Recent developments of marine ingredients for food and nutraceutical applications: a review.

Remerciements à l'éditeur pour son accord de diffusion. L'article original est aussi accessible sur le site de l'éditeur à l'adresse : http://www.halieutique.org/23201b.htmlInternational audienceIn a global context of marine biological resource overexploitation, a better upgrading of fish and shellfish biomass is a challenge for the 21st century. One of the main and promising issues will be the production of marine bio-ingredients using enzymatic hydrolysis. This paper presents the key steps in the production of enzymatic hydrolysates, such as (i) enzymatic treatment for the bioconversion of solid discards, and more particularly, use of proteases, (ii) quantification of the proteolysis extend and procedures of quality-control and (iii) identification of biological activity, using in vitro and in vivo methods. In the last part, examples of marine, commercially available functional foods or nutraceutical ingredients carrying bioactive properties are presented in order to demonstrate the interest of biotechnological exploitation of marine resources

Homologous Recombination Is Stimulated by a Decrease in dUTPase in Arabidopsis

Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis (Arabidopsis thaliana) and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants

Measuring stress-induced changes in defense phytohormones and related compounds

Measuring quantitative changes in plant hormones and derivatives is crucial to understand how reactive oxygen species trigger signaling cascades to regulate stress responses. In this chapter, we describe the liquid chromatography-mass spectrometry procedure that we use to extract and quantify salicylic acid (SA), jasmonic acid (JA), and related compounds in common extracts of Arabidopsis tissue. The method can provide quantitative data on SA, SA glucosides, and JA, as well as information on oxidized and conjugated forms of these compounds and related derivatives of benzoic acid

Phosphomimetic T335D Mutation of Hydroxypyruvate Reductase 1 Modifies Cofactor Specificity and Impacts Arabidopsis Growth in Air

International audiencePhotorespiration is an essential process in oxygenic photosynthetic organisms triggered by the oxygenase activity of Rubisco. In peroxisomes, photorespiratory HYDROXYPYRUVATE REDUCTASE1 (HPR1) catalyzes the conversion of hydroxypyruvate to glycerate together with the oxidation of a pyridine nucleotide cofactor. HPR1 regulation remains poorly understood; however, HPR1 phosphorylation at T335 has been reported. By comparing the kinetic properties of phosphomimetic (T335D), nonphosphorylatable (T335A), and wild-type recombinant Arabidopsis (Arabidopsis thaliana) HPR1, it was found that HPR1-T335D exhibits reduced NADH-dependent hydroxypyruvate reductase activity while showing improved NADPH-dependent activity. Complementation of the Arabidopsis hpr1-1 mutant by either wild-type HPR1 or HPR1-T335A fully complemented the photorespiratory growth phenotype of hpr1-1 in ambient air, whereas HPR1-T335D-containing hpr1-1 plants remained smaller and had lower photosynthetic CO2 assimilation rates. Metabolite analyses indicated that these phenotypes were associated with subtle perturbations in the photorespiratory cycle of HPR1-T335D-complemented hpr1-1 rosettes compared to all other HPR1-containing lines. Therefore, T335 phosphorylation may play a role in the regulation of HPR1 activity in planta, although it was not required for growth under ambient air controlled conditions. Furthermore, improved NADP-dependent HPR1 activities in peroxisomes could not compensate for the reduced NADH-dependent HPR1 activit

Experimental evidence of phosphoenolpyruvate resynthesis from pyruvate in illuminated leaves.

International audienceDay respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis. However, recent studies have suggested that the tricarboxylic acid pathway is rate limiting and mitochondrial pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon source for amino acid (e.g. alanine) or fatty acid synthesis, but pyruvate metabolism is not well documented, and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, we examined the capacity of pyruvate to convert back to PEP using (13)C and (2)H labeling in illuminated cocklebur (Xanthium strumarium) leaves. We show that the intramolecular labeling pattern in Glu, 2-oxoglutarate, and malate after (13)C-3-pyruvate feeding was consistent with (13)C redistribution from PEP via the PEP-carboxylase reaction. Furthermore, the deuterium loss in Glu after (2)H(3)-(13)C-3-pyruvate feeding suggests that conversion to PEP and back to pyruvate washed out (2)H atoms to the solvent. Our results demonstrate that in cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, approximately 0.5‰ of the net CO(2) assimilation rate. This is likely to involve pyruvate inorganic phosphate dikinase and the fundamental importance of this flux for PEP and inorganic phosphate homeostasis is discussed

Metabolomic characterization of the functional division of nitrogen metabolism in variegated leaves

