Teresa Dias

Nitrogen Deposition Effects on Mediterranean-type Ecosystems: An Ecological Assessment

We review the ecological consequences of N deposition on the five Mediterranean regions of the world. Seasonality of precipitation and fires regulate the N cycle in these water-limited ecosystems, where dry N deposition dominates. Nitrogen accumulation in soils and on plant surfaces results in peaks of availability with the first winter rains. Decoupling between N flushes and plant demand promotes losses via leaching and gas emissions. Differences in P availability may control the response to N inputs and susceptibility to exotic plant invasion. Invasive grasses accumulate as fuel during the dry season, altering fire regimes. California and the Mediterranean Basin are the most threatened by N deposition; however, there is limited evidence for N deposition impacts outside of California. Consequently, more research is needed to determine critical loads for each region and vegetation type based on the most sensitive elements, such as changes in lichen species composition and N cycling.

The flavonoid-rich fraction of Coreopsis tinctoria promotes glucose tolerance regain through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats

AIM OF THE STUDY Infusions of Coreopsis tinctoria Nutt. flowering tops have been used traditionally in Portugal to control hyperglycaemia and a previous study revealed that daily administration of the infusion during a 3-week period promoted the recovery of glucose tolerance by a mechanism different from inhibition of glucose absorption and direct promotion of insulin secretion. We know report the study of the ethyl acetate fraction of Coreopsis tinctoria flowers infusion aiming to confirm flavonoids as bioactive metabolites. To give one step forward into the antihyperglycaemic mechanism of action of this traditionally used plant we also studied the activity of Coreopsis tinctoria flavonoids on the pancreatic function of glucose-intolerant rats. A standard antioxidant, Trolox, was also studied for comparative purposes as the antioxidant mechanism has been frequently purposed as one of the mechanisms mediating antihyperglycaemic effects of flavonoid-rich extracts. MATERIAL AND METHODS Thirteen compounds, mainly of flavanone and chalcone flavonoidal type, have been identified in this fraction by HPLC-DAD-ESI-MS/MS, and the major one (marein) quantified by HPLC-UV. The fraction (125 mg containing 20 mg of marein/kg b.w.) and Trolox (50 mg/kg b.w.) were administered daily by oral gavage to normal and STZ (40 mg/kg b.w.)-induced glucose-intolerant Wistar rats for 3 weeks. Blood glucose levels were measured weekly by Oral Glucose Tolerance Test. Pancreatic function was evaluated by plasma lipase of treated and non-treated glucose-tolerant and- intolerant rats after the 3-week treatment period. RESULTS After 2 weeks oral treatment with Coreopsis tinctoria AcOEt fraction the animals were no longer glucose-intolerant, an effect maintained over the remaining experimental period. Additionally, plasma lipase values of glucose-intolerant animals treated with the AcOEt fraction (13.5 ± 0.84 U/L) showed a clear reduction when compared with the glucose-intolerant group (34.60 ± 1.76 U/L; P<0.001) and normoglycaemic control (8.35 ± 0.69 U/L) demonstrating recovery of pancreatic function. On the other hand, treatment with standard antioxidant Trolox had no effect on glucose homeostasis of glucose-intolerant rats. The oral treatment with Coreopsis tinctoria fraction caused no hepatotoxicity, as determined by blood alanine and aspartate transaminases, and had also no effect on glucose homeostasis and pancreatic function of normal rats. CONCLUSIONS AcOEt fraction, containing the same amount of marein as the infusion, promoted glucose tolerance regain in the rats more quickly, which means that the bioactivity is probably due to the several flavonoids present in Coreopsis tinctoria extracts and not to marein alone. The results also strongly suggest that these compounds act by promoting pancreatic cell function recovery from STZ-induced injury, possibly through a mechanism of action other than merely antioxidant mediated.

