Functional co-expression of a fungal ferulic acid esterase and a ß-1,4 endoxylanase in Festuca arundinacea (tall fescue) modifies post-harvest cell wall deconstruction.

MAIN CONCLUSION: Improved post-harvest cell wall deconstruction of tall fescue leaves has been demonstrated by in-planta co-expression of a constitutively expressed ferulic acid esterase together with a senescence-induced ß-1,4 endoxylanase. Tall fescue plants (Festuca arundinacea) constitutively expressing vacuole- or apoplast-targeted ferulic acid esterase from Aspergillus niger were retransformed with a senescence-induced and apoplast-targeted ß-1,4 endo-xylanase from Trichoderma reesei. Enzyme activities in co-expressing plants stabilized after repeated vegetative propagation, with xylanase activity in senescent leaves increasing and ferulic acid esterase activity decreasing after tillering. Plants co-expressing both enzymes in the apoplast, with the lowest levels of ferulate monomers and dimers and the lowest levels of cell wall arabinoxylans, released ten times more cell wall hydroxycinnamic acids and five times more arabinoxylan from the cell wall on autodigestion compared to expression of ferulic acid esterase or xylanase alone. These plants also showed a 31 % increase in cellulase-mediated release of reducing sugars, a 5 % point increase in in vitro dry matter digestibility and a 23 % increase in acetyl bromide-soluble lignin. However, plant growth was adversely affected by expressing FAE in the apoplast, giving plants with narrower shorted leaves, and a 71 % decrease in biomass.

Transgenic ZmMYB167 Miscanthus sinensis with increased lignin to boost bioenergy generation for the bioeconomy.

BACKGROUND: Perennial C4 grasses from the genus Miscanthus are widely regarded as leading and promising dedicated bioenergy crops due to their high biomass accumulation on marginal land with low environmental impacts and maintenance requirements over its productive life. There is an urgent socio-political and environmental need to ramp up the production of alternative, affordable and green bioenergy sources and to re-direct the net zero carbon emissions trajectory. Hence, up-scaling of Miscanthus cultivation as a source of biomass for renewable energy could play an important role to strategically address sustainable development goals for a growing bio-based economy. Certain Miscanthus sinensis genotypes are particularly interesting for their biomass productivity across a wide range of locations. As the aromatic biomass component lignin exhibits a higher energy density than cell wall polysaccharides and is generally used as an indicator for heating or calorific value, genetic engineering could be a feasible strategy to develop M. sinensis biomass with increased lignin content and thus improving the energetic value of the biomass. RESULTS: For this purpose, transgenic M. sinensis were generated by Agrobacterium-mediated transformation for expression of ZmMYB167, a MYB transcription factor known for regulating lignin biosynthesis in C3 and C4 grasses. Four independent transgenic ZmMYB167 Miscanthus lines were obtained. Agronomic traits such as plant height, tillering and above-ground dry weight biomass of the transgenic plants were not different to that of wild-type control plants. Total lignin content of the transgenic plants was ~ 15-24% higher compared with control plants. However, the structural carbohydrates, glucan and xylan, were decreased by ~ 2-7% and ~ 8-10%, respectively, in the transgenic plants. Moreover, expression of ZmMYB167 in transgenic plants did not alter lignin composition, phenolic compounds or enzymatic saccharification efficiency yields but importantly improved total energy levels in Miscanthus biomass, equivalent to 10% higher energy yield per hectare. CONCLUSIONS: This study highlights ZmMYB167 as a suitable target for genetic lignin bioengineering interventions aimed at advancing and developing lignocellulosic biomass supply chains for sustainable production of renewable bioenergy.

Expression of a Trichoderma reesei ß-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses.

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H(2)O(2) production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H(2)O(2).

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.).

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. ß-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.

Targeting expression of a fungal ferulic acid esterase to the apoplast, endoplasmic reticulum or golgi can disrupt feruloylation of the growing cell wall and increase the biodegradability of tall fescue (Festuca arundinacea).

In the cell walls of grasses, ferulic acid is esterified to arabinoxylans and undergoes oxidative reactions to form ferulates dimers, trimers and oligomers. Feruloylation of arabinoxylan is considered important not only because it leads to cross-linked xylans but also because ferulates may act as a nucleating site for the formation of lignin and hence link arabinoxylans to lignin by forming a lignin-ferulate-arabinoxylan complex. Such cross-linking is among the main factors inhibiting the release of fermentable carbohydrates from grasses either for ruminant nutrition or for biofuel production. We have found that significant reductions in the levels of monomeric and dimeric phenolics can be achieved in the growing cell walls during plant development in leaves of Festuca arundinacea by constitutive intracellular targeted expression of Aspergillus niger ferulic acid esterase (FAEA). We propose that this occurred by directly disrupting ester bonds linking phenolics to cell wall polysaccharides by apoplast targeting or by preventing excessive feruloylation of cell wall carbohydrates prior to their incorporation into the cell wall, by targeting to the Golgi membrane system. Plants with lower cell wall ferulate levels, which showed increased digestibility and increased rates of cellulase-mediated release of fermentable sugars, were identified. Targeting FAE to the Golgi was found to be more effective than targeting to the ER, which supports the current theories of the Golgi as the site of feruloylation of arabinoxylans. It is concluded that targeting FAEA expression to the Golgi or apoplast is likely to be an effective strategy for improving wall digestibility in grass species used for fodder or cellulosic ethanol production.

