A cell wall reference profile for Miscanthus bioenergy crops highlights compositional and structural variations associated with development and organ origin.

Miscanthus spp. are promising lignocellulosic energy crops, but cell wall recalcitrance to deconstruction still hinders their widespread use as bioenergy and biomaterial feedstocks. Identification of cell wall characteristics desirable for biorefining applications is crucial for lignocellulosic biomass improvement. However, the task of scoring biomass quality is often complicated by the lack of a reference for a given feedstock. A multidimensional cell wall analysis was performed to generate a reference profile for leaf and stem biomass from several miscanthus genotypes harvested at three developmentally distinct time points. A comprehensive suite of 155 monoclonal antibodies was used to monitor changes in distribution, structure and extractability of noncellulosic cell wall matrix glycans. Glycan microarrays complemented with immunohistochemistry elucidated the nature of compositional variation, and in situ distribution of carbohydrate epitopes. Key observations demonstrated that there are crucial differences in miscanthus cell wall glycomes, which may impact biomass amenability to deconstruction. For the first time, variations in miscanthus cell wall glycan components were comprehensively characterized across different harvests, organs and genotypes, to generate a representative reference profile for miscanthus cell wall biomass. Ultimately, this portrait of the miscanthus cell wall will help to steer breeding and genetic engineering strategies for the development of superior energy crops.

A metabolomic study in oats (Avena sativa) highlights a drought tolerance mechanism based upon salicylate signalling pathways and the modulation of carbon, antioxidant and photo-oxidative metabolism.

Although a wealth of information is available on the induction of one or several drought-related responses in different species, little is known of how their timing, modulation and crucially integration influence drought tolerance. Based upon metabolomic changes in oat (Avena sativa L.), we have defined key processes involved in drought tolerance. During a time course of increasing water deficit, metabolites from leaf samples were profiled using direct infusion-electrospray mass spectroscopy (DI-ESI-MS) and high-performance liquid chromatography (HPLC) ESI-MS/MS and analysed using principal component analysis (PCA) and discriminant function analysis (DFA). The involvement of metabolite pathways was confirmed through targeted assays of key metabolites and physiological experiments. We demonstrate an early accumulation of salicylic acid (SA) influencing stomatal opening, photorespiration and antioxidant defences before any change in the relative water content. These changes are likely to maintain plant water status, with any photoinhibitory effect being counteracted by an efficient antioxidant capacity, thereby representing an integrated mechanism of drought tolerance in oats. We also discuss these changes in relation to those engaged at later points, consequence of the different water status in susceptible and resistant genotypes.

Polyphenol oxidase in leaves: is there any significance to the chloroplastic localization?

Polyphenol oxidase (PPO) catalyses the oxidation of monophenols and/or o-diphenols to o-quinones with the concomitant reduction of oxygen to water which results in protein complexing and the formation of brown melanin pigments. The most frequently suggested role for PPO in plants has been in defence against herbivores and pathogens, based on the physical separation of the chloroplast-localized enzyme from the vacuole-localized substrates. The o-quinone-protein complexes, formed as a consequence of cell damage, may reduce the nutritional value of the tissue and thereby reduce predation but can also participate in the formation of structural barriers against invading pathogens. However, since a sufficient level of compartmentation-based regulation could be accomplished if PPO was targeted to the cytosol, the benefit derived by some plant species in having PPO present in the chloroplast lumen remains an intriguing question. So is there more to the chloroplastic location of PPO? An interaction between PPO activity and photosynthesis has been proposed on more than one occasion but, to date, evidence either for or against direct involvement has been equivocal, and the lack of identified chloroplastic substrates remains an issue. Similarly, PPO has been suggested to have both pro- and anti-oxidant functions. Nevertheless, several independent lines of evidence suggest that PPO responds to environmental conditions and could be involved in the response of plants to abiotic stress. This review highlights our current understanding of the in vivo functions of PPO and considers the potential opportunities it presents for exploitation to increase stress tolerance in food crops.

Polyphenol oxidase-mediated protection against oxidative stress is not associated with enhanced photosynthetic efficiency.

