Short-term changes in carbon-isotope discrimination identify transitions between C3 and C4 carboxylation during Crassulacean acid metabolism

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Abstract
Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO2 collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO2 exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO2 (Phase I), discrimination (Δ) ranged from 4.4 to 6.6‰, equivalent to an effective carbon-isotope ratio (δ13C) of −12.3 to −14.5‰ versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO2 uptake and refixation of respiratory CO2, characteristic of CAM in the Bromeliaceae. When Δ for the relative proportion of external (p a ) and internal (p i) CO2 is taken into account, calculated p i/p a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of Δ during Phase II, early in the light period, showed the transition between C4 and C3 pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as Δ increased from 10.5 to 21.2‰ During decarboxylation in the light period (Phase III), CO2 leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of Δ calculated from on-line measurements (64.4‰) showed that the CO2 lost was considerably enriched in 13C, and this was confirmed by direct analysis of the CO2 diffusing out into a CO2-free atmosphere (δ 13C = + 51.6‰ versus PDB). Instantaneous discrimination was characteristic of the C3 pathway during Phase IV (late in the light period), but a reduction in Δ showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that “double carboxylation” involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.