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The periderm is basic for land plants due to its protective role during radial growth, which is achieved by the polymers deposited in the cell walls. In most trees, like holm oak, the periderm is frequently replaced by subsequent internal periderms yielding a heterogeneous outer bark made of a mixture of periderms and phloem tissues, known as rhytidome. Exceptionally, cork oak forms a persistent or long-lived periderm which results in a homogeneous outer bark of thick phellem cell layers known as cork. Here we use the outer bark of cork oak, holm oak, and their natural hybrids’ to analyse the chemical composition, the anatomy and the transcriptome, and further understand the mechanisms underlying periderm development. The inclusion of hybrid samples showing rhytidome-type and cork-type barks is valuable to approach to cork and rhytidome development, allowing an accurate identification of candidate genes and processes. The present study underscores that biotic stress and cell death signalling are enhanced in rhytidome-type barks whereas lipid metabolism and cell cycle are enriched in cork-type barks. Development-related DEGs, showing the highest expression, highlight cell division, cell expansion, and cell differentiation as key processes leading to cork or rhytidome-type barks.

Background The periderm is a protective barrier crucial for land plant survival, but little is known about genetic factors involved in its development and regulation. Using a transcriptomic approach in the cork oak (Q. suber) periderm, we previously identified an RS2-INTERACTING KH PROTEIN (RIK) homologue of unknown function containing a K homology (KH)-domain RNA-binding protein, as a regulatory candidate gene in the periderm. Results To gain insight into the function of RIK in the periderm, potato (S. tuberosum) tuber periderm was used as a model: the full-length coding sequence of RIK, hereafter referred to as StRIK, was isolated, the transcript profile analyzed and gene silencing in potato performed to analyze the silencing effects on periderm anatomy and transcriptome. The StRIK transcript accumulated in all vegetative tissues studied, including periderm and other suberized tissues such as root and also in wounded tissues. Downregulation of StRIK in potato by RNA interference (StRIK-RNAi) did not show any obvious effects on tuber periderm anatomy but, unlike Wild type, transgenic plants flowered. Global transcript profiling of the StRIK-RNAi periderm did show altered expression of genes associated with RNA metabolism, stress and signaling, mirroring the biological processes found enriched within the in silico co-expression network of the Arabidopsis orthologue. Conclusions The ubiquitous expression of StRIK transcript, the flower associated phenotype and the differential expression of StRIK-RNAi periderm point out to a general regulatory role of StRIK in diverse plant developmental processes. The transcriptome analysis suggests that StRIK might play roles in RNA maturation and stress response in the periderm This work was supported by the Ministerio de Educación y Ciencia (AGL2006–07342: identification of the gene and obtention of the transgenic lines), the Ministerio de Innovación y Ciencia [AGL2009–13745, FPI grant to P.B.: Transcript profile of the gene in potato, selection of the transgenic lines], the Ministerio de Economía y Competitividad [AGL2012–36725; AGL2015–67495-C2–1-R: analysis of the periderm anatomy, periderm transcriptome and cell localization of the protein], Departament d’Universitats, Investigació i Societat de la Informació de Catalunya [PhD grant to M.S.], the University of Girona [PhD grant to S.F.-P] and The European Regional Development Fund (ERDF)

Both suberin and its associated waxes contribute to the formation of apoplastic barriers that protect plants from the environment. Some transcription factors have emerged as regulators of the suberization process. The potato StNAC103 gene was reported as a repressor of suberin polyester and suberin-associated waxes deposition because its RNAi-mediated downregulation (StNAC103-RNAi) over-accumulated suberin and associated waxes in the tuber phellem concomitantly with the induction of representative biosynthetic genes. Here, to explore if other genes of the large NAC gene family participate to this repressive function, we extended the silencing to other NAC members by targeting the conserved NAC domain of StNAC103 (StNAC103-RNAi-c). Transcript profile of the StNAC103-RNAi-c phellem indicated that StNAC101 gene was an additional potential target. In comparison with StNAC103-RNAi, the silencing with StNAC103-RNAi-c construct resulted in a similar effect in suberin but yielded an increased load of associated waxes in tuber phellem, mainly alkanes and feruloyl esters. Globally, the chemical effects in both silenced lines are supported by the transcript accumulation profile of genes involved in the biosynthesis, transport and regulation of apoplastic lipids. In contrast, the genes of polyamine biosynthesis were downregulated. Altogether these results point out to StNAC101 as a candidate to repress the suberin-associated waxes., This work was supported by the Ministerio de Educación y Ciencia (AGL2006-07342, FPI grant to O.S.), the Ministerio de Innovación y Ciencia [AGL2009-13745, FPI grant to P.B.], the Ministerio de Economía y Competitividad [AGL2012-36725; AGL2015-67495-C2-1-R], funding, European Regional Development Fund, Departament d’Universitats, Investigació i Societat de la Informació of Catalonia [PhD grant to R.V. and M.S.], the University of Girona [PhD grant to D.C.-A. and S.F.-P. and the grant SING11/1].

