Methodological pipeline for monitoring post-harvest quality of leafy vegetables.

Plants are primary source of nutrients for humans. However, the nutritional value of vegetables tends to decrease once organ and tissue sinks are detached from the plant. Minimal processing of leafy vegetables involves cutting and washing before packaging and storage. These processing procedures result in stressful conditions and post-harvest disorders senescence-related can also occur. The aim of this work is to define a methodological pipeline to evaluate the "quality" changes of fresh cut leafy vegetables over their shelf-life. At this purpose, intra-species variability has been investigated considering two varieties of Lactuca sativa (var. longifolia and capitata), showing different susceptibility to browning. Since browning mainly depends on phenol oxidation, redox parameters as well as the activity of the enzymes involved in phenol biosynthesis and oxidation have been monitored over storage time. At the same time, the metabolic changes of the lettuce leaves have been estimated as response patterns to chemical sensors. The obtained sensor outputs were predictive of browning-related biological features in a cultivar-dependent manner. The integration of the results obtained by this multivariate methodological approach allowed the identification of the most appropriate quality markers in lettuce leaves from different varieties. This methodological pipeline is proposed for the identification and subsequent monitoring of post-harvest quality of leafy vegetables.

Prototypical versus contemporary Mediterranean Diet.

BACKGROUND AND AIMS: To investigate the evolution of the Mediterranean Diet (MD) in a delimited area of Southern Italy, by comparing the diet adopted 60-70 years ago (Prototypical Mediterranean Diet, PMD) with the contemporary one (Contemporary Mediterranean Diet, CMD), and to verify to what extent they fitted the recommendations of the Italian and the USDA dietary guidelines. METHODS: We recruited a total of 106 participants, divided in two groups. PMD group included 52 women aged >80 years, with a good cognitive function and full independence in basic and instrumental activities of daily living. CMD group included 20 men and 34 women aged 50-60 years. Food intake was assessed by administering the EPIC food frequency questionnaire to each participant, and an additional survey to the PMD subjects only. RESULTS: Both PMD and CMD showed adequate intakes of macronutrients, although some deficiencies related to micronutrient requirements were evident. CMD showed a slightly greater use of animal products, processed and sugary foods, and higher intakes of simple sugars, animal proteins (49.6 vs 28.3 g/day), animal lipids (37.8 vs 20.1 g/day), saturated fats (25.0 vs 15.8 g/day) and cholesterol (305.0 vs 258.5 g/day). PMD showed many similarities to the original version of the MD in terms of macronutrients distribution and food choices. CONCLUSION: The documented evolution of the dietary habits over a 70 years timespan suggests that nowadays Mediterranean regions adhere less strictly to the original MD, although nutrients intakes are adequate to LARN and USDA recommendations.

Galactone-γ-lactone-dependent ascorbate biosynthesis alters wheat kernel maturation.

Kernel development and maturation involve several well-characterised events, such as changes in ascorbate (ASC) metabolism, protein synthesis and storage, programmed cell death (PCD) of starchy endosperm and tissue dehydration. Despite many studies focusing on these events, whether and how they are metabolically related to each other, remains to be elucidated. In the present investigation, the changes in ASC-related metabolism, PCD occurrence, kernel filling and dehydration have been analysed during kernel maturation, over a 3-year period in plants grown under normal conditions and in plants displaying modified ASC synthesis. The obtained results suggest that ASC plays a pivotal role in the network of events characterising kernel maturation. During this process, a decrease in ASC content occurs. When ASC biosynthesis is improved in the kernel, by feeding the plants with its immediate precursor, L-galactone-γ-lactone (GL), the decrease in ASC, observed during kernel maturation, is delayed. As a consequence, ascorbate peroxidase (APX) activity is also enhanced. Moreover, a delay in the ASC decrease permits a delay in PCD occurring in kernel storage tissues and in kernel dehydration. Interestingly, the data emerging from the present investigation suggest that the delay in the decrease in ASC content and APX activity also improves kernel filling. The relevance of the ascorbate-dependent redox regulation for kernel productivity is discussed.

Redox regulation in plant programmed cell death.

Programmed cell death (PCD) is a genetically controlled process described both in eukaryotic and prokaryotic organisms. Even if it is clear that PCD occurs in plants, in response to various developmental and environmental stimuli, the signalling pathways involved in the triggering of this cell suicide remain to be characterized. In this review, the main similarities and differences in the players involved in plant and animal PCD are outlined. Particular attention is paid to the role of reactive oxygen species (ROS) as key inducers of PCD in plants. The involvement of different kinds of ROS, different sites of ROS production, as well as their interaction with other molecules, is crucial in activating PCD in response to specific stimuli. Moreover, the importance is stressed on the balance between ROS production and scavenging, in various cell compartments, for the activation of specific steps in the signalling pathways triggering this cell suicide process. The review focuses on the complexity of the interplay between ROS and antioxidant molecules and enzymes in determining the most suitable redox environment required for the occurrence of different forms of PCD.

