Search

Key message Our Induced Somatic Sector Analysis and protein–protein interaction experiments demonstrate that Eucalyptus grandis IAA13 regulates xylem fibre and vessel development, potentially via EgrIAA13 modules involving ARF2, ARF5, ARF6 and ARF19. Abstract Auxin is a crucial phytohormone regulating multiple aspects of plant growth and differentiation, including regulation of vascular cambium activity, xylogenesis and its responsiveness towards gravitropic stress. Although the regulation of these biological processes greatly depends on auxin and regulators of the auxin signalling pathway, many of their specific functions remain unclear. Therefore, the present study aims to functionally characterise Eucalyptus grandis AUX/INDOLE-3-ACETIC ACID 13 (EgrIAA13), a member of the auxin signalling pathway. In Eucalyptus and Populus, EgrIAA13 and its orthologs are preferentially expressed in the xylogenic tissues and downregulated in tension wood. Therefore, to further investigate EgrIAA13 and its function during xylogenesis, we conducted subcellular localisation and Induced Somatic Sector Analysis experiments using overexpression and RNAi knockdown constructs of EgrIAA13 to create transgenic tissue sectors on growing stems of Eucalyptus and Populus. Since Aux/IAAs interact with Auxin Responsive Factors (ARFs), in silico predictions of IAA13-ARF interactions were explored and experimentally validated via yeast-2-hybrid experiments. Our results demonstrate that EgrIAA13 localises to the nucleus and that downregulation of EgrIAA13 impedes Eucalyptus xylem fibre and vessel development. We also observed that EgrIAA13 interacts with Eucalyptus ARF2, ARF5, ARF6 and ARF19A. Based on these results, we conclude that EgrIAA13 is a regulator of Eucalyptus xylogenesis and postulate that the observed phenotypes are likely to result from alterations in the auxin-responsive transcriptome via IAA13-ARF modules such as EgrIAA13-EgrARF5. Our results provide the first insights into the regulatory role of EgrIAA13 during xylogenesis.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from ArabidopsisDAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Wood (secondary xylem) formation is regulated by auxin, which plays a pivotal role as an integrator of developmental and environmental cues. However, our current knowledge of auxin-signaling during wood formation is incomplete. Our previous genome-wide analysis of

Lignin is a major lock for lignocellulose valorizationin biorefineries, prompting a need tofind newligninolytic systems. Termites are efficient lignocellulose degraders, and a large part ofthis ability comes from its anaerobic gut microbiome. However, thepotential of termite gut microbiomes to degrade lignin underanaerobicconditions has yet to be elucidated. By applying wetchemistry, multidimensional NMR spectroscopy, and quantitative13C-IS py-GC-MS, we determined the chemical and structuralcharacteristics of wheat straw digested by the gut microbiomes ofhigher termitesNasutitermes ephratae,Nasutitermes lujae,Micro- cerotermes parvus, andTermes hospesimplemented in anaerobicbioreactors. Interestingly, all gut microbiomes managed to removelignin (up to 37%), although hemicellulose (mean 51%) andcellulose(mean 41%) were degraded more efficiently. Important structural differences, indicative of ligninolytic action, were discerned in the residual lignin. For the studied termite gut microbiomes, a slight decrease of S/G ratio was observed upon digestion, whereas lignin’s interunit linkages and Cα-oxidized moieties, including benzaldehyde and hydroxypropiovanillone/syringone substructures, accumulated. Additionally, tricin terminal units were clearly depleted, along with decreases inp-coumarate and ferulate pendant units. At least 80% of the observed delignification is estimated to be due to the removal of“true”lignin, with the remainder being explained by the removal of hydroxycinnamic acids and tricin. Collectively, ourfindings suggest a partial deconstruction of lignin, with“peripheral”lignin subunits being preferentially targeted. The present work thus provides new insights into the anaerobic deconstruction of lignin by termite gut microbiomes.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors (CBFs) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs, taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.

Argania spinosa L. Skeels, belonging to the Argania genus of the Sapotaceae family, is a species native to Morocco and Algeria. Due to its perfect adaptation to soil and climate, this tree plays an important ecological role in a constantly threatened encroached desert region. To understand the biological role of polyphenols in making the argan tree adapts to its natural habitat, we conducted a comparative study in two of the tree development stations: Tindouf located in South-western Algeria and Stidia located in Northwest Algeria. High performance liquid chromatography (HPLC) and Gas chromatography-mass spectrometry (GC-MS) used for the analysis of flavonoids led to the identification of six flavonols (two types of myricetin, rutin, hyperoside, quercetin, kaempferol) in the leaves of Tindouf argan tree, and two molecules (myricetin and quercetin) in the leaves of Stidia argan tree. Other molecules presented are few. The determination of flavonoids by spectrophotometry revealed the richness of Tindouf argan in these compounds (20%) compared to that of Stidia argan tree (8.7%). Histolocalisation of the flavonoids, tannins and anthraquinone in the leaves and stems of the tree was done using fluorescence microscope to understand the role of these molecules in the protection of this tree in its environment. Key words: Aragania spinosa L. (Skeels), histolocalisation, polyphenols, high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS).

