Impacts of in-vitro zebularine treatment on genome-wide DNA methylation and transcriptomic profiles in Salix purpurea L.

Renewable energy resources such as biomass are crucial for a sustainable global society. Trees are a major source of lignocellulosic biomass, which can vary in response to different environmental factors owing to epigenetic regulation, such as DNA C-methylation. To investigate the effects of DNA methylation on plant development and wood formation, and its impacts on gene expression, with a focus on secondary cell wall (SCW)-associated genes, Salix purpurea plantlets were cloned from buds derived from a single hybrid tree for both treatment and control conditions. For the treatment condition, buds were exposed to 50 µM zebularine in vitro and a combined strategy of whole-genome bisulfite sequencing (WGBS) and RNA-seq was employed to examine the methylome and transcriptome profiles of different tissues collected at various time points under both conditions. Transcriptomic and methylome data revealed that most of the promoter and gene body demethylation had no marked effects on the expression profiles of genes. Nevertheless, gene expression tended to decrease with the increased methylation levels of genes with highly methylated promoters. Results indicated that demethylation is less evident in centromeric regions and sex chromosomes. Promoters of secondary cell wall-associated genes, such as 4-coumarate-CoA ligase-like and Rac-like GTP-binding protein RHO, were differentially methylated in the secondary xylem samples collected from two-month potted treated plants compared to control samples. Our results provide novel insights into DNA methylation and gene expression landscapes and a basis for investigating the epigenetic regulation of wood formation in S. purpurea as a model plant for bioenergy species.

A comparative transcriptomic approach to understanding the formation of cork.

KEY MESSAGE: 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.

Extensive recent secondary contacts between four European white oak species.

Historical trajectories of tree species during the late Quaternary have been well reconstructed through genetic and palaeobotanical studies. However, many congeneric tree species are interfertile, and the timing and contribution of introgression to species divergence during their evolutionary history remains largely unknown. We quantified past and current gene flow events between four morphologically divergent oak species (Quercus petraea, Q. robur, Q. pyrenaica, Q. pubescens), by two independent inference methods: diffusion approximation to the joint frequency spectrum (∂a∂i) and approximate Bayesian computation (ABC). For each pair of species, alternative scenarios of speciation allowing gene flow over different timescales were evaluated. Analyses of 3524 single nucleotide polymorphisms (SNPs) randomly distributed in the genome, showed that these species evolved in complete isolation for most of their history, but recently came into secondary contact, probably facilitated by the most recent period of postglacial warming. We demonstrated that: there was sufficient genetic differentiation before secondary contact for the accumulation of barriers to gene flow; and current European white oak genomes are a mosaic of genes that have crossed species boundaries and genes impermeable to gene flow.

Comprehensive genome-wide analysis of the Aux/IAA gene family in Eucalyptus: evidence for the role of EgrIAA4 in wood formation.

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 roles of key Aux/IAA gene regulators of the auxin response in these processes, we identified and characterized members of 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 groups are represented. High-throughput expression profiling of different organs and tissues highlighted several Aux/IAA genes expressed in vascular cambium and/or developing xylem, some showing differential expression in response to developmental (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.

Genome-wide analysis of the lignin toolbox of Eucalyptus grandis.

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.

The Eucalyptus grandis R2R3-MYB transcription factor family: evidence for woody growth-related evolution and function.

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.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Revealing the transitory and local effect of zebularine on development and on proteome dynamics of Salix purpurea.

