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 (Brassicacampestris 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.