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Enhancing Poplar Traits for Energy Applications

Final Report Summary - ENERGYPOPLAR (Enhancing poplar traits for energy applications)

Since lignin is the main factor limiting saccharification for fermentation, the team has identified new elite poplar clones and developed new transgenic poplars combining genes improving yield with those that reduce lignin. ENERGYPOPLAR has established several short rotation crops (SRC) field trials, planting trees at high density for maximum biomass production, and has identified high yield genotypes being studied further to elucidate key genetic control points underlying wood production.

Over the course of the project, significant breakthrough findings have been obtained:

(i) an effector protein controlling the development of the ectomycorrhizal symbiosis has been characterised,
(ii) a transcriptional factor controlling poplar root stem cell division has been identified,
(iii) five transcription factors with a putative role in lignification and yield have been identified and altered for improved saccharification,
(iv) calibration models have been constructed to measure poplar lignin,
(v) a saccharification protocol to process woody stems has been established,
(vi) lignin and cell wall carbohydrates have been measured through wet chemical analyses in candidate clones. These high-throughput tools to determine cellulose and lignin content and saccharification potential have been optimised using 300 genotypes of a F2 tree pedigree with varying cell wall compositions.

The research has created new information on saccharification variation from a range of poplar genotypes and has generated up to 200 new indicators for clone breeding and improvement for global biofuel production, including mapping quantitative trait loci (QTL) for saccharification. Through conventional breeding, as well as genetic engineering, the project has identified gene markers for a deeper understanding of cell wall parameters determining saccharification potential.

A key achievement is the discovery of natural mutant alleles in genes related to lignin synthesis through next generation sequencing. This major finding opens new possibilities for selection of lower lignin-content poplars in natural populations, without making use of genetically modified poplars. This should provide an efficient basis for optimising the saccharification potential of selected poplar lines by targeted alteration of their cell wall composition. The most promising trees will then be used in experimental crosses producing elite offspring, further enhancing their potential to become biofuel crops of tomorrow.

A key focus has been the below ground aspects of biomass yield, including the role of ectomycorrhizal fungal symbionts in shaping an efficient root system, which may have beneficial effects on nutrient uptake and as a consequence, improved tree yield. Progress has been made on novel transgenic trees for improved yield and quality, through targeting developmental processes including stem cell division, root and canopy architecture. Phenotyping analysis has been undertaken to develop material for future field trials beyond this project.

Maintaining a high microbial biodiversity in biofuel SRC monocultures is a major challenge. Alongside its genetic work on poplars, the project has also established tools for assessing environmental sustainability of poplar SRC, conducting molecular surveys of soil microbial taxa by high-throughput sequencing and full life cycle assessment (LCA) of biofuels from poplar biomass in different scenarios.

A detailed life cycle inventory (LCI) and LCA analysis of 24 SRC poplar biofuel supply chains representing 4 locations in Europe (northern, southern, eastern, western), 3 time horizons (2010, 2020, 2030) and short rotation or very short rotation management has therefore been performed and will be available in the public domain. This analysis confirmed that research into the structure of poplar biomass should focus on manipulating the cell wall composition to allow for easier conversion requiring lower inputs of process chemicals and enzymes.

Project context and objectives:

ENERGYPOPLAR aimed to unravel genetic mechanisms controlling growth yield and cell wall structure and composition, in order to design new SRC poplar tree cultivars with enhanced agronomical traits for industrial production of bioethanol. Crucial in bioethanol production is the lignocellulosic material - the plant biomass composed of cellulose, hemicellulose, and lignin: the higher the (hemi)cellulose yield, the more efficient the fuel. So lower-lignin varieties provide more efficient biofuel crops. ENERGYPOPLAR therefore aimed at better understanding mechanisms regulating growth of roots and shoots and synthesis of cell wall lignocellulosic polymers, as well as establishing assays to demonstrate genetic and genomic traits of high or low-lignin trees.

ENERGYPOPLAR brought together an interdisciplinary group of 10 public and private partners from 6 European countries with expertise in tree biotechnology and breeding, wood decay and fermentation, environmental science, as well as scientists engaged in commercial-scale development of lignocellulose to ethanol. Together they have been working towards step-change discoveries that will enable more efficient bioethanol production. The ultimate goal of the project that kicked off in May 2008 was to develop poplars as a second-generation bioenergy crop, suitable for large-scale deployment in Europe. The project integrated an ambitious systems biology approach in Populus to optimise the yield, composition, and structure of cell walls for biofuel production and to develop novel genotypes of Populus with enhanced growth traits under low-input environments.

The specific objectives of the project were as follows:

(i) providing a better understanding of fundamental mechanisms determining optimised yield in poplar hybrids - identifying several master transcription factors and other candidate genes regulating biomass yield, including those involved in nutrient acquisition and ectomycorrhizal symbiotic interactions,
(ii) providing a better understanding of mechanisms that regulate the synthesis of cell wall lignocellulosic polymers,
(iii) developing high throughput, cross-validated assays for lignocellulosic quality and lignocellulose saccharification potential,
(iv) establishing a platform for rapid gene discovery and testing to identify novel transcripts for traits of interest,
(v) Establishing tools for environmental sustainability assessments of SRC Populus growing systems with respect to soil microbial diversity, greenhouse gas (GHG) mitigation, and water use efficiency,
(vi) disseminating the results and transfer technology to the energy industry, land-based sector and to appropriate policy makers.

More generally, the over-arching aims of the ENERGYPOPLAR project remain highly relevant to the wider development of this bioenergy industry.

Project results:

WP1 - Optimised yield for bioenergy poplar

The overall objective of WP1 was to understand the genetic and genomic determinants of optimised yield in energy-domesticated poplars and to test new genotypes suitable for breeding and gene stacking in WP3. Both forward and reverse genetic approaches have been used and the science to some extent has been modified as the project has progressed to ensure that best use was made of the emerging technologies available from high throughput genotyping and sequencing using next generation sequencing. We have developed an Illumina genotyping chip following the re-sequencing of 50 genotypes of black poplar, P. nigra and the discovery of several million SNPs from which 12 000 probes were selected that targeted genes for yield, water use efficiency and saccharification QTL as data emerged from WP1, 2, and 4 and in collaboration with WP3.

