Profiling and optimized extraction of bioactive polyphenolic compounds from young, red-fleshed apple using eco-friendly deep eutectic solvents.

Red-fleshed apple cultivars with an enhanced content of polyphenolic compounds have attracted increasing interest due to their promising health benefits. Here, we have analysed the polyphenolic content of young, red-fleshed apples (RFA) and optimised extraction conditions of phenolics by utilising natural deep eutectic solvents (NDES). We also compare the antioxidant, neuroprotective and antimicrobial activities of NDES- and methanol-extracted phenolics from young RFA. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was used for phenolics identification and quantification. Besides young RFA, ripe red-fleshed, young and ripe white-fleshed apples were analysed, revealing that young RFA possess the highest phenolic content (2078.4 ± 4.0 mg gallic acid equivalent/100 g), and that ripe white-fleshed apples contain the least amount of phenolics (545.0 ± 32.0 mg gallic acid equivalent/100 g). The NDES choline chloride-glycerol containing 40 % w/w H2O gave similar yields at 40 °C as methanol. In addition, the polyphenolics profile, and bioactivities of the NDES extract from young RFA were comparable that of methanol extracts. Altogether, our data show that NDES extracts of young RFA are a promising source of bioactive polyphenolics with potential applications in diverse sectors, e.g., for functional food production, smart material engineering and natural therapies.

Non-targeted discovery of high-value bio-products in Nicotiana glauca L: a potential renewable plant feedstock.

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography-mass spectrometry (GC-MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.

Biochemical and in silico characterization of glycosyltransferases from red sweet cherry (Prunus avium L.) reveals their broad specificity toward phenolic substrates.

Polyphenolic compounds are a class of phytonutrients that play important roles in plants and contribute to human health when incorporated into our diet through fruit consumption. A large proportion occur as glycoconjugates but the enzymes responsible for their glycosylation are poorly characterized. Here, we report the biochemical and structural characterization of two glycosyltransferases from sweet cherry named PaUGT1 and PaUGT2. Both are promiscuous glucosyltransferases active on diverse anthocyanidins and flavonols, as well as phenolic acids in the case of PaUGT1. They also exhibit weaker galactosyltransferase activity. The expression of the gene encoding PaUGT1, the most active of the two proteins, follows anthocyanin accumulation during fruit ripening, suggesting that this enzyme is the primary glycosyltransferase involved in flavonoid glycosylation in sweet cherry. It can potentially be used to synthesize diverse glycoconjugates of flavonoids for integration into bioactive formulations, and for generating new fruit cultivars with enhanced health-promoting properties using breeding methods.

Polyphenol characterisation and diverse bioactivities of native Australian lilly pilly (Syzygium paniculatum) extract.

Magenta lilly pilly (Syzygium paniculatum) is an Australian native tree that produces berry fruits that are rich in phytochemicals reportedly beneficial to human health. Here we explored the biological activities of polyphenol-enriched extracts from the magenta lilly pilly fruit, benchmarking it against traditional sources including purple sweet potato and blackberry. We show that the extracts exert potent antioxidant and neuroprotective properties as well as antimicrobial activity against Staphylococcus aureus. The phenolic composition of lilly pilly was investigated using liquid chromatography coupled to mass spectrometry (HPLC-DAD-MS), revealing anthocyanins to be the primary component in high abundance compared to traditional anthocyanin-containing plants. Three anthocyanins from lilly pilly, along with their glycosylation patterns and stability, were characterised. Altogether, our results demonstrate the potential to exploit magenta lilly pilly fruits as a high-yielding source of phenolics with beneficial biological properties of potential interest for multiple downstream applications.

Game-changing alternatives to conventional fungicides: small RNAs and short peptides.

Fungicide use is one of the core elements of intensive agriculture because it is necessary to fight pathogens that would otherwise cause large production losses. Oomycete and fungal pathogens are kept under control using several active compounds, some of which are predicted to be banned in the near future owing to serious concerns about their impact on the environment, non-targeted organisms, and human health. To avoid detrimental repercussions for food security, it is essential to develop new biomolecules that control existing and emerging pathogens but are innocuous to human health and the environment. This review presents and discusses the use of novel low-risk biological compounds based on small RNAs and short peptides that are attractive alternatives to current contentious fungicides.

