Adaptation Mechanisms Of Phaeodactylum Tricornutum Under Multiple Environmental Stresses

In the context of global change,the oceans are undergoing multifaceted changes in physical and chemical properties,including ocean acidification,seawater warming,and enhanced UVB radiation in the upper mixed layer.These changes significantly affect the growth,reproduction,metabolism and survival of various marine organisms,and consequently,the balance of marine ecosystems and their service functions are also altered.The responses of marine organisms under these multiple environmental changes are one of research priorities in the field of marine science.In this study,to explore the molecular mechanisms of phytoplankton under multiple environmental changes,we conducted a long-term experimental evolution experiment by culturing a model marine diatom Phaeodactylum tricornutum in ocean acidification and/or warming conditions for more than two years,including the control（CO₂:400 ppmv;Temperature:15°C,LCLT）,ocean acidification（CO₂:1000 ppmv;Temperature:15°C,HCLT）,ocean warming（CO₂:400 ppmv;Temperature:20°C,LCHT）,ocean acidification+warming（CO₂:1000 ppmv;Temperature:20°C,HCHT）.We investigated their physiological and molecular responses to ocean acidification and/or warming by applying an integrated approach of transcriptomics,epigenetics（DNA methylation）and algal physiology.The combined effects of ocean acidification,warming and enhanced UVB radiation on P.tricornutum were also examined in the present study.The main findings are listed as below:In this study,significant changes were observed at the gene transcriptional level in P.tricornutum,which have grown in ocean acidification,warming and acidification combined warming conditions（approximately 1600-1700 generations of growth）for two years.The results showed that genes involved in carbon metabolism pathways such as 3-phosphoglyceraldehyde dehydrogenase were significantly down-regulated,while genes involved in fatty acid metabolism-related genes such as acetyl coenzyme A carboxylase were significantly up-regulated.In long-term warming adapted cultures,the expression of photosynthesis-related genes such as light-trapping chlorophyll-binding proteins were significantly suppressed,resulting in a 72%decrease in maximum photosynthetic rate at warming.In the long-term acidification and warming adapted cultures,the proteasomal pathway of P.tricornutum was significantly down-regulated and more genes in carbon metabolism pathway,such as glutamate dehydrogenase were significantly up-or down-regulated compared with that under ocean acidification or warming alone.The results demonstrated a constraint of acidification on warming,that is the genes significantly up-or down-regulated under warming conditions alone were less stimulated or inhibited in the warming combined with acidification condition,such as phosphoenolpyruvate carboxylase in photosynthesis.In parallel with the transcriptomics analysis,P.tricornutum cultures under four culture conditions（control,ocean acidification,warming,ocean acidification+warming）were also collected for DNA methylation analysis analysis at the end of the2-years long-term adaptation experiments.The results showed that the genome-wide DNA methylation level of P.tricornutum in all four culture conditions（control,ocean acidification,warming,ocean acidification+warming）was low,about 0.5%,with m CG being the predominant methylation type.Correlation analysis between DNA methylation and gene expression showed that m CG showed a positive correlation with gene expression,while m CHG showed the opposite trend.Differentially methylated genes covered by differentially methylated regions（DMRs）were enriched in key pathways such as central carbon metabolism,amino acid metabolism,ribosome biogenesis,terpene biosynthesis and degradation of misfolded proteins,and the transcript levels of differentially expressed genes in these pathways were mostly down-regulated,so we hypothesized that DNA methylation in P.tricornutum under long-term stress culture may affect gene expression to adapt to environmental changes.However,the adaptations of any organism to environmental change come with costs,i.e.,,trade-offs.To investigate the trade-offs associated with the adaptations of diatom to ocean acidification and/or warming,P.tricornutum populations that have grown in ocean acidification and/or warming conditions for 3.5 years were exposed to two levels of UVB radiation（0.1 W/m² and 0.2 W/m²）for 2 weeks,and various physiological parameters were measured.The results showed that the high intensity of UVB radiation（0.2 W/m²）had a negative impact on the physiology of the algae,for example,led to a significant reduction in growth rate of 75.8%.At the same time,it was found that long-term adaptation to warming could alleviate the negative effects of UVB radiation,suggesting an antagonistic interaction between warming and UVB.However,these antagonistic interactions were also found to be modulated by ocean acidification.In summary,our study demonstrated that P.tricornutum exhibited adaptive evolution to ocean acidification and/or warming with substantial changes at gene transcriptional level and DNA methylation level.And DNA methylation can act cooperatively with gene transcription to contribute to these adaptations.Furthermore,our results showed long-term adaptation to sea surface warming and rising CO₂ may alter this diatom’s sensitivity to elevated UVB radiation in the environment.Our study provides new evidence for the response of marine phytoplankton to multiple environmental changes and contributes to a better understanding of the impact of climate change on ecosystems.