Dedicated biomass crops can enhance biodiversity in the arable landscape.

Suggestions that novel, non-food, dedicated biomass crops used to produce bioenergy may provide opportunities to diversify and reinstate biodiversity in intensively managed farmland have not yet been fully tested at the landscape scale. Using two of the largest, currently available landscape-scale biodiversity data sets from arable and biomass bioenergy crops, we take a taxonomic and functional trait approach to quantify and contrast the consequences for biodiversity indicators of adopting dedicated biomass crops on land previously cultivated under annual, rotational arable cropping. The abundance and community compositions of biodiversity indicators in fields of break and cereal crops changed when planted with the dedicated biomass crops, miscanthus and short rotation coppiced (SRC) willow. Weed biomass was consistently greater in the two dedicated biomass crops than in cereals, and invertebrate abundance was similarly consistently higher than in break crops. Using canonical variates analysis, we identified distinct plant and invertebrate taxa and trait-based communities in miscanthus and SRC willows, whereas break and cereal crops tended to form a single, composite community. Seedbanks were shown to reflect the longer term effects of crop management. Our study suggests that miscanthus and SRC willows, and the management associated with perennial cropping, would support significant amounts of biodiversity when compared with annual arable crops. We recommend the strategic planting of these perennial, dedicated biomass crops in arable farmland to increase landscape heterogeneity and enhance ecosystem function, and simultaneously work towards striking a balance between energy and food security.

Association mapping in Salix viminalis L. (Salicaceae) - identification of candidate genes associated with growth and phenology.

Willow species (Salix) are important as short-rotation biomass crops for bioenergy, which creates a demand for faster genetic improvement and breeding through deployment of molecular marker-assisted selection (MAS). To find markers associated with important adaptive traits, such as growth and phenology, for use in MAS, we genetically dissected the trait variation of a Salix viminalis (L.) population of 323 accessions. The accessions were sampled throughout northern Europe and were established at two field sites in Pustnäs, Sweden, and at Woburn, UK, offering the opportunity to assess the impact of genotype-by-environment interactions (G × E) on trait-marker associations. Field measurements were recorded for growth and phenology traits. The accessions were genotyped using 1536 SNP markers developed from phenology candidate genes and from genes previously observed to be differentially expressed in contrasting environments. Association mapping between 1233 of these SNPs and the measured traits was performed taking into account population structure and threshold selection bias. At a false discovery rate (FDR) of 0.2, 29 SNPs were associated with bud burst, leaf senescence, number of shoots or shoot diameter. The percentage of accession variation (Radj2) explained by these associations ranged from 0.3% to 4.4%, suggesting that the studied traits are controlled by many loci of limited individual impact. Despite this, a SNP in the EARLY FLOWERING 3 gene was repeatedly associated (FDR < 0.2) with bud burst. The rare homozygous genotype exhibited 0.4-1.0 lower bud burst scores than the other genotype classes on a five-grade scale. Consequently, this marker could be promising for use in MAS and the gene deserves further study. Otherwise, associations were less consistent across sites, likely due to their small Radj2 estimates and to considerable G × E interactions indicated by multivariate association analyses and modest trait accession correlations across sites (0.32-0.61).

High yielding biomass genotypes of willow (Salix spp.) show differences in below ground biomass allocation.

Willows (Salix spp.) grown as short rotation coppice (SRC) are viewed as a sustainable source of biomass with a positive greenhouse gas (GHG) balance due to their potential to fix and accumulate carbon (C) below ground. However, exploiting this potential has been limited by the paucity of data available on below ground biomass allocation and the extent to which it varies between genotypes. Furthermore, it is likely that allocation can be altered considerably by environment. To investigate the role of genotype and environment on allocation, four willow genotypes were grown at two replicated field sites in southeast England and west Wales, UK. Above and below ground biomass was intensively measured over two two-year rotations. Significant genotypic differences in biomass allocation were identified, with below ground allocation differing by up to 10% between genotypes. Importantly, the genotype with the highest below ground biomass also had the highest above ground yield. Furthermore, leaf area was found to be a good predictor of below ground biomass. Growth environment significantly impacted allocation; the willow genotypes grown in west Wales had up to 94% more biomass below ground by the end of the second rotation. A single investigation into fine roots showed the same pattern with double the volume of fine roots present. This greater below ground allocation may be attributed primarily to higher wind speeds, plus differences in humidity and soil characteristics. These results demonstrate that the capacity exists to breed plants with both high yields and high potential for C accumulation.

