Mechanical stimulation in wheat triggers age- and dose-dependent alterations in growth, development and grain characteristics.

BACKGROUND AND AIMS: Wheat crops are exposed to a range of mechanical stimulations in their natural environment, yet we know very little about their response to such conditions. The aim of this study was to better understand the effect of mechanical stimulation on wheat growth and development, stem mechanical properties and grain measures. We focused on the following questions: (1) Does plant age affect the response to mechanical stimulation? (2) Is there a minimum threshold for the perception of mechanical stimuli? (3) Is the effect of manual brushing different to natural wind stimulation? METHODS: For age- and dose-response experiments, wheat plants were grown under controlled glasshouse conditions with brushing treatments applied using a purpose-built rig. The results of the controlled experiments are compared with those from an outside experiment where wheat plants were exposed to natural wind, with or without additional brushing. Detailed phenotypic measurements were conducted and treatment effects on grain characteristics were determined using micro-computed tomography imaging. KEY RESULTS: Two-week-old wheat plants were particularly sensitive to mechanical stimulation by controlled brushing treatments. Amongst others, plants exhibited a large reduction in height and grain yield, and an increase in tillers, above-ground biomass and stiffness of stem segments. Plants responded significantly to doses as small as one daily brushstroke. Outdoor experiments by and large confirmed results from controlled environment experiments. CONCLUSIONS: The morphological and developmental response to mechanical brushing treatment, in relation to vegetative above-ground biomass and grain yield, is dependent on plant age as well as the dose of the treatments. This study shows that mechanical stimulation of wheat impacts on a multitude of agriculturally relevant traits and provides a much needed advancement of our understanding of wheat thigmomorphogenesis and the potential applications of mechanical conditioning to control relevant traits.

Genomic index selection provides a pragmatic framework for setting and refining multi-objective breeding targets in Miscanthus.

BACKGROUND: Miscanthus has potential as a biomass crop but the development of varieties that are consistently superior to the natural hybrid M. × giganteus has been challenging, presumably because of strong G × E interactions and poor knowledge of the complex genetic architectures of traits underlying biomass productivity and climatic adaptation. While linkage and association mapping studies are starting to generate long lists of candidate regions and even individual genes, it seems unlikely that this information can be translated into effective marker-assisted selection for the needs of breeding programmes. Genomic selection has emerged as a viable alternative, and prediction accuracies are moderate across a range of phenological and morphometric traits in Miscanthus, though relatively low for biomass yield per se. METHODS: We have previously proposed a combination of index selection and genomic prediction as a way of overcoming the limitations imposed by the inherent complexity of biomass yield. Here we extend this approach and illustrate its potential to achieve multiple breeding targets simultaneously, in the absence of a priori knowledge about their relative economic importance, while also monitoring correlated selection responses for non-target traits. We evaluate two hypothetical scenarios of increasing biomass yield by 20 % within a single round of selection. In the first scenario, this is achieved in combination with delaying flowering by 44 d (roughly 20 %), whereas, in the second, increased yield is targeted jointly with reduced lignin (-5 %) and increased cellulose (+5 %) content, relative to current average levels in the breeding population. KEY RESULTS: In both scenarios, the objectives were achieved efficiently (selection intensities corresponding to keeping the best 20 and 4 % of genotypes, respectively). However, the outcomes were strikingly different in terms of correlated responses, and the relative economic values (i.e. value per unit of change in each trait compared with that for biomass yield) of secondary traits included in selection indices varied considerably. CONCLUSIONS: Although these calculations rely on multiple assumptions, they highlight the need to evaluate breeding objectives and explicitly consider correlated responses in silico, prior to committing extensive resources. The proposed approach is broadly applicable for this purpose and can readily incorporate high-throughput phenotyping data as part of integrated breeding platforms.

Collecting wild Miscanthus germplasm in Asia for crop improvement and conservation in Europe whilst adhering to the guidelines of the United Nations' Convention on Biological Diversity.

