Chlamydomonas Reinhardtii Cells Research Articles

Design of new strategy for green algal photo-hydrogen production: Spectral-selective photosystem I activation and photosystem II deactivation

A new strategy in photosynthetic hydrogen (photo-H2) production from green algae was developed based on theory and successfully demonstrated. The new strategy applied a spectral-selective photosystem I (PSI) activating/photosystem II (PSII) deactivating radiation (or PSI light) that would drive a steady flow of electrons in the electron transport chain for delivery to hydrogenase for photo-H2 production, but would reduce oxygen production through water photolysis below the respiratory oxygen consumption so that an anoxic condition would be maintained as required by hydrogenase. Implementing the strategy by using a PSI light (692nm peak, 680–700nm) on Chlamydomonas reinhardtii cells resulted in relatively sustained photo-H2 production (total of 0.108mLH2mg−1Chl, exceeding 0.066mLH2mg−1Chl under white light). The strategy also proved successful and convenient in allowing cells to alternately switch between photo-H2 production and a recovery period by simply turning on or off the PSI light.

Inhibition of photosystem 2 in starch-enriched Chlamydomonas reinhardtii cells prevents the efficient induction of H2 production in sulfur-depleted cultures

In sulfur-deprived Chlamydomonas reinhardtii cells the activity of photosystem 2 (PSII) has been shown to have a crucial role in the photosynthetic production of H2, since it allows the synthesis of internal reserves such as starch. In the present investigation, the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was added in starch-enriched and sulfur-depleted C. reinhardtii cultures 1) at the very end of the aerobic phase, and 2) soon after the culture started to evolve H2. In the former case, production of H2 on a volumetric basis was completely down-regulated, although starch mobilization was enhanced. In vitro tests showed that the hydrogenase enzyme was active, although its efficiency of utilization in vivo was lowered very soon in the experiment. When an inhibitor of Rubisco such as glycolaldehyde was added under the same conditions, no substantial improvement in H2 production rates was noted. These findings indicate that, aside starch storage, PSII plays an active role in the induction of the H2 production process.

Activation of the Carbon Concentrating Mechanism by CO2 Deprivation Coincides with Massive Transcriptional Restructuring in Chlamydomonas reinhardtii

A CO(2)-concentrating mechanism (CCM) is essential for the growth of most eukaryotic algae under ambient (392 ppm) and very low (<100 ppm) CO(2) concentrations. In this study, we used replicated deep mRNA sequencing and regulatory network reconstruction to capture a remarkable scope of changes in gene expression that occurs when Chlamydomonas reinhardtii cells are shifted from high to very low levels of CO(2) (≤100 ppm). CCM induction 30 to 180 min post-CO(2) deprivation coincides with statistically significant changes in the expression of an astonishing 38% (5884) of the 15,501 nonoverlapping C. reinhardtii genes. Of these genes, 1088 genes were induced and 3828 genes were downregulated by a log(2) factor of 2. The latter indicate a global reduction in photosynthesis, protein synthesis, and energy-related biochemical pathways. The magnitude of transcriptional rearrangement and its major patterns are robust as analyzed by three different statistical methods. De novo DNA motif discovery revealed new putative binding sites for Myeloid oncogene family transcription factors potentially involved in activating low CO(2)-induced genes. The (CA)(n) repeat (9 ≤ n ≤ 25) is present in 29% of upregulated genes but almost absent from promoters of downregulated genes. These discoveries open many avenues for new research.

Measurement of the Microscopic Dynamics of Photoprotection in Living Cells of Green Algae

Photosynthetic organisms reduce inhibition of photosystem II (PSII) in variable light conditions by using a suite of photoprotective mechanisms called nonphotochemical quenching (NPQ), in which excess absorbed light is dissipated safely as heat. The primary, rapidly reversible component of NPQ is energy-dependent quenching (qE), which is induced by a low pH in the thylakoid lumen. Measuring the change in chlorophyll excited state lifetime in PSII due to specific physical processes involved in qE has been difficult because of multiple NPQ components and the difficulty of measuring ultrafast processes on a sample whose photophysical decay pathways change in response to actinic light. Using time-resolved fluorescence, we measured the lifetime of excited chlorophylls in PSII of live cells of the green alga Chlamydomonas reinhardtii as they adapted to variable light conditions. The average trajectory to a qE site, τqE, was measured to be 276 ps for any quenched PSIIs in wild-type cells. This result provides direct evidence that PSII has two primary conformations - unquenched and quenched - and that it switches between these two states based on the pH of the lumen. The appearance of the fast lifetime had a 200 ms delay and 400 ms rise time. We attribute these timescales to changes in the lumen pH, with the first timescale being the time for protons to begin building up in the lumen and the second timescale being the time for the lumen pH to drop to the pKa of the pH sensing residues of PSII. We anticipate that these methods will be useful for probing the photophysical mechanism of qE quenching and using the dynamics of NPQ induction as a probe of chloroplast dynamics.