This has been an effective collaboration and the resources were shared with a second FP7 project, NOVELTREE. This genotyping chip places us in a strong position to take maximum benefit from this resource in future projects, combined with additional funds to now complete Genome Wide Association Studies in black poplar.

- Task 1.1 Establishment of field trials with different species and GMO clones under SRC

Prior to the on-set of the project, and with the exception of the United Kingdom (UK), rather few studies had been undertaken with poplar clones in SRC culture, since the vast majority of work in continental Europe focused on poplar as a forest product rather than an energy tree. One of the key tasks of 1.1 was to bring together disparate collections of suitable poplar material from the partnership and to supply these for diverse studies across WP1, WP2, WP3, and WP4.

The following material was made available to the consortium of ENERGYPOPLAR.

Elite genotypes

- A collection of 22 elite poplar genotypes selected in SRC field experiments in UK (SOTON).
- An INRA collection of 10 P. x euramericana (P. deltoides x P. nigra) commercial clones with different ability to produce tension wood.
- A collection of 30 P. x euramericana (P. deltoides x P. nigra) elite genotypes from the French poplar breeding programme managed by the GIS Peuplier (INRA, Cemagref and FCBA).
- Three interspecific pedigrees showing hybrid vigour (heterosis) for growth potential (INRA).

Mapping pedigrees

From the five poplar mapping populations, two of them were made available to ENERGY-POPLAR project and allowed identification of genomic regions specifically involved in ligno-cellulosic quality, water-use and nitrogen-use efficiency.

1. POP1 (Fam331), an F2 P. trichocarpa x P. deltoides mapping population (SOTON) was made available in a current on-going SRC field experiment in UK (Headley site).
2. POP2, an F1 P. deltoides x P. trichocarpa mapping population from INRA composed of more than 1200 genotypes was made available for the project.

Populus nigra diversity population

Recent analysis of nucleotide diversity has revealed that significant levels of linkage disequilibrium in natural populations of Populus nigra should enable identification of favourable alleles in different genes of interest thanks to an association genetic approach. A first association population of P. nigra composed of 480 clones sampled in natural populations from different European countries has been evaluated in a field experiment in Belgium during the Sixth Framework Programme (FP6) POPYOMICS project.

This collection covers 11 degrees of latitude from Spanish populations along the Ebro river (41 degrees North) to populations in Netherlands (52 degrees North). This first field trial was harvested during winter 2006 / 2007 and wood samples were collected by SOTON on 2500 trees representing 450 different clones. These samples were made available to the project to evaluate lignin and cellulose contents and saccharification potential thanks to a near infrared spectroscopy (NIRS) calibration model.

Transgenic lines

GM poplar lines developed within the consortium were wide-ranging and supplied by INRA, SLU, STT and VIB. These included anti-sense caffeic acid O-methyl transferase (COMt), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl-coenzyme A reductase (CCR) and caffeoyl CoA 3-O-methyltransferase (CCoAOMT) cDNA plants, used to investigate how lignin modification impacted above-ground growth in coppice culture, below ground growth and interactions with ectomycorrhizal fungi (ECM), and saccharification potential. A set of new GM clones supplied by STT and SLU included 10 with altered expression of key transcription factors for meristematic activity associated with above ground growth enhancement.

New field trials were devoted to evaluation of novel genotypes for biomass production under SRC conditions, ligno-cellulosic quality of the woody biomass produced and for saccharification potential. These novel genotypes correspond to:

(1) elite genotypes already identified for their high productivity level and their water-use efficiency,
(2) a large collection of P. nigra genotypes from which the best clones could be further used as parent of interspecific hybrids in task 3.3
(3) transgenic lines with modified wood chemistry which have shown superior growth in greenhouse conditions.

In order to get phenotypic evaluation of the corresponding GM lines, a semi-outdoor experiment in large pots was conducted by UGOE since 2008. Changes in regulation procedures induced important delay in the establishment of a field trial at VIB in Belgium, but eventually this permission was given and the site provided valuable data for ENERGYPOPLAR.

Since a central aim of this WP was to optimise yield and since this requires testing of novel ENERGYPOPLAR genotypes under field conditions and an in-depth understanding of the fundamental controls on traits determining water- and nutrient-use efficiency of these plants, UGOE established outdoor growth boxes, in which the outdoor performance of promising genotypes from other partners (INRA, VIB, STT) were characterised. The growth boxes were filled with soil and installed in a caged area outdoors for which a permission to grow transgenic poplars had been granted. These experimental facilities permit rapid testing of novel transgenic genotypes under relatively realistic conditions including competition with neighbouring plants, extensive space of the plants for root development, natural conditions of mycorrhizal colonisation and exposure to natural pests and pathogens. During the time course of the project about 15 novel genotypes were tested.

Many of those showing improved growth in the greenhouse did not realise this potential under free-air conditions. This underlines the importance of rapid testing facilities. These experiments, in addition to transgenic field plantations installed by INRA, were also used to study the natural colonisation by ectomycorrhizal fungi.

- Task 1.2 Through genetical genomics identify an area of the Populus genome determining yield and identify a set of underlying genes

QTL mapping identified regions of genetic control for biomass yield. We mapped consistent QTL across multiple coppice cycles and identified five robust QTL clusters on linkage groups III, IV, X, XIV, and XIX, calling these Poplar biomass loci (PBL). In total 20 % of the variation in final harvest biomass yield was explained by mapped QTL. We also investigated the genetic correlations between yield related traits to identify early diagnostic indicators of yield showing that early biomass was a reasonable predictor of coppice yield and that leaf size, cell number and stem and sylleptic number were also valuable traits. These have important links with the work in task 1.3 where meristematic activity and cell production are also identified from transcriptional network analysis as being key targets in identifying genes underlying biomass yield.

We mapped QTL in this outbreeding inbred F2 explaining a relatively large trait variation for numerous traits associated with biomass yield, typically with few QTL explaining the largest %age of trait variation. There was nearly a 30-fold variation in biomass yield for the CC2-4 biomass harvest with genotypic mean values ranging from 0.58 Kg to 16.3 Kg. The degree of variation for the CC1-1 harvest was far greater with nearly a 100-fold range in yield and the rank order of genotypes differed between the two. Biomass at both CC1-1 and CC2-4 was skewed towards lower values, a result also reported previously.

