An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ���missing-self��� recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 �� 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

Inducible endothelial cell-specific gene expression in transgenic mouse embryos and adult mice

Utilizing both the TET-OFF and TET-ON systems in combination with transcriptional control elements of the Tie-2 gene, we have established a series of transgenic activator and responder mice for TET-regulated endothelial cell-specific transgene expression in double transgenic mouse embryos and in adult mice. TET-regulated expression of LacZ reporter genes could be achieved in virtually all endothelia in mid gestation stage mouse embryos. In contrast in adult mice, using the very same Tie-2 tTA activator mouse strain, we observed striking differences of TET-induced gene expression from various inducible expression constructs in different vascular beds. Non-endothelial expression was never detected. The prominent differences in completeness of TET-induced endothelial expression highlight the still underestimated critical role of the responder mouse lines for uniform TET-induced gene expression in heterogeneous cell populations such as endothelial cells. Interestingly, in double transgenic mice inducibly expressing several different adhesion molecules, no adverse effects were observed even though these proteins were robustly expressed on endothelial cells in adult tissues. These transgenic model systems provide versatile tools for the TET-regulated manipulation of endothelial cell-specific gene expression in the entire embryonic vasculature and distinct vascular beds in adult mice.

An investigation of phenolic glycoside and condensed tannin homeostasis in <i>Populus</i> by salicyl alcohol feeding to cell cultures and by transgenic manipulation of the sucrose transporter, <i>PTSUT4, in planta</i>

Secondary metabolites play an important role in plant protection against biotic and abiotic stress. In Populus, phenolic glycosides (PGs) and condensed tannins (CTs) are two such groups of compounds derived from the common phenylpropanoid pathway. The basal levels and the inducibility of PGs and CTs depend on genetic as well as environmental factors, such as soil nitrogen (N) level. Carbohydrate allocation, transport and sink strength also affect PG and CT levels. A negative correlation between the levels of PGs and CTs was observed in several studies. However, the molecular mechanism underlying such relation is not known. We used a cell culture system to understand negative correlation of PGs and CTs. Under normal culture conditions, neither salicin nor higher-order PGs accumulated in cell cultures. Several factors, such as hormones, light, organelles and precursors were discussed in the context of aspen suspension cells��� inability to synthesize PGs. Salicin and its isomer, isosalicin, were detected in cell cultures fed with salicyl alcohol, salicylaldehyde and helicin. At higher levels (5 mM) of salicyl alcohol feeding, accumulation of salicins led to reduced CT production in the cells. Based on metabolic and gene expression data, the CT reduction in salicin-accumulating cells is partly a result of regulatory changes at the transcriptional level affecting carbon partitioning between growth processes, and phenylpropanoid CT biosynthesis. Based on molecular studies, the glycosyltransferases, GT1-2 and GT1-246, may function in glycosylation of simple phenolics, such as salicyl alcohol in cell cultures. The uptake of such glycosides into vacuole may be mediated to some extent by tonoplast localized multidrug-resistance associated protein transporters, PtMRP1 and PtMRP6. In Populus, sucrose is the common transported carbohydrate and its transport is possibly regulated by sucrose transporters (SUTs). SUTs are also capable of transporting simple PGs, such as salicin. Therefore, we characterized the SUT gene family in Populus and investigated, by transgenic analysis, the possible role of the most abundantly expressed member, PtSUT4, in PG-CT homeostasis using plants grown under varying nitrogen regimes. PtSUT4 transgenic plants were phenotypically similar to the wildtype plants except that the leaf area-to-stem volume ratio was higher for transgenic plants. In SUT4 transgenics, levels of non-structural carbohydrates, such as sucrose and starch, were altered in mature leaves. The levels of PGs and CTs were lower in green tissues of transgenic plants under N-replete, but were higher under N-depleted conditions, compared to the levels in wildtype plants. Based on our results, SUT4 partly regulates N-level dependent PG-CT homeostasis by differential carbohydrate allocation.

DISSECTION OF STRESS RESPONSE NETWORKS REGULATING MULTIPLE STRESSES IN RICE

Important food crops like rice are constantly exposed to various stresses that can have devastating effect on their survival and productivity. Being sessile, these highly evolved organisms have developed elaborate molecular machineries to sense a mixture of stress signals and elicit a precise response to minimize the damage. However, recent discoveries revealed that the interplay of these stress regulatory and signaling molecules is highly complex and remains largely unknown. In this work, we conducted large scale analysis of differential gene expression using advanced computational methods to dissect regulation of stress response which is at the heart of all molecular changes leading to the observed phenotypic susceptibility. One of the most important stress conditions in terms of loss of productivity is drought. We performed genomic and proteomic analysis of epigenetic and miRNA mechanisms in regulation of drought responsive genes in rice and found subsets of genes with striking properties. Overexpressed genesets included higher number of epigenetic marks, miRNA targets and transcription factors which regulate drought tolerance. On the other hand, underexpressed genesets were poor in above features but were rich in number of metabolic genes with multiple co-expression partners contributing majorly towards drought resistance. Identification and characterization of the patterns exhibited by differentially expressed genes hold key to uncover the synergistic and antagonistic components of the cross talk between stress response mechanisms. We performed meta-analysis on drought and bacterial stresses in rice and Arabidopsis, and identified hundreds of shared genes. We found high level of conservation of gene expression between these stresses. Weighted co-expression network analysis detected two tight clusters of genes made up of master transcription factors and signaling genes showing strikingly opposite expression status. To comprehensively identify the shared stress responsive genes between multiple abiotic and biotic stresses in rice, we performed meta-analyses of microarray studies from seven different abiotic and six biotic stresses separately and found more than thirteen hundred shared stress responsive genes. Various machine learning techniques utilizing these genes classified the stresses into two major classes' namely abiotic and biotic stresses and multiple classes of individual stresses with high accuracy and identified the top genes showing distinct patterns of expression. Functional enrichment and co-expression network analysis revealed the different roles of plant hormones, transcription factors in conserved and non-conserved genesets in regulation of stress response.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