Willow (Salix spp. L.) species are fast-growing trees and shrubs that have attracted emergent attention for their potential as feedstocks for bioenergy and biofuel production, as well as for pharmaceutical and phytoremediation applications. This economic and environmental potential has propelled the creation of several genetic and genomic resources for Salix spp. Furthermore, the recent availability of an annotated genome for Salix purpurea has pinpointed novel candidate genes underlying economically relevant traits. However, functional studies have been stalled by the lack of rapid and efficient coupled regeneration-transformation systems for Salix purpurea and Salix spp. in general. In this report, we describe a fast and highly efficient hairy root transformation protocol for S. purpurea. It was effective for different explant sources and S. purpurea genotypes, with efficiencies between 63.4% and 98.7%, and the screening of the transformed hairy roots was easily carried out using the fluorescent marker DsRed. To test the applicability of this hairy root transformation system for gene functional analysis, we transformed hairy roots with the vector pGWAY-SpDRM2, where the gene SpDRM2 encoding a putative Domain Rearranged Methyltransferase (DRM) was placed under the control of the CaMV 35S constitutive promoter. Indeed, the transgenic hairy roots obtained exhibited significantly increased expression of SpDRM2 as compared to controls, demonstrating that this protocol is suitable for the medium/high-throughput functional characterization of candidate genes in S. purpurea and other recalcitrant Salix spp.

Although lignin is essential to ensure the correct growth and development of land plants, it may be an obstacle to the production of lignocellulosics-based biofuels, and reduces the nutritional quality of crops used for human consumption or livestock feed. The need to tailor the lignocellulosic biomass for more efficient biofuel production or for improved plant digestibility has fostered considerable advances in our understanding of the lignin biosynthetic pathway and its regulation. Most of the described regulators are transcriptional activators of lignin biosynthesis, but considerably less attention has been devoted to the repressors of this pathway. We provide a comprehensive overview of the molecular factors that negatively impact on the lignification process at both the transcriptional and post-transcriptional levels., SCOPUS: re.j, info:eu-repo/semantics/published

International audience; Wood production in fast-growing Eucalyptus grandis trees is highly dependent on both potassium (K) fertilization and water availability but the molecular processes underlying wood formation in response to the combined effects of these two limiting factors remain unknown. E. grandis trees were submitted to four combinations of K-fertilization and water supply. Weighted gene co-expression network analysis and MixOmics-based co-regulation networks were used to integrate xylem transcriptome, metabolome and complex wood traits. Func- tional characterization of a candidate gene was performed in transgenic E. grandis hairy roots. This integrated network-based approach enabled us to identify meaningful biological pro- cesses and regulators impacted by K-fertilization and/or water limitation. It revealed that modules of co-regulated genes and metabolites strongly correlated to wood complex traits are in the heart of a complex trade-off between biomass production and stress responses. Nested in these modules, potential new cell-wall regulators were identified, as further con- firmed by the functional characterization of EgMYB137. These findings provide new insights into the regulatory mechanisms of wood formation under stressful conditions, pointing out both known and new regulators co-opted by K-fertilization and/or water limitation that may potentially promote adaptive wood traits.

Re-engineering of transcriptional networks regulating secondary cell wall formation may allow the improvement of plant biomass in widely grown plantation crops such as Eucalyptus. However, there is currently a scarcity of freely available standardized biological parts (e.g., Phytobricks) compatible with Type IIS assembly approaches from forest trees, and there is a need to accelerate transcriptional network inference in nonmodel biomass crops. Here we describe the design and synthesis of a versatile three-panel biological parts collection of 221 secondary cell wall-related Eucalyptus grandis transcription factor coding sequences and 65 promoters that are compatible with GATEWAY, Golden Gate, MoClo, and GoldenBraid DNA assembly methods and generally conform to accepted Phytobrick syntaxes. This freely available resource is intended to accelerate synthetic biology applications in multiple plant biomass crops and enable reconstruction of secondary cell wall transcriptional networks using high-throughput assays such as DNA affinity purification sequencing (DAP-seq) and enhanced yeast one-hybrid (eY1H) screening.

Aerenchyma formation in sugarcane roots is controlled by an ethylene-related RAV transcription factor. It binds to the promoter of an endogalacturonase and apparently controls cell separation., The development of lysigenous aerenchyma starts with cell expansion and degradation of pectin from the middle lamella, leading to cell wall modification, and culminating with cell separation. Here we report that nutritional starvation of sugarcane induced gene expression along sections of the first 5 cm of the root and between treatments. We selected two candidate genes: a RAV transcription factor, from the ethylene response factors superfamily, and an endopolygalacturonase (EPG), a glycosyl hydrolase related to homogalacturonan hydrolysis from the middle lamella. epg1 and rav1 transcriptional patterns suggest they are essential genes at the initial steps of pectin degradation during aerenchyma development in sugarcane. Due to the high complexity of the sugarcane genome, rav1 and epg1 were sequenced from 17 bacterial artificial chromosome clones containing hom(e)ologous genomic regions, and the sequences were compared with those of Sorghum bicolor. We used one hom(e)olog sequence from each gene for transactivation assays in tobacco. rav1 was shown to bind to the epg1 promoter, repressing β-glucuronidase activity. RAV repression upon epg1 transcription is the first reported link between ethylene regulation and pectin hydrolysis during aerenchyma formation. Our findings may help to elucidate cell wall degradation in sugarcane and therefore contribute to second-generation bioethanol production.

