Classe de Ciências

info:eu-repo/semantics/publishedVersio

As Bio na Era Digital – Metodologias, Aspectos Técnicos e Éticos

info:eu-repo/semantics/publishedVersio

Classe de Ciências

info:eu-repo/semantics/publishedVersio

Classe de Ciências

info:eu-repo/semantics/publishedVersio

Genome restructuring in rye affects the expression, organization and disposition of homologous rDNA loci

Research ArticleThe standard rye cultivar ‘Imperial’ and a structural variant carrying an intact 1R chromosome and two telocentric 1R chromosomes (short and long arms) were used to investigate expression patterns of homologous rDNA loci, and the influence of chromosome structural change on their interphase organisation and relative disposition. Sequential silver staining and in situ hybridization with the rDNA probe pTa71, established a correspondence between the expression and organization patterns of rDNA domains in metaphase and interphase cells. In most cells of the cultivar Imperial, nucleolar organizer region (NOR) silver staining on metaphase chromosomes with equivalent numbers of rDNA genes revealed a size heteromorphism between homologousrDNA loci, resulting from their differential expression. NOR heteromorphism in the structural variant line was significantly reduced. The preferential activity of one NOR over its homologue was found to be random within cells and independent of parental origin. Nucleotypic modifications mediated by changes in the 1R chromosome structure include increased proximity between homologous rDNA loci in interphase, and an increase in the frequency of cells with intra-nucleolar ribosomal condensed chromatin. These results seem to indicate a ‘sequence recognition’ process for the regulation of homologous loci

Behind Brain Metastases Formation: Cellular and Molecular Alterations and Blood Brain Barrier Disruption

Breast cancer (BC) brain metastases is a life-threatening condition to which accounts the poor understanding of BC cells’ (BCCs) extravasation into the brain, precluding the development of preventive strategies. Thus, we aimed to unravel the players involved in the interaction between BCCs and blood–brain barrier (BBB) endothelial cells underlying BBB alterations and the transendothelial migration of malignant cells. We used brain microvascular endothelial cells (BMECs) as a BBB in vitro model, under conditions mimicking shear stress to improve in vivo-like BBB features. Mixed cultures were performed by the addition of fluorescently labelled BCCs to distinguish individual cell populations. BCC–BMEC interaction compromised BBB integrity, as revealed by junctional proteins (β-catenin and zonula occludens-1) disruption and caveolae (caveolin-1) increase, reflecting paracellular and transcellular hyperpermeability, respectively. Both BMECs and BCCs presented alterations in the expression pattern of connexin 43, suggesting the involvement of the gap junction protein. Myosin light chain kinase and phosphorylated myosin light chain were upregulated, revealing the involvement of the endothelial cytoskeleton in the extravasation process. β4-Integrin and focal adhesion kinase were colocalised in malignant cells, reflecting molecular interaction. Moreover, BCCs exhibited invadopodia, attesting migratory properties. Collectively, hub players involved in BC brain metastases formation were unveiled, disclosing possible therapeutic targets for metastases prevention.This work was funded by the Portuguese Foundation for Science and Technology (FCT), Portugal, grant numbers PTDC/MED-ONC/29402/2017, UIDP/04138/2020 and UIDB/04138/2020. We also acknowledge FCT financial support of J.G.-P. (SFRH/BD/145522/2019) and of A.R.G. (2020.07115.BD). We acknowledge the Faculty of Sciences of the University of Lisbon’s Microscopy Facility, a node of the Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122). We also acknowledge Luís Marques (Faculty of Sciences, University of Lisbon) for the excellent technical support with image acquisition.info:eu-repo/semantics/publishedVersio

An in‑planta comparative study of Plasmopara viticola proteome reveals diferent infection strategies towards susceptible and Rpv3‑mediated resistance hosts

Plasmopara viticola, an obligate biotrophic oomycete, is the causal agent of one of the most harmful grapevine diseases, downy mildew. Within this pathosystem, much information is gathered on the host, as characterization of pathogenicity and infection strategy of a biotrophic pathogen is quite challenging. Molecular insights into P. viticola development and pathogenicity are just beginning to be uncovered, mainly by transcriptomic studies. Plasmopara viticola proteome and secretome were only predicted based on transcriptome data. In this study, we have identified the in-planta proteome of P. viticola during infection of a susceptible ('Trincadeira') and a Rpv3-mediated resistance ('Regent') grapevine cultivar. Four hundred and twenty P. viticola proteins were identified on a label-free mass spectrometry-based approach of the apoplastic fluid of grapevine leaves. Overall, our study suggests that, in the compatible interaction, P. viticola manipulates salicylic-acid pathway and isoprenoid biosynthesis to enhance plant colonization. Furthermore, during the incompatible interaction, development-associated proteins increased while oxidoreductases protect P. viticola from ROS-associated plant defence mechanism. Up to our knowledge this is the first in-planta proteome characterization of this biotrophic pathogen, thus this study will open new insights into our understanding of this pathogen colonization strategy of both susceptible and Rpv3-mediated resistance grapevine genotypes.info:eu-repo/semantics/publishedVersio

