CYTOPLASMIC MALATE LEVELS IN MAIZE ROOT-TIPS DURING K+ ION UPTAKE DETERMINED BY C-13-NMR SPECTROSCOPY

C-13-NMR spectroscopy was used to determine the level of cytoplasmic malate in maize root tips that exhibited different rates of malate synthesis. Intracellular malate was C-13-labeled at carbons 1 and 4 by perfusing root tips with 5 mM H13CO3-. This labeling reflects the activities of phosphoenolpyruvate carboxylase and malate dehydrogenase (production of [4- C-13]malate), and fumarase (scrambling of C-13-label between C1 and C4 of malate). In vivo C-13-NMR spectra contained a clearly resolved resonance from cytoplasmic [4- C-13]malate, while the resonance from cytoplasmic [1- C-13]malate overlapped with others. After 90 min of H13CO3- treatment, C-13-labeling of organic acid pools had reached steady-state. Thereafter, the ratios [C-13]malate/[C-12+C-13]malate and [1- C-13]malate/[4- C-13]malate in tissue extracts remained constant; evidence is presented that these ratios were the same for both cytoplasmic and total cellular malate. Hence, the intensity of the cytoplasmic [4- C-13]malate signal was proportional to the amount of cytoplasmic malate in root tips. Potassium sulfate stimulated malate synthesis in maize root tips, relative to root tips perfused with HCO3- alone; total cellular malate doubled after approx. 1 h of 5 mM K2SO4-treatment. Cytoplasmic malate increased from approx. 3.5 mM to approx. 7.5 mM within 45 min of the onset of K2SO4-treatment, declining slightly thereafter. The possible effects of these changing cytoplasmic malate concentrations on the enzymes involved in malate metabolism are discussed.