Response to UV-C radiation in topo I-deficient carrot cells with low ascorbate levels.

In animal cells, recent studies have emphasized the role played by DNA topoisomerase I (topo I) both as a cofactor of DNA repair complexes and/or as a damage sensor. All these functions are still unexplored in plant cells, where information concerning the relationships between DNA damage, PCD induction, and topo I are also limited. The main goal of this study was to investigate the possible responses activated in topo I-depleted plant cells under oxidative stress conditions which induce DNA damage. The carrot (Daucus carota L.) AT1-beta/22 cell line analysed in this study (characterized by an antisense-mediated reduction of top1beta gene expression of approximately 46% in association with a low ascorbate content) was more sensitive to UV-C radiation than the control line, showing consistent cell death and high levels of 8-oxo-dG accumulation. The topo I-depleted cells were also highly susceptible to the cross-linking agent mitomycin C. The death response was associated with a lack of oxidative burst and there were no changes in ascorbate metabolism in response to UV-C treatment. Electron and fluorescence microscopy suggested the presence of three forms of cell death in the UV-C-treated AT1-beta/22 population: necrosis, apoptotic-like PCD, and autophagy. Taken together, the data reported here support a reduced DNA repair capability in carrot topo I-deficient cells while the putative relationship between topo I-depletion and ascorbate impairment is also discussed.

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system.

The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O(2) (-) and H(2)O(2)) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O(2) (-) production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl(3) assays, which monitor H(2)O(2) production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl(3)) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H(2)O(2) is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H(2)O(2) in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H(2)O(2) was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H(2)O(2) produced by this in vitro culture system is destined for use during lignin biosynthesis.

A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds.

The ascorbate and glutathione systems have been studied during the first stages of germination in orthodox seeds of the gymnosperm Pinus pinea L. (pine). The results indicate that remarkable changes in the content and redox balance of these metabolites occur in both the embryo and endosperm; even if with different patterns for the two redox pairs. Dry seeds are devoid of the ascorbate reduced form (ASC) and contain only dehydroascorbic acid (DHA). By contrast, glutathione is present both in the reduced (GSH) and in the oxidized (GSSG) forms. During imbibition the increase in ASC seems to be mainly caused by the reactivation of its biosynthesis. On the other hand, the GSH rise occurring during the first 24 h seems to be largely due to GSSG reduction, even if GSH biosynthesis is still active in the seeds. The enzymes of the ascorbate--glutathione cycle also change during germination, but in different ways. ASC peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities progressively rise both in the embryo and in endosperm. These changes are probably required for counteracting production of reactive oxygen species caused by recovery of oxidative metabolism. The two enzymes involved in the ascorbate recycling, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and DHA reductase (EC 1.8.5.1), show different behaviour: the DHA reductase activity decreases, while that of AFR reductase remains unchanged. The relationship between ascorbate and glutathione metabolism and their relevance in the germination of orthodox seeds are also discussed.

Combined cadmium and ozone treatments affect photosynthesis and ascorbate-dependent defences in sunflower.

• The combined effects of the two pollutants, cadmium and ozone, on sunflower (Helianthus annuus) metabolism are analysed here. • Photosysnthetic processes and ascorbate metabolism were studied in sunflower plants grown for 15 d in the presence of cadmium and exposed to acute O3 treatments. • CO2 assimilation rate was reduced in plants subjected to Cd(II) and/or O3 treatments, but no alterations in stomatal conductance and Fv  : Fm ratio were observed. Rubisco activity was significantly reduced only in plants grown in the presence of cadmium indicating that the photosynthetic process is mainly altered by this factor. Photochemical quenching and the quantum efficiency of PSII in steady-state conditions were significantly depressed and nonphotochemical quenching increased in stressed plants. Cd(II) and O3 also strongly affected ascorbate metabolism. • The changes in ascorbate redox state and the increase in ascorbate-redox enzymes strongly supported an ascorbate over-utilization in Cd(II) and/or O3 -treated plants. However, the increase in ascorbate-based detoxification mechanisms did not provide complete protection against the oxidative stress imposed by the two pollutants, since an increase in lipid peroxidation and protein oxidation accompanied a decrease in photosynthesis under pollutant exposure.

