A field-compatible method for measuring alternative respiratory pathway activities in vivo using stable O ₂ isotopes

Plants can alter rates of electron transport through the alternative oxidase (AOX) pathway in response to environmental cues, thus modulating respiratory efficiency, but the ¹⁸O discrimination method necessary for measuring electron partitioning in vivo has been restricted to laboratory settings. To overcome this limitation, we developed a field-compatible analytical method. Series of plant tissue subsamples were incubated in 12mL septum-capped vials for 0.5-4h before aliquots of incubation air were injected into 3.7mL evacuated storage vials. Vials were stored for up to 10 months before analysis by mass spectrometry. Measurements were corrected for unavoidable contamination. Additional mathematical tools were developed for detecting and addressing non-linearity (whether intrinsic or due to contamination) in the data used to estimate discrimination values. Initial contamination in the storage vials was 0.03±0.01atm; storing the gas samples at -17°C eliminated further contamination effects over 10 months. Discrimination values obtained using our offline incubation and computation method replicated previously reported results over a range of 10-31‰, with precision generally better than ±0.5‰. Our method enables large-scale investigations of plant alternative respiration along natural environmental gradients under field conditions.