G-fibre cell wall development in willow stems during tension wood induction.

Willows (Salix spp.) are important as a potential feedstock for bioenergy and biofuels. Previous work suggested that reaction wood (RW) formation could be a desirable trait for biofuel production in willows as it is associated with increased glucose yields, but willow RW has not been characterized for cell wall components. Fasciclin-like arabinogalactan (FLA) proteins are highly up-regulated in RW of poplars and are considered to be involved in cell adhesion and cellulose biosynthesis. COBRA genes are involved in anisotropic cell expansion by modulating the orientation of cellulose microfibril deposition. This study determined the temporal and spatial deposition of non-cellulosic polysaccharides in cell walls of the tension wood (TW) component of willow RW and compared it with opposite wood (OW) and normal wood (NW) using specific antibodies and confocal laser scanning microscopy and transmission electron microscopy. In addition, the expression patterns of an FLA gene (SxFLA12) and a COBRA-like gene (SxCOBL4) were compared using RNA in situ hybridization. Deposition of the non-cellulosic polysaccharides (1-4)-ß-D-galactan, mannan and de-esterified homogalacturonan was found to be highly associated with TW, often with the G-layer itself. Of particular interest was that the G-layer itself can be highly enriched in (1-4)-ß-D-galactan, especially in G-fibres where the G-layer is still thickening, which contrasts with previous studies in poplar. Only xylan showed a similar distribution in TW, OW, and NW, being restricted to the secondary cell wall layers. SxFLA12 and SxCOBL4 transcripts were specifically expressed in developing TW, confirming their importance. A model of polysaccharides distribution in developing willow G-fibre cells is presented.

Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar.

Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow.

X-ray micro-computed tomography in willow reveals tissue patterning of reaction wood and delay in programmed cell death.

BACKGROUND: Variation in the reaction wood (RW) response has been shown to be a principle component driving differences in lignocellulosic sugar yield from the bioenergy crop willow. The phenotypic cause(s) behind these differences in sugar yield, beyond their common elicitor, however, remain unclear. Here we use X-ray micro-computed tomography (µCT) to investigate RW-associated alterations in secondary xylem tissue patterning in three dimensions (3D). RESULTS: Major architectural alterations were successfully quantified in 3D and attributed to RW induction. Whilst the frequency of vessels was reduced in tension wood tissue (TW), the total vessel volume was significantly increased. Interestingly, a delay in programmed-cell-death (PCD) associated with TW was also clearly observed and readily quantified by µCT. CONCLUSIONS: The surprising degree to which the volume of vessels was increased illustrates the substantial xylem tissue remodelling involved in reaction wood formation. The remodelling suggests an important physiological compromise between structural and hydraulic architecture necessary for extensive alteration of biomass and helps to demonstrate the power of improving our perspective of cell and tissue architecture. The precise observation of xylem tissue development and quantification of the extent of delay in PCD provides a valuable and exciting insight into this bioenergy crop trait.

Secondary cell wall composition and candidate gene expression in developing willow (Salix purpurea) stems.

The properties of the secondary cell wall (SCW) in willow largely determine the suitability of willow biomass feedstock for potential bioenergy and biofuel applications. SCW development has been little studied in willow and it is not known how willow compares with model species, particularly the closely related genus Populus. To address this and relate SCW synthesis to candidate genes in willow, a tractable bud culture-derived system was developed in Salix purpurea, and cell wall composition and RNA-Seq transcriptome were followed in stems during early development. A large increase in SCW deposition in the period 0-2 weeks after transfer to soil was characterised by a big increase in xylan content, but no change in the frequency of substitution of xylan with glucuronic acid, and increased abundance of putative transcripts for synthesis of SCW cellulose, xylan and lignin. Histochemical staining and immunolabeling revealed that increased deposition of lignin and xylan was associated with xylem, xylem fibre cells and phloem fibre cells. Transcripts orthologous to those encoding xylan synthase components IRX9 and IRX10 and xylan glucuronyl transferase GUX1 in Arabidopsis were co-expressed, and showed the same spatial pattern of expression revealed by in situ hybridisation at four developmental stages, with abundant expression in proto-xylem, xylem fibre and ray parenchyma cells and some expression in phloem fibre cells. The results show a close similarity with SCW development in Populus species, but also give novel information on the relationship between spatial and temporal variation in xylan-related transcripts and xylan composition.

