Chlamydomonas reinhardtii and photosynthesis: genetics to genomics

Genetic and physiological features of the green alga Chlamydomonas reinhardtii have provided a useful model for elucidating the function, biogenesis and regulation of the photosynthetic apparatus. Combining these characteristics with newly developed molecular technologies for engineering Chlamydomonas and the promise of global analyses of nuclear and chloroplast gene expression will add a new perspective to views on photosynthetic function and regulation.     

The effect of triacontanol on growth, photosynthesis and photorespiration in Chlamydomonas reinhardtii and Anacystis nidulans

Chlamydomonas reinhardtii Dangerad 11–32(90) (−), which exhibits C3 properties, and Anacystis nidulans (Strain no. UTEX 625), which exhibits C4 properties, were used to study the effects of triacontanol on growth, photosynthesis and photorespiration. Photosynthetic rate was measured as CO2 uptake and the O2 inhibition of photosynthesis was used as a measure of photorespiration. Triacontanol dissolved in chloroform and dispersed in Tween-20 and triacontanol colloidally dispersed in an aqueous solution of sodium tallow alkyl sulfate were tested. Chlamydomonas cultures increased significantly in cell number after 4 days, and in chlorophyll content after 3 days of treatment with 2.3 × 10⁻⁸ M TRIA in chloroform/Tween-20. In cultures of Anacystis the chlorophyll content became significantly higher 3 days after treatment with 2.3 × 10⁻⁹ M TRIA and the cell number was noticeably higher than the controls.
CO₂ uptake by triacontanol-treated Chlamydomonas cultures was about the same in both 2 and 21% O2, and the O2 inhibition was significantly reduced as compared with the controls. Photosynthesis in Anacystis was O2-insensitive under the experimental condition used. When Anacystis was treated with triacontanol there was no change in the rate of CO2 uptake and no change in the O2 sensitivity of its CO2 uptake. It appears that triacontanol affects some process which regulated the balance between photosynthesis and photorespiration, but other processes which result in increased growth are probably also affected.     

Acclimation of Chlamydomonas reinhardtii to different growth irradiances

We report on the changes the photosynthetic apparatus of Chlamydomonas reinhardtii undergoes upon acclimation to different light intensity. When grown in high light, cells had a faster growth rate and higher biomass production compared with low and control light conditions. However, cells acclimated to low light intensity are indeed able to produce more biomass per photon available as compared with high light-acclimated cells, which dissipate as heat a large part of light absorbed, thus reducing their photosynthetic efficiency. This dissipative state is strictly dependent on the accumulation of LhcSR3, a protein related to light-harvesting complexes, responsible for nonphotochemical quenching in microalgae. Other changes induced in the composition of the photosynthetic apparatus upon high light acclimation consist of an increase of carotenoid content on a chlorophyll basis, particularly zeaxanthin, and a major down-regulation of light absorption capacity by decreasing the chlorophyll content per cell. Surprisingly, the antenna size of both photosystem I and II is not modulated by acclimation; rather, the regulation affects the PSI/PSII ratio. Major effects of the acclimation to low light consist of increased activity of state 1 and 2 transitions and increased contributions of cyclic electron flow.     

Identification and validation of bioactive small molecule target through phenotypic screening

For effective bioactive small molecule discovery and development into new therapeutic drug, a systematic screening and target protein identification is required. Different from the conventional screening system, herein phenotypic screening in combination with multi-omics-based target identification and validation (MOTIV) is introduced. First, phenotypic screening provides visual effect of bioactive small molecules in the cell or organism level. It is important to know the effect on the cell or organism level since small molecules affect not only a single target but the entire cellular mechanism within a cell or organism. Secondly, MOTIV provides systemic approach to discover the target protein of bioactive small molecule. With the chemical genomics and proteomics approach of target identification methods, various target protein candidates are identified. Then network analysis and validations of these candidates result in identifying the biologically relevant target protein and cellular mechanism. Overall, the combination of phenotypic screening and MOTIV will provide an effective approach to discover new bioactive small molecules and their target protein and mechanism identification.     

