José Gomes-Laranjo

Morphological, biochemical and molecular characterization of Anabaena , Aphanizomenon and Nostoc strains (Cyanobacteria, Nostocales) isolated from Portuguese freshwater habitats

Studies of cyanobacterial nostocacean taxa are important to the global scientific community, mainly because a significant number of beneficial strains that belong to the order Nostocales fix atmospheric nitrogen, thus contributing to the fertility of agricultural soils worldwide, while others behave as nuisance microorganisms in aquatic ecosystems due to their involvement in toxic bloom events. However, in spite of their ecological importance and environmental concerns, their identification and taxonomy are still problematic and doubtful, often being based on current morphological and physiological studies, which generate confusing classification systems and usually vary under different conditions. Therefore, the present research aimed to investigate through a polyphasic approach differences in morphological, biochemical and genotypic features of three nostocacean cyanobacterial strains isolated from central-western Portuguese shallow freshwater bodies. Morphometric, genetic (16S rRNA, nifH and hetR fragments) and biochemical (fatty acid methyl ester; FAME profiles) data were used to characterize the strains. Morphological analysis and sequencing of 16S rRNA fragments showed that the strains belonged to Anabaena cylindrica (UTAD_A212), Aphanizomenon gracile (UADFA16) and Nostoc muscorum (UTAD_N213) species. These strains showed clear distinct morphological and genetic features, allowing easy allocation to their respective genera. The same happened by using partial sequences of hetR and nifH genes, in spite of the scarcity of deposited sequences. Biochemical characterization showed that the FAME profiles obtained were consistent with both morphological and molecular analyses. It was suggested that the ratio of monounsaturated to polyunsaturated FAMEs, together with the unsaturation index, could be used as genus-specific chemotaxonomic biomarkers.

Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber calli.

The effects of the herbicides 1,1-dimethyl-4,4-bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1 microM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.

Exposure of the cyanobacterium Nostoc muscorum from Portuguese rice fields to Molinate (Ordram(®)): Effects on the antioxidant system and fatty acid profile.

Herbicide contamination of aquatic ecosystems is a serious global environmental concern. Several herbicides enhance the intracellular formation of reactive oxygen species, and can lead to the damage of macromolecules and to a decrease of oxidant defenses in a wide range of non-target microorganisms including cyanobacteria. The effects of molinate (a thiocarbamate herbicide used for controlling grassy weeds in rice fields) on the activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione S-transferase were evaluated in Nostoc muscorum, a freshwater cyanobacterium with a significant spread in Portuguese rice fields. These were determined in N. muscorum cultures acutely (72h) exposed to concentrations ranging from 0.75 to 2mM of molinate. This study also analyzed the effects of molinate on: (1) the nonenzymatic antioxidant contents (reduced and oxidized glutathione, carotenoids, and proline), (2) the oxidative cell damage measured in terms of lipid peroxidation (MDA level) and electrolyte leakage (intactness of plasma membrane), and (3) the total fatty acid profile. The results showed that the activities of all antioxidant enzymes decreased dramatically with the rising concentration of molinate after 72h. Time-dependent and concentration-dependent increase in MDA and enhanced cell membrane leakage were indicative of lipid peroxidation, formation of free radicals and oxidative damage. Compared to control, 72-h herbicide exposure increased lipid peroxidation by 5.4%, 19% and 28% with 0.75, 1.5 and 2mM of molinate, respectively. Similarly, herbicide stress induced an increase in electrolyte leakage (5.8%, 29.5% and 30.2% above control, with 0.75, 1.5 and 2mM of molinate, respectively). The increased production of proline at higher molinate concentrations (the values rose above control by 45%, 95% and 156% with 0.75, 1.5 and 2mM, respectively) indicated the involvement of this osmoprotectant in a free radical scavenging mechanism. Moreover, a radical decline in both glutathione pool, carotenoids and saturated fatty acids were also observed. The results of the present study lead us to conclude that: (1) both enzymatic and nonenzymatic antioxidative defense system of N. muscorum are dramatically affected by molinate, (2) the herbicide induces peroxidation, (3) it contributes to an increase of the unsaturation level of cell membrane fatty acids. These evidences should be taken in account when using N. muscorum as an environmental indicator species in studies of herbicide biotransformation and biomarker response as well as in environmental monitoring programmes.