Functional screening of willow alleles in Arabidopsis combined with QTL mapping in willow (Salix) identifies SxMAX4 as a coppicing response gene.

Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short-rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time-consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone-related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot-branching traits. Single-nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%-99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait.

Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.

Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production.

Genetic strategies for dissecting complex traits in biomass willows (Salix spp.).

Willows are highly diverse catkin-bearing trees and shrubs of the genus Salix. They occur in many growth forms, from tall trees to creeping alpines, and successfully occupy a wide variety of ecological niches. Shrubby willows (sub-genus Vetrix) have many characteristics that render them suited to cultivation in much faster growth cycles than conventional forestry. They respond well to coppicing, can be propagated vegetatively as cuttings and achieve rapid growth with low fertilizer inputs. As a result, willows grown as short rotation coppice are now among the leading commercially grown biomass crops in temperate regions. However, although willows have a long history of cultivation for traditional uses, their industrial use is relatively recent and, compared with major arable crops, they are largely undomesticated. Breeding programmes initiated to improve willow as a biomass crop achieved a doubling of yields within a period of <15 years. These advances were made by selecting for stem characteristics (height and diameter) and coppicing response (shoot number and shoot vigour), as well as resistance to pests, diseases and environmental stress, with little or no knowledge of the genetic basis of these traits. Genetics and genomics, combined with extensive phenotyping, have substantially improved our understanding of the basis of biomass traits in willow for more targeted breeding via marker-assisted selection. Here, we present the strategy we have adopted in which a genetic-based approach was used to dissect complex traits into more defined components for molecular breeding and gene discovery.

Using Arabidopsis to study shoot branching in biomass willow.

The success of the short-rotation coppice system in biomass willow (Salix spp.) relies on the activity of the shoot-producing meristems found on the coppice stool. However, the regulation of the activity of these meristems is poorly understood. In contrast, our knowledge of the mechanisms behind axillary meristem regulation in Arabidopsis (Arabidopsis thaliana) has grown rapidly in the past few years through the exploitation of integrated physiological, genetic, and molecular assays. Here, we demonstrate that these assays can be directly transferred to study the control of bud activation in biomass willow and to assess similarities with the known hormone regulatory system in Arabidopsis. Bud hormone response was found to be qualitatively remarkably similar in Salix spp. and Arabidopsis. These similarities led us to test whether Arabidopsis hormone mutants could be used to assess allelic variation in the cognate Salix spp. hormone genes. Allelic differences in Salix spp. strigolactone genes were observed using this approach. These results demonstrate that both knowledge and assays from Arabidopsis axillary meristem biology can be successfully applied to Salix spp. and can increase our understanding of a fundamental aspect of short-rotation coppice biomass production, allowing more targeted breeding.

Reaction wood - a key cause of variation in cell wall recalcitrance in willow.

BACKGROUND: The recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered. RESULTS: A pot trial was designed to test if the 'RW response' varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site. CONCLUSIONS: Our findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a 'RW response') indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.

Investigation of tension wood formation and 2,6-dichlorbenzonitrile application in short rotation coppice willow composition and enzymatic saccharification.

BACKGROUND: Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment. RESULTS: Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin. CONCLUSIONS: Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.

Meeting the challenge of food and energy security.

Growing crops for bioenergy or biofuels is increasingly viewed as conflicting with food production. However, energy use continues to rise and food production requires fuel inputs, which have increased with intensification. Focussing on the question of food or fuel is thus not helpful. The bigger, more pertinent, challenge is how the increasing demands for food and energy can be met in the future, particularly when water and land availability will be limited. Energy crop production systems differ greatly in environmental impact. The use of high-input food crops for liquid transport fuels (first-generation biofuels) needs to be phased out and replaced by the use of crop residues and low-input perennial crops (second/advanced-generation biofuels) with multiple environmental benefits. More research effort is needed to improve yields of biomass crops grown on lower grade land, and maximum value should be extracted through the exploitation of co-products and integrated biorefinery systems. Policy must continually emphasize the changes needed and tie incentives to improved greenhous gas reduction and environmental performance of biofuels.

Genetic improvement of willow for bioenergy and biofuels.