BACKGROUND Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM. METHODS We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. RESULTS A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. CONCLUSIONS The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

Depletion of regulatory T cells augments a vaccine-induced T effector cell response against the liver-stage of malaria but fails to increase memory

Regulatory T cells (T(reg)) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4(+)CD25(+) T(reg) were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3(+)CD25(-) T(reg). To obtain more insights in the specific function of T(reg) during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC). Using this system we demonstrate here that the number of CSP-specific T cells increases when T(reg) are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.

The Rice Transcription Factor WRKY53 Suppresses Herbivore-Induced Defenses by Acting as a Negative Feedback Modulator of Mitogen-Activated Protein Kinase Activity

The mechanisms by which herbivore-attacked plants activate their defenses are well studied. By contrast, little is known about the regulatory mechanisms that allow them to control their defensive investment and avoid a defensive overshoot. We characterized a rice (Oryza sativa) WRKY gene, OsWRKY53, whose expression is rapidly induced upon wounding and induced in a delayed fashion upon attack by the striped stem borer (SSB) Chilo suppressalis. The transcript levels of OsWRKY53 are independent of endogenous jasmonic acid but positively regulated by the mitogen-activated protein kinases OsMPK3/OsMPK6. OsWRKY53 physically interacts with OsMPK3/OsMPK6 and suppresses their activity in vitro. By consequence, it modulates the expression of defensive, MPK-regulated WRKYs and thereby reduces jasmonic acid, jasmonoyl-isoleucine, and ethylene induction. This phytohormonal reconfiguration is associated with a reduction in trypsin protease inhibitor activity and improved SSB performance. OsWRKY53 is also shown to be a negative regulator of plant growth. Taken together, these results show that OsWRKY53 functions as a negative feedback modulator of MPK3/MPK6 and thereby acts as an early suppressor of induced defenses. OsWRKY53 therefore enables rice plants to control the magnitude of their defensive investment during early signaling.

Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, ��H is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Characterization of tumor-associated Foxp3+ regulatory T cells and de-novo induction by the tumor microenvironment

Regulatory T cells expressing the fork-head box transcription factor 3 (Foxp3) play a central role in the dominant control of immunological tolerance. Compelling evidence obtained from both animal and clinical studies have now linked the expansion and accumulation of Foxp3+ regulatory T cells associated with tumor lesions to the failure of immune-mediated tumor rejection. However, further progress of the field is hampered by the gap of knowledge regarding their phenotypic, functional, and the developmental origins in which these tumor-associated Foxp3+ regulatory T cells are derived. Here, we have characterized the general properties of tumor-associated Foxp3+ regulatory T cells and addressed the issue of tumor microenvironment mediated de-novo induction by utilizing a well known murine tumor model MCA-205 in combination with our BAC Foxp3-GFP reporter mice and OT-II TCR transgenic mice on the RAG deficient background (RAG OT-II). De-novo induction defines a distinct mechanism of converting non-regulatory precursor cells to Foxp3+ regulatory T cells in the periphery as opposed to the expansion of pre-existing regulatory T cells formed naturally during thymic T cell development. This mechanism is of particularly importance to how tumors induce tumor-antigen-specific suppressor cells to subvert anti-tumor immune responses. Our study has found that tumor-associated Foxp3+ regulatory T cells are highly activated, undergo vigorous proliferation, are more potent by in-vitro suppression assays, and express higher levels of membrane-bound TGF-��1 than non-tumor regulatory T cells. With Foxp3-GFP reporter mice or RAG OT-II TCR transgenic mice, we show that tumor tissue can induce detectable de-novo generation of Foxp3+ regulatory T cells of both polyclonal or antigen specific na��ve T cells. This process was not only limited for subcutaneous tumors but for lung tumors as well. Furthermore, this process required the inducing antigen to be co-localized within the tumor tissue. Examination of tumor tissue revealed an abundance of myeloid CD11b+ antigen-presenting cells that were capable of inducing Foxp3+ regulatory T cells. Taken together, these findings elucidate the general attributes and origins of tumor-associated Foxp3+ regulatory T cells in the tumor microenvironment and in their role in the negative regulation of tumor immunity.^

Control of the master virulence regulatory gene atxA in Bacillus anthracis

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact atxA transcript levels, and the timing and steady state level of atxA expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the atxA promoter. The AbrB-binding site has been described, but additional cis-acting control sequences have not been defined. Using transcriptional lacZ fusions, electrophoretic mobility shift assays, and Western blot analysis, the cis-acting elements and trans-acting factors involved in regulation of atxA in B. anthracis strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a trans-acting protein(s) other than AbrB negatively impacts atxA transcription when it binds specifically to a 9-bp palindrome within atxA promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the trans-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner. The identity of the trans-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the trans-acting repressor protein(s) indirectly controls B. anthracis development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both atxA- and pXO2-61-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper B. anthracis development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on atxA expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of atxA. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an abrB-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an abrB-null mutant. In addition, the abrB-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the atxA promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the abrB-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an abrB-null mutant. Overall, this work highlights the complex regulatory network that governs expression of atxA and provides an additional role for AtxA in B. anthracis development.

