Received August 14, 2006
Accepted November 8, 2006
Discrimination in the Dark. Resolving the Interplay between Metabolic and Physical Constraints to PEPC Activity during the CAM Cycle
Howard Griffiths *, Asaph B. Cousins , Murray R. Badger , and Susanne von Caemmerer
Physiological Ecology Group, Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, U.K.; Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia
Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia
Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia; ARC CoE, Plant Energy Biology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia
* Corresponding author; email: hg230{at}cam.ac.uk.
A model defining carbon isotope discrimination (
13C) for crasssulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (Phase IV of CAM), throughout the dark period (Phase I) and into the light (Phase II). Measurements of CO2 responses curves throughout the dark period revealed changing PEP carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance and online 13C signal: all declined at the start of the dark period, then increased to a maximum two hours before dawn. Measurements of 13C were higher than predicted from the ratio of intercellular to external CO2 (pi/pa) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, gi, was 0.044 mol m-2 s-1 bar-1. A higher estimate of gi (0.085 mol m-2 s-1 bar-1) was needed to account for the modeled and measured 18O discrimination throughout the dark period. The differences in estimates of gi from the two isotope measurements, and an offset of -5.5 
between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion pathlength and/or or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase 13C discrimination during night time CO2 fixation whilst reducing 13C during Phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.
