The olfactory secretome varies according to season in female sheep and goat.

BACKGROUND: Small ungulates (sheep and goat) display a seasonal breeding, characterised by two successive periods, sexual activity (SA) and sexual rest (SR). Odours emitted by a sexually active male can reactivate the ovulatory cycle of anoestrus females. The plasticity of the olfactory system under these hormonal changes has never been explored at the peripheral level of odours reception. As it was shown in pig that the olfactory secretome (proteins secreted in the nasal mucus) could be modified under hormonal control, we monitored its composition in females of both species through several reproductive seasons, thanks to a non-invasive sampling of olfactory mucus. For this purpose, two-dimensional gel electrophoresis (2D-E), western-blot with specific antibodies, MALDI-TOF and high-resolution (nano-LC-MS/MS) mass spectrometry, RACE-PCR and molecular modelling were used. RESULTS: In both species the olfactory secretome is composed of isoforms of OBP-like proteins, generated by post-translational modifications, as phosphorylation, N-glycosylation and O-GlcNAcylation. Important changes were observed in the olfactory secretome between the sexual rest and the sexual activity periods, characterised in ewe by the specific expression of SAL-like proteins and the emergence of OBPs O-GlcNAcylation. In goat, the differences between SA and SR did not come from new proteins expression, but from different post-translational modifications, the main difference between the SA and SR secretome being the number of isoforms of each protein. Proteomics data are available via ProteomeXchange with identifier PXD014833. CONCLUSION: Despite common behaviour, seasonal breeding, and genetic resources, the two species seem to adapt their olfactory equipment in SA by different modalities: the variation of olfactory secretome in ewe could correspond to a specialization to detect male odours only in SA, whereas in goat the stability of the olfactory secretome could indicate a constant capacity of odours detection suggesting that the hallmark of SA in goat might be the emission of specific odours by the sexually active male. In both species, the olfactory secretome is a phenotype reflecting the physiological status of females, and could be used by breeders to monitor their receptivity to the male effect.

A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax.

Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes.

Nuclear genome sequence of the plastid-lacking cryptomonad Goniomonas avonlea provides insights into the evolution of secondary plastids.

BACKGROUND: The evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea-the first for any goniomonad-to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily. RESULTS: We sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~ 92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida. CONCLUSION: We found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic "rewiring" that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

Convergent evolution of polysaccharide debranching defines a common mechanism for starch accumulation in cyanobacteria and plants.

Starch, unlike hydrosoluble glycogen particles, aggregates into insoluble, semicrystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a preexisting cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semicrystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably, both the plant and cyanobacterial enzymes have evolved through convergent evolution, showing novel yet identical substrate specificities from a preexisting enzyme that originally displayed the much narrower substrate preferences required for glycogen catabolism.

Identification of new potential molecular actors related to fiber quality in flax through Omics.

One of the biggest challenges for a more widespread utilization of plant fibers is to better understand the different molecular factors underlying the variability in fineness and mechanical properties of both elementary and scutched fibers. Accordingly, we analyzed genome-wide transcription profiling from bast fiber bearing tissues of seven different flax varieties (4 spring, 2 winter fiber varieties and 1 winter linseed) and identified 1041 differentially expressed genes between varieties, of which 97 were related to cell wall metabolism. KEGG analysis highlighted a number of different enriched pathways. Subsequent statistical analysis using Partial Least-Squares Discriminant Analysis showed that 73% of the total variance was explained by the first 3 X-variates corresponding to 56 differentially expressed genes. Calculation of Pearson correlations identified 5 genes showing a strong correlation between expression and morphometric data. Two-dimensional gel proteomic analysis on the two varieties showing the most discriminant and significant differences in morphometrics revealed 1490 protein spots of which 108 showed significant differential abundance. Mass spectrometry analysis successfully identified 46 proteins representing 32 non-redundant proteins. Statistical clusterization based on the expression level of genes corresponding to the 32 proteins showed clear discrimination into three separate clusters, reflecting the variety type (spring-/winter-fiber/oil). Four of the 32 proteins were also highly correlated with morphometric features. Examination of predicted functions for the 9 (5 + 4) identified genes highlighted lipid metabolism and senescence process. Calculation of Pearson correlation coefficients between expression data and retted fiber mechanical measurements (strength and maximum force) identified 3 significantly correlated genes. The genes were predicted to be connected to cell wall dynamics, either directly (Expansin-like protein), or indirectly (NAD(P)-binding Rossmann-fold superfamily protein). Taken together, our results have allowed the identification of molecular actors potentially associated with the determination of both in-planta fiber morphometrics, as well as ex-planta fiber mechanical properties, both of which are key parameters for elementary fiber and scutched fiber quality in flax.

Cell adaptation of the extremophilic red microalga Galdieria sulphuraria to the availability of carbon sources.

