Transcriptomic and Physiological Analyses Reveal Potential Genes Involved in Photoperiod-Regulated ß-Carotene Accumulation Mechanisms in the Endocarp of Cucumber (Cucumis sativus L.) Fruit.

The accumulation of carotenoids in plants is a key nutritional quality in many horticultural crops. Although the structural genes encoding the biosynthetic enzymes are well-characterized, little is known regarding photoperiod-mediated carotenoid accumulation in the fruits of some horticultural crops. Herein, we performed physiological and transcriptomic analyses using two cucumber genotypes, SWCC8 (XIS-orange-fleshed and photoperiod-sensitive) and CC3 (white-fleshed and photoperiod-non-sensitive), established under two photoperiod conditions (8L/16D vs. 12L/12D) at four fruit developmental stages. Day-neutral treatments significantly increased fruit ß-carotene content by 42.1% compared to short day (SD) treatments in SWCC8 at 40 DAP with no significant changes in CC3. Day-neutral condition elevated sugar levels of fruits compared to short-day treatments. According to GO and KEGG analyses, the predominantly expressed genes were related to photosynthesis, carotenoid biosynthesis, plant hormone signaling, circadian rhythms, and carbohydrates. Consistent with ß-carotene accumulation in SWCC8, the day-neutral condition elevated the expression of key carotenoid biosynthesis genes such as PSY1, PDS, ZDS1, LYCB, and CHYB1 during later stages between 30 to 40 days of fruit development. Compared to SWCC8, CC3 showed an expression of DEGs related to carotenoid cleavage and oxidative stresses, signifying reduced ß-carotene levels in CC3 cucumber. Further, a WGCNA analysis revealed co-expression between carbohydrate-related genes (pentose-phosphatase synthase, ß-glucosidase, and trehalose-6-phosphatase), photoperiod-signaling genes (LHY, APRR7/5, FKF1, PIF3, COP1, GIGANTEA, and CK2) and carotenoid-biosynthetic genes, thus suggesting that a cross-talk mechanism between carbohydrates and light-related genes induces ß-carotene accumulation. The results highlighted herein provide a framework for future gene functional analyses and molecular breeding towards enhanced carotenoid accumulation in edible plant organs.

Alterations in the Gut Microbiota and Metabolomics of Seafarers after a Six-Month Sea Voyage.

Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of Firmicutes/Bacteroidetes at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of Holdemanella and Plesiomonas were significantly increased, while the abundance of Bacteroides was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. IMPORTANCE Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.

Identification of 5-Fluoro-5-Deoxy-Ribulose as a Shunt Fluorometabolite in Streptomyces sp. MA37.

A fluorometabolite, 5-fluoro-5-deoxy-D-ribulose (5-FDRul), from the culture broth of the soil bacterium Streptomyces sp. MA37, was identified through a combination of genetic manipulation, chemo-enzymatic synthesis and NMR comparison. Although 5-FDRul has been chemically synthesized before, it was not an intermediate or a shunt product in previous studies of fluorometalism in S. cattleya. Our study of MA37 demonstrates that 5-FDRul is a naturally occurring fluorometabolite, rendering it a new addition to this rare collection of natural products. The genetic inactivation of key biosynthetic genes involved in the fluorometabolisms in MA37 resulted in the increased accumulation of unidentified fluorometabolites as observed from 19F-NMR spectral comparison among the wild type (WT) of MA37 and the mutated variants, providing evidence of the presence of other new biosynthetic enzymes involved in the fluorometabolite pathway in MA37.

Characterization of mucilages extracted from different flaxseed (Linum usitatissiumum L.) cultivars: A heteropolysaccharide with desirable functional and rheological properties.