BACKGROUND AND AIMS: Germplasm with diverse, agronomically relevant traits forms the foundation of a successful plant breeding programme. Since 1993, the United Nations has been advocating the implementation of the Convention on Biological Diversity (CBD) and the subsequent 2002 Bonn Guidelines as international best practice on germplasm collection and use. In 2006, a European team made an expedition to Asia to collect wild germplasm of Miscanthus, a C4 perennial rhizomatous grass, for breeding an environmentally adaptable, resilient and high-yielding bioenergy crop. We outline general aspects of germplasm collection, conservation, breeding and biomass production evaluation while following the CBD's guidelines, respecting biodiversity and conservation needs, and the ethical use of genetic resources. METHODS: Effective protocols, quarantine, methods for collecting seed and rhizomes, and a genebank for conservation were established. Versatile informatics and database architecture were used to assist in selection, flowering synchronization, crossing, evaluation, phenotyping and data integration. Approaches were formulated to comply with the CBD guidelines. KEY RESULTS: A total of 303 accessions of M. sinensis, M. sacchariflorus and M. floridulus were collected from 158 geographically and environmentally diverse locations. These species were shown to accumulate different amounts of aerial biomass due to combinations of stem count, height and thickness. Progeny from one interspecies cross accumulated more biomass in early trials and has shown double the yield performance in years 3-4 compared with the existing commercial cultivar M. × giganteus. An example of an F1 hybrid has already demonstrated the long-term potential of exploiting this collection for a breeding programme. CONCLUSIONS: By conforming to the CBD principles, the authors' international collaboration provides a practical example of implementing the CBD. The collection widened the genetic diversity of Miscanthus available to allow for breeding of novel hybrids that exhibit more diverse traits to increase yield and resilience for growth on marginal land and in climate-challenged environments.

Genetic relationships between spring emergence, canopy phenology, and biomass yield increase the accuracy of genomic prediction in Miscanthus.

Miscanthus has potential as a bioenergy crop but the rapid development of high-yielding varieties is challenging. Previous studies have suggested that phenology and canopy height are important determinants of biomass yield. Furthermore, while genome-wide prediction was effective for a broad range of traits, the predictive ability for yield was very low. We therefore developed models clarifying the genetic associations between spring emergence, consequent canopy phenology and dry biomass yield. The timing of emergence was a moderately strong predictor of early-season elongation growth (genetic correlation >0.5), but less so for growth later in the season and for the final yield (genetic correlation <0.1). In contrast, early-season canopy height was consistently more informative than emergence for predicting biomass yield across datasets for two species in Miscanthus and two growing seasons. We used the associations uncovered through these models to develop selection indices that are expected to increase the response to selection for yield by as much as 21% and improve the performance of genome-wide prediction by an order of magnitude. This multivariate approach could have an immediate impact in operational breeding programmes, as well as enable the integration of crop growth models and genome-wide prediction.

Genome-wide association studies and prediction of 17 traits related to phenology, biomass and cell wall composition in the energy grass Miscanthus sinensis.

• Increasing demands for food and energy require a step change in the effectiveness, speed and flexibility of crop breeding. Therefore, the aim of this study was to assess the potential of genome-wide association studies (GWASs) and genomic selection (i.e. phenotype prediction from a genome-wide set of markers) to guide fundamental plant science and to accelerate breeding in the energy grass Miscanthus. • We generated over 100,000 single-nucleotide variants (SNVs) by sequencing restriction site-associated DNA (RAD) tags in 138 Micanthus sinensis genotypes, and related SNVs to phenotypic data for 17 traits measured in a field trial. • Confounding by population structure and relatedness was severe in naïve GWAS analyses, but mixed-linear models robustly controlled for these effects and allowed us to detect multiple associations that reached genome-wide significance. Genome-wide prediction accuracies tended to be moderate to high (average of 0.57), but varied dramatically across traits. As expected, predictive abilities increased linearly with the size of the mapping population, but reached a plateau when the number of markers used for prediction exceeded 10,000-20,000, and tended to decline, but remain significant, when cross-validations were performed across subpopulations. • Our results suggest that the immediate implementation of genomic selection in Miscanthus breeding programs may be feasible.

Variation in canopy duration in the perennial biofuel crop Miscanthus reveals complex associations with yield.

