Genotype-dependent sulfur depletion is linked to antioxidant dysfunction and growth responses to As, Cd, and Pb in Alnus glutinosa

Alnus glutinosa L. (black alder) is a promising phytoremediation candidate due to its N fixation, high biomass, and adaptability, yet responses to As, Cd, and Pb remain poorly understood. Three alder genotypes were exposed to increasing metal(loid) concentrations under controlled conditions hydroponically, evaluating growth, metal(loid) and elemental accumulation, antioxidant enzymes and secondary metabolites. Generalized Linear Model and Principal Component analyses revealed substantial genotype- and metal-dependent effects, with Cd causing the strongest inhibition and oxidative stress, while low-dose As and Pb induced hormetic responses (~ 20% shoot height, ~ 10% chlorophyll increases). Cd showed root accumulation with low shoot translocation; Pb was almost exclusively root sequestered. Notably, S levels declined universally. Cumulative results suggest that S depletion forms part of a broader stress-response pattern in which metal-specific uptake and tissue partitioning are followed by genotype-specific antioxidant impairments, potentially through putative effects on the ascorbate-glutathione cycle. Random Forest analysis identified glutathione-S-transferase and chlorophyll as strong metal-type predictors, while lipid peroxidation and ascorbate peroxidase best distinguished genotypes. Results support a hierarchical response pattern in which metal-specific accumulation and nutrient disruption are followed by downstream oxidative stress and growth inhibition, although the underlying sulfur-related mechanisms are inferred and require further validation. These findings highlight the potential importance of S metabolism's critical role in A. glutinosa metal(loid) tolerance and inform future phytoremediation breeding strategies.