Thermo-priming triggers species-specific physiological and transcriptome responses in Mediterranean seagrasses.

Heat-priming improves plants' tolerance to a recurring heat stress event. The underlying molecular mechanisms of heat-priming are largely unknown in seagrasses. Here, ad hoc mesocosm experiments were conducted with two Mediterranean seagrass species, Posidonia oceanica and Cymodocea nodosa. Plants were first exposed to heat-priming, followed by a heat-triggering event. A comprehensive assessment of plant stress response across different levels of biological organization was performed at the end of the triggering event. Morphological and physiological results showed an improved response of heat-primed P. oceanica plants while in C. nodosa both heat- and non-primed plants enhanced their growth rates at the end of the triggering event. As resulting from whole transcriptome sequencing, molecular functions related to several cellular compartments and processes were involved in the response to warming of non-primed plants, while the response of heat-primed plants involved a limited group of processes. Our results suggest that seagrasses acquire a primed state during the priming event, that eventually gives plants the ability to induce a more energy-effective response when the thermal stress event recurs. Different species may differ in their ability to perform an improved heat stress response after priming. This study provides pioneer molecular insights into the emerging topic of seagrass stress priming and may benefit future studies in the field.

Seagrass genomes reveal ancient polyploidy and adaptations to the marine environment.

We present chromosome-level genome assemblies from representative species of three independently evolved seagrass lineages: Posidonia oceanica, Cymodocea nodosa, Thalassia testudinum and Zostera marina. We also include a draft genome of Potamogeton acutifolius, belonging to a freshwater sister lineage to Zosteraceae. All seagrass species share an ancient whole-genome triplication, while additional whole-genome duplications were uncovered for C. nodosa, Z. marina and P. acutifolius. Comparative analysis of selected gene families suggests that the transition from submerged-freshwater to submerged-marine environments mainly involved fine-tuning of multiple processes (such as osmoregulation, salinity, light capture, carbon acquisition and temperature) that all had to happen in parallel, probably explaining why adaptation to a marine lifestyle has been exceedingly rare. Major gene losses related to stomata, volatiles, defence and lignification are probably a consequence of the return to the sea rather than the cause of it. These new genomes will accelerate functional studies and solutions, as continuing losses of the 'savannahs of the sea' are of major concern in times of climate change and loss of biodiversity.

Ocean acidification impairs seagrass performance under thermal stress in shallow and deep water.

Despite the effects of ocean acidification (OA) on seagrasses have been widely investigated, predictions of seagrass performance under future climates need to consider multiple environmental factors. Here, we performed a mesocosm study to assess the effects of OA on shallow and deep Posidonia oceanica plants. The experiment was run in 2021 and repeated in 2022, a year characterized by a prolonged warm water event, to test how the effects of OA on plants are modulated by thermal stress. The response of P. oceanica to experimental conditions was investigated at different levels of biological organization. Under average seawater temperature, there were no effects of OA in both shallow and deep plants, indicating that P. oceanica is not limited by current inorganic carbon concentration, regardless of light availability. In contrast, under thermal stress, exposure of plants to OA increased lipid peroxidation and decreased photosynthetic performance, with deep plants displaying higher levels of heat stress, as indicated by the over-expression of stress-related genes and the activation of antioxidant systems. In addition, warming reduced plant growth, regardless of seawater CO2 and light levels, suggesting that thermal stress may play a fundamental role in the future development of seagrass meadows. Our results suggest that OA may exacerbate the negative effects of future warming on seagrasses.

Signs of local adaptation by genetic selection and isolation promoted by extreme temperature and salinity in the Mediterranean seagrass Posidonia oceanica.