Volatile communication between Chlamydomonas reinhardtii cells under salt stress

Although it is known that volatile organic compounds (VOCs) can be released by lower plants, their release and ecological functions are not much studied, especially in aquatic systems under abiotic stress. In the present study, we determined the compositions of VOCs released from Chlamydomonas reinhardtii under both neutral (NaCl) and alkali (Na2CO3) salt stresses. Hexanal was the newly released abundant compound under NaCl stress, while we found 3,4-dimethyl-hexane and 5-methyl-2-heptene under Na2CO3 stress. Under both stress conditions, longifolene was the significantly increased compound. When C. reinhardtii cells were exposed to the VOCs from the salt-stressed cells, the cell density was reduced, but the content of chlorophyll, Fv/Fm and antioxidant enzyme activity were increased. This might indicate that those VOCs can transfer stress signal between C. reinhardtii cells and tell the receptor to prepare against reactive oxygen species (ROS) damage ahead of the stress coming.

Induction of reactive oxygen species inchlamydomonas reinhardtiiin response to contrasting trace metal exposures

The toxicity of metals to organisms is, in-part, related to the formation of reactive oxygen species (ROS) in cells and subsequent oxidative stress. ROS are by-products of normal respiration and photosynthesis processes in organisms, but environmental factors, like metal exposure, can stimulate excess production. Metals involved in several different mechanisms such as Haber-Weiss cycling and Fenton-type reactions can produce ROS. Some metals, such as Cd, may contribute to oxidative stress indirectly by depleting cellular antioxidants. We investigated the measurement of ROS as a sensitive biomarker of metal toxicity (that could possibly be implemented in a biotic ligand model for algae) and we compared ROS induction in response to several contrasting transition metals (Cu, V, Ni, Zn, and Cd). We also compared the ROS response to glutathione and growth toxicity endpoints measured in a previous study. The cell-permeable dye, 2'7'dichlorodihydrofluorescein diacetate, was used as a probe to detect formation of ROS in Chlamydomonas reinhardtii cells. Metal-exposed cells were incubated with the fluorescent dye in a 96-well plate and monitored over 5.5 h. A dose-response of ROS formation was observed with Cu exposure in the range of 20-500 nM. Cu produced more ROS compared with either Zn or Cd (both nonredox active metals). The redox-active metal V produced increased ROS with increased concentration. The measurement of ROS may be a useful indicator of Cu toxicity, but the signal to noise ratio was better for the glutathione endpoint assay.

Using single cell cultivation system for on-chip monitoring of the interdivision timer in Chlamydomonas reinhardtii cell cycle

Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12-15 core and 4-9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.

Atypical Subunit Composition of the Chlorophycean Mitochondrial F1FO-ATP Synthase and Role of Asa7 Protein in Stability and Oligomycin Resistance of the Enzyme

