BackgroundWhen cultivated under stress conditions, many microalgae species accumulate both starch and oil (triacylglycerols). The model green microalga Chlamydomonas reinhardtii has recently emerged as a model to test genetic engineering or cultivation strategies aiming at increasing lipid yields for biodiesel production. Blocking starch synthesis has been suggested as a way to boost oil accumulation. Here, we characterize the triacylglycerol (TAG) accumulation process in Chlamydomonas and quantify TAGs in various wild-type and starchless strains.ResultsIn response to nitrogen deficiency, Chlamydomonas reinhardtii produced TAGs enriched in palmitic, oleic and linoleic acids that accumulated in oil-bodies. Oil synthesis was maximal between 2 and 3 days following nitrogen depletion and reached a plateau around day 5. In the first 48 hours of oil deposition, a ~80% reduction in the major plastidial membrane lipids occurred. Upon nitrogen re-supply, mobilization of TAGs started after starch degradation but was completed within 24 hours. Comparison of oil content in five common laboratory strains (CC124, CC125, cw15, CC1690 and 11-32A) revealed a high variability, from 2 μg TAG per million cell in CC124 to 11 μg in 11-32A. Quantification of TAGs on a cell basis in three mutants affected in starch synthesis (cw15sta1-2, cw15sta6 and cw15sta7-1) showed that blocking starch synthesis did not result in TAG over-accumulation compared to their direct progenitor, the arginine auxotroph strain 330. Moreover, no significant correlation was found between cellular oil and starch levels among the twenty wild-type, mutants and complemented strains tested. By contrast, cellular oil content was found to increase steeply with salt concentration in the growth medium. At 100 mM NaCl, oil level similar to nitrogen depletion conditions could be reached in CC124 strain.ConclusionA reference basis for future genetic studies of oil metabolism in Chlamydomonas is provided. Results highlight the importance of using direct progenitors as control strains when assessing the effect of mutations on oil content. They also suggest the existence in Chlamydomonas of complex interplays between oil synthesis, genetic background and stress conditions. Optimization of such interactions is an alternative to targeted metabolic engineering strategies in the search for high oil yields.

Brassica napus L. (oilseed rape) was grown from seeds on a reconstituted soil contaminated with cadmium (100 mg Cd kg-1 dry soil), resulting in a marked chlorosis of the leaves which was investigated using a combination of biochemical, biophysical and physiological methods. Spectroscopic and chromatographic analyses of the photosynthetic pigments indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthesis pathway. In addition, mineral deficiency and oxidative stress were apparently not involved in the pigment loss. Leaf chlorosis was attributable to a marked decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size, suggesting that Cd interfered with chloroplast replication and cell division. Relatively little Cd was found in the chloroplasts and the properties of the photosynthetic apparatus (electron transport, protein composition, chlorophyll antenna size, chloroplast ultrastructure) were not affected appreciably in plants grown on Cd-polluted soil. Depth profiling of photosynthetic pigments by phase-resolved photoacoustic spectroscopy revealed that the Cd-induced decrease in pigment content was very pronounced at the leaf surface (stomatal guard cells) compared to the leaf interior (mesophyll). This observation was consistent with light transmission and fluorescence microscopy analyses, which revealed that stomata density in the epidermis was noticeably reduced in Cd-exposed leaves. Concomitantly, the stomatal conductance estimated from gas-exchange measurements was strongly reduced with Cd. When plants were grown in a high-CO2 atmosphere (4,000 microliters CO2 l-1), the inhibitory effect of Cd on growth was not cancelled, suggesting that the reduced availability of CO2 at the chloroplast level associated with the low stomatal conductance was not the main component of Cd toxicity in oilseed rape.

