Development of a two-step screening ESI-TOF-MS method for rapid determination of significant stress-induced metabolome modifications in plant leaf extracts: the wound response in Arabidopsis thaliana as a case study.

To study the stress-induced effects caused by wounding under a new perspective, a metabolomic strategy based on HPLC-MS has been devised for the model plant Arabidopsis thaliana. To detect induced metabolites and precisely localise these compounds among the numerous constitutive metabolites, HPLC-MS analyses were performed in a two-step strategy. In a first step, rapid direct TOF-MS measurements of the crude leaf extract were performed with a ballistic gradient on a short LC-column. The HPLC-MS data were investigated by multivariate analysis as total mass spectra (TMS). Principal components analysis (PCA) and hierarchical cluster analysis (HCA) on principal coordinates were combined for data treatment. PCA and HCA demonstrated a clear clustering of plant specimens selecting the highest discriminating ions given by the complete data analysis, leading to the specific detection of discrete-induced ions (m/z values). Furthermore, pool constitution with plants of homogeneous behaviour was achieved for confirmatory analysis. In this second step, long high-resolution LC profilings on an UPLC-TOF-MS system were used on pooled samples. This allowed to precisely localise the putative biological marker induced by wounding and by specific extraction of accurate m/z values detected in the screening procedure with the TMS spectra.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

Discordances between phylogenetic and morphological patterns in alpine leaf beetles attest to an intricate biogeographic history of lineages in postglacial Europe.

Pleistocene glacial and interglacial periods have moulded the evolutionary history of European cold-adapted organisms. The role of the different mountain massifs has, however, not been accurately investigated in the case of high-altitude insect species. Here, we focus on three closely related species of non-flying leaf beetles of the genus Oreina (Coleoptera, Chrysomelidae), which are often found in sympatry within the mountain ranges of Europe. After showing that the species concept as currently applied does not match barcoding results, we show, based on more than 700 sequences from one nuclear and three mitochondrial genes, the role of biogeography in shaping the phylogenetic hypothesis. Dating the phylogeny using an insect molecular clock, we show that the earliest lineages diverged more than 1 Mya and that the main shift in diversification rate occurred between 0.36 and 0.18 Mya. By using a probabilistic approach on the parsimony-based dispersal/vicariance framework (MP-DIVA) as well as a direct likelihood method of state change optimization, we show that the Alps acted as a cross-roads with multiple events of dispersal to and reinvasion from neighbouring mountains. However, the relative importance of vicariance vs. dispersal events on the process of rapid diversification remains difficult to evaluate because of a bias towards overestimation of vicariance in the DIVA algorithm. Parallels are drawn with recent studies of cold-adapted species, although our study reveals novel patterns in diversity and genetic links between European mountains, and highlights the importance of neglected regions, such as the Jura and the Balkanic range.

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice.

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

Genotype-level interactions determine the degree of reduction of leaf rust on wheat by seed application of beneficial Pseudomonas spp.

Some bacteria have the capacity to reduce incidence and severity of plant diseases either by inhibiting the pathogen or by modulating the resistance response of the plant. Plants dispose of different resistance mechanisms that are influenced by the biotic and abiotic environment. The present experiments explored the effects of biocontrol strains of Pseudomonas fluorescens on the resistance of wheat varieties against brown rust disease caused by Puccinia triticina. Root inoculation with biocontrol pseudomonads reduced the disease severity on the leaves. The plant response depended on the genotype of both the microbes and the wheat varieties, suggesting a straight interaction at the molecular level.

A fetal sheep liver extract containing immunostimulatory substances including LPS acts as leukocyte activator in cells of LPS responder and non responder mice.

