Plant Genomics
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Our group is interested in the mechanisms underlying sexual reproduction and early embryogenesis with a particular focus on the role of the male gametes. Contrary to previous assumptions recent studies have shown that male gametes both in the plant and animal kingdom carry complex sets of RNA molecules, including not only mRNAs but also small RNAs. We are particularly interested in the role of these two RNA classes before, during and after double fertilization as it occurs in higher plants. Using Arabidopsis thaliana as our primary experimental model we are addressing specific questions like: (1) What are the functions of small RNA and DNA methylation pathways in sperm cells? (2) Do sperm cell derived RNAs play a role after fertilization? (3) Do conserved core sets of genetic modules underlie common characteristics of male gametes across kingdoms?. In a second research line we are analyzing the symbiotic transcriptome of the model legume Medicago truncatula, both for nodule and mycorrhizal symbiosis, leading to the creation of a gene expression database that will allow the in-depth analysis of the symbiontic genetic programme.
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Funding
Fundação para a Ciência e a Tecnologia
(FCT) Research Grant (PTDC/AGR-GPL/103778/2008)
Collaborators
Rob Martienssen, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
Keith Slotkin, Ohio State University, Columbus, OH, USA
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- Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium melilotiPublication . Santos, Mario R.; Cosme, Ana M.; Becker, Jorg D.; Medeiros, Joao M C.; Mata, Marcia F.; Moreira, Leonilde M.The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell.
- Arabidopsis Tetraspanins Are Confined to Discrete Expression Domains and Cell Types in Reproductive Tissues and Form Homo- and Heterodimers When Expressed in YeastPublication . Boavida, L. C.; Qin, P.; Broz, M.; Becker, J. D.; McCormick, S.Tetraspanins are evolutionary conserved transmembrane proteins present in all multicellular organisms. In animals, they are known to act as central organizers of membrane complexes and thought to facilitate diverse biological processes, such as cell proliferation, movement, adhesion, and fusion. The genome of Arabidopsis (Arabidopsis thaliana) encodes 17 members of the tetraspanin family; however, little is known about their functions in plant development. Here, we analyzed their phylogeny, protein topology, and domain structure and surveyed their expression and localization patterns in reproductive tissues. We show that, despite their low sequence identity with metazoan tetraspanins, plant tetraspanins display the typical structural topology and most signature features of tetraspanins in other multicellular organisms. Arabidopsis tetraspanins are expressed in diverse tissue domains or cell types in reproductive tissues, and some accumulate at the highest levels in response to pollination in the transmitting tract and stigma, male and female gametophytes and gametes. Arabidopsis tetraspanins are preferentially targeted to the plasma membrane, and they variously associate with specialized membrane domains, in a polarized fashion, to intercellular contacts or plasmodesmata. A membrane-based yeast (Saccharomyces cerevisiae) two-hybrid system established that tetraspanins can physically interact, forming homo- and heterodimer complexes. These results, together with a likely genetic redundancy, suggest that, similar to their metazoan counterparts, plant tetraspanins might be involved in facilitating intercellular communication, whose functions might be determined by the composition of tetraspanin complexes and their binding partners at the cell surface of specific cell types.
- BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathwaysPublication . Marsch-Martinez, Nayelli; Greco, Raffaella; Becker, Jorg D.; Dixit, Shital; Bergervoet, Jan H. W.; Karaba, Aarati Aarati; de Folter, Stefan; Pereira, AndyThe BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leaves
- Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root NodulesPublication . Limpens, Erik; Moling, Sjef; Hooiveld, Guido; Pereira, Patrícia A.; Bisseling, Ton; Becker, Jörg D.; Küster, HelgeLegumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital "in situ". This digital "in situ" offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.