In vivo interactions between photosynthesis,mitorespiration, and chlororespiration in Chlamydomonas reinhardtii

Interactions between photosynthesis, mitochondrial respiration (mitorespiration), and chlororespiration have been investigated in the green alga Chlamydomonas reinhardtii using flash illumination and a bare platinum electrode. Depending on the physiological status of algae, flash illumination was found to induce either a fast (t(1/2) approximately 300 ms) or slow (t(1/2) approximately 3 s) transient inhibition of oxygen uptake. Based on the effects of the mitorespiratory inhibitors myxothiazol and salicyl hydroxamic acid (SHAM), and of propyl gallate, an inhibitor of the chlororespiratory oxidase, we conclude that the fast transient is due to the flash-induced inhibition of chlororespiration and that the slow transient is due to the flash-induced inhibition of mitorespiration. By measuring blue-green fluorescence changes, related to the redox status of the pyridine nucleotide pool, and chlorophyll fluorescence, related to the redox status of plastoquinones (PQs) in C. reinhardtii wild type and in a photosystem I-deficient mutant, we show that interactions between photosynthesis and chlororespiration are favored when PQ and pyridine nucleotide pools are reduced, whereas interactions between photosynthesis and mitorespiration are favored at more oxidized states. We conclude that the plastid oxidase, similar to the mitochondrial alternative oxidase, becomes significantly engaged when the PQ pool becomes highly reduced, and thereby prevents its over-reduction.     

Effects of calcium channel blockers on phototaxis and motility of Chlamydomonas reinhardtii

The effects of the four calcium channel blockers flunarizine, verapamil, diltiazem and nimodipine on motility and phototaxis of Chlamydomonas reinhardtio have been tested with a fully automated and computerized population system. Flunarizine inhibits motility transiently by causing the detachement of the flagella which, however, are regenerated during some hours. Phototaxis is inhibited to the same extent, but this is simply the result of the decreased motility and, hence, a non-specific effect. Verapamil causes also a detachement of the flagella with following regeneration, but in addition motility and phototaxis are inhibited by this drug to different extents, indicating the involvement of calcium channels in both processes. Diltiazem and nimodipine inhibit phototaxis without impairing motility, indicating that both processes are regulated in different ways. If diltiazem and nimodipine are applied simultaneously, no additive inhibitory effect can be observed. However, the combination of both blockers with verapamil causes and additive inhibitory effect as if verapamil is applied alone. By increasing the external calcium concentration from 10^-4 M to 10^-3 M the optimum of positive phototaxis is shifted to higher fluence rates. This shifting occurs also in the presence of channel blockers, but the strength of the positive reaction is influenced. These results point to the involvement of calcium channels in both phototaxis and motility, but simultaneously demonstrate the different sensitivity of the two processes to these drugs.Abbreviations DIL diltiazem (=benzothiazepine) - FLU flunarizine (=(E)-1-(bis-(4-fluorophenyl(methyl)-4-(3-phenyl-2-propenyl)piperazinex2HCl) - NIM nimodipine (=1,4-dihydropyridine) - VER verapamil (=diphenylalkylamine) CaM, calmodulin - PDE phosphodiesterase - DMSO dimethylsulfoxide     

Pressure reduction affects growth and morphology of Chlamydomonas reinhardtii

Cellular perception of pressure is a largely unknown field in microalgae research although it should be addressed for optimization of a photobioreactor design regarding typically occurring pressure cycles. Also for the purpose of using microalgae as basic modules for material cycles in controlled ecological life support systems, the absence of pressure in outer space or the low absolute pressures on other planets is an abiotic factor that needs to be considered for design of integrated microalgae‐based modules. The aim of this work is to study the effects of lowered pressure and pressure changes on photosynthesis as well as morphology. Two Chlamydomonas reinhardtii wild‐type strains were exposed to controlled pressure patterns during batch cultivations. Sudden pressure changes should test for existing threshold values for cell survival to mimic such events during space missions. Algae were grown inside a 2 L photobioreactor with an integrated vacuum pump ensuring constant pressures down to 700 mbar. Cultivation samples were analyzed for OD₇₅₀, cell dry weight, and morphology via light microscope. Chlamydomonas reinhardtii CC‐1690 cells showed decreased growth rates, higher carbon dioxide uptake rates, and unchanged oxygen production rates at lower pressures. For sudden pressures changes in the range of 300 mbar no fatal threshold was determined. This study shows that pressure reduction affects growth, gas exchange rates, and morphology. Within the tested pressure range no fatal threshold value was reached.     

Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii

The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA‐ and siRNA‐based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one‐step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G‐Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon‐counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids 1) and the flagellar protein CDPK3 (calcium‐dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.     

Effects of increasing doses of UV-B radiation on photosynthesis and motility inChlamydomonas reinhardtii

Effects of increasing doses of UV-B radiation on photosynthetic efficiency (PE) and motility inChlamydomonas reinhardtii were investigated. The exposure time ranged from 10 to 120 min at 0.73 W/m² UV-B radiation. A decrease in compensation points (CP) was followed by an increase in PE on extending the UV-B treatment up to 60 min. The subsequent increase in PE was accompanied by increasing CPs. Enhanced doses of UV-B radiation thus have a stimulatory effect on the PE inC. reinhardtii. No inhibitory effects could be detected. No statistically significant differences in motility could be observed due to an extremely high variation of values.     

Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis

Articular cartilage, which is mainly composed of collagen II, enables smooth skeletal movement. Degeneration of collagen II can be caused by various events, such as injury, but degeneration especially increases over the course of normal aging. Unfortunately, the body does not fully repair itself from this type of degeneration, resulting in impaired movement. Microfracture, an articular cartilage repair surgical technique, has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However, the therapeutic outcomes of all these techniques vary in different patients depending on their age, health, lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage, both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone, or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs), which are able to self-renew and differentiate into multiple cell types, provides a potentially valuable cell resource for drug screening in a “more relevant” cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.     

Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-XL

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Small molecule inhibitors of Bcl-X_L function have been discovered from diverse structure classes using rational drug design as well as high-throughput screening (HTS) approaches. However, most of the BH3 mimetics that have been identified via screening based on fluorescence polarization displayed an affinity for their presumed protein targets that is far lower than that of BH3-only proteins. Therefore, it is important to establish a simple and inexpensive secondary platform for hit validation which is pertinent to current efforts for developing compounds that mimic the action of BH3-only proteins as novel anticancer agents. These considerations prompted us to explore the differential scanning fluorimetry (DSF) method that is based on energetic coupling between ligand binding and protein unfolding. We have systematically tested known Bcl-X_L/Bcl-2 inhibitors using DSF and have revealed distinct subsets of inhibitors. More importantly, we report that some of these inhibitors interacted selectively with glutathione S-transferase tagged Bcl-X_L, whereas certain inhibitors exhibited marked selectivity towards native untagged Bcl-X_L. Therefore, we propose that the affinity tag may cause a significant conformational switch in the Bcl-X_L, which results in the selectivity for certain subsets of small molecule inhibitors. This finding also implies that the previous screens involving tagged proteins need to be carefully reexamined while further investigations must ensure that the right conformation of protein is used in future screens.     

Combined effect of temperature and bleaching herbicides on photosynthesis,pigment and fatty acid composition of Chlamydomonas reinhardtii

Norflurazon (Nf) and fluridone (Fd) are phytoene desaturase inhibitor herbicides that are widely used for the control of grasses and invasive aquatic weeds, respectively. These herbicides enter aquatic environments where they can negatively affect non-target plant species (e.g. algae). Their toxicity towards algae may be modified by abiotic factors such as light intensity, temperature, pH and nutrients. Investigating the effect of low temperature on the toxicity of Nf and Fd is particularly important because both temperature and herbicides affect some of the same physiological process (e.g. carotenoid biosynthesis). Here we demonstrate that Nf reduced photosynthesis in the green alga Chlamydomonas reinhardtii more strongly at 15 than at 25ºC, while Fd showed stronger effects at 25 than at 15ºC. Neither herbicide significantly inhibited photosynthesis at 8ºC. Although the overall pigment content decreased with lower temperature, there was an increase in photo-protective carotenoids relative to chlorophylls at both 15 and 8ºC in the absence of herbicides. Moreover, most of the measured pigments decreased markedly in the presence of Nf and Fd at 15 and 25ºC, including β-carotene which fell to below detection limits. The fatty acid composition was modified by temperature and the level of unsaturation noticeably increased at 15 compared with 25ºC. At 8ºC, however, despite a 2.4 times decrease in fatty acid content, the unsaturation level was similar to 25ºC acclimated cells. Monounsaturated fatty acids increased concomitant with a decrease in polyunsaturated fatty acid in the 2.5 µM Nf treatment at 25ºC. Differences in the effect of Nf and Fd on photosynthesis at 15 and 25ºC can be attributed to the marked decrease in carotenoids, which play an important role in photoprotection. At 8ºC, the apparent lack of inhibitory effects compared with control cultures could be due to enhanced photoprotection and/or decreased uptake of herbicides by the alga.     

