Resumo em Inglês:

The expressed sequence tags (ESTs) produced in the Forests project provide an invaluable opportunity to assess the Eucalyptus transcriptome. Besides providing information on the different proteins produced by this plant, it is possible to infer gene expression profiles because non-normalized cDNA libraries were used. The EST frequency from any gene is correlated to the transcript levels in the tissues from which the cDNA libraries were constructed. The goal of this work was to identify Eucalyptus genes that showed either differential expression pattern or were ubiquitously expressed in the tissues sampled in the Forests project. Six robust statistical tests and very restrictive rules were applied to gain confidence in the in silico data aiming to avoid false positives. Several genes with interesting expression profiles were identified and some of them were validated by RT-PCR.

Resumo em Inglês:

Post-transcriptional gene silencing (PTGS) is a conserved surveillance mechanism that identifies and cleaves double-stranded RNA molecules and their cellular cognate transcripts. The RNA silencing response is actually used as a powerful technique (named RNA interference) for potent and specific inhibition of gene expression in several organisms. To identify gene products in Eucalyptus sharing similarities with enzymes involved in the PTGS pathway, we queried the expressed sequence tag database of the Brazilian Eucalyptus Genome Sequence Project Consortium (FORESTs) with the amino acid sequences of known PTGS-related proteins. Among twenty-six prospected genes, our search detected fifteen assembled sequences encoding products presenting high level of similarity (E value < 10-40) to proteins involved in PTGS in plants and other organisms. We conclude that most of the genes known to be involved in the PTGS pathway are represented in the FORESTs database.

Resumo em Inglês:

MADS-box genes encode a family of transcription factors which control diverse developmental processes in flowering plants ranging from root to flower and fruit development. Members of the MADS-box gene family share a highly conserved sequence of approximately 180 nucleotides that encodes a DNA-binding domain. We used bioinformatics tools to investigate the information generated by the Eucalyptus Expressed Sequence Tag (FORESTs) genome project in order to identify and annotate MADS-box genes. The comparative phylogenetic analysis of the Eucalyptus MADS-box genes with Arabidopsis homologues allowed us to group them into one of the well-known subfamilies. Trends in gene expression of these putative Eucalyptus MADS-box genes were investigated by hierarchical clustering analysis. Among 24 MADS-box genes identified by our analysis, 12 are expressed in vegetative organs. Out of these, five are expressed predominately in wood. Understanding of the molecular mechanisms performed by MADS-box proteins underlying Eucalyptus growth, development and stress reactions would provide important insights into tree development and could reveal means by which tree characteristics could be modified for the improvement of industrial properties.

Resumo em Inglês:

Homeobox genes encode transcriptional factors, usually involved in molecular control of plant developmental patterns. They can be divided into several classes according to conserved sequences within the homeobox region and the presence of specific additional sequences. Based on these conserved sequences, we developed a search procedure to identify possible homeobox genes in the Eucalyptus Genome Sequencing Project Consortium (FORESTs) database. We were able to identify 50 Eucalyptus sequences (EST-contigs) containing the homeodomain sequence. Phylogenetic analysis was applied to this ESTs-contigs and 44 of them were found to have similarities with one of three well-known homeobox classes: Bell, Knox and HD-Zip, and their sub-classes. However, no EST-contig grouped with the fourth important homeobox class, the PHD-finger homeobox. On the other hand, two sequences have showed pronounced similarity to the Arabidopsis thaliana Wuschel gene, considered an "atypical" homeobox gene. Hierarchical clustering analysis of the expression pattern of these putative Eucalyptus homeobox genes revealed the presence of ten distinct expression groups. Combining phylogenetic analysis and expression patterns for some of the Eucalyptus genes revealed interesting aspects about some of the potential homeobox genes, which might lead to a better understanding of the Eucalyptus biology and to biotechnological applications.

Resumo em Inglês:

Expressed Sequence Tags (ESTs) sequencing provides reliable and useful information concerning gene expression patterns in the genomic context. Our group used bioinformatics to identify and annotate 5'EST-contigs belonging to the molecular chaperones within the Eucalyptus Genome Sequencing Project Consortium (FORESTs) database. We found that 1,959 5'EST-contigs, or approximately 1.6% of the total 5'EST-contigs, encoded chaperones, emphasizing their biological importance. About 55% of the chaperones that we found were Hsp70 chaperones and its co-chaperones, 18% were Hsp90 chaperones, 15% were Hsp60 and its co-chaperone, 8% were Hsp100 chaperones, and 4% were Small Hsps. We also investigated the digital expression profile of the chaperone genes to gain information on gene expression levels in the different libraries and we found that molecular chaperones may have differential expression. The results discussed here give important hints about the role of chaperones in Eucalyptus cells.