BACKGROUND AND AIMS: Polyphenol oxidases (PPOs) catalyse the oxidation of monophenols and/or o-diphenols to highly reactive o-quinones, which in turn interact with oxygen and proteins to form reactive oxygen species (ROS) and typical brown-pigmented complexes. Hence PPOs can affect local levels of oxygen and ROS. Although the currently known substrates are located in the vacuole, the enzyme is targeted to the thylakoid lumen, suggesting a role for PPOs in photosynthesis. The current study was designed to investigate the potential involvement of PPOs in the photosynthetic response to oxidative stress. METHODS: Photosynthesis (A, Fv/Fm, ΦPSII, qN, qP, NPQ) was measured in leaves of a wild-type and a low-PPO mutant of red clover (Trifolium pratense 'Milvus') under control conditions and under a stress treatment designed to induce photooxidative stress: cold/high light (2 °C/580 µmol m(2 )s(-1)) or 0-10 µm methyl viologen. Foliar protein content and oxidation state were also determined. KEY RESULTS: Photosynthetic performance, and chlorophyll and protein content during 4 d of cold/high light stress and 3 d of subsequent recovery under control growth conditions showed similar susceptibility to stress in both lines. However, more extensive oxidative damage to protein in mutants than wild-types was observed after treatment of attached leaves with methyl viologen. In addition, PPO activity could be associated with an increased capacity to dissipate excess energy, but only at relatively low methyl viologen doses. CONCLUSIONS: The presence of PPO activity in leaves did not correspond to a direct role for the enzyme in the regulation or protection of photosynthesis under cold stress. However, an indication that PPO could be involved in cellular protection against low-level oxidative stress requires further investigation.

Genotype, development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus.

BACKGROUND AND AIMS: Species and hybrids of the genus Miscanthus contain attributes that make them front-runners among current selections of dedicated bioenergy crops. A key trait for plant biomass conversion to biofuels and biomaterials is cell-wall quality; however, knowledge of cell-wall composition and biology in Miscanthus species is limited. This study presents data on cell-wall compositional changes as a function of development and tissue type across selected genotypes, and considers implications for the development of miscanthus as a sustainable and renewable bioenergy feedstock. METHODS: Cell-wall biomass was analysed for 25 genotypes, considering different developmental stages and stem vs. leaf compositional variability, by Fourier transform mid-infrared spectroscopy and lignin determination. In addition, a Clostridium phytofermentans bioassay was used to assess cell-wall digestibility and conversion to ethanol. KEY RESULTS: Important cell-wall compositional differences between miscanthus stem and leaf samples were found to be predominantly associated with structural carbohydrates. Lignin content increased as plants matured and was higher in stem tissues. Although stem lignin concentration correlated inversely with ethanol production, no such correlation was observed for leaves. Leaf tissue contributed significantly to total above-ground biomass at all stages, although the extent of this contribution was genotype-dependent. CONCLUSIONS: It is hypothesized that divergent carbohydrate compositions and modifications in stem and leaf tissues are major determinants for observed differences in cell-wall quality. The findings indicate that improvement of lignocellulosic feedstocks should encompass tissue-dependent variation as it affects amenability to biological conversion. For gene-trait associations relating to cell-wall quality, the data support the separate examination of leaf and stem composition, as tissue-specific traits may be masked by considering only total above-ground biomass samples, and sample variability could be mostly due to varying tissue contributions to total biomass.

Bacterial endophytic community composition varies by hemp cultivar in commercially sourced seed.

The seed-endophytic bacterial community is a potentially beneficial and heritable fraction of the plant microbiome. Its utilization as a sustainable crop improvement strategy could be especially valuable for species such as hemp, where production is being scaled up and new challenges will be faced in managing crop productivity and health. However, little is known about the makeup and variation of the hemp seed microbiome. This study profiled the endophytic bacterial communities harboured by 16 hemp cultivars sourced from commercial suppliers in Europe. A 16S rDNA amplicon sequencing approach identified 917 amplicon sequence variants across samples. Taxonomic classification of sequences revealed 4 phyla and 87 genera to be represented in the dataset. Several genera were widespread while some were specific to one or a few cultivars. Flavobacterium, Pseudomonas, and Pantoea were notable in their high overall abundance and prevalence, but community composition was variable and no one taxon was universally abundant, suggesting a high degree of flexibility in community assembly. Taxonomic composition and alpha diversity differed among cultivars, though further work is required to understand the relative influence of hemp genetic factors on community structure. The taxonomic profiles presented here can be used to inform further work investigating the functional characteristics and potential plant-growth-promoting traits of seed-borne bacteria in hemp.

