Aflatoxin B1 Degradation by Ery4 Laccase: From In Vitro to Contaminated Corn.

Aflatoxins (AFs) are toxic secondary metabolites produced by Aspergillus spp. and are found in food and feed as contaminants worldwide. Due to climate change, AFs occurrence is expected to increase also in western Europe. Therefore, to ensure food and feed safety, it is mandatory to develop green technologies for AFs reduction in contaminated matrices. With this regard, enzymatic degradation is an effective and environmentally friendly approach under mild operational conditions and with minor impact on the food and feed matrix. In this work, Ery4 laccase, acetosyringone, ascorbic acid, and dehydroascorbic acid were investigated in vitro, then applied in artificially contaminated corn for AFB1 reduction. AFB1 (0.1 µg/mL) was completely removed in vitro and reduced by 26% in corn. Several degradation products were detected in vitro by UHPLC-HRMS and likely corresponded to AFQ1, epi-AFQ1, AFB1-diol, or AFB1dialehyde, AFB2a, and AFM1. Protein content was not altered by the enzymatic treatment, while slightly higher levels of lipid peroxidation and H2O2 were detected. Although further studies are needed to improve AFB1 reduction and reduce the impact of this treatment in corn, the results of this study are promising and suggest that Ery4 laccase can be effectively applied for the reduction in AFB1 in corn.

Blue and Red LED Lights Differently Affect Growth Responses and Biochemical Parameters in Lentil (Lens culinaris).

Light-emitting diodes are an attractive tool for improving the yield and quality of plant products. This study investigated the effect of different light intensity and spectral composition on the growth, bioactive compound content, and antioxidant metabolism of lentil (Lens culinaris Medik.) seedlings after 3 and 5 days of LED treatment. Two monochromatic light quality × three light intensity treatments were tested: red light (RL) and blue light (BL) at photosynthetic photon flux density (PPFD) of 100, 300, and 500 µmol m-2 s-1. Both light quality and intensity did not affect germination. At both harvest times, the length of seedling growth under BL appeared to decrease, while RL stimulated the growth with an average increase of 26.7% and 62% compared to BL and seedlings grown in the darkness (D). A significant blue light effect was detected on ascorbate reduced form, with an average increase of 35% and 50% compared to RL-grown plantlets in the two days of harvesting, respectively. The content of chlorophyll and carotenoids largely varied according to the wavelength and intensity applied and the age of the seedlings. Lipid peroxidation increased with increasing light intensity in both treatments, and a strong H2O2 formation occurred in BL. These results suggest that red light can promote the elongation of lentil seedlings, while blue light enhances the bioactive compounds and the antioxidant responses.

Inuloxin A Inhibits Seedling Growth and Affects Redox System of Lycopersicon esculentum Mill. and Lepidium sativum L.

Allelochemicals are considered an environment-friendly and promising alternative for weed management, although much effort is still needed for understanding their mode of action and then promoting their use in plant allelopathy management practices. Here, we report that Inuloxin A (InA), an allelochemical isolated from Dittrichia viscosa, inhibited root elongation and growth of seedlings of Lycopersicon esculentum and Lepidium sativum at the highest concentrations tested. InA-induced antioxidant responses in the seedlings were investigated by analysing the contents of glutathione (GSH) and ascorbate (ASC), and their oxidized forms, dehydroascorbate (DHA), and glutathione disulphide (GSSG), as well as the redox state of thiol-containing proteins. An increase in ASC, DHA, and GSH levels at high concentrations of InA, after 3 and 6 days, were observed. Moreover, the ASC/DHA + ASC and GSH/GSSG + GSH ratios showed a shift towards the oxidized form. Our study provides the first insight into how the cell redox system responds and adapts to InA phytotoxicity, providing a framework for further molecular studies.

Vitamin C in Plants: From Functions to Biofortification.

