Alginate as a feature of osmotolerance differentiation among soil bacteria isolated from wild legumes growing in Portugal

Plants are naturally colonized by bacteria that can exert beneficial effects on growth and stress tolerance. These bacteria can be used as inoculants to boost crop productivity and plants resilience, and can be especially interesting if they are able to survive to abiotic stresses, such as drought. Herein we report the mechanisms that soil bacteria resort to tolerate drought and we also explore the influence of each mechanism to the level of drought tolerance exhibited, in order to test the hypothesis that different levels of tolerance displayed by bacteria are linked to differential efficiency of osmotolerance mechanisms. For this, the biochemical and physiological responses of bacterial strains of different genera and displaying different levels of tolerance to osmotic stress (sensitive, moderately tolerant and tolerant) induced by polyethylene glycol-6000 (PEG) were studied. Betaine, trehalose and alginate content increased in the majority of the strains exposed to PEG. Betaine was the osmolyte with higher increases, evidencing the important role of this compound in the tolerance of bacteria to drought. However, betaine and trehalose levels were not significantly different among bacteria with different osmotolerance levels. Several biochemical endpoints (protein content, superoxide dismutase, catalase, glutathione-S-transferases) related to oxidative stress were assessed, since oxidative damage has been reported in drought conditions, but little information is available. The oxidative stress parameters were not sufficient to explain differences in the osmotolerance observed for the tested strains. In contrast, alginate showed significant differences among the three levels of osmotolerance, linking the level of osmotolerance with the ability of soil bacteria to synthesize and accumulate alginate intracellularly for the first time. Moreover, our results show that this ability is present in different bacteria genera. Thus, evaluating the ability to synthesize alginate might be an important cue when considering bacterial inoculants for osmotically stressful conditions.publishe

Impacts of the combined exposure to seawater acidification and arsenic on the proteome of Crassostrea angulata and Crassostrea gigas

Proteomic analysis was performed to compare the effects of Arsenic (As), seawater acidification (Low pH) and the combination of both stressors (Low pH + As) on Crassostrea angulata and Crassostrea gigas juveniles in the context of global environmental change. This study aimed to elucidate if two closely related Crassostrea species respond similarly to these environmental stressors, considering both single and combined exposures, to infer if the simultaneous exposure to both stressors induced a differentiated response. Identification of the most important differentially expressed proteins between conditions revealed marked differences in the response of each species towards single and combined exposures, evidencing species-related differences towards each experimental condition. Moreover, protein alterations observed in the combined exposure (Low pH + As) were substantially different from those observed in single exposures. Identified proteins and their putative biological functions revealed an array of modes of action in each condition. Among the most important, those involved in cellular structure (Actin, Atlastin, Severin, Gelsolin, Coronin) and extracellular matrix modulation (Ependymin, Tight junction ZO-1, Neprilysin) were strongly regulated, although in different exposure conditions and species. Data also revealed differences regarding metabolic modulation capacity (ATP β, Enolase, Aconitate hydratase) and oxidative stress response (Aldehyde dehydrogenase, Lactoylglutathione, Retinal dehydrogenase) of each species, which also depended on single or combined exposures, illustrating a different response capacity of both oyster species to the presence of multiple stressors. Interestingly, alterations of piRNA abundance in C. angulata suggested genome reconfiguration in response to multiple stressors, likely an important mode of action related to adaptive evolution mechanisms previously unknown to oyster species, which requires further investigation. The present findings provide a deeper insight into the complexity of C. angulata and C. gigas responses to environmental stress at the proteome level, evidencing different capacities to endure abiotic changes, with relevance regarding the ecophysiological fitness of each species and competitive advantages in a changing environment.Centro-01-0145-FEDER-000018info:eu-repo/semantics/publishedVersio

Assessing Cu impacts on freshwater diatoms: biochemical and metabolomic responses of Tabellaria flocculosa (Roth) Kutzing

