Conservative versus flexible physiological and metabolic acclimation of Norway spruce and European beech under elevated CO2, nitrogen supply, and water availability.

Elevated atmospheric CO2 (EC) can enhance photosynthesis, biomass accumulation and water-use efficiency (WUE), but responses depend on water and nutrient availability and differ among tree species. Multifactorial studies addressing these interactions remain scarce. We examined interactive effects of EC (~700 μmol mol-1), nitrogen supply (nitrogen addition, N+; no nitrogen addition, N-) and water availability (well-watered, WW; drought-stressed, DS) on physiological and metabolic responses of more anisohydric European beech (Fagus sylvatica), which maintains stomatal opening longer during drought, and more isohydric Norway spruce (Picea abies), which closes stomata earlier to stabilise leaf water status. Across both species, EC produced the largest shifts in gas exchange and metabolite profiles, while water and nitrogen availability strongly modified these responses. EC generally increased net CO2 assimilation and WUE and reduced stomatal conductance, although responses differed between species and treatments; WUE was highest under EC×DS×N+. Species divergence was most evident in biochemical acclimation. In beech, EC markedly reduced photosynthetic capacity, reflected in lower Rubisco carboxylation capacity (VCmax) and electron transport rate (Jmax) across water and nitrogen treatments. This response was accompanied by lower succinate and foliar N and by resource-dependent shifts in amino acids, phenolic and stress-related metabolites, consistent with greater metabolic reorganization and photosynthetic downregulation. In contrast, spruce maintained comparatively stable VCmax and Jmax under EC, including under DS and N- conditions, together with comparatively stable primary metabolism and sustained phenolic-based defence. Overall, beech showed greater physiological and metabolic flexibility but stronger downregulation of photosynthetic capacity under EC, whereas spruce maintained a more conservative physiological strategy and greater biochemical stability. These contrasting responses highlight species-specific acclimation to CO2 enrichment and its modulation by water and nitrogen availability.