This is likely the outcome of inbreeding depression and the resultant high genetic load associated with an inbred F2 population derived from out-breeding species. The largest variation in any trait for the CC2-4 data was seen for total basal diameter where there was more than 150 fold difference between the genotypic mean min and max values. Height showed the least variation with a minimum value of 0.98 m and a maximum of 6.99 m. These trends were similar for the CC1-1 data. The number of coppice stems varied from 1 to 24 and there was considerable variance in the consistency of the diameter of each stem, with some genotypes having a clearly identified leader and others having many stems of more uniform size.

In all work on this population in both the United States (US) and UK, LG X has been universally mapped in relation to biomass yield suggesting that this is a highly robust QTL with consistent effect across environments and growth practices. LG X contains multiple collocating QTL for both height and diameter. It is possible that this represents the location of a gene for the activity of the cambial meristem region.

A major challenge in bridging the gap between QTL and the underlying, causative DNA polymorphism is the lack of resolution associated with QTL mapping, especially in forest tree species where multi-generation inbred populations cannot be developed. It is for this reason that it has recently been proposed that QTL mapping be used as a pre-screening method to direct subsequent fine mapping in a natural population (i.e. association mapping), where historic recombination is utilised to offer far greater mapping resolution in the case of poplar down to the individual gene level.

Here task 1.2 we investigated the genetic correlations between yield related traits to identify early diagnostic indicators of yield showing that early biomass was a reasonable predictor of coppice yield and that leaf size, cell number, and stem and sylleptic number were also valuable traits. These have important links with the work in task 1.3 where meristematic activity and cell production are also identified from transcriptional network analysis as being key targets in identifying genes underlying biomass yield. Following this, we focused on one of the five PBL identified on linkage group X and we have taken a genetical genomics approach to elucidate the functional genomic aspects of this QTL further.

Although PBL3 is a useful QTL for bioenergy yield, it still represents a significant number of genes more than 500 and the task in this part of the research was to reduce this number of genes by a fine mapping approach. In order to achieve this we chose a low throughput SNP genotyping (SNPLEX) technology that enabled us to map a random set of 21 SNPs markers from across the whole linkage group and then this was followed up with an intensive second set of SNPs marker from the QTL core of 21 genes, as depicted below. This approach has revealed a set of differentially expressed genes from the biomass extremes where four high biomass and four low biomass genotypes were selected, replicated in three biological reps and subjected to transcriptome profiling using the AFFYMETRIX poplar gene chip.

This identified a set of 196 differentially expressed genes. 44 of these differentially expressed genes appear within the biomass QTL and 7 candidate genes for high biomass were taken forward to a reverse genetics approach using RNAi and over-expression technologies for five gene models and we then observed the phenotype. Street and co-workers proposed that candidate genes can be selected by identifying genes with differential expression between genotypes at the extremes of a phenotypic trait distribution. Here, the assumption is that these genotypes are fixed for the alleles contributing positive and negative effects on the phenotype, and additionally that gene expression plays an important role in determining phenotype.

Alternatively the list of genes within a QTL hotspot (or individual QTL CI) can be examined and a short list determined based on available annotation information. Although we examined the functional annotation of genes in identified QTL hotspots, with many hundreds of genes existing in each, this is not a viable exercise. This is especially true considering the complexity of, and number of contributing traits to, biomass production.

We are therefore undertook work to examine differences in gene expression between the population extremes for biomass yield and have combined this with a fine mapping approach using the low through-put SNPLEX technology. Combining these approaches has been extremely fruitful in that we have moved from several hundred candidate genes to 197 differentially expressed genes, 44 found in biomass QTL and only one gene, a WRK transcription factor, found within the linkage group X, of particular interest here.

- Task 1.3 Optimise shot function and yield. Functional analysis of engineered trees for optimised yield

The overall aim of Task 1.3 was to use a bioinformatics and literature-based approach to identify early in the project, candidate genes for biomass yield and manipulate these using transgenic approaches. These lines were also relevant to WP2 and where appropriate, material was transferred across the project for additional analyses.

STT have in parallel with SLU performed bioinformatics analysis of expression data. Genes giving increased growth in SweTrees internal gene mining have been analysed and co-regulated genes identified. Based on this data mining, we have selected three transcription factors to be over-expressed. The three novel transcription factors were over-expressed in hybrid aspen and their ability to enhance cell division was investigated. Of these PttAIL1 was found to increase cell division in the cambium. Cloning and transformation of these genes has been done. From that are plants produced and they are analysed for their increased growth. Further analysed are done with FTIR to make a characterisation on their wood quality.

An approach was explored as a complement to breeding approach in ENERGYPOPLAR proposal to identify means to enhance biomass production in poplar trees that relied on the use of functional genomics to identify candidate genes that could have the potential to increase above ground biomass in poplar. The strategy was based on the use of large amount of microarray data that describes the changes in the expression of a large number of genes in poplar deposited at the Umea Plant Science Center microarray database. This gene expression data was analysed using bioinformatic approach called as modular gene expression analysis strategy to firstly develop transcriptional networks.

Following this, the transcriptional network data was analysed to identify key transcription factors (genes that regulate the expression of other downstream targets) that could regulate the expression of known cell cycle genes. Once identified, the next step was to clone the full length cDNAs and place them under the control of a strong promoter to increase their expression and then introduce them into poplar to increase biomass via enhancement of cell division activity. To date we have identified five transcription factors that enhance cell division activity and therefore have the potential to increase the aboveground biomass.

- Task 1.4 Optimise root function for nutrient capture and carbon sequestration using GM Populus trees available to the consortium

The overall aim of task 1.4 was to investigate the potential to optimise root growth and function for the more efficient production of bioethanol from bioenergy trees. Within this task ENERGYPOPLAR considered nutrient use efficiency at a molecular and whole-tree level. The project investigated several transgenic and elite Populus lines available to the project but previously unconsidered for detailed root responses and finally investigated the complex interactions with symbiotic ectomycorrhizal (EM) associations from a molecular and whole plant perspective. Their inter-relationship with lateral and adventitious root formation was investigated.

Important goals of UGOE were the analyses of the nutrient-use efficiency and drought tolerance of poplars. Screening of the d13C signature was carried out on GM poplars, altered genes coding enzymes of the lignification pathway, grown in a field plantation at INRA (Orleans). This analysis did not reveal significant changes compared with the wildtype (P. x canescens).