Metals are a recognised threat to aquatic organisms but the impact of metals such as copper (Cu) on benthic freshwater diatoms is poorly understood, even if diatoms are commonly used as water quality indicators. Our study aimed to elucidate the cellular targets of Cu toxicity and the mechanisms cells resort to counteract toxicity and to increase tolerance to Cu. A concerted approach analysing the biochemical, physiological and metabolome alterations in diatom cells was conducted by exposing the freshwater diatom Tabellaria flocattosa to 0, 0.3, 6 and 10 mu g Cu/L. Cu was already toxic to T. flocculosa at concentrations common in environments and which are not usually considered to be contaminated (0.3 mu g Cu/L). Under Cu impact, the metabolome of T. flocculosa changed significantly, especially at high concentrations (6 and 10 mu g Cu/L). Cu toxicity was counteracted by increasing extracellular immobilization (EPS, frustulins), antioxidant (SOD, CAT) and detoxifying (GSTs) enzymes activity and low molecular weight antioxidants (GSH). These mechanisms were fuelled by higher energy production (increased ETS activity). At the highest Cu concentration (10 mu g/L), these processes were specially enhanced in an attempt to restrain the oxidative stress generated by high intracellular Cu concentrations. However, these mechanisms were not able to fully protect cells, and damage in membranes and proteins increased. Moreover, the decrease of hydroxylamine and unsaturated fatty acids and the increase of saturated fatty acids, 2-palmiloylglycerol, glycerol and diterpenoid compounds should be tested as new specific markers of Cu toxicity in future studies. This information can support the prediction of diatom behaviour in different Cu contamination levels, including highly impacted environments, such as mining scenarios, and may assist in environmental risk assessment policies and restoration programs. (C) 2018 Elsevier B.V. All rights reserved

Airborne exposure of Rhizobium leguminosarum strain E20-8 to volatile monoterpenes: effects on cells challenged by cadmium

Volatile organic compounds (VOCs) are produced by plants, fungi, bacteria and animals. These compounds are metabolites originated mainly in catabolic reactions and can be involved in biological processes. In this study, the airborne effects of five monoterpenes (α-pinene, limonene, eucalyptol, linalool, and menthol) on the growth and oxidative status of the rhizobial strain Rhizobium leguminosarum E20-8 were studied, testing the hypothesis that these VOCs could influence Rhizobium growth and tolerance to cadmium. The tested monoterpenes were reported to have diverse effects, such as antibacterial activity (linalool, limonene, α-pinene, eucalyptol), modulation of antioxidant response or antioxidant properties (α-pinene and menthol). Our results showed that non-stressed cells of Rhizobium E20-8 have different responses (growth, cell damage and biochemistry) to monoterpenes, with α-pinene and eucalyptol increasing colonies growth. In stressed cells the majority of monoterpenes failed to minimize the detrimental effects of Cd and increased damage, decreased growth and altered cell biochemistry were observed. However, limonene (1 and 100 mM) and eucalyptol (100 nM) were able to increase the growth of Cd-stressed cells. Our study evidences the influence at-a-distance that organisms able to produce monoterpenes may have on the growth and tolerance of bacterial cells challenged by different environmental conditions.publishe

Aspectos da tolerância salina em Pisum sativum L.: influência da nutrição azotada