Wood, which represents the most abundant lignocellulosic biomass on earth, fulfils key roles in trees, and is also a raw material for multiple end-uses by mankind. The differentiation of this complex vascular tissue starts with cell division in the vascular cambium and is characterized by a massive deposition of lignified secondary cell walls mainly in fibres and vessels. A transcriptional network underlying this differentiation process ensures a tight regulation of the expression of thousands of genes both at the spatial and temporal levels. Most of our current knowledge of this hierarchical network was extrapolated from studies performed in Arabidopsis. Here, we review recent findings on the regulation of wood formation in angiosperm trees species highlighting conserved and distinct mechanisms with Arabidopsis. We provide examples shedding light on the central role of auxin and its cross-talk with other hormones at different stages of secondary xylem differentiation. Functional studies of trees' wood-associated transcription factors revealed diversified functions as compared to their Arabidopsis orthologs. Sophisticated mechanisms of alternative splicing and cross-regulation between the two distinct groups of top level NAC-domain master regulators were uncovered. These findings underlie the high level of complexity of wood formation in trees and provide a framework for future lines of research in this exiting research field.

Although eucalypts are the most planted hardwood trees worldwide, the majority of them are frost sensitive. The recent creation of frost-tolerant hybrids such as Eucalyptus gundal plants (E. gunnii × E. dalrympleana hybrids), now enables the development of industrial plantations in northern countries. Our objective was to evaluate the impact of cold on the wood structure and composition of these hybrids, and on the biosynthetic and regulatory processes controlling their secondary cell-wall (SCW) formation. We used an integrated approach combining histology, biochemical characterization and transcriptomic profiling as well as gene co-expression analyses to investigate xylem tissues from Eucalyptus hybrids exposed to cold conditions. Chilling temperatures triggered the deposition of thicker and more lignified xylem cell walls as well as regulation at the transcriptional level of SCW genes. Most genes involved in lignin biosynthesis, except those specifically dedicated to syringyl unit biosynthesis, were up-regulated. The construction of a co-expression network enabled the identification of both known and potential new SCW transcription factors, induced by cold stress. These regulators at the crossroads between cold signalling and SCW formation are promising candidates for functional studies since they may contribute to the tolerance of E. gunnii × E. dalrympleana hybrids to cold.

International audience; Wood, also called secondary xylem, is a specialized vascular tissue constituted by different cell types that undergo a differentiation process involving deposition of thick, lignified secondary cell walls. The mechanisms needed to control the extent of lignin deposition depending on the cell type and the differentiation stage are far from being fully understood. We found that the Eucalyptus transcription factor EgMYB1, which is known to repress lignin biosynthesis, interacts specifically with a linker histone variant, EgH1.3. This interaction enhances the repression of EgMYB1' s target genes, strongly limiting the amount of lignin deposited in xylem cell walls. The expression profiles of EgMYB1 and EgH1.3 overlap in xylem cells at early stages of their differentiation as well as in mature parenchymatous xylem cells, which have no or only thin lignified secondary cell walls. This suggests that a complex between EgMYB1 and EgH1.3 integrates developmental signals to prevent premature or inappropriate lignification of secondary cell walls, providing a mechanism to fine-tune the differentiation of xylem cells in time and space. We also demonstrate a role for a linker histone variant in the regulation of a specific developmental process through interaction with a transcription factor, illustrating that plant linker histones have other functions beyond chromatin organization.

The R2R3-MYB family, one of the largest transcription factor families in higher plants, controls a wide variety of plant-specific processes including, notably, phenylpropanoid metabolism and secondary cell wall formation. We performed a genome-wide analysis of this superfamily in Eucalyptus, one of the most planted hardwood trees world-wide. A total of 141 predicted R2R3-MYB sequences identified in the Eucalyptus grandis genome sequence were subjected to comparative phylogenetic analyses with Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Vitis vinifera. We analysed features such as gene structure, conserved motifs and genome location. Transcript abundance patterns were assessed by RNAseq and validated by high-throughput quantitative PCR. We found some R2R3-MYB subgroups with expanded membership in E. grandis, V. vinifera and P. trichocarpa, and others preferentially found in woody species, suggesting diversification of specific functions in woody plants. By contrast, subgroups containing key genes regulating lignin biosynthesis and secondary cell wall formation are more conserved across all of the species analysed. In Eucalyptus, R2R3-MYB tandem gene duplications seem to disproportionately affect woody-preferential and woody-expanded subgroups. Interestingly, some of the genes belonging to woody-preferential subgroups show higher expression in the cambial region, suggesting a putative role in the regulation of secondary growth.

Strong constitutive promoters, such as CaMV35S, are widely used for plant transformation, but undesirable phenotypic changes have been reported when used to drive biotic stress tolerance and/or for modifying lignin content. The promoter of the eucalyptus cinnamoyl CoA reductase (CCR), a key enzyme of the lignin biosynthetic pathway, was shown to be preferentially expressed in vascular tissues both in herbaceous and woody transgenic plants but not eucalyptus. In this work, we transformed Eucalyptus globulus with the EgCCR promoter governing both β-glucuronidase (GUS) and GFP activity patterns. No statistical differences were found between the survival rate and percentage of GUS positive shoots between eucalyptus transformed with either the constitutive CaMV35S or with the EgCCR promoter. The EgCCR transformed plantlets exhibited high GUS expression levels associated with the vascular tissues opening the possibility of targeting vascular-associated traits such as lignin content or vascular pathogen resistance in adult elite plants of eucalyptus while avoiding the undesirable pleiotropic effects caused by strong constitutive promoters.