Purified fractions from a fetal sheep liver extract (FSLE) were investigated, in a murine model, for induction of leukocyte stimulating activities. The fractions FSLE-1 and FSLE-2 induced splenocyte proliferation in vitro in C57Bl/10ScSn (LPS responder) mice comparable to LPS, and in C57Bl/10ScCr (LPS non responder) mice. They also stimulated the release of nitrogen radicals in bone marrow-derived macrophages (BMDM) from several mouse inbred strains including both C57Bl/10ScSn and C57Bl/10ScCr mice. Stimulation of NO production could be blocked by L-NMMA, an inhibitor of iNOS, and enhanced by the simultaneous addition of IFN-gamma. Moreover, stimulation of macrophages by FSLE-1 and FSLE-2 induced a cytostatic effect of the activated macrophages for Abelson 8-1 tumor cells. The stimulatory activity of the purified fractions is partially due to trace amounts of LPS derived from the fetal liver extract which was enriched during purification. Our results may help to explain the beneficial effect of the extract in patients which has been observed clinically.

Does a shift in host plants trigger speciation in the Alpine leaf beetle Oreina speciosissima (Coleoptera, Chrysomelidae)?

Background: Within the Coleoptera, the largest order in the animal kingdom, the exclusively herbivorous Chrysomelidae are recognized as one of the most species rich beetle families. The evolutionary processes that have fueled radiation into the more than thirty-five thousand currently recognized leaf beetle species remain partly unresolved. The prominent role of leaf beetles in the insect world, their omnipresence across all terrestrial biomes and their economic importance as common agricultural pest organisms make this family particularly interesting for studying the mechanisms that drive diversification. Here we specifically focus on two ecotypes of the alpine leaf beetle Oreina speciosissima (Scop.), which have been shown to exhibit morphological differences in male genitalia roughly corresponding to the subspecies Oreina speciosissima sensu stricto and Oreina speciosissima troglodytes. In general the two ecotypes segregate along an elevation gradient and by host plants: Oreina speciosissima sensu stricto colonizes high forb vegetation at low altitude and Oreina speciosissima troglodytes is found in stone run vegetation at higher elevations. Both host plants and leaf beetles have a patchy geographical distribution. Through use of gene sequencing and genome fingerprinting (AFLP) we analyzed the genetic structure and habitat use of Oreina speciosissima populations from the Swiss Alps to examine whether the two ecotypes have a genetic basis. By investigating a wide range of altitudes and focusing on the structuring effect of habitat types, we aim to provide answers regarding the factors that drive adaptive radiation in this phytophagous leaf beetle.Results: While little phylogenetic resolution was observed based on the sequencing of four DNA regions, the topology and clustering resulting from AFLP genotyping grouped specimens according to their habitat, mostly defined by plant associations. A few specimens with intermediate morphologies clustered with one of the two ecotypes or formed separate clusters consistent with habitat differences. These results were discussed in an ecological speciation framework.Conclusions: The question of whether this case of ecological differentiation occurred in sympatry or allopatry remains open. Still, the observed pattern points towards ongoing divergence between the two ecotypes which is likely driven by a recent shift in host plant use.

Characterisation of "fou2", a constitutive wound-activated mutant and analysis of the proteome and leaf physiology in the region proximal to wounds in Arabidopsis