Resumo em Inglês:

Antioxidant metabolism protects cells from oxidative damage caused by reactive oxygen species (ROS). In plants, several enzymes act jointly to maintain redox homeostasis. Moreover, isoform diversity contributes to the fine tuning necessary for plant responses to both exogenous and endogenous signals influencing antioxidant metabolism. This study aimed to provide a comprehensive view of the major classes of antioxidant enzymes in the woody species Eucalyptus grandis. A careful survey of the FORESTs data bank revealed 36 clusters as encoding antioxidant enzymes: six clusters encoding ascorbate peroxidase (APx) isozymes, three catalase (CAT) proteins, three dehydroascorbate reductase (DHAR), two glutathione reductase (GR) isozymes, four monodehydroascorbate reductase (MDHAR), six phospholipid hydroperoxide glutathione peroxidases (PhGPx), and 12 encoding superoxide dismutases (SOD) isozymes. Phylogenetic analysis demonstrated that all clusters (identified herein) grouped with previously characterized antioxidant enzymes, corroborating the analysis performed. With respect to enzymes involved in the ascorbate-glutathione cycle, both cytosolic and chloroplastic isoforms were putatively identified. These sequences were widely distributed among the different ESTs libraries indicating a broad gene expression pattern. Overall, the data indicate the importance of antioxidant metabolism in eucalyptus.

Resumo em Inglês:

The Eucalyptus Genome Sequencing Project (FORESTs), an initiative from the Brazilian ONSA consortium (Organization for Nucleotide Sequencing and Analysis), has achieved the sequencing of 123.889 EST clones from 18 different cDNA libraries. We have investigated the FORESTs data set to identify EST clusters potentially encoding thioredoxins (TRX). Two types of thioredoxin families described in plants, chloroplastic (TRXm/f/x/y) and cytosolic (TRXh), have been found in the transcriptome. Putative typical TRXs have been identified in fifteen clusters, four m-type, seven h-type, two f-type, one cluster for each x/y-types and one putative homologue of the TDX gene from Arabidopsis thaliana. One cluster presents an atypical active site WCMPS, different from the conserved WCGPC present in the other 15 clusters, and corresponds to a subgroup of cytosolic thioredoxins. Except in specific libraries from callus, roots, seedlings and wood tissues, thioredoxin deduced ESTs are found in all remaining libraries. According to the calculated frequencies of ESTs, chloroplastic thioredoxins are preferentially present in green tissues such as leaves whilst cytoplasmic thioredoxins are more general but demonstrate elevated frequencies in seedlings and flower tissues. TRX frequency patterns in the Eucalyptus transcriptome seem to indicate a good coherence with data from Arabidopsis thaliana gene expression.

Resumo em Inglês:

This work was aimed at locating Eucalyptus ESTs corresponding to the PROTOX or PPO enzyme (Protoporphyrinogen IX oxidase, E.C. 1.3.3.4) directly related to resistance to herbicides that promote oxidative stress, changing the functionality of this enzyme. PROTOX, which is the site of action of diphenyl-ether (oxyfluorfen, lactofen, fomesafen), oxadiazole (oxadiazon and oxadiargyl), and aryl triazolinone (sulfentrazone and carfentrazone) herbicides, acts on the synthesis route of porphyrins which is associated with the production of chlorophyll a, catalases, and peroxidases. One cluster and one single read were located, with e-values better than e-70, associated to PROTOX. The alignment results between amino acid sequences indicated that this enzyme is adequately represented in the ESTs database of the FORESTs project.

Resumo em Inglês:

This work was aimed at locating Eucalyptus ESTs corresponding to the GS enzyme (Glutamine Synthetase, EC = 6.3.1.2) and to the D1 protein, which are directly related to resistance to herbicides that promote oxidative stress. Glutamine Synthetase corresponds to the site of action of the herbicide glufosinate. Herbicides that belong to groups such as ureas, uracils, triazines and triazinones act on the D1-Qb complex (receptor of electrons from the Photosystem II) by inactivating it. The clusters EGEQRT3302E01.g, EGEQRT3001F12.b; EGEZLV1203B04.g; EGBGFB1211H06.g and EGEZLV1205F09.g enclosed complete sequences (with 356 amino acids) of the Glutamine Synthetase enzyme. The cluster EGEQSL1054G06.g is a consensus of four reads and enclosed a complete sequence of D1 Protein (with 353 amino acids). The comparison of the sequences of Protein D1 from different species showed that the substitutions of serine (S) by glycine (G) or serine (S) by threonine (T) at the position 264 could produce plants resistant to herbicides that act on electron flow on Photosystem II. The sequence of amino acids corresponding to the cluster EGEQSL1054G06.g had a serine in position 264 indicating sensitivity of the Eucalyptus plants to herbicides that act on this site.