Adaptation to local conditions is known to occur in seagrasses; however, knowledge of the genetic basis underlying this phenomenon remains scarce. Here, we analysed Posidonia oceanica from six sites within and around the Stagnone di Marsala, a semi-enclosed coastal lagoon where salinity and temperature exceed the generally described tolerance thresholds of the species. Sea surface temperatures (SSTs) were measured and plant samples were collected for the assessment of morphology, flowering rate and for screening genome-wide polymorphisms using double digest restriction-site-associated DNA sequencing. Results demonstrated more extreme SSTs and salinity levels inside the lagoon than the outer lagoon regions. Morphological results showed significantly fewer and shorter leaves and reduced rhizome growth of P. oceanica from the inner lagoon and past flowering events were recorded only for a meadow farthest away from the lagoon. Using an array of 51,329 single nucleotide polymorphisms, we revealed a clear genetic structure among the study sites and confirmed the genetic isolation and high clonality of the innermost site. In all, 14 outlier loci were identified and annotated with several proteins including those relate to plant stress response, protein transport and regulators of plant-specific developmental events. Especially, five outlier loci showed maximum allele frequency at the innermost site, likely reflecting adaptation to the extreme temperature and salinity regimes, possibly due to the selection of more resistant genotypes and the progressive restriction of gene flow. Overall, this study helps us to disentangle the genetic basis of seagrass adaptation to local environmental conditions and may support future works on assisted evolution in seagrasses.

A trait-based framework for seagrass ecology: Trends and prospects.

In the last three decades, quantitative approaches that rely on organism traits instead of taxonomy have advanced different fields of ecological research through establishing the mechanistic links between environmental drivers, functional traits, and ecosystem functions. A research subfield where trait-based approaches have been frequently used but poorly synthesized is the ecology of seagrasses; marine angiosperms that colonized the ocean 100M YA and today make up productive yet threatened coastal ecosystems globally. Here, we compiled a comprehensive trait-based response-effect framework (TBF) which builds on previous concepts and ideas, including the use of traits for the study of community assembly processes, from dispersal and response to abiotic and biotic factors, to ecosystem function and service provision. We then apply this framework to the global seagrass literature, using a systematic review to identify the strengths, gaps, and opportunities of the field. Seagrass trait research has mostly focused on the effect of environmental drivers on traits, i.e., "environmental filtering" (72%), whereas links between traits and functions are less common (26.9%). Despite the richness of trait-based data available, concepts related to TBFs are rare in the seagrass literature (15% of studies), including the relative importance of neutral and niche assembly processes, or the influence of trait dominance or complementarity in ecosystem function provision. These knowledge gaps indicate ample potential for further research, highlighting the need to understand the links between the unique traits of seagrasses and the ecosystem services they provide.

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.

2b-RAD Genotyping of the Seagrass Cymodocea nodosa Along a Latitudinal Cline Identifies Candidate Genes for Environmental Adaptation.

Plant populations distributed along broad latitudinal gradients often show patterns of clinal variation in genotype and phenotype. Differences in photoperiod and temperature cues across latitudes influence major phenological events, such as timing of flowering or seed dormancy. Here, we used an array of 4,941 SNPs derived from 2b-RAD genotyping to characterize population differentiation and levels of genetic and genotypic diversity of three populations of the seagrass Cymodocea nodosa along a latitudinal gradient extending across the Atlantic-Mediterranean boundary (i.e., Gran Canaria-Canary Islands, Faro-Portugal, and Ebro Delta-Spain). Our main goal was to search for potential outlier loci that could underlie adaptive differentiation of populations across the latitudinal distribution of the species. We hypothesized that such polymorphisms could be related to variation in photoperiod-temperature regime occurring across latitudes. The three populations were clearly differentiated and exhibited diverse levels of clonality and genetic diversity. Cymodocea nodosa from the Mediterranean displayed the highest genotypic richness, while the Portuguese population had the highest clonality values. Gran Canaria exhibited the lowest genetic diversity (as observed heterozygosity). Nine SNPs were reliably identified as outliers across the three sites by two different methods (i.e., BayeScan and pcadapt), and three SNPs could be associated to specific protein-coding genes by screening available C. nodosa transcriptomes. Two SNPs-carrying contigs encoded for transcription factors, while the other one encoded for an enzyme specifically involved in the regulation of flowering time, namely Lysine-specific histone demethylase 1 homolog 2. When analyzing biological processes enriched within the whole dataset of outlier SNPs identified by at least one method, "regulation of transcription" and "signalling" were among the most represented. Our results highlight the fundamental importance signal integration and gene-regulatory networks, as well as epigenetic regulation via DNA (de)methylation, could have for enabling adaptation of seagrass populations along environmental gradients.

