The thioredoxin superfamily in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The thioredoxin (TRX) superfamily includes redox proteins such as thioredoxins, glutaredoxins (GRXs) and protein disulfide isomerases (PDI). These proteins share a common structural motif named the thioredoxin fold. They are involved in disulfide oxido-reduction and/or isomerization. The sequencing of the Arabidopsisgenome revealed an unsuspected multiplicity of TRX and GRX genes compared to other organisms. The availability of full Chlamydomonasgenome sequence offers the opportunity to determine whether this multiplicity is specific to higher plant species or common to all photosynthetic eukaryotes. We have previously shown that the multiplicity is more limited in Chlamydomonas for TRX and GRX families. We extend here our analysis to the PDI family. This paper presents a comparative analysis of the TRX, GRX and PDI families present in Arabidopsis,Chlamydomonas and Synechocystis. The putative subcellular localization of each protein and its relative expression level, based on EST data, have been investigated. This analysis provides a large overview of the redox regulatory systems present in Chlamydomonas. The data are discussed in view of recent results suggesting a complex cross-talk between the TRX, GRX and PDI redox regulatory networks.     

Strategies to facilitate transgene expression in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The unicellular green alga Chlamydomonas reinhardtii has been identified as a promising organism for the production of recombinant proteins. While during the last years important improvements have been developed for the production of proteins within the chloroplast, the expression levels of transgenes from the nuclear genome were too low to be of biotechnological importance. In this study, we integrated endogenous intronic sequences into the expression cassette to enhance the expression of transgenes in the nucleus. The insertion of one or more copies of intron sequences from the Chlamydomonas RBCS2 gene resulted in increased expression levels of a Renilla-luciferase gene used as a reporter. Although any of the three RBCS2 introns alone had a positive effect on expression, their integration in their physiological number and order created an over-proportional stimulating effect observed in all transformants. The secretion of the luciferase protein into the medium was achieved by using the export sequence of the Chlamydomonas ARS2 gene in a cell wall deficient strain and Renilla-luciferase could be successfully concentrated with the help of attached C-terminal protein tags. Similarly, a codon adapted gene variant for human erythropoietin (crEpo) was expressed as a protein of commercial relevance. Extracellular erythropoietin produced in Chlamydomonas showed a molecular mass of 33 kDa probably resulting from post-translational modifications. Both, the increased expression levels of transgenes by integration of introns and the isolation of recombinant proteins from the culture medium are important steps towards an extended biotechnological use of this alga. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhancement of photorespiration in immobilized <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Immobilization of Chlamydomonas reinhardtii in alginate increases its photorespiration rate. In the immobilized cells, the photorespiratory enzyme, phosphoglycolate phosphatase, was 75% higher than in freely suspended cells. Thus, the immobilized cells produced glycolate at twice the rate than in freely suspended cells when treated with aminooxyacetate (a transaminase inhibitor). With immobilized cells in a batch reactor, 270mol glycolatemg^–1 Chl was produced after 12h.Revisions requested 27 October 2004; Revisions received 13 December 2004     

Nitrogen deprivation results in photosynthetic hydrogen production in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The unicellular green alga Chlamydomonas reinhardtii is able to use photosynthetically provided electrons for the production of molecular hydrogen by an [FeFe]-hydrogenase HYD1 accepting electrons from ferredoxin PetF. Despite the severe sensitivity of HYD1 towards oxygen, a sustained and relatively high photosynthetic hydrogen evolution capacity is established in C. reinhardtii cultures when deprived of sulfur. One of the major electron sources for proton reduction under this condition is the oxidation of starch and subsequent non-photochemical transfer of electrons to the plastoquinone pool. Here we report on the induction of photosynthetic hydrogen production by Chlamydomonas upon nitrogen starvation, a nutritional condition known to trigger the accumulation of large deposits of starch and lipids in the green alga. Photochemistry of photosystem II initially remained on a higher level in nitrogen-starved cells, resulting in a 2-day delay of the onset of hydrogen production compared with sulfur-deprived cells. Furthermore, though nitrogen-depleted cells accumulated large amounts of starch, both hydrogen yields and the extent of starch degradation were significantly lower than upon sulfur deficiency. Starch breakdown rates in nitrogen or sulfur-starved cultures transferred to darkness were comparable in both nutritional conditions. Methyl viologen treatment of illuminated cells significantly enhanced the efficiency of photosystem II photochemistry in sulfur-depleted cells, but had a minor effect on nitrogen-starved algae. Both the degradation of the cytochrome b ₆ f complex which occurs in C. reinhardtii upon nitrogen starvation and lower ferredoxin amounts might create a bottleneck impeding the conversion of carbohydrate reserves into hydrogen evolution.     

Functional characterization of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with alterations in the <Emphasis Type="Italic">atpE</Emphasis> gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphopantetheinylation in the green microalgae <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Microalgal biofuel is a promising solution to the decline of fossil fuels. However, algal fatty acid metabolism, the machinery producing the raw material for biofuels, remains poorly understood. The central unit of the fatty acid synthase (FAS) is the acyl carrier protein (ACP), which is responsible for holding the product. Fatty acid biosynthesis is initiated through posttranslational modification of the ACP by the phosphopantetheinyl transferase (PPTase). We identified two PPTases, PptC1 and PptC2, in the model alga Chlamydomonas reinhardtii by genome analysis and phylogenetic and structural comparison. Both PPTases are of Sfp-type, the archetypical PPTase type for non-ribosomal peptide and polyketide biosynthetic pathways in bacteria and cyanobacteria. In vitro analysis revealed that PptC2 has a broader substrate range than PptC1. Both PPTases were able to activate the cognate ACP of the type II FAS, while PptC2 also recognized ACP of Escherichia coli type II FAS and actinorhodin type II polyketide synthase. Besides FAS as PPTase target, the C. reinhardtii genome encodes a single type I PKS, and we hypothesize that PptC2 is responsible for its activation. Screening of the currently available microalgal genome data revealed that most green microalgae appear to carry two PPTases forming clusters with each C. reinhardtii PPTase, while microalgae of other divisions carry one or two PPTases and do not cluster in the pattern of the green algal data. This new understanding on the PPTases in microalgae shows that microalgae are already primed for biotechnological applications in contrast to other organisms. Thus, microalgae have great potential for metabolic engineering efforts in the realm of biofuel and high-value products including direct engineering of the fatty acid or secondary metabolism using the natural genomic reservoir and as biotechnological platform for heterologous expression.     

