Mechanism and dynamics of fatty acid photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-ß.

Our recent study showed that human mesenchymal stem/stromal cells (hMSCs) are activated to express tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) by exposure to TNF-α and these activated hMSCs effectively induce apoptosis in triple-negative breast cancer MDA-MB-231 (MDA) cells in vitro and in vivo. Here, we further demonstrated that activated hMSCs not only induced apoptosis of MDA cells but also reduced metastatic features in MDA cells. These activated hMSC-exposed MDA cells showed reduced tumorigenicity and suppressed formation of lung metastasis when implanted in the mammary fat pad. Surprisingly, the activated hMSC-exposed MDA cells increased TRAIL expression, resulting in apoptosis in MDA cells. Interestingly, upregulation of TRAIL in MDA cells was mediated by interferon-beta (IFN-ß) secreted from activated hMSCs. Furthermore, IFN-ß in activated hMSCs was induced by RNA and DNA released from apoptotic MDA cells in absent in melanoma 2 (AIM2) and IFN induced with helicase C domain 1 (IFIH1)-dependent manners. These observations were only seen in the TRAIL-sensitive breast cancer cell lines but not in the TRAIL-resistant breast cancer cell lines. Consistent with these results, Kaplan-Meier survival analysis also showed that lack of innate sensors detecting DNA or RNA is strongly associated with poor survival in estrogen receptor-negative breast cancer patients. In addition, cancer-associated fibroblasts (CAF) isolated from a breast cancer patient were also able to express TRAIL and IFN-ß upon DNA and RNA stimulation. Therefore, our results suggest that the crosstalk between TRAIL-sensitive cancer cells and stromal cells creates a tumor-suppressive microenvironment and further provide a novel therapeutic approach to target stromal cells within cancer microenvironment for TRAIL sensitive cancer treatment.

Lipidomic and transcriptomic analyses of Chlamydomonas reinhardtii under heat stress unveil a direct route for the conversion of membrane lipids into storage lipids.

Studying how photosynthetic cells modify membrane lipids in response to heat stress is important to understand how plants and microalgae adapt to daily fluctuations in temperature and to investigate new lipid pathways. Here, we investigate changes occurring in lipid molecular species and lipid metabolism genes during early response to heat stress in the model photosynthetic microorganism Chlamydomonas reinhardtii. Lipid molecular species analyses revealed that, after 60 min at 42 °C, a strong decrease in specific polyunsaturated membrane lipids was observed together with an increase in polyunsaturated triacylglycerols (TAGs) and diacylglycerols (DAGs). The fact that decrease in the major chloroplastic monogalactosyldiacylglycerol sn1-18:3/sn2-16:4 was mirrored by an accumulation of DAG sn1-18:3/sn2-16:4 and TAG sn1-18:3/sn2-16:4/sn3-18:3 indicated that newly accumulated TAGs were formed via direct conversion of monogalactosyldiacylglycerols to DAGs then TAGs. Lipidomic analyses showed that the third fatty acid of a TAG likely originated from a phosphatidylethanolamine or a diacylglyceryl-O-4'-(N,N,N,-trimethyl)-homoserine betaine lipid species. Candidate genes for this TAG synthesis pathway were provided through comparative transcriptomic analysis and included a phospholipase A2 homolog and the DAG acyltransferase DGTT1. This study gives insights into the molecular events underlying changes in membrane lipids during heat stress and reveals an alternative route for TAG synthesis.

Development and validation of a screening procedure of microalgae for biodiesel production: application to the genus of marine microalgae Nannochloropsis.

