed by a soluble, matrix-located peptidase.     

Isolation,Growth, Ultrastructure,and Metal Tolerance of the Green Alga,Chlamydomonas acidophila (Chlorophyta)

An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance.

Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5-4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50%, EC₅₀ were measured, after 72h of static exposure. EC₅₀s were 14.4 μM Cd²⁺, 81.3 μM Co²⁺, 141μM Cu²⁺, and 1.16 mM Zn²⁺ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas.