Cadmium interferes with auxin physiology and lignification in poplar.

Cadmium (Cd) is a phytotoxic heavy metal that causes rapid growth reduction. To investigate if Cd interferes with the metabolism of auxin, a major growth hormone in plants, poplars (Populus × canescens) expressing a heterologous GH3::GUS reporter gene were exposed to 50 µM Cd in hydroponic solutions. Growth, photosynthetic performance, lignification, peroxidase activity, auxin concentration, and GUS staining were determined in order to record the activities of GH3 enzymes in the stem apex, the elongation zone, wood in the zone of radial growth, and in roots. Cd-induced growth reductions were tissue-specific decreasing in the order: roots>wood>shoot elongation and leaf initiation, whereas Cd concentrations increased in the order: leaves

Myosin-cross-reactive antigen (MCRA) protein from Bifidobacterium breve is a FAD-dependent fatty acid hydratase which has a function in stress protection.

BACKGROUND: The aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates. RESULTS: MCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls. CONCLUSIONS: MCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection.

Jasmonate biosynthesis in legume and actinorhizal nodules.

Jasmonic acid (JA) is a plant signalling compound that has been implicated in the regulation of mutualistic symbioses. In order to understand the spatial distribution of JA biosynthetic capacity in nodules of two actinorhizal species, Casaurina glauca and Datisca glomerata, and one legume, Medicago truncatula, we determined the localization of allene oxide cyclase (AOC) which catalyses a committed step in JA biosynthesis. In all nodule types analysed, AOC was detected exclusively in uninfected cells. The levels of JA were compared in the roots and nodules of the three plant species. The nodules and noninoculated roots of the two actinorhizal species, and the root systems of M. truncatula, noninoculated or nodulated with wild-type Sinorhizobium meliloti or with mutants unable to fix nitrogen, did not show significant differences in JA levels. However, JA levels in all plant organs examined increased significantly on mechanical disturbance. To study whether JA played a regulatory role in the nodules of M. truncatula, composite plants containing roots expressing an MtAOC1-sense or MtAOC1-RNAi construct were inoculated with S. meliloti. Neither an increase nor reduction in AOC levels resulted in altered nodule formation. These data suggest that jasmonates are not involved in the development and function of root nodules.

Limitation of nocturnal ATP import into chloroplasts seems to affect hormonal crosstalk, prime defense, and enhance disease resistance in Arabidopsis thaliana.

When grown under short-day conditions at low light, leaves of an Arabidopsis thaliana (accession Col-0) mutant with defects in the two genes encoding plastid ATP/ADP antiporters (so-called ntt1-2 null mutants) display a variety of physiological changes. These include the formation of necrotic lesions and the accumulation of hydrogen peroxide in the leaves. Here, we show that, under short-day conditions, leaves of the ntt1-2 mutant display enhanced resistance to Hyaloperonospora arabidopsidis, Botrytis cinerea, and Pseudomonas syringae pv. tomato DC3000. Resistance to these pathogens was associated with constitutively elevated levels of the plant hormone salicylic acid and, eventually, jasmonic acid, and constitutive or primed activation after pathogen attack of various defense genes that are dependent on these hormones. In addition, the antagonistic crosstalk between the salicylic acid and jasmonic acid signaling pathways seems to be affected in ntt1-2. Because the enhanced resistance of ntt1-2 to H. arabidopsidis was not seen when the mutant was grown under long-day conditions, our findings argue that nocturnal ATP import into chloroplasts is crucial to keep A. thaliana from runaway activation of pathogen resistance.

Phosphoenolpyruvate provision to plastids is essential for gametophyte and sporophyte development in Arabidopsis thaliana.

Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1(+/-)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems, and aberrant exine formation in pollen indicate that cue1/eno1(+/-) disrupts multiple pathways. While diminished fatty acid biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/-) contained increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts, ENO1 might be the only enzyme missing for a complete plastidic glycolysis.

The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology.

• Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)-12-oxo-phytodienoic acid (cis-(+)-OPDA), were isolated from the moss Physcomitrella patens. • Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13-hydroperoxy linolenic acid (13-HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12-hydroperoxy arachidonic acid (12-HPETE). • In protonema and gametophores the occurrence of cis-(+)-OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis-(+)-OPDA was detected. • Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.

A bisallylic mini-lipoxygenase from cyanobacterium Cyanothece sp. that has an iron as cofactor.

Lipoxygenases are enzymes that are found ubiquitously in higher animals and plants, but have only recently been identified in a number of bacteria. The genome of the diazotrophic unicellular cyanobacterium Cyanothece sp. harbors two genes with homology to lipoxygenases. Here we describe the isolation of one gene, formerly named csplox2. It was cloned, and the protein was expressed in Escherichia coli and purified. The purified enzyme belongs to the group of prokaryotic mini lipoxygenases, because it had a molecular mass of 65 kDa. Interestingly, it catalyzed the conversion of linoleic acid, the only endogenously found polyunsaturated fatty acid, primarily to the bisallylic hydroperoxide 11R-hydroperoxyoctadecadienoic acid. This product had previously only been described for the manganese lipoxygenase from the take all fungus, Gaeumannomyces graminis. By contrast, CspLOX2 was shown to be an iron lipoxygenase. In addition, CspLOX2 formed a mixture of typical conjugated lipoxygenase products, e.g. 9R- and 13S-hydroperoxide. The conversion of linoleic acid took place with a maximum reaction rate of 31 s(-1). Incubation of the enzyme with [(11S)-(2)H]linoleic acid led to the formation of hydroperoxides that had lost the deuterium label, thus suggesting that CspLOX2 catalyzes antarafacial oxygenation as opposed to the mechanism of manganese lipoxygenase. CspLOX2 could also oxidize diarachidonylglycerophosphatidylcholine with similar specificity as the free fatty acid, indicating that binding of the substrate takes place with a "tail-first" orientation. We conclude that CspLOX2 is a novel iron mini-lipoxygenase that catalyzes the formation of bisallylic hydroperoxide as the major product.

PpoC from Aspergillus nidulans is a fusion protein with only one active haem.

In Aspergillus nidulans Ppos [psi (precocious sexual inducer)-producing oxygenases] are required for the production of so-called psi factors, compounds that control the balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different haem-containing domains are predicted: a fatty acid haem peroxidase/dioxygenase domain in the N-terminal region and a P450 haem-thiolate domain in the C-terminal region. Whereas PpoA was shown to use both haem domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerization), we found that PpoC apparently only harbours a functional haem peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyses mainly the dioxygenation of linoleic acid (18:2Delta9Z,12Z), yielding 10-HPODE (10-hydroperoxyoctadecadienoic acid). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-KODE (10-keto-octadecadienoic acid) and 10-HODE (10-hydroxyoctadecadienoic acid). In parallel, decomposition of 10-HPODE into 10-ODA (10-octadecynoic acid) and volatile C-8 alcohols that are, among other things, responsible for the characteristic mushroom flavour. Besides these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE into the respective epoxy alcohols: 12,13-epoxy-8-HOME (where HOME is hydroxyoctadecenoic acid) and 12,13-epoxy-10-HOME. By using site-directed mutagenesis we demonstrated that both enzymes share a similar mechanism for the oxidation of 18:2Delta9Z,12Z; they both use a conserved tyrosine residue for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain.

Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles.

Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10-15 mM nitrate.

Upgrading root physiology for stress tolerance by ectomycorrhizas: insights from metabolite and transcriptional profiling into reprogramming for stress anticipation.

Ectomycorrhizas (EMs) alleviate stress tolerance of host plants, but the underlying molecular mechanisms are unknown. To elucidate the basis of EM-induced physiological changes and their involvement in stress adaptation, we investigated metabolic and transcriptional profiles in EM and non-EM roots of gray poplar (Populus x canescens) in the presence and absence of osmotic stress imposed by excess salinity. Colonization with the ectomycorrhizal fungus Paxillus involutus increased root cell volumes, a response associated with carbohydrate accumulation. The stress-related hormones abscisic acid and salicylic acid were increased, whereas jasmonic acid and auxin were decreased in EM compared with non-EM roots. Auxin-responsive reporter plants showed that auxin decreased in the vascular system. The phytohormone changes in EMs are in contrast to those in arbuscular mycorrhizas, suggesting that EMs and arbuscular mycorrhizas recruit different signaling pathways to influence plant stress responses. Transcriptome analyses on a whole genome poplar microarray revealed activation of genes related to abiotic and biotic stress responses as well as of genes involved in vesicle trafficking and suppression of auxin-related pathways. Comparative transcriptome analysis indicated EM-related genes whose transcript abundances were independent of salt stress and a set of salt stress-related genes that were common to EM non-salt-stressed and non-EM salt-stressed plants. Salt-exposed EM roots showed stronger accumulation of myoinositol, abscisic acid, and salicylic acid and higher K(+)-to-Na(+) ratio than stressed non-EM roots. In conclusion, EMs activated stress-related genes and signaling pathways, apparently leading to priming of pathways conferring abiotic stress tolerance.

Methods for the analysis of oxylipins in plants.

Plant oxylipins comprise a highly diverse and complex class of molecules that are derived from lipid oxidation. The initial oxidation of unsaturated fatty acids may either occur by enzymatic or chemical reactions. A large variety of oxylipin classes are generated by an array of alternative reactions further converting hydroperoxy fatty acids. The structural diversity of oxylipins is further increased by their occurrence either as free fatty acid derivatives or as esters in complex lipids. Lipid peroxidation is common to all biological systems, appearing in developmentally regulated processes and as a response to environmental changes. The oxylipins formed may perform various biological roles; some of them have signaling functions. In order to elucidate the roles of oxylipins in a given biological context, comprehensive analytical assays are available for determining the oxylipin profiles of plant tissues. This review summarizes indirect methods to estimate the general peroxidation state of a sample and more sophisticated techniques for the identification, structure determination and quantification of oxylipins.

(1)O2-mediated retrograde signaling during late embryogenesis predetermines plastid differentiation in seedlings by recruiting abscisic acid.

Plastid development in seedlings of Arabidopsis thaliana is affected by the transfer of (1)O(2)-mediated retrograde signals from the plastid to the nucleus and changes in nuclear gene expression during late embryogenesis. The potential impact of these mechanisms on plastid differentiation is maintained throughout seed dormancy and becomes effective only after seed germination. Inactivation of the 2 nuclear-encoded plastid proteins EXECUTER1 and EXECUTER2 blocks (1)O(2)-mediated retrograde signaling before the onset of dormancy and impairs normal plastid formation in germinating seeds. This long-term effect of (1)O(2) retrograde signaling depends on the recruitment of abscisic acid (ABA) during seedling development. Unexpectedly, ABA acts as a positive regulator of plastid formation in etiolated and light-grown seedlings.

Truffles regulate plant root morphogenesis via the production of auxin and ethylene.

Truffles are symbiotic fungi that form ectomycorrhizas with plant roots. Here we present evidence that at an early stage of the interaction, i.e. prior to physical contact, mycelia of the white truffle Tuber borchii and the black truffle Tuber melanopsorum induce alterations in root morphology of the host Cistus incanus and the nonhost Arabidopsis (Arabidopsis thaliana; i.e. primary root shortening, lateral root formation, root hair stimulation). This was most likely due to the production of indole-3-acetic acid (IAA) and ethylene by the mycelium. Application of a mixture of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and IAA fully mimicked the root morphology induced by the mycelium for both host and nonhost plants. Application of the single hormones only partially mimicked it. Furthermore, primary root growth was not inhibited in the Arabidopsis auxin transport mutant aux1-7 by truffle metabolites while root branching was less effected in the ethylene-insensitive mutant ein2-LH. The double mutant aux1-7;ein2-LH displayed reduced sensitivity to fungus-induced primary root shortening and branching. In agreement with the signaling nature of truffle metabolites, increased expression of the auxin response reporter DR5GFP in Arabidopsis root meristems subjected to the mycelium could be observed, confirming that truffles modify the endogenous hormonal balance of plants. Last, we demonstrate that truffles synthesize ethylene from l-methionine probably through the alpha-keto-gamma-(methylthio)butyric acid pathway. Taken together, these results establish the central role of IAA and ethylene as signal molecules in truffle/plant interactions.

The alpha-subunit of the heterotrimeric G-protein affects jasmonate responses in Arabidopsis thaliana.

Heterotrimeric G-proteins have been implicated in having a role in many plant signalling pathways. To understand further the role of G-proteins, a preliminary experiment was performed to assess the impact of the G alpha subunit loss-of-function mutation gpa1-1 on the Arabidopsis transcriptome. The analysis indicated that the G alpha subunit may play a role in response to jasmonic acid (JA). Consistent with this, G alpha mutants showed a reduced response to JA in inhibition of chlorophyll accumulation and root growth, whilst G alpha gain-of-function plants overexpressing G alpha showed the opposite phenotype. The levels of JA and related compounds were unaffected in the gpa1-1 mutant, as was autoregulation of the Allene Oxide Synthase (AOS) gene that encodes a key enzyme for JA biosynthesis. In contrast, further analyses using G alpha loss- and gain-of-function Arabidopsis lines indicated that G alpha positively modulates the expression of the Vegetative Storage Protein (VSP) gene. This indicates that the G alpha subunit regulates a subset of JA-regulated genes defining a branch point in this signalling pathway in Arabidopsis. Further analysis of the impact of G alpha loss of function upon the JA-regulated transcriptome using Arabidopsis full genome arrays indicated that up to 29% of genes that are >2-fold regulated by JA in the wild type are misregulated in the G alpha mutant. This supports the observation that a significant proportion of, but not all, JA-regulated gene expression is mediated by G alpha.

Fatty acid metabolism in the ectomycorrhizal fungus Laccaria bicolor.

Here, the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolorwas explored with the aim of constructing a genome-wide inventory of genes involved in fatty acid metabolism. Sixty-three genes of the major pathways were annotated and validated by the detection of the corresponding transcripts. Seventy-one per cent belonged to multigene families of up to five members. In the mycelium of L. bicolor, 19 different fatty acids were detected, including at low concentrations palmitvaccenic acid (16:1(11Z)), which is known to be a marker for arbuscular mycorrhizal fungi. The pathways of fatty acid biosynthesis and degradation in L. bicolor were reconstructed using lipid composition, gene annotation and transcriptional analysis. Annotation results indicated that saturated fatty acids were degraded in mitochondria, whereas degradation of modified fatty acids was confined to peroxisomes. Fatty acid synthase (FAS) was the second largest protein annotated in L. bicolor. Phylogenetic analysis indicated that L. bicolor, Ustilago maydis and Coprinopsis cinerea have a vertebrate-like type I FAS encoded as a single protein, whereas in other basidiomycetes, including the human pathogenic basidiomycete Cryptococcus neoformans, and in most ascomycetes FAS is composed of the two structurally distinct subunits α and ß.

MarVis: a tool for clustering and visualization of metabolic biomarkers.

BACKGROUND: A central goal of experimental studies in systems biology is to identify meaningful markers that are hidden within a diffuse background of data originating from large-scale analytical intensity measurements as obtained from metabolomic experiments. Intensity-based clustering is an unsupervised approach to the identification of metabolic markers based on the grouping of similar intensity profiles. A major problem of this basic approach is that in general there is no prior information about an adequate number of biologically relevant clusters. RESULTS: We present the tool MarVis (Marker Visualization) for data mining on intensity-based profiles using one-dimensional self-organizing maps (1D-SOMs). MarVis can import and export customizable CSV (Comma Separated Values) files and provides aggregation and normalization routines for preprocessing of intensity profiles that contain repeated measurements for a number of different experimental conditions. Robust clustering is then achieved by training of an 1D-SOM model, which introduces a similarity-based ordering of the intensity profiles. The ordering allows a convenient visualization of the intensity variations within the data and facilitates an interactive aggregation of clusters into larger blocks. The intensity-based visualization is combined with the presentation of additional data attributes, which can further support the analysis of experimental data. CONCLUSION: MarVis is a user-friendly and interactive tool for exploration of complex pattern variation in a large set of experimental intensity profiles. The application of 1D-SOMs gives a convenient overview on relevant profiles and groups of profiles. The specialized visualization effectively supports researchers in analyzing a large number of putative clusters, even though the true number of biologically meaningful groups is unknown. Although MarVis has been developed for the analysis of metabolomic data, the tool may be applied to gene expression data as well.

Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450.

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C(16) and C(18) fatty acids at pH approximately 7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.

The Arabidopsis patatin-like protein 2 (PLP2) plays an essential role in cell death execution and differentially affects biosynthesis of oxylipins and resistance to pathogens.

We previously reported that patatin-like protein 2 (PLP2), a pathogen-induced patatin-like lipid acyl hydrolase, promotes cell death and negatively affects Arabidopsis resistance to the fungus Botrytis cinerea and to the bacteria Pseudomonas syringae. We show here that, on the contrary, PLP2 contributes to resistance to Cucumber mosaic virus, an obligate parasite inducing the hypersensitive response. These contrasted impacts on different pathosystems were also reflected by differential effects on defense gene induction. To examine a possible link between PLP2 lipolytic activity and oxylipin metabolism, gene expression profiling was performed and identified B. cinerea among these pathogens as the strongest inducer of most oxylipin biosynthetic genes. Quantitative oxylipin profiling in wild-type and PLP2-modified, Botrytis-challenged plants established the massive accumulation of oxidized fatty acid derivatives in infected leaves. Several compounds previously described as modulating plant tissue damage and issued from the alpha-dioxygenase pathway were found to accumulate in a PLP2-dependent manner. Finally, the contribution of PLP2 to genetically controlled cell death was evaluated using PLP2-silenced or -overexpressing plants crossed with the lesion mimic mutant vascular-associated death 1 (vad1). Phenotypic analysis of double-mutant progeny showed that PLP2 expression strongly promotes necrotic symptoms in vad1 leaves. Collectively, our data indicate that PLP2 is an integral component of the plant cell death execution machinery, possibly providing fatty acid precursors for the biosynthesis of specific oxylipins and differentially affecting resistance to pathogens with distinct lifestyles.

Attacks by a piercing-sucking insect (Myzus persicae Sultzer) or a chewing insect (Leptinotarsa decemlineata Say) on potato plants (Solanum tuberosum L.) induce differential changes in volatile compound release and oxylipin synthesis.

Plant defensive strategies bring into play blends of compounds dependent on the type of attacker and coming from different synthesis pathways. Interest in the field is mainly focused on volatile organic compounds (VOCs) and jasmonic acid (JA). By contrast, little is known about the oxidized polyunsaturated fatty acids (PUFAs), such as PUFA-hydroperoxides, PUFA-hydroxides, or PUFA-ketones. PUFA-hydroperoxides and their derivatives might be involved in stress response and show antimicrobial activities. Hydroperoxides are also precursors of JA and some volatile compounds. In this paper, the differential biochemical response of a plant against insects with distinct feeding behaviours is characterized not only in terms of VOC signature and JA profile but also in terms of their precursors synthesized through the lipoxygenase (LOX)-pathway at the early stage of the plant response. For this purpose, two leading pests of potato with distinct feeding behaviours were used: the Colorado Potato Beetle (Leptinotarsa decemlineata Say), a chewing herbivore, and the Green Peach Aphid (Myzus persicae Sulzer), a piercing-sucking insect. The volatile signatures identified clearly differ in function with the feeding behaviour of the attacker and the aphid, which causes the smaller damages, triggers the emission of a higher number of volatiles. In addition, 9-LOX products, which are usually associated with defence against pathogens, were exclusively activated by aphid attack. Furthermore, a correlation between volatiles and JA accumulation and the evolution of their precursors was determined. Finally, the role of the insect itself on the plant response after insect infestation was highlighted.

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways.

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 alpha-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, alpha-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred alpha-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from alpha-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.