Targeted Deletion Of Functionally Validated Enhancers Defines Their Role in Mouse Limb Development

Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription ��� the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development. The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse 280 and 1442 (M280 and M1442, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined lacZ expression. In M280-lacZ mice, expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. M1442-lacZ mice exhibited lacZ expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the M280 and M1442 enhancers by gene targeting. Intercrosses between M280 -/+ and M1442 -/+, respectively, generated M280 and M1442 null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both M280 and M1442. In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in M280 and M1442 embryos from E13.5-E15.5, transient development defects were not detected. These results demonstrate that M280 and M1442 are not required for mouse limb development. Though M280 is not required for embryonic limb development it is required for the development and/or maintenance of body size ��� adult M280 mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers in situ to understand their contribution to development.

Genomic analyses of flowering transition and inflorescence development in grapevine = An��lisis gen��micos de la transici��n floral y del desarrollo de la inflorescencia en la vid

The general objective of this work is to analyze the regulatory processes underlying flowering transition and inflorescence and flower development in grapevine. Most of these crucial developmental events take place within buds growing during two seasons in two consecutive years. During the first season, the shoot apical meristem within the bud differentiates all the basic elements of the shoot including flowering transition in lateral primordia and development of inflorescence primordia. These events practically end with bud dormancy. The second season, buds resume shoot growth associated to flower formation and development. In grapevine, the lateral meristems can give rise either to tendril or inflorescence primordia that are homologous organs. With this purpose, we performed global transcriptome analyses along the bud annual cycle and during inflorescence and tendril development. In addition, we approach the genomic analysis of the MIKC type MADS-box gene family in grapevine to identify all its members and assign them putative biological functions. Regarding buds developmental cycle, the results indicate that the main factors explaining the global gene expression differences were the processes of bud dormancy and active growth as well as stress responses. Non dormant buds exhibited up-regulation in functional categories typical of actively proliferating and growing cells (photosynthesis, cell cycle regulation, chromatin assembly) whereas in dormant ones the main functional categories up-regulated were associated to stress response pathways together with transcripts related to starch catabolism. Major transcriptional changes during the dormancy period were associated to the para/endodormancy, endo/ecodormancy and ecodormancy/bud break transitions. Global transcriptional analyses along tendril and inflorescence development suggested that these two homologous organs share a common transcriptional program related to cell proliferation functions. Both structures showed a progressive decrease in the expression of categories such as cell-cycle, auxin metabolism/signaling, DNA metabolism, chromatin assembly and a cluster of five transcripts belonging to the GROWTH-REGULATING FACTOR (GRF) transcription factor family, that are known to control cell proliferation in other species and determine the size of lateral organs. However, they also showed organ specific transcriptional programs that can be related to their differential organ structure and function. Tendrils showed higher transcription of genes related to photosynthesis, hormone signaling and secondary metabolism than inflorescences, while inflorescences have higher transcriptional activity for genes encoding transcription factors (especially those belonging to the MADS-box gene family). Further analysis along inflorescence development evidenced the relevance of additional functions likely related to processes of flower development such as fatty acid and lipid metabolism, jasmonate signaling and oxylipin biosynthesis. The transcriptional analyses performed highlighted the relevance of several groups of transcriptional regulators in the developmental processes studied. The expression profiles along bud development revealed significant differences for some MADS-box subfamilies in relation to other plant species, like the members of the FLC and SVP subfamilies suggesting new roles for these groups in grapevine. In this way, it was found that VvFLC2 and VvAGL15.1 could participate, together with some members of the SPL-L family, in dormancy regulation, as was shown for some of them in other woody plants. Similarly, the expression patterns of the VvFLC1, VvFUL, VvSOC1.1 (together with VvFT, VvMFT1 and VFL) genes could indicate that they play a role in flowering transition in grapevine, in parallel to their roles in other plant systems. The expression levels of VFL, the grapevine LEAFY homolog, could be crucial to specify the development of inflorescence and flower meristems instead of tendril meristems. MADS-box genes VvAP3.1 and 2, VvPI, VvAG1 and 3, VvSEP1-4, as well as VvBS1 and 2 are likely associated with the events of flower meristems and flower organs differentiation, while VvAP1 and VvFUL-L (together with VvSOC1.1, VvAGL6.2) could be involved on tendril development given their expression patterns. In addition, the biological function ofVvAP1 and VvTFL1A was analyzed using a gene silencing approach in transgenic grapevine plants. Our preliminary results suggested a possible role for both genes in the initiation and differentiation of tendrils. Finally, the genomic analysis of the MADS-box gene family in grapevine revealed differential features regarding number and expression pattern of genes putatively involved in the flowering transition process as compared to those involved in the specification of flower and fruit organ identity. Altogether, the