Probing the role of cell wall feruloylation during maize development by differential expression of an apoplast targeted fungal ferulic acid esterase.

While many aspects of the growth of maize are well understood, the role of cell wall feruloylation particularly during internode elongation has not been firmly established, but results so far indicate that it has significant implications for both biofuel feedstock conversion and for crop yield. The growth of the cell wall is achieved by synthesis, integration and cross-linking between wall polymers. As ferulate oxidative coupling of arabinoxylan side chains constitutes a significant type of cross-link in grass cell walls, it is expected to have a crucial role in plant growth. Making use of plants expressing an apoplast targeted Aspergillus niger FAEA under the control of either a constitutive or an inducible promoter, the role of cell wall feruloylation in maize internode expansion was investigated. Analysis of FAEA expressing plants showed that where FAEA was targeted to the apoplast under a constitutive promoter, plants varied in stature either from semi-dwarf plants with a 40-60% height reduction, to extreme dwarf mutants with over 90% reduction in plant heights compared to controls. Results indicate that disruption of cell wall feruloylation by FAEA occurs before the start of rapid internode expansion is initiated and affects the normal course of internode elongation, resulting in short internodes and dwarfed plants. In contrast, when under the inducible Lm See1 senescence promoter, FAEA activity was found to be low up to the VT stage of development but increased significantly at the VR stage as plants began to senesce, strongly suggesting that normal cell wall feruloylation is required for the process of internode expansion. In addition, with apoplast targeted expression of FAEA under control of the senescence enhanced promoter it was possible to demonstrate decreased cell wall feruloylation without affecting internode expansion or other aspects of plant development.

Women, alcohol and femininity: a discourse analysis of women heavy drinkers' accounts.

Qualitative interviews were conducted with 24 women who were heavy drinkers, as part of a larger, longitudinal study of heavy drinking in the West Midlands of England. Critical discourse analysis was used to analyse the interviews, and resulted in the identification of two main discursive constructions: drink as self-medication, and drink as pleasure and leisure. However, women need to resist and negotiate stigmatizing subject positions of the ;woman drinker' in order both to justify their drinking and to protect their moral status as 'good women'.

Modified expression of ZmMYB167 in Brachypodium distachyon and Zea mays leads to increased cell wall lignin and phenolic content.

One of the challenges to enable targeted modification of lignocellulosic biomass from grasses for improved biofuel and biochemical production lies within our limited understanding of the transcriptional control of secondary cell wall biosynthesis. Here, we investigated the role of the maize MYB transcription factor ZmMYB167 in secondary cell wall biosynthesis and how modified ZmMYB167 expression in two distinct grass model species affects plant biomass and growth phenotypes. Heterologous expression of ZmMYB167 in the C3 model system Brachypodium led to mild dwarf phenotypes, increased lignin (~7% to 13%) and S-lignin monomer (~11% to 16%) content, elevated concentrations of cell wall-bound p-coumaric acid (~15% to 24%) and reduced biomass sugar release (~20%) compared to controls. Overexpression of ZmMYB167 in the C4 model system Zea mays increased lignin (~4% to 13%), p-coumaric acid (~8% to 52%) and ferulic acid (~13% to 38%) content but did not affect plant growth and development nor biomass recalcitrance. Taken together, modifying ZmMYB167 expression represents a target to alter lignin and phenolic content in grasses. The ZmMYB167 expression-induced discrepancies in plant phenotypic and biomass properties between the two grass model systems highlight the challenges and opportunities for MYB transcription factor-based genetic engineering approaches of grass biomass.

Breeding progress and preparedness for mass-scale deployment of perennial lignocellulosic biomass crops switchgrass, miscanthus, willow and poplar.

Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output-input energy ratios: namely Panicum virgatum (switchgrass), species of the genera Miscanthus (miscanthus), Salix (willow) and Populus (poplar). For each crop, we report on the size of germplasm collections, the efforts to date to phenotype and genotype, the diversity available for breeding and on the scale of breeding work as indicated by number of attempted crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic data sets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed-based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5 years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass-scale deployment of PBCs.

Expression of a fungal ferulic acid esterase increases cell wall digestibility of tall fescue (Festuca arundinacea).

