Foliar uptake of atmospheric elemental Hg (Hg(0)) significantly impacts Hg deposition and terrestrial cycling, yet the specific Hg species in leaves that govern its mobility, toxicity and post-senescence fates remain poorly characterized. Using Hg(0)-exposed poplar (Populus × euramericana) under controlled conditions and wild Erigeron annuus from a Hg mining area, this study investigated Hg species within the operationally soluble fraction in leaves, a mobile pool with translocation and transformation potential. Size exclusion chromatography (SEC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) revealed that soluble Hg primarily existed as high-molecular-weight species across the leaf total soluble fraction, formic acid extract, and chloroplast stroma, with an elution peak close to that of catalase (molecular weight: 240 kDa). Ultrafiltration also confirmed that 97.5% of soluble Hg was in the >100 kDa fraction (540 ± 186 ng/g leaf fresh weight). Protein-bound Hg was further identified as an important form via proteinase K digestion and SEC-ICP-MS analysis. Through gel electrophoretic separation and mass spectrometry, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as the dominant Hg-binding protein, likely attributed to Hg's strong binding for protein thiol groups. These findings revealed that high-molecular-weight Hg species, rather than low-molecular-weight Hg complexes, represent a major component of the soluble Hg pool. The localization of Rubisco in chloroplasts restricts Hg mobility within plants. Furthermore, as a key photosynthetic enzyme, Rubisco's binding to Hg provides a molecular link between atmospheric Hg uptake and potential photosynthetic impairment, requiring further research on Hg-protein interactions and their physiological consequences.