results obtained allow identifying putative candidate genes and pathways regulating grapevine reproductive developmental processes paving the way to future experiments demonstrating specific gene biological functions. RESUMEN El objetivo general de este trabajo es analizar los procesos regulatorios subyacentes a la inducci��n floral as�� como al desarrollo de la inflorescencia y la flor en la vid. La mayor parte de estos eventos cruciales tienen lugar en las yemas a lo largo de dos estaciones de crecimiento consecutivas. Durante la primera estaci��n, el meristemo apical contenido en la yema diferencia los elementos b��sicos del p��mpano, lo cual incluye la inducci��n de la floraci��n en los meristemos laterales y el subsiguiente desarrollo de primordios de inflorescencia. Estos procesos pr��cticamente cesan con la entrada en dormici��n de la yema. En la segunda estaci��n, se reanuda el crecimiento del p��mpano acompa��ado por la formaci��n y desarrollo de las flores. En la vid, los meristemos laterales pueden dar lugar a primordios de inflorescencia o de zarcillo que son considerados ��rganos hom��logos. Con este objetivo llevamos a cabo un estudio a nivel del transcriptoma de la yema a lo largo de su ciclo anual, as�� como a lo largo del desarrollo de la inflorescencia y del zarcillo. Adem��s realizamos un an��lisis gen��mico de la familia MADS de factores transcripcionales (concretamente aquellos del tipo MIKC) para identificar todos sus miembros y tratar de asignarles posibles funciones biol��gicas. En cuanto al ciclo de desarrollo de la yema, los resultados indican que los principales factores que explican las diferencias globales en la expresi��n g��nica fueron los procesos de dormici��n de la yema y el crecimiento activo junto con las respuestas a diversos tipos de estr��s. Las yemas no durmientes mostraron un incremento en la expresi��n de genes contenidos en categor��as funcionales t��picas de c��lulas en proliferaci��n y crecimiento activo (como fotos��ntesis, regulaci��n del ciclo celular, ensamblaje de cromatina), mientras que en las yemas durmientes, las principales categor��as funcionales activadas estaban asociadas a respuestas a estr��s, as�� como con el catabolismo de almid��n. Los mayores cambios observados a nivel de transcriptoma en la yema coincidieron con las transiciones de para/endodormici��n, endo/ecodormici��n y ecodormici��n/brotaci��n. Los an��lisis transcripcionales globales a lo largo del desarrollo del zarcillo y de la inflorescencia sugirieron que estos dos ��rganos hom��logos comparten un programa transcripcional com��n, relacionado con funciones de proliferaci��n celular. Ambas estructuras mostraron un descenso progresivo en la expresi��n de genes pertenecientes a categor��as funcionales como regulaci��n del ciclo celular, metabolismo/se��alizaci��n por auxinas, metabolismo de ADN, ensamblaje de cromatina y un grupo de cinco tr��nscritos pertenecientes a la familia de factores transcripcionales GROWTH-REGULATING FACTOR (GRF), que han sido asociados con el control de la proliferaci��n celular y en determinar el tama��o de los ��rganos laterales en otras especies. Sin embargo, tambi��n pusieron de manifiesto programas transcripcionales que podr��an estar relacionados con la diferente estructura y funci��n de dichos ��rganos. Los zarcillos mostraron mayor actividad transcripcional de genes relacionados con fotos��ntesis, se��alizaci��n hormonal y metabolismo secundario que las inflorescencias, mientras que ��stas presentaron mayor actividad transcripcional de genes codificantes de factores de transcripci��n (especialmente los pertenecientes a la familia MADS-box). An��lisis adicionales a lo largo del desarrollo de la inflorescencia evidenciaron la relevancia de otras funciones posiblemente relacionadas con el desarrollo floral, como el metabolismo de l��pidos y ��cidos grasos, la se��alizaci��n mediada por jasmonato y la bios��ntesis de oxilipinas. Los an��lisis transcripcionales llevados a cabo pusieron de manifiesto la relevancia de varios grupos de factores transcripcionales en los procesos estudiados. Los perfiles de expresi��n estudiados a lo largo del desarrollo de la yema mostraron diferencias significativas en algunas de las subfamilias de genes MADS con respecto a otras especies vegetales, como las observadas en los miembros de las subfamilias FLC y SVP, lo cual sugiere que podr��an desempe��ar nuevas funciones en la vid. En este sentido, se encontr�� que los genes VvFLC2 y VvAGL15.1 podr��an participar, junto con algunos miembros de la familia SPL-L, en la regulaci��n de la dormici��n. De un modo similar, los patrones de expresi��n de los genes VvFLC1, VvFUL, VvSOC1.1 (junto con VvFT, VvMFT1 y VFL) podr��a indicar que desempe��an un papel en la regulaci��n de la inducci��n de la floraci��n en la vid, como se ha observado en otros sistemas vegetales. Los niveles de expresi��n de VFL, el hom��logo en vid del gen LEAFY de A. thaliana podr��an ser cruciales para la especificaci��n del desarrollo de meristemos de inflorescencia y flor en lugar de meristemos de zarcillo. Los genes VvAP3.1 y 2, VvPI, VvAG1 y 3, VvSEP1-4, as�� como VvBS1 y 2 parecen estar asociados con los eventos de diferenciaci��n de meristemos y ��rganos florales, mientras que VvAP1 y VvFUL-L (junto con VvSOC1.1 y VvAGL6.2) podr��an estar implicados en el desarrollo del zarcillo dados sus patrones de expresi��n. Adicionalmente, se analiz�� la funci��n biol��gica de los genes VvAP1 y VvTFL1A por medio de una estrategia de silenciamiento g��nico. Los datos preliminares sugieren un posible papel para ambos genes en la iniciaci��n y diferenciaci��n de los zarcillos. Finalmente, el an��lisis gen��mico de la familia MADS en vid evidenci�� diferencias con respecto a otras especies vegetales en cuanto a n��mero de miembros y patr��n de expresi��n en genes supuestamente implicados en la inducci��n de la floraci��n, en comparaci��n con aquellos relacionados con la especificaci��n de identidad de ��rganos florales y desarrollo del fruto. En conjunto, los resultados obtenidos han permitido identificar posibles rutas y genes candidatos a participar en la regulaci��n de los procesos de desarrollo reproductivo de la vid, sentando las bases de futuros experimentos encaminados a conocer la funciones biol��gicas de genes espec��ficos.

