Phototactic responses of Chlamydomonas reinhardtii in electric fields

Crude mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers were purified on a 23% (v/v) continuous Percoll gradient. Microbodies and damaged mitochondria banded on top of the gradient, whereas the purified mitochondria handed close to the bottom. The purified mitochondria showed improved membrane integrities, specific enzyme activities and respiratory properties (higher rate, respiratory control, ADP/O ratio) than the crude mitochondria. Purified mitochondria could he stored for 24 h on ice in a phosphate buffer with only small loss of activity.     

Phototropin from Chlamydomonas reinhardtii is functional in Arabidopsis thaliana

Phototropin, a plant blue light photoreceptor, mediates important blue light responses such as phototropism, chloroplast positioning and stomatal opening in higher plants. In Arabidopsis thaliana, two phototoropins, phototropin 1 and 2, are known. Recently, in the unicellular green alga, Chlamydomonas reinhardtii, a phototropin homolog was identified. It exhibits photochemical properties similar to those of higher plant phototropins and is involved in multiple steps of the sexual life cycle of Chlamydomonas. Here, we expressed Chlamydomonas phototropin in Arabidopsis to examine whether it is active in a distantly related plant species. The Arabidopsis mutant deficient in both phototropin 1 and 2 was transformed with a vector containing Chlamydomonas phototropin cDNA fused to a cauliflower mosaic virus 35S promoter. The resulting lines were classified into high, medium and low expressers based on RNA gel blot and immunoblot analyses. Typical phototropin responses were restored in high expression lines. These results demonstrate that Chlamydomonas phototropin is functional in higher plants. Hence, the basic mechanism of phototropin action is highly conserved, even though its apparent physiological functions are quite diverse.     

Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. C hlamydomonas reinhardtii flagella use an asymmetric beat stroke, similar to a breast stroke, to propel cells forward. To generate the asymmetric beat stroke, dynein activity must be regulated both along the length and around the circumference of the flagella. If all dyneins were active at the same time, the flagella would exist in a state of rigor. The dyneins are located in two rows along the length of the doublet microtubules. The inner dynein arms are heterogeneous in composition with at least eight heavy chains and various intermediate and light chains arranged in an elaborate morphology that repeats every 96 nm (Kagami and Kamiya, 1992; Mastronarde et al., 1992). In contrast, the outer dynein arms are biochemically and morphologically homogeneous (Huang et al., 1979; Mitchell and Rosenbaum, 1985; Kamiya, 1988); each outer dynein arm contains three dynein heavy chains and 10 intermediate and light chains. The inner and outer arms appear to have different functions in the formation of the beat stroke; the inner arms generate the waveform of the beat stroke, whereas the outer arms provide additional force to the waveform (Brokaw and Kamiya, 1987).Previous workers had shown that dynein regulation is imposed, in part, by activities of the radial spokes and the central pair complex. Mutant strains that are missing or have altered radial spokes or central pair complexes are paralyzed even if they have a full complement of dyneins (Adams et al., 1981; Piperno et al., 1981). Many extragenic suppressors of this paralysis phenotype do not restore the missing structures, but rather suppress by altering either inner arm or outer arm region structures (Huang et al., 1982a ; Piperno et al., 1992; Porter et al., 1992, 1994). These data suggest that direct or indirect interactions exist between the dynein arms and the radial spokes or central pair complexes.Over 80 proteins in Chlamydomonas flagella are phosphorylated (Piperno et al., 1981), which makes dynein regulation by phosphorylation an attractive model. Hasegawa et al. (1987) showed that a higher percentage of demembranated axonemes reactivate with ATP after treatments that lower cAMP levels or inhibit cAMP-dependent protein kinase (cAPK)¹. In flagella from other organisms, cAMP has an opposite role (for reviews see Tash and Means, 1983; Tash, 1989). An increased frequency of reactivation also occurs after the NP-40–soluble components are extracted from the axonemes, which suggests that the cAPK, target phosphoproteins, and endogenous phosphatases are all integral axonemal components (Hasegawa et al., 1987). In quantitative sliding disintegration assays, the inner dynein arm activity of axonemes that are missing the radial spokes is increased in the presence of pharmacological or specific peptide inhibitors of cAPK (Smith and Sale, 1992; Howard et al., 1994). Reconstitution experiments with axonemes that are missing the radial spokes suggest that radial spokes normally function to activate the inner dynein arms by inhibiting a cAPK (Smith and Sale, 1992; Howard et al., 1994). It is not known if the cAPK directly phosphorylates inner dynein arm components or phosphorylates another axonemal component that then acts on the inner dynein arms (Howard et al., 1994).The f (originally called I1) inner arms are biochemically the best studied inner dynein arm complex. This complex is comprised of two dynein heavy chains and three intermediate chains of 140, 138, and 110 kD; it can be purified by sucrose density centrifugation (Piperno and Luck, 1981; Smith and Sale, 1991; Porter et al., 1992) or ion-exchange chromatography (Kagami and Kamiya, 1992). The purified complex has low ATPase activity and only rarely translocates microtubules in vitro (Smith and Sale, 1991; Kagami and Kamiya, 1992). Deep-etch EM of the purified f inner arm shows a two-headed complex that is connected to a common base by thin stalks (Smith and Sale, 1991). Longitudinal EM image analyses have shown that this complex is located just proximally of the first radial spoke in each 96-nm repeating unit (Piperno et al., 1990; Mastronarde et al., 1992). Mutations at three different loci (PF9/ IDA1, IDA2, and IDA3) result in the complete loss of the f complex (Kamiya et al., 1991; Kagami and Kamiya, 1992; Porter et al., 1992). The PF9/IDA1 locus encodes a dynein heavy chain that is believed to be one of the two heavy chains that are components of the f complex (Porter, 1996).We undertook a new approach to identify axonemal components involved in dynein regulation; we isolated and characterized mutant strains that were unable to perform phototaxis. In Chlamydomonas, phototaxis is a behavior by which cells orient to the direction of incident light. Light direction is detected by the eyespot, an asymmetrically located organelle, and a signal is transmitted to the flagella using voltage-gated ion channels (Harz and Hegemann, 1991). For cells to perform phototaxis, the waveforms of the two flagella are altered coordinately. The trans flagellum, which is located farther from the eyespot, beats with a larger front amplitude than the cis flagellum to turn the cell toward the light (Rüffer and Nultsch, 1991). It seemed likely that the alterations in the beat amplitudes needed for correct phototactic behavior could be caused by differential dynein regulation in the cis and trans flagella. Therefore, we hypothesized that there should be a class of phototactic mutant strains that is not able to perform phototaxis because of defects in the regulation of dyneins. Three of the eight phototactic mutant strains that we characterized had biochemical defects in the f class of inner dynein arms. One of these strains, pf9-4, was missing the entire f complex, and the other two strains, mia1-1 and mia2-1, exhibited a novel f class inner dynein arm biochemical phenotype. These observations suggest that the f inner dynein arm is a target for regulation during phototaxis.     