Evaluation of olive oil mill wastewater toxicity on the mitochondrial bioenergetics after treatment with Candida oleophila

In a previous work the ability of Candida oleophila to use phenolic compounds as sole carbon and energy source at high concentrations without an additional carbon source was reported. C. oleophila grown in bioreactor batch cultures in a diluted and sterilized olive oil mill wastewater (OMW) caused a significant decrease in the total tannins content but no significant alteration was observed in phenolic acid and fatty acid content. Both treated and untreated OMWs were tested to evaluate the capacity in interfering with mitochondrial bioenergetics. Mitochondrial respiration was not affected by treated OMW on the range of used concentrations, contrary to the untreated OMW. Furthermore, mitochondrial membrane potential and respiratory complexes were always significantly less affected by treated OMW in comparison with untreated OMW. However, supplementary treatment should be applied before OMW could be considered non-toxic.

Comparative toxicity of bentazon and molinate on growth, photosynthetic pigments, photosynthesis, and respiration of the Portuguese ricefield cyanobacterium Nostoc muscorum

Bentazon and molinate are selective herbicides recommended for integrated weed management in rice. Their toxicity on growth and some biochemical and physiological parameters of Nostoc muscorum, an abundant cyanobacterium in Portuguese rice fields, was evaluated under laboratory conditions during time‐ and concentration‐dependent exposure for 72 h. Results showed that toxic concentrations (0.75–2 mM) of both herbicides have pleiotropic effects on the cyanobacterium. Molinate was more toxic than bentazon to growth, respiration, chlorophyll‐a, carotenoids, and phycobiliproteins contents. Protein content was increased by both herbicides although the effect was particularly evident with higher concentrations of molinate (1.5–2 mM). The herbicides had contrasting effects on carbohydrates content: molinate increased this organic fraction whereas bentazon decreased it. Photosynthesis and respiration were inhibited by both herbicides.

Bentazon triggers the promotion of oxidative damage in the Portuguese ricefield cyanobacterium Anabaena cylindrica: Response of the antioxidant system

Rice fields are frequently exposed to environmental contamination by herbicides and cyanobacteria, as primary producers of these aquatic ecosystems, are adversely affected. Anabaena cylindrica is a cyanobacterium with a significantly widespread occurrence in Portuguese rice fields. This strain was studied throughout 72 h in laboratory conditions for its stress responses to sublethal concentrations (0.75–2 mM) of bentazon, a selective postemergence herbicide recommended for integrated weed management in rice, with special reference to oxidative stress, role of proline and intracellular antioxidant enzymes in herbicide‐induced free radicals detoxification. Activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione S‐transferase (GST) increased in a time‐ and herbicide dose‐response manner and were higher than those in the control samples after 72 h. A time‐ and concentration‐dependent increase of malondialdehyde (MDA) levels and the enhanced cell membrane leakage following bentazon exposure are indicative of lipid peroxidation, free radicals formation, and oxidative damage, while increased amounts of SOD, CAT, APX, GST, and proline indicated their involvement in free radical scavenging mechanisms. The appreciable decline in the reduced glutathione (GSH) pool after 72 h at higher bentazon concentrations could be explained by the reduction of the NADPH‐dependent glutathione reductase (GR) activity. The obtained results suggested that the alterations of antioxidant systems in A. cylindrica might be useful biomarkers of bentazon exposure. As the toxic mechanism of bentazon is a complex phenomenon, this study also adds relevant findings to explain the oxidative stress pathways of bentazon promoting oxidative stress in cyanobacteria.

Water Use Strategies of Plants Under Drought Conditions

The growing concerns about water scarcity have focused more attention on water management in agriculture and promotion of water conservation through improved water use efficiency (WUE). Depending on the main purpose of the study, WUE can be estimated at multiple scales, from leaf to whole plant, crop, yield, and ecosystem levels. Drought resistance and WUE are not synonymous and their association is often misunderstood. Effectively, two water use strategies may be employed by woody plants under drought conditions. The prodigal water use behavior is beneficial in conditions where water supply is interrupted for short periods only. The conservative water use is favorable in conditions where a long dry period prevails and is associated with high capacity for drought resistance and slow growth rates. In this chapter, we also examine how human manipulation such as breeding and agricultural management techniques will offer new opportunities to improve plant water use under drought conditions.

