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

Malate synthesis by CO(2) fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH(13)CO(3) as well as with NaH(14)CO(3). The distribution of (14)C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of (13)C, 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 (14)C 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-(13)C]-, [4-(13)C]-, and [1,4-(13)C] 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.