Browsing by Author "Becker, Jörg D"
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- 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.
- Expression-based and co-localization detection of arabinogalactan protein 6 and arabinogalactan protein 11 interactors in Arabidopsis pollen and pollen tubesPublication . Costa, Mário; Nobre, Margarida Sofia; Becker, Jörg D; Masiero, Simona; Amorim, Maria Isabel; Pereira, Luís Gustavo; Coimbra, SílviaArabinogalactan proteins (AGPs) are cell wall proteoglycans that have been shown to be important for pollen development. An Arabidopsis double null mutant for two pollen-specific AGPs (agp6 agp11) showed reduced pollen tube growth and compromised response to germination cues in vivo. A microarray experiment was performed on agp6 agp11 pollen tubes to search for genetic interactions in the context of pollen tube growth. A yeast two-hybrid experiment for AGP6 and AGP11 was also designed.
- FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nucleiPublication . Borges, Filipe; Gardner, Rui; Lopes, Telma; Calarco, Joseph P; Boavida, Leonor C; Slotkin, R; Martienssen, Robert A; Becker, Jörg DBackground: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.
- Identification of differentially expressed genes in the heart precursor cells of the chick embryoPublication . Bento, Margaret; Correia, Elizabeth; Tavares, Ana T; Becker, Jörg D; Belo, José AGenetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their targets and pathways they are involved with, remain largely unknown. Previous studies in the avian embryo showed that at HH4 Cerberus (cCer) transcripts are detected in the anterior endomesoderm including the heart precursor cells and later in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression in a population of cells that consistently exit from the anterior primitive streak region, from as early as stage HH3+, and that later will populate the heart. Using this promoter element as a tool allowed us to identify novel genes previously not known to potentially play a role in heart development. In order to identify and study genes expressed and involved in the correct development and differentiation of the vertebrate heart precursor cell (HPC) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 700 transcripts differentially expressed in the heart forming regions (HFR). Bioinformatic tools allowed us to filter the large amount of data generated from this approach and to select a few transcripts for in vivo validation. Whole-mount in situ hybridization and sectioning of selected genes showed heart and vascular expression patterns for these transcripts during early chick development. We have developed an effective strategy to specifically identify genes that are differentially expressed in the HPC lineages. Within this set we have identified several genes that are expressed in the heart, blood and vascular lineages, which are likely to play a role in their development. These genes are potential candidates for future functional studies on early embryonic patterning.
- Inhibition of glutamine synthetase by phosphinothricin leads to transcriptome reprograming in root nodules of Medicago truncatulaPublication . Seabra, Ana R; Pereira, Patrícia A; Becker, Jörg D; Carvalho, Helena GGlutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.
- LegumeGRN: a gene regulatory network prediction server for functional and comparative studiesPublication . Wang, Mingyi; Verdier, Jerome; Benedito, Vagner A; Tang, Yuhong; Murray, Jeremy D; Ge, Yinbing; Becker, Jörg D; Carvalho, Helena; Rogers, Christian; Udvardi, Michael; He, JiBuilding accurate gene regulatory networks (GRNs) from high-throughput gene expression data is a long-standing challenge. However, with the emergence of new algorithms combined with the increase of transcriptomic data availability, it is now reachable. To help biologists to investigate gene regulatory relationships, we developed a web-based computational service to build, analyze and visualize GRNs that govern various biological processes. The web server is preloaded with all available Affymetrix GeneChip-based transcriptomic and annotation data from the three model legume species, i.e., Medicago truncatula, Lotus japonicus and Glycine max. Users can also upload their own transcriptomic and transcription factor datasets from any other species/organisms to analyze their in-house experiments. Users are able to select which experiments, genes and algorithms they will consider to perform their GRN analysis. To achieve this flexibility and improve prediction performance, we have implemented multiple mainstream GRN prediction algorithms including co-expression, Graphical Gaussian Models (GGMs), Context Likelihood of Relatedness (CLR), and parallelized versions of TIGRESS and GENIE3. Besides these existing algorithms, we also proposed a parallel Bayesian network learning algorithm, which can infer causal relationships (i.e., directionality of interaction) and scale up to several thousands of genes. Moreover, this web server also provides tools to allow integrative and comparative analysis between predicted GRNs obtained from different algorithms or experiments, as well as comparisons between legume species. The web site is available at http://legumegrn.noble.org.
- Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signaturePublication . Becker, Jörg D; Takeda, Seiji; Borges, Filipe; Dolan, Liam; Feijó, José ACurrent views on the control of cell development are anchored on the notion that phenotypes are defined by networks of transcriptional activity. The large amounts of information brought about by transcriptomics should allow the definition of these networks through the analysis of cell-specific transcriptional signatures. Here we test this principle by applying an analogue to comparative anatomy at the cellular level, searching for conserved transcriptional signatures, or conserved small gene-regulatory networks (GRNs) on root hairs (RH) and pollen tubes (PT), two filamentous apical growing cells that are a striking example of conservation of structure and function in plants.
- Transcriptomic comparison between two Vitis vinifera L. varieties (Trincadeira and Touriga Nacional) in abiotic stress conditionsPublication . Rocheta, Margarida; Coito, João L; Ramos, Miguel J N; Carvalho, Luísa; Becker, Jörg D; Carbonell-Bejerano, Pablo; Amâncio, SaraPredicted climate changes announce an increase of extreme environmental conditions including drought and excessive heat and light in classical viticultural regions. Thus, understanding how grapevine responds to these conditions and how different genotypes can adapt, is crucial for informed decisions on accurate viticultural actions. Global transcriptome analyses are useful for this purpose as the response to these abiotic stresses involves the interplay of complex and diverse cascades of physiological, cellular and molecular events. The main goal of the present work was to evaluate the response to diverse imposed abiotic stresses at the transcriptome level and to compare the response of two grapevine varieties with contrasting physiological trends, Trincadeira (TR) and Touriga Nacional (TN).