Energy crops can provide a sustainable source of power and fuels, and mitigate the negative effects of CO2 emissions associated with fossil fuel use. Miscanthus is a perennial C4 energy crop capable of producing large biomass yields whilst requiring low levels of input. Miscanthus is largely unimproved and therefore there could be significant opportunities to increase yield. Further increases in yield will improve the economics, energy balance, and carbon mitigation of the crop, as well as reducing land-take. One strategy to increase yield in Miscanthus is to maximize the light captured through an extension of canopy duration. In this study, canopy duration was compared among a diverse collection of 244 Miscanthus genotypes. Canopy duration was determined by calculating the number of days between canopy establishment and senescence. Yield was positively correlated with canopy duration. Earlier establishment and later senescence were also both separately correlated with higher yield. However, although genotypes with short canopy durations were low yielding, not all genotypes with long canopy durations were high yielding. Differences of yield between genotypes with long canopy durations were associated with variation in stem and leaf traits. Different methodologies to assess canopy duration traits were investigated, including visual assessment, image analysis, light interception, and different trait thresholds. The highest correlation coefficients were associated with later assessments of traits and the use of quantum sensors for canopy establishment. A model for trait optimization to enable yield improvement in Miscanthus and other bioenergy crops is discussed.

Identification of genes involved in cell wall biogenesis in grasses by differential gene expression profiling of elongating and non-elongating maize internodes.

Despite the economic importance of grasses as food, feed, and energy crops, little is known about the genes that control their cell wall synthesis, assembly, and remodelling. Here a detailed transcriptome analysis that allowed the identification of genes involved in grass cell wall biogenesis is provided. Differential gene expression profiling, using maize oligonucleotide arrays, was used to identify genes differentially expressed between an elongating internode, containing cells exhibiting primary cell wall synthesis, and an internode that had just ceased elongation and in which many cells were depositing secondary cell wall material. This is one of only a few studies specifically aimed at the identification of cell wall-related genes in grasses. Analysis identified new candidate genes for a role in primary and secondary cell wall biogenesis in grasses. The results suggest that many proteins involved in cell wall processes during normal development are also recruited during defence-related cell wall remodelling events. This work provides a platform for studies in which candidate genes will be functionally tested for involvement in cell wall-related processes, increasing our knowledge of cell wall biogenesis and its regulation in grasses. Since several grasses are currently being developed as lignocellulosic feedstocks for biofuel production, this improved understanding of grass cell wall biogenesis is timely, as it will facilitate the manipulation of traits favourable for sustainable food and biofuel production.

The Effect of Red & Blue Rich LEDs vs Fluorescent Light on Lollo Rosso Lettuce Morphology and Physiology.

The challenges of feeding an increasing population, an increasingly urban population and within an increasingly challenging global environment have focused ideas on new ways to grow food. Growing food in a controlled environment (CE) is not new but new technologies such as broad-spectrum LEDs and robotics are generating new opportunities. Growth recipes can be tailored to plant species in a CE and plasticity in plant responses to the environment may be utilized to make growth systems more efficient for improved yield and crop quality. Light use efficiency within CE must consider energy requirements, yield and impacts on quality. We hypothesized that understanding how plants change their morphology and physiology in response to light will allow us to identify routes to make light more efficient for delivery of high-quality produce. We focused on responses to light in Lollo rosso lettuce which produces compact, crinkly and highly pigmented leaves. We compared the spectra of the commonly used artificial light sources in indoor farming (compact fluorescence tubes, FL, and broad-spectrum light-emitting diodes, LEDs) at two irradiance levels (270 and 570 µmol m-2 s-1). We discovered LEDs (λP: 451, 634, and 665 nm) produced the same amount of produce for half the incident energy of FL (T5). At higher irradiances LEDs produced 9% thicker leaves, 13% larger rosettes and 15% greater carotenoid content. Leaves differed in light absorptance with plants grown under lower FL absorbing 30% less of mid-range wavelengths. We show that the relative efficiencies of LED and FL is a function of the irradiances compared and demonstrate the importance of understanding the asymptotes of yield and quality traits. Increasing our understanding of structural and biochemical changes that occur under different combination of wavelengths may allow us to better optimize light delivery, select for different ranges of plasticity in crop plants and further optimize light recipes.

Producing Enhanced Yield and Nutritional Pigmentation in Lollo Rosso Through Manipulating the Irradiance, Duration, and Periodicity of LEDs in the Visible Region of Light.