Cytoprotective effect of Coreopsis tinctoria extracts and flavonoids on tBHP and cytokine-induced cell injury in pancreatic MIN6 cells

ETHNOPHARMACOLOGICAL RELEVANCE [corrected] Coreopsis tinctoria flowering tops infusion is traditionally used in Portugal for treating the symptoms of diabetes. Recent studies have revealed its antihyperglycemic activity when administered for 3 weeks to a STZ-induced glucose intolerance model in the rat and glucose tolerance regain was even clearer and pancreatic function recovery was achieved when administering Coreopsis tinctoria flavonoid-rich AcOEt fraction. In this study we aimed to evaluate the protective effect of Coreopsis tinctoria flowering tops aqueous extract, AcOEt fraction and the pure compounds marein and flavanomarein, against beta-cell injury, in a mouse insulinoma cell line (MIN6) challenged with pro-oxidant tert-butyl-hydroperoxide (tBHP) or cytokines. MATERIALS AND METHODS The protective effects of Coreopsis tinctoria flowering tops extracts and pure compounds were evaluated through pre-incubating MIN6 cells with samples followed by treatment with tBHP (400 μM for 2 h) after which viability was determined through ATP measurements. In order to assess whether plant extracts were involved in decreasing reactive oxygen species, superoxide anion production was determined through a lucigenin-enhanced chemiluminescent method. Lastly, the direct influence of Coreopsis tinctoria extracts and main compounds on cell survival/apoptosis was determined measuring caspase 3 and 7 cleavage induced by cytokines. RESULTS Coreopsis tinctoria flowering tops extracts (25-100 μg/mL) and pure compounds (200-400 μM), when pre-incubated with MIN6 cells did not present any cytotoxicity, instead they increased cell viability in a dose dependent manner when challenged with tBHP. Treatment with this pro-oxidant also showed a rise in superoxide radical anion formation in MIN6 cells. This increase was significantly reduced by treatment with superoxide dismutase enzyme (SOD) but not by pre-treatment with Coreopsis tinctoria flowering tops extracts. Caspase 3/7 activation measurements show that Coreopsis tinctoria flowering tops extracts, as well as marein and flavanomarein, significantly inhibit apoptosis. CONCLUSIONS Coreopsis tinctoria extracts and pure compounds show cytoprotection that seems to be due to inhibition of the apoptotic pathway, and not through a decrease on superoxide radical production.

Assessment of Critical Levels of Atmospheric Ammonia for Lichen Diversity in Cork-Oak Woodland, Portugal

The effect of atmospheric ammonia on ecosystems has been the subject of ongoing research. Its adverse effects as an air pollutant are well characterised, and may be even more widespread than previously thought (see Aber et al. 2003; Erisman et al. 2003; Krupa 2003; Purvis et al. 2003). The most important sources of NH3 in Europe are agricultural activities, mainly crop fertilization and cattle management (Galloway et al. 2003; EPER 2004). Livestock housing facilities are recognised to be large point sources of NH3 emissions. Close to such facilities, atmospheric NH3 concentrations are very high, decreasing rapidly with distance over a few hundreds of meters to a few kilometres (Sutton et al. 1998). Measurement of atmospheric NH3 in the vicinity of livestock housing include those by Pitcairn et al. (1998, 2003), with reported values in Scotland of 24 –59 μg m−3 close to source, which declined to background values of 1.6 –5 μg m−3 at 1 km. In order to assess the range of effects of NH3 in natural ecosystems, that can be used for effective NH3 mitigation policies (Dragosits et al. 2006), one can rely on two distinct approaches: (i) direct measurements of atmospheric NH3 concentrations, which provide an estimate of dry NH3-N deposition, but require intensive and costly operations; (ii) monitoring of effects on the biotic component. The latter approach should be carried out using groups of biota that are more sensitive to the pollutant of interest. Lichens have been reported as the most sensitive group to NH3 emissions (e.g. Wolseley et al. 2006a; van Herk 1999). Lichens are symbiotic organisms widely used as biomonitors of environmental changes (e.g. Nimis et al. 1991; Vokou et al. 1999; Geebelen and Hoffmann 2001; Giordani et al. 2002; Pirintsos and Loppi 2003; Geiser and Neitlich 2007). Monitoring atmospheric pollutants using lichens may be undertaken in three ways: (1) measuring variations in lichens diversity and/or abundance, (2) using variations in physiological parameters, and/or using lichens as accumulators of pollutants (Branquinho 2001), and (3) considering functional groups related to nutrients tolerance, such as the division between nitrophytic/oligotrophic (or nitrophytic/ acidophitic) groups (see van Dobben and ter Braak 1999; Ruisi et al. 2005; Wolseley et al. 2006b).

