Cellular respiration via the alternative oxidase pathway (AOP) leads to a considerable loss in efficiency. Compared to the cytochrome pathway (COP), AOP produces 0-50% as much ATP per carbon (C) respired. Relative partitioning between the pathways can be measured in vivo based on their differing isotopic discriminations against 18 O in O 2 . Starting from published methods, we have refined and tested a new protocol to improve measurement precision and efficiency. The refinements detect an effect of tissue water content (P < 0.0001), which we have removed, and yield precise discrimination endpoints in the presence of pathway-specific respiratory inhibitors [CN − and salicylhydroxamic acid (SHAM)], which improves estimates of AOP/COP partitioning. Fresh roots of Pinus sylvestris were sealed in vials with a CO 2 trap. The air was replaced to ensure identical starting conditions. Headspace air was repeatedly sampled and isotopically analyzed using isotope-ratio mass spectrometry. The method allows high-precision measurement of the discrimination against 18 O in O 2 because of repeated measurements of the same incubation vial. COP and AOP respiration discriminated against 18 O by 15.1 ± 0.3‰ and 23.8 ± 0.4‰, respectively. AOP contributed to root respiration by 23 ± 0.2% of the total in an unfertilized stand. In a second, nitrogen-fertilized, stand AOP contribution was only 14 ± 0.2% of the total. These results suggest the improved method can be used to assess the relative importance of COP and AOP activities in ecosystems, potentially yielding information on the role of each pathway for the carbon use efficiency of organisms.
<p>The measurement of <em>&#948;</em><sup>2</sup>H of non-exchangeable-H (<em>&#948;</em><sup>2</sup>H<sub>n</sub>) in organic matter (OM) by isotope-ratio mass spectrometry is often hampered by the difficulties in controlling H isotope exchange of the exchangeable H fraction (f<sub>ex</sub>) and removal of residual moisture. The determination of <em>&#948;</em><sup>2</sup>H<sub>n&#160;</sub>in organic matter requires control of the isotopic composition of f<sub>ex</sub>. This can be achieved through dual water H isotope exchange experiments. However, these experiments are laborious, sensitive to the method used (e.g. prior sample treatment, temperature, time) and are costly. This has resulted in a wide range of reported f<sub>ex </sub>for known isotopic references. Moreover, it is not always clear that samples are completely dry following the H exchange experiments, leading to even larger variations. The <em>&#948;</em><sup>2</sup>H<sub>n&#160;</sub>data is typically used in ecological studies for source attribution due to the large observed separation between contributing end members. However, it is not clear to what degree the analytical errors in <em>&#948;</em><sup>2</sup>H determined by incomplete H isotope exchange of f<sub>ex&#160;</sub>and the residual moisture impact the source attribution. Here we developed a simple offline sample preparation system, the Isobox, and a protocol for the measurement of <em>&#948;</em><sup>2</sup>H<sub>n&#160;</sub>in natural OM as well as pure organic compounds. We performed dual water H isotopic exchange experiments with both liquid and vapor water at near 0 and 105&#176;C respectively. We analyzed three keratin reference materials (KHS, CBS and USGS42), two amino acids (Isoleucine and Threonine), along with caffeine (USGS62) and polyethylene (IAEA-CH-7) as drying references. We have also used natural samples of demineralized soil and green algae to create known mixtures to test these methods and their analytical uncertainty impact on the source attribution. We show that the liquid water exchange experiments led to f<sub>ex </sub>close to the theoretical expectations for both keratin and pure compounds. Depending on the research question careful determination with controlled dual water procedure for the determination of <em>&#948;</em><sup>2</sup>H<sub>n&#160;</sub>may be required. However, simple sample treatment with exposure to a single isotopically known water can be used to derive <em>&#948;</em><sup>2</sup>H for source attribution. The offline sample preparation system and equilibration method we developed is simple, accurate and cost effective and can be implemented in virtually any laboratory for the analysis of a wider range of OM types.</p>
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