In yeast, mammals, and land plants, mitochondrial F(1)F(O)-ATP synthase (complex V) is a remarkable enzymatic machinery that comprises about 15 conserved subunits. Peculiar among eukaryotes, complex V from Chlamydomonadales algae (order of chlorophycean class) has an atypical subunit composition of its peripheral stator and dimerization module, with nine subunits of unknown evolutionary origin (Asa subunits). In vitro, this enzyme exhibits an increased stability of its dimeric form, and in vivo, Chlamydomonas reinhardtii cells are insensitive to oligomycins, which are potent inhibitors of proton translocation through the F(O) moiety. In this work, we showed that the atypical features of the Chlamydomonadales complex V enzyme are shared by the other chlorophycean orders. By biochemical and in silico analyses, we detected several atypical Asa subunits in Scenedesmus obliquus (Sphaeropleales) and Chlorococcum ellipsoideum (Chlorococcales). In contrast, complex V has a canonical subunit composition in other classes of Chlorophytes (Trebouxiophyceae, Prasinophyceae, and Ulvophyceae) as well as in Streptophytes (land plants), and in Rhodophytes (red algae). Growth, respiration, and ATP levels in Chlorophyceae were also barely affected by oligomycin concentrations that affect representatives of the other classes of Chlorophytes. We finally studied the function of the Asa7 atypical subunit by using RNA interference in C. reinhardtii. Although the loss of Asa7 subunit has no impact on cell bioenergetics or mitochondrial structures, it destabilizes in vitro the enzyme dimeric form and renders growth, respiration, and ATP level sensitive to oligomycins. Altogether, our results suggest that the loss of canonical components of the complex V stator happened at the root of chlorophycean lineage and was accompanied by the recruitment of novel polypeptides. Such a massive modification of complex V stator features might have conferred novel properties, including the stabilization of the enzyme dimeric form and the shielding of the proton channel. In these respects, we discuss an evolutionary scenario for F(1)F(O)-ATP synthase in the whole green lineage (i.e., Chlorophyta and Streptophyta).

Structural and functional changes of PSI-LHCI supercomplexes of Chlamydomonas reinhardtii cells grown under high salt conditions

The eVect of high salt concentration (100 mM NaCl) on the organization of photosystem I-light harvesting complex I supercomplexes (PSI-LHCI) of Chlamydomonas reinhardtii was studied. The electron transfer activity was reduced by 39% in isolated PSI-LHCI supercomplexes. The visible circular dichroism (CD) spectra associated with strongly coupled chlorophyll (Chl) dimers were reduced in intensity, indicating that pigment-pigment interactions were disrupted. This data is consistent with results from Xuorescence streak camera spectroscopy, which suggest that red-shifted pigments in the PSI-LHCI antenna had been lost. Denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI reaction center proteins PsaD, PsaE and PsaF were reduced due to salt stress. PsaE is almost completely absent under high salt conditions. It is known that the membrane-extrinsic subunits PsaD and E form the ferredoxin-docking site. Our results indicate that the PSI-LHCI supercomplex is damaged by reactive oxygen species at high salt concentration, with particular impact on the ferredoxin-docking site and the PSILHCI interface.

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.

Temperature Dependence of the Reduction of P700+ by Tightly Bound Plastocyanin in Vivo

The kinetics of reduction of P(700)(+), the stably oxidized electron donor of Photosystem I, by plastocyanin (PC) has been investigated by pump-probe optical spectroscopy in living cells of the green alga Chlamydomonas reinhardtii, between 277 and 318 K. The reduction of P(700)(+) in vivo is described by two kinetic components with lifetimes of 6 +/- 0.5 and 56 +/- 1 micros at room temperature. The rapid reduction phase, which is attributed to reduction of P(700)(+) by prebound PC, is thermally activated with an apparent activation barrier of 105-115 meV. The analysis of the in vivo reaction is consistent with (i) reduced PC and PS I forming a relatively tight binary complex that does not undergo kinetically limiting conformational reconfiguration and (ii) the activation barrier being determined principally by enthalpic contributions to the free energy change. Under the approximation that entropic contributions to the free energy change associated with this electron transfer reaction are negligible, a lower boundary value of the reorganization energy is estimated to be 0.54-0.63 eV, which is on the lower range of the distribution for intraprotein electron transfer reactions. This low activation barrier is discussed in terms of the optimization of primary donor reduction.

Electron-tomographic analysis of intraflagellar transport particle trains in situ

Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.

Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii

We aimed to clarify if heat pretreatment could protect Chlamydomonas reinhardtii cells from gamma rays DNA damaging action. It was studied whether: (1) heat pretreatment could accelerate DNA DSB rejoining; (2) chloroplast chaperones (HSP70B, HSP90C) could be involved in protection from radiation-induced DNA DSB. It was obtained that heat pretreatment (37–42 °C) induced minor DNA DSB levels which might be insufficient as signals for DNA DSB repair induction. No correlation between chaperones overproduction and DNA DSB rejoining was shown. These are probably the first data that HSP70B and HSP90C do not protect DNA against radiation-induced damage in a plant model system.

METAL-INDUCED REACTIVE OXYGEN SPECIES PRODUCTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)(1).

Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation-sensitive reporter dye, 5-(and-6)-carboxy-2',7'-dihydrodifluorofluorescein diacetate (H2 DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10(-6) to 10(-9) M, except in the case of Fe(III), which was effective at 10(-18) M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.

