Danieli Cristina Gonçalves

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.

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

Comparative proteomics and metallomics studies in Arabidopsis thaliana leaf tissues: Evaluation of the selenium addition in transgenic and nontransgenic plants using two-dimensional difference gel electrophoresis and laser ablation imaging

The main goal of this work is to evaluate some differential protein species in transgenic (T) and nontransgenic (NT) Arabidopsis thaliana plants after their cultivation in the presence or absence of sodium selenite. The transgenic line was obtained through insertion of CaMV 35S controlling nptII gene. Comparative proteomics through 2D‐DIGE is carried out in four different groups (NT × T; NT × Se‐NT (where Se is selenium); Se‐NT × Se‐T, and T × Se‐T). Although no differential proteins are achieved in the T × Se‐T group, for the others, 68 differential proteins (by applying a regulation factor ≥1.5) are achieved, and 27 of them accurately characterized by ESI‐MS/MS. These proteins are classified into metabolism, energy, signal transduction, disease/defense categories, and some of them are involved in the glycolysis pathway—Photosystems I and II and ROS combat. Additionally, laser ablation imaging is used for evaluating the Se and sulfur distribution in leaves of different groups, corroborating some results obtained and related to proteins involved in the glycolysis pathway. From these results, it is possible to conclude that the genetic modification also confers to the plant resistance to oxidative stress.

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360±30nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.

Human Hsp70/Hsp90 Organizing Protein (Hop) D456G Is a Mixture of Monomeric and Dimeric Species

Hop is a tetratricopeptide repeat domain (TPR)-containing co-chaperone that is able to directly associate with both Hsp70 and Hsp90. Previous data showed that the TPR2A-domain is the primary site for dimerization and that the TPR2B-domain may also play a role in dimerization. We present Hop-D456G, a mutant within the TPR2B-domain, that is a mixture of monomeric and dimeric species.

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.

αB‐Crystallin interacts and attenuates the tyrosine phosphatase activity of Shp2 in cardiomyocytes under mechanical stress

The small heat shock protein αB‐Crystallin (CryAB, HspB5) and SH2 domain‐containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock‐down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.