Monya M. Costa

Physiological responses of Zostera marina and Cymodocea nodosa to light-limitation stress

The effects of light-limitation stress were investigated in natural stands of the seagrasses Zostera marina and Cymodocea nodosa in Ria Formosa coastal lagoon, southern Portugal. Three levels of light attenuation were imposed for 3 weeks in two adjacent meadows (2–3 m depth), each dominated by one species. The response of photosynthesis to light was determined with oxygen electrodes. Chlorophylls and carotenoids were determined by high-pressure liquid chromatography (HPLC). Soluble protein, carbohydrates, malondialdehyde and phenol contents were also analysed. Both species showed evident signs of photoacclimation. Their maximum photosynthetic rates were significantly reduced with shading. Ratios between specific light harvesting carotenoids and the epoxidation state of xanthophyll cycle carotenoids revealed significantly higher light harvesting efficiency of C. nodosa, a competitive advantage in a low light environment. The contents of both soluble sugars and starch were considerably lower in Z. marina plants, particularly in the rhizomes, decreasing even further with shading. The different carbohydrate energy storage strategies found between the two species clearly favour C. nodosas resilience to light deprivation, a condition enhanced by its intrinsic arrangement of the pigment pool. On the other hand, Z. marina revealed a lower tolerance to light reduction, mostly due to a less plastic arrangement of the pigment pool and lower carbohydrate storage. Our findings indicate that Z. marina is close to a light-mediated ecophysiological threshold in Ria Formosa.

Establishing research strategies, methodologies and technologies to link genomics and proteomics to seagrass productivity, community metabolism, and ecosystem carbon fluxes

A complete understanding of the mechanistic basis of marine ecosystem functioning is only possible through integrative and interdisciplinary research. This enables the prediction of change and possibly the mitigation of the consequences of anthropogenic impacts. One major aim of the European Cooperation in Science and Technology (COST) Action ES0609 “Seagrasses productivity. From genes to ecosystem management,” is the calibration and synthesis of various methods and the development of innovative techniques and protocols for studying seagrass ecosystems. During 10 days, 20 researchers representing a range of disciplines (molecular biology, physiology, botany, ecology, oceanography, and underwater acoustics) gathered at The Station de Recherches Sous-marines et Océanographiques (STARESO, Corsica) to study together the nearby Posidonia oceanica meadow. STARESO is located in an oligotrophic area classified as “pristine site” where environmental disturbances caused by anthropogenic pressure are exceptionally low. The healthy P. oceanica meadow, which grows in front of the research station, colonizes the sea bottom from the surface to 37 m depth. During the study, genomic and proteomic approaches were integrated with ecophysiological and physical approaches with the aim of understanding changes in seagrass productivity and metabolism at different depths and along daily cycles. In this paper we report details on the approaches utilized and we forecast the potential of the data that will come from this synergistic approach not only for P. oceanica but for seagrasses in general.

Essential Oil Composition and Glandular Trichomes of Marrubium vulgare L. Growing Wild in Algeria

Abstract A scanning electron microscopic (SEM) study of the aerial parts of Marrubium vulgare L. grown wild in Algeria showed a dense woolly indumentum of stellate, nonglandular, point-shaped trichomes that completely hide both the adaxial and abaxial leaf epidermes, as well as the stem and flower surfaces bearing the glandular trichomes. Peltate and two types of capitate trichomes constitute the morphological distinct glandular trichomes that are spread over the vegetative and reproductive organs. The essential oils of M. vulgare were obtained by hydrodistillation and distillation-extraction from the aerial parts collected during the flowering (F) and vegetative phases (V), and analyzed by GC and GC/MS. The essential oils were obtained in a yield of ≤ 0.05% (v/w). Eugenol was the main component in the F oil (50%) but comprised only 16% in the V oil. β-Bisabolene was the dominant component of the V oil (29%) and the second main component in the F oil (11%).

Depth-specific fluctuations of gene expression and protein abundance modulate the photophysiology in the seagrass Posidonia oceanica

Here we present the results of a multiple organizational level analysis conceived to identify acclimative/adaptive strategies exhibited by the seagrass Posidonia oceanica to the daily fluctuations in the light environment, at contrasting depths. We assessed changes in photophysiological parameters, leaf respiration, pigments, and protein and mRNA expression levels. The results show that the diel oscillations of P. oceanica photophysiological and respiratory responses were related to transcripts and proteins expression of the genes involved in those processes and that there was a response asynchrony between shallow and deep plants probably caused by the strong differences in the light environment. The photochemical pathway of energy use was more effective in shallow plants due to higher light availability, but these plants needed more investment in photoprotection and photorepair, requiring higher translation and protein synthesis than deep plants. The genetic differentiation between deep and shallow stands suggests the existence of locally adapted genotypes to contrasting light environments. The depth-specific diel rhythms of photosynthetic and respiratory processes, from molecular to physiological levels, must be considered in the management and conservation of these key coastal ecosystems.