Factores transcripcionales de la clase DOF en Brachypodium distachyon: caracterizaci��n molecular de BdDOF24 durante la germinaci��n de las semillas

La semilla es el ��rgano que garantiza la propagaci��n y continuidad evolutiva de las plantas espermatofitas y constituye un elemento indispensable en la alimentaci��n humana y animal. La semilla de cereales acumula en el endospermo durante la maduraci��n, mayoritariamente, almid��n y prote��nas de reserva. Estas reservas son hidrolizadas en la germinaci��n por hidrolasas sintetizadas en la aleurona en respuesta a giberelinas (GA), siendo la principal fuente de energ��a hasta que la pl��ntula emergente es fotosint��ticamente activa. Ambas fases del desarrollo de la semilla, est��n reguladas por una red de factores de transcripci��n (TF) que unen motivos conservados en cis- en los promotores de sus genes diana. Los TFs son prote��nas que han desempe��ado un papel central en la evoluci��n y en el proceso de domesticaci��n, siendo uno de los principales mecanismos de regulaci��n g��nica; en torno al 7% de los genes de plantas codifican TFs. Atendiendo al motivo de uni��n a DNA, ��stos, se han clasificado en familias. La familia DOF (DNA binding with One Finger) participa en procesos vitales exclusivos de plantas superiores y sus ancestros cercanos (algas, musgos y helechos). En las semillas de las Triticeae (subfamilia Pooideae), se han identificado varias prote��nas DOF que desempe��an un papel fundamental en la regulaci��n de la expresi��n g��nica. Brachypodium distachyon es la primera especie de la subfamilia Pooideae cuyo genoma (272 Mbp) ha sido secuenciado. Su peque��o tama��o, ciclo de vida corto, y la posibilidad de ser transformado por Agrobacterium tumefaciens (pl��smido Ti), hacen que sea el sistema modelo para el estudio de cereales de la tribu Triticeae con gran importancia agron��mica mundial, como son el trigo y la cebada. En este trabajo, se han identificado 27 genes Dof en el genoma de B. distachyon y se han establecido las relaciones evolutivas entre estos genes Dof y los de cebada (subfamilia Pooideae) y de arroz (subfamilia Oryzoideae), construyendo un ��rbol filogen��tico en base al alineamiento m��ltiple del dominio DOF. La cebada contiene 26 genes Dof y en arroz se han anotado 30. El an��lisis filogen��tico establece cuatro grupos de genes ort��logos (MCOGs: Major Clusters of Orthologous Genes), que est��n validados por motivos conservados adicionales, adem��s del dominio DOF, entre las secuencias de las prote��nas de un mismo MCOG. El estudio global de expresi��n en diferentes ��rganos establece un grupo de nueve genes BdDof expresados abundantemente y/o preferencialmente en semillas. El estudio detallado de expresi��n de estos genes durante la maduraci��n y germinaci��n muestra que BdDof24, ort��logo putativo a BPBF-HvDOF24 de cebada, es el gen m��s abundante en las semillas en germinaci��n de B. distachyon. La regulaci��n transcripcional de los genes que codifican hidrolasas en la aleurona de las semillas de cereales durante la post���germinaci��n ha puesto de manifiesto la existencia en sus promotores de un motivo tripartito en cis- conservado GARC (GA-Responsive Complex), que unen TFs de la clase MYB-R2R3, DOF y MYBR1-SHAQKYF. En esta tesis, se ha caracterizado el gen BdCathB de Brachypodium que codifica una proteasa tipo catepsina B y es ort��logo a los genes Al21 de trigo y HvCathB de cebada, as�� como los TFs responsables de su regulaci��n transcripcional BdDOF24 y BdGAMYB (ort��logo a HvGAMYB). El an��lisis in silico del promotor BdCathB ha identificado un motivo GARC conservado, en posici��n y secuencia, con sus ort��logos en trigo y cebada. La expresi��n de BdCathB se induce durante la germinaci��n, as�� como la de los genes BdDof24 y BdGamyb. Adem��s, los TFs BdDOF24 y BdGAMYB interaccionan en el sistema de dos h��bridos de levadura e in planta en experimentos de complementaci��n bimolecular fluorescente. En capas de aleurona de cebada, BdGAMYB activa el promotor BdCathB, mientras que BdDOF24 lo reprime; este resultado es similar al obtenido con los TFs ort��logos de cebada BPBF-HvDOF24 y HvGAMYB. Sin embargo, cuando las c��lulas de aleurona se transforman simult��neamente con los dos TFs, BdDOF24 tiene un efecto aditivo sobre la trans-activaci��n mediada por BdGAMYB, mientras que su ort��logo BPBF-HvDOF24 produce el efecto contrario, revirtiendo el efecto de HvGAMYB sobre el promotor BdCathB. Las diferencias entre las secuencias deducidas de las prote��nas BdDOF24 y BPBF-HvDOF24 podr��an explicar las funciones opuestas que desempe��an en su interacci��n con GAMYB. Resultados preliminares con l��neas de inserci��n de T-DNA y de sobre-expresi��n estable de BdGamyb, apoyan los resultados obtenidos