Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method.

Cool-season pasture grasses contain arabinoxylans (AX) as their major cell wall hemicellulosic polysaccharide. AX structural differences may influence enzymatic degradability, but this relationship has not been fully explored in the AX from the vegetative tissues of cool-season forages, primarily because only limited AX structural characterization has been performed in pasture grasses. Structural profiling of forage AX is a necessary foundation for future work assessing enzymatic degradability and may also be useful for assessing forage quality and suitability for ruminant feed. The main objective of this study was to optimize and validate a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method for the simultaneous quantification of 10 endoxylanase-released xylooligosaccharides (XOS) and arabinoxylan oligosaccharides (AXOS) in cool-season forage cell wall material. The following analytical parameters were determined or optimized: chromatographic separation and retention time (RT), internal standard suitability, working concentration range (CR), limit of detection (LOD), limit of quantification (LOQ), relative response factor (RRF), and quadratic calibration curves. The developed method was used to profile the AX structure of four cool-season grasses commonly grown in pastures (timothy, Phleum pratense L.; perennial ryegrass, Lolium perenne L.; tall fescue, Schedonorus arundinaceus (Schreb.) Dumort.; and Kentucky bluegrass, Poa pratensis L.). In addition, the cell wall monosaccharide and ester-linked hydroxycinnamic acid contents were determined for each grass. The developed method revealed unique structural aspects of the AX structure of these forage grass samples that complemented the results of the cell wall monosaccharide analysis. For example, xylotriose, representing an unsubstituted portion of the AX polysaccharide backbone, was the most abundantly-released oligosaccharide in all the species. Perennial rye samples tended to have greater amounts of released oligosaccharides compared to the other species. This method is ideally suited to monitor structural changes of AX in forages as a result of plant breeding, pasture management, and fermentation of plant material.

Improved methods for mare milk analysis: Extraction and quantification of mare milk carbohydrates and assessment of FTIR-based macronutrient quantification.

Accurately determining the macronutrient profile of mare milk is a precursor to studying how milk composition affects foals' growth and development. This study optimized and validated an extraction and quantification method for mare milk oligosaccharides, which make up a portion of the carbohydrate fraction of mare milk. Mare milk was extracted with chloroform and methanol, and oligosaccharides were selectively isolated from the carbohydrate fraction using porous-graphitized carbon solid-phase-extraction (SPE). Good recovery rates for milk oligosaccharides (between 70 and 100%) were achieved with the optimized method. This study also compared the use of Fourier-Transform infrared (FTIR) spectroscopy versus wet chemistry quantification methods for protein, fat, and lactose. The FTIR method produced statistically equivalent protein contents to the wet chemistry method, along with substantial savings in both analyst time and consumable consumption. FTIR analysis slightly underestimated the fat content of mare milk relative to the official wet chemistry method, with the difference between the methods increasing at higher fat contents. FTIR also overestimated the lactose content of mare milk and appeared to generate "lactose" values that included the milk oligosaccharides and thus represented the total carbohydrate (lactose and milk oligosaccharides) content of mare milk.

2D-HSQC-NMR-Based Screening of Feruloylated Side-Chains of Cereal Grain Arabinoxylans.

Arabinoxylans of commelinid monocots are characterized by high contents of ferulic acid that is incorporated into arabinose-bearing side-chains of varying complexity. Species-related differences in the feruloylated side-chain profiles of grain arabinoxylans are observed and lead to differences in arabinoxylan functionality. Here, a semi-quantitative assay based on 1H-13C-correlation NMR spectroscopy (HSQC experiment) was developed to profile feruloylated side-chains of cereal grain arabinoxylans. Following acidic liberation of the feruloylated side-chains from the xylan backbone and a clean-up step using C18 solid phase extraction, the feruloylated oligosaccharides FA (5-O-trans-feruloyl-L-arabinofuranose), FAX (ß-d-xylopyranosyl-(1 → 2)-5-O-trans-feruloyl-l-arabinofuranose) and FAXG (α-l-galactopyranosyl-(1 → 2)-ß-d-xylopyranosyl-(1 → 2)-5-O-trans-feruloyl-l-arabinofuranose) were analyzed by HSQC-NMR. Marker signals were identified for each compound, and experimental conditions such as solvent and internal standard as well as measurement and processing conditions were optimized for a semi-quantitative determination. The approach was validated with respect to accuracy, precision, limit of detection, and limit of quantification. The newly developed approach was applied to several cereal samples including oats, popcorn maize, wheat, and wild rice. Data were compared to an HPLC-DAD/MS approach published earlier by our group, demonstrating that the results of the HSQC approach were comparable to the more time-consuming and technically more challenging HPLC-DAD/MS method.