Introduction: DNA methylation plays major roles in the epigenetic regulation of gene expression, transposon and transcriptional silencing, and DNA repair, with implications in developmental processes and phenotypic plasticity. Relevantly for woody species, DNA methylation constitutes a regulative layer in cell wall dynamics associated with xylogenesis. The use of methyltransferase and/or demethylase inhibitors has been proven informative to shed light on the methylome dynamics behind the regulation of these processes. Methods: The present work employs the cytidine analog zebularine to inhibit DNA methyltransferases and induce DNA hypomethylation in Salix purpurea plantlets grown in vitro and in soil. An integrative approach was adopted to highlight the effects of zebularine on proteomic dynamics, revealing age-specific (3 weeks of in vitro culture and 1 month of growth in soil) and tissue-specific (stem and root) effects. Results and discussion: After 3 weeks of recovery from zebularine treatment, a decrease of 5-mC levels was observed in different genomic contexts in the roots of explants that were exposed to zebularine, whereas a functionally heterogeneous subset of protein entries was differentially accumulated in stem samples, including entries related to cell wall biosynthesis, tissue morphogenesis, and hormonal regulation. Significant proteomic remodeling was revealed in the development from in vitro to in-soil culture, but no significant changes in 5-mC levels were observed. The identification of tissue-specific proteomic hallmarks in combination with hypomethylating agents provides new insights into the role of DNA methylation and proteome in early plant development in willow species. Proteomic data are available via ProteomeXchange with identifier PXD045653. WGBS data are available under BioProject accession PRJNA889596.

A Taxonomically-verified and Vouchered Checklist of the Vascular Plants of the Republic of Guinea.

The Checklist of the Vascular Plants of the Republic of Guinea (CVPRG) is a specimen-based, expert-validated knowledge product, which provides a concise synthesis and overview of current knowledge on 3901 vascular plant species documented from Guinea (Conakry), West Africa, including their accepted names and synonyms, as well as their distribution and status within Guinea (indigenous or introduced, endemic or not). The CVPRG is generated automatically from the Guinea Collections Database and the Guinea Names Backbone Database, both developed and maintained at the Royal Botanic Gardens, Kew, in collaboration with the staff of the National Herbarium of Guinea. A total of 3505 indigenous vascular plant species are reported of which 3328 are flowering plants (angiosperms); this represents a 26% increase in known indigenous angiosperms since the last floristic overview. Intended as a reference for scientists documenting the diversity and distribution of the Guinea flora, the CVPRG will also inform those seeking to safeguard the rich plant diversity of Guinea and the societal, ecological and economic benefits accruing from these biological resources.

Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions.

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.

Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries.

BACKGROUND: Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. RESULTS: We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. CONCLUSIONS: The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.

In Medicago truncatula, water deficit modulates the transcript accumulation of components of small RNA pathways.

BACKGROUND: Small RNAs (sRNAs) are 20-24 nucleotide (nt) RNAs and are involved in plant development and response to abiotic stresses. Plants have several sRNA pathways implicated in the transcriptional and post-transcriptional silencing of gene expression. Two key enzyme families common to all pathways are the Dicer-like (DCL) proteins involved in sRNAs maturation and the Argonautes (AGOs) involved in the targeting and functional action of sRNAs. Post-transcriptional silencing mediated by AGOs may occur by cleavage or translational repression of target mRNA's, while transcriptional silencing may be controlled by DNA methylation and chromatin remodeling. Thus far, these gene families have not been characterized in legumes, nor has their involvement in adaptation to water deficit been studied. RESULTS: A bioinformatic search in Medicago truncatula genome databases, using Arabidopsis thaliana AGO and DCL cDNA and protein sequences, identified three sequences encoding for putative Dicer-like genes and twelve sequences encoding for putative Argonaute genes. Under water deficit conditions and mainly in roots, MtDCL1 and MtAGO1, two enzymes probably involved in the processing and activation of microRNAs (miRNAs), increased their transcript levels. mir162 which target DCL1 mRNA and mir168 which target AGO1 mRNA reduced their expression in the roots of plants subjected to water deficit. Three putative genes, MtDCL3, MtAGO4b and MtAGO4c probably involved in DNA methylation mechanisms, increased their mRNA levels. However, the mRNA levels of MtAGO6 reduced, which probably encodes a protein with functions similar to MtAGO4. MtAGO7 mRNA levels increased and possibly encodes a protein involved in the production of trans-acting small interfering RNAs. The transcript abundance of MtAGO12a, MtAGO12b and MtAGO12c reduced under water deprivation. Plants recovered from water deprivation reacquire the mRNA levels of the controls. CONCLUSIONS: Our work demonstrates that in M. truncatula the transcript accumulation of the components of small RNA pathways is being modulated under water deficit. This shows that the transcriptional and post-transcriptional control of gene expression mediated by sRNAs is probably involved in plant adaptation to abiotic environmental changes. In the future this will allow the manipulation of these pathways providing a more efficient response of legumes towards water shortage.

