Spectral and Kinetic Analysis of the Energy Coupling in the PS I–LHC I Supercomplex from the Green Alga Chlamydomonas reinhardtii at 77 K

Energy transfer processes in the chlorophyll antenna of the PS I–LHCI supercomplexes from the green alga Chlamydomonas reinhardtii have been studied at 77 K using transient absorption spectroscopy with multicolor excitation in the 640–670 nm region. Comparison of the kinetic data obtained at low and room temperatures indicates that the slow ∼ ∼100 ps excitation equilibration phase that is characteristic of energy coupling of the LHCI peripheral antenna to the PS I core at physiological temperatures (Melkozernov AN, Kargul J, Lin S, Barber J and Blankenship RE (2004) J Phys Chem B 108: 10547–10555) is not observed in the excitation dynamics of the PS I–LHCI supercomplex at 77 K. This suggests that at low temperatures the peripheral antenna is energetically uncoupled from the PS I core antenna. Under these conditions the observed kinetic phases on the time scales from subpicoseconds to tens of picoseconds represent the superposition of the processes occurring independently in the PS I core antenna and the Chl a/b containing LHCI antenna. In the PS I–LHCI supercomplex with two uncoupled antennas the excitation is channeled to the excitation sinks formed at low temperature by clusters of red pigments. A better spectral resolution of the transient absorption spectra at 77 K results in detection of two ΔA bands originating from the rise of photobleaching on the picosecond time scale of two clearly distinguished pools of low energy absorbing Chls in the PS I–LHCI supercomplex. The first pool of low energy pigments absorbing at 687 nm is likely to originate from the red pigments in the LHCI where the Lhca1 protein is most abundant. The second pool at 697 nm is suggested to result either from the structural interaction of the LHCI and the PS I core or from other Lhca proteins in the antenna. The kinetic data are discussed based on recent structural models of the PS I–LHCI. It is proposed that the uncoupling of pigment pools may be a control mechanism that regulates energy flow in Photosystem I.     

Identification of target mRNAs for the clock-controlled RNA-binding protein Chlamy 1 from Chlamydomonas reinhardtii

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the green alga Chlamydomonas reinhardtii, a clock-controlled RNA-binding protein (Chlamy 1) was identified recently, which represents an analog of the circadian trans-acting factor CCTR from the phylogenetically diverse alga Gonyaulax polyedra. In order to identify in C. reinhardtii target mRNAs that can be recognized by Chlamy 1, gel mobility-shift assays and UV-crosslinking experiments were carried out, and revealed that this protein interacts specifically with the 3' untranslated regions of several mRNAs and recognizes them all via a common cis-acting element, composed of at least seven UG repeats. By using competition assays, it was found that the affinity of Chlamy 1 is highest for mRNAs whose products are key components of nitrogen and CO2 metabolism. Since the activities of enzymes involved in nitrogen metabolism vary in a temporal pattern that is opposite in phase to that of Chlamy 1 binding activity, the protein may repress the translation of the cognate mRNAs.     

Chloroplast-encoded PsbT is required for efficient biogenesis of photosystem II complex in the green alga Chlamydomonas reinhardtii

The small hydrophobic polypeptide PsbT is associated with the photosystem II (PSII) reaction center (D1/D2 heterodimer). Here, we report the effect of the deletion of PsbT on the biogenesis of PSII complex during light-induced greening of y-1 mutants of the green alga Chlamydomonas reinhardtii. The y-1 is unable to synthesize chlorophylls in the dark but do so in the light. The dark-grown y-1 cells accumulated no major PSII proteins but a small amount of PsbT. Upon illumination, PsbT was immediately synthesized while chlorophylls, major PSII proteins, and O(2)-evolving activity increased after a 1-h lag. The y-1 cells without PsbT accumulated chlorophylls and PSI protein at a similar rate, whereas the accumulation of PSII complex was specifically retarded during greening. The absence of PsbT did not affect the synthesis of PSII proteins. These results indicate that PsbT is required for the efficient biogenesis of PSII complex.     

Uptake of HCO3? and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.     