Resumo em Inglês:

A major goal of plant genome research is to recognize genes responsible for important traits. Resistance genes are among the most important gene classes for plant breeding purposes being responsible for the specific immune response including pathogen recognition, and activation of plant defence mechanisms. These genes are quite abundant in higher plants, with 210 clusters found in Eucalyptus FOREST database presenting significant homology to known R-genes. All five gene classes of R-genes with their respective conserved domains are present and expressed in Eucalyptus. Most clusters identified (93) belong to the LRR-NBS-TIR (genes with three domains: Leucine-rich-repeat, Nucleotide-binding-site and Toll interleucine 1-receptor), followed by the serine-threonine-kinase class (49 clusters). Some new combinations of domains and motifs of R-genes may be present in Eucalyptus and could represent novel gene structures. Most alignments occurred with dicots (94.3%), with emphasis on Arabidopsis thaliana (Brassicaceae) sequences. All best alignments with monocots (5.2%) occurred with rice (Oryza sativa) sequences and a single cluster aligned with the gymnosperm Pinus sylvestris (0.5%). The results are discussed and compared with available data from other crops and may bring useful evidences for the understanding of defense mechanisms in Eucalyptus and other crop species.

Resumo em Inglês:

Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs) in the FORESTs Genome Project database (<A HREF="https://forests.esalq.usp.br">https://forests.esalq.usp.br</A>) related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature, respectively. The alignment of EPSPS sequences allowed the identification of conserved regions that can be used to design specific primers for additional sequencings.

Resumo em Inglês:

This study assessed the abundance of microsatellites, or simple sequence repeats (SSR), in 19 Eucalyptus EST libraries from FORESTs, containing cDNA sequences from five species: E. grandis, E. globulus, E. saligna, E. urophylla and E. camaldulensis. Overall, a total of 11,534 SSRs and 8,447 SSR-containing sequences (25.5% of total ESTs) were identified, with an average of 1 SSR/2.5 kb when considering all motifs and 1 SSR/3.1 kb when mononucleotides were not included. Dimeric repeats were the most abundant (41.03%), followed by trimerics (36.11%) and monomerics (19.59%). The most frequent motifs were A/T (87.24%) for monomerics, AG/CT (94.44%) for dimerics, CCG/CGG (37.87%) for trimerics, AAGG/CCTT (18.75%) for tetramerics, AGAGG/CCTCT (14.04%) for pentamerics and ACGGCG/CGCCGT (6.30%) for hexamerics. According to sequence length, Class II or potentially variable markers were the most commonly found, followed by Class III. Two sequences presented high similarity to previously published Eucalyptus sequences from the NCBI database, EMBRA_72 and EMBRA_122. Local blastn search for transposons did not reveal the presence of any transposable elements with a cut-off value of 10-50. The large number of microsatellites identified will contribute to the refinement of marker-assisted mapping and to the discovery of novel markers for virtually all genes of economic interest.

Resumo em Inglês:

The main goal of our research was to search for SSRs in the Eucalyptus EST FORESTs database (using a software for mining SSR-motifs). With this objective, we created a database for cataloging Eucalyptus EST-derived SSRs, and developed a bioinformatics tool, named Satellyptus, for finding and analyzing microsatellites in the Eucalyptus EST database. The search for microsatellites in the FORESTs database containing 71,115 Eucalyptus EST sequences (52.09 Mb) revealed 20,530 SSRs in 15,621 ESTs. The SSR abundance detected on the Eucalyptus ESTs database (29% or one microsatellite every four sequences) is considered very high for plants. Amongst the categories of SSR motifs, the dimeric (37%) and trimeric ones (33%) predominated. The AG/CT motif was the most frequent (35.15%) followed by the trimeric CCG/CGG (12.81%). From a random sample of 1,217 sequences, 343 microsatellites in 265 SSR-containing sequences were identified. Approximately 48% of these ESTs containing microsatellites were homologous to proteins with known biological function. Most of the microsatellites detected in Eucalyptus ESTs were positioned at either the 5’ or 3’ end. Our next priority involves the design of flanking primers for codominant SSR loci, which could lead to the development of a set of microsatellite-based markers suitable for marker-assisted Eucalyptus breeding programs.