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.

m6A RNA Methylation in Marine Plants: First Insights and Relevance for Biological Rhythms.

Circadian regulations are essential for enabling organisms to synchronize physiology with environmental light-dark cycles. Post-transcriptional RNA modifications still represent an understudied level of gene expression regulation in plants, although they could play crucial roles in environmental adaptation. N6-methyl-adenosine (m6A) is the most prevalent mRNA modification, established by "writer" and "eraser" proteins. It influences the clockwork in several taxa, but only few studies have been conducted in plants and none in marine plants. Here, we provided a first inventory of m6A-related genes in seagrasses and investigated daily changes in the global RNA methylation and transcript levels of writers and erasers in Cymodocea nodosa and Zostera marina. Both species showed methylation peaks during the dark period under the same photoperiod, despite exhibiting asynchronous changes in the m6A profile and related gene expression during a 24-h cycle. At contrasting latitudes, Z. marina populations displayed overlapping daily patterns of the m6A level and related gene expression. The observed rhythms are characteristic for each species and similar in populations of the same species with different photoperiods, suggesting the existence of an endogenous circadian control. Globally, our results indicate that m6A RNA methylation could widely contribute to circadian regulation in seagrasses, potentially affecting the photo-biological behaviour of these plants.

Adaptive responses along a depth and a latitudinal gradient in the endemic seagrass Posidonia oceanica.

Seagrass meadows provide important ecosystem services and are critical for the survival of the associated invertebrate community. However, they are threatened worldwide by human-driven environmental change. Understanding the seagrasses' potential for adaptation is critical to assess not only their ability to persist under future global change scenarios, but also to assess the persistence of the associated communities. Here we screened a wild population of Posidonia oceanica, an endemic long-lived seagrass in the Mediterranean Sea, for genes that may be target of environmental selection, using an outlier and a genome-wide transcriptome analysis. We identified loci where polymorphism or differential expression was associated with either a latitudinal or a bathymetric gradient, as well as with both gradients in an effort to identify loci associated with temperature and light. We found the candidate genes underlying growth and immunity to be divergent between populations adapted to different latitudes and/or depths, providing evidence for local adaptation. Furthermore, we found evidence of reduced gene flow among populations including adjacent populations. Reduced gene flow, combined with low sexual recombination, small effective population size, and long generation time of P. oceanica raises concerns for the long-term persistence of this species, especially in the face of rapid environmental change driven by human activities.

Seagrass collapse due to synergistic stressors is not anticipated by phenological changes.

Seagrasses are globally declining and often their loss is due to synergies among stressors. We investigated the interactive effects of eutrophication and burial on the Mediterranean seagrass, Posidonia oceanica. A field experiment was conducted to estimate whether shoot survival depends on the interactive effects of three levels of intensity of both stressors and to identify early changes in plants (i.e., morphological, physiological and biochemical, and expression of stress-related genes) that may serve to detect signals of imminent shoot density collapse. Sediment burial and nutrient enrichment produced interactive effects on P. oceanica shoot survival, as high nutrient levels had the potential to accelerate the regression of the seagrass exposed to high burial (HB). After 11 weeks, HB in combination with either high or medium nutrient enrichment caused a shoot loss of about 60%. Changes in morphology were poor predictors of the seagrass decline. Likewise, few biochemical variables were associated with P. oceanica survival (the phenolics, ORAC and leaf δ34S). In contrast, the expression of target genes had the highest correlation with plant survival: photosynthetic genes (ATPa, psbD and psbA) were up-regulated in response to high burial, while carbon metabolism genes (CA-chl, PGK and GADPH) were down-regulated. Therefore, die-offs due to high sedimentation rate in eutrophic areas can only be anticipated by altered expression of stress-related genes that may warn the imminent seagrass collapse. Management of local stressors, such as nutrient pollution, may enhance seagrass resilience in the face of the intensification of extreme climate events, such as floods.