Asymmetric localization of Arabidopsis SYP124 syntaxin at the pollen tube apical and sub-apical zones is involved in tip growth

<p>Abstract</p> <p>Background</p> <p>The continuous polarized vesicle secretion in pollen tubes is essential for tip growth but the location of endo- and exocytic sub-domains remains however controversial. In this report we aimed to show that <it>Arabidopsis thaliana </it>syntaxins are involved in this process and contribute to spatially define exocytosis and membrane recycling.</p> <p>Results</p> <p>Using GFP-fusion constructs, we imaged the distribution of pollen-specific (AtSYP124) and non-pollen syntaxins (AtSYP121 and AtSYP122) in transiently transformed <it>Nicotiana tabacum </it>pollen tubes. All three proteins associate with the plasma membrane and with apical vesicles indicating a conserved action mechanism for all SYPs. However, the GFP tagged SYP124 showed a specific distribution with a higher labelling at the plasma membrane flanks, 10-25 μm behind the apex. This distribution is affected by Ca²⁺fluxes as revealed by treatment with Gd³⁺(an inhibitor of extracellular Ca²⁺influx) and TMB-8 (an inhibitor of intracellular Ca²⁺release). Both inhibitors decreased growth rate but the distribution of SYP124 at the plasma membrane was more strongly affected by Gd³⁺. Competition with a related dominant negative mutant affected the specific distribution of SYP124 but not tip growth. In contrast, co-expression of the phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4) or of the small GTPase Rab11 perturbed polarity and the normal distribution of GFP-SYP but did not inhibit the accumulation in vesicles or at the plasma membrane.</p> <p>Conclusions</p> <p>The results presented suggest that in normal growing pollen tubes, a net exocytic flow occurs in the flanks of the tube apex mediated by SYP124. The specific distribution of SYP124 at the plasma membrane is affected by changes in Ca²⁺levels in agreement with the importance of this ion for exocytosis. Apical growth and the specific localization of SYP124 were affected by regulators of membrane secretion (Ca²⁺, PIP5K4 and Rab11) but competition with a dominant negative mutant affected only SYP distribution. These data thus suggest that syntaxins alone do not provide the level of specificity that is required for apical growth and that additional signalling and functional mechanisms are required.</p

Revisiting Vitis vinifera Subtilase Gene Family: A Possible Role in Grapevine Resistance against Plasmopara viticola

Subtilisin-like proteases, also known as subtilases, are a very diverse family of serine peptidases present in many organisms. In grapevine, there are hints of the involvement of subtilases in defense mechanisms, but their role is not yet understood. The first characterization of the subtilase gene family was performed in 2014. However, simultaneously, the grapevine genome was re-annotated and several sequences were re-annotated or retrieved. We have performed a re-characterization of this family in grapevine and identified 82 genes coding for 97 putative proteins, as result of alternative splicing. All the subtilases identified present the characteristic S8 peptidase domain and the majority of them also have a pro-domain I9 inhibitor, a protease-associated (PA) domain, and a signal peptide for targeting to the secretory pathway. Phylogenetic studies revealed six subtilase groups denominated VvSBT1 to VvSBT6. As several evidences have highlighted the participation of plant subtilases in response to biotic stimulus, we have investigated subtilase participation in grapevine resistance to Plasmopara viticola, the causative agent of downy mildew. Fourteen grapevine subtilases presenting either high homology to P69C from tomato, SBT3.3 from Arabidopsis thaliana or located near the Resistance to P. viticola (RPV) locus were selected. Expression studies were conducted in the grapevine-P. viticola pathosystem with resistant and susceptible cultivars. Our results may indicate that some of grapevine subtilisins are potentially participating in the defense response against this biotrophic oomycete.info:eu-repo/semantics/publishedVersio

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

<p>Abstract</p> <p>Background</p> <p>Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility.</p> <p>Results</p> <p>A plant cell suspension culture of <it>Medicago sativa </it>was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by <it>Medicago sativa </it>cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, <it>Medicago sativa </it>cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H₂DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation.</p> <p>Conclusions</p> <p>Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for <it>Medicago sativa </it>cells, a safe range of 1-5 nM should not be exceeded for biological applications.</p