Flaxseed mucilage is composed of water soluble heteropolysaccharides of high molecular weight representing about 3 to 9% of the total seed. In the present study mucilages were extracted from six Indian flaxseed cultivars and their chemical, functional, microstructural and rheological properties were investigated. The extracted mucilages differed significantly (P < 0.05) in their yield (5.56-6.54%), ash (4.80-7.23%), protein (7.68-12.33%), pentose (0.48-0.80 mg/ml) and total sugar (1.58-3.06 mg/ml) contents. Copper (18.87-148.08 mg/kg) and zinc (15.43-53.43 mg/kg) were found to be the most abundant minerals in mucilages. "LC-2023" cv. mucilage exhibited lower values for lead, chromium and cadmium. The mucilage solutions exhibited high foaming (>40%) and solubility (64.5-69.15% at 80 °C) characteristics. Rheological data revealed shear rate dependent behaviour of aqueous mucilage solutions irrespective of cultivar type and concentration used. Frequency sweep tests demonstrated that at high frequency range investigated, storage modulus was higher than loss modulus thereby suggesting viscoelastic fluid behaviour of flaxseed mucilage. All mucilages exhibited endothermic as well exothermic transitions with high decomposition onset temperatures. Mucilage is of special research interest owing to its desirable functional properties, so the outcomes of the present study could be used to identify cultivars for producing mucilages for a desired end use.

Quantification of Soluble Sugars and Sugar Alcohols by LC-MS/MS.

Sugars are simple carbohydrates composed primarily of carbon, hydrogen, and oxygen. They play a central role in metabolism as sources of energy and as building blocks for synthesis of structural and nonstructural polymers. Many different techniques have been used to measure sugars, including refractometry, colorimetric and enzymatic assays, gas chromatography, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. In this chapter we describe a method that combines an initial separation of sugars by high-performance anion-exchange chromatography (HPAEC) with detection and quantification by tandem mass spectrometry (MS/MS). This combination of techniques provides exquisite specificity, allowing measurement of a diverse range of high- and low-abundance sugars in biological samples. This method can also be used for isotopomer analysis in stable-isotope labeling experiments to measure metabolic fluxes.

Enantioresolution and quantification of monosaccharides by comprehensive two-dimensional gas chromatography.

This work presents the development of a simple and efficient analytical protocol for the direct enantioselective resolution of sugars. A racemic mixture of the C3 sugar d,l-glyceraldehyde and the C5 monosaccharides d,l-arabinose, d,l-ribose, d,l-xylose, and d,l-lyxose was subjected to derivatization with trifluoroacetic anhydride, and corresponding derivatives were separated on a ß-cyclodextrin column with excellent resolution factors. Even though each aldopentose shows beside the linear form four predominant cyclic hemiacetals being the α- and ß-furanose along with the α- and ß-pyranose, we show that the overall enantiomeric excess of each compound can be precisely determined. Moreover, the measured detection limit for derivatized aldopentoses ranges from 0.015 to 0.019pmol on the column, while the quantification limit varies from 0.5 to 0.64pmol on the column.

Structure and antioxidant activity of a novel polysaccharide derived from Amanita caesarea.

A heteropolysaccharide was isolated from the fruiting bodies of Amanita caesarea using a diethylaminoethyl-cellulose column, Sephacryl S­300 gel column and Sephadex G­200 column. The Amanita caesarea polysaccharide was predominantly composed of α-D-glucose and α-D-lyxose at a ratio of 2:1, and it had a molecular weight of 19,329 Da. The structural features of the Amanita caesarea polysaccharide were investigated by a combination of total hydrolysis, methylation analysis, gas chromatography-mass spectrometry, and infrared spectra and nuclear magnetic resonance spectroscopy. The results showed that Amanita caesarea polysaccharide (termed AC­1) had a backbone of 1,4­linked α­D­glucose and 1,3,6­linked α­D­glucose, with branches of one 1­linked α­D­lyxose residue. The antioxidant activity of AC­1 was evaluated by two biochemical methods, 2,2-azino-bis diammonium (ABTS+) radical scavenging activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH-) radical scavenging activity. The uncontrolled production of free radicals is involved in various diseases, including cancer, atherosclerosis and degenerative aging processes. The results indicated that the Amanita caesarea polysaccharide exhibits strong antioxidant activity, thus, it may be a useful natural product antioxidant.

Distinct Geographical Distribution of the Miscanthus Accessions with Varied Biomass Enzymatic Saccharification.