Examination of chlorophyll fluorescence in sulfur-deprived cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F ₀ and F _m) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F ₀ and F _m) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b ₅₅₉.     

Alanine racemase from the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Summary. Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 μmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.     

Development of an alcohol-inducible gene expression system for recombinant protein expression in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Microalgae have been widely considered for the production of valuable products, such as lipid-based biofuel, value-added pigments, and anti-photo aging reagents. More recently, microalgae have been considered an alternative host for recombinant protein production because of their economic benefits and ecofriendly characteristics. Additionally, many microalgal strains identified to date are generally recognized as safe (GRAS); therefore, the use of microalgae-based technology is promising. However, basic studies on the genetic engineering of microalgae are rare, despite their importance. Particularly, inducible promoter systems that can be applied for strain engineering or recombinant protein production are rarely studied; hence, a number of challenging issues remain unsolved. Therefore, in this study, we focused on the development of a convenient and compact-inducible promoter system that can be used in microalgae. Based on previous success with plant systems, we employed the alcohol-inducible AlcR-P_alcA system, which originates from the filamentous fungus, Aspergillus nidulans. This system comprises only two components, a regulatory protein, AlcR, and an inducible promoter, P_alcA. Therefore, construction and transformation of the gene cassettes can be easily performed. Ethanol-dependent gene expression was observed in the transformants with no significant growth retardation or inducer consumption observed in the cells cultivated under optimized conditions.     

Efficient expression of epidermal growth factor in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> CC400

The application of the green alga Chlamydomonas reinhardtii as a bioreactor is not adequate because of the difficulties caused by efficiency expressing foreign genes. To improve this efficiency a plasmid containing the epidermal growth factor (EGF) gene and a bleomycin resistance gene (ble) was constructed. We amplified the EGF gene according to the codon usage of C. reinhardtii. The vector carrying 2 expression cassettes for EGF gene and ble gene was constructed by adding rbc promoter and rbc terminator. Transformants, selected on Tris-acetate-phosphate medium containing 15 mg/L bleomycin, were screened by PCR and confirmed by Southern blotting, which showed that 3 transgenic C. reinhardtii cells contained only one copy of EGF gene integrated in different 3 sites of C. reinhardtii CC400 genome. Then EGF protein content of 3 transformants was determined by EGF precoated ELISA, indicating that EGF gene was first expressed, although at a low level, in algal cells. The presented study, as an example for expressing heterologous gene in green alga, provided feasibility to improve the efficiency of transformation of C. reinhardtii.     

Effect of dibromothymoquinone on Chlorophyll <Emphasis Type="Italic">a</Emphasis> Fluorescence in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells incubated in complete or sulfur-depleted medium

The effect of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. Dibromothymoquinone was shown to affect differently control cells incubated in complete medium and S-starved cells. The fluorescence yield in the control suspension considerably increased in the presence of the inhibitor. Presumably, this can be due to inactivation of protein kinase, as a result of which part of light-harvesting complex II that could have diffused from the stacking zone of the membrane into the lamellar zone towards photosystem I remains close to photosystem II. In S-starved cells, whose photosynthetic apparatus is in state 2, the fluorescence level declines in the presence of dibromothymoquinone. The JIP testing of induction curves (O-J-I-P fluorescence transient) suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to the control, while in the starved cells it acts predominantly as an electron acceptor.     

Whole cell solid-state NMR study of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> microalgae

In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically ¹³C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents. The number of resonances detected with these robust and broadly applicable experiments appears to be surprisingly sparse. Various constituents, notably galactolipids abundant in organelle membranes, carbohydrates from the cell wall, and starch from storage grains could be unambiguously assigned. Moreover, the dominant crystal form of starch could be determined in situ. This work illustrates the feasibility and caveats of using solid-state NMR to study intact non-specifically ¹³C labelled micro-organisms.     

Chemotactic Behavior of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Is Altered During Gametogenesis

Chemotactic behavior of Chlamydomonas reinhardtii is altered during the sexual life cycle. Unlike vegetative cells and noncompetent pregametes, mature gametes did not show chemotaxis to ammonium. Loss of chemotaxis to ammonium in mating-competent cells is controlled by gamete-specific genes that are common for both mating-type gametes. Change of chemotaxis mode requires the sequential action of the two environmental signals: removal of ammonium from the medium and light. The mutants lrg1, lrg3, and lrg4 affected in the light-dependent step of sexual differentiation exhibited the loss of chemotaxis to ammonium in the absence of light. These data indicate that there are common components in the signaling pathways that control change of chemotactic behavior and forming of mating competence in gametes. Received: 14 May 2002 / Accepted: 21 June 2002     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Analysis of Chromatin Structure in the Control Regions of the <Emphasis Type="Italic">Chlamydomonas HSP70A</Emphasis> and <Emphasis Type="Italic">RBCS2</Emphasis> Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of ~700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.