Short-term UV-B exposure induces metabolic and anatomical changes in peel of harvested lemons contributing in fruit protection against green mold.

UV-B radiation (UVBR) is a small fraction of the solar spectrum from 280 to 315nm. UVBR produces photomorphogenic acclimation responses in plants, modulating their cellular structure and physiology. Here, changes in the peel of harvested lemons after short time exposure to UVBR were analyzed and its potential effects against fungal infection were studied. In the flavedo, UVBR treatment induced variations in the respiratory profiles and increased the phenolic compound contents. Final products of the flavonoid pathway (flavones, flavonols and anthocyanins) increased more markedly than their precursors (flavanones and dihydroflavonols). The increased accumulation of soluble phenolics in the flavedo of treated lemons is associated with the high antioxidant activity found in the flavedo of these samples. Supporting the biochemical determinations, anatomical observations showed abundant intravacuolar deposits of phenolic compounds and an increase in the cell wall thickness in UVBR-treated samples. Metabolic and anatomical modifications associated to UVBR improved natural defenses against Penicillium digitatum, the causal agent of green mold disease. Our results suggest that mature postharvest lemons exposed to the artificial radiation showed phenotypic plasticity, allowing an acclimation response to UVBR which confers fruit resistance to pathogens. Thus, combination of UVBR with other treatments could represent an important improvement to control postharvest diseases on citrus.

Cellular damage induced by a sequential oxidative treatment on Penicillium digitatum.

AIM: To investigate the cellular damage on Penicillium digitatum produced by a sequential oxidative treatment (SOT), previously standardized in our laboratory, to prevent the conidia growth. Lethal SOT consists of 2-min preincubation with 10 ppm NaClO followed by 2-min incubation with 6 mmol l(-1) CuSO(4) and 100 mmol l(-1) H(2)O(2) at 25°C. METHODS AND RESULTS: After the application of lethal SOT or sublethal SOT (decreasing only the H(2)O(2) concentration), we analysed several conidia features such as germination, oxygen consumption, ultrastructure and integrity of the cellular wall and membrane. Also, we measured the production of reactive oxygen species (ROS) and the content of thiobarbituric acid-reactive species (TBARS). With the increase of H(2)O(2) concentration in the SOT, germination and oxygen consumption of conidia became inhibited, while the membrane permeability, ROS production and TBARS content of conidia increased. Several studies revealed ultrastructural disorganization in P. digitatum conidia after lethal SOT, showing severe cellular damage without apparent damage to the cell wall. In addition, mycelium of P. digitatum was more sensitive than conidia to the oxidative treatment, because growth ceased and permeability of the membranes increased after exposure of the mycelium to a SOT with only 50 mmol l(-1) H(2)O(2) compared to a SOT of 100 mmol l(-1) for these effects to occur on conidia. CONCLUSION: Our insights into cellular changes produced by the lethal SOT are consistent with the mode of action of the oxidant compounds, by producing both alteration of membrane integrity and intracellular damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results allow the understanding of SOT effects on P. digitatum, which will be useful to develop a reliable treatment to control postharvest diseases in view of its future application in packing houses.

Cadmium induces changes in sucrose partitioning, invertase activities, and membrane functionality in roots of Rangpur lime (Citrus limonia L. Osbeck).

Cadmium (Cd) uptake effects on sucrose content, invertase activities, and plasma membrane functionality were investigated in Rangpur lime roots ( CITRUS LIMONIA L. Osbeck). Cadmium accumulation was significant in roots but not in shoots and leaves. Cadmium produced significant reduction in roots DW and increment in WC. Leaves and shoots did not show significant differences on both parameters. Sucrose content was higher in control roots than in Cd-exposed ones. Apoplastic sucrose content was much higher in Cd-exposed roots than in control ones. Cd-exposed roots showed a significant decrease in both cell wall-bound and cytoplasmic (neutral) invertase activities; while the vacuolar isoform did not show any change. Alterations in lipid composition and membrane fluidity of Cd-exposed roots were also observed. In Cd-exposed roots phospholipid and glycolipid contents decreased about 50 %, while sterols content was reduced about 22 %. Proton extrusion was inhibited by Cd. Lipid peroxidation and proton extrusion inhibition were also detected by histochemical analysis. This work's findings demonstrate that Cd affects sucrose partitioning and invertase activities in apoplastic and symplastic regions in Rangpur lime roots as well as the plasma membrane functionality and H (+)-ATPase activity.