- Comparative analysis of transcriptomic responses to sub-lethal levels of six environmentally relevant pesticides in Saccharomyces cerevisiaePublication . Gil, Fátima N.; Gonçalves, Alina C.; Becker, Jörg D.; Viegas, Cristina A.Accidental spills and misuse of pesticides may lead to current and/or legacy environmental contamination and may pose concerns regarding possible risks towards non-target microbes and higher eukaryotes in ecosystems. The present study was aimed at comparing transcriptomic responses to effects of sub-lethal levels of six environmentally relevant pesticide active substances in the Saccharomyces cerevisiae eukaryotic model. The insecticide carbofuran, the fungicide pyrimethanil and the herbicides alachlor, S-metolachlor, diuron and methyl(4-chloro-2-methylphenoxy)acetate were studied. Some are currently used agricultural pesticides, while others are under restricted utilization or banned in Europe and/or North America albeit being used in other geographical locations. In the present work transcriptional profiles representing genome-wide responses in a standardized yeast population upon 2 h of exposure to concentrations of each compound exerting equivalent toxic effects, i.e., inhibition of growth by 20% relative to the untreated control cells, were examined. Hierarchical clustering and Venn analyses of the datasets of differentially expressed genes pointed out transcriptional patterns distinguishable between the six active substances. Functional enrichment analyses allowed predicting mechanisms of pesticide toxicity and response to pesticide stress in the yeast model. In general, variations in transcript numbers of selected genes assessed by Real-Time quantitative reverse transcription polymerase chain reaction confirmed microarray data and correlated well with growth inhibitory effects. A possible biological relevance of mechanistic predictions arising from these comparative transcriptomic analyses is discussed in the context of better understanding potential modes of action and adverse side-effects of pesticides.
- Comparative Transcriptomic Analysis of the Burkholderia cepacia Tyrosine Kinase bceF Mutant Reveals a Role in Tolerance to Stress, Biofilm Formation, and VirulencePublication . Ferreira, A. S.; Silva, I. N.; Oliveira, V. H.; Becker, J. D.; Givskov, M.; Ryan, R. P.; Fernandes, F.; Moreira, L. M.The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.
- Comparative transcriptomics of Arabidopsis sperm cellsPublication . Borges, Filipe Filipe; Gomes, Gabriela; Gardner, Rui; Moreno, Nuno; McCormick, Sheila; Feijo, Jose A. Jose A.; Becker, Joeg D Joeg Dn flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins
- Cyclooxygenase-2 (COX-2) expression by in vitro produced bovine embryos. Preliminary results = Expressão da ciclo-oxigenase-2 (COX-2) em embriões bovinos produzidos in vitro. Resultados preliminaresPublication . Marques, C.C.; Horta, A.E.; Vasques, M.I.; Baptista, M.C.; Becker, J.D.; Pimenta, J.; Pereira, R.M.Expression of the two isoforms of cyclooxygenase enzyme, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), by in vitro produced bovine embryos was examined. Cumulus-oocyte complexes were recovered from ovaries of slaughtered animals and subsequently in vitro matured and in vitro fertilized. Presumptive zygotes were cultured in serum containing medium (TCM 199+10% bovine superovulated oestrus serum) on a granulosa cell monolayer for 12 days.
- Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodellingPublication . Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G.; Becker, Jörg D.; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G.Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.
- Elucidating how the saprophytic fungus Aspergillus nidulans uses the plant polyester suberin as carbon sourcePublication . Martins, Isabel; Hartmann, Diego O; Alves, Paula C; Martins, Celso; Garcia, Helga; Leclercq, Céline C; Ferreira, Rui; He, Ji; Renaut, Jenny; Becker, Jörg D; Silva Pereira, CristinaLipid polymers in plant cell walls, such as cutin and suberin, build recalcitrant hydrophobic protective barriers. Their degradation is of foremost importance for both plant pathogenic and saprophytic fungi. Regardless of numerous reports on fungal degradation of emulsified fatty acids or cutin, and on fungi-plant interactions, the pathways involved in the degradation and utilisation of suberin remain largely overlooked. As a structural component of the plant cell wall, suberin isolation, in general, uses harsh depolymerisation methods that destroy its macromolecular structure. We recently overcame this limitation isolating suberin macromolecules in a near-native state.