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 va rious conditions in the field. The only reliable method at present to measu re alternative oxidase (AOX) activity is through measurement of changes in delta O-18(O-2), which to date has only been used in laboratory environment s. 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 s amples are enclosed in airtight cuvettes and O-2 consumption is monitored. Gas samples from the cuvette are stored in evacuated gas containers until m easurement of delta O-18(O-2). We have validated this method using differin g plant material to assess AOX activity. Fractionation factors were calcula ted from delta O-18(O-2) measurements, which could be measured with an accu racy and precision to 0.1 parts per thousand and 0.3 parts per thousand res pectively. Potential sources of error are discussed and quantified. Our met hod provides results similar to those obtained with laboratory incubations on-line to a mass spectrometer but greatly increases the potential for adop tion of the stable isotope method.