Environmental metabolomics reveals acclimatization mechanisms of European beech trees to natural altitudinal, light, and seasonal gradients

We explored how European beech (Fagus sylvatica L.) acclimatizes to natural environmental gradients of altitude (400-1100 m a.s.l.), within-canopy light (sun vs shade leaves), and season (July vs September) in Central European mountain forests using complementary non-targeted and targeted metabolomics. A set of 836 non-targeted KEGG-mapped metabolites, multivariate analyses (PERMANOVA, PCA, sPLS, Random Forest), and targeted profiling revealed three consistent patterns. (i) Across the metabolome, season explained the most variance, followed by canopy position and then altitude. (ii) From July to September, lipid-associated metabolite pools increased (notably pathways related to alpha-linolenic acid and ascorbate), whereas phytochemical pools decreased, with lower monoterpenoid pathway scores in late season. (iii) A pronounced interaction of altitude × season was evident for amino acids: branched-chain and related amino acids increased at high altitude in September, with the strongest responses in sun-acclimated leaves. Targeted analyses corroborated these trends for amino acids and phenolic compounds and showed associations between pathway-level shifts and leaf traits (e.g., RuBisCO content, leaf mass per area, intrinsic water-use efficiency). Collectively, the data indicate that interactive environmental gradients-dominated by seasonal progression-shape beech leaf metabolite abundances, highlighting pathway-level adjustments consistent with acclimatization to varying temperature, water, nutrient, and light conditions along altitude.