An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

In situ measurements of alternative respiratory pathway activity are needed to provide insight into the energy efficiency of plant metabolism under various conditions in the field. The only reliable method at present to measure alternative oxidase (AOX) activity is through measurement of changes in delta(18)O(O(2)), which to date has only been used in laboratory environments. We have developed a cuvette system to measure partitioning of electrons to AOX that is suitable for off-line use and for field experiments. Plant samples are enclosed in airtight cuvettes and O(2) consumption is monitored. Gas samples from the cuvette are stored in evacuated gas containers until measurement of delta(18)O(O(2)). We have validated this method using differing plant material to assess AOX activity. Fractionation factors were calculated from delta(18)O(O(2)) measurements, which could be measured with an accuracy and precision to 0.1 per thousand and 0.3 per thousand, respectively. Potential sources of error are discussed and quantified. Our method provides results similar to those obtained with laboratory incubations on-line to a mass spectrometer but greatly increases the potential for adoption of the stable isotope method.