Enhanced formation of methane in plant cell cultures by inhibition of cytochrome c oxidase

The claim of methane (CH₄) formation in plants has caused much controversy and debate within the scientific community over the past 4 years. Here, using both stable isotope and concentration measurements, we demonstrate that CH₄ formation occurs in plant cell cultures that were grown in the dark under sterile conditions. Under non‐stress conditions the plant cell cultures produced trace amounts [0.3–0.6 ng g^?1 dry weight (DW) h^?1] of CH₄ but these could be increased by one to two orders of magnitude (up to 12 ng g^?1 DW h^?1) when sodium azide, a compound known to disrupt electron transport flow at the cytochrome c oxidase (complex IV) in plant mitochondria, was added to the cell cultures. The addition of other electron transport chain (ETC) inhibitors did not result in significant CH₄ formation indicating that a site‐specific disturbance of the ETC at complex IV causes CH₄ formation in plant cells. Our study is an important first step in providing more information on non‐microbial CH₄ formation from living plants particularly under abiotic stress conditions that might affect the electron transport flow at the cytochrome c oxidase in plant mitochondria.