Corrigendum: Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance.

[This corrects the article on p. 1903 in vol. 8, PMID: 29250082.].

Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance.

It is widely known that numerous adaptive responses of drought-stressed plants are stimulated by chemical messengers known as phytohormones. Jasmonic acid (JA) is one such phytohormone. But there are very few reports revealing its direct implication in drought related responses or its cross-talk with other phytohormones. In this study, we compared the morpho-physiological traits and the root proteome of a wild type (WT) rice plant with its JA biosynthesis mutant coleoptile photomorphogenesis 2 (cpm2), disrupted in the allene oxide cyclase (AOC) gene, for insights into the role of JA under drought. The mutant had higher stomatal conductance, higher water use efficiency and higher shoot ABA levels under severe drought as compared to the WT. Notably, roots of cpm2 were better developed compared to the WT under both, control and drought stress conditions. Root proteome was analyzed using the Tandem Mass Tag strategy to better understand this difference at the molecular level. Expectedly, AOC was unique but notably highly abundant under drought in the WT. Identification of other differentially abundant proteins (DAPs) suggested increased energy metabolism (i.e., increased mobilization of resources) and reactive oxygen species scavenging in cpm2 under drought. Additionally, various proteins involved in secondary metabolism, cell growth and cell wall synthesis were also more abundant in cpm2 roots. Proteome-guided transcript, metabolite, and histological analyses provided further insights into the favorable adaptations and responses, most likely orchestrated by the lack of JA, in the cpm2 roots. Our results in cpm2 are discussed in the light of JA crosstalk to other phytohormones. These results together pave the path for understanding the precise role of JA during drought stress in rice.

Biochemical and Structural Analyses of Two Cryptic Esterases in Bacteroides intestinalis and their Synergistic Activities with Cognate Xylanases.

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s-1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.

Perennial Grain and Oilseed Crops.

Historically, agroecosystems have been designed to produce food. Modern societies now demand more from food systems-not only food, fuel, and fiber, but also a variety of ecosystem services. And although today's farming practices are producing unprecedented yields, they are also contributing to ecosystem problems such as soil erosion, greenhouse gas emissions, and water pollution. This review highlights the potential benefits of perennial grains and oilseeds and discusses recent progress in their development. Because of perennials' extended growing season and deep root systems, they may require less fertilizer, help prevent runoff, and be more drought tolerant than annuals. Their production is expected to reduce tillage, which could positively affect biodiversity. End-use possibilities involve food, feed, fuel, and nonfood bioproducts. Fostering multidisciplinary collaborations will be essential for the successful integration of perennials into commercial cropping and food-processing systems.

Structural Transformation of 8-5-Coupled Dehydrodiferulates by Human Intestinal Microbiota.