Molecular Mechanisms behind the Physiological Resistance to Intense Transient Warming in an Iconic Marine Plant.

The endemic Mediterranean seagrass Posidonia oceanica is highly threatened by the increased frequency and intensity of heatwaves. Meadows of the species offer a unique opportunity to unravel mechanisms marine plants activate to cope transient warming, since their wide depth distribution impose divergent heat-tolerance. Understanding these mechanisms is imperative for their conservation. Shallow and deep genotypes within the same population were exposed to a simulated heatwave in mesocosms, to analyze their transcriptomic and photo-physiological responses during and after the exposure. Shallow plants, living in a more unstable thermal environment, optimized phenotype variation in response to warming. These plants showed a pre-adaptation of genes in anticipation of stress. Shallow plants also showed a stronger activation of heat-responsive genes and the exclusive activation of genes involved in epigenetic mechanisms and in molecular mechanisms that are behind their higher photosynthetic stability and respiratory acclimation. Deep plants experienced higher heat-induced damage and activated metabolic processes for obtaining extra energy from sugars and amino acids, likely to support the higher protein turnover induced by heat. In this study we identify transcriptomic mechanisms that may facilitate persistence of seagrasses to anomalous warming events and we discovered that P. oceanica plants from above and below the mean depth of the summer thermocline have differential resilience to heat.

Long-term acclimation to reciprocal light conditions suggests depth-related selection in the marine foundation species Posidonia oceanica.

Phenotypic differences among populations of the same species reflect selective responses to ecological gradients produced by variations in abiotic and biotic factors. Moreover, they can also originate from genetic differences among populations, due to a reduced gene flow. In this study, we examined the extent of differences in photo-acclimative traits of Posidonia oceanica (L.) Delile clones collected above and below the summer thermocline (i.e., -5 and -25 m) in a continuous population extending along the water depth gradient. During a reciprocal light exposure and subsequent recovery in mesocosms, we assessed degree of phenotypic plasticity and local adaptation of plants collected at different depths, by measuring changes in several traits, such as gene expression of target genes, photo-physiological features, and other fitness-related traits (i.e., plant morphology, growth, and mortality rates). Samples were also genotyped, using microsatellite markers, in order to evaluate the genetic divergence among plants of the two depths. Measures collected during the study have shown a various degree of phenotypic changes among traits and experimental groups, the amount of phenotypic changes observed was also dependent on the type of light environments considered. Overall plants collected at different depths seem to be able to acclimate to reciprocal light conditions in the experimental time frame, through morphological changes and phenotypic buffering, supported by the plastic regulation of a reduced number of genes. Multivariate analyses indicated that plants cluster better on the base of their depth origin rather than the experimental light conditions applied. The two groups were genetically distinct, but the patterns of phenotypic divergence observed during the experiment support the hypothesis that ecological selection can play a role in the adaptive divergence of P. oceanica clones along the depth gradient.

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.

Physiological and molecular evidence of differential short-term heat tolerance in Mediterranean seagrasses.

The increase in extreme heat events associated to global warming threatens seagrass ecosystems, likely by affecting key plant physiological processes such as photosynthesis and respiration. Understanding species' ability to acclimate to warming is crucial to better predict their future trends. Here, we study tolerance to warming in two key Mediterranean seagrasses, Posidonia oceanica and Cymodocea nodosa. Stress responses of shallow and deep plants were followed during and after short-term heat exposure in mesocosms by coupling photo-physiological measures with analysis of expression of photosynthesis and stress-related genes. Contrasting tolerance and capacity to heat acclimation were shown by shallow and deep P. oceanica ecotypes. While shallow plants acclimated through respiratory homeostasis and activation of photo-protective mechanisms, deep ones experienced photosynthetic injury and impaired carbon balance. This suggests that P. oceanica ecotypes are thermally adapted to local conditions and that Mediterranean warming will likely diversely affect deep and shallow meadow stands. On the other hand, contrasting mechanisms of heat-acclimation were adopted by the two species. P. oceanica regulates photosynthesis and respiration at the level of control plants while C. nodosa balances both processes at enhanced rates. These acclimation discrepancies are discussed in relation to inherent attributes of the two species.

