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- Influência do aumento da concentração do CO2 atmosférico e da temperatura do ar no desenvolvimento da cultura do caféPublication . Semedo, J.N.; Pais, I.P.; Rodrigues, A.P.; Leitão, A.E.; Ribeiro, A.; Dubberstein, D.; Partelli, F.L.; DaMatta, F. M.; Lidon, F.C.; Scotti-Campos, P.; Ramalho, J.C.O clima condiciona fortemente a produtividade agrícola e mesmo alterações moderadas a severas das condições ambientais poderão afetar a produção, levando a perdas económicas e impactos sociais. O previsível aumento da concentração de CO2 atmosférico, associado a alterações nos padrões de pluviosidade, ao aumento na duração e intensidade da seca, bem como a um aumento generalizado das temperaturas, são realidades cada vez mais presentes em todos os cenários agrícolas. As interações complexas entre estes diferentes fatores alterarão as respostas das plantas com potencial impacto acrescido na produtividade e qualidade dos produtos finais. O aumento da concentração atmosférica de diferentes gases com efeito de estufa, com destaque para o CO2, tem ocorrido em simultâneo com o aumento da temperatura do ar. Desde o início da revolução industrial no séc. XVIII, a concentração de CO2 aumentou de ca. 280 μL CO2 L-1, tendo ultrapassado 400 μL CO2 L-1 em 2013, sendo previsível que possa atingir valores entre 421 e 936 μL CO2 L-1 no final do século. Adicionalmente, previsões recentes para este século apontam para aumentos da temperatura ao nível da superfície do planeta que poderão ir de 0,3-1,7 ºC, até um extremo de 2,6-4,8 ºC. Este eventual aumento de temperatura levará a alterações drásticas nos teores de humidade do ar e consequentemente nos regimes de pluviosidade. Estas circunstâncias poderão promover condições de seca mais frequentes e extremas. Contrastando com o impacto negativo da redução da disponibilidade hídrica ou do aumento da temperatura, o aumento do valor de CO2 per se pode ter um papel positivo, pois estimula a produção. A cultura do café é uma das mais importantes culturas de rendimento do mundo, estando presente em mais de 80 países da região tropical e sendo suportada por 2 espécies, Coffea arabica L. (café tipo Arábica) e Coffea canephora Pierre ex A. Froehner, (café tipo Robusta). Neste contexto torna-se premente o estudo dos mecanismos (com destaque para os ecofisiológicos) envolvidos na aclimatação das plantas, a um ambiente em permanente mudança. Recentes projeções indicam perdas significativas da área de cultivo de Coffea sp (particularmente de C. arabica), mas estudos recentes mostraram que o aumento dos valores de CO2 na atmosfera têm um efeito claramente mitigador do impacto de temperaturas supra-óptimas, moderando os impactos antes estimados com base em modelos que não têm em linha de conta este efeito benéfico do CO2. O conhecimento proveniente de estudos multidisciplinares e a obtenção de indicadores ecofisiológicos auxiliará na seleção de indivíduos mais tolerantes e servirá de ferramentas para o melhoramento de novas plantas com uma maior capacidade de adaptação.
- Antioxidant and antimicrobial potential of the Bifurcaria bifurcata epiphytic bacteriaPublication . Horta, André; Pinteus, Susete; Alves, Celso; Fino, Nádia; Silva, Joana; Fernandez, Sara; Rodrigues, Américo; Pedrosa, RuiSurface-associated marine bacteria are an interesting source of new secondary metabolites. The aim of this study was the isolation and identification of epiphytic bacteria from the marine brown alga, Bifurcaria bifurcata, and the evaluation of the antioxidant and antimicrobial activity of bacteria extracts. The identification of epiphytic bacteria was determined by 16S rRNA gene sequencing. Bacteria extracts were obtained with methanol and dichloromethane (1:1) extraction. The antioxidant activity of extracts was performed by quantification of total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Antimicrobial activities were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans. A total of 39 Bifurcaria bifurcata-associated bacteria were isolated and 33 were identified as Vibrio sp. (48.72%), Alteromonas sp. (12.82%), Shewanella sp. (12.26%), Serratia sp. (2.56%), Citricoccus sp. (2.56%), Cellulophaga sp. (2.56%), Ruegeria sp. (2.56%) and Staphylococcus sp. (2.56%). Six (15.38%) of the 39 bacteria Bifurcaria bifurcata-associated bacteria presented less than a 90% Basic Local Alignment Search Tool (BLAST) match, and some of those could be new. The highest antioxidant activity and antimicrobial activity (against B. subtilis) was exhibited by strain 16 (Shewanella sp.). Several strains also presented high antimicrobial activity against S. aureus, mainly belonging to Alteromonas sp. and Vibrio sp. There were no positive results against fungi and Gram-negative bacteria. Bifurcaria bifurcata epiphytic bacteria were revealed to be excellent sources of natural antioxidant and antimicrobial compounds.