Transcription factors play a fundamental role in plants by orchestrating temporal and spatial gene expression in response to environmental stimuli. Several R2R3-MYB genes of the Arabidopsis subgroup 4 (Sg4) share a C-terminal EAR motif signature recently linked to stress response in angiosperm plants. It is reported here that nearly all Sg4 MYB genes in the conifer trees Picea glauca (white spruce) and Pinus taeda (loblolly pine) form a monophyletic clade (Sg4C) that expanded following the split of gymnosperm and angiosperm lineages. Deeper sequencing in P. glauca identified 10 distinct Sg4C sequences, indicating over-representation of Sg4 sequences compared with angiosperms such as Arabidopsis, Oryza, Vitis, and Populus. The Sg4C MYBs share the EAR motif core. Many of them had stress-responsive transcript profiles after wounding, jasmonic acid (JA) treatment, or exposure to cold in P. glauca and P. taeda, with MYB14 transcripts accumulating most strongly and rapidly. Functional characterization was initiated by expressing the P. taeda MYB14 (PtMYB14) gene in transgenic P. glauca plantlets with a tissue-preferential promoter (cinnamyl alcohol dehydrogenase) and a ubiquitous gene promoter (ubiquitin). Histological, metabolite, and transcript (microarray and targeted quantitative real-time PCR) analyses of PtMYB14 transgenics, coupled with mechanical wounding and JA application experiments on wild-type plantlets, allowed identification of PtMYB14 as a putative regulator of an isoprenoid-oriented response that leads to the accumulation of sesquiterpene in conifers. Data further suggested that PtMYB14 may contribute to a broad defence response implicating flavonoids. This study also addresses the potential involvement of closely related Sg4C sequences in stress responses and plant evolution.

article in press; International audience; Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes.

Key message A holistic approach, molecular and eco-physiological, has provided a better understanding of the response of eucalyptus and poplar genotypes to salt stress. Different tolerance mechanisms with varying degrees of effectiveness as well as differences in the response of genes linked to xylem differentiation have been identified. Abstract We studied the behavior of four eucalyptus genotypes (Eucalyptus camaldulensis Dehnh ‘169’; E. grandis Hill ex Maidenï¿½× E. urophylla S.T. Blake ‘5E’; Eucalyptus globulus Labill ‘Anselmo’ and ‘Odiel’) and four poplar genotypes (Populus alba L. ‘PO 10-10-20’ and ‘J 1-3-18’, P. tremula L.ï¿½× P. alba ‘7171-B4’ and P.ï¿½× canadensis Moench.: ‘Oudenberg’) in relation to their response to saline conditions and their capacity to grow in short rotation for biomass production. For this purpose, plants were grown under greenhouse conditions and subjected to two different saline concentrations of NaCl, one moderate (50�mM) and one severe (125�mM), as well as a control treatment. Growth, as well as several functional, morphological and biochemical parameters were considered. We also performed an expression analysis of genes that encode enzymes and transcription factors involved in wood formation. The four eucalyptus genotypes showed a very high survival rate under both moderate and severe salt treatments, as did both white poplar genotypes (‘PO 10-10-20’ and ‘J 1-3-18’). All of them displayed a tolerant behavior toward salinity stress. In contrast, the poplar hybrids (‘7171-B4’ and ‘Oudenberg’) exhibited medium-tolerance or sensitive behavior. Possible tolerance mechanisms based on stomatal control, water use efficiency, capacity of dilute toxic ions through decreasing the specific leaf area and higher root/aerial biomass ratios were detected. These mechanisms were deemed to have varying degrees of effectiveness. A molecular approach identified changes in the expression of genes linked to xylem differentiation, the more tolerant genotypes being those with fewer modifications. These findings could contribute towards enabling the cultivation of fast-growing species in short rotation on marginal land affected by salinity for the production of lignocellulosic biomass. The response variability detected could lead to advances in breeding for tolerance to this type of stress. � 2016, Springer-Verlag Berlin Heidelberg.

The knowledge of the gene families mostly impacting cell wall digestibility variations would significantly increase the efficiency of marker-assisted selection when breeding maize and grass varieties with improved silage feeding value and/or with better straw fermentability into alcohol or methane. The maize genome sequence of the B73 inbred line was released at the end of 2009, opening up new avenues to identify the genetic determinants of quantitative traits. Colocalizations between a large set of candidate genes putatively involved in secondary cell wall assembly and QTLs for cell wall digestibility (IVNDFD) were then investigated, considering physical positions of both genes and QTLs. Based on available data from six RIL progenies, 59 QTLs corresponding to 38 non-overlapping positions were matched up with a list of 442 genes distributed all over the genome. Altogether, 176 genes colocalized with IVNDFD QTLs and most often, several candidate genes colocalized at each QTL position. Frequent QTL colocalizations were found firstly with genes encoding ZmMYB and ZmNAC transcription factors, and secondly with genes encoding zinc finger, bHLH, and xylogen regulation factors. In contrast, close colocalizations were less frequent with genes involved in monolignol biosynthesis, and found only with the [i]C4H2[/i], [i]CCoAOMT5[/i], and [i]CCR1[/i] genes. Close colocalizations were also infrequent with genes involved in cell wall feruloylation and cross-linkages. Altogether, investigated colocalizations between candidate genes and cell wall digestibility QTLs suggested a prevalent role of regulation factors over constitutive cell wall genes on digestibility variations.