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.

The transcriptome comparison of two oak species reveals possible candidates accounting for the exceptionally thick and pure cork oak phellem, such as those involved in secondary metabolism and phellogen activity. Cork oak, Quercus suber, differs from other Mediterranean oaks such as holm oak (Quercus ilex) by the thickness and organization of the external bark. While holm oak outer bark contains sequential periderms interspersed with dead secondary phloem (rhytidome), the cork oak outer bark only contains thick layers of phellem (cork rings) that accumulate until reaching a thickness that allows industrial uses. Here we compare the cork oak outer bark transcriptome with that of holm oak. Both transcriptomes present similitudes in their complexity, but whereas cork oak external bark is enriched with upregulated genes related to suberin, which is the main polymer responsible for the protective function of periderm, the upregulated categories of holm oak are enriched in abiotic stress and chromatin assembly. Concomitantly with the upregulation of suberin-related genes, there is also induction of regulatory and meristematic genes, whose predicted activities agree with the increased number of phellem layers found in the cork oak sample. Further transcript profiling among different cork oak tissues and conditions suggests that cork and wood share many regulatory mechanisms, probably reflecting similar ontogeny. Moreover, the analysis of transcripts accumulation during the cork growth season showed that most regulatory genes are upregulated early in the season when the cork cambium becomes active. Altogether our work provides the first transcriptome comparison between cork oak and holm oak outer bark, which unveils new regulatory candidate genes of phellem development.

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.

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.

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.

The presence of Cronobacter or Salmonella in powdered infant formulae represents a serious health risk when fed to newborn infants. The detection of these pathogens by standard food analysis methods takes around 3–6 days to provide an accurate result. In order to reduce the time of analysis to less than 24 h, a real-time PCR-based method for the simultaneous detection of both Cronobacter spp. and Salmonella spp. in powdered infant formula was designed combining two existing primer-probe sets, and included an internal amplification control to validate the negative results. Inclusivity and exclusivity of the PCR were tested with 70 strains of Cronobacter, 13 strains of Salmonella enterica, and 75 non-Cronobacter non-Salmonella strains. The method was validated against the reference standard methods ISO/TS 22964: 2006 for Cronobacter and ISO 6579: 2002 for Salmonella using powdered infant formula samples, but also with other powdered products which could represent a health risk for infants younger than 6 months, such as follow-up formulae and hydrolyzed cereals. The detection limit of the new method was set at 1 cfu in 10 g of food for Cronobacter and 1 cfu in 25 g of food for Salmonella after 18 h enrichment in buffered peptone water, fulfilling the requirements of the European Commission.

In this study, a real-time polymerase chain reaction (PCR)-based method was designed for the fast detection of Cronobacter spp. (a newly proposed genus formerly known as Enterobacter sakazakii) in infant formula. The real-time PCR was positively tested with 70 Cronobacter strains, including members of all the species of this genus, and 88 non-Cronobacter strains. This new PCR-based system was validated against the reference standard ISO/TS 22964: 2006 (ISO International Organization for Standardization 2006) using 70 food matrices including powdered infant formula, follow-up formula, and hydrolyzed cereals for infants. The detection limit of the technique was found to be of 1 cfu in 10 g of food, fulfilling the requirements of the European Commission. The time of analysis, which comprises around 3–6 days using the reference method, is considerably reduced to less than 24 h using the real-time PCR-based system hereby described, allowing food industry a faster release of the stocks for commercialization. Moreover, this method includes an internal amplification control, co-amplified during each PCR run to verify the results.

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 102 genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.