- A dual function of SnRK2 kinases in the regulation of SnRK1 and plant growthPublication . Belda-Palazón, Borja; Adamo, Mattia; Valerio, Concetta; Ferreira, Liliana J.; Confraria, Ana; Reis-Barata, Elena; Rodrigues, Américo; Meyer, Christian; Rodriguez, Pedro L.; Baena-González, ElenaAdverse environmental conditions trigger responses in plants that promote stress tolerance and survival at the expense of growth1. However, little is known of how stress signalling pathways interact with each other and with growth regulatory components to balance growth and stress responses. Here, we show that plant growth is largely regulated by the interplay between the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1) protein kinase and the abscisic acid (ABA) phytohormone pathway. While SnRK2 kinases are main drivers of ABA-triggered stress responses, we uncover an unexpected growth-promoting function of these kinases in the absence of ABA as repressors of SnRK1. Sequestration of SnRK1 by SnRK2 containing complexes inhibits SnRK1 signalling, thereby allowing target of rapamycin (TOR) activity and growth under optimal conditions. On the other hand, these complexes are essential for releasing and activating SnRK1 in response to ABA, leading to the inhibition of TOR and growth under stress. This dual regulation of SnRK1 by SnRK2 kinases couples growth control with environmental factors typical for the terrestrial habitat and is likely to have been critical for the water-to-land transition of plants.
- In vitro break of dormancy of axillary buds from woody species (Persea indica and Arbutus unedo) by sectioning with a laser beamPublication . Rodrigues, Américo; Sérgio, Pedro M.; Teixeira, Manuel R.; Pais, Maria S.The effect of laser cut of explants on the break of dormancy of axillary buds and the development of micropropagated Persea indica and Arbutus unedo nodal segments was investigated. A continuous CO2 wave laser was used. A regular cut was obtained with a laser power of 75 W and a corresponding cutting speed of 0.01 m s−1. Using these parameters, the surfaces of the segments were cut consistently with little or no disruption of the neighboring cells and tissues, in contrast to the irregular cut made with a surgical blade, especially at the level of vascular vessels. For both species studied, when the sectioning was performed in November or December, the laser cut promoted break of dormancy of axillary buds and a faster growth of the resulting shoots. When sectioning was carried out in February, the laser cut had little or no effect on axillary bud development.
- Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteinsPublication . Park, Sang-Youl; Fung, Pauline; Nishimura, Noriyuki; Jensen, Davin R.; Fujii, Hiroaki; Zhao, Yang; Lumba, Shelley; Santiago, Julia; Rodrigues, Américo; Chow, Tsz-fung F.; Alfred, Simon E.; Bonetta, Dario; Finkelstein, Ruth; Provart, Nicholas J.; Desveaux, Darrell; Rodriguez, Pedro L.; McCourt, Peter; Zhu, Jian-Kang; Schroeder, Julian I.; Volkman, Brian F.; Cutler, Sean R.Type 2C protein phosphatases (PP2Cs) are vitally involved in abscisic acid (ABA) signaling. Here, we show that a synthetic growth inhibitor called pyrabactin functions as a selective ABA agonist. Pyrabactin acts through PYRABACTIN RESISTANCE 1 (PYR1), the founding member of a family of START proteins called PYR/PYLs, which are necessary for both pyrabactin and ABA signaling in vivo. We show that ABA binds to PYR1, which in turn binds to and inhibits PP2Cs. We conclude that PYR/PYLs are ABA receptors functioning at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting PP2Cs. Our results illustrate the power of the chemical genetic approach for sidestepping genetic redundancy.