Blue Light Regulation of Cell Division in Chlamydomonas reinhardtii

A delay in cell division was observed when synchronized cultures of the unicellular green alga Chlamydomonas reinhardtii growing under heterotrophic conditions were exposed to white light during the second half of the growth period. This effect was also observed when photosynthesis was blocked by addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Light pulses of 10 minutes were sufficient to induce a delay in cell division in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A delay in cell division was induced by blue light but not by illumination with red or far-red light. The equal intensity action spectrum revealed two peaks at 400 and 500 nm.     

Heavy-Metal Regulation of Thioredoxin Gene Expression in Chlamydomonas reinhardtii

Heavy metals are highly toxic compounds for cells. In this report we demonstrate that the expression of Chlamydomonas reinhardtii thioredoxins (TRX) m and h is induced by heavy metals. Upon exposure of the cells to Cd and Hg, a strong accumulation of both messengers was observed. Western-blot experiments revealed that among these two TRXs, only TRX h polypeptides accumulated in response to the toxic cations. A biochemical analysis indicated that heavy metals inhibit TRX activity, presumably by binding at the level of their active site. Sequence analysis of the C. reinhardtii TRX h promoter revealed the presence of cis-acting elements related to cadmium induction. The origins and purposes of this regulation are discussed. Our data suggest, for the first time to our knowledge, a possible implication of TRXs in defense mechanisms against heavy metals.     

