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- Programa de Monitorização dos Ecossistemas Terrestre e Estuarino na envolvente à CTRSU de S. João da Talha - Relatório AnualPublication . Sim-Sim, Manuela; Gordo, Leonel; Brotas, Vanda; Caçador, Isabel; Garcia, César; Neves, Ana; Silva, Teresa; Urriza, Carolina BeltranA equipa responsável por este relatório realizou o programa de monitorização entre Outubro de 1998 e Outubro de 2012, o que permitiu uma boa caracterização dos ecossistemas e evidenciar as principais tendências sazonais e/ou anuais das comunidades biológicas. No presente relatório são apresentados os resultados das diferentes componentes inseridas no programa de monitorização dos ecossistemas terrestre (Flora Epifítica) e estuarino (Fitoplâncton, Vegetação de Sapal, Macroinvertebrados e Ictiofauna). As metodologias seguidas foram idênticas aos períodos anteriores, de modo a permitir uma série temporal das vários componentes estudados do ecossistema.
- Investigating the mechanisms underlying phytoprotection by plant growth-promoting rhizobacteria in Spartina densiflora under metal stressPublication . Paredes-Páliz, K.; Rodríguez-Vázquez, R.; Duarte, Bernardo; Caviedes, M. A.; Mateos-Naranjo, E.; Redondo-Gómez, S.; Caçador, Isabel; Rodríguez-Llorente, I. D.; Pajuelo, E.Pollution of coasts by toxic metals and metalloids is a worldwide problem for which phytoremediation using halophytes and associated microbiomes is becoming relevant. Metal(loid) excess is a constraint for plant establishment and development, and plant growth promoting rhizobacteria (PGPR) mitigate plant stress under these conditions. However, mechanisms underlying this effect remain elusive. The effect of toxic metal(loid)s on activity and gene expression of ROS-scavenging enzymes in roots of the halophyte Spartina densiflora grown on real polluted sediments in a greenhouse experiment was investigated. Sediments of the metal-polluted joint estuary of Tinto and Odiel rivers and control, unpollutred samples from the Piedras estuary were collected and submitted to ICP-OES. Seeds of S. densiflora were collected from the polluted Odiel marshes and grown in polluted and unpolluted sediments. Rhizophere biofilm-forming bacteria were selected based on metal tolerance and inoculated to S. densiflora and grown for 4 months. Fresh or frozen harvested plants were used for enzyme assays and gene expression studies, respectively. Metal excess induced SOD (five-fold increase), whereas CAT and ascorbate peroxidase displayed minor induction (twofold). A twofold increase of TBARs indicated membrane damage. Our results showed that metal-resistant PGPR (P. agglomerans RSO6 and RSO7 and B. aryabhattai RSO25) contributed to alleviate metal stress, as deduced from lower levels of all antioxidant enzymes to levels below those of non-exposed plants. The oxidative stress index (OSI) decreased between 50 and 75% upon inoculation. The results also evidenced the important role of PAL, involved in secondary metabolism and/or lignin synthesis, as a pathway for metal stress management in this halophyte upon inoculation with appropriate PGPR, since the different inoculation treatments enhanced PAL expression between 3.75- and five-fold. Our data confirm, at the molecular level, the role of PGPR in alleviating metal stress in S. densiflora and evidence the difficulty of working with halophytes for which little genetic information is available.
- Growth, chlorophyll fluorescence and mineral nutrition in the halophyte Tamarix gallica cultivated in combined stress conditions: Arsenic and NaClPublication . Sghaier, Dhouha Belhaj; Duarte, Bernardo; Bankaji, Insaf; Caçador, Isabel; Sleimi, NoomeneTrace metal elements can cause various environmental and health issues due to their accumulation and integration in the food chain. In the present study, we determined the major toxic effects of arsenic on physiological behaviour of plants. For this propose, several combinations of high salinity and arsenic (As) concentrations were applied to the halophytic shrub, Tamarix gallica, by growing for three months with an irrigation solution supplemented with different concentrations of As (0, 200, 500 and 800M) with and without 200mM NaCl. The effect of the combined stress conditions on growth, physiological patterns and biochemical parameters were also assessed. The results demonstrated that T. gallica is a tolerant plant regarding arsenic. The photosynthesis apparatus Fo, Fm and Fv fluorescence, as well as Fv/Fm were not affected by As nor by As combined with salt. Likewise, pigment and nutrient (K+, Ca2+ and Mg2+) contents were not affected either. However, the study results revealed that As adversely and significantly influenced the growth with increasing the concentration of As. Despite shoots growth reduction, the present research demonstrates that T. gallica is able to cope with high external concentrations of As (under 500μM) alone or in combination with NaCl.