Is a ferroxidase involved in the high-affinity iron uptake in Chlamydomonas reinhardtii?

In phosphorus deficient soils and under smallscale farming systems, the development of efficient management strategies for P fertilizers is crucial to sustain food production. A field experiment was conducted on a P-fixing Acrisol in western Kenya to study possibilities of replenishing soil P with seasonal additions of small rates of P fertilizers. Triple superphosphate was applied at 0, 10, 25, 50 and 150 kg P ha^–1 for 5 consecutive maize growing seasons followed by 4 seasons of residual crops. Maize yields and soil P fractions were determined. Although maize responded to additions of 10 kg P ha^–1 with a cumulative grain yield of 16.8 Mg ha^–1, at the end of the experiment, compared to 8.8 Mg ha^–1 in the non-P fertilized plots, soil labile P did not increase correspondingly. Seasonal additions of 150 kg P ha^–1 increased maize yields to a cumulative value of 39 Mg ha^–1 at the end of the experiment, and increased all soil inorganic P fractions. At the third season of residual phase, treatment with a cumulative addition of 750 kg P ha^–1 gave the highest yields compared to treatments in the same residual stage, but these yields were considered less than the maximum yield of the season. This indicates that the large build up of soil P was not available for crop uptake. The inorganic P fraction extracted by NaHCO₃ was the most affected by changes in management, increasing during the input phase and decreasing after interruption of P addition, for all P rates. The decrease in this pool during the residual phase could be explained by the maize uptake. This study showed that seasonal additions of 25 kg P ha^–1 can increase maize yield with gradual replenishment of soil P.     

Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a model organism for various studies in biology. CC-124 is a laboratory strain widely used as a wild type. However, this strain is known to carry agg1 mutation, which causes cells to swim away from the light source (negative phototaxis), in contrast to the cells of other wild-type strains, which swim toward the light source (positive phototaxis). Here we identified the causative gene of agg1 (AGG1) using AFLP-based gene mapping and whole genome next-generation sequencing. This gene encodes a 36-kDa protein containing a Fibronectin type III domain and a CHORD-Sgt1 (CS) domain. The gene product is localized to the cell body and not to flagella or basal body.     

The Antarctic psychrophile, Chlamydomonas subcaudata, is deficient in state I–state II transitions

State I-State II transitions were monitored in vivo and in vitro in the Antarctic, psychrophillic, green alga, Chlamydomonas subcaudata, as changes in the low-temperature (77 K) chlorophyll fluorescence emission maxima at 722 nm (F722) relative to 699 nm (F699). As expected, the control mesophillic species, Chlamydomonas reinhardtii, was able to modulate the light energy distribution between photosystem II and photosystem I in response to exposure to four different conditions: (i) dark/anaerobic conditions, (ii) a change in Mg2+ concentration, (iii) red light, and (iv) increased incubation temperature. This was correlated with the ability to phosphorylate both of its major light-harvesting polypeptides. In contrast, exposure of C. subcaudata to the same four conditions induced minimum alterations in the 77 K fluorescence emission spectra, which was correlated with the ability to phosphorylate only one of its major light-harvesting polypeptides. Thus, C. subcaudata appears to be deficient in the ability to undergo a State I-State II transition. Functionally, this is associated with alterations in the apparent redox status of the intersystem electron transport chain and with higher rates of photosystem I cyclic electron transport in the psychrophile than in the mesophile, based on in vivo P700 measurements. Structurally, this deficiency is associated with reduced levels of Psa A/B relative to D1, the absence of specific photosystem I light-harvesting polypeptides [R.M. Morgan et al. (1998) Photosynth Res 56:303-314] and a cytochrome b6/f complex that exhibits a form of cytochrome f that is approximately 7 kDa smaller than that observed in C. reinhardtii. We conclude that the Antarctic psychrophile, C. subcaudata, is an example of a natural variant deficient in State I-State II transitions.     

