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

Phosphenolpyruvate 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.