Doutoramento em BiologiaOs habitats salinos são caracterizados pela presença de quantidades de sais solúveis que são prejudiciais ao desenvolvimento da maioria das plantas. Cerca de 7% da superfície total de terra contém solos afectados pela salinidade. A tolerância salina das plantas baseia-se na manutenção de baixas concentrações citoplasmáticas, uma vez que o seu metabolismo é afectado pela presença de iões de Na+ e Cl- em excesso. Os mecanismos que as plantas utilizam para manterem as concentrações iónicas baixas no citoplasma são diversos. Existe consenso sobre quais as características fisiológicas a abordar para a obtenção de maior tolerância salina, o transporte iónico e a sua compartimentação, a síntese de solutos compatíveis, a protecção dos efeitos nocivos induzidos por condições oxidativas e os processos metabólicos considerados mais sensíveis, são frequentemente referidos A maioria das leguminosas é bastante sensível ao sal, tendência que aumenta se estiverem dependentes da fixação simbiótica de azoto atmosférico. É , geralmente, aceite que o estabelecimento e a fixação de N2 são os processos mais afectados pela salinidade. A maioria das tentativas de aumentar a tolerância salina em leguminosas não resultaram em variedades com uma tolerância salina demarcada, talvez porque os mecanismos subjacentes à tolerância salina ainda não estejam completamente compreendidos. A idealização deste trabalho teve como principais preocupações a constatação da existência de variabilidade genotípica na tolerância à salinidade em Pisum sativum e a avaliação da vulnerabilidade salina da simbiose (capítulo II); a obtenção e a selecção de genótipos de Rhizobium que sejam capazes de estabelecer uma simbiose efectiva sob condições salinas (capítulo III); a percepção da influência da nutrição azotada na tolerância salina de Pisum sativum ao longo do seu desenvolvimento, bem como a identificação dos mecanismos subjacentes a essa tolerância (capítulo IV); e a análise dos efeitos da salinidade e da nutrição azotada na composição proteica da semente (capítulo V). A avaliação da tolerância salina de quatro cultivares de Pisum sativum (‘Resal’, ‘Tristar’, ‘Combi’ e ‘Rauel’) evidenciou diferenças entre elas. ‘Resal’ aparece como a cultivar mais indicada para o cultivo sob condições salinas. A presença de 90 mM NaCl não afectou significativamente o seu crescimento vegetativo. No entanto, 90 mM NaCl provocou uma redução elevada no número de nódulos. Com o int uito de aumentar a obtenção de azoto simbioticamente fixado sob condições salinas, procedeu-se ao isolamento e à selecção de Rhizobium em diversos locais. Os isolados de S. Bernardo, Costa Nova e Vagos evidenciaram sensibilidade salina, uma vez que o crescimento in vitro a 100 mM NaCl provocou inibições de crescimento superiores a 70 %. Apenas os isolados provenientes de um local que durante parte do ano está sujeito a “stress” hídrico (Elvas) evidenciaram tolerância ao sal. Em alguns isolados de Elvas, o crescimento diminui menos de 30% em meio suplementado com 700 mM NaCl em comparação com o controlo,. A avaliação da eficiência de simbiose entre alguns isolados ou estirpes de Rhizobium e Pisum sativum abriu boas perspectivas para o estabelecimento de uma simbiose efectiva sob condições de salinidade moderada, devido à obtenção de três isolados que, simultaneamente, toleraram salinidades de 400 mM e propiciaram o maior crescimento da parte aérea. A forma de azoto disponibilizada influenciou o crescimento e a tolerância salina de Pisum sativum. A produtividade foi o parâmetro que melhor evidenciou essa influência. As plantas dependentes do nitrato foram as que demonstraram menor diminuição da produtividade. As diferenças de tolerância observadas só conseguem ser explicadas pela existência simultânea de mais do que um mecanismo, cujas intensidades parecem variar ao longo do tempo. À osmoregulação, conseguida parcialmente pela acumulação de Na+, Cl- e K+, associa-se a exclusão de Na+ da parte aérea. Embora a síntese proteica seja reconhecida como uma actividade celular sensível ao sódio, com excepção das plantas dependentes da fixação simbiótica de N2, a salinidade provocou o aumento do conteúdo proteico nas sementes. Facto que não será alheio à manutenção de baixas concentrações de Na + e Cl- neste órgão. Os perfis dos polipeptídicos mais abundantes não foram muito influenciados pelas condições de crescimento impostas. Contudo, a salinidade induziu o aparecimento de novos polipeptídeos que poderão conferir a este órgão maior tolerância ao aumento de níveis de Na+ e Cl- detectados nas plantas a crescer sob 90 mM NaCl. Em conclusão, este trabalho facultou o melhor conhecimento das respostas de Pisum sativum à salinidade, bem como de alguns factores que condicionam estas respostas. Os resultados permitem afirmar que, sob condições controladas, é possível o cultivo de Pisum sativum ‘Resal’ sob salinidade moderada (90 mM) sem grande