Pigmented food are an important part of the human diet, and anthocyanins have demonstrable protection against tumor production in mouse models and beneficial effects on human liver chemistry. As such, producing pigmented crops is important for a nutritionally diverse diet. Lollo rosso lettuce is a fast-growing pigmented plant, is rich in phenolic compounds, and represents a suitable system to test optimization strategies for yield and anthocyanin production. High-energy UV wavebands are often used to stimulate increased pigmentation; however, we hypothesized that optimizing visible wavebands would deliver both yield and quality improvements. Growing Lollo rosso under irradiances between 5 and 180 W m-2 using visible waveband LEDs produced 0.4 g fresh weight per W m-2 in the linear portion of the curve between 5 and 40 W m-2 and achieved an approximate asymptote of 20 g fresh weight at around 100-120 W m-2 for yield. Anthocyanin content increased linearly with irradiance. We attempted to optimize the visible wavebands by supplementing half the asymptotic energy for 15 days with supplemental red (R) or blue (B) wavebands in the peaks of photosynthetic activity (430-460 and 630-660 nm). R and B affected rosette morphology with no significant impact on yield, but B significantly increased anthocyanin content by 94% compared to R. We therefore focused on further optimizing B by shortening the daily duration of supplemental B. The minimum B treatment that lacked significant pigment induction was 1 h. We hypothesized that short durations would be more active at different times in the diurnal cycle. Supplemental B was applied for 2 h at four different times. A night-break with B produced the highest yield and anthocyanin content. Our research demonstrates new ways to efficiently use readily available LEDs within the PAR wavebands to increase both yield and crop quality in controlled environment agriculture.

Genome biology of the paleotetraploid perennial biomass crop Miscanthus.

Miscanthus is a perennial wild grass that is of global importance for paper production, roofing, horticultural plantings, and an emerging highly productive temperate biomass crop. We report a chromosome-scale assembly of the paleotetraploid M. sinensis genome, providing a resource for Miscanthus that links its chromosomes to the related diploid Sorghum and complex polyploid sugarcanes. The asymmetric distribution of transposons across the two homoeologous subgenomes proves Miscanthus paleo-allotetraploidy and identifies several balanced reciprocal homoeologous exchanges. Analysis of M. sinensis and M. sacchariflorus populations demonstrates extensive interspecific admixture and hybridization, and documents the origin of the highly productive triploid bioenergy crop M. × giganteus. Transcriptional profiling of leaves, stem, and rhizomes over growing seasons provides insight into rhizome development and nutrient recycling, processes critical for sustainable biomass accumulation in a perennial temperate grass. The Miscanthus genome expands the power of comparative genomics to understand traits of importance to Andropogoneae grasses.

Soil & Water Assessment Tool (SWAT) simulated hydrological impacts of land use change from temperate grassland to energy crops: A case study in western UK.

When considering the large-scale deployment of bioenergy crops, it is important to understand the implication for ecosystem hydrological processes and the influences of crop type and location. Based on the potential for future land use change (LUC), the 10,280 km2 West Wales Water Framework Directive River Basin District (UK) was selected as a typical grassland dominated district, and the Soil & Water Assessment Tool (SWAT) hydrology model with a geographic information systems interface was used to investigate implications for different bioenergy deployment scenarios. The study area was delineated into 855 sub-basins and 7,108 hydrological response units based on rivers, soil type, land use, and slope. Changes in hydrological components for two bioenergy crops (Miscanthus and short rotation coppice, SRC) planted on 50% (2,192 km2) or 25% (1,096 km2) of existing improved pasture are quantified. Across the study area as a whole, only surface run-off with SRC planted at the 50% level was significantly impacted, where it was reduced by up to 23% (during April). However, results varied spatially and a comparison of annual means for each sub-basin and scenario revealed surface run-off was significantly decreased and baseflow significantly increased (by a maximum of 40%) with both Miscanthus and SRC. Evapotranspiration was significantly increased with SRC (at both planting levels) and water yield was significantly reduced with SRC (at the 50% level) by up to 5%. Effects on streamflow were limited, varying between -5% and +5% change (compared to baseline) in the majority of sub-basins. The results suggest that for mesic temperate grasslands, adverse effects from the drying of soil and alterations to streamflow may not arise, and with surface run-off reduced and baseflow increased, there could, depending on crop location, be potential benefits for flood and erosion mitigation.

Stem growth characteristics of high yielding Miscanthus correlate with yield, development and intraspecific competition within plots.

