Valorisation Potential of Invasive Acacia dealbata, A. longifolia and A. melanoxylon from Land Clearings.

Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild alkali pretreatments. For example, in A. dealbata stems treated with alkali and P. ostreatus, saccharification yield was 702.3 nmol mg-1, which is higher than the samples treated only with alkali (608.1 nmol mg-1), and 2.9-fold higher than the non-pretreated controls (243.9 nmol mg-1). By characterising biomass and pretreatments, generated data creates value for unused biomass resources, contributing to the implementation of sustainable biorefining systems. In due course, the generated value will lead to economic incentives for landowners to cut back invasive Acacia spp. more frequently, thus reducing excess biomass, which exacerbates wildfire risk.

Nutrient and drought stress: implications for phenology and biomass quality in miscanthus.

BACKGROUND AND AIMS: The cultivation of dedicated biomass crops, including miscanthus, on marginal land provides a promising approach to the reduction of dependency on fossil fuels. However, little is known about the impact of environmental stresses often experienced on lower-grade agricultural land on cell-wall quality traits in miscanthus biomass crops. In this study, three different miscanthus genotypes were exposed to drought stress and nutrient stress, both separately and in combination, with the aim of evaluating their impact on plant growth and cell-wall properties. METHODS: Automated imaging facilities at the National Plant Phenomics Centre (NPPC-Aberystwyth) were used for dynamic phenotyping to identify plant responses to separate and combinatorial stresses. Harvested leaf and stem samples of the three miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus and Miscanthus × giganteus) were separately subjected to saccharification assays, to measure sugar release, and cell-wall composition analyses. KEY RESULTS: Phenotyping showed that the M. sacchariflorus genotype Sac-5 and particularly the M. sinensis genotype Sin-11 coped better than the M. × giganteus genotype Gig-311 with drought stress when grown in nutrient-poor compost. Sugar release by enzymatic hydrolysis, used as a biomass quality measure, was significantly affected by the different environmental conditions in a stress-, genotype- and organ-dependent manner. A combination of abundant water and low nutrients resulted in the highest sugar release from leaves, while for stems this was generally associated with the combination of drought and nutrient-rich conditions. Cell-wall composition analyses suggest that changes in fine structure of cell-wall polysaccharides, including heteroxylans and pectins, possibly in association with lignin, contribute to the observed differences in cell-wall biomass sugar release. CONCLUSIONS: The results highlight the importance of the assessment of miscanthus biomass quality measures in addition to biomass yield determinations and the requirement for selecting suitable miscanthus genotypes for different environmental conditions.

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.

Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models.

BACKGROUND: Zebrafish is a clinically-relevant model of heart regeneration. Unlike mammals, it has a remarkable heart repair capacity after injury, and promises novel translational applications. Amputation and cryoinjury models are key research tools for understanding injury response and regeneration in vivo. An understanding of the transcriptional responses following injury is needed to identify key players of heart tissue repair, as well as potential targets for boosting this property in humans. RESULTS: We investigated amputation and cryoinjury in vivo models of heart damage in the zebrafish through unbiased, integrative analyses of independent molecular datasets. To detect genes with potential biological roles, we derived computational prediction models with microarray data from heart amputation experiments. We focused on a top-ranked set of genes highly activated in the early post-injury stage, whose activity was further verified in independent microarray datasets. Next, we performed independent validations of expression responses with qPCR in a cryoinjury model. Across in vivo models, the top candidates showed highly concordant responses at 1 and 3 days post-injury, which highlights the predictive power of our analysis strategies and the possible biological relevance of these genes. Top candidates are significantly involved in cell fate specification and differentiation, and include heart failure markers such as periostin, as well as potential new targets for heart regeneration. For example, ptgis and ca2 were overexpressed, while usp2a, a regulator of the p53 pathway, was down-regulated in our in vivo models. Interestingly, a high activity of ptgis and ca2 has been previously observed in failing hearts from rats and humans. CONCLUSIONS: We identified genes with potential critical roles in the response to cardiac damage in the zebrafish. Their transcriptional activities are reproducible in different in vivo models of cardiac injury.

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.

Antioxidant Potential of Tamarillo Fruits-Chemical and Infrared Spectroscopy Analysis.