Sustained H₂ production in a Chlamydomonas reinhardtii D1 protein mutant

In the present investigation, a detailed biochemical analysis of the high H? producer D1 protein mutant strain L159I-N230Y of Chlamydomonas reinhardtii, carrying a double amino acid substitution, was made. The leucine residue L159 was replaced by isoleucine, and the N230 asparagine was replaced by tyrosine. The performance of this strain was compared to that of the cc124 strain. The mutant showed a sustained capacity to donate electrons by means of direct biophotolysis for H? production, as demonstrated by the higher efficiency of utilization of the hydrogenase enzyme when carried out under anaerobic conditions. The latter property was maintained also under sulfur deprivation. Furthermore, when compared to the cc124, the mutant showed a higher amount of D1 protein content, a higher carbohydrate storage capacity and a sustained PSII direct contribution to the H? production during sulfur deprivation. The addition of DCMU to the cells showed that as much as 7.0 mL H? liter of culture h?1 were produced by means of direct biophotolysis. The maximum apparent light-to-hydrogen conversion efficiency expressed on PAR (photosynthetically active radiation) reached 3.22%, while PSII efficiency to perform direct biophotolysis was calculated to be 2.03%. These values are significantly higher than what has been reported in the literature.     

Negative Impact on Growth and Photosynthesis in the Green Alga Chlamydomonas reinhardtii in the Presence of the Estrogen 17α-Ethynylestradiol

It is well known that estrogenic compounds affect development of fertilized eggs of many species of birds, fish and amphibians through disrupted activity of carbonic anhydrase (CA). The most potent activity comes from the most commonly occurring synthetic sterol, 17α-Ethynylestradiol (EE2). Less is known about the responses of aquatic phytoplankton to these compounds. Here we show for the first time that, in comparision to the control, the addition of 7 µM EE2 reduced the growth rate of the green alga Chlamydomonas reinhardtii by 68% for cells grown at high CO₂. When cells were grown in ambient air (low C_i) with a fully activated carbon concentrating mechanism through the induction of CA activity, the growth rates were reduced by as much as 119%. A reduced growth rate could be observed at EE2 concentrations as low as 10 pM. This was accompanied by a reduced maximum capacity for electron transport in photosystem II as determined by a lower F_V/F_M for low C_i-grown cells, which indicates the involvement of CAH3, a CA specifically located in the thylakoid lumen involved in proton pumping across the thylakoid membranes. These results were in agreement with an observed reduction in the chloroplastic affinity for C_i as shown by a strong increase in the Michaelis-Menten K_0.5 for HCO₃ ⁻. In itself, a lowering of the growth rate of a green alga by addition of the sterol EE2 warrants further investigation into the potential environmental impact by the release of treated waste water.     

CO2 and O2 dynamics in human-impacted watersheds in the state of São Paulo, Brazil

We studied the spatial and temporal variation in O₂ and dissolved inorganic carbon (DIC) forms concentrations in ten subtropical watersheds located in the state of São Paulo, Brazil, with different degrees of impact by urbanization and land-use changes. Additionally, we used stable carbon isotopic composition of DIC to explain observed patterns. We found that land-cover changes and watershed geology are the main drivers of DIC distribution. Land-cover/use changes influence the riverine DIC in two ways: by replacing the original Cerrado 3 (C3)-type forest vegetation by C4-type vegetation composed of grasses (pasture), and by sugarcane. Most domestic sewage is dumped untreated into rivers in the state of São Paulo. Consequently, in the most densely populated watersheds, sewage is an important source of labile carbon and consequently of DIC to rivers. In terms of geology, although silicate weathering that produces kaolinite is the main type of weathering in the watersheds, the weathering of carbonate cements present in the geological formations of the western portion of the state of São Paulo are also an important source of DIC to rivers.     

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt −

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt ^– were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 M), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.Abbreviations HSM high-salt medium This study was supported by the Deutsche Forschungsgemeinschaft. O. A. Sineshchekov was supported by a Research Fellowship from the Alexander von Humboldt Foundation. The authors wish to thank U. Powalowski (Botanisches Institut, Universität zu Köln) for help with electron microscopy.     

