Inmaculada Garrido

Defence response of tomato seedlings to oxidative stress induced by phenolic compounds from dry olive mill residue

ADOR is an aqueous extract obtained from the dry olive mill residue (DOR) which contains the majority of its soluble phenolic compounds, which are responsible for its phytotoxic properties. Some studies have shown that ADOR negatively affects seed germination. However, to date, few studies have been carried out on the effect of ADOR on the oxidative stress of the plant. It is well known that saprobe fungi can detoxify these phenolic compounds and reduce the potential negative effects of ADOR on plants. To gain a better understanding of the phytotoxic effects and oxidative stress caused by this residue, tomato seeds were germinated in the presence of ADOR, treated and untreated with Coriolopsis rigida, Trametes versicolor, Pycnoporus cinnabarinus and Penicillium chrysogenum-10 saprobe fungi. ADOR sharply reduced tomato seed germination and also generated high levels of malondialdehyde (MDA), O(2)(-) and H(2)O(2). However, bioremediated ADOR did not negatively affect germination and reduced MDA, O(2)(-) and H(2)O(2) content in different ways depending on the fungus used. In addition, the induced defense response was studied by analyzing the activity of both antioxidant enzymes (superoxide dismutase (SOD), catalase, ascorbate peroxidasa, glutathione reductase (GR), peroxidases and coniferil alcohol peroxidasa) and detoxification enzymes (glutathione-S-transferase (GST)). Our findings suggest that, because ADOR is capable of inducing oxidative stress, tomato seedlings trigger a defense response through SOD, GR, and GST activity and through antioxidant and lignification processes. On the other hand, the bioremediation of ADOR plays an important role in counteracting the oxidative stress induced by the untreated residue.

Redox Activities and ROS, NO and Phenylpropanoids Production by Axenically Cultured Intact Olive Seedling Roots after Interaction with a Mycorrhizal or a Pathogenic Fungus

Roots of intact olive seedlings, axenically cultured, were alternatively placed in contact with Rhizophagus irregularis (mycorrhizal) or Verticillim dahliae (pathogenic) fungi. MeJA treatments were also included. In vivo redox activities in the apoplast of the intact roots (anion superoxide generation, superoxide dismutase and peroxidase activities) were measured. All our results showed that apoplastic redox activities of intact seedling roots in contact with the compatible mycorrhizal fungus were clearly attenuated in comparison with the pathogenic fungus or treated with MeJA, even at the early stages of treatment used. Total phenolics, flavonoids and phenylpropanoid glycosides were also quantified. Roots in contact with the mycorrhizal fungus did not enhance the biosynthesis of phenolic compounds with respect to controls, while those in contact with the pathogenic one significantly enhanced the biosynthesis of all phenolic fractions measured. Reactive oxygen species and nitric oxid accumulation in roots were examined by fluorescence microscopy. All of them presented much higher accumulation in roots in contact with the pathogenic than with the mycorrhizal fungus. Altogether these results indicate that intact olive seedling roots clearly differentiated between mycorrhizal and pathogenic fungi, attenuating defense reactions against the first to facilitate its establishment, while inducing a strong and sustained defense reaction against the second. Both reactive oxygen and nitrogen species seemed to be involved in these responses from the first moments of contact. However, further investigations are required to clarify the proposed crosstalk between them and their respective roles in these responses since fluorescence images of roots revealed that reactive oxygen species were mainly accumulated in the apoplast (congruently with the measured redox activities in this compartment) while nitric oxid was mainly stored in the cytosol.

Comparison of RAPD, ISSR, and AFLP Molecular Markers to Reveal and Classify Orchardgrass (Dactylis glomerata L.) Germplasm Variations.

Three different DNA-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) markers, were used for fingerprinting Dactylis glomerata genotypes and for detecting genetic variation between the three different subspecies. In this study, RAPD assays produced 97 bands, of which 40 were polymorphic (41.2%). The ISSR primers amplified 91 bands, and 54 showed polymorphism (59.3%). Finally, the AFLP showed 100 bands, of which 92 were polymorphic (92%). The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Three cluster analyses were performed to express–in the form of dendrograms–the relationships among the genotypes and the genetic variability detected. All DNA-based techniques used were able to amplify all of the genotypes. There were highly significant correlation coefficients between cophenetic matrices based on the genetic distance for the RAPD, ISSR, AFLP, and combined RAPD-ISSR-AFLP data (0.68, 0.78, 0.70, and 0.70, respectively). Two hypotheses were formulated to explain these results; both of them are in agreement with the results obtained using these three types of molecular markers. We conclude that when we study genotypes close related, the analysis of variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for RAPD, ISSR and AFLP markers. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationship among genotypes of Dactylis glomerata.