Vitamin C (l-ascorbic acid) is an excellent free radical scavenger, not only for its capability to donate reducing equivalents but also for the relative stability of the derived monodehydroascorbate radical. However, vitamin C is not only an antioxidant, since it is also a cofactor for numerous enzymes involved in plant and human metabolism. In humans, vitamin C takes part in various physiological processes, such as iron absorption, collagen synthesis, immune stimulation, and epigenetic regulation. Due to the functional loss of the gene coding for l-gulonolactone oxidase, humans cannot synthesize vitamin C; thus, they principally utilize plant-based foods for their needs. For this reason, increasing the vitamin C content of crops could have helpful effects on human health. To achieve this objective, exhaustive knowledge of the metabolism and functions of vitamin C in plants is needed. In this review, the multiple roles of vitamin C in plant physiology as well as the regulation of its content, through biosynthetic or recycling pathways, are analyzed. Finally, attention is paid to the strategies that have been used to increase the content of vitamin C in crops, emphasizing not only the improvement of nutritional value of the crops but also the acquisition of plant stress resistance.

Effect of different light-emitting diode (LED) irradiation on the shelf life and phytonutrient content of broccoli (Brassica oleracea L. var. italica).

Broccoli (Brassica oleracea L. var. italica) is largely cultivated in southern Italy. It is an important source of phytonutrients, which are partially lost during postharvest storage. The aim of this work was to evaluate the overall effect of five different low-intensity light-emitting diodes (LEDs) on the quality parameters of broccoli florets over 20 d of cold storage. The level of ascorbic acid, chlorophylls, carotenoids, phenolic compounds and soluble proteins, as well as colour analysis, were evaluated. Green LED increased the chlorophyll and ascorbic acid content; white, red and yellow LEDs had a positive effect on the redox status of broccoli. Globally, only green LED had a statistically significant positive effect when considering all analysed parameters and could be proposed to prolong the shelf life of broccoli during cold storage.

Changes in lignin biosynthesis and monomer composition in response to benzothiadiazole and root-knot nematode Meloidogyne incognita infection in tomato.

Benzothiadiazole (BTH) acts as a priming agent in plant defence leading to a reduction in penetration and development of the root-knot nematode Meloidogyne incognita in susceptible tomato roots. Changes in lignin biosynthesis in the susceptible tomato cv. Roma following nematode infection and/or BTH treatment were investigated in comparison to the resistant cv. Rossol. Both untreated and BTH-treated susceptible infected roots (galls) showed an increased level of expression of lignin synthesis-related genes (PAL, C4H, HCT and F5H) at early times during infection (2-4 days post inoculation). Peroxidase (soluble and cell-wall bound, POX) enzyme activities increased after inoculation with M. incognita and the priming effect of BTH treatment was evident at later stages of infection (7 days post inoculation). As expected, the induction of PAL and POXs and lignin synthesis-related genes was faster and greater in resistant roots after infection. Histochemical analysis revealed accumulation of higher lignin levels at later infection stages in BTH-treated galls compared to untreated ones. Furthermore, the monomer composition of lignin indicated a different composition in guaiacyl (G) and syringyl (S) units in BTH-treated galls compared to untreated galls. The increase in G units made G/S ratio similar to that in the resistant genotype. Overall, lignin played a critical role in tomato defence to M. incognita in response to BTH.

Characterization of a Novel Mitochondrial Ascorbate Transporter From Rat Liver and Potato Mitochondria.

The Mitochondrial Ascorbic Acid Transporter (MAT) from both rat liver and potato mitochondria has been reconstituted in proteoliposomes. The protein has a molecular mass in the range of 28-35 kDa and catalyzes saturable, temperature and pH dependent, unidirectional ascorbic acid transport. The transport activity is sodium independent and it is optimal at acidic pH values. It is stimulated by proton gradient, thus supporting that ascorbate is symported with H+. It is efficiently inhibited by the lysine reagent pyridoxal phosphate and it is not affected by inhibitors of other recognized plasma and mitochondrial membranes ascorbate transporters GLUT1(glucose transporter-1) or SVCT2 (sodium-dependent vitamin C transporter-2). Rat protein catalyzes a cooperative ascorbate transport, being involved two binding sites; the measured K0.5 is 1.5 mM. Taking into account the experimental results we propose that the reconstituted ascorbate transporter is not a GLUT or SVCT, since it shows different biochemical features. Data of potato transporter overlap the mammalian ones, except for the kinetic parameters non-experimentally measurable, thus supporting the MAT in plants fulfills the same transport role.

