Florencio E. Podestá

Purification and characterization of an NAD-dependent malate dehydrogenase from leaves of the crassulacean acid metabolism plant Aptenia cordifolia

An NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) was purified and characterized from leaves of Aptenia cordifolia L. f. (Schwant). This plant performs crassulacean acid metabolism (CAM), as indicated by: (a) elevated levels of phosphoenolpyruvate carboxylase (PEPC) and NAD(P) malic enzyme; (b) regulation of PEPC compatible with its function during the night; (c) characteristic day/night changes in titratable acidity; and (d) gas exchange profile consistent with that shown by CAM plants. These features remained unchanged by water availability or salt stress, suggesting constitutive CAM. The purified MDH showed a subunit molecular mass of 39.4 kDa, a native mass of 83 kDa (dimer) and a pI of 5.8. It cross-reacted with antibodies against cytosolic malate dehydrogenase (cMDH) from pineapple. Maximum activities for oxaloacetate (OAA) reduction or malate oxidation were observed at pH 7.0 and between pH 7.2 and 8.4, respectively. The enzyme was inhibited by excess OAA, in a pH-dependent manner. A discontinuity was observed in Arrhenius plots at 33 °C, with an activation energy twice as high below this temperature. Although immunologically related, some physical and kinetic dissimilarities between the A. cordifolia and pineapple enzymes suggest that diverse CAM metabolic subtypes may require different MDH isozymes to carry out OAA reduction.

Physiological aspects of fruit ripening: The mitochondrial connection

Fruit ripening is a genetically programmed process which leads to an assortment of physiological and metabolic changes that irreversibly alter its characteristics. Depending on the species, fruit maturation can be either climacteric or non-climacteric. In both cases there is a metabolic shift from normal development conditions toward the fully mature state, but climacteric fruit is characterized by a sharp increase in respiration. In non-climacteric fruit, that generally does not display this feature, respiration changes can be affected by processes related to postharvest storage. This review describes some of the many ways in which mitochondrial metabolism is implicated in this crucial reproductive stage, such as the connection between ethylene production and respiration rate, the involvement of alternative oxidase (AOX) and plant uncoupling mitochondrial protein (PUMP) during the ripening and the common alterations of this organelle in fruits affected by different stress conditions.

Cloning, expression, purification and physical and kinetic characterization of the phosphoenolpyruvate carboxylase from orange (Citrus sinensis osbeck var. Valencia) fruit juice sacs.

Phosphoenolpyruvate (PEP) carboxylase (PEPCase) from orange fruit juice sacs has been cloned and heterogously expressed in high yield. The purified recombinant enzyme displays properties typical of plant PEPCase, including activation by sugar phosphates and inhibition by malate and citrate. Malate inhibition is weak in the physiological pH range, and the enzyme is also poorly affected by Glu and Asp, known inhibitors of C(3) plants PEPCases. However, it is strongly inhibited by citrate. Orange fruit PEPCase phosphorylation by mammalian protein kinase A decreased inhibition by malate. The enzyme presents an unusual high molecular mass in the absence of PEP, while in its presence it displays a more common tetrameric arrangement. The overall properties of the enzyme suggest that it is suited for organic acid synthesis and NADH reoxidation in the mature fruit. The present study provides the first analysis of a recombinant fruit PEPCase.

Proteomic and metabolomic profiling of Valencia orange fruit after natural frost exposure

The aim of this study was to evaluate the response of orange fruit (Citrus sinensis var. Valencia Late) to freezing stress in planta, both immediately after the natural event and after a week, in order to understand the biochemical and molecular basis of the changes that later derive in internal and external damage symptoms. Using two-dimensional differential gel electrophoresis to analyze exposed and non-exposed fruit, 27 differential protein spots were detected in juice sacs and flavedo, among all comparisons made. Also, primary and secondary metabolites relative contents were analyzed in both tissues by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, respectively. Proteins and compounds involved in regulatory functions, iron metabolism, oxidative damage and carbohydrate metabolism were the most affected. Interestingly, three glycolytic enzymes were induced by cold, and there was an increase in fermentation products (volatiles); all of that suggests that more energy generation might be required from glycolysis to counter the cold stress. Moreover, a notable increase in sugar levels was observed after frost, but it was not at the expense of organic acids utilization. Consequently, these results suggest a probable redistribution of photoassimilates in the frost-exposed plants, tending to restore the homeostasis altered by that severe type of stress. Isosinensetin was the most cold-sensitive secondary metabolite because it could not be detected at all after the frost, constituting a possible tool to early diagnose freezing damage.

Substrate Binding to NADP-Malic Enzyme from Maize Leaves as Determined by Intrinsic Fluorescence Quenching

NADP-malic enzyme catalyzes the decarboxylating step of the C4 pathway of photosynthesis at the bundle-sheath chloroplasts in maize leaves (1). The enzyme purified from different C4 plants exhibits a homotetrameric structure, although variations in the oligomeric state have been reported (2).