An automated GCxGC‐TOF‐MS protocol for batch‐wise extraction and alignment of mass isotopomer matrixes from differential 13C‐labelling experiments: a case study for photoautotrophic‐mixotrophic grown Chlamydomonas reinhardtii cells

Two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC-TOF-MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC-TOF-MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of (13)C-labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC-TOF-MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/RI2-reference spectra and new search algorithms. Using those techniques we analysed the dynamics of (13)CO(2) and (13)C-acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions.

Additive Effect of Mutations Affecting the Rate of Phylloquinone Reoxidation and Directionality of Electron Transfer within Photosystem I†

Optical pump-probe spectroscopy in the nanosecond-microsecond timescale has been used to study the electron transfer reactions taking place within the Photosystem I reaction center of intact Chlamydomonas reinhardtii cells. The biphasic kinetics of phylloquinone (PhQ) reoxidation were investigated in double mutants that combine a mutation (PsaA-Y696F) near the primary acceptor chlorophyll, ec3A, with those near PhQA (PsaA-S692A, PsaA-W697F). The PsaA-S692A and PsaA-W697F mutations selectively lengthened the 200 ns lifetime component observed in the wild-type (WT). The reverse similar 20 ns component was unaltered in the single mutant, both in terms of lifetime and relative amplitude. However, both double mutants possessed a reverse similar 20 ns component (PhQB(-) reoxidation) with increased amplitude compared with the WT and the individual PhQA mutants. The component assigned to PhQA(-) reoxidation was slowed, like the individual PhQA mutants, and of lower amplitude, as observed in the single ec3A mutant. Hence, the effects of these mutations are almost entirely additive, providing strong support for the previously proposed bidirectional electron transfer model, which attributes the reverse similar 20 and reverse similar 200 ns phases to reoxidation of PhQB or PhQA, respectively. Moreover, in all the mutants investigated, it was also possible to observe an intermediate (approximately 180 ns) component, as previously reported for mutants of the PhQ(A) binding pocket (Biochim. Biophys. Acta [2006] 1757, 1529-1538), which we have tentatively attributed to forward electron transfer between the iron-sulfur clusters FX and FA/B.

Effect of dibromothymoquinone on Chlorophyll a Fluorescence in Chlamydomonas reinhardtii cells incubated in complete or sulfur-depleted medium

The influence of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. The reagent affected differently control cells incubated in complete medium and S-starved cells. Thus, the fluorescence yield in the control essentially increased in the presence of dibromothymoquinone, which can be due to the inactivation of light-harvesting complex II protein kinase, followed by the suppression of membrane transition from high-fluorescence state 1 to low-fluorescence state 2. On the contrary, S-starved cells with membranes in state 2 showed a lower fluorescence yield in the presence of dibromothymoquinone than without it. The JIP test of OJIP fluorescence transients suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to control, while in starved cells, it acts predominantly as an electron acceptor.

Alginate-immobilized Chlamydomonas reinhardtii cell beads as a promising means of removing cadmium from aqueous media

Alginate-immobilized Chlamydomonas reinhardtii cell beads as a promising means of removing cadmium from aqueous media

In vivo analysis of chlorophyll a fluorescence induction

Quantitative characteristics of photosynthetic electron transport were evaluated in vivo on the basis of the multi-exponential analysis of OJIP fluorescence transients induced by saturating actinic light. The OJIP fluorescence curve F(t), measured in Chlamydomonas reinhardtii cells, was transformed into the (1 - F(O)/F(t)) x (F(V)/F(M))(-1) transient, which is shown to relate to PS 2 closure. We assumed that kinetics of PS 2 closure during OJIP rise reflects time-separated processes related to the establishment of redox equilibrium at the PS 2 acceptor side (OJ), PQ pool (JI), and beyond Cyt b/f (IP). Three-exponential fitting was applied to (1 - F(O)/F(t)) x (F(V)/F(M))(-1) transient to obtain lifetimes and amplitudes of the OJ, JI, and IP components of PS 2 closure, which were used to calculate overall rates of reduction and re-oxidation of the PS 2 acceptor side, PQ pool, and intermediates beyond Cyt b/f complex. The results, obtained in the presence of inhibitors, oxidative reagents, and under different stress conditions prove the suggested model and characterize the introduced parameters as useful indicators of photosynthetic function.