Jean-Charles Leplé

Field and pulping performances of transgenic trees with altered lignification

The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula × Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

Transgenic poplars: expression of chimeric genes using four different constructs

SummaryLeaf or stem explants of a hybrid poplar clone (Populus tremula X Populus alba), sensitive to Agrobacterium tumefaciens, were co-cultivated either by an octopine or a nopaline disarmed A. tumefaciens modified strain. Transformed poplar shoots were readily regenerated from explants. The protocol was improved using the nopaline disarmed strain C58/pMP90 with the binary vector pBI121. This protocol was then used to test three other vectors. The first one, possessing a nptII gene fused to the CaMV 19S promoter, permitted regeneration of transformed shoots in presence of 50 to 100 mg/l kanamycin. The two other vectors carried an additional nptII gene under the control of the CaMV 35S or CaMV 35S promoter with a double enhancer sequence (CaMV 70). CaMV 70 promoter provided consistently higher level of gene expression than the other promoters in both callus and leaf tissues.

Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase.

Significance In the transition from a fossil-based to a bio-based economy, bioethanol will be generated from the lignocellulosic biomass of second-generation biofuel crops, such as poplar. The lignin polymers in the plant cell walls represent the main factor determining the recalcitrance of biomass to enzymatic processing. We have grown genetically modified poplars, down-regulated for cinnamoyl-CoA reductase (CCR), an enzyme in the lignin biosynthetic pathway, in field trials in Belgium and France. We show that wood samples derived from the transgenic trees are more easily processed into ethanol. However, strong down-regulation also affected biomass yield. In conclusion, CCR down-regulation may become a successful strategy to improve biomass processing if the yield penalty can be overcome. Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR–down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome.

Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl CoA reductase deficiency)

A molecular marker compound, derived from lignin by the thioacidolysis degradative method, for structures produced when ferulic acid is incorporated into lignin in angiosperms (poplar, Arabidopsis, tobacco), has been structurally identified as 1,2,2-trithioethyl ethylguaiacol [1-(4-hydroxy-3-methoxyphenyl)-1,2,2-tris(ethylthio)ethane]. Its truncated side chain and distinctive oxidation state suggest that it derives from ferulic acid that has undergone bis-8-O-4 (cross) coupling during lignification, as validated by model studies. A diagnostic contour for such structures is found in two-dimensional (13)C-(1)H correlated (HSQC) NMR spectra of lignins isolated from cinnamoyl CoA reductase (CCR)-deficient poplar. As low levels of the marker are also released from normal (i.e. non-transgenic) plants in which ferulic acid may be present during lignification, notably in grasses, the marker is only an indicator for CCR deficiency in general, but is a reliable marker in woody angiosperms such as poplar. Its derivation, together with evidence for 4-O-etherified ferulic acid, strongly implies that ferulic acid is incorporated into angiosperm lignins. Its endwise radical coupling reactions suggest that ferulic acid should be considered an authentic lignin precursor. Moreover, ferulic acid incorporation provides a new mechanism for producing branch points in the polymer. The findings sharply contradict those reported in a recent study on CCR-deficient Arabidopsis.

Wood formation in Angiosperms

Wood formation is a complex biological process, involving five major developmental steps, including (1) cell division from a secondary meristem called the vascular cambium, (2) cell expansion (cell elongation and radial enlargement), (3) secondary cell wall deposition, (4) programmed cell death, and (5) heartwood formation. Thanks to the development of genomic studies in woody species, as well as genetic engineering, recent progress has been made in the understanding of the molecular mechanisms underlying wood formation. In this review, we will focus on two different aspects, the lignification process and the control of microfibril angle in the cell wall of wood fibres, as they are both key features of wood material properties.

An alternative approach for gene transfer in trees using wild-type Agrobacterium strains.

Micropropagated shoots of three forest tree species, poplar (Populus tremula × P.alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra × J. regia), were inoculated each with six different wild-typeAgrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with anAgrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carryingneo (kanamycin resistance) anduidA (β-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to expressneo anduidA genes. These results suggest that wild-typeAgrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.

Expression of the mutantArabidopsis thaliana acetolactate synthase gene confers chlorsulfuron resistance to transgenic poplar plants

The mutant acetolactate synthase (crs1-1) gene fromArabidopsis thaliana, which confers resistance to the herbicide chlorsulfuron, was transferred to a hybrid poplar (Populus tremula×P. alba) using twoAgrobacterium-mediated transformation methods (co-inoculation and co-cultivation). Two different constructs were used. In one, the mutantcrs1-1 gene was placed under the control of its own promoter, and, in the other, this gene was under the control of the duplicated cauliflower mosaic virus 35S promoter (70 promoter). The transformation efficiency ranged from 22 to 32% of the tumours in co-inoculation and from 67 to 77% of the stem explants in co-cultivation experiments. The usefulness of the herbicide chlorsulfuron as a selectable marker gene was also demonstrated. Successful genetic transformation was verified by Southern and northern analyses and enzyme activity. Plants carrying thecrs1-1 mutant gene under the control of the 70 promoter showed high levels of transcription and activity whereas plants carrying the nativecrs1-1 gene showed low levels of expression. However, transgenic plants expressing each of the chimaericcrs1-1 genes are completely resistant to high doses of chlorsulfuron in greenhouse tests.

Genome-Wide Analysis of LIM Gene Family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa

Abstract In Eukaryotes, LIM proteins act as developmental regulators in basic cellular processes such as regulating the transcription or organizing the cytoskeleton. The LIM domain protein family in plants has mainly been studied in sunflower and tobacco plants, where several of its members exhibit a specific pattern of expression in pollen. In this paper, we finely characterized in poplar six transcripts encoding these proteins. In Populus trichocarpa genome, the 12 LIM gene models identified all appear to be duplicated genes. In addition, we describe several new LIM domain proteins deduced from Arabidopsis and rice genomes, raising the number of LIM gene models to six for both species. Plant LIM genes have a core structure of four introns with highly conserved coding regions. We also identified new LIM domain proteins in several other species, and a phylogenetic analysis of plant LIM proteins reveals that they have undergone one or several duplication events during the evolution. We gathered several LIM protein members within new monophyletic groups. We propose to classify the plant LIM proteins into four groups: αLIM1, βLIM1, γLIM2, and δLIM2, subdivided according to their specificity to a taxonomic class and/or to their tissue-specific expression. Our investigation of the structure of the LIM domain proteins revealed that they contain many conserved motifs potentially involved in their function.

Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole‐genome shotgun (WGS) approach, without the use of costly and time‐consuming methods, such as fosmid or BAC clone‐based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS‐FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired‐end and mate‐pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired‐end reads and contaminants detected, resulting in a total of 17 910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.