Optimising parameters for pilot scale steam explosion and continuous pressurised disc refining of Miscanthus and sugarcane bagasse for xylose and xylo-oligosaccharide release.

The first comparative pre-treatment study of Miscanthus (Mxg) and sugarcane bagasse (SCB) using steam explosion (SE) and pressurised disc refining (PDR) pretreatment to optimise xylose and xylo-oligosaccharide release is described. The current investigation aimed to 1) Develop optimised batch-wise steam explosion parameters for Mxg and SCB, 2) Scale from static batch steam explosion to dynamic continuous pressurised disc refining, 3) Identify, understand, and circumvent scale-up production hurdles. Optimised SE parameters released 82% (Mxg) and 100% (SCB) of the available xylan. Scaling to PDR, Miscanthus yielded 85% xylan, highlighting how robust scouting assessments for boundary process parameters can result in successful technical transfer. In contrast, SCB technical transfer was not straightforward, with significant differences observed between the two processes, 100% (SE) and 58% (PDR). This report underlines the importance of feedstock-specific pretreatment strategies to underpin process development, scale-up, and optimisation of carbohydrate release from biomass.

Latitudinal variation in ambient UV-B radiation is an important determinant of Lolium perenne forage production, quality, and digestibility.

Few studies to date have considered the responses of agriculturally important forage grasses to UV-B radiation. Yet grasses such as Lolium perenne have a wide current distribution, representing exposure to a significant variation in ambient UV-B. The current study investigated the responses of L. perenne (cv. AberDart) to a simulated latitudinal gradient of UV-B exposure, representing biologically effective UV-B doses at simulated 70, 60, 50, 40, and 30° N latitudes. Aspects of growth, soluble compounds, and digestibility were assessed, and results are discussed in relation to UV-B effects on forage properties and the implications for livestock and bio-ethanol production. Aboveground biomass production was reduced by approximately 12.67% with every 1 kJ m(-2) day(-1) increase in biologically weighted UV-B. As a result, plants grown in the highest UV-B treatment had a total biomass of just 13.7% of controls. Total flavonoids were increased by approximately 76% by all UV-B treatments, while hydroxycinnamic acids increased in proportion to the UV-B dose. Conversely, the digestibility of the aboveground biomass and concentrations of soluble fructans were reduced by UV-B exposure, although soluble sucrose, glucose, and fructose concentrations were unaffected. These results highlight the capacity for UV-B to directly affect forage productivity and chemistry, with negative consequences for digestibility and bioethanol production. Results emphasize the need for future development and distribution of L. perenne varieties to take UV-B irradiance into consideration.

Enhanced UV-B and elevated CO(2) impacts sub-arctic shrub berry abundance, quality and seed germination.

This study investigated the effects of long-term-enhanced UV-B, and combined UV-B with elevated CO(2) on dwarf shrub berry characteristics in a sub-arctic heath community. Germination of Vaccinium myrtillus was enhanced in seeds produced at elevated UV-B, but seed numbers and berry size were unaffected. Elevated UV-B and CO(2) stimulated the abundance of V. myrtillus berries, whilst UV-B alone stimulated the berry abundance of V. vitis-idaea and Empetrum hermaphroditum. Enhanced UV-B reduced concentrations of several polyphenolics in V. myrtillus berries, whilst elevated CO(2) increased quercetin glycosides in V. myrtillus, and syringetin glycosides and anthocyanins in E. hermaphroditum berries. UV-B × CO(2) interactions were found for total anthocyanins, delphinidin-3-hexoside and peonidin-3-pentosidein in V. myrtillus berries but not E. hermaphroditum. Results suggest positive impacts of UV-B on the germination of V. myrtillus and species-specific impacts of UV-B × elevated CO(2) on berry abundance and quality. The findings have relevance and implications for human and animal consumers plus seed dispersal and seedling establishment.