International audienceMany horticultural and natural plant species have variegated leaves, that is, patchy leaves with green and non-green or white areas. Specific studies on the metabolism of variegated leaves are scarce and although white (non-green) areas have been assumed to play the role of a 'nitrogen store', there is no specific studies showing the analysis of nitrogenous metabolites and the dynamics of nitrogen assimilation. Here, we examined the metabolism of variegated leaves of Pelargonium × hortorum. We show that white areas have a larger N : C ratio, more amino acids, with a clear accumulation of arginine. Metabolomic analyses revealed clear differences in the chemical composition, suggesting contrasted metabolic commitments such as an enhancement of alkaloid biosynthesis in white areas. Using isotopic labelling followed by nuclear magnetic resonance or liquid chromatography/mass spectrometry, we further showed that in addition to glutamine, tyrosine and tryptophan, N metabolism forms ornithine in green area and huge amounts of arginine in white areas. Fine isotopic measurements with isotope ratio mass spectrometry indicated that white and green areas exchange nitrogenous molecules but nitrogen export from green areas is quantitatively much more important. The biological significance of the metabolic exchange between leaf areas is briefly discussed

Manganese concentration affects chloroplast structure and the photosynthetic apparatus in Marchantia polymorpha

International audienceManganese (Mn) is an essential metal for plant growth. The most important Mn-containing enzyme is the Mn4CaO5 cluster that catalyzes water oxidation in photosystem II (PSII). Mn deficiency primarily affects photosynthesis, whereas Mn excess is generally toxic. Here, we studied Mn excess and deficiency in the liverwort Marchantia polymorpha, an emerging model ideally suited for analysis of metal stress since it accumulates rapidly toxic substances due to the absence of well-developed vascular and radicular systems and a reduced cuticle. We established growth conditions for Mn excess and deficiency and analyzed the metal content in thalli and isolated chloroplasts. In vivo super-resolution fluorescence microscopy and transmission electron microscopy revealed changes in the organization of the thylakoid membrane under Mn excess and deficiency. Both Mn excess and Mn deficiency increased the stacking of the thylakoid membrane. We investigated photosynthetic performance by measuring chlorophyll fluorescence at room temperature and 77 K, measuring P700 absorbance, and studying the susceptibility of thalli to photoinhibition. Nonoptimal Mn concentrations changed the ratio of PSI to PSII. Upon Mn deficiency, higher non-photochemical quenching was observed, electron donation to PSI was favored, and PSII was less susceptible to photoinhibition. Mn deficiency seemed to favor cyclic electron flow around PSI, thereby protecting PSII in high light. The results presented here suggest an important role of Mn in the organization of the thylakoid membrane and photosynthetic electron transport

Recent developments of marine ingredients for food and nutraceutical applications: a review.

Remerciements à l'éditeur pour son accord de diffusion. L'article original est aussi accessible sur le site de l'éditeur à l'adresse : http://www.halieutique.org/23201b.htmlInternational audienceIn a global context of marine biological resource overexploitation, a better upgrading of fish and shellfish biomass is a challenge for the 21st century. One of the main and promising issues will be the production of marine bio-ingredients using enzymatic hydrolysis. This paper presents the key steps in the production of enzymatic hydrolysates, such as (i) enzymatic treatment for the bioconversion of solid discards, and more particularly, use of proteases, (ii) quantification of the proteolysis extend and procedures of quality-control and (iii) identification of biological activity, using in vitro and in vivo methods. In the last part, examples of marine, commercially available functional foods or nutraceutical ingredients carrying bioactive properties are presented in order to demonstrate the interest of biotechnological exploitation of marine resources

Uracil salvage is necessary for early Arabidopsis development.

International audienceUridine nucleotides can be formed by energy-consuming de novo synthesis or by the energy-saving recycling of nucleobases resulting from nucleotide catabolism. Uracil phosphoribosyltransferases (UPRTs; EC 2.4.2.9) are involved in the salvage of pyrimidines by catalyzing the formation of uridine monophosphate (UMP) from uracil and phosphoribosylpyrophosphate. To date, UPRTs are described as non-essential, energy-saving enzymes. In the present work, the six genes annotated as UPRTs in the Arabidopsis genome are examined through phylogenetic and functional complementation approaches and the available T-DNA insertion mutants are characterized. We show that a single nuclear gene encoding a protein targeted to plastids, UPP, is responsible for almost all UPRT activity in Arabidopsis. The inability to salvage uracil caused a light-dependent dramatic pale-green to albino phenotype, dwarfism and the inability to produce viable progeny in loss-of-function mutants. Plastid biogenesis and starch accumulation were affected in all analysed tissues, with the exception of stomata. Therefore we propose that uracil salvage is of major importance for plant development

Metabolic adjustments in response to ATP spilling by the small DX protein in a Streptomyces strain

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms . However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces , the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong Erm E promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health