International audience; Comparative phylogenetic analyses of the R2R3-MYB transcription factor family revealed that five subgroups were preferentially found in woody species and were totally absent from Brassicaceae and monocots (Soler et al., 2015). Here, we analyzed one of these subgroups (WPS-I) for which no gene had been yet characterized. Most Eucalyptus members of WPS-I are preferentially expressed in the vascular cambium, the secondary meristem responsible for tree radial growth. We focused on EgMYB88, which is the most specifically and highly expressed in vascular tissues, and showed that it behaves as a transcriptional activator in yeast. Then, we functionally characterized EgMYB88 in both transgenic Arabidopsis and poplar plants overexpressing either the native or the dominant repression form (fused to the Ethylene-responsive element binding factor-associated Amphiphilic Repression motif, EAR). The transgenic Arabidopsis lines had no phenotype whereas the poplar lines overexpressing EgMYB88 exhibited a substantial increase in the levels of the flavonoid catechin and of some salicinoid phenolic glycosides (salicortin, salireposide, and tremulacin), in agreement with the increase of the transcript levels of landmark biosynthetic genes. A change in the lignin structure (increase in the syringyl vs. guaiacyl, S/G ratio) was also observed. Poplar lines overexpressing the EgMYB88 dominant repression form did not show a strict opposite phenotype. The level of catechin was reduced, but the levels of the salicinoid phenolic glycosides and the S/G ratio remained unchanged. In addition, they showed a reduction in soluble oligolignols containing sinapyl p-hydroxybenzoate accompanied by a mild reduction of the insoluble lignin content. Altogether, these results suggest that EgMYB88, and more largely members of the WPS-I group, could control in cambium and in the first layers of differentiating xylem the biosynthesis of some phenylpropanoid-derived secondary metabolites including lignin.

Cryptogein is a proteinaceous elicitor secreted by the oomycete Phytophthora cryptogea, which induces a hypersensitive response in tobacco plants. We have previously reported that in tobacco BY-2 cells treated with cryptogein, most of the genes of the phenylpropanoid pathway were upregulated and cell wall-bound phenolics accumulated. Both events were Ca(2+) dependent. In this study, we designed a microarray covering a large proportion of the tobacco genome and monitored gene expression in cryptogein-elicited BY-2 cells to get a more complete view of the transcriptome changes and to assess their Ca(2+) dependence. The predominant functional gene categories affected by cryptogein included stress- and disease-related proteins, phenylpropanoid pathway, signaling components, transcription factors and cell wall reinforcement. Among the 3819 unigenes whose expression changed more than fourfold, 90% were Ca(2+) dependent, as determined by their sensitivity to lanthanum chloride. The most Ca(2+)-dependent transcripts upregulated by cryptogein were involved in defense responses or the oxylipin pathway. This genome-wide study strongly supports the importance of Ca(2+)-dependent transcriptional regulation of regulatory and defense-related genes contributing to cryptogein responses in tobacco.

In the view of the economic importance of grapevine and the increasing threaten represented by vascular diseases, transgenic grapevine with enhanced tolerance could represent an attractive opportunity. Hitherto, constitutive promoters have been used generally to study the effects of transgene expression in grapevine. Given the fact that constitutive gene expression may be harmful to the host plant, affecting plant growth and development, the use of tissue -specific promoters restricting gene expression to tissues of interest and at given developmental stages could be more appropriate. For this purpose, we decided to study in grapevine the activity of the Eucalyptus gunnii CCR promoter that was previously reported to be vascular-preferential. We transformed grapevine with the "Sonication assisted Agrobacterium-mediated transformation" (SAAT) method and a construct where both GUS and GFP (green fluorescent protein) marker genes were under control of the EgCCR promoter. High GUS and GFP activities were found to be associated with the newly formed vascular tissues in stems, leaves and petioles of transformed grapevine, suggesting a preferential activity of the EgCCR promoter in the vascular tissues of grapevine. These results suggest the tissue-specificity of this promoter from eucalyptus is conserved in grapevine and that it could be used to drive expression of defense genes in order to enhance resistance against vascular pathogens.

Plant cells use calcium-based signalling pathways to transduce biotic and/or abiotic stimuli into adaptive responses. However, little is known about the coupling between calcium signalling, transcriptional regulation and the downstream biochemical processes. To understand these relationships better, we challenged tobacco BY-2 cells with cryptogein and evaluated how calcium transients (monitored through the calcium sensor aequorin) impact (1) transcript levels of phenylpropanoid genes (assessed by RT-qPCR); and (2) derived-phenolic compounds (analysed by mass spectrometry). Most genes of the phenylpropanoid pathway were up-regulated by cryptogein and cell wall-bound phenolic compounds accumulated (mainly 5-hydroxyferulic acid). The accumulation of both transcripts and phenolics was calcium-dependent. The transcriptional regulation of phenylpropanoid genes was correlated in a non-linear manner with stimulus intensity and with components of the cryptogein-induced calcium signature. In addition, calmodulin inhibitors increased the sensitivity of cells to low concentrations of cryptogein. These results led us to propose a model of coupling between the cryptogein signal, calcium signalling and the transcriptional response, exerting control of transcription through the coordinated action of two decoding modules exerting opposite effects.