Isolation and Characterisation of Quercitrin as a Potent Anti-Sickle Cell Anaemia Agent from Alchornea cordifolia.

Alchornea cordifolia Müll. Arg. (commonly known as Christmas Bush) has been used traditionally in Africa to treat sickle cell anaemia (a recessive disease, arising from the S haemoglobin (Hb) allele), but the active compounds are yet to be identified. Herein, we describe the use of sequential fractionation coupled with in vitro anti-sickling assays to purify the active component. Sickling was induced in HbSS genotype blood samples using sodium metabisulphite (Na2S2O5) or through incubation in 100% N2. Methanol extracts of A. cordifolia leaves and its sub-fractions showed >70% suppression of HbSS erythrocyte sickling. The purified compound demonstrated a 87.2 ± 2.39% significant anti-sickling activity and 93.1 ± 2.69% erythrocyte sickling-inhibition at 0.4 mg/mL. Nuclear magnetic resonance (NMR) spectra and high-resolution mass spectroscopy identified it as quercitrin (quercetin 3-rhamnoside). Purified quercitrin also inhibited the polymerisation of isolated HbS and stabilized sickle erythrocytes membranes. Metabolomic comparisons of blood samples using flow-infusion electrospray-high resolution mass spectrometry indicated that quercitrin could convert HbSS erythrocyte metabolomes to be like HbAA. Sickling was associated with changes in antioxidants, anaerobic bioenergy, and arachidonic acid metabolism, all of which were reversed by quercitrin. The findings described could inform efforts directed to the development of an anti-sickling drug or quality control assessments of A. cordifolia preparations.

Biorefining Potential of Wild-Grown Arundo donax, Cortaderia selloana and Phragmites australis and the Feasibility of White-Rot Fungi-Mediated Pretreatments.

Arundo donax, Cortaderia selloana and Phragmites australis are high-biomass-producing perennial Poalean species that grow abundantly and spontaneously in warm temperate regions, such as in Mediterranean-type climates, like those of Southern Europe, Western United States coastal areas, or in regions of South America, South Africa and Australia. Given their vigorous and spontaneous growth, biomass from the studied grasses often accumulates excessively in unmanaged agro-forestry areas. Nonetheless, this also creates the demand and opportunity for the valorisation of these biomass sources, particularly their cell wall polymers, for biorefining applications. By contrast, a related crop, Miscanthus × giganteus, is a perennial grass that has been extensively studied for lignocellulosic biomass production, as it can grow on low-input agricultural systems in colder climates. In this study Fourier transform mid-infrared spectroscopy (FTIR), high-performance anion-exchange chromatography (HPAEC) and lignin content determinations were used for a comparative compositional characterisation of A. donax, C. selloana and P. australis harvested from the wild, in relation to a trial field-grown M. × giganteus high-yielding genotype. A high-throughput saccharification assay showed relatively high sugar release values from the wild-grown grasses, even with a 0.1M NaOH mild alkali pretreatment. In addition to this alkaline pretreatment, biomass was treated with white-rot fungi (WRF), which preferentially degrade lignin more readily than holocellulose. Three fungal species were used: Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor. Our results showed that neutral sugar contents are not significantly altered, while some lignin is lost during the pretreatments. Furthermore, sugar release upon enzymatic saccharification was enhanced, and this was dependent on the plant biomass and fungal species used in the treatment. To maximise the potential for lignocellulose valorisation, the liquid fractions from the pretreatments were analysed by high performance liquid chromatography - photodiode array detection - electrospray ionisation tandem mass spectrometry (HPLC-PDA-ESI-MS n ). This study is one of the first to report on the composition of WRF-treated grass biomass, while assessing the potential relevance of breakdown products released during the treatments, beyond more traditional sugar-for-energy applications. Ultimately, we expect that our data will help promote the valorisation of unused biomass resources, create economic value, while contributing to the implementation of sustainable biorefining systems.

