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.

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.

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.

Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis.

• The staygreen (SGR) gene encodes a chloroplast-targeted protein which promotes chlorophyll degradation via disruption of light-harvesting complexes (LHCs). • Over-expression of SGR in Arabidopsis (SGR-OX) in a Columbia-0 (Col-0) background caused spontaneous necrotic flecking. To relate this to the hypersensitive response (HR), Col-0, SGR-OX and RNAi SGR (SGRi) lines were challenged with Pseudomonas syringae pv tomato (Pst) encoding the avirulence gene avrRpm1. Increased and decreased SGR expression, respectively, accelerated and suppressed the kinetics of HR-cell death. In Col-0, SGR transcript increased at 6 h after inoculation (hai) when tissue electrolyte leakage indicated the initiation of cell death. • Excitation of the chlorophyll catabolite pheophorbide (Pheide) leads to the formation of toxic singlet oxygen ((1)O(2)). Pheide was first detected at 6 hai with Pst avrRpm1 and was linked to (1)O(2) generation and correlated with reduced Pheide a oxygenase (PaO) protein concentrations. The maximum quantum efficiency of photosystem II (F(v)/F(m)), quantum yield of electron transfer at photosystem II (φPSII), and photochemical quenching (qP) decreased at 6 hai in Col-0 but not in SGRi. Disruption of photosynthetic electron flow will cause light-dependent H(2)O(2) generation at 6 hai. • We conclude that disruption of LHCs, possibly influenced by SGR, and absence of PaO produce phototoxic chlorophyll catabolites and oxidative stress leading to the HR.

Could Miscanthus replace maize as the preferred substrate for anaerobic digestion in the United Kingdom? Future breeding strategies.

Fodder maize is the most commonly used crop for biogas production owing to its high yields, high concentrations of starch and good digestibility. However, environmental concerns and possible future conflict with land for food production may limit its long-term use. The bioenergy grass, Miscanthus, is a high-yielding perennial that can grow on marginal land and, with 'greener' environmental credentials, may offer an alternative. To compete with maize, the concentration of non-structural carbohydrates (NSC) and digestibility may need to be improved. Non-structural carbohydrates were quantified in 38 diverse genotypes of Miscanthus in green-cut biomass in July and October. The aim was to determine whether NSC abundance could be a target for breeding programmes or whether genotypes already exist that could rival maize for use in anaerobic digestion systems. The saccharification potential and measures of N P and K were also studied. The highest concentrations of NSC were in July, reaching a maximum of 20% DW. However, the maximum yield was in October with 300-400 g NSC plant-1 owing to higher biomass. The digestibility of the cell wall was higher in July than in October, but the increase in biomass meant yields of digestible sugars were still higher in October. Nutrient concentrations were at least twofold higher in July compared to November, and the abundance of potassium showed the greatest degree of variation between genotypes. The projected maximum yield of NSC was 1.3 t ha-1 with significant variation to target for breeding. Starch accumulated in the highest concentrations and continued to increase into autumn in some genotypes. Therefore, starch, rather than sugars, would be a better target for breeding improvement. If harvest date was brought forward to autumn, nutrient losses in non-flowering genotypes would be comparable to an early spring harvest.

Fructan synthesis, accumulation and polymer traits. II. Fructan pools in populations of perennial ryegrass (Lolium perenne L.) with variation for water-soluble carbohydrate and candidate genes were not correlated with biosynthetic activity and demonstrated constraints to polymer chain extension.

Differences have been shown between ryegrass and fescue within the Festulolium subline introgression family for fructan synthesis, metabolism, and polymer-size traits. It is well-established that there is considerable variation for water-soluble carbohydrate and fructan content within perennial ryegrass. However there is much still to be discovered about the fructan polymer pool in this species, especially in regard to its composition and regulation. It is postulated that similar considerable variation for polymer traits may exist, providing useful polymers for biorefining applications. Seasonal effects on fructan content together with fructan synthesis and polymer-size traits have been examined in diverse perennial ryegrass material comprising contrasting plants from a perennial ryegrass F2 mapping family and from populations produced by three rounds of phenotypic selection. Relationships with copy number variation in candidate genes have been investigated. There was little evidence of any variation in fructan metabolism across this diverse germplasm under these conditions that resulted in substantial differences in the complement of fructan polymers present in leaf tissue at high water-soluble carbohydrate concentrations. The importance of fructan synthesis during fructan accumulation was unclear as fructan content and polymer characteristics in intact plants during the growing season did not reflect the capacity for de novo synthesis. However, the retention of fructan in environmental conditions favoring high sink/low source demand may be an important component of the high sugar trait and the roles of breakdown and turnover are discussed.