Proteomic Analysis Identifies Markers of Exposure to Cadmium Sulphide Quantum Dots (CdS QDs).

The use of cadmium sulphide quantum dot (CdS QD)-enabled products has become increasingly widespread. The prospect of their release in the environment is raising concerns. Here we have used the yeast model Saccharomyces cerevisiae to determine the potential impact of CdS QD nanoparticles on living organisms. Proteomic analyses and cell viability assays performed after 9 h exposure revealed expression of proteins involved in oxidative stress and reduced lethality, respectively, whereas oxidative stress declined, and lethality increased after 24 h incubation in the presence of CdS QDs. Quantitative proteomics using the iTRAQ approach (isobaric tags for relative and absolute quantitation) revealed that key proteins involved in essential biological pathways were differentially regulated over the time course of the experiment. At 9 h, most of the glycolytic functions increased, and the abundance of the number of heat shock proteins increased. This contrasts with the situation at 24 h where glycolytic functions, some heat shock proteins as well as oxidative phosphorylation and ATP synthesis were down-regulated. It can be concluded from our data that cell exposure to CdS QDs provokes a metabolic shift from respiration to fermentation, comparable to the situation reported in some cancer cell lines.

Structural analysis and biological activity of cell wall polysaccharides extracted from Panax ginseng marc.

Ginseng marc is a major by-product of the ginseng industry currently used as animal feed or fertilizer. This fibrous, insoluble waste stream is rich in cell wall polysaccharides and therefore a potential source of ingredients for functional food with health-promoting properties. However, the extraction of these polysaccharides has proved problematic and their exact composition remains unknown. Here we have analysed the composition, structure and biological activity of polysaccharides from ginseng root, stem and leaf marc fractionated using a chelator and alkali solutions. The pectic fraction has been extracted from root marc in high abundance and can activate the production of interleukine-1α and the hematopoietic growth factor by RAW 264.7 murine macrophage cells, which are important immune regulators of T-cells during inflammatory responses and infection processes. Our study reveals the potential to increase the value of ginseng marc by generating carbohydrate-based products with a higher value than animal feed.

Asexual Female Gametogenesis Involves Contact with a Sexually-Fated Megaspore in Apomictic Hieracium.

Apomixis results in asexual seed formation where progeny are identical to the maternal plant. In ovules of apomictic species of the Hieracium subgenus Pilosella, meiosis of the megaspore mother cell generates four megaspores. Aposporous initial (AI) cells form during meiosis in most ovules. The sexual pathway terminates during functional megaspore (FM) differentiation, when an enlarged AI undergoes mitosis to form an aposporous female gametophyte. Then, the mitotically programmed FM dies along with the three other megaspores by unknown mechanisms. Transcriptomes of laser-dissected AIs, ovule cells, and ovaries from apomicts and AI-deficient mutants were analyzed to understand the pathways involved. The steps leading to AI mitosis and sexual pathway termination were determined using antibodies against arabinogalactan protein epitopes found to mark both sexual and aposporous female gametophyte lineages at inception. At most, four AIs differentiated near developing megaspores. The first expanding AI cell to contact the FM formed a functional AI that underwent mitosis soon after megaspore degeneration. Transcriptome analyses indicated that the enlarged, laser-captured AIs were arrested in the S/G2 phase of the cell cycle and were metabolically active. Further comparisons with AI-deficient mutants showed that AIs were enriched in transcripts encoding homologs of genes involved in, and potentially antagonistic to, known FM specification pathways. We propose that AI and FM cell contact provides cues required for AI mitosis and megaspore degeneration. Specific candidates to further interrogate AI-FM interactions were identified here and include Hieracium arabinogalactan protein family genes.

Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan.

Mixed-linkage (1,3;1,4)-ß-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-ß-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-ß-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.

Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity.

Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P'1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.

Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata.

The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

Flexible and Responsive Chiral Nematic Cellulose Nanocrystal/Poly(ethylene glycol) Composite Films with Uniform and Tunable Structural Color.