Identifying Anti-Oxidant Biosynthesis Genes in Pearl Millet [Pennisetum glaucum (L.) R. Br.] Using Genome-Wide Association Analysis.

Pearl millet [Pennisetum glaucum (L.) R Br.] is an important staple food crop in the semi-arid tropics of Asia and Africa. It is a cereal grain that has the prospect to be used as a substitute for wheat flour for celiac patients. It is an important antioxidant food resource present with a wide range of phenolic compounds that are good sources of natural antioxidants. The present study aimed to identify the total antioxidant content of pearl millet flour and apply it to evaluate the antioxidant activity of its 222 genotypes drawn randomly from the pearl millet inbred germplasm association panel (PMiGAP), a world diversity panel of this crop. The total phenolic content (TPC) significantly correlated with DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity (% inhibition), which ranged from 2.32 to 112.45% and ferric-reducing antioxidant power (FRAP) activity ranging from 21.68 to 179.66 (mg ascorbic acid eq./100 g). Genome-wide association studies (GWAS) were conducted using 222 diverse accessions and 67 K SNPs distributed across all the seven pearl millet chromosomes. Approximately, 218 SNPs were found to be strongly associated with DPPH and FRAP activity at high confidence [-log (p) > 3.0-7.4]. Furthermore, flanking regions of significantly associated SNPs were explored for candidate gene harvesting. This identified 18 candidate genes related to antioxidant pathway genes (flavanone 7-O-beta-glycosyltransferase, GDSL esterase/lipase, glutathione S-transferase) residing within or near the association signal that can be selected for further functional characterization. Patterns of genetic variability and the associated genes reported in this study are useful findings, which would need further validation before their utilization in molecular breeding for high antioxidant-containing pearl millet cultivars.

Feeding behavior, life history, and virus transmission ability of Bemisia tabaci Mediterranean species (Hemiptera: Aleyrodidae) under elevated CO2.

The continuous rise of CO2 concentrations in the atmosphere is reducing plant nutritional quality for herbivores and indirectly affects their performance. The whitefly (Bemisia tabaci, Gennadius) is a major worldwide pest of agricultural crops causing significant yield losses. This study investigated the plant-mediated indirect effects of elevated CO2 on the feeding behavior and life history of B. tabaci Mediterranean species. Eggplants were grown under elevated and ambient CO2 concentrations for 3 weeks after which plants were either used to monitor the feeding behavior of whiteflies using the Electrical Penetration Graph technique or to examine fecundity and fertility of whiteflies. Plant leaf carbon, nitrogen, phenols and protein contents were also analyzed for each treatment. Bemisia tabaci feeding on plants exposed to elevated CO2 showed a longer phloem ingestion and greater fertility compared to those exposed to ambient CO2 suggesting that B. tabaci is capable of compensating for the plant nutritional deficit. Additionally, this study looked at the transmission of the virus Tomato yellow leaf curl virus (Begomovirus) by B. tabaci exposing source and receptor tomato plants to ambient or elevated CO2 levels before or after virus transmission tests. Results indicate that B. tabaci transmitted the virus at the same rate independent of the CO2 levels and plant treatment. Therefore, we conclude that B. tabaci Mediterranean species prevails over the difficulties that changes in CO2 concentrations may cause and it is predicted that under future climate change conditions, B. tabaci would continue to be considered a serious threat for agriculture worldwide.

Pilot-scale production of xylo-oligosaccharides and fermentable sugars from Miscanthus using steam explosion pretreatment.