Very long chain aliphatic compounds occur in the suberin polymer and associated wax. Up to now only few genes involved in suberin biosynthesis have been identified. This is a report on the isolation of a potato (Solanum tuberosum) 3-ketoacyl-CoA synthase (KCS) gene and the study of its molecular and physiological relevance by means of a reverse genetic approach. This gene, called StKCS6, was stably silenced by RNA interference (RNAi) in potato. Analysis of the chemical composition of silenced potato tuber periderms indicated that StKCS6 down-regulation has a significant and fairly specific effect on the chain length distribution of very long-chain fatty acids (VLCFAs) and derivatives, occurring in the suberin polymer and peridermal wax. All compounds with chain lengths of C(28) and higher were significantly reduced in silenced periderms, whereas compounds with chain lengths of C(26) and lower accumulated. Thus, StKCS6 is preferentially involved in the formation of suberin and wax lipidic monomers with chain lengths of C(28) and higher. As a result, peridermal transpiration of the silenced lines was about 1.5-times higher than that of the wild type. Our results convincingly show that StKCS6 is involved in both suberin and wax biosynthesis and that a reduction of the monomeric carbon chain lengths leads to increased rates of peridermal transpiration.

Suberin is a cell wall lipid polyester found in the cork cells of the periderm offering protection against dehydration and pathogens. Its biosynthesis and assembly, as well as its contribution to the sealing properties of the periderm, are still poorly understood. Here, we report on the isolation of the coding sequence CYP86A33 and the molecular and physiological function of this gene in potato (Solanum tuberosum) tuber periderm. CYP86A33 was down-regulated in potato plants by RNA interference-mediated silencing. Periderm from CYP86A33-silenced plants revealed a 60% decrease in its aliphatic suberin load and greatly reduced levels of C18:1 ω-hydroxyacid (approximately 70%) and α,ω-diacid (approximately 90%) monomers in comparison with wild type. Moreover, the glycerol esterified to suberin was reduced by 60% in the silenced plants. The typical regular ultrastructure of suberin, consisting of dark and light lamellae, disappeared and the thickness of the suberin layer was clearly reduced. In addition, the water permeability of the periderm isolated from CYP86A33-silenced lines was 3.5 times higher than that of the wild type. Thus, our data provide convincing evidence for the involvement of ω-functional fatty acids in establishing suberin structure and function.

Cork (phellem) is a multilayered dead tissue protecting plant mature stems and roots and plant healing tissues from water loss and injuries. Cork cells are made impervious by the deposition of suberin onto cell walls. Although suberin deposition and cork formation are essential for survival of land plants, molecular studies have rarely been conducted on this tissue. Here, we address this question by combining suppression subtractive hybridization together with cDNA microarrays, using as a model the external bark of the cork tree (Quercus suber), from which bottle cork is obtained. A suppression subtractive hybridization library from cork tree bark was prepared containing 236 independent sequences; 69% showed significant homology to database sequences and they corresponded to 135 unique genes. Out of these genes, 43.5% were classified as the main pathways needed for cork biosynthesis. Furthermore, 19% could be related to regulatory functions. To identify genes more specifically required for suberin biosynthesis, cork expressed sequence tags were printed on a microarray and subsequently used to compare cork (phellem) to a non-suberin-producing tissue such as wood (xylem). Based on the results, a list of candidate genes relevant for cork was obtained. This list includes genes for the synthesis, transport, and polymerization of suberin monomers such as components of the fatty acid elongase complexes, ATP-binding cassette transporters, and acyltransferases, among others. Moreover, a number of regulatory genes induced in cork have been identified, including MYB, No-Apical-Meristem, and WRKY transcription factors with putative functions in meristem identity and cork differentiation.

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.

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.

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.

The discovery that AC elements coordinated the regulation of genes belonging to the entire lignin biosynthetic pathway was the first breakthrough in understanding how lignin biosynthesis is regulated. Since then, tremendous progress has been made in the identification and characterization of many transcription factors (TFs) that regulate the genes of the phenylpropanoid branch pathway leading to lignin. A major breakthrough consisted in the discovery of a hierarchical transcriptional network regulating the biosynthesis of lignified secondary walls (SWs) in Arabidopsis. The NAC TFs (VND/NST/SND) work as the first layer of master switches activating the whole SW biosynthetic network through the regulation of a cascade of downstream TFs. Among these, MYB46/83 act as a second layer of master switches. Recent findings, however, reveal that the regulation of SW formation is far more complex than initially thought, involving both positive and negative regulators, dual function regulators, feedback loops, combinatorial complexes and cross talk between pathways. Finally, because of the great potential that lignocellulosic biomass represents for the production of bioenergy, there is a great interest in further elucidating the molecular mechanisms underlying the regulation of lignified SW and subsequently applying this knowledge to improve their saccharification potential for the generation of biofuels.

