Pathway of Malic Acid Synthesis in Response to Ion Uptake in Wheat and Lupin Roots: Evidence from Fixation of ¹³C and ¹⁴C ¹ Free

Malate synthesis by CO₂ fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH¹³CO₃ as well as with NaH¹⁴CO₃. The distribution of ¹⁴C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of ¹³C, gas chromatography-mass spectrometry. In long-term experiments (2 to 12 hours), both methods showed that the [1-C] and [4-C] positions of malic acid are approximately equally labeled, in agreement with former findings. Short-term experiments (15, 30 seconds) showed that ¹⁴C is confined initially to the [4-C] position of malate but then is distributed quickly to the [1-C] atom. Neither labeling pattern nor rate of randomization was influenced by salt treatment. Analysis of malate from roots by gas chromatography-mass spectrometry, a procedure which was tested against in vitro-prepared [1-¹³C]-, [4-¹³C]-, and [1,4-¹³C] malate, gave strong evidence for the existence of only singly labeled malate molecules. These data suggest that only one carboxylation step, catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, is responsible for malic acid synthesis in roots and that malate label is randomized by a fumarase-like reaction, presumably in mitochondria.