This study investigated pilot-scale production of xylo-oligosaccharides (XOS) and fermentable sugars from Miscanthus using steam explosion (SE) pretreatment. SE conditions (200 °C; 15 bar; 10 min) led to XOS yields up to 52 % (w/w of initial xylan) in the hydrolysate. Liquid chromatography-mass spectrometry demonstrated that the solubilised XOS contained bound acetyl- and hydroxycinnamate residues, physicochemical properties known for high prebiotic effects and anti-oxidant activity in nutraceutical foods. Enzymatic hydrolysis of XOS-rich hydrolysate with commercial endo-xylanases resulted in xylobiose yields of 380 to 500 g/kg of initial xylan in the biomass after only 4 h, equivalent to ~74 to 90 % conversion of XOS into xylobiose. Fermentable glucose yields from enzymatic hydrolysis of solid residues were 8 to 9-fold higher than for untreated material. In view of an integrated biorefinery, we demonstrate the potential for efficient utilisation of Miscanthus for the production of renewable sources, including biochemicals and biofuels.

Evidence of sequestration of triclabendazole and associated metabolites by extracellular vesicles of Fasciola hepatica.

Fascioliasis is a neglected zoonotic disease that infects humans and ruminant species worldwide. In the absence of vaccines, control of fascioliasis is primarily via anthelminthic treatment with triclabendazole (TCBZ). Parasitic flatworms, including Fasciola hepatica, are active secretors of extracellular vesicles (EVs), but research has not been undertaken investigating EV anthelmintic sequestration. Adult F. hepatica were cultured in lethal and sub-lethal doses of TCBZ and its active metabolites, in order to collect EVs and evaluate their morphological characteristics, production and anthelmintic metabolite content. Transmission electron microscopy demonstrated that F. hepatica exposed to TCBZ and its metabolites produced EVs of similar morphology, compared to non-TCBZ exposed controls, even though TCBZ dose and/or TCBZ metabolite led to measurable structural changes in the treated F. hepatica tegument. qNano particle analysis revealed that F. hepatica exposed to TCBZ and its metabolites produced at least five times greater EV concentrations than non-TCBZ controls. A combined mass spectrometry and qNano particle analysis confirmed the presence of TCBZ and the TCBZ-sulphoxide metabolite in anthelmintic exposed EVs, but limited TCBZ sulphone was detectable. This data suggests that EVs released from adult F. hepatica have a biological role in the sequestration of TCBZ and additional toxic xenobiotic metabolites.

Identification of an extensive gene cluster among a family of PPOs in Trifolium pratense L. (red clover) using a large insert BAC library.

BACKGROUND: Polyphenol oxidase (PPO) activity in plants is a trait with potential economic, agricultural and environmental impact. In relation to the food industry, PPO-induced browning causes unacceptable discolouration in fruit and vegetables: from an agriculture perspective, PPO can protect plants against pathogens and environmental stress, improve ruminant growth by increasing nitrogen absorption and decreasing nitrogen loss to the environment through the animal's urine. The high PPO legume, red clover, has a significant economic and environmental role in sustaining low-input organic and conventional farms. Molecular markers for a range of important agricultural traits are being developed for red clover and improved knowledge of PPO genes and their structure will facilitate molecular breeding. RESULTS: A bacterial artificial chromosome (BAC) library comprising 26,016 BAC clones with an average 135 Kb insert size, was constructed from Trifolium pratense L. (red clover), a diploid legume with a haploid genome size of 440-637 Mb. Library coverage of 6-8 genome equivalents ensured good representation of genes: the library was screened for polyphenol oxidase (PPO) genes.Two single copy PPO genes, PPO4 and PPO5, were identified to add to a family of three, previously reported, paralogous genes (PPO1-PPO3). Multiple PPO1 copies were identified and characterised revealing a subfamily comprising three variants PPO1/2, PPO1/4 and PPO1/5. Six PPO genes clustered within the genome: four separate BAC clones could be assembled onto a predicted 190-510 Kb single BAC contig. CONCLUSION: A PPO gene family in red clover resides as a cluster of at least 6 genes. Three of these genes have high homology, suggesting a more recent evolutionary event. This PPO cluster covers a longer region of the genome than clusters detected in rice or previously reported in tomato. Full-length coding sequences from PPO4, PPO5, PPO1/5 and PPO1/4 will facilitate functional studies and provide genetic markers for plant breeding.

Desirable plant cell wall traits for higher-quality miscanthus lignocellulosic biomass.

