Glutathione produced by γ-glutamyl cysteine synthetase acts downstream of hydrogen to positively influence lateral root branching.

Hydrogen gas (H2) mediation of lateral root (LR) branching was previously described. However, related signaling pathway is largely unexplored. In this study, we discovered that application with H2 using hydrogen-rich water, mimicking the responses of exogenous glutathione (GSH), not only enhanced GSH synthesis, but also induced tomato LR development. The changes in the transcripts of auxin signaling-related genes and cell cycle regulatory genes were matched with above phenotypes. The addition of H2 could trigger higher transcript levels of SlGSH1 and SlGSH2, encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS), confirming the stimulation of GSH synthesis. These responses were greatly abolished when the inhibitor of γ-ECS was applied. The inhibition in lateral root primordium development, especially in emergence stage, was also observed. Genetic evidence revealed that the defects in GSH production and lateral rooting in Arabidopsis cad2-1, a γ-ECS defective mutant, were obviously abolished in the presence of GSH compared to those in the presence of H2. Further evidence revealed that mRNA levels of target genes elicited by H2 in wild-type, were differentially impaired in mutant plants. Together, above data clearly demonstrated that γ-ECS-dependent GSH production might be closely associated with H2 control of LR branching.