We showed that P. x canescens is relatively drought sensitive; however colonisation with mycorrhizal fungi improved the performance of this genotype under drought stress. We found that the anatomy of the vascular system of P. x canescens was very sensitive to small changes in water availability. The vessel lumina became larger with better water supply and were less sensitive to changes in water availability when the roots were associated with EM fungi. This resulted probably in a better water and nutrient supply, which in return caused higher growth rates. These results underline the importance of EM associations for maintaining yield under drought stress. It is therefore also crucial to ensure that genetic modification or breeding do not exert negative effects on the capability of poplars to form associations with benefical fungi.

In addition the nutrient use efficiency of P. tremula (species adapted to dry conditions and marginal soils) was compared with that of P. trichocarpa (species adapted to riparian condition, which are usually nutrient-rich). We expected that hybrid aspen would be more efficiently using nitrogen for biomass production than P. trichocarpa because of its adaptation to less nutrient-rich conditions. However, we found that whole-plant nitrogen use efficiency and the stem carbon-to-nitrogen balance were lower in hybrid aspen than in P. trichocarpa.

In response to elevated N, hybrid aspen showed stronger stimulation of biomass production than in P. trichocarpa. Stem volume of fertilised hybrid aspen was strongly increased, however, this was mainly caused by decreases in density. Our results suggest that P. trichocarpa, a poplar species adapted to fluctuating N supply, is less responsive to differences in N availability than aspen that occurs in low N environments and that poplars with different growth rates strongly differ in N allocation and utilisation. Based on the data, we suggest that the relationships between nutrition and wood production should be tested to employ only those genotypes for bioenergy production that combine high growth and high nutrient use efficiency.

Transcriptome analyses indicated highly specific temporal induction of the PtAIL1 (Aintegumenta Like 1) transcription factor of the AP2 family during adventitious root formation. Transgenic Populus overexpressing PtAIL1 resulted in an increased number of adventitious roots, whereas RNAi mediated downregulation of PtAIL1 expression led to a delay in adventitious root formation. Microarray analysis showed that the expression of 15 genes including transcription factors such as Agamous Like 16 and MYB36 were upregulated in the stem tissues generating root primordia in PtAIL1 over-expressing plants whereas their expression was reduced in the RNAi lines. These results demonstrate that PtAIL1 is a positive regulator acting early in the regulation of adventitious rooting and identify potential targets involved in the regulation of adventitious rooting in poplar cuttings.

Roots of most trees form a nutrient-acquiring symbiosis with mutualistic soilborne EM fungi. We identified the mycorrhiza-induced small secreted protein MiSSP7 as an effector protein in the EM fungus Laccaria bicolor S238N. MiSSP7 is necessary for the establishment of the mutualistic interaction between L. bicolor and Populus spp. host trees. It is secreted by the fungus and taken up via endocytosis into plant cells where it localises to the nucleus. We demonstrated that MiSSP7 interacts with the jasmonic acid receptor JAZ6 of Populus trichocarpa. Further, we demonstrated that PtJAZ6 interacts with a number of other nuclear proteins to form a DNA binding complex.

We showed that MiSSP7 is able to block jasmonic acid signalling likely through its interaction with JAZ receptors. L. bicolor transformants with severely reduced expression of MiSSP7 do not enter into symbiosis with poplar roots, a phenotype that can be complemented by transgenically varying the transcription of PtJAZ6 or through inhibiting jasmonic acid biosynthesis. We concluded, based on our results, that MiSSP7 is an effector protein used to promote mutualism by blocking jasmonic acid signalling (i.e. plant immunity) through the PtJAZ6 receptor during plant colonisation.

We have demonstrated that lateral root (LR) stimulation during early signal exchange between roots and ectomycorrhizal (ECM) fungi is achieved by modulation of auxin gradients through altered polar auxin transport and through activation of auxin signalling pathways in the root. However transcriptome analysis of Populus tremula x P. alba colonised by L. bicolor S238N done in collaboration with INRA-Nancy, revealed activation by the fungus of putative additional alternative pathways involving other known plant hormones such as ethylene or jasmonic acid.

During the third period, we found that the fungus secreted very low amount of auxin (indole-3-acetic acid (IAA)), concentrations as low as 10 nM, insufficient to induce LR when exogenously applied. This suggests a role for other physiologically active compounds produced and released by the fungus. To test this hypothesis, we first estimated the impact of the fungus on LR induction. Application of 50 times higher auxin concentrations than secreted by the fungus together with the fungus did not cover nor interfere with fungal LR stimulation. LR stimulation by the fungus in Arabidopsis thaliana followed a totally different timing than with exogenously applied auxin, but appeared surprisingly additive. This suggested that auxin, together with other regulators released by the fungus maybe acting together on LR formation.

Since transcriptome analysis of P. tremula x P. alba colonised by L. bicolor S238N revealed activation by the fungus of putative additional alternative pathways involving other known plant volatile hormones such as ethylene or jasmonic acid, we tested whether volatile(s) released by L. bicolor S238N may be involved on fungal-induced LR formation. We used experimental conditions that excluded exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus. Volatile(s) released by Laccaria were sufficient for LR induction suggesting that gaseous compounds released by the fungus and sensed by the plant may act on top of auxin signalling pathways responsible for LR initiation in the plant.

WP2 - Genetic improvement of poplar wood quality for saccharification

The overall objective of WP2 is to make poplar clones that have improved properties as raw material for bio-ethanol production. Bioethanol is derived from the fermentation of sugar derived from cellulose, and lignin is considered as the main bottleneck in the saccharification of wood into fermentable sugars.

There are two main strategies to make genetically improved bioenergy poplars: conventional breeding, and genetic engineering. The aim of WP2 was to make significant and fast progress in our understanding of the relation between wood composition and saccharification efficiency. To this end, high-throughput tools to determine cellulose and lignin content and saccharification potential were optimised using a series of available genotypes with varying cell wall compositions. These tools were then used to characterise mapping and association populations, genotypes with high amounts of tension wood, and novel genotypes engineered for altered cell wall composition.

- Task 2.1 established field trials with different species, clones, triploids and existing transgenic poplar with modified lignin, under SRC culture. This Task is the same as task 1.1 but is presented here for clarity. WP2 analysed the same trees as in WP1, but in WP2 the relation between lignin / cellulose ratios and saccharification was studied. Field trials have been establish with poplars from mapping pedigrees, poplars collected for association genetics, transgenic poplars and poplars bended for the production of tension wood.