High yielding perennial grasses are utilized as biomass for the bioeconomy and to displace fossil fuels. Many such grasses, including Miscanthus, are largely undomesticated. The main Miscanthus crop is a naturally occurring hydrid M. × giganteus (Mxg). All above ground biomass from Miscanthus is harvested. Stem traits correlate strongly with yield and therefore understanding the seasonal progression of stem growth should identify routes for improved yield. If such studies utilized high yielding commercial genotypes growing in plots the conclusions are likely to be more commercially relevant. Stem elongation was measured from five high yielding genotypes, 10 plants per plot from 20 plots in a replicated field trial over 4 years. Richards growth function produced an accurate fit to stem elongation. Differentials, double differentials and integrals of the parameterized function produced six growth characteristics, describing growth rate, timing and duration of the logarithmic growth phase and area under the growth curve. Maximum growth rate was correlated with yield and compensatory interactions were identified, for example plants with higher maximal growth rates had shorter durations of logarithmic growth. Plant position within plots of lower yielding genotypes did not affect growth characteristics but had a significant effect on late season growth characteristics in higher yielding genotypes. Two high yielding genotypes were compared over 3 years and growth parameterized using four different factors. The inverse correlation between maximum growth rate and duration of logarithmic growth was consistent across years and factors in both genotypes except when parameterized using temperature and only in Mxg. This suggested that different limitations to growth were exerted on the two genotypes which may help explain the exceptional performance of the Mxg genotype. We discuss the implications of the identified complex interactions in growth characteristics for approaches to maximize seasonal yield in perennial biomass crops.

Soil N2O emissions with different reduced tillage methods during the establishment of Miscanthus in temperate grassland.

An increase in renewable energy and the planting of perennial bioenergy crops is expected in order to meet global greenhouse gas (GHG) targets. Nitrous oxide (N2O) is a potent greenhouse gas, and this paper addresses a knowledge gap concerning soil N2O emissions over the possible "hot spot" of land use conversion from established pasture to the biofuel crop Miscanthus. The work aims to quantify the impacts of this land use change on N2O fluxes using three different cultivation methods. Three replicates of four treatments were established: Miscanthus x giganteus (Mxg) planted without tillage; Mxg planted with light tillage; a novel seed-based Miscanthus hybrid planted with light tillage under bio-degradable mulch film; and a control of uncultivated established grass pasture with sheep grazing. Soil N2O fluxes were recorded every 2 weeks using static chambers starting from preconversion in April 2016 and continuing until the end of October 2017. Monthly soil samples were also taken and analysed for nitrate and ammonium. There was no significant difference in N2O emissions between the different cultivation methods. However, in comparison with the uncultivated pasture, N2O emissions from the cultivated Miscanthus plots were 550%-819% higher in the first year (April to December 2016) and 469%-485% higher in the second year (January to October 2017). When added to an estimated carbon cost for production over a 10 year crop lifetime (including crop management, harvest, and transportation), the measured N2O conversion cost of 4.13 Mg CO2-eq./ha represents a 44% increase in emission compared to the base case. This paper clearly shows the need to incorporate N2O fluxes during Miscanthus establishment into assessments of GHG balances and life cycle analysis and provides vital knowledge needed for this process. This work therefore also helps to support policy decisions regarding the costs and benefits of land use change to Miscanthus.

Measured and modelled effect of land-use change from temperate grassland to Miscanthus on soil carbon stocks after 12 years.

Soil organic carbon (SOC) is an important carbon pool susceptible to land-use change (LUC). There are concerns that converting grasslands into the C4 bioenergy crop Miscanthus (to meet demands for renewable energy) could negatively impact SOC, resulting in reductions of greenhouse gas mitigation benefits gained from using Miscanthus as a fuel. This work addresses these concerns by sampling soils (0-30 cm) from a site 12 years (T12) after conversion from marginal agricultural grassland into Miscanthus x giganteus and four other novel Miscanthus hybrids. Soil samples were analysed for changes in below-ground biomass, SOC and Miscanthus contribution to SOC (using a 13C natural abundance approach). Findings are compared to ECOSSE soil carbon model results (run for a LUC from grassland to Miscanthus scenario and continued grassland counterfactual), and wider implications are considered in the context of life cycle assessments based on the heating value of the dry matter (DM) feedstock. The mean T12 SOC stock at the site was 8 (±1 standard error) Mg C/ha lower than baseline time zero stocks (T0), with assessment of the five individual hybrids showing that while all had lower SOC stock than at T0 the difference was only significant for a single hybrid. Over the longer term, new Miscanthus C4 carbon replaces pre-existing C3 carbon, though not at a high enough rate to completely offset losses by the end of year 12. At the end of simulated crop lifetime (15 years), the difference in SOC stocks between the two scenarios was 4 Mg C/ha (5 g CO2-eq/MJ). Including modelled LUC-induced SOC loss, along with carbon costs relating to soil nitrous oxide emissions, doubled the greenhouse gas intensity of Miscanthus to give a total global warming potential of 10 g CO2-eq/MJ (180 kg CO2-eq/Mg DM).