Regulation by ammonium of nitrate and nitrite assimilation in Chlamydomonas reinhardtii

The inhibitor of mRNA synthesis, 6-methylpurine, inhibited nitrate reductase derepression in either ammonium-grown or methylammonium-treated wild-type cells of Chlamydomonas reinhardtii, but not in nitrogen-starved cells. In contrast, 6-methylpurine did not inhibit nitrate reductase synthesis in the methylammonium-resistant mutant 2170 (ma-1) either grown on ammonium, treated with methylammonium or nitrogen starved, but did inhibit the continuous synthesis of nitrate reductase, which required the presence of nitrate in the media. In both wild-type and mutant 2170 grown on ammonium and transferred to nitrate media, cycloheximide immediately prevented nitrate reductase derepression when added either at the beginning or at different times of induction treatment. Unlike wild-type cells, mutant 2170 was able to take up either nitrate or nitrite simultaneously with ammonium in whose presence nitrate and nitrite reductases were synthesized. However, synthesis of nitrate reductase was progressively inhibited in the mutant cells when the intracellular ammonium levels were raised as a result of an increase in either the external pH or the extracellular ammonium concentrations. The results rule out the existence of maturase-like proteins in Chlamydomonas and indicate that ammonium has a double effect on the regulation of nitrate reductase synthesis: (a) it prevents nitrate reductase mRNA production; and (b) it controls negatively the expression of this mRNA.     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter- or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys¹⁷⁸, whereas a second residue, Cys²⁴⁷, becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.     

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation

The mitochondrial respiratory chain in plants, some protists and many fungi consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). Although AOX has been proposed to play essential roles in nutrient stress tolerance of plants and protists, the effects of sulfur (S) deprivation, on AOX are largely unknown. The unicellular green alga Chlamydomonas reinhardtii reacts to S limitation conditions with the induced expression of many genes. In this work, we demonstrated that exposure of C. reinhardtii to S deprivation results in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, S-deprived C. reinhardtii cells display the enhanced AOX1 capacity. Moreover, nitrate assimilation regulatory protein (NIT2) is involved in the control of the AOX1 gene expression in the absence of S. Together, the results clearly indicate that AOX1 relates to S limitation stress responses and is regulated in a NIT2-dependent manner, probably together with yet-unknown regulatory factor(s).     

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys²²⁷ and Cys³⁶¹. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.     

Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.     

Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii

As we had found previously that thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-kappaB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-kappaB-alpha (IkappaB-alpha) was decreased and the nuclear translocation of NF-kappaB was increased. The thapsigargin-induced activation of NF-kappaB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-kappaB. Lipopolysaccharide (LPS)-induced activation of NF-kappaB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IkappaB-alpha. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-kappaB pathway.     

Regulation of electron-transport pathways in cells of Chlamydomonas reinhardtii under stress conditions

The kinetics of interactions between electron-transport pathways in the thylakoid membrane was examined. A mathematical model was proposed to describe the kinetics of redox transitions in photosystem II, proton concentration changes in the chloroplast stroma, and the plastoquinone pool reduction due to photosynthetic and chlororespiratory pathways. A kinetic mechanism is considered that redirects electron flows between photosynthetic and chlororespiratory pathways in response to the increased NADPH content under mineral deficiency. According to the simulation model, the electron transport flows via different routes are switched over in a stepwise manner. The results of numerical simulations are qualitatively consistent with experimental data for Chlamydomonas reinhardtii cells subjected to mineral deprivation.     

Regulation of cyclic electron flow in Chlamydomonas reinhardtii under fluctuating carbon availability

The chloroplast must rapidly and precisely adjust photosynthetic ATP and NADPH output to meet changing metabolic demands imposed by fluctuating environmental conditions. Cyclic electron flow (CEF) around photosystem I is thought to contribute to this adjustment by providing ATP in excess of that supplied by linear electron low, balancing chloroplast energy budget when relative demand for ATP is high. We assessed the kinetics and energy production of CEF activation in Chlamydomonas reinhardtii under rapid changes of organic and inorganic carbon availability. Comparisons of transient electric field and chlorophyll fluorescence measurements indicated CEF was activated under conditions where ATP demand is expected to be high, consistent with a role in balancing the cellular ATP/NADPH budget under fluctuating environmental or metabolic conditions. CEF activation was not correlated with antenna state transitions, both in wild-type and the state transition mutant stt7-9, suggesting that CEF is rapidly regulated by allosteric or redox modulators. Comparing the CEF under ambient and high CO₂ conditions suggests an increase in required energy output of approximately 1ATP/CO₂ fixed, nearly sufficient to power proposed mechanistic models for the carbon-concentrating mechanism. Additionally, we see three-fold higher CEF rates in cells under steady-state conditions than cells under similar conditions with inhibited photosystem II, and up to five times higher in cells with severe depletion of inorganic carbon, implying that CEF has larger energetic capacity than predicted from some previous work.