- The objective of Task 2.2 was to generate novel poplar genotypes with altered lignification. This was achieved by overexpressing or RNAi-downregulating five transcription factors influencing cell wall properties. These TFs were selected based on bio-informatics analysis of the expression of poplar genes represented on the POP2 micro-array to build a transcriptional network. Two of these five genes were cloned by STT, while the other three genes by SLU. Transgenic poplars overexpressing these genes have been generated.

SLU has identified extra transcription factors that have a high probability of altering key cell wall properties, and transgenic poplars with altered expression of these genes have been made. Detailed analyses of the transgenic poplars with altered levels of expression for six transcription factors has been performed by STT in collaboration with SLU, focusing on growth and cell wall properties. Positive trends were observed, but the commercial utilisation of these plants will require more work

For both PCBER and DIRIGENT, VIB made recombinant proteins for enzymatic activity assays. Substrates and products of the PCBER enzyme have been identified by comparing xylem from WT and transgenic lines. Purification followed by MS, UV and NMR analysis allowed for a putative structure of the substrate to be proposed. Data on substrate specificity was also obtained by in vitro enzymatic activity assays. Experimental evidence for the function of PCBER, the most abundant protein in poplar wood has been found by VIB.

- Task 2.3 aimed at optimising NIRS and Fourier Transform Infrared Spectroscopy (FT-IR) protocols for high-throughput analysis of poplar wood samples derived from SRC culture. In the collaboration between SOTON, STT and UGOE, two validated calibration models for lignin predictions have been developed. STT and UGOE have shared the same poplar woody material received from SOTON (Family 331). STT used 139 samples of this family for calibration of FT-IR. Lignin content was measured by SOTON as Klason-lignin. Also the saccharification potential of these samples was measured by SOTON / Imperial College. STT has now developed a stable model and is capable of estimating the lignin content with FT-IR.

UGOE has measured lignin contents with a modified acetyl bromide assay in wood samples obtained from SOTON. In addition, the same set of wood samples was used for FTIR-ATR analysis. Spectra from the FTIR-ATR measurements were obtained for a total of 139 samples. Approximately half of the samples were used to develop a calibration model.

This model shows a good calibration for prediction of lignin content with the acetyl bromide method. UGOE has also estimated energy content from FTIR. FT-IR and saccharification-protocols can now be used in the consortium for SRC poplar wood analyses.

As golden sample reference set, the elite clones from INRA have been measured with wet chemistry methods and with FTIR in collaborations between Imperial, STT and UGOE: the golden sample reference set has been distributed and analysed by FTIR with good results, based on replicate analysis.

The set has also been measured with wet chemistry analysis for lignin content and saccharification potential. The FTIR worked well. A saccharification prediction model was successfully developed, showing the use and quality of the reference set.

- Task 2.4 was dedicated to the analysis of lignin and cellulose quality and quantity of poplars grown in the greenhouse and in field plantations made in task 2.1 and a range of genotypes already available to the project. There were two objectives: assessing lignin and cellulose in poplar mapping and association populations on the one hand and in tension wood and triploids on the other. They noticed little variation in lignin content between these 153 genotypes, so that no relation between saccharification and lignin could be found. A positive relation between saccharification and galactose and glucose was found, but a negative relation between saccharification and biomass and xylose. SOTON was also able to locate one QTL for saccharification, which comprises 86 genes, accounting for ca. 13 % of the variation.

Because we have not been successful in planting triploids in the field, VIB has obtained triploid poplars and the corresponding parental lines from outdoor grown material from the INBO (Belgium). No differences in lignin and cellulose content could be detected between triploid poplars and the corresponding parents in three biological repeats, neither in saccharification potential. However, measurements were extremely variable between biological but also technical replicates, making it difficult to draw hard conclusions on the relationship between triploidy and cell wall composition. In addition to the work performed on poplar, the lignin content and saccharification potential were analysed in a set of Arabidopsis lines characterised by different ploidy levels (2n, 4n, 6n, and 8n).

Furthermore, UGOE assessed lignin and cellulose quality and quantity in transgenic poplars, whose greenhouse potential was improved by reduced isoprene emission. Furthermore, the cell wall properties were analysed in wood of poplars adapted to a dry, hot climate under osmotic stress.

- In Task 2.5 selected wood samples from the genotypes described in tasks 2.1 2.2 and 2.4 (tension wood) and genotypes from mapping populations (3-year SRC shoots) were saccharified. Both Imperial College and VIB have established saccharification protocols for SRC woody stems (deliverable 2.1) based on NREL LAP09. Different aspects have been studied, such as particle size, grinding, bark removal/separation and shortening enzymatic saccharification time. Saccharification has also been done on 153 genotypes of Family 331, an F2 mapping population, by SOTON according to both the NREL-protocol and the VIB-saccharification-protocol, providing useful insight into protocol differences.

Imperial College was the main partner performing saccharification assays in a high-throughput manner. Based on the obtained data, Imperial College was able to make a correlation matrix between cell wall composition and saccharification potential, and found that lignin content is negatively correlated with saccharification potential.

31 elite clones have been analysed by Imperial and an approximately 3x range in average glucose release across these genotypes was observed. There were no outstanding releasers, but some gave encouraging results.

VIB itself has done saccharification-analyses on PCBER and DIRIGENT-downregulated poplars. These lines showed differences in metabolites but had no significantly altered saccharification-potential compared to wild type. Also poplars modified in a monolignol biosynthesis gene (CCR) grown in the greenhouse, were saccharified. These gave a substantial increase of glucose released after saccharification. Saccharification-analyses of the same CCR deficient-lines, grown in the field, also showed an increase in glucose-release.

Using the established protocols, the saccharification-potential has been determined for the POP1 mapping population and part of the P. nigra association population. A large variation exists in saccharification potential between the different genotypes. Lignin and cell wall carbohydrates for POP1 have also been measured by wet chemical analyses. Importantly, the high and low saccharification genotypes of the POP1 family were used in a bulk segregant approach for transcriptomics where gene expression analysis revealed a suite of genes associated with high saccharification. This list of 200 genes was further refined by analysis of those located within and beyond saccharification QTL providing information for future exploitation using targeted reverse genetics.

