Gene co-expression network analysis for the selection of candidate early warning indicators of heat and nutrient stress in Posidonia oceanica.

The continuous worldwide seagrasses decline calls for immediate actions in order to preserve this precious marine ecosystem. The main stressors that have been linked with decline in seagrasses are 1) the increasing ocean temperature due to climate change and 2) the continuous inputs of nutrients (eutrophication) associated with coastal human activities. To avoid the loss of seagrass populations, an "early warning" system is needed. We used Weighed Gene Co-expression Network Analysis (WGCNA), a systems biology approach, to identify potential candidate genes that can provide an early warning signal of stress in the Mediterranean iconic seagrass Posidonia oceanica, anticipating plant mortality. Plants were collected from both eutrophic (EU) and oligotrophic (OL) environments and were exposed to thermal and nutrient stress in a dedicated mesocosm. By correlating the whole-genome gene expression after 2-weeks exposure with the shoot survival percentage after 5-weeks exposure to stressors, we were able to identify several transcripts that indicated an early activation of several biological processes (BP) including: protein metabolic process, RNA metabolic process, organonitrogen compound biosynthetic process, catabolic process and response to stimulus, which were shared among OL and EU plants and among leaf and shoot apical meristem (SAM), in response to excessive heat and nutrients. Our results suggest a more dynamic and specific response of the SAM compared to the leaf, especially the SAM from plants coming from a stressful environment appeared more dynamic than the SAM from a pristine environment. A vast list of potential molecular markers is also provided that can be used as targets to assess field samples.

DNA methylation dynamics in a coastal foundation seagrass species under abiotic stressors.

DNA methylation (DNAm) has been intensively studied in terrestrial plants in response to environmental changes, but its dynamic changes in a temporal scale remain unexplored in marine plants. The seagrass Posidonia oceanica ranks among the slowest-growing and longest-living plants on Earth, and is particularly vulnerable to sea warming and local anthropogenic pressures. Here, we analysed the dynamics of DNAm changes in plants collected from coastal areas differentially impacted by eutrophication (i.e. oligotrophic, Ol; eutrophic, Eu) and exposed to abiotic stressors (nutrients, temperature increase and their combination). Levels of global DNAm (% 5-mC) and the expression of key genes involved in DNAm were assessed after one, two and five weeks of exposure. Results revealed a clear differentiation between plants, depending on environmental stimuli, time of exposure and plants' origin. % 5-mC levels were higher during the initial stress exposure especially in Ol plants, which upregulated almost all genes involved in DNAm. Contrarily, Eu plants showed lower expression levels, which increased under chronic exposure to stressors, particularly to temperature. These findings show that DNAm is dynamic in P. oceanica during stress exposure and underlined that environmental epigenetic variations could be implicated in the regulation of acclimation and phenotypic differences depending on local conditions.

Local environment modulates whole-transcriptome expression in the seagrass Posidonia oceanica under warming and nutrients excess.

The intensification of anomalous events of seawater warming and the co-occurrence with local anthropogenic stressors are threatening coastal marine habitats, including seagrasses, which form extensive underwater meadows. Eutrophication highly affects coastal environments, potentially summing up to the widespread effects of global climate changes. In the present study, we investigated for the first time in seagrasses, the transcriptional response of different plant organs (i.e., leaf and shoot apical meristem, SAM) of the Mediterranean seagrass Posidonia oceanica growing in environments with a different history of nutrient enrichment. To this end, a mesocosm experiment exposing plants to single (nutrient enrichment or temperature increase) and multiple stressors (nutrient enrichment plus temperature increase), was performed. Results revealed a differential transcriptome regulation of plants under single and multiple stressors, showing an organ-specific sensitivity depending on plants' origin. While leaf tissues were more responsive to nutrient stress, SAM revealed a higher sensitivity to temperature treatments, especially in plants already impacted in their native environment. The exposure to stress conditions induced the modulation of different biological processes. Plants living in an oligotrophic environment were more responsive to nutrients compared to plants from a eutrophic environment. Evidences that epigenetic mechanisms were involved in the regulation of transcriptional reprogramming were also observed in both plants' organs. These results represent a further step in the comprehension of seagrass response to abiotic stressors pointing out the importance of local pressures in a global warming scenario.