Nannochloropsis has emerged as a promising alga for biodiesel production. However, the genus consists of 6 species and hundreds of strains making strain selection a challenge. Furthermore, oil productivity is instrumental to economic viability of any algal strain for industrial production, which is dependent on growth rate and oil content. In most cases, these two parameters have been studied independently. Thus, the goal of this study is to provide a combined method for evaluating strain performance in specially designed photobioreactors together with an in-depth lipidomic analyses. The nine strains of Nannochloropsis tested showed considerable variations in productivity and lipidomics highlighting the importance of strain selection. Finally, Nannochloropsis gaditana CCMP527 and Nannochloropsis salina CCMP537 emerged as the two most promising strains, with an oil content of 37 and 27 dry wt% after 11-day nitrogen starvation, respectively, resulting in TAG productivity of 13×10(-3) and 18×10(-3) kg m(-3) d(-1), respectively.

Comparison of various microalgae liquid biofuel production pathways based on energetic, economic and environmental criteria.

In view of the increasing demand for bioenergy, in this study, the techno-economic viabilities for three emerging pathways to microalgal biofuel production have been evaluated. The three processes evaluated are the hydrothermal liquefaction (HTL), oil secretion and alkane secretion. These three routes differ in their lipid extraction procedure and the end-products produced. This analysis showed that these three processes showed various advantages: possibility to convert the defatted microalgae into bio-crude via HTL thus increasing the total biodiesel yield; better energetic and environmental performance for oil secretion and an even increased net energy ratio (NER) for alkane secretion. However, great technological breakthroughs are needed before planning any scale-up strategy such as continuous wet biomass processing and heat exchange optimization for the HTL pathway and effective and sustainable excretion for both secretion pathways.

Investigation of fatty acids accumulation in Nannochloropsis oculata for biodiesel application.

Lipids production of the marine microalga species Nannochloropsis oculata was deeply investigated by studying under continuous light the effects of different nitrogen starvation strategies in photobioreactors of various thicknesses. Operating parameters like incident photons flux density (PFD), initial nitrogen (progressive starvation strategy) or biomass concentrations (sudden starvation strategy) were examined, with a detailed analysis of their effects on the quality and production kinetics of total (TL) and triglycerides (TG). In addition to the already known effect of nitrogen starvation to trigger reserve lipids accumulation (mainly TG), it was demonstrated the relevance of the light received per cell affecting TG content and productivities, as well as fatty acids (FA) profiles. With appropriate optimization, N. oculata was confirmed as an interesting candidate for biodiesel application, with high FA accumulation (up to around 50%DW with 43%DW in TG-FA), high productivity (maximum 3.6×10(-3)kg(TG-FA)m(-2)d(-1)) and a TG-FA profile close to palm oil.

An economic, sustainability, and energetic model of biodiesel production from microalgae.

A new process evaluation methodology of microalgae biodiesel has been developed. Based on four evaluation criteria, i.e. the net energy ratio (NER), biodiesel production costs, greenhouse gases (GHG) emission rate and water footprint, the model compares various technologies for each step of the process, from cultivation to oil upgrading. An innovative pathway (hybrid raceway/PBR cultivation system, belt filter press for dewatering, wet lipid extraction, oil hydrotreating and anaerobic digestion of residues) shows good results in comparison to a reference pathway (doubled NER, lower GHG emission rate and water footprint). The production costs are still unfavourable (between 1.94 and 3.35 €/L of biodiesel). The most influential parameters have been targeted through a global sensitivity analysis and classified: (i) lipid productivity, (ii) the cultivation step, and (iii) the downstream processes. The use of low-carbon energy sources is required to achieve significant reductions of the biodiesel GHG emission rate compared to petroleum diesel.

Modification of substrate specificity in single point mutants of Agrobacterium tumefaciens type II NADH dehydrogenase.

Type II NADH dehydrogenases (NDH-2) are monomeric flavoenzymes catalyzing electron transfer from NADH to quinones. While most NDH-2 preferentially oxidize NADH, some of these enzymes have been reported to efficiently oxidize NADPH. With the aim to modify the NADPH vs NADH specificity of the relatively NADH specific Agrobacterium tumefaciens NDH-2, two conserved residues (E and A) of the substrate binding domain were, respectively, mutated to Q and S. We show that when E was replaced by Q at position 203 the enzyme was able to oxidize NADPH as efficiently as NADH. Growth on a minimal medium of an Escherichia coli double mutant lacking both NDH-1 and NDH-2 was restored more efficiently when mutated proteins able to oxidize NADPH were expressed. The biotechnological interest of expressing such modified enzymes in photosynthetic organisms is discussed.

Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation.

The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

Generation and analysis of soybean plastid transformants expressing Bacillus thuringiensis Cry1Ab protoxin.

We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis).

Contact palm burns in toddlers from glass enclosed fireplaces.

We have seen an alarming increase in the incidence of pediatric palm burns associated with gas fireplaces. The increasing popularity of these units places more children at risk. Medical records of patients under the age of 5 years who sustained hand burns from contact with the glass enclosure of gas fireplaces from 1996 through 2002 were reviewed. Thirty-nine patients were identified, with a mean age of 12.8 months. A 15-fold increase in incidence was observed. Thirty-three patients suffered superficial second-degree burns that were treated conservatively. Twenty-one percent of children developed significant wound complications requiring intensive therapy including extension splinting or surgery. Pediatric burns resulting from palmar contact with the glass enclosures of gas fireplaces have emerged as an avoidable new danger within the home. Although most of these injuries heal with conservative treatment alone, many require surgery or other intensive management to regain acceptable function.

Flocculent activity of a recombinant protein from Moringa oleifera Lam. seeds.

Seeds of the tropical tree Moringa oleifera contain small storage proteins able to flocculate particles in suspension in water. The cDNA encoding one of these flocculent proteins, MO(2.1), was cloned and the recombinant protein was expressed in Escherichia coli. The flocculent activity of the purified recombinant MO(2.1)was assayed on clays and bacteria using light and confocal microscopy and GFP-overexpressing bacteria. We show that MO(2.1)is able to aggregate montmorillonite clay particles as well as gram-positive and gram-negative bacteria. We discuss the use of recombinant proteins to study flocculating properties and improve water purification processes.

Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-dehydrogenase complex to cyclic electron flow around photosystem I.

Tobacco (Nicotiana tabacum var Petit Havana) ndhB-inactivated mutants (ndhB-) obtained by plastid transformation (E.M. Horvath, S.O. Peter, T. Joët, D. Rumeau, L. Cournac, G.V. Horvath, T.A. Kavanagh, C. Schäfer, G. Peltier, P. MedgyesyHorvath [2000] Plant Physiol 123: 1337-1350) were used to study the role of the NADH-dehydrogenase complex (NDH) during photosynthesis and particularly the involvement of this complex in cyclic electron flow around photosystem I (PSI). Photosynthetic activity was determined on leaf discs by measuring CO2 exchange and chlorophyll fluorescence quenchings during a dark-to-light transition. In the absence of treatment, both non-photochemical and photochemical fluorescence quenchings were similar in ndhB- and wild type (WT). When leaf discs were treated with 5 microM antimycin A, an inhibitor of cyclic electron flow around PSI, both quenchings were strongly affected. At steady state, maximum photosynthetic electron transport activity was inhibited by 20% in WT and by 50% in ndhB-. Under non-photorespiratory conditions (2% O2, 2,500 microL x L(-1) CO2), antimycin A had no effect on photosynthetic activity of WT, whereas a 30% inhibition was observed both on quantum yield of photosynthesis assayed by chlorophyll fluorescence and on CO2 assimilation in ndhB-. The effect of antimycin A on ndhB- could not be mimicked by myxothiazol, an inhibitor of the mitochondrial cytochrome bc1 complex, therefore showing that it is not related to an inhibition of the mitochondrial electron transport chain but rather to an inhibition of cyclic electron flow around PSI. We conclude to the existence of two different pathways of cyclic electron flow operating around PSI in higher plant chloroplasts. One of these pathways, sensitive to antimycin A, probably involves ferredoxin plastoquinone reductase, whereas the other involves the NDH complex. The absence of visible phenotype in ndhB- plants under normal conditions is explained by the complement of these two pathways in the supply of extra-ATP for photosynthesis.

