In vivo phosphoenolpyruvate carboxylase activity is controlled by CO and O mole fractions and represents a major flux at high photorespiration rates.

Phosphenolpyruvate carboxylase (PEPC)-catalysed fixation of bicarbonate to C acids is commonly believed to represent a rather small flux in illuminated leaves. In addition, its potential variation with O and CO is not documented and thus is usually neglected in gas-exchange studies. Here, we used quantitative NMR analysis of sunflower leaves labelled with CO (99% C) under controlled conditions and measured the amount of C found in the four C-atom positions in malate, the major product of PEPC activity. We found that amongst malate C-isotopomers present after labelling, most molecules were labelled at both C-1 and C-4, showing the incorporation of C at C-4 by PEPC fixation and subsequent redistribution to C-1 by fumarase (malate-fumarate equilibrium). In addition, absolute quantification of C content showed that PEPC fixation increased at low CO or high O , and represented up to 1.8 μmol m s , that is, 40% of net assimilation measured by gas exchange under high O /CO conditions. Our results show that PEPC fixation represents a quantitatively important CO -fixing activity that varies with O and/or CO mole fraction and this challenges the common interpretation of net assimilation in C plants, where PEPC activity is often disregarded or considered to be constant at a very low rate.