Resumo em Inglês:

Eucalyptus ESTs libraries were screened for genes involved in lignin biosynthesis. This search was performed under the perspective of recent revisions on the monolignols biosynthetic pathway. Eucalyptus orthologues of all genes of the phenylpropanoid pathway leading to lignin biosynthesis reported in other plant species were identified. A library made with mRNAs extracted from wood was enriched for genes involved in lignin biosynthesis and allowed to infer the isoforms of each gene family that play a major role in wood lignin formation. Analysis of the wood library suggests that, besides the enzymes of the phenylpropanoids pathway, chitinases, laccases, and dirigent proteins are also important for lignification. Colocalization of several enzymes on the endoplasmic reticulum membrane, as predicted by amino acid sequence analysis, supports the existence of metabolic channeling in the phenylpropanoid pathway. This study establishes a framework for future investigations on gene expression level, protein expression and enzymatic assays, sequence polymorphisms, and genetic engineering.

Resumo em Inglês:

The occurrence of quasi-repetitive glycine-rich peptides has been reported in different organisms. Glycine-rich regions are proposed to be involved in protein-protein interactions in some mammalian protein families. In plants, a set of glycine-rich proteins (GRPs) was characterized several years ago, and since then a wealth of new GRPs have been identified. GRPs may have very diverse sub-cellular localization and functions. The only common feature among all different GRPs is the presence of glycine-rich repeat domains. The expression of genes encoding GRPs is developmentally regulated, and also induced, in several plant genera, by physical, chemical and biological factors. In addition to the highly modulated expression, several GRPs also show tissue-specific localization. GRPs specifically expressed in xylem, phloem, epidermis, anther tapetum and roots have been described. In this paper, the structural and functional features of these proteins in Eucalyptus are summarized. Since this is the first description of GRPs in this species, particular emphasis has been given to the expression pattern of these genes by analyzing their abundance and prevalence in the different cDNA-libraries of the Eucalyptus Genome Sequencing Project Consortium (ForEST). The comparison of GRPs from Eucalyptus and other species is also discussed.

Resumo em Inglês:

Boron (B) is a low mobility plant micronutrient whose molecular mechanisms of absorption and translocation are still controversial. Many factors are involved in tolerance to Boron excess or deficiency. Recently, the first protein linked to boron transport in biological systems, BOR1, was characterized in Arabidopsis thaliana. This protein is involved in boron xylem loading and is similar to bicarbonate transporters found in animals. There are indications that BOR1 is a member of a conserved protein family in plants. In this work, FORESTS database was used to identify sequences similar to this protein family, looking for a probable BOR1 homolog in eucalypt. We found five consensus sequences similar to BOR1; three of them were then used in multiple alignment analysis. Based on amino acid similarity and in silico expression patterns, a consensus sequence was identified as a candidate BOR1 homolog, helping deeper experimental assays that could identify the function of this protein family in Eucalyptus.

Resumo em Inglês:

In this work we apply a bioinformatics approach to determine the most important enzymes of the metabolic network of Eucalyptus to determine the coverage of the genome in the FORESTS library. We conclude that the library does not cover completely the metabolism of the organism. However, some important pathways could be analyzed, especially the lignin synthesis. We found that four of the most important enzymes predicted are involved in this pathway.

Resumo em Inglês:

Transposable elements (TE) are major components of eukaryotic genomes and involved in cell regulation and organism evolution. We have analyzed 123,889 expressed sequence tags of the Eucalyptus Genome Project database and found 124 sequences representing 76 TE in 9 groups, of which copia, MuDR and FAR1 groups were the most abundant. The low amount of sequences of TE may reflect the high efficiency of repression of these elements, a process that is called TE silencing. Frequency of groups of TE in Eucalyptus libraries which were prepared with different tissues or physiologic conditions from seedlings or adult plants indicated that developing plants experience the expression of a much wider spectrum of TE groups than that seen in adult plants. These are preliminary results that identify the most relevant TE groups involved with Eucalyptus development, which is important for industrial wood production.

Resumo em Inglês:

Abscisic acid (ABA) regulates stress responses in plants, and genomic tools can help us to understand the mechanisms involved in that process. FAPESP, a Brazilian research foundation, in association with four private forestry companies, has established the FORESTs database (<A HREF="https://forests.esalq.usp.br">https://forests.esalq.usp.br</A>). A search was carried out in the Eucalyptus expressed sequence tag database to find ESTs involved with 9-cis epoxycarotenoid dioxygenase (NCED), the regulatory enzyme for ABA biosynthesis, using the basic local BLAST alignment tool. We found four clusters (EGEZLV2206B11.g, EGJMWD2252H08.g, EGBFRT3107F10.g, and EGEQFB1200H10.g), which represent similar sequences of the gene that produces NCED. Data showed that the EGBFRT3107F10.g cluster was similar to the maize (Zea mays) NCED enzyme, while EGEZLV2206B11.g and EGJMWD2252H08.g clusters were similar to the avocado (Persea americana) NCED enzyme. All Eucalyptus clusters were expressed in several tissues, especially in flower buds, where ABA has a special participation during the floral development process.