- Yttrium and rare earth elements fractionation in salt marsh halophyte plantsPublication . Brito, Pedro; Malvar, Margarida; Galinha, Catarina; Caçador, Isabel; Canário, João; Araújo, M. Fátima; Raimundo, JoanaSalt marshes act as natural deposits of different metals (e.g. heavy-metals), while halophyte plants are known to retain and accumulate them in the different tissues. Scarce data exists on accumulation, partition and fractionation of YREE in these plants. To study the relationship between halophyte plants and YREE, contents of these metals were determined by ICP-MS in sediment, and in the different plants organs, from Rosário's salt marsh, in Tagus estuary (SW Europe). Results show significant differences (p < 0.001) in YREE contents between sediments. In non-colonised sediment Y was lower (5.0-18 mg·kg-1) compared to the Sarcocornia fruticosa and Spartina maritima sediment cores (19-26 and 20-26 mg·kg-1, respectively). The same was observed for ΣREE, with lower values in non-colonised sediment (32-138 mg·kg-1), while colonised ones presented higher contents (146-174 and 151-190 mg·kg-1, for S. fruticosa and S. maritima, respectively). These significant differences (p < 0.05) are explained by the sediments' nature. Yttrium and ΣREE Al-normalised ratios in non-colonised sediment ranged from 1.5 to 2.3 and 11 to 13, respectively. The colonised sediments revealed significant higher ratios (particularly for ΣREE/Al ratios; p < 0.001), varying from Y/Al: 1.8-2.3 and ΣREE: 13-16 for S. fruticosa, and Y/Al: 1.4-2.3 and ΣREE: 12-18, for S. maritima. Results suggest that these plants may promote YREE enrichment in the sediments. No differences in fractionation patterns among sediments and in both species roots were found, but fractionation was different from those in the sediment, with similar middle-REE (MREE) enrichment and no light-REE (LREE) and heavy-REE (HREE) fractionation. No evidence of YREE transfer to aboveground organs was observed. Different fractionation patterns in stems and leaves were registered, with clear enrichment of LREE relative to HREE and an increase in the MREE enrichment. Therefore, these plants showed low ability to accumulate and translocate YREE but may promote its enrichment in the sediments.
- The interplay between membrane lipids and phospholipase A family members in grapevine resistance against Plasmopara viticolaPublication . Laureano, Gonçalo; Figueiredo, Joana; Cavaco, Ana Rita; Duarte, Bernardo; Caçador, Isabel; Malhó, Rui; Sousa Silva, Marta; Matos, Ana Rita; Figueiredo, AndreiaGrapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most important diseases in modern viticulture. The search for sustainable disease control measure is of extreme importance, thus becoming imperative to fully characterize the mechanisms leading to an incompatible interaction. We have previously shown that lipid signalling events play an important role in grapevine's response to this pathogen, namely through changes in linolenic acid content, lipid peroxidation and jasmonic acid synthesis. Here, we have characterized the modulation of lipid metabolism in leaves from two V. vinifera cultivars (resistant and susceptible to P. viticola) in the first hours after pathogen inoculation. Prior to pathogen inoculation both genotypes present an inherently different fatty acid composition that is highly modulated in the resistant genotype after pathogen challenge. Such changes involve modulation of phospholipase A activity suggesting that the source of lipids mobilized upon pathogen infection are the chloroplast membranes. This work thus provides original evidence on the involvement of lipid signalling and phospholipases in grapevine immune responses to pathogen infection. The results are discussed considering the implications on the plant's physiological status and the use of discriminating lipid/fatty acids pattern in future selection procedures of cultivars.