Ecological impacts of atmospheric pollution and interactions with climate change in terrestrial ecosystems of the mediterranean basin: Current research and future directions

Mediterranean Basin ecosystems, their unique biodiversity, and the key services they provide are currently at risk due to air pollution and climate change, yet only a limited number of isolated and geographically-restricted studies have addressed this topic, often with contrasting results. Particularities of air pollution in this region include high O3 levels due to high air temperatures and solar radiation, the stability of air masses, and dominance of dry over wet nitrogen deposition. Moreover, the unique abiotic and biotic factors (e.g., climate, vegetation type, relevance of Saharan dust inputs) modulating the response of Mediterranean ecosystems at various spatiotemporal scales make it difficult to understand, and thus predict, the consequences of human activities that cause air pollution in the Mediterranean Basin. Therefore, there is an urgent need to implement coordinated research and experimental platforms along with wider environmental monitoring networks in the region. In particular, a robust deposition monitoring network in conjunction with modelling estimates is crucial, possibly including a set of common biomonitors (ideally cryptogams, an important component of the Mediterranean vegetation), to help refine pollutant deposition maps. Additionally, increased attention must be paid to functional diversity measures in future air pollution and climate change studies to establish the necessary link between biodiversity and the provision of ecosystem services in Mediterranean ecosystems. Through a coordinated effort, the Mediterranean scientific community can fill the above-mentioned gaps and reach a greater understanding of the mechanisms underlying the combined effects of air pollution and climate change in the Mediterranean Basin.

Recovery of oral glucose tolerance by wistar rats after treatment with Coreopsis tinctoria infusion

Infusions of Coreopsis tinctoria flowering tops have traditionally been used in Portugal to control hyperglycaemia but no pharmacological or toxicological studies have been reported until now. The chalcones marein and okanin were isolated from the aqueous extract, together with the 2S‐3′,4′,7,8‐tetrahydroxyflavanone. The content of marein in extracts was determined by HPLC‐UV and the radical scavenging capacity evaluated by the DPPH method (EC50 = 21 µg/mL). Glucose intolerance was induced by a single intraperitoneal injection of streptozotocin in saline (40 mg/Kg). After three weeks of oral treatment with C. tinctoria extract (500 mg/Kg/day) the animals were no longer glucose‐intolerant (p > 0.05). Additionally, this oral treatment caused no hepatotoxicity in the rats, as determined by blood alanine and aspartate transaminases. A single administration of extract had no effect on oral glucose tolerance in normal Wistar rats. The extract also had no effect on insulin secretion by MIN6 cells. In conclusion, C. tinctoria infusion is able to abolish the streptozotocin‐induced glucose‐intolerance in rats after three weeks of oral treatment by a mechanism other than induction of insulin secretion. The recovery of β‐pancreatic function mediated by an antioxidant mechanism is a possibility that deserves further investigation. Copyright

Heterogeneity of soil surface ammonium concentration and other characteristics, related to plant specific variability in a Mediterranean-type ecosystem

Heterogeneity and dynamics of eight soil surface characteristics essential for plants--ammonium and nitrate concentrations, water content, temperature, pH, organic matter, nitrification and ammonification rates--were studied in a Mediterranean-type ecosystem on four occasions over a year. Soil properties varied seasonally and were influenced by plant species. Nitrate and ammonium were present in the soil at similar concentrations throughout the year. The positive correlation between them at the time of greatest plant development indicates that ammonium is a readily available nitrogen source in Mediterranean-type ecosystems. The results presented here suggest that plant cover significantly affects soil surface characteristics.

