Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called "BRACHYTIL", for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

Monolignol glucosides are thought to be implicated in the lignin biosynthesis pathway as storage and/or transportation forms of cinnamyl alcohols between the cytosol and the lignifying cell walls. The hydrolysis of these monolignol glucosides would involve β-glucosidase activities. In Arabidopsis (Arabidopsis thaliana), in vitro studies have shown the affinity of β-GLUCOSIDASE45 (BGLU45) and BGLU46 for monolignol glucosides. BGLU45 and BGLU46 genes are expressed in stems. Immunolocalization experiments showed that BGLU45 and BGLU46 proteins are mainly located in the interfascicular fibers and in the protoxylem, respectively. Knockout mutants for BGLU45 or BGLU46 do not have a lignin-deficient phenotype. Coniferin and syringin could be detected by ultra-performance liquid chromatography-mass spectrometry in Arabidopsis stems. Stems from BGLU45 and BGLU46 mutant lines displayed a significant increase in coniferin content without any change in coniferyl alcohol, whereas no change in syringin content was observed. Other glucosylated compounds of the phenylpropanoid pathway were also deregulated in these mutants, but to a lower extent. By contrast, BGLU47, which is closely related to BGLU45 and BGLU46, is not implicated in either the general phenylpropanoid pathway or in the lignification of stems and roots. These results confirm that the major in vivo substrate of BGLU45 and BGLU46 is coniferin and suggest that monolignol glucosides are the storage form of monolignols in Arabidopsis, but not the direct precursors of lignin.

Field trials with lignin-modified transgenic trees / Gilles Pilate, Annabelle Dejardin and Jean-Charles Leple -- Lignification and lignin manipulations in conifers / Armin Wagner, Lloyd Donaldson and John Ralph -- Molecular biology of lignification in grasses / Michael J. Harrington ... [et al.] -- Cytochrome P450s in lignin biosynthesis / Annette Alber and Jurgen Ehlting -- Role of plant laccases in lignin polymerization / Serge Berthet ... [et al.] -- Transcriptional regulation of the lignin biosynthetic pathway revisited: new players and insights / Jacqueline Grima-Pettenati ... [et al.] -- Lignins and abiotic stresses / Mireille Cabane, Dany Afif and Simon Hawkins -- Fungal strategies for lignin degradation / Jean Claude Sigoillot ... [et al.] -- Evolutionary history of lignins / Esther Novo-Uzal ... [et al.].

Laccases are ubiquitous oxidases present in animals, plants, bacteria, and fungi. In plant species, they occur as large multigenic families. The involvement of peroxidases in lignification is supported by a wealth of literature data. In contrast, the role of laccases in this major plant process is less firmly established. The large number of plant laccases, which argues for a variety of functions in plant development, makes the identification of lignin-specific laccases a challenge. However, in the past decade, the development of new genetic technologies and tools has played a central role towards resolving this issue. In addition, the plant model, Arabidopsis thaliana, has recently provided novel insights about the occurrence of laccases involved in stem lignification. Information about lignin-related laccases is also available from other species, such as poplar, or other organs and tissues, such as seed coats. This review brings a short and cutting edge survey of laccases and lignification.

Deciphering the genetic determinants involved in cell wall assembly is a strategic issue for breeding programs that target both ruminant feeding and biofuel production. The Arabidopsis thaliana model system has great potentials to elucidate the genetic determinants involved in cell wall component biosynthesis and those involved in the regulation cascades allowing their coordinated assembly. QTL for biomass quality related traits (cell wall content, lignin content, and cell wall degradability) were mapped in the three Arabidopsis RIL progenies Bay0 × Shahdara, Bur0 × Col0, and Blh1 × Col0. Overall, 40 QTL were detected for these traits, explaining up to 33 and 12% of the observed phenotypic variation for lignin content and cell wall degradability respectively. Major QTL hotspots were mapped on chromosome 1 (position 5 Mbp), chromosome 4 (position 1 Mbp), and chromosome 5 (position 3 Mbp). A putative candidate gene set (82 genes) was considered including those previously described as involved in cell wall phenolic component biosynthesis, their regulation factors, and genes involved in lignified tissue patterning. Colocalisations observed (according to the reference sequence of Col0) between the detected QTL and these candidate genes did not prioritize any of the three gene groups (monolignol biosynthesis, transcription factors, lignified tissue patterning). Colocalizations were thus observed for 57% of monolignol biosynthesis related genes, 55% of the transcription factors considered, and 66% of genes considered to be involved in lignified tissue patterning and assembly. Colocalizations were observed for at least one member of all investigated gene families, except WRKY transcription factors. Colocalizations were also shown with several miRNA putatively involved in the regulation of lignifying tissue assembly. Taking into account the QTL shown in the Bur0 × Col0 progeny, allelic variations were shown in the MYB32, MYB58, MYB75, GRAS SCARECROW, AtC3H14 zinc finger, SHINE2, and IFL1 genes and in the AtMIR397a. Given that the list of candidate genes is not complete, and because the QTL support intervals encompassed genes of still unknown function, it is still not clear whether one of the selected candidates is responsible for the effect of a detected QTL. Mutant investigation and positional cloning steps are likely essential to clearly determine the causal mechanism involved in cell wall degradability variation.

Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge regarding its lignins and no data on lignin-related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose-to-ethanol conversion process. Down-regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically induced population of Brachypodium mutants (Bd213 background) for red culm coloration. We identified two mutants (Bd4179 and Bd7591), with mutations in the BdCAD1 gene. The mature stems of these mutants displayed reduced CAD activity and lower lignin content. Their lignins were enriched in 8O4- and 4O5-coupled sinapaldehyde units, as well as resistant inter-unit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end groups. Moreover, the amount of sinapic acid ester-linked to cell walls was measured for the first time in a lignin-related CAD grass mutant. Functional complementation of the Bd4179 mutant with the wild-type BdCAD1 allele restored the wild-type phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than wild-type plants. Here, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the degree of branching of lignins through incorporation of sinapaldehyde. These changes make saccharification of cells walls pre-treated with alkaline easier without compromising plant growth.

While it is established that peroxidases are involved in the polymerization of lignins, it is not yet clear whether laccases participate to lignification. In order to address this issue, we studied,laccase T-DNA insertion mutants in the model plant, Arabidopsis thaliana. We identified two genes, AtLAC4 and AtLAC17, particularly expressed in stems. AtLAC17 was more specific to fibers while AtLAC4 was expressed in vascular bundles and fibers. We produced a double mutant, lac4 lad 7, by crossing a lad 7 mutant with a lac4 mutant. The analyses of the single and double mutants provided the first evidence that laccases are involved in the constitutive lignification of Arabidopsis stems. The lower lignin levels of the double mutant resulted in higher saccharification yields. These findings suggest that the genetic engineering of lignin-specific laccases is a potentially innovative and promising tool to facilitate the lignocellulose-to-bioethanol conversion process.

Lignins have a major impact on the agro-industrial uses of plants. Until now, most of the strategies considered for lignin reduction have targeted the monolignol pathway since the genes involved in these metabolic steps have been identified in many plants. Less is known about the other steps and in particular on lignin polymerization in the cell wall. While it is established that peroxidases are involved in the polymerization of lignin precursors, it is not yet clear whether laccases (EC 1.10.3.2) participate in constitutive lignification. In order to address this issue, laccase genes (AtLAC4 and AtLAC17) that are highly expressed in Arabidopsis stems were studied. AtLAC17 was specifically expressed in the interfascicular fibers while AtLAC4 was expressed in vascular bundles and interfascicular fibers. Arabidopsis T-DNA insertion mutants were selected and characterized. Two double mutants were obtained by crossing the AtLAC17 (lac17) mutant with two AtLAC4 mutants (lac4-1 and lac4-2). The single and double mutants displayed normal growth, except the lac4-2 lac17 mutant that sometimes had a semi-dwarf phenotype and collapsed vessels. While the single mutants had moderately reduced lignin levels, the stems of lac4-1 lac17 and lac4-2 lac17 had lignin content reduced by 20% and 40%, respectively. This lower lignin level improved their saccharification yield. Thioacidolysis revealed that disrupting AtLAC17 mainly affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with AtLAC17 restored the normal lignin profile. This study provides evidence that both AtLAC4 and AtLAC17 contribute to the constitutive lignification of Arabidopsis stems and that AtLAC17 is involved in the deposition of G lignin units in fibers, suggesting a role in early lignification (Berthet et al, in press). The double mutants cannot be obtained for species that are propagated vegetatively such as poplar. In order to produce plants with lower laccase activity and reduced lignin content, we therefore used a miRNA strategy. The overexpression of two miRNA (miR397 and miR408) targeting several laccase genes was tested in different plants including Arabidopsis and poplar. These miRNAs were expressed constitutively under the control of the CaMV 35S promoter or of lignin-specific promoters such as CAD and C4L in transgenic Arabidopsis and poplar. Results obtained using of this miRNA strategy in Arabidopsis and preliminary results for poplar will be presented.

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.

Transgenic biotechnology can assist forest tree improvement programs but it may also raise environmental safety concerns. The environmental effects of genetically modified transgenic trees (GMTs) have been studied in many countries during the last 15 years. Today there is an urgent need of putting together this scattered knowledge to build-up a European knowledge platform for addressing GMTs in plantations. The main aims of Working Group 2 (WG2) of the European Cooperation in Science and Technology (COST) Action FP0905 “Biosafety of transgenic forest trees” (http://www.cost-action-fp0905.eu/) are (1) to discuss, based on scientific facts, whether current containment strategies are appropriate or need to be improved for GMTs, (2) to define a common protocol to track the transgene from the laboratory to the final product, and (3) to assess the possible impacts of GMTs on the environment. The potential risks of GMTs, the fate of recombinant material and the potential relevance of recombinant genes on plant´s omics are other main aspects to be considered and compared to similar processes with endogenous genes in conventional breeding. The group involves experts from public research, government and independent regulatory sectors across COST and non-COST member countries. The activities of this group have been organized into three Task Groups focusing on (1) risk assessment studies and guidance documents, (2) the monitoring of the transgenes and recombinant plant material, and (3) the impact of GMTs on exposed ecosystems. As a first step of WG2 activity, a database of guidance documents from national and transnational sources dealing with impacts and risk assessments of GMTs is being created to identify common and case-specific issues on biosafety. It is expected that the information gained will facilitate (1) a science-based understanding of the impacts of GMTs on the environment in comparison with that of traditionally tree breeding, and (2) future socio-economic and cost/benefits analyses of GMTs in plantations.

Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its beta-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure.

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the last steps of monolignol biosynthesis. In Arabidopsis, one CCR gene (CCR1, At1g15950) and two CAD genes (CAD C At3g19450 and CAD D At4g34230) are involved in this pathway. A triple cad c cad d ccr1 mutant, named ccc, was obtained. This mutant displays a severe dwarf phenotype and male sterility. The lignin content in ccc mature stems is reduced to 50% of the wild-type level. In addition, stem lignin structure is severely affected, as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde, sinapaldehyde, and ferulic acid. Male sterility is due to the lack of lignification in the anther endothecium, which causes the failure of anther dehiscence and of pollen release. The ccc hypolignified stems accumulate higher amounts of flavonol glycosides, sinapoyl malate and feruloyl malate, which suggests a redirection of the phenolic pathway. Therefore, the absence of CAD and CCR, key enzymes of the monolignol pathway, has more severe consequences on the phenotype than the individual absence of each of them. Induction of another CCR (CCR2, At1g80820) and another CAD (CAD1, At4g39330) does not compensate the absence of the main CCR and CAD activities. This lack of CCR and CAD activities not only impacts lignification, but also severely affects the development of the plants. These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

In order to determine the mechanism of the earlier copolymerization steps of two main lignin precursors, sinapyl (S) alcohol and coniferyl (G) alcohol, microscale in vitro oxidations were carried out with a PRX34 Arabidopsis thaliana peroxidase in the presence of H2O2. This plant peroxidase was found to have an in vitro polymerization activity similar to the commonly used horseradish peroxidase. The selected polymerization conditions lead to a bulk polymerization mechanism when G alcohol was the only phenolic substrate available. In the same conditions, the presence of S alcohol at a 50/50 S/G molar ratio turned this bulk mechanism into an endwise one. A kinetics monitoring (size-exclusion chromatography and liquid chromatography–mass spectrometry) of the different species formed during the first 24h oxidation of the S/G mixture allowed sequencing the bondings responsible for oligomerization. Whereas G homodimers and GS heterodimers exhibit low reactivity, the SS pinoresinol structure act as a nucleating site of the polymerization through an endwise process. This study is particularly relevant to understand the impact of S units on lignin structure in plants and to identify the key step at which this structure is programmed.

Overexpressed in transgenic plants, protease inhibitors showed insecticidal effects against several insect taxa. We transformed potato internodes with the mustard trypsin inhibitor mti-2 gene. Among the 35 independent transgenic potato lines obtained via Agrobacterium tumefasciens transformation, four (DM6, DM7, DM11, and DM19) were selected for their high level of MTI-2 (at least to 30% of trypsin activity inhibition). Feeding assays were carried out to evaluate their effects on the green-peach aphid, Myzus persicae (Sternorrhyncha: Aphididae). Prereproductive period, nymphal mortality, adult fecundity, and doubling time of M. persicae populations were monitored on nontransformed potato plants (NT) and the four selected DM lines. Compared to NT plants, DM19 did not induce any effect on M. persicae. In contrast, DM7 and DM11 increased nymphal survival by approximately 20%. DM6 and DM11 lines slightly enhanced M. persicae daily fecundity and intrinsic rate of natural increase, leading to a reduction of the doubling time of the populations by 1 day. DM6 did not impact nymphal mortality, whereas with the DM11 almost all the nymphs survived. Potato plants transformed with the mti-2 gene variably affected the life history of M. persicae but did not show any insecticidal effect on the aphid.

The deposition of lignin during plant-pathogen interactions is thought to play a role in plant defence. However, the function of lignification genes in plant disease resistance is poorly understood. In this article, we provide genetic evidence that the primary genes involved in lignin biosynthesis in Arabidopsis, CAD-C and CAD-D, act as essential components of defence to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato, possibly through the salicylic acid defence pathway. Thus, in contrast with cellulose synthesis, whose alteration leads to an increase in disease resistance, alteration of the cell wall lignin content leads directly or indirectly to defects in some defence components.

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. In tomato plant, rubbing applied to a young internode inhibit elongation of the rubbed internode and his neighboring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidases (POD), 24 hours after rubbing of the forth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Taken together, our results suggest that decrease in rubbed internode length is a consequence of IAA oxidation, increases in enzyme activities (PAL, CAD and POD), and cell wall rigidification associated with induction of lignification process.