Linking gene expression to productivity to unravel long- and short-term responses of seagrasses exposed to CO2 in volcanic vents

Ocean acidification is a major threat for marine life but seagrasses are expected to benefit from high CO2. In situ (long-term) and transplanted (short-term) plant incubations of the seagrass Cymodocea nodosa were performed near and away the influence of volcanic CO2 vents at Vulcano Island to test the hypothesis of beneficial effects of CO2 on plant productivity. We relate, for the first time, the expression of photosynthetic, antioxidant and metal detoxification-related genes to net plant productivity (NPP). Results revealed a consistent pattern between gene expression and productivity indicating water origin as the main source of variability. However, the hypothesised beneficial effect of high CO2 around vents was not supported. We observed a consistent long- and short-term pattern of gene down-regulation and 2.5-fold NPP decrease in plants incubated in water from the vents and a generalized up-regulation and NPP increase in plants from the vent site incubated with water from the Reference site. Contrastingly, NPP of specimens experimentally exposed to a CO2 range significantly correlated with CO2 availability. The down-regulation of metal-related genes in C. nodosa leaves exposed to water from the venting site suggests that other factors than heavy metals, may be at play at Vulcano confounding the CO2 effects.

Genome-wide transcriptional reprogramming in the seagrass Cymodocea nodosa under experimental ocean acidification.

Here, we report the first use of massive‐scale RNA‐sequencing to explore seagrass response to CO2‐driven ocean acidification (OA). Large‐scale gene expression changes in the seagrass Cymodocea nodosa occurred at CO2 levels projected by the end of the century. C. nodosa transcriptome was obtained using Illumina RNA‐Seq technology and de novo assembly, and differential gene expression was explored in plants exposed to short‐term high CO2/low pH conditions. At high pCO2, there was a significant increased expression of transcripts associated with photosynthesis, including light reaction functions and CO2 fixation, and also to respiratory pathways, specifically for enzymes involved in glycolysis, in the tricarboxylic acid cycle and in the energy metabolism of the mitochondrial electron transport. The upregulation of respiratory metabolism is probably supported by the increased availability of photosynthates and increased energy demand for biosynthesis and stress‐related processes under elevated CO2 and low pH. The upregulation of several chaperones resembling heat stress‐induced changes in gene expression highlighted the positive role these proteins play in tolerance to intracellular acid stress in seagrasses. OA further modifies C. nodosa secondary metabolism inducing the transcription of enzymes related to biosynthesis of carbon‐based secondary compounds, in particular the synthesis of polyphenols and isoprenoid compounds that have a variety of biological functions including plant defence. By demonstrating which physiological processes are most sensitive to OA, this research provides a major advance in the understanding of seagrass metabolism in the context of altered seawater chemistry from global climate change.

Epiphytes Modulate Posidonia oceanica Photosynthetic Production, Energetic Balance, Antioxidant Mechanisms, and Oxidative Damage

Epiphytes impose physical barriers to light penetration into seagrass leaves causing shading, which may decrease the production of oxygen reactive species (ROS), but also constitute a physical aggression that may trigger the production of ROS, leading to oxidative damage. Here we investigate the effects of epiphytes on Posidonia oceanica under both interactive perspectives, light attenuation and oxidative stress. Specifically the role of epiphytes in net photosynthesis, chlorophyll a and b, photoprotection (Violaxanthin+Anteraxanthin+Zeaxanthin cycle), soluble sugar and starch contents, enzymatic (ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR)) and global (trolox equivalent antioxidant capacity (TEAC)) and oxygen radical antioxidant capacity (ORAC)) antioxidant responses, phenolics and oxidative damage (malondialdehyde) are tested. Leaves with epiphytes showed higher chlorophyll b and lower content in VAZ cycle carotenoids. Epiphyte shading was the probable reason for the lower VAZ de-epoxidation-ratio of leaves with epiphytes. In spite of being shaded, leaves with epiphytes showed higher antioxidant levels, indicating that epiphytes trigger the production of ROS. Both ORAC and TEAC and also APX and DHAR activities were higher in leaves with epiphytes, indicating that this response was related with its presence. Malondialdehyde concentrations also suggest oxidative damage caused by epiphytes. We conclude that the epiphyte load causes oxidative stress in P. oceanica and the mechanisms to scavenge ROS were not completely effective to avoid cell damage.

