Hydrogen Production in Chlamydomonas: Photosystem II-Dependent and -Independent Pathways Differ in Their Requirement for Starch Metabolism

Under sulfur deprivation conditions, the green alga Chlamydomonas reinhardtii produces hydrogen in the light in a sustainable manner thanks to the contribution of two pathways, direct and indirect. In the direct pathway, photosystem II (PSII) supplies electrons to hydrogenase through the photosynthetic electron transport chain, while in the indirect pathway, hydrogen is produced in the absence of PSII through a photosystem I-dependent process. Starch metabolism has been proposed to contribute to both pathways by feeding respiration and maintaining anoxia during the direct pathway and by supplying reductants to the plastoquinone pool during the indirect pathway. At variance with this scheme, we report that a mutant lacking starch (defective for sta6) produces similar hydrogen amounts as the parental strain in conditions of sulfur deprivation. However, when PSII is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, conditions where hydrogen is produced by the indirect pathway, hydrogen production is strongly reduced in the starch-deficient mutant. We conclude that starch breakdown contributes to the indirect pathway by feeding electrons to the plastoquinone pool but is dispensable for operation of the direct pathway that prevails in the absence of DCMU. While hydrogenase induction was strongly impaired in the starch-deficient mutant under dark anaerobic conditions, wild-type-like induction was observed in the light. Because this light-driven hydrogenase induction is DCMU insensitive and strongly inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, we conclude that this process is regulated by the proton gradient generated by cyclic electron flow around PSI.In the context of economical and environmental concerns around fossil fuel depletion and global warming, the interest in hydrogen as an energy carrier for the future has considerably grown. Because molecular hydrogen is scarce on our planet, the development of a hydrogen economy strongly depends on our ability to propose clean and sustainable technologies of hydrogen production. In this context, the ability of some photosynthetic microorganisms, and particularly cyanobacteria and microalgae, to convert solar energy into hydrogen has been considered as very promising (Ghirardi et al., 2000; Rupprecht et al., 2006). When cells of the unicellular green alga Chlamydomonas reinhardtii are illuminated after adaptation to anaerobic conditions, electrons originating from water splitting at PSII are driven by the photosynthetic electron transport chain to ferredoxin and to a reversible iron hydrogenase, thereby enabling the production of molecular hydrogen from water and solar energy. Because both hydrogenase activity and expression are highly sensitive to the presence of O₂ (Happe et al., 1994; Ghirardi et al., 1997; Happe and Kaminski, 2002) and because O₂ is produced at PSII, hydrogen photoproduction stops after a few minutes of illumination. Melis et al. (2000) proposed an experimental protocol based on sulfur (S) deprivation, allowing long-term hydrogen production. This protocol relies on a two-stage process: during a first stage, oxygenic photosynthesis drives production of biomass and carbohydrate stores, and during a second anaerobic stage, the hydrogenase is induced and hydrogen is produced. Sulfur starvation has two important effects regarding hydrogen production: (1) a massive accumulation of starch that defines a common response to nutrient starvation and (2) a gradual drop in PSII activity (Wykoff et al., 1998). Once the rate of photosynthetic O₂ evolution drops below the rate of respiration, anaerobic conditions are reached, enabling the induction of hydrogenase and the production of significant amounts of hydrogen for several days. In parallel to hydrogen production, starch is degraded (Melis et al., 2000; Melis, 2007).The importance of starch fermentation in hydrogen production has been recognized early from the pioneering work of Gibbs and coworkers (Gfeller and Gibbs, 1984; Gibbs et al., 1986). Based on the observation that starchless C. reinhardtii mutants sta6 and sta7 are strongly affected in their ability to produce hydrogen, Posewitz et al. (2004) proposed that starch metabolism plays a central role in C. reinhardtii hydrogen production. Actually, two different pathways can supply reductants (i.e. reduced ferredoxin) for hydrogen production in the light, a direct pathway involving PSII and an indirect PSII-independent pathway that relies on a nonphotochemical reduction of plastoquinones (PQs; Fouchard et al., 2005; Melis, 2007). Starch catabolism was proposed to play a role in both pathways (Melis, 2007) by (1) sustaining mitochondrial respiration and allowing the maintenance of anaerobic conditions for the PSII-dependent direct pathway and (2) by supplying electrons to the chlororespiratory pathway and to the hydrogenase through a PSI-dependent process during the indirect pathway (Fouchard et al., 2005; Mus et al., 2005; Melis, 2007). Such a dual role of starch was first confirmed by the study of a Rubisco-deficient mutant (CC2653), unable to accumulate starch and to produce hydrogen in conditions of S deprivation (White and Melis, 2006), but was recently challenged by the study of another Rubisco-less mutant (CC2803), which was reported to produce significant amounts of hydrogen in S starvation conditions, although not accumulating starch (Hemschemeier et al., 2008). These conflicting results obtained on two different Rubisco-deficient mutants prompted us to reexamine the contribution of starch to both direct and indirect pathways of hydrogen production. For this purpose, we complemented the initial work of Posewitz et al. (2004) by revisiting the ability of C. reinhardtii mutants deficient in starch metabolism to produce hydrogen. We thus tested the ability to produce hydrogen in a starchless strain carrying defect in the structural gene encoding the small subunit of ADP-Glc pyrophosphorylase (AGPase; sta6; Zabawinski et al., 2001). We found that sta6 mutant produces significant hydrogen amounts in condition of S deprivation but shows a strongly reduced PSII-independent hydrogen production. We conclude that while the PSII-independent hydrogen production pathway strictly relies on starch catabolism, the PSII-dependent pathway may require either starch or acetate as a respiratory substrate to maintain anaerobiosis.     

