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

Abstract

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.

Evidence for methane production by saprotrophic fungi

“…Less well known are the findings of Ghyczy et al 9,12 who demonstrated that endothelial cells from rat liver produced CH 4 when exposed to site-specific inhibitors of the electron transport chain (ETC). A similar observation has been made for plant cell cultures grown under sterile and oxic conditions 13 . Hence, both plant and animal cells have been suggested to release CH 4 nonmicrobially.…”

“…CH 4 per h. However, if the duration of the incubation was at least 24 h this was sufficient to detect CH 4 emissions, which were well above analytical thresholds. The increase in 13 CH 4 concentrations in samples where 13 C labelled glucose was used as a substrate indicated that the CH 4 formed was essentially the result of metabolism of substrate by fungi. All incubations were carried out under sterile and oxic conditions, that is, the O 2 mixing ratio in the flasks was always above 10% when CH 4 production occurred, except for the first experiment where fungi were cultured on grass (Fig.…”

“…2 Growth experiments using 13 C-labelled glucose. Fungally derived CH 4 production on 13 C-labelled glucose substrate is presented in Fig. 3.…”

“…Whiskerman et al 13 who investigated sterile plant cell cultures suggested that CH 4 formation in living plants might be related to a site-specific disturbance of the ETC within the mitochondria. This is in accordance with the findings of Ghyczy et al 9,12 who demonstrated that endothelial cells from rat liver produced CH 4 when exposed to site-specific inhibitors of the ETC.…”

“…Interestingly, this has recently been considered by Bruhn et al 8 for the formation of CH 4 from living plants. To test this hypothesis for fungi (P. sapidus) we employed methionine, which had only the carbon atom of the thio-methyl group (-S-13 CH 3 ) labelled with 13 C. Immediately following addition of 13 C-methionine or unlabelled methionine the headspace gas of all samples showed δ 13 C(CH 4 When the amount of 13 CH 4 was related to the total amount of CH 4 production 4.9 and 3.9% of headspace-CH 4 originated from the methyl group of methionine, respectively. These results suggest that the carbon atom of the sulphur-bound methyl group of methionine is a precursor of CH 4 generated by P. sapidus.…”