Evaluating high time-resolved changes in carbon isotope ratio of respired CO2 by a rapid in-tube incubation technique.

Recent insights into fractionation during dark respiration and rapid dynamics in isotope signatures of leaf- and ecosystem-respired CO(2) indicate the need for new methods for high time-resolved measurements of the isotopic signature of respired CO(2) (delta(13)C(res)). We present a rapid and simple method to analyse delta(13)C(res) using an in-tube incubation technique and an autosampler for small septum-capped vials. The effect of storage on the delta(18)O and delta(13)C ratios of ambient CO(2) concentrations was tested with different humidity and temperatures. delta(13)C ratios remained stable over 72 h, whereas delta(18)O ratios decreased after 24 h. Storage at 4 degrees C improved the storage time for delta(18)O. Leaves or leaf discs were incubated in the vials, flushed with CO(2)-free air and respired CO(2) was automatically sampled within 5 min on a microGas autosampler interfaced to a GV-Isoprime isotope ratio mass spectrometer. Results were validated by simultaneous on-line gas-exchange measurements of delta(13)C(res) of attached leaves. This method was used to evaluate the short-term (5-60 min) and diurnal dynamics of delta(13)C(res) in an evergreen oak (Quercus ilex) and a herb (Tolpis barbata). An immediate depletion of 2-4 per thousand from the initial delta(13)C(res) value occurred during the first 30 min of darkening. Q. ilex exhibited further a substantial diurnal enrichment in delta(13)C(res) of 8 per thousand, followed by a progressive depletion during the night. In contrast, T. barbata did not exhibit a distinct diurnal pattern. This is in accordance with recent theory on fractionation in metabolic pathways and may be related to the different utilisation of the respiratory substrate in the fast-growing herb and the evergreen oak. These data indicate substantial and rapid dynamics (within minutes to hours) in delta(13)C(res), which differed between species and probably the growth status of the plant. The in-tube incubation method enables both high time-resolved analysis and extensive sampling across different organs, species and functional types.