Ammonium as a driving force of plant diversity and ecosystem functioning: Observations based on 5 years' manipulation of n dose and form in a mediterranean ecosystem

Enhanced nitrogen (N) availability is one of the main drivers of biodiversity loss and degradation of ecosystem functions. However, in very nutrient-poor ecosystems, enhanced N input can, in the short-term, promote diversity. Mediterranean Basin ecosystems are nutrient-limited biodiversity hotspots, but no information is available on their medium- or long-term responses to enhanced N input. Since 2007, we have been manipulating the form and dose of available N in a Mediterranean Basin maquis in south-western Europe that has low ambient N deposition (<4 kg N ha−1 yr−1) and low soil N content (0.1%). N availability was modified by the addition of 40 kg N ha−1 yr−1 as a 1∶1 NH4Cl to (NH4)2SO4 mixture, and 40 and 80 kg N ha−1 yr−1 as NH4NO3. Over the following 5 years, the impacts on plant composition and diversity (richness and evenness) and some ecosystem characteristics (soil extractable N and organic matter, aboveground biomass and % of bare soil) were assessed. Plant species richness increased with enhanced N input and was more related to ammonium than to nitrate. Exposure to 40 kg NH4 +-N ha−1 yr−1 (alone and with nitrate) enhanced plant richness, but did not increase aboveground biomass; soil extractable N even increased under 80 kg NH4NO3-N ha−1 yr−1 and the % of bare soil increased under 40 kg NH4 +-N ha−1 yr−1. The treatment containing less ammonium, 40 kg NH4NO3-N ha−1 yr−1, did not enhance plant diversity but promoted aboveground biomass and reduced the % of bare soil. Data suggest that enhanced NHy availability affects the structure of the maquis, which may promote soil erosion and N leakage, whereas enhanced NOx availability leads to biomass accumulation which may increase the fire risk. These observations are relevant for land use management in biodiverse and fragmented ecosystems such as the maquis, especially in conservation areas.

The opposing effects of the flavonoids isoquercitrin and sissotrin, isolated from Pterospartum tridentatum, on oral glucose tolerance in rats.

The effect of an aqueous extract of Pterospartum tridentatum on the blood glucose levels of normal Wistar rats was investigated in a situation of oral glucose challenge. The extract at 300 mg/kg showed an antihyperglycaemic effect in the first 30 min after glucose challenge but then the blood glucose levels rose above those of the control group, indicating the presence of compounds with different effects on glucose tolerance. Nine compounds of isoflavone and flavonol skeletons were identified in the extract by HPLC‐ESI‐MSn, four of them being identified for the first time in this species. The isoflavone sissotrin and the flavonol derivative, isoquercitrin, were selected for the oral glucose tolerance test. Isoquercitrin (100 mg/kg) showed time‐dependent antihyperglycaemic activity by delaying the post‐oral glucose load glycaemic peak at 30 min, as did the sodium‐dependent glucose transporter inhibitor phloridzin (100 mg/kg). In contrast, sissotrin (100 mg/kg) showed an opposite effect, impairing glucose tolerance. In conclusion, these preliminary results indicate that the effect of the extract on blood glucose may be either antihyperglycaemic or hyperglycaemic. Additionally, as far as is known, these are the first in vivo results on the acute antihyperglycaemic potential of isoquercitrin. Copyright

Down-regulation of plant defence in a resident spider mite species and its effect upon con- and heterospecifics.

Abstract Herbivorous spider mites occurring on tomato plants (Solanum lycopersicum L.) cope with plant defences in various manners: the invasive Tetranychus evansi reduces defences below constitutive levels, whereas several strains of T. urticae induce such defences and others suppress them. In the Mediterranean region, these two species co-occur on tomato plants with T. ludeni, another closely related spider mite species. Unravelling how this third mite species affects plant defences is thus fundamental to understanding the outcome of herbivore interactions in this system. To test the effect of T. ludeni on tomato plant defences, we measured (1) the activity of proteinase inhibitors, indicating the induction of plant defences, in those plants, and (2) mite performance on plants previously infested with each mite species. We show that the performance of T. evansi and T. ludeni on plants previously infested with T. ludeni or T. evansi was better than on clean plants, indicating that these two mite species down-regulate plant defences. We also show that plants attacked by these mite species had lower activity of proteinase inhibitors than clean plants, whereas herbivory by T. urticae increased the activity of these proteins and resulted in reduced spider mite performance. This study thus shows that the property of down-regulation of plant defences below constitutive levels also occurs in T. ludeni.