- Impact of heat and cold events on the energetic metabolism of the C3 halophyte Halimione portulacoidesPublication . Duarte, Bernardo; Santos, D.; Marques, J. C.; Caçador, IsabelAccording to the newest predictions, it is expected that the Mediterranean systems experience more frequent and longer heat and cold treatments events. Salt marshes will be no exception. Halimione portulacoides is a widely distributed halophyte highly adapted to harsh environments. Plants exposed to heat stress showed a reduction in the maximum electron transport rates and increase in the rate of RC closure, as indicated by the increase in M0. Alongside there was also a reduction in the quinone pool size while compared to the plants maintained in the control condition. In contrast plants exposed to low temperatures didn't show any signs of damage on the ETC. Heat-exposed individuals experienced a reduction of connectivity between the PS II antennae with simultaneous inhibition of the electron transport. This was more evident in the donor side of the PS II, Being this a consequence of the damages in the oxygen-evolving complex. Also if both PS I and PS II energy fluxes are observed, there are evident differences in the thermal tolerance of both photosystems. While compared to the control group, cold exposed plants showed an increased PS I efficiency (δR0) indicating a tolerance of this photosystem to low temperatures. Nevertheless, the excessive redox potential generated by light harvesting and inefficient processing was not dissipated correctly and consequently causing a oxidative stress situation. In the present study only heat exposed plants showed a significant activation of the xanthophyll cycle. Alongside with this mechanism and similarly to what was observed for cold treated plants, it could be observed an increase in auroxanthin content, an efficient energy quencher under stress conditions. The coupled activation of the xanthophyll cycle along with a higher auroxanthin synthesis suggests that heat-treated individuals had higher needs to dissipate excessive energy than the cells exposed to cold treatment. In both cases appears to exist an efficient ROS scavenging mechanism. According to our data, heat and cold treatment events can have serious impacts on H. portulacoides photobiology reducing its primary productivity. At the ecosystem level, these climatic events could pose a serious threat to the survival of this species in the new climatic reality that our planet is facing.
- Spartina versicolor Fabre: Another case of Spartina trans-Atlantic introduction?Publication . Baumel, A.; Rousseau-Gueutin, M.; Sapienza-Bianchi, C.; Gareil, A.; Duong, N.; Rousseau, H.; Coriton, O.; Amirouche, R.; Sciandrello, S.; Duarte, Bernardo; Caçador, Isabel; Castillo, J. M.; Ainouche, M.Intercontinental introductions are widespread in the genus Spartina, with important ecological and evolutionary consequences. The native or introduced status of Spartina species is then critical with regard to biodiversity assessment, especially for vulnerable Mediterranean coastline ecosystems. Spartina versicolor was first recorded in southern France in 1849, then successively in various places on the European and North-African Mediterranean and Atlantic coasts. This species is considered to be either a European native or an invasive species introduced from North America which has a high morphological similarity to the Atlantic American species Spartina patens. We performed extensive sampling of S. versicolor in Europe and North Africa (from natural populations and herbarium collections) and compared these samples to other European and American Spartina species (including S. patens). Chromosome counts were reported for the first time and revealed that S. versicolor is tetraploid (2n = 4x = 40). Phylogenetic analyses based on chloroplast and nuclear ribosomal DNA sequences did not reveal any molecular variation within S. versicolor. In this species, a single haplotype, that is identical to one haplotype of S. patens, was found in the four chloroplast and the nuclear ribosomal ITS regions investigated. In addition, simple sequence repeat markers were used and revealed a low level of genetic diversity within S. versicolor, suggesting that the introduction of S. versicolor occurred from a narrow genetic pool of S. patens from North America.
