Class III peroxidases constitute a plant-specific multigene family, where 73 genes have been identified in Arabidopsis thaliana. These genes are members of the reactive oxygen species (ROS) regulatory network in the whole plant, but more importantly, at the root level. In response to abiotic stresses such as cold, heat, and salinity, their expression is significantly modified. To learn more about their transcriptional regulation, an integrative phenotypic, genomic, and transcriptomic study was executed on the roots of A. thaliana Pyrenean populations. Initially, the root phenotyping highlighted 3 Pyrenean populations to be tolerant to cold (Eaux), heat (Herr), and salt (Grip) stresses. Then, the RNA-seq analyses on these three populations, in addition to Col-0, displayed variations in CIII Prxs expression under stressful treatments and between different genotypes. Consequently, several CIII Prxs were particularly upregulated in the tolerant populations, suggesting novel and specific roles of these genes in plant tolerance against abiotic stresses.

Exogenous low pH stress causes cell death in root cells, limiting root development, and agricultural production. Different lines of evidence suggested a relationship with cell wall (CW) remodeling players. We investigated whether class III peroxidase (CIII Prx) total activity, CIII Prx candidate gene expression, and reactive oxygen species (ROS) could modify CW structure during low pH-induced cell death in Arabidopsis thaliana roots. Wild-type roots displayed a good spatio-temporal correlation between the low pH-induced cell death and total CIII Prx activity in the early elongation (EZs), transition (TZs), and meristematic (MZs) zones. In situ mRNA hybridization showed that AtPrx62 transcripts accumulated only in roots treated at pH 4.6 in the same zones where cell death was induced. Furthermore, roots of the atprx62-1 knockout mutant showed decreased cell mortality under low pH compared to wild-type roots. Among the ROS, there was a drastic decrease in O2●− levels in the MZs of wild-type and atprx62-1 roots upon low pH stress. Together, our data demonstrate that AtPrx62 expression is induced by low pH and that the produced protein could positively regulate cell death. Whether the decrease in O2●− level is related to cell death induced upon low pH treatment remains to be elucidated.

ABSTRACTThe recent explosion of transcriptomics and proteomic data have resulted in vast amounts of datasets without connection and sometime too large to be easily analysed. Integration between datasets and analysis of an extracted datasets are limiting factors which need to be solved in order to make full use of the data and to connect data.ExpressWeb is an online web tool that combines a Taylor clustering of expressed data sets to extract gene network with gene annotations to visualise the co-expression network. Data sets can become from personal or publically experiments. ExpressWeb allows to easily compute clustering on expression data and provides friendly and useful visualisation tools as heatmaps, graphs and networks, generating output images which can be used for scientific publications.

We present a new database, specifically devoted to ROS homeostasis regulated proteins. This database replaced our previous database, the PeroxiBase, which was focused only on various peroxidase families. The addition of 20 new protein families related with ROS homeostasis justifies the new name for this more complex and comprehensive database as RedoxiBase. Besides enlarging the focus of the database, new analysis tools and functionalities have been developed and integrated through the web interface, with which the users can now directly access to orthologous sequences and see the chromosomal localization of sequences when available. OrthoMCL tool, completed with a post-treatment process, provides precise predictions of orthologous gene groups for the sequences present in this database. In order to explore and analyse orthogroups results, taxonomic visualization of organisms containing sequence of a specific orthogroup as well as chromosomal distribution of the orthogroup with one or two organisms have been included.

Following the publication of this article [1] the authors noted that the image in Fig. 1 was incorrect.

Lectins are fundamental to plant life and have important roles in cell-to-cell communication; development and defence strategies. At the cell surface; lectins are present both as soluble proteins (LecPs) and as chimeric proteins: lectins are then the extracellular domains of receptor-like kinases (LecRLKs) and receptor-like proteins (LecRLPs). In this review; we first describe the domain architectures of proteins harbouring G-type; L-type; LysM and malectin carbohydrate-binding domains. We then focus on the functions of LecPs; LecRLKs and LecRLPs referring to the biological processes they are involved in and to the ligands they recognize. Together; LecPs; LecRLKs and LecRLPs constitute versatile recognition systems at the cell surface contributing to the detection of symbionts and pathogens; and/or involved in monitoring of the cell wall structure and cell growth.

