Calcium-dependent hydrogen peroxide mediates hydrogen-rich water-reduced cadmium uptake in plant roots

Hydrogen gas (H₂) has a possible signaling role in many developmental and adaptive plant responses, including mitigating the harmful effects of cadmium (Cd) uptake from soil. We used electrophysiological and molecular approaches to understand how H₂ ameliorates Cd toxicity in pak choi (Brassica campestris ssp. chinensis). Exposure of pak choi roots to Cd resulted in a rapid increase in the intracellular H₂ production. Exogenous application of hydrogen-rich water (HRW) resulted in a Cd-tolerant phenotype, with reduced net Cd uptake and accumulation. We showed that this is dependent upon the transport of calcium ions (Ca²⁺) across the plasma membrane and apoplastic generation of hydrogen peroxide (H₂O₂) by respiratory burst oxidase homolog (BcRbohD). The reduction in root Cd uptake was associated with the application of exogenous HRW or H₂O₂ This reduction was abolished in the iron-regulated transporter1 (Atirt1) mutant of Arabidopsis (Arabidopsis thaliana), and pak choi pretreated with HRW showed decreased BcIRT1 transcript levels. Roots exposed to HRW had rapid Ca²⁺ influx, and Cd-induced Ca²⁺ leakage was alleviated. Two Ca²⁺ channel blockers, gadolinium ion (Gd³⁺) and lanthanum ion (La³⁺), eliminated the HRW-induced increase in BcRbohD expression, H₂O₂ production, and Cd²⁺ influx inhibition. Collectively, our results suggest that the Cd-protective effect of H₂ in plants may be explained by its control of the plasma membrane-based NADPH oxidase encoded by RbohD, which operates upstream of IRT1 and regulates root Cd uptake at both the transcriptional and functional levels. These findings provide a mechanistic explanation for the alleviatory role of H₂ in Cd accumulation and toxicity in plants.