diminuição de produtividade, desde que às plantas sejam fornecidos níveis adequados de azoto na forma de nitrato. Todavia, este trabalho também suscitou algumas questões: será que o estudo da tolerância salina de outras cultivares de Pisum sativum poderá dilatar o intervalo de tolerância da espécie? Será que a selecção de novos isolados de Rhizobium poderá permitir o aumento da eficiência de nodulação em Pisum sativum ? Quais os solutos orgânicos que as células de Pisum sativum acumulam de modo a ajustarem osmoticamente o citoplasma? Qual o gradiente de concentração de sódio e cloro através do tonoplasto em cada um dos órgãos da planta? Qual a resposta das plantas, sob stress salino a uma nutrição mista de azoto diatómico fixado simbioticamente e de menores níveis de nitrato?Saline habitats are those containing soluble salts concentrations that impair glicophytes growth. Nearly 10% of the total land surface is covered with different types of salt affected soils. Plants maintain low cytoplasmic sodium ion concentrations as a mean to achieve salt tolerance, once the excess of Na+ and Cl- ions affect their metabolism. Plants usually, rely on different mechanisms to maintain low cytoplasmic ion concentrations. There as been reasonable consensus on which physiological characteristics must be improved, to achieve salt tolerance. The attention is focused on ion transport and compartimentation, synthesis of compatible solutes, protection against deleterious oxidative effects, and metabolic processes that are salt sensitive. Most of legume plants are quite sensitive to salt, and this sensitivity is even grater when relying on dinitrogen fixation. Usually, nodule establishment and N2 fixation are considered the most salt sensitive phase in the symbiotic process. Most of reported attempts to enhance legume salt tolerance didn’t result in salt tolerant varieties, probably because the mechanisms that afford salt tolerance are difficult to understand. The main purposes of the present work are: the existence of genotipic difference among Pisum sativum and simbyosis salt sensitivity evaluation (chapter II); Rhizobium isolation and screening for N2 fixation efficiency under saline conditions (chapter III); nitrogen nutrition influence on Pisum sativum growth under salt stress and the mechanisms on which they rely to achieve salt tolerance (chapter IV); and salinity and nitrogen nutrition effects on seed protein composition (chapter V). The salt tolerance evaluation of four Pisum sativum cultivars resulted in significant differences among them. ‘Resal’ showed the highest tolerance with no significant vegetative growth reduction up to 90 mM NaCl in the soil. Rhizobium isolation and screening in different Portuguese locations was performed with the purpose to improve N2 fixation under salt conditions. Costa Nova, S. Bernardo and Vagos isolates showed saline sensitivity, since in vitro growth at 100 mM NaCl was reduced more than 70%. Only Rhizobium isolates from a water stressed site (Elvas), showed salt tolerance, growing under 700 mM NaCl. The symbiosis evaluation between Pisum sativum and Rhizobium isolates showed the possibility of an effective symbiosis under salt conditions. The molecular nitrogen form made available to plants influenced Pisum sativum growth and salt tolerance, and especially productivity. Nitrate dependent plants productivity was less affected by salinity. The observed salt tolerance differences can only be explained by more than one mechanism acting together, whose intensities changed during plant growth. The osmotic regulation was achieved, at least partially, by Na+, K+ and Cl- accumulation, and by Na+ shoot exclusion. Although, protein synthesis is generally considered a salt sensitive process, salinity caused increased protein seed content, exception being made by N2 fixation dependent plants. The maintenance of low seed Na+ and Cl- concentrations would account, to some extent, to this result. Protein SDS -PAGE separation revealed that growth conditions didn’t affect considerably seed polypeptides. Nevertheless, salinity induced new polypeptides which may, some how, improve seed salt tolerance. This work made possible a better knowledge of Pisum sativum responses to salinity and of factors that influenced those responses. Under mild salt conditions (90 mM NaCl) Pisum sativum ‘Resal’ can be grown without significant productivity reductions if, supplied with nitrate. But this work also raised some questions: the saline tolerance evaluation of other Pisum sativum cultivars could expand the species saline tolerance? Rhizobium isolation and screening in other sites could increase nodulation and N2 fixation efficiency under salinity? Which compatible solutes does Pisum sativum accumulate in the cytoplasm? Which is the Na+ and Cl- gradient concentration through tonoplast in each plant organ? How does plant respond to a mixed nitrate and N2 fixation nitrogen nutrition

