Eduardo Leal Oliveira Camargo

Xylem transcription profiles indicate potential metabolic responses for economically relevant characteristics of Eucalyptus species

BackgroundEucalyptus is one of the most important sources of industrial cellulose. Three species of this botanical group are intensively used in breeding programs: E. globulus, E. grandis and E. urophylla. E. globulus is adapted to subtropical/temperate areas and is considered a source of high-quality cellulose; E. grandis grows rapidly and is adapted to tropical/subtropical climates; and E. urophylla, though less productive, is considered a source of genes related to robustness. Wood, or secondary xylem, results from cambium vascular differentiation and is mostly composed of cellulose, lignin and hemicelluloses. In this study, the xylem transcriptomes of the three Eucalyptus species were investigated in order to provide insights on the particularities presented by each of these species.ResultsData analysis showed that (1) most Eucalyptus genes are expressed in xylem; (2) most genes expressed in species-specific way constitutes genes with unknown functions and are interesting targets for future studies; (3) relevant differences were observed in the phenylpropanoid pathway: E. grandis xylem presents higher expression of genes involved in lignin formation whereas E. urophylla seems to deviates the pathway towards flavonoid formation; (4) stress-related genes are considerably more expressed in E. urophylla, suggesting that these genes may contribute to its robustness.ConclusionsThe comparison of these three transcriptomes indicates the molecular signatures underlying some of their distinct wood characteristics. This information may contribute to the understanding of xylogenesis, thus increasing the potential of genetic engineering approaches aiming at the improvement of Eucalyptus forest plantations productivity.

Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens.

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants.

Identification of four Eucalyptus genes potentially involved in cell wall biosynthesis and evolutionarily related to SHINE transcription factors

Recently, a new Arabidopsis thaliana master regulator of plant cell wall biosynthesis was characterized. It was named SHINE transcription factor (SHINE TF). This work searched for homologous genes in Eucalyptus grandis genome draft. RNAseq data, phylogeny analysis and qRT-PCR experiments were performed to complement SHINE gene analysis. By similarity searches using A. thaliana SHINE genes, four sequences were identified in Eucalyptus. Two of them contain all conserved motifs and characteristic features of this family, being assumed as true SHINE TFs and named EgrSHN1 and EgrSHN2. The other two sequences contain an incomplete ‘mm’ motif and were not considered true SHINE TFs, being further referred as Egr33m and Egr40m. Expression analysis revealed that EgrSHN1 is more expressed in flowers than in leaves and immature xylem, and both EgrSHN1 and EgrSHN2 are absent from adult xylem RNAseq libraries. This expression profile is similar to A. thaliana orthologues. On the other hand, Egr33m and Egr40m expression was detected in adult xylems. The phylogenetic studies indicate that both EgrSHNs were originated by gene duplication events which, together with gene loss, are hypothesized as common events in SHINE evolution. In conclusion, it is possible that the overexpression of SHINE genes in Eucalyptus xylem can generate information about wood formation processes, allowing an effective increase in forest plantation productivity.

Flavonoid supplementation affects the expression of genes involved in cell wall formation and lignification metabolism and increases sugar content and saccharification in the fast-growing eucalyptus hybrid E. urophylla x E. grandis

BackgroundEucalyptus species are the most widely planted hardwood species in the world and are renowned for their rapid growth and adaptability. In Brazil, one of the most widely grown Eucalyptus cultivars is the fast-growing Eucalyptus urophylla x Eucalyptus grandis hybrid. In a previous study, we described a chemical characterization of these hybrids when subjected to flavonoid supplementation on 2 distinct timetables, and our results revealed marked differences between the wood composition of the treated and untreated trees.ResultsIn this work, we report the transcriptional responses occurring in these trees that may be related to the observed chemical differences. Gene expression was analysed through mRNA-sequencing, and notably, compared to control trees, the treated trees display differential down-regulation of cell wall formation pathways such as phenylpropanoid metabolism as well as differential expression of genes involved in sucrose, starch and minor CHO metabolism and genes that play a role in several stress and environmental responses. We also performed enzymatic hydrolysis of wood samples from the different treatments, and the results indicated higher sugar contents and glucose yields in the flavonoid-treated plants.ConclusionsOur results further illustrate the potential use of flavonoids as a nutritional complement for modifying Eucalyptus wood, since, supplementation with flavonoids alters its chemical composition, gene expression and increases saccharification probably as part of a stress response.