en expresi��n transitoria. Adem��s las l��neas homocigotas knock-out para el gen BdGamyb presentan alteraciones en anteras y polen y no producen semillas viables. ABSTRACT The seed is the plant organ of the spermatophytes responsible for the dispersion and survival in the course of evolution. In addition, it constitutes one of the most importan elements of human food and animal feed. The main reserves accumulated in the endosperm of cereal seeds through the maturation phase of development are starch and proteins. Its degradation by hydrolases synthetized in aleurone cells in response to GA upon germination provides energy, carbon and nitrogen to the emerging seedling before it acquires complete photosynthetic capacity. Both phases of seed development are controlled by a network of transcription factors (TFs) that interact with specific cis- elements in the promoters of their target genes. TFs are proteins that have played a central role during evolution and domestication, being one of the most important regulatory mechanisms of gene expression. Around 7% of genes in plant genomes encode TFs. Based on the DNA binding motif, TFs are classified into families. The DOF (DNA binding with One Finger) family is involved in specific processes of plants and its ancestors (algae, mosses and ferns). Several DOF proteins have been described to play important roles in the regulation of genes in seeds of the Triticeae tribe (Pooideae subfamily). Brachypodium distachyon is the first member of the Pooideae subfamily to be sequenced. Its small size and compact structured genome (272 Mbp), the short life cycle, small plant size and the possibility of being transformed with Agrobacterium tumefaciens (Ti-plasmid) make Brachypodium the model system for comparative studies within cereals of the Triticeae tribe that have big economic value such as wheat and barley. In this study, 27 Dof genes have been identified in the genome of B. distachyon and the evolutionary relationships among these Dof genes and those frome barley (Pooideae subfamily) and those from rice (Oryzoideae subfamily) have been established by building a phylogenetic tree based on the multiple alignment of the DOF DNA binding domains. The barley genome (Hordeum vulgare) contains 26 Dof genes and in rice (Oryza sativa) 30 genes have been annotated. The phylogenetic analysis establishes four Major Clusters of Orthologous Genes (MCOGs) that are supported by additional conserved motives out of the DOF domain, between proteins of the same MCOG. The global expression study of BdDof genes in different organs and tissues classifies BdDof genes into two groups; nine of the 27 BdDof genes are abundantly or preferentially expressed in seeds. A more detailed expression analysis of these genes during seed maturation and germination shows that BdDof24, orholog to barley BPBF-HvDof24, is the most abundantly expressed gene in germinating seeds. Transcriptional regulation studies of genes that encode hydrolases in aleurone cells during post-germination of cereal seeds, have identified in their promoters a tripartite conserved cis- motif GARC (GA-Responsive Complex) that binds TFs of the MYB-R2R3, DOF and MYBR1-SHAQKYF families. In this thesis, the characterization of the BdCathB gene, encoding a Cathepsin B-like protease and that is ortholog to the wheat Al21 and the barley HvCathB genes, has been done and its transcriptional regulation by the TFs BdDOF24 and BdGAMYB (ortholog to HvGAMYB) studied. The in silico analysis of the BdCathB promoter sequence has identified a GARC motif. BdCathB expression is induced upon germination, as well as, those of BdDof24 and BdGamyb genes. Moreover, BdDOF24 and BdGAMYB interact in yeast (Yeast 2 Hybrid System, Y2HS) and in planta (Bimolecular Fluorecence Complementation, BiFC). In transient assays in aleurone cells, BdGAMYB activates the BdCathB promoter, whereas BdDOF24 is a transcriptional repressor, this result is similar to that obtained with the barley orthologous genes BPBF-HvDOF24 and HvGAMYB. However, when aleurone cells are simultaneously transformed with both TFs, BdDOF24 has an additive effect to the trans-activation mediated by BdGAMYB, while its ortholog BPBF-HvDOF24 produces an opposite effect by reducing the HvGAMYB activation of the BdCathB promoter. The differences among the deduced protein sequences between BdDOF24 and BPBF-HvDOF24 could explain their opposite functions in the interaction with GAMYB protein. Preliminary results of T-DNA insertion (K.O.) and stable over-expression lines of BdGamyb support the data obtained in transient expression assays. In addition, the BdGamyb homozygous T-DNA insertion (K.O.) lines have anther and pollen alterations and they do not produce viable seeds.