Miscanthus is a leading bioenergy candidate for biofuels, and it thus becomes essential to characterize the desire natural Miscanthus germplasm accessions with high biomass saccharification. In this study, total 171 natural Miscanthus accessions were geographically mapped using public database. According to the equation [P(H/L| East) = P(H/L∩East)/P(East)], the probability (P) parameters were calculated on relationships between geographical distributions of Miscanthus accessions in the East of China, and related factors with high(H) or low(L) values including biomass saccahrification under 1% NaOH and 1% H2SO4 pretreatments, lignocellulose features and climate conditions. Based on the maximum P value, a golden cutting line was generated from 42°25' N, 108°22' E to 22°58' N, 116°28' E on the original locations of Miscanthus accessions with high P(H|East) values (0.800-0.813), indicating that more than 90% Miscanthus accessions were originally located in the East with high biomass saccharification. Furthermore, the averaged insolation showed high P (H|East) and P(East|H) values at 0.782 and 0.754, whereas other climate factors had low P(East|H) values, suggesting that the averaged insolation is unique factor on Miscanthus distributions for biomass saccharification. In terms of cell wall compositions and wall polymer features, both hemicelluloses level and cellulose crystallinity (CrI) of Miscanthus accessions exhibited relative high P values, suggesting that they should be the major factors accounting for geographic distributions of Miscanthus accessions with high biomass digestibility.

Digestion of Termiticide Bait Matrices by the Pest Termite Reticulitermes flavipes (Isoptera: Rhinotermitidae).

Termites are highly effective digesters of wood lignocellulose, which is a central factor contributing to their global status as pests of wooden structures. For the same reason, termite baits that combine cellulosic matrices with slow-acting insecticides are both effective and popular as a reduced-risk approach for termite control. This study took a novel approach for assessing digestibility of termite bait matrices and matrix components to gain potentially new insights into bait attractiveness and efficacy. The rationale behind this study is that termite baits that are more digestible should have more nutritional value to termites and thus encourage maximal feeding and trophallactic transfer of active ingredients through termite colonies. Studies were done using in vitro digestion assays with termite gut protein extracts followed by colorimetric detection of released glucose and pentose monosaccharides from test substrates. The substrates tested included two commercial bait matrices (Recruit IV and Recruit II HD), two matrix components (compressed and toasted compressed cellulose), and two natural pine woods as positive controls (southern yellow and northern pine). Overall results show equal or greater monosaccharide availability for some commercial matrices than standard pine lignocelluloses, suggesting sufficient nutritional value for the proprietary matrices. Another more prominent trend was significant intercolony variation in digestibility across substrates, possibly resulting from differences in microbiota composition, long-term diet adaptation, or both. These findings thus illuminate new nutrition-based factors that can potentially impact bait feeding, trophallactic exchange, and efficacy.

Chiral separation of D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent and a phenylboronic acid complex.

A novel method was developed for D/L-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of D/L isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300 nM), and the repeatabilities of the migration times and peak area responses were 0.8 % and 7.9 % (relative standard deviation; n = 6), respectively. The method was applied to the determination of D/L- galactose in red seaweed.

Chlorogenic acid-arabinose hybrid domains in coffee melanoidins: Evidences from a model system.

Arabinose from arabinogalactan side chains was hypothesized as a possible binding site for chlorogenic acids in coffee melanoidins. To investigate this hypothesis, a mixture of 5-O-caffeoylquinic acid (5-CQA), the most abundant chlorogenic acid in green coffee beans, and (α1 → 5)-L-arabinotriose, structurally related to arabinogalactan side chains, was submitted to dry thermal treatments. The compounds formed during thermal processing were identified by electrospray ionization mass spectrometry (ESI-MS) and characterized by tandem MS (ESI-MS(n)). Compounds composed by one or two CQAs covalently linked with pentose (Pent) residues (1-12) were identified, along with compounds bearing a sugar moiety but composed exclusively by the quinic or caffeic acid moiety of CQAs. The presence of isomers was demonstrated by liquid chromatography online coupled to ESI-MS and ESI-MS(n). Pent1-2CQA were identified in coffee samples. These results give evidence for a diversity of chlorogenic acid-arabinose hybrids formed during roasting, opening new perspectives for their identification in melanoidin structures.