Antigenic relationships between chloroplast and cytosolic fructose-1,6-bisphosphatases.

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.

Cloning, structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme.

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in lambda gt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3'-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39,253 daltons (Da), showing 82-88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb) KpnI-SacI fragment of the lambda gt11 derivative was subcloned between the KpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates of Escherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, the E. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of pea (Pisum sativum L.) thioredoxin f, a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase.

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (Mr) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an Mr of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at - 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP(+)-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa Mr = 6324 M(-1) · cm(-1)). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.

Regulation by ca of a cytosolic fructose-1,6-bisphosphatase from spinach leaves.

Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg(2+) or Mn(2+), and was free of interfering enzyme activities. Ca(2+) was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg(2+) or Mn(2+) was used as cofactor. In the presence of 5 millimolar Mg(2+), Ca(2+) appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca(2+). However, with Mn(2+) (60 micromolar) as cofactor, Ca(2+) exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca(2+) behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg(2+) or Mn(2+) as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg(2+) as cofactor, but this effect was low with Mn(2+). From these results we suggest that Ca(2+) plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase.

Quantitative determinations of chemical compounds with nutritional value from Inca crops: Chenopodium quinoa ('quinoa').

Quantitative determinations of total and soluble proteins, total and free sugars, starch, total lipids, tanins, ash (Ca, Na, K, Fe, and P), and caloric value were carried out on quinoa flour. Results show that the amount of soluble proteins was higher than the standard value for wheat and maize and was very close to that of barley's. The yield of free sugars like glucose (4.55%), fructose (2.41%) and sucrose (2.39%) were also of importance. Iron and calcium levels were higher than the reported values for maize and barley. The same occurred for the caloric value (435.5 Kcal/100 g). The content of saponins was also examined since its effect on red blood cells of group A and O has been related as a potential problem of the Andes population. From the chemical analysis a more complete view about quinoa as human food was presented.

Biochemical method for molecular weight determinations of proteins and other macromolecules.

The invertase of Ricinus communis complexes with proteins, polyvinylpyrrolidone, polyethylene glycol, heparin and dextran sulfate. This association produces an increase of invertase activity. The minimal concentration of activator giving the maximal activation was attained at the same molarity for a given amount of enzyme for all macromolecules studied. These conditions are used for the molecular weight determination of the activating substance. The method may be used for the molecular weight determination of polymeric substances with a molecular weight in the range from 5000 to 1000,000 Da.

Purification and characterization of Ricinus communis invertase.

An invertase from Ricinus communis leaves was purified 4,400-fold. The preparation was homogeneous by criteria of gel electrophoresis, gel permeation, adsorption, and ionic exchange chromatography. One optimum pH at 3.5 was observed with crude invertase; however, purified preparations showed two optima, at pH 3.5 and 5.5. Addition of bovine serum albumin restored one maximum at pH 3.5 and elicited a 30% activation of the invertase. The effect was caused by many other proteins and by heparin, dextran sulfate, and polyvinylpyrrolidone. Fructose, fructose 1,6-diphosphate, maleic, trans-aconitic, malic, and ascorbic acids were simple competitive inhibitors of the purified enzyme. Glucose was a noncompetitive inhibitor. The activation by proteins suppressed these inhibitory effects. The minimum concentration of activator necessary to reach the maximal activation or "point of optimal activation" was always reached at a concentration of 1 X 10(-6) M, independently of the nature of the activator, when 8.6 X 10(-12) mol of enzyme were used. Apparent molecular weight determinations of the enzyme in the presence and absence of activator and molecular weight determinations based on determinations of the point of optimal activation suggested that the purified enzyme is a heptamer (Mr of 77,900, Stokes radius 32 A, frictional ration f/fo 1.1, partial specific volume 0.749 ml/g) and that the activated form is a trimer consisting of two enzyme subunits and one activator molecule. The activation was lost by dilution of the trimer. The enzyme subunit, as isolated by gel filtration in the presence of sodium dodecyl sulfate (Mr 11,000) was inactive but quickly regained activity upon removal of sodium dodecyl sulfate.