Soil nitrous oxide flux following land-use reversion from Miscanthus and SRC willow to perennial ryegrass.

Decarbonization of the world's energy supply is essential to meet the targets of the 2016 Paris climate change agreement. One promising opportunity is the utilization of second generation, low input bioenergy crops such as Miscanthus and Short Rotation Coppice (SRC) willow. Research has previously been carried out on the greenhouse gas (GHG) balance of growing these feedstocks and land-use changes involved in converting conventional cropland to their production; however, there is almost no body of work understanding the costs associated with their end of life transitions back to conventional crops. It is likely that it is during crop interventions and land-use transitions that significant GHG fluxes might occur. Therefore, in this study, we investigated soil GHG fluxes over 82 weeks during transition from Miscanthus and SRC willow into perennial ryegrass in west Wales, UK. This study captured soil GHG fluxes at a weekly time step, alongside monthly changes in soil nitrogen and labile carbon and reports the results of regression modelling of suspected drivers. Methane fluxes were typically trivial; however, nitrous oxide (N2O) fluxes were notably affected, reverted plots produced significantly more N2O than retained controls and Miscanthus produced significantly higher fluxes overall than willow plots. N2O costs of reversion appeared to be contained within the first year of reversion when the Miscanthus plots produced an average pregrass flux of 0.13 mg N2O m-2 hr-1 while for willow, this was 0.03 mg N2O m-2 hr-1. Total N2O emission from reversion increased the carbon cost over the lifetime of the Miscanthus from 6.50 to 9.91 Mg CO2 eq. ha-1 while for the willow, this increase was from 9.61 to 10.42 Mg CO2 eq. ha-1. Despite these significant increases, the carbon cost of energy contained in these perennial crops remained far lower than the equivalent carbon cost of energy in coal.

Consensus, uncertainties and challenges for perennial bioenergy crops and land use.

Perennial bioenergy crops have significant potential to reduce greenhouse gas (GHG) emissions and contribute to climate change mitigation by substituting for fossil fuels; yet delivering significant GHG savings will require substantial land-use change, globally. Over the last decade, research has delivered improved understanding of the environmental benefits and risks of this transition to perennial bioenergy crops, addressing concerns that the impacts of land conversion to perennial bioenergy crops could result in increased rather than decreased GHG emissions. For policymakers to assess the most cost-effective and sustainable options for deployment and climate change mitigation, synthesis of these studies is needed to support evidence-based decision making. In 2015, a workshop was convened with researchers, policymakers and industry/business representatives from the UK, EU and internationally. Outcomes from global research on bioenergy land-use change were compared to identify areas of consensus, key uncertainties, and research priorities. Here, we discuss the strength of evidence for and against six consensus statements summarising the effects of land-use change to perennial bioenergy crops on the cycling of carbon, nitrogen and water, in the context of the whole life-cycle of bioenergy production. Our analysis suggests that the direct impacts of dedicated perennial bioenergy crops on soil carbon and nitrous oxide are increasingly well understood and are often consistent with significant life cycle GHG mitigation from bioenergy relative to conventional energy sources. We conclude that the GHG balance of perennial bioenergy crop cultivation will often be favourable, with maximum GHG savings achieved where crops are grown on soils with low carbon stocks and conservative nutrient application, accruing additional environmental benefits such as improved water quality. The analysis reported here demonstrates there is a mature and increasingly comprehensive evidence base on the environmental benefits and risks of bioenergy cultivation which can support the development of a sustainable bioenergy industry.

Evolutionary hierarchies of conserved blocks in 5'-noncoding sequences of dicot rbcS genes.