Brittle leaf disease (BLD) of date palms was first described in the south of Tunisia in 1960. Since then, it progressively spread out to reach epidemic proportions in 1986. Both biotic and abiotic factors have been suggested to be possible causal agents of this disease. However, the research efforts aimed at identifying the causal agent of this disease have still been unsuccessful. In order to get an insight in the molecular mechanisms involved in BLD of date palm, we have constructed suppression subtractive hybridization libraries from affected (ABLD) and healthy leaves (HBLD). This report describes the cloning, sequencing and characterization of 74 independent cDNA derived from the ABLD and the HBLD subtractive libraries. BLAST analysis showed that the ESTs from HBLD library were mainly related to photosynthesis, protein synthesis and ion transport whereas the EST sequences from ABLD were related to stress responsive genes, metabolism associated, protein synthesis and signal transduction.

An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.

Plant organisms contain a large number of genes belonging to numerous multigenic families whose evolution size reflects some functional constraints. Sequences from eight multigenic families, involved in biotic and abiotic responses, have been analyzed in Eucalyptus grandis and compared with Arabidopsis thaliana. Two transcription factor families APETALA 2 (AP2)/ethylene responsive factor and GRAS, two auxin transporter families PIN-FORMED and AUX/LAX, two oxidoreductase families (ascorbate peroxidases [APx] and Class III peroxidases [CIII Prx]), and two families of protective molecules late embryogenesis abundant (LEA) and DNAj were annotated in expert and exhaustive manner. Many recent tandem duplications leading to the emergence of species-specific gene clusters and the explosion of the gene numbers have been observed for the AP2, GRAS, LEA, PIN, and CIII Prx in E. grandis, while the APx, the AUX/LAX and DNAj are conserved between species. Although no direct evidence has yet demonstrated the roles of these recent duplicated genes observed in E. grandis, this could indicate their putative implications in the morphological and physiological characteristics of E. grandis, and be the key factor for the survival of this nondormant species. Global analysis of key families would be a good criterion to evaluate the capabilities of some organisms to adapt to environmental variations.

International audience; Auxin plays a pivotal role in various plant growth and development processes, including vascular differentiation.The modulation of auxin responsiveness through the auxin perception and signaling machinery is believed to be a major regulatory mechanism controlling cambium activity and wood formation. To gain more insights into the rolesof key Aux/IAA gene regulators of the auxin response in these processes, we identified and characterized membersof the Aux/IAA family in the genome of Eucalyptus grandis, a tree of worldwide economic importance. We found that the gene family in Eucalyptus is slightly smaller than that in Populus and Arabidopsis, but all phylogenetic groupsare represented. High-throughput expression profiling of different organs and tissues highlighted several Aux/IAAgenes expressed in vascular cambium and/or developing xylem, some showing differential expression in response todevelopmental (juvenile vs. mature) and/or to environmental (tension stress) cues. Based on the expression profiles,we selected a promising candidate gene, EgrIAA4, for functional characterization. We showed that EgrIAA4 protein is localized in the nucleus and functions as an auxin-responsive repressor. Overexpressing a stabilized version of EgrIAA4 in Arabidopsis dramatically impeded plant growth and fertility and induced auxin-insensitive phenotypes such as inhibition of primary root elongation, lateral root emergence and agravitropism. Interestingly, the lignified secondary walls of the interfascicular fibers appeared very late, whereas those of the xylary fibers were virtually undetectable, suggesting that EgrIAA4 may play crucial roles in fiber development and secondary cell wall deposition.

The identification of genes controlling lignin content is of crucial importance for breeding maize with improved feeding value and yielding more bioenergy after fermentation into alcohol or methane. Up to now, the gene families mostly impacting observed variations in cell wall lignin content have not yet been identified. However, genes involved in monolignol biosynthesis and polymerization, and the MYB and NAC transcription factors that regulate their activities, are promising candidates. In order to test this hypothesis, colocalizations between genes of these families and QTLs for lignin content originating from nine RIL families were investigated, based on gene and QTL physical positions. Ninety-three lignin QTLs were thus collected which corresponded to 44 non-overlapping positions. Among these positions, 11 were jointly shared by three or four RIL progenies, while lignin QTLs were observed for only one progeny in 16 positions. In addition to the few genes described in maize, candidate genes involved in monolignol biosynthesis and regulation of secondary wall lignification were searched for as orthologs of genes with such functions from several species including Arabidopsis, eucalyptus, poplar, and rice. Altogether, 50 ZmMYB-, 49 ZmNAC-, and 95 monolignol-related genes were considered as putative candidates. ZmMYB genes were found colocalizing with lignin QTLs in 25 positions, ZmNAC in 22 positions, and both ZmMYB and ZmNAC genes were present together in 12 QTL positions. Finally, 74 % of ZmMYB and 63 % of ZmNAC putatively involved in secondary wall regulation colocalized with or were close to lignin QTLs. Similarly, 66 % of genes involved in monolignol biosynthesis colocalized with QTL positions, whereas fewer colocalizations were observed for laccases and peroxidases putatively involved in lignin polymerization. In addition, the maize ortholog of the EgMYB2/AtMYB46 secondary cell wall master regulator was not highlighted in this study, possibly indicating significant divergences in the regulation of lignification between grasses and dicotyledonous plants. In contrast, based on gene number and colocalizations with lignin QTLs, orthologs of EgMYB1/AtMYB4 genes seem to have a greater importance in maize and grasses than in dicotyledonous species during secondary wall biosynthesis and deposition.

