WP 1 - Identification and functional characterisation of CGs as potential regulators of wood properties relevant for bioenergy production.
The overall objective is to identify and characterise candidate genes (CGs) (focusing on transcription factors, miRNAs and their respective partners and/or targets) involved in the regulation of wood properties important for bioenergy in poplar (P) and eucalypts (E).
Task 1.1 In silico identification of CGs from compendia of expression data
In the first task, we will analyse in silico transcriptomic data available publicly or within the consortium as well as new resources generated here to select a reservoir of regulatory CGs potentially involved in secondary wall (SW) formation in E & P. CGs subsequently will be mapped onto E & P genetic maps (WP3).
Subtask 1.1.1 Development of new transcriptomic resources
As stated in the background section, several partners (P1, P2, P3, P5, P8) have built databases both in poplar (P2, P5) and eucalypts (P1, P8) (see section 5 related projects for details) based on large in-house collections of ESTs and transcriptomic data already generated (and/or in the process of being developed in parallel projects). These databases are essential for the success of task 1.1. To supplement these data, new resources will be generated efficiently with deep sequencing methodologies: Xylem transcriptomes and sRNA libraries of juvenile/mature wood of E. globulus and E. gunnii, transcriptome of particular cell layers of xylem of P. tremula, sRNA libraries for tension wood of P. deltoides X P. trichocarpa.
Subtask 1 1.2 Integration and data mining of transcriptomic resources
The first step will be to integrate newly generated sequencing data with existing data through the participation of the bioinformatic platforms (P1,GenotoulBioinfo, FR; P8,INESC-ID, Portugal). We will then combine annotation with virtual northern data to select a subset of TF and miRNAs expressed specifically or preferentially in xylem and differentially under contrasted wood conditions. The availability of the recently released whole genome sequence of E. grandis and poplar will facilitate comparative phylogenetic analysis between the two genera and prediction of miRNA genes and their targets. Taking advantage on the large number of genotypes used for library construction for both genera and on the re-sequencing projects for P and E (see WP3), a list of potential polymorphisms (SNP) will be obtained in silico from these CGs that will be further exploited for mapping and genotyping in WP3.
Task 1.2 Transcript profiling of selected CGs
To refine the number of CGs (selected in task 1.1), we will select the most pertinent ones using high-throughput expression profiling tools in contrasted wood samples (juvenile vs mature, opposite vs tension, distinct wood tissues/cellular types…) from different E and P species and genotypes. Expression profiling of MiRNA, predicted miRNA-targets and TF genes will be performed using the most efficient technology available (e.g. lParaflo®technology, oligochip, high-throughput qPCR [Fluidigm®]). For both genera, integration and comparative analyses of all acquired data will allow selection of a handful number of GCs expressed in specific wood samples, cell types, and/or environmental conditions. The most promising CGs (10-20) will be shared between partners for functional characterisation in task 1.4.
Task 1.3 Identification of the transcriptional interactome
Due to the growing importance of the interactome in transcriptional regulation, we will use methods, such as 2-Yeast hybrid system, to identify protein partners of the most interesting TF selected in task 1.2, including wood-related master regulators already characterised by P1 (Goicoechea et al, 2005; Legay et al, 2007). The Proteic interactants will be considered as new potential CGs.
Task 1.4 Functional characterization of a limited number of selected CGs
The function of a selected number of CGs identified will be characterised in planta with an efficient method developed by P2 to generate in planta differentiation of wood tissues (cambial cells, xylem and phloem) from transgenic calli (Figure 1). This procedure, developed on poplar, enables obtaining transgenic wood zones of about 50 newly differentiated xylem cells within 3 weeks and we will implement it in eucalypts.
Several approaches are envisaged to modify the expression of selected CGs (overexpression, RNAi, amiRNA, dominant repressor). For RNAi studies, a novel vector system developed by DNA Cloning that allows simultaneous cloning of hundreds of different CGs will be tested. The construction of these binary vectors will be shared between partners (P1, P2, P5, P8, P11). Between 10 and 20 constructs will be generated for transformation of both poplar and eucalyptus and the different transformations will be shared between P1, P4 and P8 for E; P2, P5 and P11 for P. About 15 independent transgenic calli will be produced for each binary construct, thus, a approximately 300 transgenic lines will be generated for each genus. Transgenic wood zones from E and P (and/or young poplar transgenic plants) will be phenotyped in WP2. To further analyze transgenic lines, we will establish a collection of transgenic calli and/or plantlets using cryopreservation techniques mastered by P4 who has an extensive infrastructure for cryogenic storage (Harvengt et al. 2004).
Task 1.5 Environmental regulation of CGs and impact on biomass production and wood quality
To enable an efficient, rational process for the selection of new sustainable lines for bioenergy production, this task will provide insights into the environmental effects on the expression of CGs (selected in task 1.1/1.2) and assess how environmental conditions impact the production of wood biomass and quality.
Subtask 1.5.1 Selection of eucalyptus and poplar genotypes.
P and E genotypes will be selected (by P10 and P13) based on performance traits (Sixto et al.2007) and will include poplar clones of interest for bioenergy in France, Spain, Portugal, and Germany and for which P2, P4, P5 & P7 have expertise as well as transgenic lines affected in the development of vascular tissues (P11, P12) (Jing et al 2004).
Subtask 1.5.2 Environmental and seasonal studies of CG expression.
The environmental factors tested correspond to common agents affecting growth in plantations of both genera species, mainly fertilization and availability of inorganic N in soil (P11), water (P11), but also abiotic stresses such as cold (P12), high temperature (P11), and salinity (P13). Seasonal variation of CGs expression will be studied (P12) to get a complete view of the involvement of CGs in growth and differentiation. CG expression will be analysed by qRT PCR in xylem samples and in other organs. Plant biomass and important architectural components (plant height, stem diameter, leaf size, and internodal length) will be evaluated and samples will be supplied for wood properties (WP2).