Comparative sensitivity of Crassostrea angulata and Crassostrea gigas embryo-larval development to As under varying salinity and temperature

Oysters are a diverse group of marine bivalves that inhabit coastal systems of the world's oceans, providing a varietyofecosystemservices,andrepresentamajorsocioeconomic resource.However,oysterreefshavebecome inevitably impacted from habitat destruction, overfishing, pollution and disease outbreaks that have pushed these structures to the break of extinction. In addition, the increased frequency of climate change related events promise to further challenge oyster species survival worldwide. Oysters' early embryonic development is likely the most vulnerable stage to climate change related stressors (e.g. salinity and temperature shifts) as well as to pollutants (e.g. arsenic), and therefore can represent the most important bottleneck that define populations' survival in a changing environment. In light of this, the present study aimed to assess two important oyster species, Crassostrea angulata and Crassostrea gigas embryo-larval development, under combinations of salinity (20, 26 and 33), temperature (20, 24 and 28°C) and arsenic (As) exposure(0,30,60,120,240,480,960and1920μg.AsL−1),toinferondifferentoysterspeciescapacitytocope with these environmental stressors under the eminent threat of climate change and increase of pollution worldwide. Results showed differences in each species range of salinity and temperature for successful embryonic development. For C angulata, embryo-larval development was successful at a narrower range of both salinity and temperature, compared to C. gigas. Overall, As induced higher toxicity to C. angulata embryos, with calculated EC50 values at least an order of magnitude lower than those calculated for C. gigas. The toxicity of As (measured as median effective concentration, EC50) showed to be influenced by both salinity and temperature in both species. Nonetheless, salinity had a greater influence on embryos' sensitivity to As. This pattern was mostly noticed for C. gigas, with lower salinity inducing higher sensitivity to As. Results were discussed considering the existing literature and suggest that C. angulata populations are likely to become more vulnerable under near future predictions for temperature rise, salinity shifts and pollution.publishe

A multifactorial approach to untangle graphene oxide (GO) nanosheets effects on plants: plant growth-promoting bacteria inoculation, bacterial survival, and drought

Drought is a limiting factor for agricultural productivity. Climate change threatens to expand the areas of the globe subjected to drought, as well as to increase the severity and duration of water shortage. Plant growth-promoting bacteria (PGPB) are widely studied and applied as biostimulants to increase plant production and to enhance tolerance to abiotic and biotic constraints. Besides PGPB, studies on the potential of nanoparticles to be used as biostimulants are also thriving. However, many studies report toxicity of tested nanoparticles in bacteria and plants in laboratory conditions, but few studies have reported effects of nanoparticles towards bacterial cells and communities in the soil. The combined application of nanoparticles and PGPB as biostimulant formulations are poorly explored and it is important to unravel the potentialities of their combined application as a way to potentiate food production. In this study, Rhizobium sp. E20-8 and graphene oxide (GO) nanosheets were applied on container-grown maize seedlings in watered and drought conditions. Bacterial survival, seedling growth (dry weight), and biochemical endpoints (photosynthetic pigments, soluble and insoluble carbohydrates, proline, lipid peroxidation, protein, electron transport system, and superoxide dismutase) were evaluated. Results showed that the simultaneous exposure to GO and Rhizobium sp. E20-8 was able to alleviate the stress induced by drought on maize seedlings through osmotic and antioxidant protection by GO and mitigation of GO effects on the plant's biochemistry by Rhizobium sp. E20-8. These results constitute a new lead on the development of biostimulant formulations to improve plant performance and increase food production in water-limited conditions.publishe

Are the effects induced by increased temperature enhanced in Mytilus galloprovincialis submitted to air exposure?