Résumé : Les jasmonates (JA), une famille d'hor1none végétale, jouent un rôle central dans la réponse à la blessure, et aux attaques d'insectes et de pathogènes. Les JA sont principalement dérivés d'un acide gras, l'acide linolénique. L'addition par une lipoxygénase d'une molécule d'oxygène à l'acide linolénique initie la synthèse de JA. Cependant les mécanismes régulant l'activation de la biosynthèse de JA ne sont pas encore connus. C'est pour cette raison que dans ce travail, nous avons caractérisé chez Arabidopsis thaliana (l'Arabette des Dames) un mutant fou2 dont l'activité lipoxygénase est plus élevée que celle d'une plante sauvage. Les niveaux de JA sont constitutivement plus élevés et l'activation de la synthèse de JA après blessure est fortement plus induite chez fou2 que chez le type sauvage. En outre, fou2 est plus résistant au pathogène Botrytis cinerea et à la chenille Spodoptera littoralis. Afin de comprendre quel mécanisme chez fou2 génére ce phénotype, nous avons cloné le gène responsable du phénotype de fou2. Le mutant fou2 porte une mutation dans le gène d'un canal à deux pores transportant probablement du potassium, du lumen de la vacuole végétale vers le compartiment cytosolique. L'analyse du protéome de fou2 a permis d'identifier une expression plus élevée de sept protéines régulées par les JA ou le stress. La découverte de l'implication d'un canal dans le phénotype de fou2 renforce l'hypothèse que les flux de cations pourraient être impliqués dans les étapes précoces de la synthèse des JA. Nous avons également étudié le protéome et la physiologie d'une feuille blessée, Pour évaluer les changements d'expression protéique en réponse à la blessure et contrôlés par les JA, nous avons quantifié l'expression de 5937 protéines chez une plante d'Arabidopsis sauvage et chez un mutant incapable de synthétiser des JA. Parmi ces 5937 protéines, nous avons identifié 99 protéines régulées par la blessure chez le type sauvage. Nous avons observé pour 65% des protéines dont l'expression protéique changeait après blessure une bonne corrélation entre la quantité de transcrits et de protéines. Plusieurs enzymes de la voie des chorismates impliquées dans la biosynthèse des acides aminés phénoliques étaient induites par les JA après blessure. Une quantification des acides aminés a montré que les niveaux d'acides aminés phénoliques augmentaient significativement après blessure. La blessure induisait aussi des changements dans l'expression de protéines impliquées dans la réponse au stress et particulièrement au stress oxydatif. Nous avons quantifié l'état réduit et oxydé du glutathion, un tripeptide qui, sous sa forme réduite, est l'antioxydant majeur des cellules. Nous avons trouvé une quantité significativement plus élevée de glutathion oxydé chez le type sauvage blessé que chez la plante aus blessée. Ce résultat suggère que la génération d'un stress oxydatif et la proportion relative de glutathions réduits et oxydés sont contrôlés par les JA après blessure. Abstract : Plants possess a family of potent fatty acid-derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri?unsaturated fatty acid a-linolenic-acid (18:3). Addition of an oxygen molecule to 18:3 by 13-lipoxygenases (13-LOX) initiates JA biosynthesis. Actually components regulating the activation of JA biosynthesis are poorly defined. Therefore we characterized in Arabidopsis thaliana the fatty acid Qxygenation upregulated 2 (fou2) mutant, which was previously isolated in a screen for mutants with an enhanced 13-LOX activity. As a consequence of this increased 13-LOX activity, JA levels in fou2 are higher than in wild type (WT) and wounding strongly increased JA biosynthesis compared to WT. fou2 was more resistant to the fungus Botrytis cinerea and the generalist caterpillar Spodaptera littomlis, The fou2 mutant carries a missense mutation in the Two Pore Channel 1 gene (TPCJ), which encodes a vacuolar cation channel transporting probably K* into the cytosol. Patchclamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA- inducible proteins. The discovery of the implication of a channel in the fou2 phenotype strenghtens the hypothesis that cation fluxes might be implicated in early steps of JA synthesis. We further concentrated on the proteome and leaf physiology in the region proximal to wounds in Arabidopsis using the WT and the aos JA-biosynthesis deficient mutant in order to find JA- induced proteins changes. We used two successive proteomic methods to assess protein changes in response to wounding Arabidopsis leaves, two dimensional electrophoresis (2DE) and linear trap quadrupole ion-trap mass spectrometry. In total 5937 proteins were quantified. We identified 99 wound-regulated proteins in the WT. Most these proteins were also wound-regulated at the transcript level showing a good correlation between transcript and protein abundance. We identified several wound-regulated enzymes involved in amino acid biosynthesis and confirmed this result by amino acid quantification. Proteins involved in stress reponses were upregulated, particularly in redox species regulation. We found a significantly higher quantity of oxidized glutathione in wounded WT relative to wounded aos leaves. This result suggests that levels of reduced glutathione are controlled by JA after wounding.