Eucalyptus transcriptome analysis revealed molecular chaperones highly expressed in xylem

Background Plant development is very plastic, being coupled to environmental cues. As sessile organisms, plants must be able to respond rapidly to environmental stresses such as changes in temperature and salinity, heavy metals and water deficit. Efficient stress response systems are prerequisites for plant survival and productivity [1]. Molecular chaperones (or Heat Shock Proteins – HSP) compose a ubiquitous class of proteins involved in cellular protein quality control (PQC) and homeostasis. They play a critical role in folding and degradation of polypeptides, and therefore, in maintenance and modulation of cellular pathways, which are dependent of function (correct folding) and availability (stability and degradation) of involved proteins, under normal and stress conditions [2]. Genetics and proteomics studies of wood formation have highlighted some chaperones up-regulated in xylem of Eucalyptus, Pinus and Populus species, stating that they may play an important role in cell wall formation and xylem development [3,4]. Different species of Eucalyptus are known for their superior performance in growth, wood quality and resistance to different types of stress [5]. Such characteristics are probably driven by distinct gene expression coordination in xylogenesis. Eucalyptus grandis is one of the most planted species in the world due to its rapid growth, wide adaptability and wood quality. Eucalyptus globulus wood has higher S/G ratio which provides high yields in cellulose extraction [6]. Lignin extraction consumes large quantities of chemicals and energy, and many efforts have been made to improve this process by modifying lignin content or composition in trees, in order to reduce lignin content or make it easier to extract. Results have been achieved by supplementation and genetic modification [7,8]. This study aims to identify chaperones possibly involved in wood formation and quality of wood for pulp and paper industries.

An integrated database of Eucalyptus spp. genome project

Background The species of the genus Eucalyptus are the most planted for the fiber crop in the world. They are mainly utilized for timber, pulp and paper production. Brazil, helped by the favorable weather conditions, appears as a big producer and exporter of eucalyptus derivates. In 2002, the Brazilian network research of the Eucalyptus Genome (Genolyptus) was established with the goal of integrating several academic and private institutions currently working with eucalyptus genomics in Brazil. This project generated around 200.000 ESTs from several tissues and conditions. Consequently, several individual projects have been implemented generating other transcriptome databases, in special, using RNA-Seq technology. In 2010, a draft genome (http://eucalyptusdb.bi.up. ac.za) of the specie E. grandis was produced by researches of the Joint Genome Institute (DOE-JGI) and the Eucalyptus Genome Network (EUCAGEN). The main goal of this work is to develop an Eucalyptusdatabase (http://www.lge.ibi.unicamp.br/genolyptus) integrating public and private data in a friendly and secure web interface with bioinformatics tools that allowing the users perform complex searches.

Exploring a new model of cell wall regulation: identification and expression of two putative SHINEs transcription factors in Eucalyptus

