Final Report Summary - HEATGRASS (Tolerance to HEAT stress induced by climate change in the seaGRASS Posidonia oceanica) The general objective of the HEATGRASS project was to investigate how the increase in the frequency and intensity of summer heat waves affect the valuable Posidonia oceanica meadows in the Mediterranean Sea, as a basis to forecast and to assess how they will respond under the effects of climate change. The project, based on two mesocosms experiments using plants from the extremes of main spatial thermal gradients (bathymetric and latitudinal), was widened to include another important Mediterranean seagrass species (i.e. Cymodocea nodosa) in order to allow a comparison based on the different life strategies characteristic of the two species.HEATGRASS has produced research results of high quality, presented its outcome at major international conferences and meetings and published in national newspapers (Italian and Spanish) and in high impact scientific journals. More results others in the process of publishing and new outcomes will continue to emerge after the end of the project thanks to the high amount of results and transcriptomic resources generated.Within the above mentioned general objective, the project followed specific objectives that are numbered from 1 to 4 and described below together with a summary of main results and conclusions: Obj 1- To analyse the stress responses and tolerance mechanisms of P. oceanica over the course and recovery period of a simulated heat wave in a mesocosms system. This first objective was approached in both mesocosm experiments of the project and the main results were:• Heat stress activated in P. oceanica a complex transcriptomic response to re-establish cell homeostasis and to protect and repair damaged proteins and membranes. This response enabled the species to overcome the simulated heat waves without evidencing signs of lethal injury and favouring its recovery after stress ending. Responses at the level of gene expression reflected the molecular basis underlying the physiological mechanisms of heat tolerance in the species.• Heat reversibly affected key physiological processes, such as photosynthesis and respiration, which altered the carbon balance of plants reducing their energetic resources in the long term. Heated plants enhanced the turnover of main components of the photosynthetic apparatus, activated photoprotective mechanisms, and promoted respiratory homeostasis as the main mechanisms of heat tolerance. Besides, heated plants experienced growth inhibition to divert resources from growth to support the costs associated with the maintenance of an intense heat response.• The exposure to a heat wave induced massive flowering in P. oceanica likely as stress responsive mechanism also showed by higher plants. This allows to enhance genetic mix by sexual reproduction, to ensure a progeny when in adverse conditions and/or to escape from the adverse thermal conditions.• Heat stress responses in P. oceanica and the species’ ability to activate heat tolerance mechanisms differed among genotypes from contrasting thermal environments (see specific objectives 2 and 3 below) reflecting their local adaptation to the thermal conditions under which they growth.In conclusion, our findings support the hypothesis that P. oceanica is able to tolerate anomalous heat events although with some plant constraints. These alterations have the potential to alter the ecological functions and socio economical services that P. oceanica meadows play in the Mediterranean coastal area. However, heat sensitivity and the capacity of individuals to activate tolerance mechanisms during a heat wave depended on the thermal conditions under which plants developed, stressing their local adaptation and suggesting that populations will differentially respond to this climatic threat. These findings have an important impact on research, management and monitoring of P. oceanica meadows in the context of climate change; with direct implications for restoration (e.g. transplanting, genetic rescue) and conservation prioritisation within European coastal waters and for the establishment of specific actions for climate change adaptation by EU member states.Obj 2-To assess differences in the adaptive responses of P. oceanica genotypes from thermally contrasting depths within single populations. To approach this objective, shallow and deep P. oceanica and C. nodosa plants where experimentally exposed to short-term heat stress and subsequently allowed to recover. The main results were:• Shallow and deep P. oceanica genotypes manifested contrasting tolerance and capacity to acclimate to heat. Shallow plants evidenced thermal acclimation by reestablishing the balance between leaf respiration and photosynthesis, whereas the leaf carbon balance of deep plants progressively decreased due to photosynthetic injury and inability to attain respiratory homeostasis.• Shallow P. oceanica plants activated a more complete and intense transcriptomic response than deep plants and evidenced higher constitutive levels and stronger responsiveness of genes involved in plant thermo-tolerance. They, in addition, showed the exclusive capacity to activate the molecular mechanisms underlying their photosynthetic stability and respiratory acclimation capacity to heat.