Fatty Acid and Phenolic Compound Concentrations in Eight Different Monovarietal Virgin Olive Oils from Extremadura and the Relationship with Oxidative Stability

Olive oils have been shown to be more resistant to oxidation than other vegetable fats, mainly due to their fatty acid (FA) profile which is rich in oleic acid and to their high content of antioxidants, principally phenols and tocopherols. This has situated virgin olive oils (VOOs) among the fats of high nutritional quality. However, it is important to stress that the oil’s commercial category (olive oil, virgin olive oil, extra-virgin olive oil), the variety of the source plant, and the extraction-conservation systems all decisively influence the concentration of these antioxidants and the oil’s shelf-life. The present work studied the fatty acid (FA) and phenolic composition and the oxidative stability (OS) of eight olive varieties grown in Extremadura (Arbequina, Cornicabra, Manzanilla Cacereña, Manzanilla de Sevilla, Morisca, Pico Limón, Picual, and Verdial de Badajoz), with the olives being harvested at different locations and dates. The Cornicabra, Picual, and Manzanilla Cacereña VOOs were found to have high oleic acid contents (>77.0%), while the VOOs of Morisca and Verdial de Badajoz had high linoleic acid contents (>14.5%). Regarding the phenol content, high values were found in the Cornicabra (633 mg·kg−1) and Morisca (550 mg·kg−1) VOOs, and low values in Arbequina (200 mg·kg−1). The OS was found to depend upon both the variety and the date of harvesting. It was higher in the Cornicabra and Picual oils (>55 h), and lower in those of Verdial de Badajoz (26.3 h), Arbequina (29.8 h), and Morisca (31.5 h). In relating phenols and FAs with the OS, it was observed that, while the latter, particularly the linoleic content (R = −0.710, p < 0.001, n = 135), constitute the most influential factors, the phenolic compounds, especially o-diphenols, are equally influential when the oils’ linoleic content is ≥12.5% (R = 0.674, p < 0.001, n = 47). The results show that VOOs’ resistance to oxidation depends not only on the FA or phenolic profile, but also on the interaction of these compounds within the same matrix.

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Summary. Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H+ efflux rate, 1.80 μM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H+ consumption for O2•− dismutation to H2O2. Also K+ influx was strongly depressed by MeJA, even transitorily reverting to K+ efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H2O2 accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O2 uptake by roots gave similar results. These and other results for additions of H2O2 or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H2O2 being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN−- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O2•− and H2O2, O2 uptake, and peroxidase activity by roots.

Effect of some electron donors and acceptors on redox capacity and simultaneous net H+/K+ fluxes by aeroponic sunflower seedling roots: Evidence for a CN−-resistant redox chain accessible to nonpermeative redox compounds

SummaryExcised roots from aeroponic axenically 48 h dark-grown sunflower (Helianthus annuus L.) seedlings showed redox activities, being able to oxidize/reduce all the exogenously added electron donors/acceptors, that affected the H+/K+ net fluxes simultaneously measured in the medium. Trials were performed with in vivo and CN−-poisoned roots; these showed null+/K+ net flux activity but still oxidized/reduced all the e− donors/acceptors tested except NADH. NADH enhanced the rate of H+ efflux by in vivo roots, otherwise not changing any of the normal flux kinetic characteristics, suggesting that NADH donates e− and H+ to the exocellular NADH oxidoreductase activity of a CN−-sensitive redox chain in the plasmalemma of the root cells. K+ influx was not affected, probably because the NADH concentration was not very high. The e− donor HFC(hexacyanoferrate)(II) activated the H+ efflux in a very different way: maximum H+ efflux rate was maintained, but both the maximum rate plateau and the optimal pH range were extended, and hence the total H+ efflux was significantly enhanced. At the same time, the K+ influx was doubled. The different H+-efflux kinetics, together with the small but significant HCF(II) oxidation by CN−-poisoned roots, were taken as evidence that, besides the CN−-sensitive redox chain, an alternative CN−-resistant redox chain in the plasmalemma was involved in HCF(II) oxidation. The effect of the oxidized form HCF(III) on H+ and K+ fluxes was the opposite to that described for HCF(II), but the other H+ efflux kinetic characteristics were similar (the maximum rate plateau was extended so that total H+ efflux equaled that of the controls). It is proposed that HCF(III) accepts e− only from the alternative CN−-resistant redox chain. We could not measure the effect of HCI(hexachloroiridate)(IV) on H+ efflux, as the pH electrodes alone quickly reduced the compound. HCI(IV) promoted a rapid transitory K+ efflux, followed by recovery of K+ influx. The HCI(IV) reduction by in vivo or CN−-poisoned roots was extremely rapid, following similar kinetics. Thus, only the CN−-resistant redox chain was involved in both cases. The redox chain inhibitor cis-platinum(II) annulled ion fluxes in the presence of both NADH and HCF(III), and later even inverted them (a small H+ influx down the gradient would induce K+ efflux). Cis-platinum(II) did not affect HCF(III) reduction by in vivo roots, and only slightly depressed that by CN−-poisoned roots. Overall, the effects of the exogenously added e− donors/acceptors tested were consistent with the existence of a CN−-resistant redox chain in the plasmalemma of the root cells which would donate/accept e− even when the H+ and K+ fluxes were annulled by CN− or even inverted by cis-platinum(II) treatments. Thus, in the plasmalemma of in vivo roots this chain would compete for electrons with the normal CN−-sensitive one, as in plant mitochondria. The effects on the K+ flux were consistent with the current hypothesis that this contributes to counteracting the changes in membrane potential caused by redox activities and the H+ flux induced by the different redox compounds tested.