Respuesta transcripcional de Populus a bifenilos policlorados: Aspectos aplicados a la fitorremediaci��n

La degradaci��n del suelo ha adquirido una magnitud preocupante. Los m��todos tradicionales de descontaminaci��n, son costosos e insuficientes. La fitorremediaci��n representa una alternativa eficaz, de bajo coste, respetuosa con el medio ambiente, que adem��s mejora las propiedades del suelo, si bien ha habido desarrollos relevantes en la ��ltima d��cada. Desde el punto de vida cient��fico, el reto principal es descifrar las rutas metab��licas implicadas en respuesta a contaminantes y comprender su regulaci��n. Esta informaci��n es imprescindible si aspiramos a mejorar las capacidades naturales de algunas especies vegetales para remediar los suelos contaminados. Los estudios de esta Tesis se han centrado en Populus, el mejor modelo forestal disponible a ra��z de la secuenciaci��n de su genoma completo. Por otra parte, Populus tiene una gran capacidad natural para la degradaci��n de contaminantes org��nicos, lo que explica su predominio en los programas forestales de fitorremediaci��n que se desarrollan actualmente. Hemos elegido en concreto al h��brido Populus tremula x P. alba, por la facilidad con que se cultiva y su particular inter��s biotecnol��gico. La presente Tesis plantea un estudio comprehensivo de la respuesta molecular a bifenilos policlorados (PCBs), una familia de contaminantes org��nicos persistentes de particular relevancia a escala mundial. Se ha utilizado para ello una aproximaci��n transcript��mica, basada en tecnolog��a RNA-seq, para identificar los genes implicados en el metabolismo de los compuestos in planta y cuantificar sus niveles de activaci��n en distintas situaciones controladas. La tesis pretende asimismo definir el control transcripcional subyacente a la respuesta bioqu��mica frente a este tipo de contaminantes. Resulta sorprendente que dicha respuesta sea pr��cticamente desconocida a nivel molecular, a pesar de su gran potencial aplicado en el contexto de la tecnolog��a fitorremediadora. Para desarrollar este proyecto aplicamos a nuestros cultivos de chopo h��bridos concentraciones diferentes de Aroclor 1221, una mezcla de PCBs muy utilizada a nivel comercial durante d��cadas, su uso est�� prohibido hoy internacionalmente. Y tomamos muestras de RNA a dos concentraciones y dos momentos distintos de exposici��n al contaminante, generando as�� una matriz de cuatro elementos con sus controles correspondientes. Con el fin de incrementar la especificidad de nuestro an��lisis, consideramos sobre todo los genes diferencialmente expresados m��s significativos seg��n cuatro algoritmos estad��sticos distintos. Por otra parte, realizamos an��lisis funcionales con herramientas bioinform��ticas basadas en comparaciones de secuencias y en redes de co-expresi��n g��nica. La respuesta de los genes de particular inter��s fue validada mediante tecnolog��a qRT-PCR (reacci��n de la polimerasa en cadena cuantitativa en tiempo real). Se trata del primer estudio comprehensivo de la respuesta de un organismo vegetal ante la presencia de PCBs. Este estudio nos ha permitido identificar una cantidad considerable de genes estructurales y reguladores, definiendo nuevos factores de transcripci��n cuya expresi��n es proporcional a la concentraci��n de contaminante en el medio o al tiempo de exposici��n al mismo. Los an��lisis de correlaci��n nos permiten afirmar en que la respuesta metab��lica a PCBs, incluyendo posibles rutas degradadoras, participan en al menos quince factores de transcripci��n y unas cuarenta prote��nas o enzimas que resultan particularmente inducidas. Entre las familias implicadas destacan los citocromos P450, la glutati��n transferasas, las deshidrogenasas reductasas (short-chain dehydrogenase reductase) y las prote��nas MDR (multi-drug resistance). Mientras que los factores de transcripci��n encontrados pertenecen a la familia de ZF-TF, MYBs, WRKYs entre otros. Tambi��n identificamos prote��nas de funci��n desconocida que no se hab��an vinculado previamente a este tipo de respuestas en plantas, como la CSP (cold-shock domain proteins). Para estudiar su posible relaci��n con la presencia de PCBs, se caracteriz�� un gen de esta familia detectado mediante espectrometr��a de masas en t��ndem (MS/MS) a partir de mapas IEF x SDS-PAGE (isoelectro focusing x sodium dodecyl sulphate- polyacrylamide gel electrophoresis) de alta resoluci��n. Mediante qRT-PCR pudimos confirmar la inducci��n del gen correspondiente, ort��logo a PtCSP4 de P. trichocarpa (Potri.004g172600), en respuesta a Aroclor 1221. El an��lisis fenot��pico de las l��neas transg��nicas de Arabidopsis thaliana que sobre-expresaba la prote��na CSP de chopo h��brido confirm�� un papel para la misma tolerancia a PCBs, posiblemente a trav��s de mecanismos reguladores que activan prote��nas MDR. Este trabajo, adem��s de aportar datos novedosos sobre los mecanismos moleculares desencadenados por la presencia de un PCB en Populus, utilizado aqu�� como sistema modelo. Con ello se demuestra el potencial de las especies arb��reas no solo como agentes descontaminantes, ya explotado comercialmente, sino tambi��n como fuente potencial de genes interesantes. Entre los genes identificados en esta Tesis hay candidatos evidentes a participar en mecanismos de tolerancia al estr��s inducido por la contaminaci��n y tambi��n rutas metab��licas degradadores de PCBs. Precisamente la posibilidad de degradar al contaminante