A multi-layered approach to dissect photosynthetic and molecular adaptation of plants to increasing urban CO₂levels

Urban ecosystems, though limited in area, are hotspots of abiotic stress due to high CO₂ emissions, elevated temperatures, pollution, and altered hydrogeological cycle. Among these, CO₂ enrichment represents a persistent and often overlooked abiotic stress that shapes plant physiology and evolution. Our research aims to investigate how prolonged exposure to urban CO₂ levels influences plant responses at physiological, cellular, and molecular levels. Using Populus alba, we conduct comparative analyses between individuals growing in urban Florence and nearby non-urban green areas. We assess RuBisCO enzymatic activity through high-throughput assays and examine changes in chloroplast structure, leaf cuticle, and photosynthetic tissues via confocal and transmission electron microscopy. Data were then integrated with determination of enzymatic activity related to key enzymes (CAT, APX, SOD, peroxiredoxin, and glutathione reductase), to evaluate the redox state alteration due to altered CO2 levels. Chloroplast genomes are then analyzed to uncover potential structural rearrangements, mutations, and gene content changes associated with long-term CO₂ exposure. To complement these site-specific insights, we are performing a in-deep transcriptomic meta-analysis of Arabidopsis thaliana, Populus, and other plant species from publicly available datasets focused on CO₂ stress. This approach allows us to identify differentially expressed genes (DEGs), co-expression modules linked to CO₂ responses, organ-specific expression signatures, and novel candidate genes potentially involved in abiotic stress tolerance mechanisms. RNA-seq data were also coupled with metabarcoding analyses of fungal and bacterial endophytic communities to deepen the plant stress response from a holobiont perspective. This work was supported by the PRIN grant (MUR, 2022RYTHE3, Italy) and contributes to the identification of keystress-response markers and adaptive traits, supporting the selection of resilient species for urban environments and climate-adaptive planting strategies.