Intertidal mussel species are frequently exposed to changes of environmental parameters related to tidal regimes that include a multitude of stressors that they must avoid or tolerate by developing adaptive strategies. In particular, besides air exposure during low tides, intertidal mussels are also subjected to warming and, consequently, to higher risk of desiccation. However, scarce information is available regarding the responses of mussels to tidal regimes, particularly in the presence of other stressors such as increased temperature. Investigating the impacts of such combination of conditions will allow to understand the possible impacts that both factors interaction may generate to these intertidal organisms. To this end, the present study evaluated the impacts of different temperatures (18 ºC and 21 ºC) on Mytilus galloprovincialis when continuously submersed or exposed to a tidal regime for 14 days. Results showed that in mussels exposed to increased temperature under submersion conditions, the stress induced was enough to activate mussels’ antioxidant defenses (namely glutathione peroxidase, GPx), preventing oxidative damage (lipid peroxidation, LPO; protein carbonylation, PC). In mussels exposed to tides at control temperature, metabolic capacity increased (electron transport system activity, ETS), and GPx was induced, despite resulting in increased LPO levels. Moreover, the combination of tides and temperature increase led to a significant decrease of lipid (LIP) content, activation of antioxidant defenses (superoxide dismutase, SOD; GPx) and increase of oxidized glutathione (GSSG), despite these mechanisms were not sufficient to prevent increased cellular damage. Therefore, the combination of increased temperature and air exposure induced higher oxidative stress in mussels. These findings indicate that increasing global warming could be more impacting to intertidal organisms compared to organisms continuously submersed. Furthermore, our results indicate that air exposure can act as a confounding factor when assessing the impacts of different stressors in organisms living in coastal systemspublishe

Can ocean warming alter sub-lethal effects of antiepileptic and antihistaminic pharmaceuticals in marine bivalves?

The negative effects induced in marine organisms by Climate Change related abiotic factors consequences, namely ocean warming, are well-known. However, few works studied the combined impacts of ocean warming and contaminants, as pharmaceutical drugs. Carbamazepine (CBZ) and cetirizine (CTZ) occur in the marine environment, showing negative effects in marine organisms. This study aimed to evaluate the impacts of ocean warming on the effects of CBZ and CTZ, when acting individually and combined (drug vs drug), in the edible clam Ruditapes philippinarum. For that, drugs concentration, bioconcentration factors and biochemical parameters, related with clam's metabolic capacity and oxidative stress, were evaluated after 28 days exposure to environmentally relevant scenarios of these stressors. The results showed limited impacts of the drugs (single and combined) at control and warming condition. Indeed, it appeared that warming improved the oxidative status of contaminated clams (higher reduced to oxidized glutathione ratio, lower lipid peroxidation and protein carbonylation levels), especially when both drugs were combined. This may result from clam's defence mechanisms activation and reduced metabolic capacity that, respectively, increased elimination and limited production of reactive oxygen species. At low stress levels, defence mechanisms were not activated which resulted into oxidative stress. The present findings highlighted that under higher stress levels clams may be able to activate defence strategies that were sufficient to avoid cellular damages and loss of redox homeostasis. Nevertheless, low concentrations were tested in the present study and the observed responses may greatly change under increased pollution levels or temperatures. Further research on this topic is needed since marine heat waves are increasing in frequency and intensity and pollution levels of some pharmaceuticals are also increasing in coastal systems.publishe