Calcium-Dependent Hydrogen Peroxide Mediates Hydrogen-Rich Water-Reduced Cadmium Uptake in Plant Roots

Hydrogen gas (H 2 ) 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 H 2 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 H 2 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 (Ca 2+ ) across the plasma membrane and apoplastic generation of hydrogen peroxide (H 2 O 2 ) by respiratory burst oxidase homolog (BcRbohD). The reduction in root Cd uptake was associated with the application of exogenous HRW or H 2 O 2 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 Ca 2+ influx, and Cd-induced Ca 2+ leakage was alleviated. Two Ca 2+ channel blockers, gadolinium ion (Gd 3+ ) and lanthanum ion (La 3+ ), eliminated the HRW-induced increase in BcRbohD expression, H 2 O 2 production, and Cd 2+ influx inhibition. Collectively, our results suggest that the Cd-protective effect of H 2 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 H 2 in Cd accumulation and toxicity in plants.