BACKGROUND: Lignocellulosic biomass from dedicated energy crops such as Miscanthus spp. is an important tool to combat anthropogenic climate change. However, we still do not exactly understand the sources of cell wall recalcitrance to deconstruction, which hinders the efficient biorefining of plant biomass into biofuels and bioproducts. RESULTS: We combined detailed phenotyping, correlation studies and discriminant analyses, to identify key significantly distinct variables between miscanthus organs, genotypes and most importantly, between saccharification performances. Furthermore, for the first time in an energy crop, normalised total quantification of specific cell wall glycan epitopes is reported and correlated with saccharification. CONCLUSIONS: In stems, lignin has the greatest impact on recalcitrance. However, in leaves, matrix glycans and their decorations have determinant effects, highlighting the importance of biomass fine structures, in addition to more commonly described cell wall compositional features. The results of our interrogation of the miscanthus cell wall promote the concept that desirable cell wall traits for increased biomass quality are highly dependent on the target biorefining products. Thus, for the development of biorefining ideotypes, instead of a generalist miscanthus variety, more realistic and valuable approaches may come from defining a collection of specialised cultivars, adapted to specific conditions and purposes.

Shrub establishment favoured and grass dominance reduced in acid heath grassland systems cleared of invasive Rhododendron ponticum.

Rhododendron ponticum L. is a damaging invasive alien species in Britain, favouring the moist, temperate climate, and the acidic soils of upland areas. It outshades other species and is thought to create a soil environment of low pH that may be higher in phytotoxic phenolic compounds. We investigated native vegetation restoration and R. ponticum regeneration post-clearance using heathland sites within Snowdonia National Park, Wales; one site had existing R. ponticum stands and three were restoring post-clearance. Each site also had an adjacent, uninvaded control for comparison. We assessed whether native vegetation restoration was influenced post-invasion by soil chemical properties, including pH and phytotoxic compounds, using Lactuca sativa L. (lettuce) bioassays supported by liquid chromatography-mass spectroscopy (LC-MSn). Cleared sites had higher shrub and bare ground cover, and lower grass and herbaceous species cover relative to adjacent uninvaded control sites; regenerating R. ponticum was also observed on all cleared sites. No phenolic compounds associated with R. ponticum were identified in any soil water leachates, and soil leachates from cleared sites had no inhibitory effect in L. sativa germination assays. We therefore conclude that reportedly phytotoxic compounds do not influence restoration post R. ponticum clearance. Soil pH however was lower beneath R. ponticum and on cleared sites, relative to adjacent uninvaded sites. The lower soil pH post-clearance may have favoured shrub species, which are typically tolerant of acidic soils. The higher shrub cover on cleared sites may have greater ecological value than unaffected grass dominated sites, particularly given the recent decline in such valuable heathland habitats. The presence of regenerating R. ponticum on all cleared sites however highlights the critical importance of monitoring and re-treating sites post initial clearance.

Extraction, structural characterisation and evaluation of hydroxycinnamate esters of orchard grass (Dactylis glomerata) as substrates for polyphenol oxidase.

Polyphenol oxidase (PPO) activity has been reported in orchard grass (Dactylis glomerata); however, to date, no endogenous substrates have been identified. In the present study, we report the isolation and structural elucidation of PPO substrates in this species. The free phenol fraction was extracted, separated by reverse-phase chromatography and six potential substrates, including two hydroxycinnamate esters, were identified by UV spectrometry, electrospray ionisation-tandem mass spectrometry (LC-ESI-MS(n)) and 1D and 2D NMR analyses ((1)H NMR, (13)C NMR, DEPT, COSY, HMQC and HMBC). Furthermore, three caffeoylquinic acids (3-CQA, 4-CQA and 5-CQA) were identified by comparison of their spectral data (ESI-MS) with those of known compounds and literature data. Five of these compounds were demonstrated to be substrates for orchard grass PPO.

Latent and active polyphenol oxidase (PPO) in red clover (Trifolium pratense) and use of a low PPO mutant to study the role of PPO in proteolysis reduction.

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme's own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.