In the cell walls of forage grasses, ferulic acid is esterified to arabinoxylans and participates with lignin monomers in oxidative coupling pathways to generate ferulate-polysaccharide-lignin complexes that cross-link the cell wall. Such cross-links hinder cell wall degradation by ruminant microbes, reducing plant digestibility. In this study, genetically modified Festuca arundinacea plants were produced expressing an Aspergillus niger ferulic acid esterase (FAEA) targeted to the vacuole. The rice actin promoter proved to be effective for FAEA expression, as did the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin promoters. Higher levels of expression were, however, found with inducible heat-shock and senescence promoters. Following cell death and subsequent incubation, vacuole-targeted FAEA resulted in the release of both monomeric and dimeric ferulic acids from the cell walls, and this was enhanced several fold by the addition of exogenous endo-1,4-beta-xylanase. Most of the FAEA-expressing plants showed increased digestibility and reduced levels of cell wall esterified phenolics relative to non-transformed plants. It is concluded that targeted FAEA expression is an effective strategy for improving wall digestibility in Festuca and, potentially, other grass species used for fodder or cellulosic ethanol production.

Three ways of talking about health in communities targeted for regeneration: interviews with community professionals.

Interviews were held with nine peripatetic professionals-district nurses, health visitors and social workers-working in New Deal for Communities (NDC) urban areas in the English West Midlands. They spoke of health in the community in three distinct ways: health as individual and family lifestyle; the local environment and health; and ;life is a struggle for some'. Those who emphasized the individual and family lifestyle approach expressed frustration in their professional role. The two alternative discourses, while recognizing the influence of social determinants of health, were problematic in different ways, reflecting the lack of a clear alternative to the individual and family lifestyle model for public health professions.

Expression of a fungal ferulic acid esterase in suspension cultures of tall fescue (Festuca arundinacea) decreases cell wall feruloylation and increases rates of cell wall digestion.

In the cell walls of grasses ferulic acid is esterified to arabinosyl residues in arabinoxylans that can then undergo oxidative coupling reactions to form ferulate dehydrodimers, trimers and oligomers which function to cross-link cell-wall polysaccharides, limiting cell wall degradability. Fungal ferulic acid esterase can release both esterified monomeric and dimeric ferulic acids from these cell wall arabinoxylans making the cell wall more susceptible to further enzymatic attack and increasing cell wall degradability. Non-embryogenic cell suspension cultures of Festuca arundinacea expressing a Aspergillus niger ferulic acid esterase (faeA) targeted to either the apoplast, or endoplasmic reticulum under the control of a constitutive actin promoter, or to the vacuole under the control of a soybean heat shock promoter, were established and FAE activity determined in the cells and medium during a growth cycle. Analysis of the ester-linked ferulates of the cell walls showed that all three transformed cell lines had both reduced ferulate levels and increased levels of xylanase mediated release of wall phenolics on autodigestion as well as increased rates of cell wall digestion in a simulated rumen environment, when compared to control non-transformed cells.

'Hardcore drinking': portrayals of alcohol consumption in young women's and men's magazines.

Young adults and young women in particular are drinking more alcohol than ever before, with implications for risky behaviours and long-term health. This study explored the ways in which alcohol and drinking were represented in six monthly UK magazines (three targeted at young men, three at young women) across a three-month period (18 magazines). We identified three main discourses across the texts, namely the drug alcohol; masculinity and machismo; and drinking as normality. These discourses constructed women's and men's drinks and drinking behaviours in sharp contrast. Drinking was aligned with traditional masculine images, although new kinds of drinks were aligned with traditional feminine images--and derided in men's magazines. Findings highlight how gender, constructed in relation to the other, is an important aspect of representations of drinking patterns in young adults.

Manipulating the phenolic acid content and digestibility of italian ryegrass (Lolium multiflorum) by vacuolar-targeted expression of a fungal ferulic acid esterase.

In grass cell walls, ferulic acid esters linked to arabinosyl residues in arabinoxylans play a key role in crosslinking hemicellulose. Although such crosslinks have a number of important roles in the cell wall, they also hinder the rate and extent of cell wall degradation by ruminant microbes and by fungal glycohydrolyase enzymes. Ferulic acid esterase (FAE) can release both monomeric and dimeric ferulic acids from arabinoxylans making the cell wall more susceptible to further enzymatic attack. Transgenic plants of Lolium multiflorum expressing a ferulic acid esterase gene from Aspergillus niger, targeted to the vacuole under a constitutive rice actin promoter, have been produced following microprojectile bombardment of embryogenic cell cultures. The level of FAE activity was found to vary with leaf age and was highest in young leaves. FAE expression resulted in the release of monomeric and dimeric ferulic acids from cell walls on cell death and this was enhanced severalfold by the addition of exogenous beta-1,4-endoxylanase. We also show that a number of plants expressing FAE had reduced levels of cell wall esterified monomeric and dimeric ferulates and increased in vitro dry-matter digestibility compared with nontransformed plants.