Hydrogen peroxide is involved in methane-induced tomato lateral root formation

Pharmacological and molecular evidence reveals a novel role of methane (CH₄) gas in root organogenesis, the induction of lateral root (LR) formation, and this response might require hydrogen peroxide (H₂O₂) synthesis. Although plants can produce CH₄ and release this to atmosphere, the beneficial role(s) of CH₄ are not fully elucidated. In this study, the fumigation with CH₄ not only increased NADPH oxidase activity and H₂O₂ production, but also induced tomato lateral root primordial formation and thereafter LR development. However, exogenously applied argon and nitrogen failed to influence LR formation. Above responses triggered by CH₄ were sensitive to the removal of endogenous H₂O₂ with dimethylthiourea (DMTU; a membrane-permeable scavenger of H₂O₂), suggesting the hypothesis that CH₄'s effect on LR formation could be mediated by endogenous H₂O₂. Diphenylene iodonium (DPI) inhibition of the H₂O₂ generating enzyme NADPH oxidase attenuated H₂O₂ synthesis and impaired LR formation in response to CH₄, confirming the requirement of NADPH oxidase-dependent H₂O₂. Meanwhile, the alterations of endogenous H₂O₂ concentrations failed to influence CH₄ production in tomato seedlings. Molecular evidence revealed that CH₄-induced SlCDKA1, SlCYCA2;1, and SlCYCA3;1 transcripts, and -decreased SlKRP2 mRNA were impaired by DMTU or DPI. Contrasting changes in LR formation-related miR390a and miR160 transcripts and their target genes, including SlARF4 and SlARF16, were observed. Together, our pharmacological and molecular evidence suggested the requirement of H₂O₂ synthesis in CH₄-triggered tomato LR formation, partially via the regulation of cell cycle regulatory genes, miRNA-, and tasiRNA-modulated gene expression.