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3, 4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular "secretory" peroxidase activity (-73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed.

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells.

The effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.

Inhibition of galactonolactone dehydrogenase activity by lycorine.

Galactonolactone dehydrogenase, a mitochondrial enzyme catalyzing the last step in ascorbate biosynthesis, is strongly inhibited by lycorine. A concentration of 10 microM of the alkaloid fully inhibits the activity of the enzyme. The high sensitivity of this enzyme to lycorine supports the hypothesis that the lycorine specifically inhibits ascorbate biosynthesis and that all the other metabolic responses to lycorine treatment depend on this primary inhibition of ascorbate biosynthesis.

The multiplicity of enzymatic DNA reduction: a new purification procedure for a DHA reducing protein from potato tubers.

The multiplicity of DHA reducing proteins has been observed by means of a native-PAGE technique in several plant species, thus demonstrating, in accordance with recent literature, that several differently evolved proteins are likely to perform DHA reduction. Moreover, a research strategy coupling the use of native-PAGE with chromatographic separation procedure, tentatively performed in Solanum tuberosum, proved to be a useful tool for the separation and partial identification of the proteins involved in DHA reduction.

An electrophoretic procedure to detect three key enzymes of the ascorbate system.

A method for detection of ascorbic acid oxidase, ascorbic acid peroxidase and dehydroascorbic acid reductase is reported. This method allows the qualitative determination of the presence of these enzymes, also in conditions where the commonly used spectrophotometric assays are unreliable.

Changes in the Ascorbate System during Seed Development of Vicia faba L.

Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H(2)O(2) produced in the cell metabolism. There is no ascorbate oxidase in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is furnished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.

Ascorbate system in Dasypyrum villosum from different environments.

Ascorbic acid content and redox-enzymes activities of AA system are determined in four population of Dasypyrum villosum adapted to live in different environments. In D. villosum from the driest and warmest environment (Pachino), AFR reductase and AA peroxidase have activities lower than in the other populations. The results point out the role of AA peroxidase as the main "scavenger" of the H2O2 produced by cell metabolism.

Reduction of cytochrome C by ascorbic free radical.

The rate of "in vivo" reduction of cytochrome c by ascorbic acid (AA) increases from 69 nmoles of cytochrome c for minute, to 202 nanomoles when ascorbate oxidase is added. Since the AA oxidation by AA oxidase is a system to generate ascorbic free radical (AFR), data suggest that AFR is a better reducing compound than ascorbate in cytochrome c reduction. Since the addition of oxidized glutathione and human immunoglobulins (-S-S- bridge containing compounds) in the medium produces a remarkable decrease in cytochrome c reduction, it is suggested that AFR could also reduce -S-S- groups.

Further researches upon the inhibiting action of lycorine on ascorbic acid biosynthesis.

Lycorine, an alkaloid extracted from Amarillidaceae, strongly inhibits the "in vivo" conversion of galactono-gamma-lactone to ascorbic acid. Lycorine seems to act as a non-competitive inhibitor on galactono-gamma-lactone oxidase, because the alkaloid rapidly forms a stable bound with the enzyme. In fact, a short incubation period with 50 microM lycorine gets a high inhibitory effect that persists when the alkaloid is removed from the incubation medium. Considering that lycorine induces scurvy-like symptoms in ascorbic acid-synthesising animals, it is reasonable to suppose that in both plants and animals lycorine inhibits the last step in the biosynthetic pathway leading from sugar to ascorbate.

[Comparison of presence of ascorbic acid and the appearance of ascorbate peroxidase activity in embryos of Avena sativa L]. / Correlazione tra presenza di acido ascorbico e comparsa dell'attivita' ascorbico perossidasica in embrioni di Avena sativa L.

Avena sativa L. grains are devoid of ascorbic acid (AA) and of oxidative enzymes (AA oxidase and AA peroxidase), while both reducing enzymes (AFR reductase and DHA reductase) are present. AA biosynthesis in the embryos starts after 12-14 hours of germination and at the same time AA peroxidase activity is detectable. During the following 14 hours the AA peroxidase activity rises up to 28 nmoles/AA oxidated/min/mg/prot. Incubation of Avena embryos with GL (the last precursor of AA according to the Isherwood biosynthetic pathway), results in both earlier AA biosynthesis and enhanced AA peroxidase activity. A 4 hour treatment is enough to induce AA synthesis and AA peroxidase elicitation. These data suggest that the development of AA peroxidase activity is controlled by AA, but they are not sufficient to clarify how that happens. Probably AA induces the synthesis of specific m-RNAs or activates enzymic precursors present in the embryos but still not working.