Evaluation of seven promoters to achieve germline directed Cre-<Emphasis Type="Italic">lox</Emphasis> recombination in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Site-specific recombination systems, such as Cre-lox from bacteriophage P1, have become very important tools for plant genome engineering. In many cases a constitutive promoter is used to express the recombinase gene. However, for certain research and commercial applications constitutive Cre-mediated recombination may not be desirable. We have evaluated the potential of seven different germline promoter:cre fusions to remove a stably integrated lox cassette through Cre-mediated recombination in Arabidopsis thaliana. We monitored the functionality of each promoter in the germline of primary transformants by analyzing the presence of the recombined lox cassette in T₂ progeny. The selected germline promoters are involved in different developmental cues, including early stem cell identity (CLAVATA3), flower meristem identity (LEAFY, APETALA1), floral organ identity (AGAMOUS), and meiosis (SOLO DANCERS, DMC1, SWITCH1). For five out of these seven promoters we were able to show that efficient Cre-mediated recombination does, indeed, occur and that the recombination takes place at some point during germline development. Furthermore, a recombination efficiency of 100% is obtained when Cre-expression is regulated by the CLAVATA3 promoter. In addition, with these promoters, we observe much less variation in recombination frequency than previously reported for the 35S promoter. For these reasons, we believe that germline-specific Cre-lox recombination provides an additional tool to the site-specific recombination technology in plants.     

Synthesis of 3-fluoro-6-S-(2-S-pyridyl) nucleosides as potential lead cytostatic agents

The 3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-β-d-glucopyranosyl nucleoside analogs 7 were prepared via two facile synthetic routes. Their precursors, 3-fluoro-6-thio-glucopyranosyl nucleosides 5a-e, were obtained by the sequence of deacetylation of 3-deoxy-3-fluoro-β-d-glucopyranosyl nucleosides 2a-e, selective tosylation of the primary OH of 3 and finally treatment with potassium thioacetate. The desired thiolpyridine protected analogs 7a-c,f,g were obtained by the sequence of deacetylation of 5a-c followed by thiopyridinylation and/or condensation of the corresponding heterocyclic bases with the newly synthesized peracetylated 6-S-(2-S-pyridyl) sugar precursor 13, which was obtained via a novel synthetic route from glycosyl donor 12. None of the compounds 6 and 7 showed antiviral activity, but the 5-fluorouracil derivative 7c and particularly the uracil derivative 7b were endowed with an interesting and selective cytostatic action against a variety of murine and human tumor cell cultures.     

Evaluating the specificity of antisense oligonucleotide conjugates. A DNA array analysis

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.     

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon α and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells

 Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon γ (IFNγ), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3⁺CD56⁺ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNα but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis^y antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. Received: 9 December 1999 / Accepted: 18 May 2000