Jean-Pierre Salaün

Biotic interactions of marine algae

Marine algae encompass lineages that diverged about one billion years ago. Recent results suggest that they feature natural immunity traits that are conserved, as well as others that appear to be phylum- or environment-specific. In particular, marine plants resemble terrestrial plants and animals in their basic mechanisms for pathogen recognition and signaling, suggesting that these essential cell functions arose in the sea. Specific traits are based on the synthesis of unique secondary defense metabolites, often making use of the variety of halides found in the sea.

Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450.

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the omega3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10muM of these two omega3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of omega3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.

The Innate Immunity of a Marine Red Alga Involves Oxylipins from Both the Eicosanoid and Octadecanoid Pathways

The oxygenated derivatives of fatty acids, known as oxylipins, are pivotal signaling molecules in animals and terrestrial plants. In animal systems, eicosanoids regulate cell differentiation, immune responses, and homeostasis. In contrast, terrestrial plants use derivatives of C18 and C16 fatty acids as developmental or defense hormones. Marine algae have emerged early in the evolution of eukaryotes as several distinct phyla, independent from the animal and green-plant lineages. The occurrence of oxylipins of the eicosanoid family is well documented in marine red algae, but their biological roles remain an enigma. Here we address the hypothesis that they are involved with the defense mechanisms of the red alga Chondrus crispus. By investigating its association with a green algal endophyte Acrochaete operculata, which becomes invasive in the diploid generation of this red alga, we showed that (1) when challenged by pathogen extracts, the resistant haploid phase of C. crispus produced both C20 and C18 oxylipins, (2) elicitation with pathogen extracts or methyl jasmonate activated the metabolism of C20 and C18 polyunsaturated fatty acids to generate hydroperoxides and cyclopentenones such as prostaglandins and jasmonates, and (3) C20 and C18 hydroperoxides as well as methyl jasmonate did induce shikimate dehydrogenase and Phe ammonialyase activities in C. crispus and conferred an induced resistance to the diploid phase, while inhibitors of fatty acid oxidation reduced the natural resistance of the haploid generation. The dual nature of oxylipin metabolism in this alga suggests that early eukaryotes featured both animal- (eicosanoids) and plant-like (octadecanoids) oxylipins as essential components of innate immunity mechanisms.

Wounding-induced cinnamic acid hydroxylase in Jerusalem artichoke tuber

Abstract Cinnamic acid hydroxylase (CAH), which catalyses transformation of trans-cinnamic acid into p-hydroxy-cinnamic acid, is a multi-enzyme system localized on the endoplasmic reticulum. Electrons are transferred from NADPH, the preferential electron donor for the system, to cytochrome P-450 via NADPH-cytochrome P-450 reductase, an enzyme regulated by the NADPH-NADP+ ratio. The induction and subsequent changes of CAH activity during ageing are accounted for by the variations in reductase and cytochrome P-450 content. The content of cytochrome b5, already present in the dormant tuber, is markedly enhanced by wounding; its participation in electron transport from NADH to the hydroxylase is discussed.

Copper stress induces biosynthesis of octadecanoid and eicosanoid oxygenated derivatives in the brown algal kelp Laminaria digitata.

To better understand the toxicity and the orchestration of antioxidant defenses of marine brown algae in response to copper-induced stress, lipid peroxidation processes were investigated in the brown alga Laminaria digitata. The expression of genes involved in cell protection and anti-oxidant responses were monitored by semi-quantitative reverse transcriptase polymerase chain reaction and the lipid peroxidation products were further characterized by profiling oxylipin signatures using high-pressure liquid chromatography-mass spectrometry. Exposure to copper excess triggers lipoperoxide accumulation and upregulates the expression of stress related genes. It also increases the release of free polyunsaturated fatty acids, leading to an oxidative cascade through at least two distinct mechanisms. Incubations in presence of inhibitors of lipoxygenases and cycloxygenases showed that in addition to the reactive oxygen species-mediated processes, copper stress induces the synthesis of oxylipins through enzymatic mechanisms. Among complex oxylipins, cyclopentenones from C18 and C20 fatty acids such as 12-oxo-PDA and prostaglandins were detected for the first time in brown algae, as well as unique compounds such as the 18-hydroxy-17-oxo-eicosatetraenoic acid. These results suggest that lipid peroxidation participates in the toxic effects of copper and that lipid peroxidation derivatives may regulate protective mechanisms by employing plant-like octadecanoid signals but also eicosanoid oxylipins which are absent in vascular plants.

Free Fatty Acids and Methyl Jasmonate Trigger Defense Reactions in Laminaria digitata

Arachidonic acid, linolenic acid and methyl jasmonate (MeJA) were found to be strong triggers of an oxidative burst in the kelp Laminaria digitata. These findings constitute the first report of an oxidative burst in an algal system induced by free fatty acids. The source of reactive oxygen species can be at least partially inhibited by diphenylene iodonium (DPI). Treatment with arachidonic acid increases the levels of a number of free fatty acids [including myristic (C14:0), linoleic (C18:2), linolenic (C18:3) and eicosapentaeneoic (C20:5) acids] and hydroxylated derivatives [such as 15-hydroxyeicosatetraenoic acid (15-HETE), 13-hydroxyoctadecatrienoic acid (13-HOTE) and 15-hydroxyeicosapentaenoic acid (15-HEPE)]. Similar to a previous report of the function of an alginate oligosaccharide-triggered oxidative burst in the establishment of resistance in L. digitata against infection by its brown algal endophyte Laminariocolax tomentosoides, C20:4- and MeJA-induced oxidative bursts seem to be involved in establishing the same protection in L. digitata. Altogether, this study supports the notion that lipid oxidation signaling plays a key role in defense induction in marine brown algae.

Mixed-function oxygenase enzymes as tools for pollution monitoring: field studies on the French coast of the Mediterranean Sea.

1. MFO enzyme activities were measured in microsomes from whole mussels (Mytilus galloprovincialis) comber livers (Serranus cabrilla), or Posidonia oceanica etiolated tissues, and PAH contents were determined in sediments collected in coastal locations of the French Riviera and Corsica during 3 oceanographic cruises in 1987-1988. 2. BaP activities in mussel and EROD activities measured in fish were strongly correlated to the log of PAH content in sediments. The first results for CA4H in Posidonia showed significant differences related to PAH pollution levels. The increase in MFO activities measured in Corsica in summer 1988 indicated a recent petroleum contamination.

Benzo(a)pyrene hydroxylase activity in the marine mussel Mytilus galloprovincialis: a potential marker of contamination by polycyclic aromatic hydrocarbon-type compounds

Abstract The aim of this work is to optimize and standardize the incubation conditions necessary to determine benzo(a)pyrene hydroxylase (BPH) activity in mussel microsomes. Evidence is presented that the reaction is dependent on NADPH and linear with time, at least up to the incubation time used. To measure BPH activity with the highest efficiency, the concentrations of benzo(a)pyrene (70 μM) microsomal proteins (0–75 mg/0–8 ml) and NADPH (0.74 mM), and the incubation time (10 min) were optimized to avoid plateauing of the activity due to a lack of substrate or NADPH. The best compromise between signal/noise ratio and linearity of the reaction with time was considered. BPH induction in mussels treated with 3-methylcholanthrene or exposed to field contamination by polycyclic aromatic hydrocarbons (PAH)-type compounds demonstrated the ability of this technique to be a potential marker of exposure to PAH type compound.

ω-hydroxylation of Z9-octadecenoic, Z9,10-epoxystearic and 9,10-dihydroxystearic acids by microsomal cytochrome P450 systems from Vicia sativa

A microsomal fraction from etiolated Vicia sativa seedlings incubated aerobically with [1-14C]oleic acid (Z9-octadecenoic acid) or [1-14C]9,10-epoxystearic acid or [1-14C]9,10-dihydroxystearic acid catalyzed the NADPH-dependent formation of hydroxylated metabolites. The chemical structure of compounds formed from oleic, 9,10-epoxystearic or 9,10-dihydroxystearic acids was established by gas chromatography/mass spectra analysis to be 18-hydroxyoleic acid, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid, respectively. The reactions required O2 and NADPH and were inhibited by carbon monoxide. As expected for monooxygenase reactions involving cytochrome P450, inhibition could be partially reversed by light and all three reactions were inhibited by antibodies raised against NADPH-cytochrome P450 reductase from Jerusalem artichoke. The omega-hydroxylation of the three substrates was enhanced in microsomes from clofibrate induced seedlings.

Induction by manganese, ethanol, phenobarbital, and herbicides of microsomal cytochrome P-450 in higher plant tissues.

Abstract Cytochrome P -450 content and, to a lesser extent the activity of tr -cinnamic acid 4-hydroxylase, are induced in ageing Jerusalem artichoke ( Helianthus tuberosus L.) tuber cells by manganese ions, ethanol, phenobarbital, and herbicides. Manganese, ethanol, and phenobarbital induced cytochrome P -450 and also modified the time-course of its appearance. In contrast the herbicides tested stimulated the cytochrome P -450 content without modifying its time-course. The extent of induction was enhanced when the aging medium was supplemented with iron.