Recovery of photosynthesis and phtosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light

Recovery from 60 min of photoinhibitory treatment at photosynthetic photon flux densities of 500, 1400 and 2200 μMmol m^?2 s^? was followed in cells of the green alga Chlamydomonas reinhardtii grown at 125 μMmol m^?2 s^?1. These light treatments represent photoregulation, moderate photoinhibition and strong photoinhibition, respectively. Treatment in photoregulatory light resulted in an increased maximal rate of oxygen evolution (P_max) and an increased quantum yield (Φ), but a 15% decrease in F_v/F_M. Treatment at moderately photoinhibitory light resulted in a 30% decrease in F_v/F_M and an approximately equal decrease in Φ. Recovery in dim light restored F_v/F_M within 15 and 45 min after high light treatment at 500 and 1400 μMmol m^?2 s^?1, respectively. Convexity (Θ), a measure of the extent of co-limitation between PS II turnover and whole-chain electron transport, and Φ approached, but did not reach the control level during recovery after exposure to 1400 μMmol m^?2 s^?1, whereas P_max increased above the control. Treatment at 2200 μMmol m^?2 s^?1 resulted in a strong reduction of the modeled parameters Φ, Θ and P_max. Subsequent recovery was initially rapid but the rate decreased, and a complete recovery was not reached within 120 min. Based on the results, it is hypothesized that exposure to high light results in two phenomena. The first, expressed at all three light intensities, involves redistribution within the different aspects of PS II heterogeneity rather than a photoinhibitory destruction of PS II reaction centers. The second, most strongly expressed at 2200 μmol m^?2 s^?1, is a physical damage to PS II shown as an almost total loss of PS II_α and PS II Q_B-reducing centers. Thus recovery displayed two phase, the first was rapid and the only visible phase in algae exposed to 500 and 1400 μmol m^?2 s^?1. The second phase was slow and visible only in the later part of recovery in cells exposed to 2200 μmol m^?2 s^?1.     

Identification of small molecule inhibitors of beta-amyloid cytotoxicity through a cell-based high-throughput screening platform

Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of beta-amyloid (Abeta) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer's disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Abeta. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Abeta with IC(50) values of <10 microM and showed little or no toxicity at concentrations up to 30 microM, none inhibit the calpain enzyme directly.     

A small molecule microarray platform to select RNA internal loop-ligand interactions.

Herein, we report the development of a microarray platform to select RNA motif-ligand interactions that allows simultaneous screening of both RNA and chemical space. We used this platform to identify the RNA internal loops that bind 6'- N-5-hexynoate kanamycin A ( 1). Selected internal loops that bind 1 were studied in detail and commonly display an adenine across from a cytosine independent of the size of the loop. Additional preferences are also observed. For 3 x 3 nucleotide loops, there is a preference for purines, and for 2 x 2 nucleotide loops there is a preference for pyrimidines neighbored by an adenine across from a cytosine. This technique has several advantageous features for selecting RNA motif-ligand interactions: (1) higher affinity RNA motif-ligand interactions are identified by harvesting bound RNAs from lower ligand loadings; (2) bound RNAs are harvested from the array via gel extraction, mitigating kinetic biases in selections; and (3) multiple selections are completed on a single array surface. To further demonstrate that multiple selections can be completed in parallel on the same array surface, we selected the RNA internal loops from a 4096-member RNA internal loop library that bound a four-member aminoglycoside library. These experiments probed 16,384 (4 aminoglycoside x 4096-member RNA library) interactions in a single experiment. These studies allow for parallel screening of both chemical and RNA space to improve our understanding of RNA-ligand interactions. This information may facilitate the rational and modular design of small molecules targeting RNA.     

A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions

Continuous photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, is observed after incubating the cultures for about a day in the absence of sulfate and in the presence of acetate. Sulfur deprivation causes the partial and reversible inactivation of photosynthetic O(2) evolution in algae, resulting in the light-induced establishment of anaerobic conditions in sealed photobioreactors, expression of two [FeFe]-hydrogenases in the cells, and H(2) photoproduction for several days. We have previously demonstrated that sulfur-deprived algal cultures can produce H(2) gas in the absence of acetate, when appropriate experimental protocols were used (Tsygankov, A.A., Kosourov, S.N., Tolstygina, I.V., Ghirardi, M.L., Seibert, M., 2006. Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int. J. Hydrogen Energy 31, 1574-1584). We now report the use of an automated photobioreactor system to compare the effects of photoautotrophic, photoheterotrophic and photomixotrophic growth conditions on the kinetic parameters associated with the adaptation of the algal cells to sulfur deprivation and H(2) photoproduction. This was done under the experimental conditions outlined in the above reference, including controlled pH. From this comparison we show that both acetate and CO(2) are required for the most rapid inactivation of photosystem II and the highest yield of H(2) gas production. Although, the presence of acetate in the system is not critical for the process, H(2) photoproduction under photoautotrophic conditions can be increased by optimizing the conditions for high starch accumulation. These results suggest ways of engineering algae to improve H(2) production, which in turn may have a positive impact on the economics of applied systems for H(2) production.