Effect of the Form of Mineral Nitrogen on the Growth and Heterotrophic Fixation of 14С-Bicarbonate in Chlamydomonas reinhardtii

Russian Journal of Plant Physiology - The earlier-detected effect of the mineral nitrogen form in the nutrient medium (ammonium or nitrate) on the quantity of ribosomes in the cells of...     

Function of 3′ non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3 untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3 flanking sequences of psaB or extended the open reading frame into the 3 inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3 stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3 inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.     

Accumulation of ζ-carotene in Chlamydomonas reinhardtii under control of the ac5 nuclear gene

 The accumulation of different precursors of carotenoid biosynthesis in carotenoid-deficient mutants of Chlamydomonas reinhardtii was studied by HPLC-analysis. ζ-Carotene accumulated in several ac5 mutants, this character cosegregated with mutations in the ac5 gene. Two groups of ac5 mutants differing in ζ-carotene accumulation were distinguished. One (ac5–1) accumulated ζ-carotene in the dark but not in the light. The other (ac5–2) accumulated ζ-carotene under both dark and light conditions. ac5–2 strains accumulated more ζ-carotene in the dark than ac5-1 strains. Genetic data suggested that the mutations ac5–1 and ac5–2 were allelic. Pleiotropic effects of mutations in the ac5 gene included decreased levels of chlorophyll a and b and acetate requirement. The results are consistent with the presence of a defective ζ-carotene desaturase in ac5 mutants. Received: 27 October 1998 / Revision received: 1 February 1999 / Accepted: 16 February 1999     

γ-Tocopherol methyltransferase from the green alga Chlamydomonas reinhardtii: functional characterization and expression analysis

γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is a very important enzyme in tocopherol biosynthesis in all photosynthetic organisms. In this paper, we present the functional characterization and expression analysis of γ-TMT from the unicellular green alga Chlamydomonas reinhardtii. Recombinant TMT1 enzyme was purified and characterized. The size of TMT1 subunit was estimated as 37 kDa by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), in accordance with the predicted molecular size after TMT1 cDNA sequence. Recombinant TMT1 also showed an apparent molecular mass of 37 kDa in its native conformation, suggesting that native TMT1 has a monomeric structure similar to the plant TMTs already characterized. pH and temperature dependence of TMT1 activity were also similar to plant TMTs. Substrate specificity studies showed that Chlamydomonas TMT1 is responsible for the conversion of γ- and δ-tocopherol to α- and β-tocopherol, respectively. The kinetic properties of Chlamydomonas recombinant γ-TMT activity were studied and γ-TMT1 has a similar affinity for γ- and δ-tocopherol. Promoter sequence analysis and expression analysis by northern blot revealed that tmt1 expression is strongly upregulated by high light and downregulated by low temperature. This regulatory pattern of tmt1 expression supports the idea that γ- and α-tocopherol play specific roles in the adaptation to growth under low temperature and high light stress conditions.     

A Mechanism for Ultra-Slow Oscillations in the Cortical Default Network

When the brain is in its noncognitive “idling” state, functional MRI measurements reveal the activation of default cortical networks whose activity is suppressed during cognitive processing. This default or background mode is characterized by ultra-slow BOLD oscillations (∼0.05 Hz), signaling extremely slow cycling in cortical metabolic demand across distinct cortical regions. Here we describe a model of the cortex which predicts that slow cycling of cortical activity can arise naturally as a result of nonlinear interactions between temporal (Hopf) and spatial (Turing) instabilities. The Hopf instability is triggered by delays in the inhibitory postsynaptic response, while the Turing instability is precipitated by increases in the strength of the gap-junction coupling between interneurons. We comment on possible implications for slow dendritic computation and information processing.     

A large open reading frame (orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein

An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green alga Chlamydomonas reinhardtii. Besides having a short hydrophobic N-terminal domain with five putative transmembrane helices, the predicted orf1995 product is highly basic. orf1995 might be a homologue of the ycf1 gene in land plants, whose function has not yet been determined. Mutants of C. reinhardtii transformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that the orf1995 product is essential for cell survival.