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr 2؉ instead of Ca 2؉ with the aim of biosynthetically replacing the Ca 2؉ of the oxygen-evolving enzyme with Sr 2؉. Not only were the cells able to grow normally with Sr 2؉ , they actively accumulated the ion to levels higher than those of Ca 2؉ in the normal cultures. A protocol was developed to purify a fully active Sr The evolution of oxygen as a result of light-driven water oxidation is catalyzed by photosystem II (PSII) 1 in which a cluster of 4 manganese ions acts both as a device for accumulating oxidizing equivalents and as the active site. The reaction center of PSII is made up of two membrane-spanning polypeptides (D1 and D2) that bear the redox cofactors involved in the main electron transfer route. Absorption of a photon results in a charge separation between a chlorophyll molecule (P 680 ), and a pheophytin molecule. The pheophytin anion transfers the electron to a quinone, Q A , and P 680 ϩ is reduced by a tyrosine residue, Tyr Z , that in turn is reduced by the Mn 4 cluster. During the enzyme cycle, the oxidizing side of PSII goes through five different redox states that are denoted S n , n varying from 0 to 4. Oxygen is released during the S 3 to S 0 transition in which S 4 is a transient state (reviewed in Refs.

During oxygenic photosynthesis, metabolic reactions of CO 2 fixation require more ATP than is supplied by the linear electron flow operating from photosystem II to photosystem I (PSI). Different mechanisms, such as cyclic electron flow (CEF) around PSI, have been proposed to participate in reequilibrating the ATP/NADPH balance. To determine the contribution of CEF to microalgal biomass productivity, here, we studied photosynthesis and growth performances of a knockout Chlamydomonas reinhardtii mutant (pgrl1) deficient in PROTON GRADIENT REGULATION LIKE1 (PGRL1)-mediated CEF. Steady state biomass productivity of the pgrl1 mutant, measured in photobioreactors operated as turbidostats, was similar to its wild-type progenitor under a wide range of illumination and CO 2 concentrations. Several changes were observed in pgrl1, including higher sensitivity of photosynthesis to mitochondrial inhibitors, increased light-dependent O 2 uptake, and increased amounts of flavodiiron (FLV) proteins. We conclude that a combination of mitochondrial cooperation and oxygen photoreduction downstream of PSI (Mehler reactions) supplies extra ATP for photosynthesis in the pgrl1 mutant, resulting in normal biomass productivity under steady state conditions. The lower biomass productivity observed in the pgrl1 mutant in fluctuating light is attributed to an inability of compensation mechanisms to respond to a rapid increase in ATP demand.

The organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same qnon-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology.

The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.

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Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20-and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.Industrial and agricultural activities have led to substantial release of toxic heavy metals in the environment, which can constitute a major hazard for ecosystems and human health (16). Bioremediation, using bacteria or plants, is often regarded as a relatively inexpensive and efficient way of cleaning up wastes, sediments, or soils contaminated with toxic heavy metals (14). A promising way of improving bioremediation processes is to genetically engineer bacterial strains to confer increased abilities to accumulate toxic heavy metals. Attempts to enhance the metal content of bacterial cells have been made by overexpressing metal-binding peptides or proteins such as poly-histidines (23) or metallothioneins (14, 24).When exposed to toxic heavy metals, plant cells and yeasts accumulate phytochelatins (PCs), which are n repeats (n ϭ 2 to 5) of the ␥-Glu-Cys dipeptide terminated by a Gly residue. PCs are enzymatically synthesized from glutathione (GSH) by PC synthase (EC 2.3.2.15) (7) and bind heavy metals such as cadmium, silver, copper, or arsenite by forming complexes (13, 21). PC synthase genes were initially cloned from wheat, Arabidopsis thaliana and Schizosaccharomyces pombe (4, 9, 27) and recently a gene encoding a functional PC synthase was found in the nematode Caenorhabditis elegans (5, 26). PCs are clearly involved in heavy metal detoxification. Indeed, plant and yeast mutants deficient in PC synthase are hypersensitive to cadmium (9), and expression of PC synthase genes in yeast confers augmented cadmium tolerance (4,26,27).In an effort to increase the heavy metal content of bacterial cells, we expressed the A. thaliana PC synthase gene (AtPCS) in Escherichia coli. We show that expression of AtPCS allows PC synthesis resulting in a large increase in intracellular metal content when bacterial cells are grown on metal-enriched media. These results pave the way for using enzymes of the PC biosynthetic pathway to enhance the metal content of bacteria and plants and optimize bioremediation and/or phytoremediation processes of toxic metals. MATERIALS AND METHODSBacterial strains and growth conditions...