A correlation was found between the results from saccharification assays in two different labs on the same material, proving that saccharification assays are a reliable tool in selecting wood material for bioethanol production.

WP3 - Generation of novel genotypes

Our efforts to generate novel genotypes were directed along four different research lines, reflecting the four tasks declared in the description of work of the project.

- The first research line (Task 3.1) aimed at the development of locus-specific SNP markers for molecular diagnostics. A first selection of candidate genes was performed based on known function, position (near QTL) or expression. Chosen candidates were: Ptr-GT8.1 GT2-53, CCH2, CAM1, PtOMT1, Fer2, Aqua1, Thau, PinT1, OX_RED1.

These 10 genes were screened for polymorphism in 8 P. nigra accessions and 8 P. deltoides accessions. This work led to the identification of 89 SNPs in P. nigra and 137 in P. deltoides. The identified polymorphisms will be used in genetic mapping of selected traits.

We also performed experiments in five candidate genes involved in lignin biosynthesis, this time with the aim of identifying functional variants, i.e. SNPs with a potential effect on the gene product, such as missense or nonsense SNPs.

We selected the candidate genes based on previous work performed by ENERGYPOPLAR partners or other institutions, choosing genes more likely to affect lignin composition. The five selected genes were: CAD4, C3H3, Pt4CL3, CCR7 and HCT1. We performed multiplexed pooled next generation sequencing of the coding regions of the selected genes in 768 P. nigra accessions. The 768 accessions were allocated to 12 pools, each one consisting of 64 individuals.

Partitioning the accessions in 12 pools facilitated variant identification and follow-up of interesting polymorphisms in small subsamples. We identified a total of 84 SNPs. More than 50 % of the SNPs had a minor allele frequency lower than 1 %. Thirty-six of the identified SNPs cause an aminoacidic substitution and one caused a premature stop codon in the gene HCT1. Given the interest of the identified polymorphism (hereafter referred to as HCT1-729C>A) we individually tested each accession for mutation. We identified a total of 42 mutation carriers, one of which carried the mutation in homozygous state.

We performed phenotypic analysis of lignin composition and lignin content to assess differences between carriers and non-carriers. Our results indicated that homozygous line had an increase in H-units. This increase in H was charged by a decrease in G-units, while the amount of S-units remained the same. For this reason, the S/G-ratio for the homozygous carrier seemed to be increased.

Analysis of lignin content with the acetil-bromide method and of saccharification potential showed no difference between homozygous carriers, heterozygous carriers and wild type accessions. However, large variability was observed between different heterozygous individuals and between different replicates of the same individuals. In addition, it is possible that genetic background also plays a role in determining lignin content.

- Task 3.4 (Selection of superior parental trees and production of F1 hybrids) performed crosses between the homozygous carrier of HCT1-729C>A and two different heterozygous carriers (71104 and SPM12). We identified 39 homozygous carriers and 58 heterozygous carriers. Two subjects showed wild type genotype (C/C), inconsistent with the expectation from a cross of the type A/A X A/C. They were considered as genotyping errors and removed from further analysis.

No obvious visible phenotypes were observed that distinguished homozygous carriers from heterozygous carriers, although large biological variation was still observed. The H/G/S composition of the lignin was comparable to that found for young developing wood of the parental line 71030-501. Interestingly, although in both progenies the level of H units increased in the homozygous compared to the heterozygous genotypes, a clear difference was observed in the level of accumulation (18x and 10x increase for the two crosses). No significant difference in saccharification potential could be seen.

As the defective allele will be used in breeding programs as well as for gene stacking approaches, the homozygous carrier (71030-501) as well as several heterozygous carriers were transferred to in vitro cultures.

- In task 3.2 we aimed at the development of whole genome markers for genetic distance estimation, with the aim of developing genome-wide markers that could be used to predict heterosis. We performed a factorial design between 2 P. nigra males and 2 P. deltoides females, and selected 12 F1 accessions (3 for each of the four possible crosses). We chose to focus on two different sets of potential markers of heterosis:

a) large structural variants; and
b) gene expression.

In order to obtain information on large structural variants we performed next generation sequencing on the 16 accessions. The total coverage was > 200x. We then identified structural variations between the two species and followed the segregation in the offspring. We used an available software package to detect 3380 deletions and developed a novel algorithm that enabled us to detect 5877 insertions relying on paired-end signature mapping, de novo assembly and homology of inserted sequence to known transposable elements. 76 % of the identified deletions showed homology with transposable elements. By definition, all of the identified insertions showed homology with transposable elements. All of the identified structural variants can be used as genetic markers for heterosis.

Another potential source of heterosis variation in gene expression among the two parentals, cis-regulatory variation in gene expression has been often cited as a potential mechanism of heterosis. To identify potential markers of cis-regulatory gene expression, we performed RNAseq on two different tissues. We identified a total of 1685 differentially expressed genes between the two species. Of them, 897 had higher expression in P. nigra, 788 in P. deltoides. In addition, we identified 514 genes expressed in offspring with values different from midparent value. Of them, 205 had lower values in offspring and 309 had lower midparent values. The genes included in the above-mentioned lists can be considered as potential markers of heterosis, although due to the small sample size no final claim can be made.

- Task 3.3 was dedicated to generate transgenic poplars carrying multiple transgenes. All the experiments were performed on INRA 717-1B4, a female Populus tremula x P. alba clone, which we considered to be the best suited for transformation experiments. Candidate genes for gene stacking were chosen: F5H, CAD, CCR, GA20-oxidase and GS genes.

Two different strategies were used to combine beneficial genes in transgenic plants:

1. retransformation of existing transgenic lines;
2. combinatorial transformation of wild-type lines.

- The fourth research line (task 3.4) aimed at selecting superior parental trees and obtaining from them the F1 offspring. This research line was strictly interconnected with previous work (in which phenotypic evaluation of poplar accessions had been performed) and with work in the framework of ENERGYPOPLAR, in particular for obtaining a large number of carriers of the mutation identified in task 3.1.

Twenty P. nigra male and female parental genotypes were selected either on phenotypic performance or desired genotype configuration. Phenotypic selection was based on biomass production (2 clones, 1 male and one female) and resistance to Mlp leaf rust (5 clones, 3 males and two females). Parents selected for their genotype configuration were chosen as carriers of the HCT1-729 mutation (15 clones, 8 males and 7 females).