Ingested dehydrodiferulates (DFAs) are partially released from cereal dietary fiber by human colonic microbiota, but little research has explored the further microbial metabolism of 8-5-coupled DFAs. This study investigated the in vitro microbial metabolism and elucidated major metabolites of free 8-5-DFAs (benzofuran and open forms) and an esterified analogue, 8-5-DFA diethyl ester (benzofuran). Synthesized standard compounds were incubated with fresh human fecal suspensions. Metabolites were isolated and structurally elucidated using high-resolution-LC-time-of-flight-(ToF)-MS, GC-MS, and NMR. Nine metabolite structures were unambiguously characterized with NMR, and four additional metabolites were tentatively identified to reveal structural conversion motifs: propenyl side chain hydrogenation (all substrates), O-demethylation and reductive ring-opening (8-5-DFA diethyl ester and free 8-5-DFA [benzofuran]), and de-esterification (8-5-DFA diethyl ester). A pathway of microbial 8-5-DFA metabolism was proposed based on metabolite formation kinetics. Importantly, de-esterification of the 8-5-DFA diethyl ester occurred primarily after and/or concurrently with other metabolism steps. Cleavage to monomers was not observed.

Isolation and characterization of feruloylated arabinoxylan oligosaccharides from the perennial cereal grain intermediate wheat grass (Thinopyrum intermedium).

In comparison to the annual grain crops dominating current agricultural production, perennial grain species require fewer chemical and energy inputs and improve soil health and erosion control. The possibility for producing sustainable grain harvests from marginal land areas is motivating research initiatives to integrate perennial grains into commercial cropping and food processing systems. In this study, the feruloylated arabinoxylans from intermediate wheat grass (Thinopyrum intermedium, IWG), a promising perennial grain candidate in agronomic screening studies, were investigated. Insoluble fiber isolated from IWG whole grain flour was subjected to either mildly acidic (50 mM TFA, 100 °C, 2 h) or enzymatic (Driselase) hydrolysis. The liberated feruloylated arabinoxylan oligosaccharides were concentrated with Amberlite XAD-2, separated with gel chromatography (Sephadex LH-20, water), and purified with reversed-phase HPLC (C18, water-MeOH gradient). Thirteen feruloylated oligosaccharides were isolated (including eight structures described for the first time) and identified by LC-ESI-MS and NMR. Linkage-type analysis via methylation analysis, as well as the monosaccharide and phenolic acid profiles of the IWG insoluble fiber were also determined. IWG feruloylated arabinoxylans have a relatively simple structure with only short feruloylated side chains, a lower backbone substitution rate than annual rye and wheat varieties, and a moderate phenolic acid content.

Quantitative Profiling of Feruloylated Arabinoxylan Side-Chains from Graminaceous Cell Walls.

Graminaceous arabinoxylans are distinguished by decoration with feruloylated monosaccharidic and oligosaccharidic side-chains. Although it is hypothesized that structural complexity and abundance of these feruloylated arabinoxylan side-chains may contribute, among other factors, to resistance of plant cell walls to enzymatic degradation, quantitative profiling approaches for these structural units in plant cell wall materials have not been described yet. Here we report the development and application of a rapid and robust method enabling the quantitative comparison of feruloylated side-chain profiles in cell wall materials following mildly acidic hydrolysis, C18-solid phase extraction (SPE), reduction under aprotic conditions, and liquid chromatography with diode-array detection/mass spectrometry (LC-DAD/MS) separation and detection. The method was applied to the insoluble fiber/cell wall materials isolated from 12 whole grains: wild rice (Zizania aquatica L.), long-grain brown rice (Oryza sativa L.), rye (Secale cereale L.), kamut (Triticum turanicum Jakubz.), wheat (Triticum aestivum L.), spelt (Triticum spelta L.), intermediate wheatgrass (Thinopyrum intermedium), maize (Zea mays L.), popcorn (Zea mays L. var. everta), oat (Avena sativa L.) (dehulled), barley (Hordeum vulgare L.) (dehulled), and proso millet (Panicum miliaceum L.). Between 51 and 96% of the total esterified monomeric ferulates were represented in the quantified compounds captured in the feruloylated side-chain profiles, which confirms the significance of these structures to the global arabinoxylan structure in terms of quantity. The method provided new structural insights into cereal grain arabinoxylans, in particular, that the structural moiety α-l-galactopyranosyl-(1→2)-ß-d-xylopyranosyl-(1→2)-5-O-trans-feruloyl-l-arabinofuranose (FAXG), which had previously only been described in maize, is ubiquitous to cereal grains.