- HAB1–SWI3B interaction reveals a link between abscisic acid signaling and putative SWI/SNF chromatin-remodeling complexes in arabidopsisPublication . Saez, Angela; Rodrigues, Américo; Santiago, Julia; Rubio, Silvia; Rodriguez, Pedro L.Abscisic acid (ABA) has an important role for plant growth, development, and stress adaptation. HYPERSENSITIVE TO ABA1 (HAB1) is a protein phosphatase type 2C that plays a key role as a negative regulator of ABA signaling; however, the molecular details of HAB1 action in this process are not known. A two-hybrid screen revealed that SWI3B, an Arabidopsis thaliana homolog of the yeast SWI3 subunit of SWI/SNF chromatin-remodeling complexes, is a prevalent interacting partner of HAB1. The interaction mapped to the N-terminal half of SWI3B and required an intact protein phosphatase catalytic domain. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the interaction of HAB1 and SWI3B in the nucleus of plant cells. swi3b mutants showed a reduced sensitivity to ABA-mediated inhibition of seed germination and growth and reduced expression of the ABA-responsive genes RAB18 and RD29B. Chromatin immunoprecipitation experiments showed that the presence of HAB1 in the vicinity of RD29B and RAB18 promoters was abolished by ABA, which suggests a direct involvement of HAB1 in the regulation of ABA-induced transcription. Additionally, our results uncover SWI3B as a novel positive regulator of ABA signaling and suggest that HAB1 modulates ABA response through the regulation of a putative SWI/SNF chromatin-remodeling complex.
- RGA & GAI: On the characterization of two DELLA plant growth repressor proteinsPublication . Vaz, Daniela C.; Sousa, Raquel; Simões, Marco; Rodrigues, AméricoDELLA proteins are a family of nuclear proteins responsible for plant growth modulation. They act as growth repressor proteins in response to gibberellin signaling pathways. Five DELLA protein homologs were found in Arabidopsis thaliana, namely, RGA (Repressor of Gibberellic Acid), GAI (Gibberellic Acid Insensitive) and three RGA-like proteins (RGL-1, RGL-2 and RGL-3). The RGA- DELLA and GAI-DELLA protein homologs have been classified as Intrinsically Unstructured Proteins (IUPs) that undergo a disorder-to-order transition upon receptor binding. This structural change has found to be physiologically relevant for biological signaling and molecular recognition. Thus, in order to better characterize the structural features and molecular changes that govern these conformational variations of the polypeptide chain, we have produced recombinant RGADELLA and GAI-DELLA proteins in three length-versions, i.e. full-length, N-terminal and C-terminal versions. Full-length and terminal versions present different sequence motifs, attributed to different biological functions. All proteins were analyzed spectro-photometrically, via Light Scattering (LS), Circular Dichroism (CD) and intrinsic and extrinsic fluorescence (ANS binding), in order to compare spectral profiles, secondary structure propensities, levels of solvent exposure and structural compactness. Full-length and terminal variants exhibit different behaviors, spectral profiles and levels of compactness that can be related to different protein domains and ultimately to different functional implications.
- Marine invasive species for high-value products' exploration: Unveiling the antimicrobial potential of Asparagopsis armata against human pathogensPublication . Pinteus, Susete; Lemos, Marco F.L.; Simões, Marco; Alves, Celso; Silva, Joana; Gaspar, Helena; Martins, Alice; Rodrigues, Américo; Pedrosa, RuiInfectious diseases remain one of the leading global causes of death, mainly due to the increasing number of multi-resistant microorganisms. Therefore, novel antimicrobials are continuously needed. Marine organisms have already proven to be a rich source of bioactive compounds which can be used for the development of novel pharmacological drugs. Within these, seaweeds are an important resource still underexplored. Asparagopsis armata is a marine invasive seaweed that has spread along several coastlines of the world, inducing negative pressures in marine ecosystems. Therefore, it is crucial to develop strategies to counteract their impacts. In this work, A. armata was extracted with methanol and dichloromethane (1:1) and fractionated by column chromatography with different polarity solvents, providing 8 fractions (F1-F8). All the fractions were evaluated for their antimicrobial potential against important human pathogens, namely Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, and Candida albicans. Additionally, their capacity to inhibit biofilms formation and the effects on cytoplasmatic membrane and DNA integrity were also assessed. The most active fractions were further purified and submitted to GC–MS analysis. The results showed that A. armata produces compounds with a high inhibitory activity against S. aureus, P. aeruginosa, and C. albicans growth, possibly mediated by cytoplasmatic membrane disruption and DNA damage. GC–MS analysis suggested that the most active fractions were mainly composed of bromoditerpenes and fatty acids. The attained results point to the relevance of the invasive A. armata as a source of antimicrobial substances with broad-spectrum activity. The use of invasive species to obtain natural bioactive compounds presents a two-folded opportunity - high availability of the biological material for the extraction of bioactive compounds and, through specimen collection, the mitigation of the negative effects caused by invasive species, contributing to ecosystem integrity and sustainability.