Fifteen out of 21 different crosses were successful and produced between 5 and 126 seedlings. The total number of seedlings produced at the end of the growing season was 820. Mortality was often observed at cotyledonary stage and was uniform for all crosses. No specific deviation compared to usual intraspecific crosses success was observed. Progeny of the crosses between 71104 and 71030-501 and between SPM12 and 71030-501 were used in the context of task 3.1 as described above.

WP4 - Environmental and economic sustainability assessment

Optimised systems for biofuel production will have to afford sustainability benefits at all levels of the production chain involving the usage of highly productive trees with low nutrient demand and high water use efficiency, protection of biodiversity and the exploitation of beneficial soil microbes for plant protection and improved nutrition. LCAs that consider the full life cycle of biofuels from the production of raw materials (biomass, additives), processing, distribution and final combustion emissions in motor vehicles are required to evaluate the sustainability and benefits of novel feedstocks for bioenergy. WP4 defined three tasks to address the following key issues:

1. LCA to investigate environmental impact of biological and technical production processes for biofuel production from energy poplar supply chains,
2. diversity diagnosis and assessment of ecosystem services in SRC bioenergy poplar,
3. identification of novel genotypes with improved water use efficiency.

LCA to investigate environmental impact of biofuel production

Lignocellulosic biofuel is set to become an important component of European Union (EU) strategy for mitigating climate change. Imperial adopted a whole LCA approach together with techno-economic assessment (TEA) using a process engineering model to evaluate both the environmental and economic perspectives of bioethanol derived from Poplar feedstocks within the EU.

LCA and TEA were carried out to investigate:

1) bioethanol derived from the poplar biomass grown under short or very SRC or VSRC) management;
2) bioethanol produced via different processing technologies;
3) different EU regions with regional agro-ecosystem variations;
4) prospective scenarios for year 2020 and 2030 with optimised processing technology and poplar feedstock.

A process-oriented biogeochemistry model DNDC (modified in ENERGYPOPLAR for application to poplar SRC) was incorporated into environmental model in this study to simulate the biogeochemistry process and C and N cycles in poplar agro-ecosystem. This is important for the consideration of N emissions of the GHG N2O from agricultural land. The results demonstrated that the biomass yield and C pool derived from DNDC simulations were consistent with the experimental observations. The modelled daily carbon and nitrogen fluxes were subsequently used as site-specific inputs to the LCA model to develop the regional, site-specific case studies.

This scenario modelling at the EU level has demonstrated the potential to deliver both environmentally sustainable and economically viable poplar-based bioethanol market within the EU. It should be noted that an important element of the GHG balance of the biofuel supply chain is the carbon stock in soils under Poplar cultivation. In some categories the bio-ethanol fuel is neutral with regard to petrol and in others the environmental impacts are higher (e.g. aquatic ecotoxicity). Detailed analysis of the scenarios and modelling are described in full in the scientific paper prepared from this work. The TEA has indicated that the main economic cost drivers in the bioethanol supply chain are feedstock and handling and saccharification and fermentation, with the greatest contributor being purchase of raw materials (92 % of feedstock and handling is attributed to poplar purchase, and 84 % of saccharification and fermentation cost is from enzyme purchase).

Overall, by sustaining the development of advanced hybrid poplar feedstock and processing technology for biofuel production within the EU (genetic engineering, advanced breeding programme, conversion biochemistry advances) will provide climate change mitigation benefits, enhance energy security and advance the case for biofuel as a component of the EU's sustainable energy future.

Diversity diagnosis and assessment of ecosystem services in SRC bioenergy poplars

Biodiversity and functional interactions of organisms critically affect the stability and maintenance of ecosystems. In this regard fungi are an extremely important organismal group because they improve plant performance as mutualists when colonising roots, e.g. ectomycorrhizal fungi (ECM) or they may cause profound damage and yield loss as pathogens. It is, thus, evident that the fungal microbiome plays key roles in modulating biomass production and plant adaptation to changing environmental conditions. To date, the diversity of this important organismal group is still not well understood. UGOE investigated the influence of transgenic poplars grown in SRC on the underground soil microbiome. The transgenic poplar plantation was made available by INRA-Orleans (WP1). For the analysis of fungal diversity soil and root samples were collected, 454 sequencing was conducted at the high-throughput sequencing facilities at UGOE and an analysis pipeline was developed by INRA-Nancy.

In total, 811 900 sequence reads were generated by 454 pyrosequencing. After quality filtering 686 053 sequence reads were used for further analyses. 4706 to 17 994 sequences were obtained per sample. These sequences were clustered according to similarity and yielded 28 to 570 non-singleton OTUs per sample. After removal of singletons, soil contained 5944 OTUs matching with 186 fungal families; roots contained 2399 OTUs matching with 115 fungal families. These data show that in a newly installed homogeneous poplar plantation fungal diversity is extremely high. Fungal hyperdiversity is therefore not only a characteristics of natural and complex ecosystems but also of simple agroforest systems

Transgenic poplars with suppressed activities of CAD did not affect the fungal diversity or community composition, neither in soil adjacent to the roots nor in the roots themselves. In soil saprotrophic, pathogenic and endophytic fungi were the dominating groups, whereas ECMs were dominant in roots. Arbuscular mycorrhizal diversity was higher in soil than in roots.

The 454 sequencing data were validated by a combined morphotyping / rRNA Sanger sequencing (MT/rRNAseq) approach. As in other studies species richness of the root-associated ECM community was initially low and increased with plantation age. All ECM found on one-year-old poplar roots by MT/rRNAseq were also present in the 454 sequencing data set. 454 identified also a number of ECM-forming fungi in roots, which developed functional ECM only in the second year after plantation. This result supports the priority concept that ECMs present on roots have a competitive advantage over soil-localised ECM fungi.

Further MT/rRNAseq analyses on novel genetically modified poplar clones provided by STT and VIB did not provide evidence effects of the transgene on ECM interactions. Previous studies revealed positive relationships between tree biomass and ECM colonisation. This relationship was confirmed for genetically modified bioenergy poplars and supports positive ecosystem services of ECM fungi.