Global energy demand and fossil fuels impact on climate can be partially managed by an increase in the use of biofuels for transports and industries. Biodiesel production is generally preceded by a transesterification process of the green biomass triacylglycerols that generates large amounts of glycerol as a by-product. In this study, the extremophilic red microalga Galdieria sulphuraria 074W was cultivated in heterotrophy. The microalgal growth parameters and biomass composition were compared when grown on an equivalent molar concentration of carbon of either glucose or glycerol as unique carbon source. The maximal biomass reached in these two conditions was not significantly different (∼2.5 g.L-1). Fatty acid profile, protein and storage carbohydrate contents were also statistically similar, irrespectively of the metabolized carbon source. We also observed that the pigment content of G. sulphuraria cells decreased during heterotrophic growth compared to photoautotrophic cultivated cells, and that this diminution was more important in the presence of glucose than glycerol: cells were yellowish in the presence of glucose and green in the presence of glycerol. The pigmentation was restored when glucose was totally consumed in the medium, suggesting that the presence of glucose repressed pigment synthesis. Based on this observation, a transcriptome analysis was performed in order to better understand the mechanisms involved in the loss of color mediated by darkness and by glucose in G. sulphuraria. Three conditions were analyzed: heterotrophy with glycerol or glucose and phototrophy. This allowed us to understand the transcriptional response of cells to light and dark environments both at the nuclear and chloroplast levels, and to show that transcription of gene families, acquired by horizontal gene transfer, such as sugar, amino acid, or acetate transporters, were involved in the response to the availability of different (in)organic sources.

Retracing Storage Polysaccharide Evolution in Stramenopila.

Eukaryotes most often synthesize storage polysaccharides in the cytosol or vacuoles in the form of either alpha (glycogen/starch)- or beta-glucosidic (chrysolaminarins and paramylon) linked glucan polymers. In both cases, the glucose can be packed either in water-soluble (glycogen and chrysolaminarins) or solid crystalline (starch and paramylon) forms with different impacts, respectively, on the osmotic pressure, the glucose accessibility, and the amounts stored. Glycogen or starch accumulation appears universal in all free-living unikonts (metazoa, fungi, amoebozoa, etc.), as well as Archaeplastida and alveolata, while other lineages offer a more complex picture featuring both alpha- and beta-glucan accumulators. We now infer the distribution of these polymers in stramenopiles through the bioinformatic detection of their suspected metabolic pathways. Detailed phylogenetic analysis of key enzymes of these pathways correlated to the phylogeny of Stramenopila enables us to retrace the evolution of storage polysaccharide metabolism in this diverse group of organisms. The possible ancestral nature of glycogen metabolism in eukaryotes and the underlying source of its replacement by beta-glucans are discussed.

Conservation of the glycogen metabolism pathway underlines a pivotal function of storage polysaccharides in Chlamydiae.

The order Chlamydiales includes obligate intracellular pathogens capable of infecting mammals, fishes and amoeba. Unlike other intracellular bacteria for which intracellular adaptation led to the loss of glycogen metabolism pathway, all chlamydial families maintained the nucleotide-sugar dependent glycogen metabolism pathway i.e. the GlgC-pathway with the notable exception of both Criblamydiaceae and Waddliaceae families. Through detailed genome analysis and biochemical investigations, we have shown that genome rearrangement events have resulted in a defective GlgC-pathway and more importantly we have evidenced a distinct trehalose-dependent GlgE-pathway in both Criblamydiaceae and Waddliaceae families. Altogether, this study strongly indicates that the glycogen metabolism is retained in all Chlamydiales without exception, highlighting the pivotal function of storage polysaccharides, which has been underestimated to date. We propose that glycogen degradation is a mandatory process for fueling essential metabolic pathways that ensure the survival and virulence of extracellular forms i.e. elementary bodies of Chlamydiales.

Intra-platform comparison of 25-mer and 60-mer oligonucleotide Nimblegen DNA microarrays.

BACKGROUND: We performed a Nimblegen intra-platform microarray comparison by assessing two categories of flax target probes (short 25-mers oligonucleotides and long 60-mers oligonucleotides) in identical conditions of target production, design, labelling, hybridization, image analyses, and data filtering. We compared technical parameters of array hybridizations, precision and accuracy as well as specific gene expression profiles. RESULTS: Comparison of the hybridization quality, precision and accuracy of expression measurements, as well as an interpretation of differential gene expression in flax tissues were performed. Both array types yielded reproducible, accurate and comparable data that are coherent for expression measurements and identification of differentially expressed genes. 60-mers arrays gave higher hybridization efficiencies and therefore were more sensitive allowing the detection of a higher number of unigenes involved in the same biological process and/or belonging to the same multigene family. CONCLUSION: The two flax arrays provide a good resolution of expressed functions; however the 60-mers arrays are more sensitive and provide a more in-depth coverage of candidate genes potentially involved in different biological processes.