In vivo phospho enol pyruvate carboxylase activity is controlled by CO 2 and O 2 mole fractions and represents a major flux at high photorespiration rates

Summary Phosph enol pyruvate carboxylase ( PEPC )‐catalysed fixation of bicarbonate to C 4 acids is commonly believed to represent a rather small flux in illuminated leaves. In addition, its potential variation with O 2 and CO 2 is not documented and thus is usually neglected in gas‐exchange studies. Here, we used quantitative NMR analysis of sunflower leaves labelled with 13 CO 2 (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 CO 2 or high O 2 , and represented up to 1.8 μmol m −2 s −1 , that is, 40% of net assimilation measured by gas exchange under high O 2 / CO 2 conditions. Our results show that PEPC fixation represents a quantitatively important CO 2 ‐fixing activity that varies with O 2 and/or CO 2 mole fraction and this challenges the common interpretation of net assimilation in C 3 plants, where PEPC activity is often disregarded or considered to be constant at a very low rate.