Summary EgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway, whereas core phenylpropanoid genes were not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation.

The composition and structure of lignified walls has a dramatic impact on the technological value of raw materials. The chemical flexibility of the secondary cell wall has been demonstrated and it is now possible to develop strategies to optimize its composition through genetic engineering. Thanks to functional genomics, new target genes of both plant and microbial origin are rapidly becoming available for this purpose and their use will open new avenues for producing tailor-made plant products with improved properties. Moreover, the major proportion of terrestrial plant biomass comprises lignified cell walls and this reservoir of carbon should be increasingly exploited for the production of chemicals and energy within the context of sustainable development. For example, the design of plants suitable for downstream conversion processes, such as the production of bioethanol, and the exploitation of microorganisms and microbial enzymes for biomass pretreatments or for the production of novel chemicals.

The developmental tissue- and cell-specific expression pattern of two ‘lignification’ genes, caffeic acid O -methyltransferase (C-OMT) and cinnamyl alcohol dehydrogenase (CAD), was analysed by in situ hybridisation in leaf and stem samples of Eucalyptus plantlets. Both genes are expressed, in a coordinated, developmental fashion, in the same cell types—especially developing vessels—of differentiating stem xylem tissue confirming their role in lignification and demonstrating that this process is under strict developmental control. C-OMT, but not CAD, transcripts were also localised to developing xylem vessels in the midribs of leaves. Histochemical analyses revealed that, in stem xylem tissues, C-OMT and CAD are expressed in cells poor in S-type lignin (primary xylem vessels and immature secondary xylem cells) and also in cells that later become rich in S-type lignin (mature secondary xylem cells).

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the last step in the synthesis of the monomeric precursors of lignin. Here, we demonstrate that the vascular expression pattern conferred by the Eucalyptus gunnii EgCAD2 promoter in transgenic poplar (Populus tremula x Populus alba) is conserved in another perennial woody angiosperm of economic interest (Vitis vinifera L.), as well as in a model herbaceous plant (Nicotiana tabacum L.). Furthermore, promoter deletion analysis performed in both tobacco and poplar allowed us to identify the proximal region [-340/-124] as essential for vascular cambium/xylem-specific expression whereas the [-124/+117] region was shown to contain cis element-driving activity in phloem fibres. Interestingly, the [-340/-124] fragment contains an AC-rich cis-acting element present in numerous genes of the phenylpropanoid pathway expressed in xylem tissues, and known as a consensus Myb transcription factor binding site, suggesting that common Myb sites may provide a mechanism by which different steps of phenylpropanoid metabolism are coordinately regulated and expressed in vascular tissues. We have also shown in both tobacco and poplar that the EgCAD2 promoter is inducible by wounding and the cis-elements responsible for wounding responsiveness are located in the distal promoter region. Taken together, our data suggest that the mechanisms controlling developmental and wounding inducible expression of the EgCAD2 promoter are conserved among perennial woody and annual herbaceous plant species enabling us now to investigate in depth the transcriptional regulation of the EgCAD2 promoter in tobacco.

Transgenic plants severely suppressed in the activity of cinnamoyl-CoA reductase were produced by introduction of a partial sense CCR transgene into tobacco. Five transgenic lines with CCR activities ranging from 2 to 48% of wild-type values were selected for further study. Some lines showed a range of aberrant phenotypes including reduced growth, and all had changes to lignin structure making the polymer more susceptible to alkali extraction. The most severely CCR-suppressed line also had significantly decreased lignin content and an increased proportion of free phenolic groups in non-condensed lignin. These changes are likely to make the lignin easier to extract during chemical pulping. Direct Kraft pulping trials confirmed this. More lignin could be removed from the transgenic wood than from wild-type wood at the same alkali charge. A similar improvement in pulping efficiency was recently shown for poplar trees expressing an antisense cinnamyl alcohol dehydrogenase gene. Pulping experiments performed here on CAD-antisense tobacco plants produced near-identical results – the modified lignin was more easily removed during pulping without any adverse effects on the quality of the pulp or paper produced. These results suggest that pulping experiments performed in tobacco can be predictive of the results that will be obtained in trees such as poplar, extending the utility of the tobacco model. On the basis of our results on CCR manipulation in tobacco, we predict that CCR-suppressed trees may show pulping benefits. However, it is likely that CCR-suppression will not be the optimal target for genetic manipulation of pulping character due to the potential associated growth defects.

Lignin, a major component of secondary cell walls, hinders the optimal processing of wood for industrial uses. The recent availability of the Eucalyptus grandis genome sequence allows comprehensive analysis of the genes encoding the 11 protein families specific to the lignin branch of the phenylpropanoid pathway and identification of those mainly involved in xylem developmental lignification. We performed genome-wide identification of putative members of the lignin gene families, followed by comparative phylogenetic studies focusing on bona fide clades inferred from genes functionally characterized in other species. RNA-seq and microfluid real-time quantitative PCR (RT-qPCR) expression data were used to investigate the developmental and environmental responsive expression patterns of the genes. The phylogenetic analysis revealed that 38 E. grandis genes are located in bona fide lignification clades. Four multigene families (shikimate O-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeate/5-hydroxyferulate O-methyltransferase (COMT) and phenylalanine ammonia-lyase (PAL)) are expanded by tandem gene duplication compared with other plant species. Seventeen of the 38 genes exhibited strong, preferential expression in highly lignified tissues, probably representing the E. grandis core lignification toolbox. The identification of major genes involved in lignin biosynthesis in E. grandis, the most widely planted hardwood crop world-wide, provides the foundation for the development of biotechnology approaches to develop tree varieties with enhanced processing qualities.

