In vivo phosphoenolpyruvate carboxylase activity is controlled by CO2 and O2 mole fractions and represents a major flux at high photorespiration rates.

Phosphenolpyruvate carboxylase (PEPC)-catalysed fixation of bicarbonate to C4 acids is commonly believed to represent a rather small flux in illuminated leaves. In addition, its potential variation with O2 and CO2 is not documented and thus is usually neglected in gas-exchange studies. Here, we used quantitative NMR analysis of sunflower leaves labelled with 13 CO2 (99% 13 C) under controlled conditions and measured the amount of 13 C found in the four C-atom positions in malate, the major product of PEPC activity. We found that amongst malate 13 C-isotopomers present after labelling, most molecules were labelled at both C-1 and C-4, showing the incorporation of 13 C at C-4 by PEPC fixation and subsequent redistribution to C-1 by fumarase (malate-fumarate equilibrium). In addition, absolute quantification of 13 C content showed that PEPC fixation increased at low CO2 or high O2 , and represented up to 1.8 μmol m-2  s-1 , that is, 40% of net assimilation measured by gas exchange under high O2 /CO2 conditions. Our results show that PEPC fixation represents a quantitatively important CO2 -fixing activity that varies with O2 and/or CO2 mole fraction and this challenges the common interpretation of net assimilation in C3 plants, where PEPC activity is often disregarded or considered to be constant at a very low rate.