Flexibility in photosynthetic electron transport: a newly identified chloroplast oxidase involved in chlororespiration.

Besides electron transfer reactions involved in the 'Z' scheme of photosynthesis, alternative electron transfer pathways have been characterized in chloroplasts. These include cyclic electron flow around photosystem I (PS I) or a respiratory chain called chlororespiration. Recent work has supplied new information concerning the molecular nature of the electron carriers involved in the non-photochemical reduction of the plastoquinone (PQ) pool. However, until now little is known concerning the nature of the electron carriers involved in PQ oxidation. By using mass spectrometric measurement of oxygen exchange performed in the presence of 18O-enriched O2 and Chlamydomonas mutants deficient in PS I, we show that electrons can be directed to a quinol oxidase sensitive to propyl gallate but insensitive to salicyl hydroxamic acid. This oxidase has immunological and pharmacological similarities with a plastid protein involved in carotenoid biosynthesis.

Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure.

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO(2) fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO(2) concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO(2) limitation.

Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration.

In Chlamydomonas reinhardtii mutants deficient in photosystem I because of inactivation of the chloroplast genes psaA or psaB, oxygen evolution from photosystem II occurs at significant rates and is coupled to a stimulation of oxygen uptake. Both activities can be simultaneously monitored by continuous mass spectrometry in the presence of (18)O(2). The light-driven O(2) exchange was shown to involve the plastoquinone pool as an electron carrier, but not cytochrome b(6)f. Photosystem II-dependent O(2) production and O(2) uptake were observed in isolated chloroplast fractions. Photosystem II-dependent oxygen exchange was insensitive to a variety of inhibitors (azide, carbon monoxide, cyanide, antimycin A, and salicylhydroxamic acid) and radical scavengers. It was, however, sensitive to propyl gallate. From inhibitors effects and electronic requirements of the O(2) uptake process, we conclude that an oxidase catalyzing oxidation of plastoquinol and reduction of oxygen to water is present in thylakoid membranes. From the sensitivity of flash-induced O(2) exchange to propyl gallate, we conclude that this oxidase is involved in chlororespiration. Clues to the identity of the protein implied in this process are given by pharmacological and immunological similarities with a protein (IMMUTANS) identified in Arabidopsis chloroplasts.

Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress.

Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints.

Involvement of CDSP 32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants.

In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress. We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought-induced stress protein of 32 kDa. In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying). Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments. These data show for the first time in plants the induction of a thioredoxin by oxidative stress. We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought.

The gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of synechocystis PCC6803.

The physiological function of the type 1 NAD(P)H dehydrogenase (Ndh-1) of Synechocystis sp. PCC6803 has been investigated by inactivating the gene ndhH encoding a subunit of the complex. Molecular analysis of independent transformants revealed that all clones were heteroploid, containing both wild-type and mutant ndhH copies, whatever the metabolic conditions used during genome segregation, including high CO(2) concentration. By replacing the chromosomal copy of the ndhH gene by a plasmidial copy under the control of a temperature-controlled promoter, we induce a conditional phenotype, growth being only possible at high temperature. This clearly shows for the first time that an ndh gene is indispensable to the survival of Synechocystis sp. PCC6803.

Photosystem I is indispensable for photoautotrophic growth, CO2 fixation, and H2 photoproduction in Chlamydomonas reinhardtii.

Certain Chlamydomonas reinhardtii mutants deficient in photosystem I due to defects in psaA mRNA maturation have been reported to be capable of CO2 fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L. , and Mets, L. J. (1995) Nature 376, 438-441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364-367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of either psaA or psaC; these activities could be restored to the F8-psaADelta mutant by reintroduction of psaA. We observed limited O2 photoevolution in mutants lacking photosystem I; use of 18O2 indicated that this O2 evolution is coupled to O2 uptake (i.e. respiration) rather than CO2 fixation or H2 evolution. We conclude that the reported instances of CO2 fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I.