BACKGROUND: Evolutionary processes in gene regulatory regions are major determinants of organismal evolution, but exceptionally challenging to study. We explored the possibilities of evolutionary analysis of phylogenetic footprints in 5'-noncoding sequences (NCS) from 27 ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) genes, from three dicot families (Brassicaceae, Fabaceae and Solanaceae). RESULTS: Sequences of up to 400 bp encompassing proximal promoter and 5'-untranslated regions were analyzed. We conducted phylogenetic footprinting by several alternative methods: generalized Lempel-Ziv complexity (CLZ), multiple alignments with DIALIGN and ALIGN-M, and the MOTIF SAMPLER Gibbs sampling algorithm. These tools collectively defined 36 conserved blocks of mean length 12.8 bp. On average, 12.5 blocks were found in each 5'-NCS. The blocks occurred in arrays whose relative order was absolutely conserved, confirming the existence of 'conserved modular arrays' in promoters. Identities of half of the blocks confirmed past rbcS research, including versions of the I-box, G-box, and GT-1 sites such as Box II. Over 90% of blocks overlapped DNase-protected regions in tomato 5'-NCS. Regions characterized by low CLZ in sliding-window analyses were also frequently associated with DNase-protection. Blocks could be assigned to evolutionary hierarchies based on taxonomic distribution and estimated age. Lineage divergence dates implied that 13 blocks found in all three plant families were of Cretaceous antiquity, while other family-specific blocks were much younger. Blocks were also dated by formation of multigene families, using genome and coding sequence information. Dendrograms of evolutionary relations of the 5'-NCS were produced by several methods, including: cluster analysis using pairwise CLZ values; evolutionary trees of DIALIGN sequence alignments; and cladistic analysis of conserved blocks. CONCLUSION: Dicot 5'-NCS contain conserved modular arrays of recurrent sequence blocks, which are coincident with functional elements. These blocks are amenable to evolutionary interpretation as hierarchies in which ancient, taxonomically widespread blocks can be distinguished from more recent, taxon-specific ones.

Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using bacterial artificial chromosome landing with fluorescence in situ hybridization.

As part of an initiative to develop Brachypodium distachyon as a genomic "bridge" species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.

Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases.

BACKGROUND: Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice. RESULTS: Genes encoding the 11α-steroid hydroxylase enzymes from Aspergillus ochraceus (11α-SHAoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11α-SHAoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 µM progesterone) producing either 11α-hydroxyprogesterone as the sole metabolite (AH22:p11α-SHAoch) or a 7:1 mixture of 11α-hydroxyprogesterone and 6ß-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11α-SHAoch and AH22:p509C12 were comparable resulting in ≥75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11α-SHAoch gene into the yeast genome (AH22:11α-SHAoch+K) resulted in a 36% reduction in yield of 11α-hydroxyprogesterone to 174 µmol/L using 300 µM progesterone. However, increasing progesterone concentration to 955 µM and optimizing growth conditions increased 11α-hydroxyprogesterone production to 592 µmol/L product formed. CONCLUSIONS: The progesterone 11α-steroid hydroxylases from A. ochraceus and R. oryzae, both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in S. cerevisiae. It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the A. ochraceous enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11α-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.

Environmental costs and benefits of growing Miscanthus for bioenergy in the UK.

Planting the perennial biomass crop Miscanthus in the UK could offset 2-13 Mt oil eq. yr-1, contributing up to 10% of current energy use. Policymakers need assurance that upscaling Miscanthus production can be performed sustainably without negatively impacting essential food production or the wider environment. This study reviews a large body of Miscanthus relevant literature into concise summary statements. Perennial Miscanthus has energy output/input ratios 10 times higher (47.3 ± 2.2) than annual crops used for energy (4.7 ± 0.2 to 5.5 ± 0.2), and the total carbon cost of energy production (1.12 g CO2-C eq. MJ-1) is 20-30 times lower than fossil fuels. Planting on former arable land generally increases soil organic carbon (SOC) with Miscanthus sequestering 0.7-2.2 Mg C4-C ha-1 yr-1. Cultivation on grassland can cause a disturbance loss of SOC which is likely to be recovered during the lifetime of the crop and is potentially mitigated by fossil fuel offset. N2O emissions can be five times lower under unfertilized Miscanthus than annual crops and up to 100 times lower than intensive pasture. Nitrogen fertilizer is generally unnecessary except in low fertility soils. Herbicide is essential during the establishment years after which natural weed suppression by shading is sufficient. Pesticides are unnecessary. Water-use efficiency is high (e.g. 5.5-9.2 g aerial DM (kg H2O)-1, but high biomass productivity means increased water demand compared to cereal crops. The perennial nature and belowground biomass improves soil structure, increases water-holding capacity (up by 100-150 mm), and reduces run-off and erosion. Overwinter ripening increases landscape structural resources for wildlife. Reduced management intensity promotes earthworm diversity and abundance although poor litter palatability may reduce individual biomass. Chemical leaching into field boundaries is lower than comparable agriculture, improving soil and water habitat quality.