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In all trypanosomatids, includingTrypanosoma brucei, glycolysis takes place in peroxisome-like organelles called glycosomes. These are closed compartments wherein the energy and redox (NAD+/NADH) balances need to be maintained. We have characterized a T. bruceigene called FRDgencoding a protein 35% identical to Saccharomyces cerevisiaefumarate reductases. Microsequencing of FRDg purified from glycosome preparations, immunofluorescence, and Western blot analyses clearly identified this enzyme as a glycosomal protein that is only expressed in the procyclic form of T. bruceibut is present in all the other trypanosomatids studied, i.e. Trypanosoma congolense, Crithidia fasciculataandLeishmania amazonensis. The specific inactivation ofFRDggene expression by RNA interference showed that FRDg is responsible for the NADH-dependent fumarate reductase activity detected in glycosomal fractions and that at least 60% of the succinate secreted by the T. bruceiprocyclic form (in the presence of d-glucose as the sole carbon source) is produced in the glycosome by FRDg. We conclude that FRDg plays a key role in the energy metabolism by participating in the maintenance of the glycosomal NAD+/NADH balance. We have also detected a significant pyruvate kinase activity in the cytosol of the T. bruceiprocyclic cells that was not observed previously. Consequently, we propose a revised model of glucose metabolism in procyclic trypanosomes that may also be valid for all other trypanosomatids except the T. bruceibloodstream form. Interestingly, H. Gest has hypothesized previously (Gest, H. (1980) FEMS Microbiol. Lett.7, 73–77) that a soluble NADH-dependent fumarate reductase has been present in primitive organisms and evolved into the present day fumarate reductases, which are quinol-dependent. FRDg may have the characteristics of such an ancestral enzyme and is the only NADH-dependent fumarate reductase characterized to date.

Changes in metabolites (organic acids, sugars and amino acids) and subcellular pH were studied during fruit development of cherry tomato (Lycopersicon esculentum Mill. var. cerasiformae). Fructose and glucose were the major sugars, whereas citrate and malate the two major organic acids. At different stages of fruit development, vacuolar and cytoplasmic pH changes were followed by in vivo 13C and 31P NMR spectroscopy. Fruit compartments had a cytoplasmic pH around 7.1 as early as the cell-divi-sion and -expansion stages. The vacuolar pH measured by in vivo 13C NMR spectroscopy decreased from 4.5 to 3.6. Concomitantly, strong accumulation of γ-aminobutyric acid (GABA) was observed during the first 15 days after anthesis and glutamate decarboxylase (GAD) activity increased 10-fold during the first 8 days of development. The relationships between organic acid biosynthesis and storage, GABA produc-tion, and subcellular pH changes during development of cherry tomato fruit are discussed. Keywords: fruit development, GABA shunt, nuclear magnetic resonance, organic acids, pH, γ-aminobutyric acid.