Monosaccharide identification as a first step toward de novo carbohydrate sequencing: mass spectrometry strategy for the identification and differentiation of diastereomeric and enantiomeric pentose isomers.

De novo carbohydrate sequencing, including monosaccharide identification, largely remains a tremendous analytical challenge. A first step in the complete structural determination of any large polysaccharide is an accurate and robust method for analysis of the constituent monosaccharides. Herein, the first mass spectrometry-based method for the complete identification and absolute configuration determination of all 12 pentose isomers, including the d and l enantiomers for arabinose, lyxose, ribose, xylose, ribulose, and xylulose, is reported. As compared to earlier work to distinguish hexose isomers, the chiral separation of the pentose isomers was significantly more challenging. Specifically, the 12 pentoses are much more structurally similar to one another, with only the axial or equatorial orientation of two hydroxyl groups differentiating among these isomers in their five-membered ring furanose structure and smaller energetic differences between pentose conformations than between hexose conformations. Despite such inherently minimal energetic differences between the 12 pentoses, two unique fixed ligand kinetic method combinations were discovered to achieve chiral discrimination for this set of isomers. This assay can be readily applied to the identification of any isolated pentose monosaccharide using only microgram quantities and a commercial instrument and complements the method to distinguish hexose isomers. A workflow that incorporates this mass spectrometry-based method and thereby could achieve complete de novo identification of all monosaccharide building blocks in an oligo- or polysaccharide is proposed.

Selective detection of epimeric pentose saccharides at physiological pH using a fluorescent receptor.

Epimerisation between ribofuranose and arabinofuranose sugars is crucial in several biosynthetic pathways, but is typically challenging to monitor. Here, we have screened for fluorescent boronic acids that can be used as molecular probes for the specific detection of ribofuranose over arabinofuranose sugars in solution. We show excellent specificity of the fluorescent response of 3-biphenylboronic acid to ribofuranose at physiological pH. This provides a tool for in situ monitoring of carbohydrate modifying enzymes and provides a viable alternative to traditional radiolabelled assays.

Optimization of fermentable sugar production from rape straw through hydrothermal acid pretreatment.

Operational conditions for the hydrolysis of rape straw were optimized using the combined severity index (CS), which combines the effects of time, temperature, and acid concentration into a single parameter. The sugar recovery yield was 77.8% of the theoretical yield at a value of CS=1.3. A maximum concentration of xylose of 7.22 g/L was obtained when the straw was treated for 10 min at a low reaction temperature (150 °C) and high acid concentration (pH 1.17). The pentose-rich hydrolyzate exhibited a low concentration of fermentation-inhibiting compounds. The concept of CS can be conveniently and effectively applied for optimization of pretreatments.

Correlating changes that occur in chemical properties with the generation of antioxidant capacity in different sugar-amino acid Maillard reaction models.

UNLABELLED: We investigated the development of antioxidant activity relative to the change of pH, fluorescent intensity, ultraviolet (UV) absorbance (A294), browning (A420), and alpha-dicarbonyl compounds in sugar-amino acid Maillard reaction (MR) model systems comprising fructose, glucose, or ribose each with glycine (Fru-Gly, Glu-Gly, and Rib-Gly) or lysine (Fru-Lys, Glu-Lys, and Rib-Lys), respectively, which were heated at 121 °C for 5 to 90 min. For hexose models, the change in pH was shown to fit a second-order polynomial regression with A294 and A420. Antioxidant activity was significantly and positively correlated with UV absorbance (r = 0.905, P < 0.001) and browning products (r = 0.893, P < 0.001) rather than with fluorescent products or the alpha-dicarbonyl compounds. Type of sugar was most important in evoking a change in UV absorbance, browning, alpha-dicarbonyl compounds, and antioxidant activity of MR products (MRPs). In conclusion, the antioxidant activity of MRPs in six model systems was more closely associated with products derived at the intermediate-to-late stages of the reaction and influenced mostly by the type of sugar. PRACTICAL APPLICATION: We report on the different factors and their interactions that are important for understanding the functional attributes of food components that comprise the generation of Maillard browning products and the associated antioxidant activities generated during high-temperature food processing.

Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis.

Rice straw is one of the abundant lignocellulosic feed stocks in the world and has been selected for producing ethanol at an economically feasible manner. It contains a mixture of sugars (hexoses and pentoses). Biphasic acid hydrolysis was carried out with sulphuric acid using rice straw. After acid hydrolysis, the sugars, furans and phenolics were estimated. The initial concentration of sugar was found to be 16.8 g L(-1). However to increase the ethanol yield, the initial sugar concentration of the hydrolysate was concentrated to 31 g L(-1) by vacuum distillation. The concentration of sugars, phenols and furans was checked and later detoxified by over liming to use for ethanol fermentation. Ethanol concentration was found to be 12 g L(-1), with a yield, volumetric ethanol productivity and fermentation efficiency of 0.33 g L(-1)h(-1), 0.4 g g(-1) and 95%, respectively by co-culture of OVB 11 (Saccharomyces cerevisiae) and Pichia stipitis NCIM 3498.

Structural characterization and cytotoxic properties of an apiose-rich pectic polysaccharide obtained from the cell wall of the marine phanerogam Zostera marina.

Zosterin, an apiose-rich pectic polysaccharide, was extracted and purified from the sea grass Zostera marina. Structural studies conducted by gas chromatography and NMR spectroscopy on a purified zosterin fraction (AGU) revealed a typical apiogalacturonan structure comprising an alpha-1,4-d-galactopyranosyluronan backbone substituted by 1,2-linked apiofuranose oligosaccharides and single apiose residues. The average molecular mass of AGU was estimated to be about 4100 Da with a low polydispersity. AGU inhibited proliferation of A431 human epidermoid carcinoma cells with an approximate IC(50) value of 3 microg/mL (0.7 microM). In addition, AGU inhibited A431 cell migration and invasion. Preliminary experiments showed that inhibition of metalloproteases expression could play a role in these antimigration and anti-invasive properties. Autohydrolysis of AGU, which eliminated apiose and oligo-apiose substituents, led to a virtual disappearance of cytotoxic properties, thus suggesting a direct structure-function relationship with the apiose-rich hairy region of AGU.

Organ-specific defence strategies of pepper (Capsicum annuum L.) during early phase of water deficit.

Drought is one of the major factors that limits crop production and reduces yield. To understand the early response of plants under nearly natural conditions, pepper plants (Capsicum annuum L.) were grown in a greenhouse and stressed by withholding water for 1 week. Plants adapted to the decreasing water content of the soil by adjustment of their osmotic potential in root tissue. As a consequence of drought, strong accumulation of raffinose, glucose, galactinol and proline was detected in the roots. In contrast, in leaves the levels of fructose, sucrose and also galactinol increased. Due to the water deficit cadaverine, putrescine, spermidine and spermine accumulated in leaves, whereas the concentration of polyamines was reduced in roots. To study the molecular basis of these responses, a combined approach of suppression subtractive hybridisation and microarray technique was performed on the same material. A total of 109 unique ESTs were detected as responsive to drought, while additional 286 ESTs were selected from the bulk of rare transcripts on the array. The metabolic profiles of stressed pepper plants are discussed with respect to the transcriptomic changes detected, while attention is given to the differences between defence strategies of roots and leaves.

Structure and bioactivity of the polysaccharides in medicinal plant Dendrobium huoshanense.

Detailed structures of the active polysaccharides extracted from the leaf and stem cell walls and mucilage of Dendrobium huoshanense are determined by using various techniques, including chromatographic, spectroscopic, chemical, and enzymatic methods. The mucilage polysaccharide exhibits specific functions in activating murine splenocytes to produce several cytokines including IFN-gamma, IL-10, IL-6, and IL-1alpha, as well as hematopoietic growth factors GM-CSF and G-CSF. However, the deacetylated mucilage obtained from an alkaline treatment fails to induce cytokine production. The structure and bioactivity of mucilage components are validated by further fractionation. This is the first study that provides clear evidence for the structure and activity relationship of the polysaccharide in D. huoshanense.