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 C-4 acids is commonly believed to represent a rather small flux in illuminated leaves. In addition, its potential variation with O-2 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 (CO2)-C-13 (99% C-13) under controlled conditions and measured the amount of C-13 found in the four C-atom positions in malate, the major product of PEPC activity. We found that amongst malate C-13-isotopomers present after labelling, most molecules were labelled at both C-1 and C-4, showing the incorporation of C-13 at C-4 by PEPC fixation and subsequent redistribution to C-1 by fumarase (malate-fumarate equilibrium). In addition, absolute quantification of C-13 content showed that PEPC fixation increased at low CO2 or high O-2, and represented up to 1.8 mu mol m(-2) s(-1), that is, 40% of net assimilation measured by gas exchange under high O-2/CO2 conditions. Our results show that PEPC fixation represents a quantitatively important CO2-fixing activity that varies with O-2 and/or CO2 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.