Ozonated water reduces susceptibility in tomato plants to Meloidogyne incognita by the modulation of the antioxidant system.

Few studies have been carried out on the effect of ozonated water (O3 wat) on the oxidative stress of root systems and, in particular, in combination with biotic stress. The aim of this study was to determine whether aqueous ozone is effective in the control of root-knot nematode (RKN) infection and to investigate the concomitant changes in the basal defence system. A tomato cultivar susceptible to Meloidogyne incognita was treated with O3 wat as a soil drench. No negative effects were seen following ozone application in comparison with the control under the exposure conditions used. The treatment reduced significantly the nematode infection rate and induced changes in the morphology of nematode feeding sites, some of which were characterized by visible symptoms of senescence. The antioxidant response, as well as parameters of oxidative damage, were examined in untreated and O3 wat-treated galls at 2, 4 and 7 days after inoculation and compared with uninfected roots. High levels of reactive oxygen species (ROS), H2 O2 and malondialdehyde were generated in galls in response to combined abiotic and biotic stresses. Throughout the experimental period, the activities and relative transcript levels of the antioxidant enzymes catalase, superoxide dismutase and ascorbate peroxidase produced different responses when exposed to ozone treatment and/or infection. The results demonstrate how O3 wat protects tomato against the RKN M. incognita through the modulation of basal defence mechanisms.

Defense Responses to Mycotoxin-Producing Fungi Fusarium proliferatum, F. subglutinans, and Aspergillus flavus in Kernels of Susceptible and Resistant Maize Genotypes.

Developing kernels of resistant and susceptible maize genotypes were inoculated with Fusarium proliferatum, F. subglutinans, and Aspergillus flavus. Selected defense systems were investigated using real-time reverse transcription-polymerase chain reaction to monitor the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes protective from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) at 72 h postinoculation. The study was also extended to the analysis of the ascorbate-glutathione cycle and catalase, superoxide dismutase, and cytosolic and wall peroxidases enzymes. Furthermore, the hydrogen peroxide and malondialdehyde contents were studied to evaluate the oxidation level. Higher gene expression and enzymatic activities were observed in uninoculated kernels of resistant line, conferring a major readiness to the pathogen attack. Moreover expression values of PR genes remained higher in the resistant line after inoculation, demonstrating a potentiated response to the pathogen invasions. In contrast, reactive oxygen species-scavenging genes were strongly induced in the susceptible line only after pathogen inoculation, although their enzymatic activity was higher in the resistant line. Our data provide an important basis for further investigation of defense gene functions in developing kernels in order to improve resistance to fungal pathogens. Maize genotypes with overexpressed resistance traits could be profitably utilized in breeding programs focused on resistance to pathogens and grain safety.

Increase of fumonisin b2 and ochratoxin a production by black Aspergillus species and oxidative stress in grape berries damaged by powdery mildew.

Powdery mildew (PM), caused by the fungus Erysiphe necator, is one of the most widespread fungal disease of grape and may cause extensive openings on the berry surface during the infection. We evaluated the effect of damage caused by PM in grape berries on the growth of and mycotoxin production by Aspergillus and on the oxidative stress in infected berries. Berries of Vitis vinifera L. cv. Negroamaro with sound skin (SS) and those naturally infected by PM were surface sterilized and inoculated with either fumonisin B2(FB2)-producing strains of Aspergillus niger or ochratoxin A (OTA)-producing strains of Aspergillus carbonarius and incubated at 20 and 30°C. The PM berries were significantly more susceptible to both Aspergillus colonization (5 to 15 times more susceptible) and OTA and FB2 contamination (2 to 9 times more susceptible) than were SS berries. The highest toxin concentration was detected in inoculated PM berries both for OTA (9 ng/g) at 20°C and for FB2 (687 ng/g) at 30°C. In inoculated SS and PM berries, although malondialdehyde and hydrogen peroxide concentrations did not increase, the two black Aspergillus species caused a significant decrease in ascorbate content, thus inducing a pro-oxidant effect. These results indicate that grape berries affected by PM are more susceptible to black Aspergillus growth and to production and/or accumulation of FB2 and OTA. Thus, preventive control of E. necator on grape berries could reduce the mycotoxicological risk from black Aspergillus infection.