We also studied the performance of poplars with reduced isoprene emissions. Isoprene is one of the major biogenic greenhouse gases. Therefore, cultivation of poplar with reduced levels of this compound has been recommended. Under free-air conditions these poplars showed neither growth trade-off nor effects on wood properties or ECM colonisation.

Identification of novel genotypes with improved water use efficiency

QTL discovery for water use efficiency in Populus: one of the pressing issues for the development of bioenergy crops is their use of natural resources, including water, fertilisers and land. This is because they all contribute to the overall LCA of the crop and ultimately add to the carbon cost. Thus an improved understanding of the genetic basis of water in bioenergy poplar is warranted and ENERGYPOPLAR has made significant steps forward in resolving this issue.

Using a mapping population, POP1, SOTON have identified a suite of QTL for water use efficiency from carbon isotope analysis where this population was grown in three separate environmental conditions - one in relatively droughted conditions in Italy and in both coppice and SRC in the UK. We have assessed stomatal conductance (gs, a measure of stomatal opening) and leaf carbon isotope composition (d13C, an indirect indicator of leaf water use efficiency (WUE)) in the two parental Populus species, P. deltoides and P. trichocarpa and their F2 progeny.

QTL were identified for d13C on nine LG and two QTL for gs. From these QTL and gene expression from microarrays, we focused on three hotspots and 23 novel candidate gene models on LGs VI, X and XVI that are targets for future genetic and genomic analysis. We also quantified the phenotypic variation in WUE related traits in a F2 mapping population using the measurement of carbon isotope composition, for which there is extensive theoretical and empirical data to suggest that this composition is positively correlated with WUE. Here we were able to quantify phenotypic variation in the F2 population for leaf d13C, revealing considerable differences, dependent upon both genotype and the varying environments and management conditions to which the trees were subjected. In average, d13C was higher in Italy than the UK. Given the detected variation in d13C and gs between genotypes in Family 331 QTL were evident, providing some insight into the genetic basis of WUE associated traits. Our QTL accounted for a moderate amount of genetic variation.

QTL for stomatal conductance and carbon isotope composition measurements in the F2 Family 331 were determined for these WUE related traits for the first time to our knowledge in Populus. Few QTL exist for stomatal conductance for any species in the published literature, reflecting the difficulty of measuring this trait on many hundreds on individuals in replicated QTL mapping population experiments. Thus our attention was focused on carbon isotope composition to provide a robust surrogate for WUE. For several regions of the genome in particular QTL hotspots were defined where at least one QTL explained > 5 % of the variation and where multiple QTL were present.

Three QTL hotspots were mapped to LG VI, X and XVI and contained 2 and 3 d13C QTL on LG VI and X respectively and one QTL for d13C and one for gs on LG XVI. Markers linking the genetic and physical map of the F2 pedigree were used to determine gene models localised within these QTL hotspots in the Populus genome. 950, 758 and 979 genes were found within the 95 % confidence intervals to which the QTL on LG VI, X and XVI were mapped respectively.

Combining microarray data and gene lists within QTL hotspots, 46 gene models were found within the QTL and 23 on LG VI, X and XVI which were also highly expressed in response to dehydration.

In conclusion, we have revealed wide variation in stomatal behaviour, traits related to WUE and response to drought within a Populus pedigree, that has enabled three hotspots within the genome linked to water use efficiency. Our preliminary analysis has already identified a small number of candidate genes in these hotspots that provide targets for future molecular breeding and improved drought adaptation in Populus. Wider application can be possible throughout the Salicaceae to willow as recent papers have shown the construction of linkage maps of Salix aligned with the Populus genome.

Assessing natural genetic variation in traits associated with WUE in an association population of black poplar Populus nigra: SOTON assessed the natural genetic variation in carbon isotope discrimination of the association population in a common garden experiment and then undertook intensive work in control environment experiments on six and then two extreme genotypes, one from Spain and one from Italy. For the population of P. nigra, genotypic variation was clear in WUE using d13C and varied with latitude of origin. Wood d13C had lower values in populations from the North East of Europe, such as The Netherlands and Germany, and the East, such as North Italy and the Drome region in France, and consequently had the highest WUE. These trees were collected from wet environments in Europe; their latitude of origin is comparable to the conditions in the fields in Belgium. On the other hand, Spanish and Southern French populations had the highest d13C therefore the lowest WUE.

However, this study was conducted in a wet environment in Belgium and had extremely different conditions compared to their origin for genotypes from Spain or Southern France. d13C varies with weather such as precipitation and soil water regime.

This hypothesis was tested in a moderate drought experiment in a greenhouse, focusing on six genotypes originated from four locations in Europe (Spain, France, Northern Italy and the Netherlands). A progressive and moderate drought was applied and soil moisture was monitored. French, Italian and Dutch genotypes responded fairly similarly to water deficit: height growth and new leaf formation decreased, no branches were developed in either treatments and SLA did not change.

Uniquely Fr1 and Fr2 had a high leaf loss due to drought stress while the genotypes from Italy and the Netherlands did not lose leaves in response to drought. Mature leaf senescence in response to drought allows remobilising nutrients from the mature leaf towards younger leaves.

Physiologically the genotypes reacted differently in the intensity of decrease in response to drought. French and Spanish genotypes responded quickly to drought by closing their stomata only five and seven days after drought while the Italian genotype did not vary in genotypes. Genotypes reacted differently in response to drought for d13C. Spanish and French genotypes decreased d13C showing thus an increase in WUE.

A genomic study was then conducted in both well-watered and moderate drought stress conditions, using microarray analysis and real-time qPCR between two genotypes. The number of transcripts expressed in response to water deficit was very high for Ita compared to Sp2. Although it might be expected for the supposedly drought tolerant plant to express more genes in response to drought, similar findings were observed in rice. Degenkolbe et al. used two genotypes of rice: a drought tolerant and a drought sensitive, to study their genomic response to a moderate and long-term water stress. The drought tolerant genotype had less genes drought regulated than the sensitive genotype. A possible reason for the Italian genotype of P. nigra to express more genes compared to Sp2 in response to drought could be due to the damage water stress induces in Ita while Sp2 is less affected.

This study has shown that screening for variation in wild plants from diverse geographic locations under drought stress is a very useful strategy to discover genotypes naturally adapted to water deficit. Similar studies have been done with other plants such as Arabidopsis. Finding drought tolerant genotypes can subsequently be used to identify and to study drought-related genes.