Unusually Warm Summer Temperatures Exacerbate Population and Plant Level Response of Posidonia oceanica to Anthropogenic Nutrient Stress.

Posidonia oceanica is a key foundation species in the Mediterranean providing valuable ecosystem services. However, this species is particularly vulnerable towards high coastal nutrient inputs and the rising frequency of intense summer heat waves, but their combined effect in situ has received little attention so far. Here, we investigated the effects of in situ nutrient addition during an unusually warm summer over a 4-month period, comparing different morphological, physiological and biochemical population metrics of seagrass meadows growing in protected areas (Ischia) with meadows already exposed to significant anthropogenic pressure (Baia - Gulf of Pozzuoli). Our study highlights that the effects of warmer than usual summer temperatures on the population level of seagrass meadows can be exacerbated if the plants are already exposed to higher anthropogenic pressures. Morphological and population level indicators mainly changed over time, possibly impacted by season and the warmer temperatures, and displayed more pronounced reductions in seagrasses from impacted sites. The additional nutrient supply had even more deleterious effects, as shown by a decrease in approximately 67% in cover in fertilized plots at high impacted sites and 33% at low impacted sites. Moreover, while rhizome starch concentration showed a seasonal increase in plants from low impacted sites it displayed a trend of a 27% decrease in fertilized plots of the high impacted sites. Epiphyte biomass was approximately four-fold higher on leaves of plants growing in impacted sites and even doubled with the additional nutrient input. Predicting and anticipating stress in P. oceanica is of crucial importance for conservation and management efforts, given the limited colonizing and reproductive ability and extremely slow growth of this ecosystem engineer. Our results suggest that monitoring efforts should focus especially on leaf area index (LAI), carbohydrate concentrations in the rhizomes, and epiphyte cover on leaves as indicators of the onset of stress in Posidonia oceanica, which can be used by decision makers to take appropriate measures before damage to the ecosystem becomes irreversible, minimize future human interference and strengthen the resilience of these important ecosystems.

Photosynthetic Responses of Turf-forming Red Macroalgae to High CO2 Conditions.

Seaweeds are important components of near-shore ecosystems as primary producers, foundation species, and biogeochemical engineers. Seaweed communities are likely to alter under predicted climate change scenarios. We tested the physiological responses of three perennial, turf-building, intertidal rhodophytes, Mastocarpus stellatus, Osmundea pinnatifida, and the calcified Ellisolandia elongata, to elevated pCO2 over 6 weeks. Responses varied between these three species. E. elongata was strongly affected by high pCO2 , whereas non-calcified species were not. Elevated pCO2 did not induce consistent responses of photosynthesis and respiration across these three species. While baseline photophysiology differed significantly between species, we found few clear effects of elevated pCO2 on this aspect of macroalgal physiology. We found effects of within-species variation in elevated pCO2 response in M. stellatus, but not in the other species. Overall, our data confirm the sensitivity of calcified macroalgae to elevated pCO2 , but we found no evidence suggesting that elevated pCO2 conditions will have a strong positive or negative impact on photosynthetic parameters in non-calcified macroalgae.

Ecophysiological and metabolic responses to interactive exposure to nutrients and copper excess in the brown macroalga Cystoseira tamariscifolia.

Global scenarios evidence that contamination due to anthropogenic activities occur at different spatial-temporal scales, being important stressors: eutrophication, due to increased nutrient inputs; and metal pollution, mostly derived from industrial activities. In this study, we investigated ecophysiological and metabolic responses to copper and nutrient excess in the brown macroalga Cystoseira tamariscifolia. Whole plants were incubated in an indoor system under control conditions, two levels of nominal copper (0.5 and 2.0µM), and two levels of nutrient supply for two weeks. Maximal quantum yield (Fv/Fm) and maximal electron transport rate (ETRmax) increased under copper exposure. Photosynthetic pigments and phenolic compounds (PC) increased under the highest copper levels. The intra-cellular copper content increased under high copper exposure in both nutrient conditions. C. tamariscifolia from the Atlantic displayed efficient metal exclusion mechanisms, since most of the total copper accumulated by the cell was bound to the cell wall.