- Zostera noltii development probing using chlorophyll a transient analysis (JIP-test) under field conditions: Integrating physiological insights into a photochemical stress indexPublication . Duarte, Bernardo; Pedro, Sílvia; Marques, J. C.; Adão, Helena; Caçador, IsabelBeyond their undeniable role, nowadays we also must look to seagrass beds as endangered environmentswith urgent monitoring and conservation needs. In the present study Z. noltii photochemical performanceof under different stages of development/recovery was assessed and its results applied in the develop-ment of a photochemical stress index (PSI) to classify and efficiently assess the physiological conditionof seagrass beds. In order to investigate deeper into this metabolic network the JIP-test was applied,allowing to identify the reasons underlying the first signs of stress. Less developed beds low connectiv-ity between PS II antennae leading inevitably to an impairment of the energetic transport. Associatedto this also the quinone pool showed severe depletion both in number and function. Alongside the K-step presence in the Kautsky curve points to severe damage at donor side of the PS II, where the OxygenEvolving Complexes (OECs) are located. All these negative impacts increase the quantum yield of the non-photochemical reactions in stressed/less developed seagrass beds. In sum, more developed beds showproportionally higher light use efficiencies promoted by a higher number of oxidized reaction centrescoupled with an enhanced capacity in using the generated electron potential and relatively lower energydissipations. Coupling all the photochemical into an Integrated Biomarker Response (IBR) approach, aphotochemical stress index (PSI) was produced. The PSI showed that more developed sites present lowerphotochemical stress values with inverse significant correlation with biomass coverage, reinforcing theapplicability of this non-invasive index as a reflex of the seagrass bed development stage.
- Chromium Phyto-transformation in Salt Marshes: The Role of HalophytesPublication . Caçador, Isabel; Duarte, BernardoWetlands, in particular salt marshes, are very interesting field laboratories to study metal biogeochemistry, namely, Cr. Due to the heavily industrialized history of most of estuarine systems, salt marshes became large deposits of heavy metals. Due to the large affinity of Cr to the medium organic matter, the removal of Cr throughout natural or enhanced processes occurs throughout plant-mediated processes. Naturally, plants acquire during their life cycle nutrients from their sediments but also some non-nutritional elements, like Cr, and store them in their tissues. In the last decades, this natural ability attracted the attention of several projects focusing on the enhancement of this process throughout the application of transporter molecules, like LMWOA, in order to increase the sediment-plant Cr transport. Due to its chemistry, Cr presents to oxidation states, Cr (III) and Cr (VI), being this last very toxic. Thus it became important to study not only the plant accumulation capacity but also the root-mediated processes of phyto-conversion of Cr (VI) toxic form to the less toxic Cr (III). Again, halophytes acquire an important role with high conversion efficiencies. All these passive and enhanced processes point out to a promising biotechnology using halophytes as potential cleaners of Cr-contaminated sediments, using environmental-friendly and low-cost technologies.
- Leaf fatty acid remodeling in the salt-excreting halophytic grass Spartina patens along a salinity gradientPublication . Duarte, Bernardo; Matos, Ana Rita; Marques, João Carlos; Caçador, IsabelSpartina patens is a highly dispersed halophytic grass invader in Mediterranean marshes. It is also characterized by having a high degree of resistance to salinity, one of the main drivers of plant zonation in salt marshes. Nevertheless, the physiological basis behind the extreme resistance of S. patens requires more detailed studies. In the present work, we aimed to study how membrane fatty acid remodeling could contribute to the resistance of this plant to salt. Spartina patens individuals exposed to increasing levels of salinity and its leaf fatty acid profile under lipid peroxidation products evaluated under all tested concentrations. A significant increase in the relative amounts of the saturated fatty acids (SFA) was observed, namely palmitic acid (C16:0), essential for PS II functioning, and stearic (C18:0) acid. The chloroplastidial trans-hexadecenoic acid (C16:1t) as well as the polyunsaturated linoleic (C18:2) and linolenic (C18:3) acids showed significant decreases in all the salt treatments. These changes led to a reduction in the double bond index in salt-treated plants which reflects reduction of the fluidity of the chloroplast membranes, which could contribute to maintain the membrane impermeable to the toxic exogenous Na. Despite the decrease observed in the total fatty acid contents in plants exposed to high salt concentrations the amounts of lipid peroxidation products decreased highlighting the resistance of this species towards toxic exogenous salt concentrations. Membrane fatty acid remodeling could represent an efficient mechanism to maintain the photosynthetic machinery of S. patens highly efficient under salt stress.