BioRePortAP, an electronic clinical record coupled with a database: an example of its use in a single centre

AIMS To evaluate the efficacy and safety of the treatment of psoriatic arthritis (PsA) patients with tumor necrosis factor (TNF) antagonists in the Rheumatology Department of Hospital de Santa Maria using the BioRePortAP. METHODS The Portuguese Society of Rheumatology (SPR) developed an electronic medical chart coupled with a database for the follow up of PsA patients, the BioRePortAP, which was launched in May 2009. This evaluation was based on all the PsA patients that were on active treatment with TNF antagonists in September 2009 and were registered in the BioRePortAP. All the previous data on these patients were introduced in BioRePortAP using the prospective paper based follow up protocol that this Department was using since 1999. Only patients with more than 9 months of treatment were analyzed. RESULTS Forty-two patients with PsA, actively treated with anti-TNF agents in September 2009, for at least 9 months, were analyzed in BioRePortAP. Twenty-three patients were male (55%) and nineteen were female (45%). The average age of these patients was 49.8+/-10.9 years old, the average disease duration was of 10.7+/-5.6 years and the mean duration of biological therapy was of 37.8+/-27.8 months. For the 81% of patients with peripheral joint disease there was a mean reduction of more than 80% in the swollen and tender joint counts, and almost 50% in the health assessment questionnaire (HAQ) value. In the 19% of the patients with axial involvement the reduction of BASDAI and BASFI was not statistically significative. On top of that, PASI score suffered a reduction of 64%. Fourteen patients (33.3%) had to switch their TNF antagonist treatment. 58.8% of the switches were due to adverse effects and 41.2% due to therapy failure. Regarding the 56 adverse reactions registered, only one was a severe reaction. The remaining adverse reactions were not severe and 67% of them were due to infections. DISCUSSION The results of this first report of the use of the BioRePortAP in clinical practice confirm the efficacy and safety of TNF antagonist treatment in PsA. The results shown here elucidate the potential applications of BioRePortAP as a tool for efficacy and safety assessment of PsA patients treated with biotechnological drugs.

SU‐FF‐I‐79: Java‐Based Plugin for Tomographic Reconstruction for SPECT Data

Purpose: To implement a plugin for the softwareImageJ, a public domain and open source software, written in JAVA, used for image processing and analysis, able to perform the filtered backprojection (FBP) algorithm for tomographic reconstruction of SPECT data. Method and Materials: New classes were added to the softwareImageJ in order to implement the plugin. The following classes have been developed: (1) the backprojection process, in which data from projections or filtered projection are smeared back into the image matrix; (2) the tomographic reconstruction ramp filter with other windowing possibilities using loss‐pass filters such as Butterworth, Shepp‐Logan and Hamming; (3) the one‐dimensional Fourier transform to perform the projection filtering; (4) and the user interface of the plugin. The plugin was tested with Monte Carlo simulated projection data of Zubal brain phantom, NCAT phantom and patient data. Results: The plugin is able to reconstructsinograms of 8, 16 and 32‐bits. Users can choose filter/window combinations. After the user sets the parameters needed for each combination (cutoff frequency and/or order), the filter curve can be visualized. When no filtering is choosen, simple backprojection is performed. The final reconstructed image is displayed and can be visualized or analyzed using segmentation and processing tools available in ImageJ.Conclusion: The outcome of this work consists of a plugin for the softwareImageJ to reconstructimages from SPECT data as sinograms using filtered backprojection or simple backprojection. The plugin is plataform independent and runs either in Windows or Linux, freely downloadable and accessible, and based on open source code. Further implementations will allow this plugin to work with DICOM images and offer iterative methods of tomographic reconstruction. Research sponsored by FRIDA Program/LACNIC and CNPq.

Photoacclimation strategies in northeastern Atlantic seagrasses: Integrating responses across plant organizational levels

Seagrasses live in highly variable light environments and adjust to these variations by expressing acclimatory responses at different plant organizational levels (meadow, shoot, leaf and chloroplast level). Yet, comparative studies, to identify species’ strategies, and integration of the relative importance of photoacclimatory adjustments at different levels are still missing. The variation in photoacclimatory responses at the chloroplast and leaf level were studied along individual leaves of Cymodocea nodosa, Zostera marina and Z. noltei, including measurements of variable chlorophyll fluorescence, photosynthesis, photoprotective capacities, non-photochemical quenching and D1-protein repair, and assessments of variation in leaf anatomy and chloroplast distribution. Our results show that the slower-growing C. nodosa expressed rather limited physiological and biochemical adjustments in response to light availability, while both species of faster-growing Zostera showed high variability along the leaves. In contrast, the inverse pattern was found for leaf anatomical adjustments in response to light availability, which were more pronounced in C. nodosa. This integrative plant organizational level approach shows that seagrasses differ in their photoacclimatory strategies and that these are linked to the species’ life history strategies, information that will be critical for predicting the responses of seagrasses to disturbances and to accordingly develop adequate management strategies.