A rapid assay for the coupled cell free generation of oxylipins

We developed a rapid and simple assay for the coupled in vitro synthesis of oxylipins using free unsaturated fatty acids as substrates. Reactions were catalysed with extracts expressed from living plant tissues. Preliminary experiments involving the cell free transformation of fatty acid hydroperoxides revealed that storage or pretreatment of the plant extract rapidly altered its capacity to catalyse the generation of oxidised fatty acid derivatives. This could reflect changes in oxylipin generation that might take place in situ in damaged plant cells during herbivory. All subsequent experiments were performed without dilution, titration or any other modification of the plant extract prior to its addition to the assay system. The assays were used to study, for the first time, tissue-specific differences in fatty acid transformation to divinyl ethers. Root tissues from tomato efficiently catalysed the formation of corneleic and colnelenic acids from linoleic acid and linolenic acids, respectively, whereas leaf, hypocotyl and cotyledon extracts did not promote the formation of these compounds. We observed the efficient generation of 9-oxo-nonanoic acid from the substrate linolenic acid and speculate that this aldehyde could arise either from the action of hydroperoxide lyase on 9-hydroperoxylinolenic acid or by a novel route involving cleavage of colnelenic acid which was also present among the products of the reaction. A potential role of divinyl ethers as substrates for the generation of toxic aldehydes is discussed

GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling.

Wounded leaves communicate their damage status to one another through a poorly understood process of long-distance signalling. This stimulates the distal production of jasmonates, potent regulators of defence responses. Using non-invasive electrodes we mapped surface potential changes in Arabidopsis thaliana after wounding leaf eight and found that membrane depolarizations correlated with jasmonate signalling domains in undamaged leaves. Furthermore, current injection elicited jasmonoyl-isoleucine accumulation, resulting in a transcriptome enriched in RNAs encoding key jasmonate signalling regulators. From among 34 screened membrane protein mutant lines, mutations in several clade 3 GLUTAMATE RECEPTOR-LIKE genes (GLRs 3.2, 3.3 and 3.6) attenuated wound-induced surface potential changes. Jasmonate-response gene expression in leaves distal to wounds was reduced in a glr3.3 glr3.6 double mutant. This work provides a genetic basis for investigating mechanisms of long-distance wound signalling in plants and indicates that plant genes related to those important for synaptic activity in animals function in organ-to-organ wound signalling.

The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming.

We present a high-quality (>100× depth) Illumina genome sequence of the leaf-cutting ant Acromyrmex echinatior, a model species for symbiosis and reproductive conflict studies. We compare this genome with three previously sequenced genomes of ants from different subfamilies and focus our analyses on aspects of the genome likely to be associated with known evolutionary changes. The first is the specialized fungal diet of A. echinatior, where we find gene loss in the ant's arginine synthesis pathway, loss of detoxification genes, and expansion of a group of peptidase proteins. One of these is a unique ant-derived contribution to the fecal fluid, which otherwise consists of "garden manuring" fungal enzymes that are unaffected by ant digestion. The second is multiple mating of queens and ejaculate competition, which may be associated with a greatly expanded nardilysin-like peptidase gene family. The third is sex determination, where we could identify only a single homolog of the feminizer gene. As other ants and the honeybee have duplications of this gene, we hypothesize that this may partly explain the frequent production of diploid male larvae in A. echinatior. The fourth is the evolution of eusociality, where we find a highly conserved ant-specific profile of neuropeptide genes that may be related to caste determination. These first analyses of the A. echinatior genome indicate that considerable genetic changes are likely to have accompanied the transition from hunter-gathering to agricultural food production 50 million years ago, and the transition from single to multiple queen mating 10 million years ago.

Maintenance of neuronal expression of calbindin by a muscular extract in cultures of chick dorsal root ganglion cells.

Calbindin D-28K is a calcium-binding protein which is expressed by subpopulations of dorsal root ganglion cells cultured from 10-day-old (E10) chick embryos. After 7 or 10 days of culture, more than 20% of the ganglion cells are immunostained by an anticalbindin-antiserum; however, after 14 days of culture, the proportion drops to 10%. This fall can be prevented by addition of muscle extract to cultures at 10 days. Thus the transitory expression of calbindin-immunoreactivity by responsive sensory neurons would be not only induced but also maintained by a differentiation factor of muscular origin.

Leaf Mimicry: Chameleon-like Leaves in a Patagonian Vine.

Mimicry has evolved in plants for a number of traits, both floral and vegetative. The discovery of a vine that mimics the leaf shape of different hosts poses new questions about the function of leaf mimicry, interplant signalling and leaf development.