High-molecular-weight species dominate the leaf soluble mercury pool: Rubisco as the primary binding protein

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.