International audience; Auxin is a central hormone involved in a wide range of developmental processes including the specification of vascular stem cells. Auxin Response Factors (ARF) are important actors of the auxin signalling pathway, regulating the transcription of auxin-responsive genes through direct binding to their promoters. The recent availability of the Eucalyptus grandis genome sequence allowed us to examine the characteristics and evolutionary history of this gene family in a woody plant of high economic importance. With 17 members, the E. grandis ARF gene family is slightly contracted, as compared to those of most angiosperms studied hitherto, lacking traces of duplication events. In silico analysis of alternative transcripts and gene truncation suggested that these two mechanisms were preeminent in shaping the functional diversity of the ARF family in Eucalyptus. Comparative phylogenetic analyses with genomes of other taxonomic lineages revealed the presence of a new ARF clade found preferentially in woody and/or perennial plants. High-throughput expression profiling among different organs and tissues and in response to environmental cues highlighted genes expressed in vascular cambium and/or developing xylem, responding dynamically to various environmental stimuli. Finally, this study allowed identification of three ARF candidates potentially involved in the auxin-regulated transcriptional program underlying wood formation.

NAC domain transcription factors regulate many developmental processes and stress responses in plants and vary widely in number and family structure. We analysed the characteristics and evolution of the NAC gene family of Eucalyptus grandis, a fast-growing forest tree in the rosid order Myrtales. NAC domain genes identified in the E. grandis genome were subjected to amino acid sequence, phylogenetic and motif analyses. Transcript abundance in developing tissues and abiotic stress conditions in E. grandis and E. globulus was quantified using RNA-seq and reverse transcription quantitative PCR (RT-qPCR). One hundred and eighty-nine E. grandis NAC (EgrNAC) proteins, arranged into 22 subfamilies, are extensively duplicated in subfamilies associated with stress response. Most EgrNAC genes form tandem duplicate arrays that frequently carry signatures of purifying selection. Sixteen amino acid motifs were identified in EgrNAC proteins, eight of which are enriched in, or unique to, Eucalyptus. New candidates for the regulation of normal and tension wood development and cold responses were identified. This first description of a Myrtales NAC domain family reveals an unique history of tandem duplication in stress-related subfamilies that has likely contributed to the adaptation of eucalypts to the challenging Australian environment. Several new candidates for the regulation of stress, wood formation and tree-specific development are reported.

International audience; Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Changes in soluble proteins and Rubisco (E.C.4.1.1.39) contents were examined in leaves of nitrogen-deprived and nitrogen-sufficient soybeans. Rubisco content was very responsive to nitrogen stress, and this protein appeared to be the largest source of mobilizable nitrogen in the senescent leaf. Loss of soluble proteins and Rubisco was associated with a decrease in the activities of several proteolytic enzymes measured using artificial substrates: carboxypeptidase, aminopeptidase and haemoglobinase.The in vitro activity of enzyme(s) which can degrade Rubisco was investigated using endogenous Rubisco and in vitro radiolabelled Rubisco as substrates. Highest endopeptidic cleavage of endogenous Rubisco occured at pH 4; the enzyme responsible for this breakdown appeared to be a sulfhydryl-dependent proteinase. In contrast, [(14)C] Rubisco was attacked preferentially at pH 9, by a peptide hydrolase sensitive to EDTA. No increase in Rubisco-degrading activities was detected in nitrogen-deficient soybean leaves compared to control plant leaves.

Different transgenic tobacco lines down-regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down-regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down-regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.

Cinnamoyl-CoA reductase (CCR ; EC 1.2.1.44) catalyses the conversion of cinnamoyl-CoAs into their corresponding cinnamaldehydes, i.e. the first step of the phenylpropanoid pathway specifically dedicated to the monolignol biosynthetic branch. In previous work, we described the isolation and characterisation of the first cDNA encoding CCR in Eucalyptus (Lacombe, E., Hawkins, S., Van Dorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., Boudet, A.M., Grima-Pettenati, J., 1997. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant Journal 11, 429–441) and shown the role of this enzyme in controlling the carbon flux into lignins (Piquemal, J., Lapierre, C., Myton, K., O’Connell, A., Schuch, W., Grima-Pettenati, J., Boudet, A.M., 1998. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant Journal 13, 71–83). Here, we report the characterisation of two functionally and structurally distinct cDNA clones, AtCCR1 and AtCCR2 (81.6% protein sequence identity) in Arabidopsis thaliana. The two recombinant proteins expressed in Escherichia coli are able to use the three cinnamoyl-CoAs tested but with different levels of efficiency. AtCCR1 is five times more efficient with feruloyl-CoA and sinapoyl-CoA than AtCCR2. In addition, the two genes are differentially expressed during development and in response to infection. AtCCR1 is preferentially expressed in tissues undergoing lignification. In contrast, AtCCR2, which is poorly expressed during development, is strongly and transiently induced during the incompatible interaction with Xanthomonas campestris pv. campestris leading to a hypersensitive response. Altogether, these data suggest that AtCCR1 is involved in constitutive lignification whereas AtCCR2 is involved in the biosynthesis of phenolics whose accumulation may lead to resistance.