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

With the current global focus on bioenergy, forest plantations are increasingly becoming important sources for second generation biofuel, where the whole plant lignocellulosic biomass is to be mobilized. The lignocellulosic biomass is mainly composed of secondary walls (SW) possessing unique characteristics (biochemical composition and tridimensional association of polymers), which govern the intrinsic properties of wood. They especially contain high amounts of lignins, hydrophobic phenolic polymers which constitute an obstacle to the optimal utilization of plant species in paper industry and for saccharification prior to bioethanol production. Among perennial species, Eucalyptus species grow very fast and produce high yields of lignocellulosic biomass. They represent the main industrial plantations in the worldand one of the most appealing lignocellulosic feedstock for bioenergy production. Dissection of the molecular switches controlling the coordinated lignin biosynthetic genes is therefore of utmost importance to understand the molecular mechanisms underlying tissue specific deposition of lignin and be able to improve secondary cell wall properties. With the objective of improving Eucalyptus wood quality to better-fit industrial applications, we are focusing our efforts towards the identification and functional characterisation of regulatory genes controlling the biosynthesis of the cell wall polymers (mainly lignins). We performed a precise mapping and functional characterization of the cis-regulatory elements contained in the promoters of two genes encoding key and consecutive steps of the lignin biosynthetic pathway i.e. Cinnamoyl CoA reductase (CCR) and Cinnamyl Alcohol dehydrogenase (CAD) (Rahantamala et al, 2010). Our results supported a major role for the MYB transcription factors (TF) consensus sites in the control of the coordinated expression of these two genes. The functional analysis of two MYB factors (EgMYB1 and EgMYB2) preferentially expressed in Eucalyptus xylem revealed that they are able to bind specifically to these promoters and regulate transcriptionin vivo. EgMYB1 behaves as a repressor whereas EgMYB2 is an activator (Goicoechea et al, 2005, Legay et al, 2010) of the lignin biosynthetic genes but also of the secondary wall biosynthesis. Indeed, both MYBs were shown recently to be master genes regulating the entire secondary wall biosynthetic program including cellulose, xylan and lignin genes (Zhong et al, 2010; Legay et al 2010). The presence of both positive and negative regulators in Eucalyptus xylem offers the possibility of a combinatorial control of gene expression that could provide the necessary flexibility to ensure tight temporal and spatial regulation of lignin biosynthesis or secondary cell wall. To address this question and get a deeper insight the complex regulation of the SW formation in Eucalyptus, we are now studying the regulation of these two MYBs including fine spatial and temporal expression, identification of their direct targets genes and of their protein partners. We have constructed a yeast-two-hybrid library from Eucalyptus xylem that will also be instrumental for deciphering the interactants of landmark genes for the International Eucalyptus community. Thanks to the recent release of the E. grandis genome (Eucalyptus grandis Genome Project 2010, http://www.phytozome.net/eucalyptus), we have performed a genome-wide survey of the large R2R3-MYB superfamily. The phylogenetic comparison of this family with Arabidopsis, rice, poplar and grapevine showed a marked expansion of some clusters putatively involved in wood-related processes. Some R2R3 MYB genes seem to be specific of woody plants. The spatiotemporal expression patterns of members of such clusters are currently being studied. Although Auxin is known as a key regulator of cambium activity and wood formation, the Auxin response mediators [Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) transcription factors] extensively characterized in model plants, are still largely uncharacterized in tree species. We have identified 23 Aux/IAA and 17 ARF in the E. grandis genome. Comparative phylogenetic analysis revealed that several Aux/IAA and ARF subgroups have differentially expanded or contracted amongst the three dicotyledonous plants studied (Arabidopsis, Populus and Eucalyptus). Expression analysis and EST database surveys are currently underway to explore the transcript levels of each member in the different organs and tissues of Eucalyptus at key developmental stages as well as in response to hormonal treatments and to environmental stresses. Further functional genomics studies conducted on new candidate transcription factors, their regulation under the developmental or environmental stimuli will help identifying major factors underpinning the physicochemical properties of cell walls, the recalcitrance of which remains a key scientific challenge for establishing highly efficient, sustainably produced, second-generation biofuels.

Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

Background Nitrogen (N) is a main nutrient required for tree growth and biomass accumulation. In this study, we analyzed the effects of contrasting nitrogen fertilization treatments on the phenotypes of fast growing Eucalyptus hybrids (E. urophylla x E. grandis) with a special focus on xylem secondary cell walls and global gene expression patterns. Results Histological observations of the xylem secondary cell walls further confirmed by chemical analyses showed that lignin was reduced by luxuriant fertilization, whereas a consistent lignin deposition was observed in trees grown in N-limiting conditions. Also, the syringyl/guaiacyl (S/G) ratio was significantly lower in luxuriant nitrogen samples. Deep sequencing RNAseq analyses allowed us to identify a high number of differentially expressed genes (1,469) between contrasting N treatments. This number is dramatically higher than those obtained in similar studies performed in poplar but using microarrays. Remarkably, all the genes involved the general phenylpropanoid metabolism and lignin pathway were found to be down-regulated in response to high N availability. These findings further confirmed by RT-qPCR are in agreement with the reduced amount of lignin in xylem secondary cell walls of these plants. Conclusions This work enabled us to identify, at the whole genome level, xylem genes differentially regulated by N availability, some of which are involved in the environmental control of xylogenesis. It further illustrates that N fertilization can be used to alter the quantity and quality of lignocellulosic biomass in Eucalyptus, offering exciting prospects for the pulp and paper industry and for the use of short coppices plantations to produce second generation biofuels. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0256-9) contains supplementary material, which is available to authorized users.

The present study provides new insights on the role of the potato (Solanum tuberosum) suberin feruloyl transferase FHT in native and wound tissues, leading to conclusions about hitherto unknown properties of the phellogen. In agreement with the enzymatic role of FHT, it is shown that its transcriptional activation and protein accumulation are specific to tissues that undergo suberization such as the root boundary layers of the exodermis and the endodermis, along with the tuber periderm. Remarkably, FHT expression and protein accumulation within the periderm is restricted to the phellogen derivative cells with phellem identity. FHT levels in the periderm are at their peak near harvest during periderm maturation, with the phellogen becoming meristematically inactive and declining thereafter. However, periderm FHT levels remain high for several months after harvest, suggesting that the inactive phellogen retains the capacity to synthesize ferulate esters. Tissue wounding induces FHT expression and the protein accumulates from the first stages of the healing process onwards. FHT is up-regulated by abscisic acid and down-regulated by salicylic acid, emphasizing the complex regulation of suberin synthesis and wound healing. These findings open up new prospects important for the clarification of the suberization process and yield important information with regard to the skin quality of potatoes The authors thank Professor S. Prat (Centro Nacional de Biotecnologia, Madrid) and Dr E. Dominguez (Centre de Recerca en Agrigenomica CRAG, Barcelona) for providing potato ssp. andigena, for support, and for helpful experimental advice; Dr P. Suarez (CRAG) for kindly lending us the ultracentrifuge; Professor D. Ludevid, Dr S. Irar, and Dr M. P. Gonzalez (CRAG), and Dr T. Roscoe (Institut de Recherche pour le Development, Montpellier) for fruitful suggestions regarding protein and antibody production, immunolocalization, and GUS staining; and Dr J. Castro (Biology Department, University of Girona, UdG) for helpful advice on setting up the western blot conditions. We also thank Mr J. Blavia and D. Reyes (Serveis Tecnics de Recerca, UdG) and S. Gomez (Departament de Biologia, UdG) for their valuable assistance in carrying out the laboratory work. This work was supported by the Ministerio de Innovacion y Ciencia [AGL2009-13745], the Ministerio de Educacion y Ciencia [FPI grant to PB], and the Ministerio de Economia y Competitividad [AGL2012-36725]

Background Eucalyptus species are the most widely planted hardwood trees in the world representing more than 4.75 million ha in Brazil. Their high productivity, valuable wood properties and wide adaptability could allow sustainable and cost-efficient production of lignocellulosic bioenergy. The main limitation to this objective is wood recalcitrance to degradation which is linked to the structure and composition of lignified secondary cell walls. Lignin, for example, impairs the accessibility of cellulose during kraft pulping as well as during saccharification, a key step of bioethanol production. The application of nitrogen fertilizers is one strategy to increase growth rates and productivity since nitrogen is one of the most limiting nutrient for tree growth and carbon sequestration. However, the effects of nitrogen availability on wood properties and related gene expression are poorly understood. In poplar, it was recently reported that N fertilization increased aerial biomass, while in wood, fibre morphology and secondary cell wall structure and composition were modified. An increase in cellulose coupled with a decrease in lignin was observed and the mRNA profiles evaluated by microarray showed that nitrogen and tension wood have overlapping effects [1]. Moreover, a highly significant genetic correlation was observed between plant growth and lignin/cellulose composition. Quantitative trait loci co-localization identified the genomic position of potential pleiotropic regulators [2]. In order to get an insight on the regulation of nitrogen availability on wood formation in Eucalyptus, we have studied the effects of nitrogen fertilization on xylem transcriptome profiles using RNA-seq technology.