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.

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.

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.

Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots.

Transcriptional regulation in response to cadmium treatment was investigated in both roots and leaves of Arabidopsis, using the whole genome CATMA microarray containing at least 24,576 independent probe sets. Arabidopsis plants were hydroponically treated with low (5 microM) or high (50 microM) cadmium concentrations during 2, 6, and 30 hours. At each time point, Cd level was determined using ICP-AES showing that both plant tissues are able to accumulate the heavy metal. RT-PCR of eight randomly selected genes confirmed the reliability of our microarray results. Analyses of response profiles demonstrate the existence of a regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to cadmium. One of the main response observed in roots was the induction of genes involved in sulfur assimilation-reduction and glutathione (GSH) metabolism. In addition, HPLC analysis of GSH and phytochelatin (PC) content shows a transient decrease of GSH after 2 and 6 h of metal treatment in roots correlated with an increase of PC contents. Altogether, our results suggest that to cope with cadmium, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of GSH for PC biosynthesis. Interestingly, in leaves an early induction of several genes encoding enzymes involved in the biosynthesis of phenylpropanoids was observed. Finally, our results provide new insights to understand the molecular mechanisms involved in transcriptional regulation in response to cadmium exposure in plants.

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.

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.

Accumulation of plastid lipid-associated proteins (fibrillin/CDSP34) upon oxidative stress, ageing and biotic stress in Solanaceae and in response to drought in other species.

Plastid lipid-associated proteins, also termed fibrillin/CDSP34 proteins, are known to accumulate in fibrillar-type chromoplasts such as those of ripening pepper fruit, and in leaf chloroplasts from Solanaceae plants under abiotic stress conditions. It is shown here that treatments generating active oxygen species (high light combined with low temperature, gamma irradiation or methyl viologen treatment) result in potato CDSP34 gene induction and protein accumulation in leaves. Using transgenic tomato plants containing the pepper fibrillin promoter, a significant increase in promoter activity in leaves subjected to biotic stress, namely bacterial infections, was observed. In WT, a higher level of the endogenous fibrillin/CDSP34 protein is also observed after infection by E. chrysanthemi strain 3739. In addition to stress-related induction, a progressive increase in the fibrillin promoter activity is noticed during ageing in various tomato photosynthetic tissues and this increase correlates with a higher abundance of the endogenous protein in WT leaves. It is proposed that a mechanism related to oxidative events plays an essential role in the regulation of fibrillin/CDSP34 genes during stress and also during development. Using a biolistic transient expression assay, the pepper fibrillin promoter is found to be active in various dicot species, but not in monocots. Further, substantially increased levels of fibrillin/ CDSP34 proteins are shown in various dicotyledonous and monocotyledonous plants in response to water deficit.

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.

Evidence for an association of ndh B, ndh J gene products and ferredoxin-NADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase complex.

Using non-denaturing gel electrophoresis and staining with nitro-blue tetrazolium, we reveal the presence of two NAD(P)H oxidoreductase activity bands within thylakoids membranes of Solanum tuberosum L. Second dimension SDS-PAGE and Western analysis show that one of the activity bands contains several polypeptides, two of them being recognized by antibodies directed against peptides corresponding to conserved domains of chloroplastic genes products NDH B and NDH J (at 32 and 18 kDa, respectively). Both activity bands also contain a polypeptide (around 36 kDa) recognized by an antibody directed against ferredoxin-NADP(+)-reductase (FNR). We conclude from these results that both chloroplastic ndh B and ndh J gene products are components of a thylakoid NAD(P)H dehydrogenase complex. The association with FNR is suggested to allow the complex to use NADPH instead of NADH as a preferential substrate.

Characterization of a novel drought-induced 34-kDa protein located in the thylakoids of Solanum tuberosum L. plants.

Using two-dimensional electrophoresis and Coomassie Blue staining, the accumulation of a 34-kDa protein (named cdsp 34 for chloroplastic drought-induced stress protein) is shown in the thylakoids of Solanum tuberosum plants subjected to a progressive and reversible water deficit. In-vivo labeling experiments showed an increased synthesis of cdsp 34 from the early stages of drought stress (leaf relative water content around 85%) and throughout the constraint. Sequences of the N-terminal part and of four tryptic-digest peptides did not reveal significant homology between the cdsp 34 protein and other known proteins. Western blotting analysis, using a serum raised against the N-terminal part of cdsp 34, confirmed the accumulation of cdsp 34 in thylakoids upon drought stress. From immunoblot analysis of different chloroplastic subfractions, the cdsp 34 protein appears to be an extrinsic protein preferentially located in unstacked stroma thylakoids. Immunoprecipitation of in-vitro-translated products, as well as Southern analysis, showed that the cdsp 34 protein is nuclear encoded. After rewatering of water-stressed plants, the level of cdsp 34 synthesis was reduced, but remained substantially higher than in control plants. Western analysis showed the persistence of a high amount of cdsp 34 in rewatered plants for at least two weeks. Based on the abundance and on the location of cdsp 34 within thylakoids, a putative role for this novel chloroplastic protein is discussed in relation to the tolerance of the photosynthetic apparatus of higher plants to dehydration.

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves: characterization of two compartment-specific isoforms.

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO2 to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255,000 and is composed of eight identical subunits with an estimated Mr of 30,000. The chloroplastic isoenzyme (Mr 220,000) is also an octamer composed of two different subunits with Mr estimated at 27,000 and 27,500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the Mr-27,000 subunit was three amino acids shorter than that of the Mr-27,500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl-, I-, N3- and NO3-) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.

Identification of specific amphipathic alpha-helical sequence of human apolipoprotein A-IV involved in lecithin:cholesterol acyltransferase activation.

To investigate the structure-function relationship of human apolipoprotein A-IV (apoA-IV), several deletion mutants of this protein were constructed by sequentially removing pairs of 22-residue repeats, potentially having an amphipathic alpha-helical conformation. The mutants, produced as recombinant poly-histidine-tagged apolipoproteins (t-apo) in Escherichia coli, assembled with phosphatidylcholine (i.e. dimyristoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, or egg lecithin) as did native apoA-IV. Lecithin:cholesterol acyltransferase (LCAT) cofactor function, measured as cholesterol esterification occurring when t-apo-phosphatidylcholine-cholesterol complexes were incubated with purified enzyme, decreased significantly when pairs of repeats between residues 117 and 248 were deleted and most markedly when residues 117-160 were deleted. LCAT cofactor activity decreased by 90 and 75%, respectively, when egg lecithin or palmitoyloleoylphosphatidylcholine was used to form the particles with the delta aa 117-160 mutant. Thus, on the basis of deletion scanning of t-apo, residues 117-160 seem to be involved in the LCAT cofactor function of apoA-IV.