Willows (Salix spp.) are a very diverse group of catkin-bearing trees and shrubs that are widely distributed across temperate regions of the globe. Some species respond well to being grown in short rotation coppice (SRC) cycles, which are much shorter than conventional forestry. Coppicing reinvigorates growth and the biomass rapidly accumulated can be used as a source of renewable carbon for bioenergy and biofuels. As SRC willows re-distribute nutrients during the perennial cycle they require only minimal nitrogen fertilizer for growth. This results in fuel chains with potentially high greenhouse gas reductions. To exploit their potential for renewable energy, willows need to be kept free of pests and diseases and yields need to be improved without significantly increasing the requirements for fertilizers and water. The biomass composition needs to be optimized for different end-uses. Yields also need to be sustainable on land less productive for food crops to reduce conflicts over land use. Advances in understanding the physiology and growth of willow, and in the identification of genes underlying key traits, are now at the stage where they can start to be used in breeding programs to help achieve these goals.

Genetic structure and population dynamics of a heteroecious plant pathogen Melampsora larici-epitea in short-rotation coppice willow plantations.

Complex life strategies are common among plant pathogens belonging to rust fungi (Uredinales). The heteroecious willow rust Melampsora larici-epitea produces five spore stages and alternates on larch (Larix). To shed light on the epidemiology of this pathogen, amplified fragment length polymorphisms (AFLPs) were used to determine the genetic diversity and genetic structure of rust samples collected from coppice willow (Salix) plantations at three UK sites (LA, CA and MC) over three sampling dates (September 2000, July 2001 and September 2001). Of the total of 819 isolates, 465 were unique AFLP phenotypes and there was a shift in genotype diversity between the two seasons (0.67 in 2000 and 0.87-0.89 in 2001). No phenotypes were common between the two seasons within a site, suggesting that the rust did not overwinter as an asexual stage within plantations. A temporal analysis detected large amounts of genetic drift (F(S) = 0.15-0.26) between the two seasons and very small effective population sizes (N(e) = 2-3) within sites. These results all point to a new colonization of the plantations by the rust in the second season (2001). The F(ST)-analogue values were Phi(CT) = 0.121, Weir and Cockerham's theta = 0.086 and the Bayesian estimate theta(B) = 0.087-0.096. The results suggest that the sources of inoculum were somewhat localized and the same sources were mainly responsible for disease epidemics in LA and CA over the two seasons. The relatively low F(ST)-values among sites (0.055-0.13) suggest the existence of significant gene flow among the three sampled sites.

Bioenergy from plants and the sustainable yield challenge.

Bioenergy from plants, particularly from perennial grasses and trees, could make a substantial contribution to alleviation of global problems in climate change and energy security if high yields can be sustained. Here, yield traits in a range of key bioenergy crops are reviewed, from which several targets for future improvement can be identified. Some are already the focus of genetically modified (GM) and non-GM approaches. However, the efficient growth strategies of perennial bioenergy crops rely on newly assimilated and recycled carbon and remobilized nitrogen in a continually shifting balance between sources and sinks. This balance is affected by biotic (e.g. pest, disease) and abiotic (e.g. drought) stresses. Future research should focus on three main challenges: changing (photo)thermal time sensitivity to lengthen the growing season without risking frost damage or limiting remobilization of nutritional elements following senescence; increasing aboveground biomass without depleting belowground reserves required for next year's growth and thus without increasing the requirement for nutrient applications; and increasing aboveground biomass without increasing water use.

Evidence of diversity within the SnRK1b gene family of Hordeum species.

Adaptor-specific polymerase chain reaction (PCR) was used to amplify 3 different products, termed variant A, B, and C, from the seed-specific class (SnRK1b) of the sucrose nonfermenting-1-related protein kinase gene family (SnRK1) of different Hordeum species and cultivars of barley (Hordeum vulgare). Standard PCR or reverse transcription-PCR (RT-PCR) at a high temperature, using primers that differed by 1 or 2 nucleotides, was then used to amplify and clone 3 specific variants. One primer pair amplified a variant from I genome species suggesting that this could be a useful I-genome specific marker. The corresponding genes of the 3 variants (A, B, and C) were termed SnRK1b.1, 2, and 3, respectively. SnRK1b.1 and 2, showed 98% - 100% nucleotide sequence identity in the coding region, and 89% - 90% identity in the promoter region (up to 200 bp upstream of the translation start site, ATG). However, they differed in having insertions, deletions, and base pair changes at potentially important sites in the polymerase binding regions. SnRK1b.3 showed 90% nucleotide sequence identity with SnRK1b.1 in the coding region and 86% in the promoter region. This gene predominates in H-genome species within the genus Hordeum and could be a useful marker for this group.