Proteomic evaluation of wound-healing processes in potato (Solanum tuberosum L.) tuber tissue.

Proteins from potato (Solanum tuberosum L.) tuber slices, related to the wound-healing process, were separated by 2-DE and identified by an MS analysis in MS and MS/MS mode. Slicing triggered differentiation processes that lead to changes in metabolism, activation of defence and cell-wall reinforcement. Proteins related to storage, cell growth and division, cell structure, signal transduction, energy production, disease/defence mechanisms and secondary metabolism were detected. Image analysis of the 2-DE gels revealed a time-dependent change in the complexity of the polypeptide patterns. By microscopic observation the polyalyphatic domain of suberin was clearly visible by D4, indicating that a closing layer (primary suberisation) was formed by then. A PCA of the six sampling dates revealed two time phases, D0-D2 and D4-D8, with a border position between D2 and D4. Moreover, a PCA of differentially expressed proteins indicated the existence of a succession of proteomic events leading to wound-periderm reconstruction. Some late-expressed proteins (D6-D8), including a suberisation-associated anionic peroxidase, have also been identified in the native periderm. Despite this, protein patterns of D8 slices and native periderm were still different, suggesting that the processes of wound-periderm formation are extended in time and not fully equivalent. The information presented in this study gives clues for further work on wound healing-periderm formation processes.

Hairy Root Transformation: A Useful Tool to Explore Gene Function and Expression in Salix spp. Recalcitrant to Transformation.

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.

Molecular and phenotypic profiling from the base to the crown in maritime pine wood-forming tissue.

Environmental, developmental and genetic factors affect variation in wood properties at the chemical, anatomical and physical levels. Here, the phenotypic variation observed along the tree stem was explored and the hypothesis tested that this variation could be the result of the differential expression of genes/proteins during wood formation. Differentiating xylem samples of maritime pine (Pinus pinaster) were collected from the top (crown wood, CW) to the bottom (base wood, BW) of adult trees. These samples were characterized by Fourier transform infrared spectroscopy (FTIR) and analytical pyrolysis. Two main groups of samples, corresponding to CW and BW, could be distinguished from cell wall chemical composition. A genomic approach, combining large-scale production of expressed sequence tags (ESTs), gene expression profiling and quantitative proteomics analysis, allowed identification of 262 unigenes (out of 3512) and 231 proteins (out of 1372 spots) that were differentially expressed along the stem. A good relationship was found between functional categories from transcriptomic and proteomic data. A good fit between the molecular mechanisms involved in CW-BW formation and these two types of wood phenotypic differences was also observed. This work provides a list of candidate genes for wood properties that will be tested in forward genetics.

Advancing Eucalyptus Genomics: Cytogenomics Reveals Conservation of Eucalyptus Genomes.