The Evolution of Total Phenolic Compounds and Antioxidant Activities during Ripening of Grapes (Vitis vinifera L., cv. Tempranillo) Grown in Semiarid Region: Effects of Cluster Thinning and Water Deficit

A study was made of how water status (rainfed vs. irrigated) and crop load (no cluster thinning vs. cluster thinning) can together affect the grapes of Vitis vinifera cv. Tempranillo vines growing in a semiarid zone of Extremadura (Spain). The grapes were monitored at different stages of ripening, measuring the peroxidase (POX) and superoxide dismutase (SOD) antioxidant activities and the phenolic content (flavonoids and phenylpropanoids), together with other parameters. The irrigation regime was adjusted to provide 100% of crop evapotranspiration (ETc). The findings confirmed previous results that both thinning and water deficit advance ripening, while irrigation and high crop load (no thinning) lengthen the growth cycle. The SOD activity remained practically constant throughout ripening in the thinned treatments and was always lower than in the unthinned treatments, an aspect which could have been the cause of the observed greater level of lipid peroxidation in the water deficit, thinned treatment. The nonspecific peroxidase activity was very low, especially in the thinned treatments. The effect of thinning was enhanced when combined with water deficit, inducing increases in phenylpropanoids and, above all, flavonoids at the harvest stage of ripening, while leaving the polyphenol oxidase activity (PPO) unaffected.

Oxidative stress induced in sunflower seedling roots by aqueous dry olive-mill residues.

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O2·- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O2·- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O2·- and H2O2 accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H2O2 content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.

Net simultaneous hydrogen and potassium ion flux kinetics in sterile aeroponic sunflower seedling roots: Effects of potassium ion supply, valinomycin, and dicyclohexylcarbodiimide

Abstract Variation of pH and potassium ion (K+) concentration in a medium bathing 48 h sterile, aeroponic dark‐grown sunflower (Helianthus annuus L.) seedling roots were simultaneously monitored using specific non‐combined high‐sensitivity electrodes for pH and K+. Net K+ influx rates for different K+ concentrations (0.25, 0.5, 1, 2 and 5 mM) lagged by ≈ 60 min with respect to the, hydrogen ion (H+) efflux, and showed the biphasic saturable kinetics (Epsteins Systems I and II) described by other authors. However, in our close to natural conditions (H+ extrusion and K+ uptake systems are operating simultaneously), the range of K+ concentrations at which each of the two K+ uptake systems operated was also dependent on the time that the H+ extrusion system was operating too, and hence on the ?pHin,out created. The K+ concentration in the medium had negligible effects on the net root H+ efflux rate: only 5 mM K+ slightly enhanced in = 14% the maximum H+ efflux rate. Valinomycin caused a sharp K+ efflux, foll...

Apoplastic superoxide production and peroxidase activity by intact and excised axenically grown seedling roots of sunflower

Excised and cold-preincubated sunflower seedling roots were compared with intact non-preincubated roots to test the effect of the injury stress and cold preincubation on the oxidative burst measured as apoplastic superoxide (O2.−) generation and exocellular peroxidase (ECPOX) activity. Preincubated excised or intact roots released into the medium apoplastic proteins with peroxidase activity. Intact and excised roots responded to methyl jasmonate by an immediate oxidative burst that could not be induced by salicylic acid; both phytohormones also induced a slight and slow O2.− generation and ECPOX activity on excised roots, when added to the cold preincubation medium. The results with cyanide, azide, SHAM (ECPOX inhibitors) and diphenylene iodonium (inhibitor of trans-plasma membrane NAD(P)H-oxidases (NOX)–respiratory burst oxidase homologue in plants (RBOH), the trans-plasmamembrane nicotinamide adenine dinucleotide phosphate oxidase) are consistent with the hypothesis that different systems may be the origin of O2.− in intact and excised roots; ECPOX was an important component of them in both, together with NOX–RBOH in intact roots, but in excised roots the last one was replaced by an oxidase sensitive to the same inhibitors as the alternative mitochondrial oxidase. According to our hypothesis, these results could be explained if the electron flux would be deviated to different interconnected plasma membrane–redox systems, with different terminal oxidases, activated by different effectors or stresses.