Native to South America, tamarillo (Solanum betaceum Cav.) is a small tree cultivated as a fruit crop in several regions of the world. Known for its sweet and sour taste, tamarillo fruits are very nutritious due to the presence of health-beneficial components such as fiber, vitamins, and antioxidants. Despite its nutritional value, tamarillo remains poorly known in global markets. The present work aims to study the antioxidant activity of four genotypes of tamarillo. Several chemical assays were performed to assess the antioxidant components and antioxidant activity of aqueous ethanolic extracts from each genotype. Overall, the Mealhada genotype (a red cultivar) showed the most interesting results, displaying the highest amount of total phenolic, flavonoids, and anthocyanin contents, as well as higher antioxidant activity. To evaluate the composition of the extract, Fourier-transform infrared spectroscopy (FTIR) was used to characterize important components in aqueous ethanolic extracts of the fruits, having revealed the presence of high amounts of phenols (the main compounds responsible for antioxidant activity), as well as triterpenoids and polysaccharides. The present results highlight the potential nutraceutical importance of tamarillo fruits.

Spectrochemical analysis of seasonal and sexual variation of antioxidants in Corema album (L.) D. Don leaf extracts.

Bioactive phytoconstituents have been increasingly investigated for their potential human health benefits. Corema album (L.) D. Don, an Ericaceae, reportedly has antioxidant, antimicrobial and anticancer properties. Aiming at enhancing its nutraceutical potential, we performed a spectrochemical analysis of hydroethanolic extracts from C. album leaves. We report on changes in the antioxidant activity of the extracts, as well as in the accumulation of key phytoconstituents (namely phenolic compounds), in female and male samples, throughout three harvesting seasons (February, July, and October). For each extract, the antioxidant activity was assessed by different spectrophotometric methods. Simultaneously, attenuated total reflectance Fourier transform mid-infrared spectroscopy (FTIR-ATR), and high-performance liquid chromatography - electrospray ionisation - quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS), were used to identify and monitor variations in the composition of phenolic compounds in the extracts. The main compounds identified were epicatechin, laricitrin-O-hexoside isomers, and myricetin-O-hexoside isomers. Significant differences were found in the composition and relative abundance of the compounds of interest, according to sex and season. Overall, a trend was observed whereby phenolic content and antioxidant activities were higher in males and increased between the earlier and the latest harvests. Based on these results, we may conclude that late summer or early autumn harvests are preferable when aiming at the highest yearly content of bioactive compounds. Additionally, it should be considered that extracts from male individuals typically display higher antioxidant activities. Ultimately, our understanding of C. album in the context of nutraceutical applications is benefited from the quantitative and qualitative portrait provided here, thus promoting its relevance as a source of bioactive compounds.

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.

Analysis of Plant Cell Walls by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy.

Attenuated total reflectance Fourier transform mid-infrared (ATR-FTIR) spectroscopy is widely applicable for the chemical analysis of biological materials, relatively inexpensive, requires only simple sample preparation, and is of comparatively high-throughput compared to traditional wet chemical or chromatographic methods. It is particularly well suited for the nondestructive analysis of dried and finely ground plant samples for the subsequent prediction of cell wall and other compositional or processing parameters using chemometric regression models. Furthermore, analysis of mid IR spectra by nonregression methods (e.g., principal component analysis) provides a straightforward approach for multivariate comparison of the effects of experimental, processing, and environmental treatments, and genotypic and temporal differences on chemical composition including changes in cell wall composition. There is thus great potential for using ATR-FTIR in the lignocellulosic biomass industry at a number of levels. Here we describe methods for cell wall sample preparation and generation of ATR-FTIR spectra, and suggest techniques for the statistical analysis and/or chemometric pattern recognition between the analyzed samples.

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.

In vitro cytotoxicity and structure-activity relationship approaches of ent-kaurenoic acid derivatives against human breast carcinoma cell line.

In this study, ent-kaurenoic acid derivatives were obtained by microbial transformation methodologies and tested against breast cancer cell lines (MCF-7). A multivariate quantitative-structure activity relationship (QSAR) analysis was performed taking into account both microbial transformation derivatives and other analogues previously reported in literature to give some insight into the main features behind the cytotoxic activity displayed by kaurane-type diterpenes against MCF-7 cells. The partial least square regression (PLS) method was employed in the training set and the best PLS model was built with a factor describing 69.92% of variance and three descriptors (logP, εHOMO and εHOMO-1) selected by the Ordered Predictors Selection (OPS) algorithm. The QSAR model provided reasonable regression (Q2 = 0.64, R2 = 0.72, SEC = 0.29 and SEV = 0.33). The model was validated by leave-N-out cross-validation, y-randomization and external validation (R2pred = 0.89 and SEP = 0.27). The selected descriptors indicated that the activity was mainly related to electronic parameters (HOMO and HOMO-1 molecular orbital energies), as well as to logP. These findings suggest that higher activity values are directly related with both higher logP and frontier orbital energy values. The positive relationship between these orbitals and the activity suggests that the ent-kaurenoic acid analogues interaction with the target involves charge displacement, which is entirely consistent with the literature. Based on these findings, three compounds were proposed and one of them was synthesized and tested. The experimental result confirmed the activity predicted by the model.