- Loliolide, a new therapeutic option for neurological diseases? In vitro neuroprotective and anti-inflammatory activities of a monoterpenoid lactone isolated from codium tomentosumPublication . Silva, Joana; Alves, Celso; Martins, Alice; Susano, Patrícia; Simões, Marco; Guedes, Miguel; Rehfeldt, Stephanie; Pinteus, Susete; Gaspar, Helena; Rodrigues, Américo; Goettert, Márcia Inês; Alfonso, Amparo; Pedrosa, RuiParkinsons Disease (PD) is the second most common neurodegenerative disease worldwide, and is characterized by a progressive degeneration of dopaminergic neurons. Without an effective treatment, it is crucial to find new therapeutic options to fight the neurodegenerative process, which may arise from marine resources. Accordingly, the goal of the present work was to evaluate the ability of the monoterpenoid lactone Loliolide, isolated from the green seaweed Codium tomentosum, to prevent neurological cell death mediated by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y cells and their anti-inflammatory effects in RAW264.7 macrophages. Loliolide was obtained from the diethyl ether extract, purified through column chromatography and identified by NMR spectroscopy. The neuroprotective effects were evaluated by the MTT method. Cells’ exposure to 6-OHDA in the presence of Loliolide led to an increase of cells’ viability in 40%, and this effect was mediated by mitochondrial protection, reduction of oxidative stress condition and apoptosis, and inhibition of the NF-KB pathway. Additionally, Loliolide also suppressed nitric oxide production and inhibited the production of TNF- and IL-6 pro-inflammatory cytokines. The results suggest that Loliolide can inspire the development of new neuroprotective therapeutic agents and thus, more detailed studies should be considered to validate its pharmacological potential.
- Identification of Asparagopsis armata‐associated bacteria and characterization of their bioactive potentialPublication . Alves, Celso; Pinteus, Susete; Lopes, Cláudia; Fino, Nádia; Silva, Joana; Ribeiro, Joana; Rodrigues, Daniel; Francisco, João; Rodrigues, Américo; Pedrosa, Rui; Horta, AndréMacroalgae‐associated bacteria have already proved to be an interesting source of compounds with therapeutic potential. Accordingly, the main aim of this study was to characterize Asparagopsis armata‐associated bacteria community and evaluate their capacity to produce substances with antitumor and antimicrobial potential. Bacteria were selected according to their phenotype and isolated by the streak plate technique. The identification was carried out by the RNA ribosomal 16s gene amplification through PCR techniques. The antimicrobial activities were evaluated against seven microorganisms (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae, Candida albicans) by following their growth through spectrophotometric readings. Antitumor activities were evaluated in vitro on human cell lines derived from hepatocellular (HepG‐2) and breast carcinoma (MCF‐7) using the MTT method. The present work identified a total of 21 bacteria belonging to the genus Vibrio, Staphylococcus, Shewanella, Alteromonadaceae, Bacillus, Cobetia, and Photobacterium, with Vibrio being the most abundant (42.86%). The extract of Shewanella sp. ASP 26 bacterial strain induced the highest antimicrobial activity, namely against Bacillus subtilis and Staphylococcus aureus with an IC50 of 151.1 and 346.8 μg/mL, respectively. These bacteria (Shewanella sp.) were also the ones with highest antitumor potential, demonstrating antiproliferative activity on HepG‐2 cells. Asparagopsis armata‐associated bacteria revealed to be a potential source of compounds with antitumor and antibacterial activity.