• Shallow plants from both species (P. oceanica and C. nodosa) evidenced heat-acclimation but through contrasting mechanisms, the former by regulating photosynthesis and respiration at control levels and the latter by balancing both processes at enhanced rates. These interspecific differences are related to inherent biological and ecological attributes of the species.In conclusion, the results support the idea that P. oceanica genotypes are thermally adapted to local conditions at a small spatial scale, and suggest that Mediterranean warming will diversely affect deep and shallow meadow stands. Deep meadow areas are more prone to experience regression than shallow ones as a result the more frequent occurrence of extreme summer heat events. Moreover, interspecific differences in heat acclimation between the two main Mediterranean seagrass species have the potential to produce species substitution and habitat shift with important consequences in ecosystem structure and functioning.The findings have important consequences for conservation and management of P. oceanica meadows as well as for seagrass research in the framework of climate change. In particular, they are helpful to improve the accuracy of predictions about the impact of climate change on Mediterranean seagrass meadows. Besides, heat-resistant genotypes can be used for assisted colonization or restoration of deep meadow margins as a way to preserve the functionality and the socio-economic services of these valuable coastal ecosystems. The identified physiological and molecular indicators of heat stress are promising for future monitoring strategies of Mediterranean seagrass meadows in the context of global warming.Obj 3- To assess differences in the adaptive responses of P. oceanica genotypes from contrasting localities along a latitudinal cline. This objective was approached experimentally exposing northern and southern P. oceanica and C. nodosa genotypes to a simulated heat wave and subsequently allowed to recover, and the main results were:• P. oceanica and C. nodosa genotypes from contrasting latitudes displayed contrasting heat acclimation capacities. Irrespective of the species, plants from warmer localities were less sensitive to heat than those from colder ones, although all of them completely recovered after heat stress cessation. Inter- and intraspecific differences in long-term heat responses were consistent with short-term responses detected in shallow and deep genotypes. • The photosynthetic sensitivity to heat and the ability for respiratory heat acclimation determined plant fitness along the heat wave exposure. Heat affected plants’ growth but did not increase mortality in either of the species or their populations. Heated plants activated antioxidant defence mechanisms but with an intensity that depended on the species and the thermal origin of plants.• Warming promoted sexual reproduction in P. oceanica plants form cold waters (northern latitudes). Flowering intensity in blooming genotypes was correlated with their genetic makeup, in particular with allelic richness and heterozygosity.In conclusion, our results demonstrate that P. oceanica was able to overcome a realistic heat wave but with a stress level that depended on the thermal conditions to which plants are adapted. In consequence, populations from higher latitudes (i.e. colder conditions) are more likely to experience regression in the coming decades as consequence of the intensification of summer heat waves. However, the massive flowering event induced by heat in these plants might have important consequences on the genetic diversity and resilience of their populations in future, long-term scenarios of climate change.These findings have important implications in the ecological and evolutionary context of the species in future scenarios of climate change. They also have strong implications in the management of P. oceanica meadows and are useful for an adequate monitoring of their evolution during and after extreme heat wave events. Moreover, our findings will be highly relevant to formulating environmental policies and adopting conservation measures to sustainable safeguarding European seagrass meadows.Obj 4- To select specific genes associated with tolerance and resilience of P. oceanica to thermal stress. This particular objective was approached using the transcriptional resources produced in both experiments of the project, and although the bioinformatic analysis of some of these resources are still in progress, the main results were:• Heat shock transcriptional factors (HSFs) were genes exclusively activated in heat-tolerant genotypes and favoured the activation of a complete and intense heat transcriptional reprogramming. Many of the genes uniquely activated in heat-tolerant genotypes encoded proteins involved in conferring thermotolerance (e.g. heat shock proteins, antioxidants), while most common heat responsive genes showed higher constitutive expression levels as well as higher expression levels in comparison to the more sensitive genotypes.• Genes codifying for pentatricopeptide repeat proteins (PPR) are also potentially involved in the physiological heat tolerance of P. oceanica. PPR proteins have profound effects on chloroplast and mitochondria function and consequently, on photosynthesis, respiration and hence, on P. oceanica responses to heat stress.• Genes involved in epigenetic mechanisms (e.g. histone methylation) are also putative candidate genes involved in P. oceanica thermotolerance. These genes were exclusively induced by heat in tolerant genotypes and are crucial in activating the plants’ immediate response to stress, as well as in the establishment of short- and long-term adaptation.In conclusion, our results provide evidence that plants growing in warm environments are more tolerant to heat than those from cold waters thanks to a more complete and intense transcriptomic response that allows their physiological acclimation to heat waves. The transcriptomic response to heat of P. oceanica genotypes from contrasting thermal environments provided valuable information about the key molecular mechanisms involved in thermal tolerance. Epigenetic mechanisms seems to have played a crucial role in the local adaptation of P. oceanica genotypes and consequently in their ability to activate molecular responses that enable their successful acclimation to heat. The massive transcriptomic resources generated in the project will continue shedding light about the mechanisms that the species adopt to contrast the threat of global warming. These findings, as well as future ones derived from the transcriptomic data, have strong implications for the management and conservation of P. oceanica meadows and are useful environmental tools to monitor Mediterranean meadows in the scope of global warming. They can be used as early warning indicators in monitoring programmes and can also assist to identify which meadows are more sensitive to heat to reinforce their monitoring and stablish adequate conservation plans.