The fabrication of responsive photonic structures from cellulose nanocrystals (CNCs) that can operate in the entire visible spectrum is challenging due to the requirements of precise periodic modulation of the pitch size of the self-assembled multilayer structures at the length scale within the wavelength of the visible light. The surface charge density of CNCs is an important factor in controlling the pitch size of the chiral nematic structure of the dried solid CNC films. The assembly of poly(ethylene glycol) (PEG) together with CNCs into smaller chiral nematic domains results in solid films with uniform helical structure upon slow drying. Large, flexible, and flat photonic composite films with uniform structure colors from blue to red are prepared by changing the composition of CNCs and PEG. The CNC/PEG(80/20) composite film demonstrates a reversible and smooth structural color change between green and transparent in response to an increase and decrease of relative humidity between 50% and 100% owing to the reversible swelling and dehydration of the chiral nematic structure. The composite also shows excellent mechanical and thermal properties, complementing the multifunctional property profile.

Quantitative Proteomic Analysis of Four Developmental Stages of Saprolegnia parasitica.

Several water mold species from the Saprolegnia genus infect fish, amphibians, and crustaceans in natural ecosystems and aquaculture farms. Saprolegnia parasitica is one of the most severe fish pathogens. It is responsible for millions of dollars of losses to the aquaculture industry worldwide. Here, we have performed a proteomic analysis, using gel-based and solution (iTRAQ) approaches, of four defined developmental stages of S. parasitica grown in vitro, i.e., the mycelium, primary cysts, secondary cysts and germinated cysts, to gain greater insight into the types of proteins linked to the different stages. A relatively high number of kinases as well as virulence proteins, including the ricin B lectin, disintegrins, and proteases were identified in the S. parasitica proteome. Many proteins associated with various biological processes were significantly enriched in different life cycle stages of S. parasitica. Compared to the mycelium, most of the proteins in the different cyst stages showed similar enrichment patterns and were mainly related to energy metabolism, signal transduction, protein synthesis, and post-translational modifications. The proteins most enriched in the mycelium compared to the cyst stages were associated with amino acid metabolism, carbohydrate metabolism, and mitochondrial energy production. The data presented expand our knowledge of metabolic pathways specifically linked to each developmental stage of this pathogen.

Quantitative proteomics links metabolic pathways to specific developmental stages of the plant-pathogenic oomycete Phytophthora capsici.

The oomycete Phytophthora capsici is a plant pathogen responsible for important losses to vegetable production worldwide. Its asexual reproduction plays an important role in the rapid propagation and spread of the disease in the field. A global proteomics study was conducted to compare two key asexual life stages of P. capsici, i.e. the mycelium and cysts, to identify stage-specific biochemical processes. A total of 1200 proteins was identified using qualitative and quantitative proteomics. The transcript abundance of some of the enriched proteins was also analysed by quantitative real-time polymerase chain reaction. Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts. The proteins enriched in the mycelium are mainly associated with glycolysis, the tricarboxylic acid (or citric acid) cycle and the pentose phosphate pathway, providing the energy required for the biosynthesis of cellular building blocks and hyphal growth. In contrast, the proteins that are predominant in cysts are essentially involved in fatty acid degradation, suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production. The data provide a better understanding of P. capsici biology and suggest potential metabolic targets at the two different developmental stages for disease control.

(1,3;1,4)-ß-Glucan Biosynthesis by the CSLF6 Enzyme: Position and Flexibility of Catalytic Residues Influence Product Fine Structure.

Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-ß-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4)-linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-ß-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-ß-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.

Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P3 with the pleckstrin homology domain of an oomycete cellulose synthase.

Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P3 is found to bind at a specific site located at ß1, ß2 and ß1-ß2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogen-bond interactions with Lys88, Lys100 and Arg102 in the binding site.

Phenylcoumaran benzylic ether reductase prevents accumulation of compounds formed under oxidative conditions in poplar xylem.

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.

Role of pathogen-derived cell wall carbohydrates and prostaglandin E2 in immune response and suppression of fish immunity by the oomycete Saprolegnia parasitica.

Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i.e., induction of interleukin-1ß1 [IL-1ß1], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglandin [corrected] E2 (PGE2) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE2 was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-γ] and the IFN-γ-inducible protein [γ-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development.

Transcript and metabolite profiling for the evaluation of tobacco tree and poplar as feedstock for the bio-based industry.

The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Functional characterization of the pleckstrin homology domain of a cellulose synthase from the oomycete Saprolegnia monoica.

Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.