confiere particular inter��s a este tipo de estudios frente a la fitorremediaci��n de metales pesados y otros contaminantes elementales. La comparaci��n de los datos generados en este estudio con estudios an��logos que se realicen en el futuro con otras especies y xenobi��ticos, contribuir��n a definir mejor la respuesta de las plantas ante la contaminaci��n org��nica y mejorar su potencial descontaminante. ABSTRACT Soil degradation has acquired a disturbing magnitude. Traditional methods of decontamination are expensive and insufficient. Phytoremediation represent an effective alternative, low cost, respectful of the environment, that also improves soil properties, although there have been relevant developments in the last decade. From a life scientist, the challenge is to decipher the major metabolic pathways involved in response to pollutants and understand their regulation. This information is essential if we desire to enhance the natural abilities of some plant species to remediate contaminated soils. This thesis studies have focused on Populus, the best available forestry model following the sequencing of the entire genome. Moreover, Populus has a natural ability to degrade organic pollutants, which explains its predominance in phytoremediation forestry programs currently being developed. We have chosen specifically to hybrid Populus tremula x P. alba, the ease with which it is grown and its particular biotechnological interest. This thesis presents a comprehensive study of the molecular response to polychlorinated biphenyls (PCBs), a family of persistent organic pollutants of particular relevance worldwide. It has been used for a transcriptomic approach using RNA-seq technology, to identify genes involved in the metabolism of compounds in plant and quantify their levels of activation in different controlled situations. The thesis also aims to define the underlying transcriptional control the biochemical response to these pollutants. It is surprising that the response is virtually unknown at the molecular level, despite its great potential applied in the context of phytoremediation technology. To develop this project we applied our hybrid poplar crops different concentrations of Aroclor 1221, a mixture of PCBs widely used commercially for decades, its use is now banned internationally. And we RNA samples at two different concentrations and times of exposure to the pollutant, generating an array of four elements with their corresponding controls. In order to increase the specificity of our analysis, we consider mainly the most significant differentially expressed genes in four different statistical algorithms. Moreover, functional analyzes conducted with bioinformatics tools based on sequence comparisons and networks gene co-expression. The response of genes of particular interest was validated by qRT-PCR (polymerase reaction chain in real-time quantitative. This is the first comprehensive study of the response of a plant organism in the presence of PCBs. This study allowed us to identify a considerable amount of structural and regulatory genes, defining new transcription factors whose expression is proportional to the concentration of contaminant in the middle or at the time of exposure. Correlation analyzes allow us to affirm that the metabolic response to PCBs, including possible degradative pathways, at least fifteen involved in transcription factors and forty proteins or enzymes which are particularly induced. Among the families involved include cytochromes P450, the glutathione transferases, dehydrogenases reductases (short -chain dehydrogenase reductase) and MDR proteins (multi - drug resistance). While transcription factors belong to the family found ZF-TF, MYBs, WRKYs among others. We also identify proteins of unknown function that had not been previously linked to such responses in plants such as CSP (cold- shock domain proteins). To study their possible relationship with the presence of PCBs, a gene in this family was characterized and was detected by tandem mass spectrometry (MS/MS) from maps IEF x SDS -PAGE (sodium dodecyl isoelectro x sulphate- polyacrylamide gel electrophoresis) of high resolution. By qRT -PCR could confirm the induction of the corresponding gene, ortholog to PtCSP4 of P. trichocarpa (Potri.004g172600), in response to Aroclor 1221. Phenotypic analysis of transgenic Arabidopsis thaliana lines over- expressing the protein CSP poplar hybrid confirmed a role for PCBs same tolerance, possibly through regulatory mechanisms activated MDR proteins. This work, in addition to providing new data on the molecular mechanisms triggered by the presence of PCBs in Populus, used here as a model system. Thus the potential of tree species not only as decontamination agents, and commercially exploited, but also as a potential source of interesting genes is shown. Among the genes identified in this thesis there are evident candidates to participate in tolerance mechanisms to stress induced by pollution and degrading metabolic pathways of PCBs. Precisely the possibility of degrading the pollutant attaches particular interest to this type of study off the phytoremediation of heavy metals and other elemental pollutants. The comparison of the data generated in this study with similar studies carried out in the future with other species and xenobiotics contribute to better define the response of plants to organic pollution and improve their decontamination potential.