The genus Eucalyptus encloses several species with high ecological and economic value, being the subgenus Symphyomyrtus one of the most important. Species such as E. grandis and E. globulus are well characterized at the molecular level but knowledge regarding genome and chromosome organization is very scarce. Here we characterized and compared the karyotypes of three economically important species, E. grandis, E. globulus, and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta, and E. occidentalis, through an integrative approach including genome size estimation, fluorochrome banding, rDNA FISH, and BAC landing comprising genes involved in lignin biosynthesis. All karyotypes show a high degree of conservation with pericentromeric 35S and 5S rDNA loci in the first and third pairs, respectively. GC-rich heterochromatin was restricted to the 35S rDNA locus while the AT-rich heterochromatin pattern was species-specific. The slight differences in karyotype formulas and distribution of AT-rich heterochromatin, along with genome sizes estimations, support the idea of Eucalyptus genome evolution by local expansions of heterochromatin clusters. The unusual co-localization of both rDNA with AT-rich heterochromatin was attributed mainly to the presence of silent transposable elements in those loci. The cinnamoyl CoA reductase gene (CCR1) previously assessed to linkage group 10 (LG10) was clearly localized distally at the long arm of chromosome 9 establishing an unexpected correlation between the cytogenetic chromosome 9 and the LG10. Our work is novel and contributes to the understanding of Eucalyptus genome organization which is essential to develop successful advanced breeding strategies for this genus.

Antimalarial activity of medicinal plants used in traditional medicine in S. Tomé and Príncipe islands.

The present study investigates the antimalarial activity of 13 medicinal plants used in traditional medicine in S. Tomé and Príncipe (STP) islands in the Gulf of Guinea, aiming at identifying the most effective plants for further research. Fieldwork was carried out with the collaboration of 37 traditional healers from both islands, during an ethnobotanical study, which was conducted from 1993 to 1999. Our results indicate that the traditional healers in STP use several medicinal plants against fever and/or 'malaria' which reveal strong antiparasitic activity in vitro: four of the plant extracts have evident antiplasmodial activity against chloroquine resistant Plasmodium falciparum, with IC(50) values <10 microg/ml, and also revealed hepatic schizontocidal activity (<5-35 microg/ml). In vivo, the extracts caused partial reduction of Plasmodium berghei parasitaemia in mice.

Screening and identification of neuroprotective compounds relevant to Alzheimer׳s disease from medicinal plants of S. Tomé e Príncipe.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer׳s disease (AD) neuropathology is strongly associated with the activation of inflammatory pathways, and long-term use of anti-inflammatory drugs reduces the risk of developing the disease. In S. Tomé e Príncipe (STP), several medicinal plants are used both for their positive effects in the nervous system (treatment of mental disorders, analgesics) and their anti-inflammatory properties. The goal of this study was to determine whether a phenotypic, cell-based screening approach can be applied to selected plants from STP (Voacanga africana, Tarenna nitiduloides, Sacosperma paniculatum, Psychotria principensis, Psychotria subobliqua) in order to identify natural compounds with multiple biological activities of interest for AD therapeutics. MATERIALS AND METHODS: Plant hydroethanolic extracts were prepared and tested in a panel of phenotypic screening assays that reflect multiple neurotoxicity pathways relevant to AD-oxytosis in hippocampal nerve cells, in vitro ischemia, intracellular amyloid toxicity, inhibition of microglial inflammation and nerve cell differentiation. HPLC fractions from the extract that performed the best in all of the assays were tested in the oxytosis assay, our primary screen, and the most protective fraction was analyzed by mass spectrometry. The predominant compound was purified, its identity confirmed by ESI mass spectrometry and NMR, and then tested in all of the screening assays to determine its efficacy. RESULTS: An extract from the bark of Voacanga africana was more protective than any other plant extract in all of the assays (EC50s≤2.4 µg/mL). The HPLC fraction from the extract that was most protective against oxytosis contained the alkaloid voacamine (MW=704.90) as the predominant compound. Purified voacamine was very protective at low doses in all of the assays (EC50s≤3.4 µM). CONCLUSION: These findings validate the use of our phenotypic screening, cell-based assays to identify potential compounds to treat AD from plant extracts with ethnopharmacological relevance. Our study identifies the alkaloid voacamine as a major compound in Voacanga africana with potent neuroprotective activities in these assays.

Genome-wide characterization and expression profiling of the AUXIN RESPONSE FACTOR (ARF) gene family in Eucalyptus grandis.

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.