The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea.

Seagrasses colonized the sea on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet. Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes, genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae and that is important for ion homoeostasis, nutrient uptake and O2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming, to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants.

Insights into the transcriptome of the marine copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi.

Diatoms dominate productive regions in the oceans and have traditionally been regarded as sustaining the marine food chain to top consumers and fisheries. However, many of these unicellular algae produce cytotoxic oxylipins that impair reproductive and developmental processes in their main grazers, crustacean copepods. The molecular mode of action of diatoms and diatom oxylipins on copepods is still unclear. In the present study we generated two Expressed Sequence Tags (ESTs) libraries of the copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi and the cryptophyte Rhodomonas baltica as a control, using suppression subtractive hybridization (SSH). Our aim was to investigate differences in the transcriptome between females fed toxic and non-toxic food and identify differentially expressed genes and biological processes targeted by this diatom. We produced 947 high quality ESTs from both libraries, 475 of which were functionally annotated and deposited in GenBank. Clustering and assembling of ESTs resulted in 376 unique transcripts, 200 of which were functionally annotated. Functional enirchment analysis between the two SSH libraries showed that ESTs belonging to biological processes such as response to stimuli, signal transduction, and protein folding were significantly over-expressed in the S. marinoi-fed C. helgolandicus compared to R. baltica-fed C. helgolandicus library. These findings were confirmed by RT-qPCR analysis. In summary, 2 days of feeding on S. marinoi activated a generalized Cellular Stress Response (CSR) in C. helgolandicus, by over-expressing genes of molecular chaperones and signal transduction pathways that protect the copepod from the immediate effects of the diatom diet. Our results provide insights into the response of copepods to a harmful diatom diet at the transcriptome level, supporting the hypothesis that diatom oxylipins elicit a stress response in the receiving organism. They also increase the genomic resources for this copepod species, whose importance could become ever more relevant for pelagic ecosystem functioning in European waters due to global warming.

Acclimation to different depths by the marine angiosperm Posidonia oceanica: transcriptomic and proteomic profiles.

For seagrasses, seasonal and daily variations in light and temperature represent the mains factors driving their distribution along the bathymetric cline. Changes in these environmental factors, due to climatic and anthropogenic effects, can compromise their survival. In a framework of conservation and restoration, it becomes crucial to improve our knowledge about the physiological plasticity of seagrass species along environmental gradients. Here, we aimed to identify differences in transcriptomic and proteomic profiles, involved in the acclimation along the depth gradient in the seagrass Posidonia oceanica, and to improve the available molecular resources in this species, which is an important requisite for the application of eco-genomic approaches. To do that, from plant growing in shallow (-5 m) and deep (-25 m) portions of a single meadow, (i) we generated two reciprocal Expressed Sequences Tags (EST) libraries using a Suppressive Subtractive Hybridization (SSH) approach, to obtain depth/specific transcriptional profiles, and (ii) we identified proteins differentially expressed, using the highly innovative USIS mass spectrometry methodology, coupled with 1D-SDS electrophoresis and labeling free approach. Mass spectra were searched in the open source Global Proteome Machine (GPM) engine against plant databases and with the X!Tandem algorithm against a local database. Transcriptional analysis showed both quantitative and qualitative differences between depths. EST libraries had only the 3% of transcripts in common. A total of 315 peptides belonging to 64 proteins were identified by mass spectrometry. ATP synthase subunits were among the most abundant proteins in both conditions. Both approaches identified genes and proteins in pathways related to energy metabolism, transport and genetic information processing, that appear to be the most involved in depth acclimation in P. oceanica. Their putative rules in acclimation to depth were discussed.