Antioxidant activities of chestnut nut of Castanea sativa Mill. (cultivar 'Judia') as function of origin ecosystem.

The antioxidant properties of different ecotypes of chestnut nut (cv. Judia) were studied. Total phenolics and flavonoids were also determinated. Total phenolics amount ranged from 9.6mg/g of GAE (hottest ecotype, Murça) to 19.4mg/g of GAE (coldest ecotype, Valpaços). Gallic and ellagic acid were the predominant compounds and Valpaços had the highest values while, Murça had the lowest ones. The antioxidant capacity of ethanolic extracts were evaluated through several biochemical essays: ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity, FRAP (ferric reducing/antioxidant power) and inhibition of oxidative haemolysis in erythrocytes. In order to evaluate the antioxidant efficiency of each ecotype, the EC50 values were calculated. Once again Valpaços revealed the best antioxidant properties, presenting much lower EC50 values. Climatic conditions influence seems to be a limiting factor for production of phenolic compounds and consequently for the antioxidant properties of chestnut nuts.

Isolation of chestnut chloroplasts: Membrane potentials of chestnut and spinach thylakoids

Typical chestnut thylakoid extracts isolated by mechanical disruption of leaf tissues had an equivalent of 0.28 kg m−3 chlorophyll (Chl) which is six times less than in thylakoids obtained from spinach, although Chl content in leaves was only half as small. According to optical microscopy, the vesicles showed a good integrity, exhibiting at 21 °C a high capacity of photon-induced potential membrane generation, which was demonstrated by the almost full 9-amino-6-chloro-2-methoxyacridine fluorescence quenching in a hyper-saline medium containing 150 mM KCl and having osmotic potential of −1.5 MPa. The half-time of the thylakoid potential generation was 11.7 s with the time of dissipation around 8.9 s. In such conditions, spinach thylakoids showed an increased swelling and also differences in the half-time generation which was almost four times faster than was observed in chestnut. However, when spinach thylakoids were incubated in a typical hypo-saline medium without KCl with osmotic potential −0.8 MPa, no additional swelling was observed. Consequently the half-time of potential dissipation was 35 s. Studies with nigericin suggested a chestnut thylakoid ΔpH significantly smaller than that observed in spinach, which was confirmed by the measurements of the ATP driven pumping activity.

Mapping and transcriptomic approches implemented for understanding disease resistance to Phytophthora cinammomi in Castanea sp

The European chestnut, Castanea sativa Mill, covers a total area of 2.53 million hectares two million of which are chestnut forests, i.e. forests where chestnut is the dominant tree species, being the remaining 0.53 million hectares devoted to fruit production (20.9% of the total chestnut-growing area). Chestnut fruit production in Europe declined considerably during the XX century to the current 200,000 t (almost 300 million euros). This decline arose mainly due to serious diseases and changes in the structure of society. Towards the end of the century, there was a sharp increase in chestnut demand which triggered new planting and the restoration of old orchards throughout Europe, although ink disease (Phytophthora spp.), canker blight (Cryphonectria parasitica (Murrill) M.E. Barr.) and more recently Dryocosmus kuriphilus Yasumatsu, still represent major threats to the species. In Europe, chestnut breeding has been focused on the improvement of cultivars and rootstocks through selection and hybridisation with Asian species resistant to ink disease. Considerable success was obtained using this approach, however little information was acquired until now on the genetic basis of resistance in chestnut. n nA set of interspecific crosses were established between European chestnut and Asian species and two separate full-sib pedigrees have been produced, SC (C. sativa x C. crenata) and SM (C.sativa x C. mollissima). The goal is to perform DNA marker:trait association analysis to identify QTLs related to ink disease metrics and also to identify putative resistance genes to P cinnamomi using a transcriptomic approach. An overview of the study will be presented including the genotyping of the mapping population (parents and progenies) with nuclear SSR markers designed for Castanea mollissima and provided by the American team of Fagaceae Genomics Project, for the construction of the first genetic linkage map. The methodologies implemented for the determination of the metrics of resistance of mapping population, for further identification of QTLs, will also be presented.