Summary In the context of climate change, the water‐use efficiency (WUE) of highly productive tree varieties, such as eucalypts, has become a major issue for breeding programmes. This study set out to dissect the genetic architecture of carbon isotope composition (δ13C), a proxy of WUE, across several environments. A family of Eucalyptus urophylla × E. grandis was planted in three trials and phenotyped for δ13C and growth traits. High‐resolution genetic maps enabled us to target genomic regions underlying δ13C quantitative trait loci (QTLs) on the E. grandis genome. Of the 15 QTLs identified for δ13C, nine were stable across the environments and three displayed significant QTL‐by‐environment interaction, suggesting medium to high genetic determinism for this trait. Only one colocalization was found between growth and δ13C. Gene ontology (GO) term enrichment analysis suggested candidate genes related to foliar δ13C, including two involved in the regulation of stomatal movements. This study provides the first report of the genetic architecture of δ13C and its relation to growth in Eucalyptus. The low correlations found between the two traits at phenotypic and genetic levels suggest the possibility of improving the WUE of Eucalyptus varieties without having an impact on breeding for growth.

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.

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.

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

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.

Arbuscular mycorrhizal (AM) fungi are involved in one of the most widespread plant-fungus interactions. A number of studies on the population dynamics of AM fungi have used mitochondrial (mt) DNA sequences, and yet mt AM fungus genomes are poorly known. To date, four mt genomes of three species of AM fungi are available, among which are two from Rhizophagus irregularis. In order to study intra- and interstrain mt genome variability of R. irregularis, we sequenced and de novo assembled four additional mt genomes of this species. We used 454 pyrosequencing and Illumina technologies to directly sequence mt genomes from total genomic DNA. The mt genomes are unique within each strain. Interstrain divergences in genome size, as a result of highly polymorphic intergenic and intronic sequences, were observed. The polymorphism is brought about by three types of variability generating element (VGE): homing endonucleases, DNA polymerase domain-containing open reading frames and small inverted repeats. Based on VGE positioning, mt sequences and nuclear markers, two subclades of R. irregularis were characterized. The discovery of VGEs highlights the great intraspecific plasticity of the R. irregularis mt genome. VGEs allow the design of powerful mt markers for the typing and monitoring of R. irregularis strains in genetic and population studies.

SUMMARY GECA is a fast, user-friendly and freely-available tool for representing gene exon/intron organization and highlighting changes in gene structure among members of a gene family. It relies on protein alignment, completed with the identification of common introns in the corresponding genes using CIWOG. GECA produces a main graphical representation showing the resulting aligned set of gene structures, where exons are to scale. The important and original feature of GECA is that it combines these gene structures with a symbolic display highlighting sequence similarity between subsequent genes. It is worth noting that this combination of gene structure with the indications of similarities between related genes allows rapid identification of possible events of gain or loss of introns, or points to erroneous structural annotations. The output image is generated in a portable network graphics format which can be used for scientific publications.

The PeroxiBase (http://peroxibase.toulouse.inra.fr/) is a specialized database devoted to peroxidases' families, which are major actors of stress responses. In addition to the increasing number of sequences and the complete modification of the Web interface, new analysis tools and functionalities have been developed since the previous publication in the NAR database issue. Nucleotide sequences and graphical representation of the gene structure can now be included for entries containing genomic cross-references. An expert semi-automatic annotation strategy is being developed to generate new entries from genomic sequences and from EST libraries. Plus, new internal and automatic controls have been included to improve the quality of the entries. To compare gene structure organization among families' members, two new tools are available, CIWOG to detect common introns and GECA to visualize gene structure overlaid with sequence conservation. The multicriteria search tool was greatly improved to allow simple and combined queries. After such requests or a BLAST search, different analysis processes are suggested, such as multiple alignments with ClustalW or MAFFT, a platform for phylogenetic analysis and GECA's display in association with a phylogenetic tree. Finally, we updated our family specific profiles implemented in the PeroxiScan tool and made new profiles to consider new sub-families.

During sporulation, Bacillus subtilis undergoes successive morphological changes that can be arrested at various stages by mutations in many genes. One of these, spoIIGB, encodes a transcriptional factor, sigma E, which is necessary to proceed beyond stage II and to differentiate the cell in two compartments, the forespore and the mother cell. Mutations were introduced in an open reading frame located immediately downstream of spoIIGB. They block sporulation at stage III and define a new gene, spoIIIG, encoding a 260-amino-acid polypeptide highly similar to bacterial sigma-factors. A promoter was identified in the spoIIGB-spoIIIG interval by transcriptional fusion to lacZ. It is turned on 1 hr after the start of sigma E synthesis and is specifically activated in the forespore. The tandemly arranged spoIIGB and spoIIIG genes appear to encode homologous proteins that modulate transcription in a sequential fashion during sporulation.

Summary: The prototrophic clones formed after fusion of mixed protoplasts from two polyauxotrophic strains of Bacillus subtilis have been counted both as L-form colonies and as bacterial colonies, by plating on selective medium in the presence and in the absence of methicillin. On average, one hundred times as many prototrophic colonies were counted when cell wall regeneration was prevented by the antibiotic. Thus genetic inactivation, which occurs regularly in bacterial exfusants, may be dependent on cell wall regeneration.