Background Eucalyptus forests are a competitive and efficient alternative to convert carbon from the atmosphere in cellulose, an important source for paper manufacture and bioenergy production. To obtain transgenic Eucalyptus with important traits improved it is necessary to make modifications in genes that affect the final phenotype. One interesting gene that follows this requisite was recently found: this is the AtSHN2 gene (Arabidopsis thaliana SHINE 2). AtSHN2 codifies to a Transcription Factor known as “Arabidopsis SHINE/WAX INDUCER”. Instead of inducing drought tolerance in transgenic rice (Oryza sativa), AtSHN2 overexpression causes: i) 34% increase in the cellulose content; ii) 45% reduction in lignin content and iii) increase in wood digestibly (elevated S:G ratio) with no compromise in plant strength and performance [1]. The discovery of AtSHN2 function in plant cell wall formation, led Ambavaram and collaborators [1] to perform other studies and ultimately to propose the following model: AtSHN2 regulates positively MYB transcription factors (TF) related to cellulose synthesis and it downregulates MYBTF’s related to lignin formation. At the same time, SHINE can repress NAC TFthat controls MYB expression[1]. As a consequence of the interesting phenotype achieved through AtSHN2 overexpression in rice, this work focused on the identification and analyses of AtSHN orthologues in Eucalyptus. Bioinformatics tools were used to search for AtSHN similar genes in Eucalyptus. Moreover, the expression profile of the corresponding genes in Eucalyptus was evaluated to prove their role as AtSHN. To carry it on, the expression experiments were done with flower, leaf and xylem. If the Eucalyptus putativeSHINE’s has the same function of the AtSHN’s,, gene expression in flower tissues will be the highest [2]. This is because it is known that AtSHN’s genes are preferentially expressed in abscission and dehiscence zones, a phenomenon that usually occurs in lots of flower tissues.

Eucalyptus Cell Wall Architecture: Clues for Lignocellulosic Biomass Deconstruction

The architecture, composition, and chemical properties of wood cell walls have a direct influence on the process that occurs prior to fermentation in second-generation biofuel production. The understanding of the construction patterns of cell wall types is the key to the new era of second-generation biofuels. Eucalyptus species are great candidates for this purpose since these species are among the fastest growing hardwood trees in the world and they have been improved for biomass production. We applied the glycome profiling and other combined techniques to study xylem cell walls of three economically important species (Eucalyptus globulus, Eucalyptus grandis, and Eucalyptus urophylla). Glycome profiling analyses revealed that species differ in the same key aspects of cell wall polymer linkages, with E. globulus and E. urophylla presenting contrasting phenotypes, and E. grandis with intermediate characteristics. E. urophylla is known for high recalcitrance, that is probably determined by the strong associations between lignin and cell wall polymers, and also lignin content. On the other hand, E. globulus cell wall polymers are loosely linked, so its cell wall can be easily deconstructed. We have shown in this work that the composition of cell walls differs in quantity and quality among the Eucalyptus species and such variations in composition influence the process of lignocellulosic feedstock assessment. However, the greatest influence relies on the amount and type of associations between cell wall polymers. A high yield of cellulose, from any biomass source, directly depends on the cell wall architecture.

Effects of nitrogen fertilization on global xylem transcript profiling of Eucalyptus urophylla x grandis evaluated by RNA-seq technology

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.

EUCANEXT: an integrated database for the exploration of genomic and transcriptomic data from Eucalyptus species

Abstract Tree species of the genus Eucalyptus are the most valuable and widely planted hardwoods in the world. Given the economic importance of Eucalyptus trees, much effort has been made towards the generation of specimens with superior forestry properties that can deliver high-quality feedstocks, customized to the industrýs needs for both cellulosic (paper) and lignocellulosic biomass production. In line with these efforts, large sets of molecular data have been generated by several scientific groups, providing invaluable information that can be applied in the development of improved specimens. In order to fully explore the potential of available datasets, the development of a public database that provides integrated access to genomic and transcriptomic data from Eucalyptus is needed. EUCANEXT is a database that analyses and integrates publicly available Eucalyptus molecular data, such as the E. grandis genome assembly and predicted genes, ESTs from several species and digital gene expression from 26 RNA-Seq libraries. The database has been implemented in a Fedora Linux machine running MySQL and Apache, while Perl CGI was used for the web interfaces. EUCANEXT provides a user-friendly web interface for easy access and analysis of publicly available molecular data from Eucalyptus species. This integrated database allows for complex searches by gene name, keyword or sequence similarity and is publicly accessible at http://www.lge.ibi.unicamp.br/eucalyptusdb. Through EUCANEXT, users can perform complex analysis to identify genes related traits of interest using RNA-Seq libraries and tools for differential expression analysis. Moreover, all the bioinformatics pipeline here described, including the database schema and PERL scripts, are readily available and can be applied to any genomic and transcriptomic project, regardless of the organism. Database URL: http://www.lge.ibi.unicamp.br/eucalyptusdb