Yolanda Gogorcena

Antioxidant Defenses against Activated Oxygen in Pea Nodules Subjected to Water Stress.

The involvement of activated oxygen in the drought-induced damage of pea (Pisum sativum L. cv Frilene) nodules was examined. To this purpose, various pro-oxidant factors, antioxidant enzymes and related metabolites, and markers of oxidative damage were determined in nodules of well-watered (nodule water potential approximately -0.29 MPa) and water-stressed (nodule water potential approximately -2.03 MPa) plants. Water-stressed nodules entered senescence as evidenced by the 30% decrease in leghemoglobin and total soluble protein. Drought also caused a decrease in the activities of catalase (25%), ascorbate peroxidase (18%), dehydroascorbate reductase (15%), glutathione reductase (31%), and superoxide dismutase (30%), and in the contents of ascorbate (59%), reduced (57%) and oxidized (38%) glutathione, NAD+ and NADH (43%), NADP+ (31%), and NADPH (17%). The decline in the antioxidant capacity of nodules may result from a restricted supply of NAD(P)H in vivo for the ascorbate-glutathione pathway and from the Fe-catalyzed Fenton reactions of ascorbate and glutathione with activated oxygen. The 2-fold increase in the content of “catalytic Fe” would also explain the augmented levels of lipid peroxides (2.4-fold) and oxidatively modified proteins (1.4-fold) found in water-stressed nodules because of the known requirement of lipid and protein oxidation for a transition catalytic metal.

Evaluation of the Antioxidant Capacity, Phenolic Compounds, and Vitamin C Content of Different Peach and Nectarine [Prunus persica (L.) Batsch] Breeding Progenies

Antioxidant capacity and contents of total phenolics, anthocyanins, flavonoids, and vitamin C were evaluated in 218 genotypes from 15 peach and nectarine breeding progenies. Significant differences were found among progenies on the fruit antioxidant profile, corroborated by the high contribution showed by cross to the phenotypic variance of each phytochemical trait analyzed (16-45%). Phytochemical profile varied depending on peach/nectarine and yellow/white flesh color qualitative traits. On the other hand, no significant effect of year was found on the bioactive profile of peaches and nectarines. Antioxidant capacity was linearly correlated to total phenolic content, but correlation varied depending on the progeny. No correlation was found for vitamin C versus any other phytochemical trait. The results suggest the importance of genetic background on the antioxidant profile of peaches and nectarines and stress its relevance for the ultimate objective of this work: selecting new peach and nectarine genotypes rich in bioactive compounds to benefit consumers health.

Effects of Cd and Pb in sugar beet plants grown in nutrient solution: induced Fe deficiency and growth inhibition

Effects of Cd and Pb toxicity were investigated in sugar beet (Beta vulgaris L.) grown in hydroponics under growth-chamber-controlled conditions. Chemical speciation calculations were used to estimate the chemical species in equilibrium. Cd, used as chloride salt or chelated to EDTA, decreased fresh and dry mass of both root and shoot, and increased root / shoot ratios. Plants developed few brownish roots with short laterals. Cd decreased N, P, Mg, K, Mn, Cu and Zn uptake, and facilitated Ca uptake. Leaves of plants treated with 10 or 50 μM Cd-EDTA and 10 μM CdCl2 developed symptoms of Fe deficiency. These symptoms included decreased leaf chlorophyll (Chl) and carotenoid concentrations, increased carotenoid / Chl and Chl a/b ratios, de-epoxidation of violaxanthin cycle pigments, and decreased photosynthetic rates and PSII efficiency. Plants treated with 50 μM CdCl2, however, had decreased growth but did not show marked leaf Fe-deficiency symptoms. All Cd treatments increased Fe(III)-chelate reductase activity in root tips, although Fe concentrations in shoots were similar to those found in control plants. Pb chelated with EDTA induced visual symptoms only at concentrations of 2 mM. Leaves of Pb-treated plants remained green and their edges were rolled inwards. Pb increased root fresh and dry mass with no changes in shoot mass, therefore increasing the root / shoot ratio. Changes in plant nutrient concentrations with Pb were only minor, although leaf Cu levels approached critical deficiency levels. No symptoms of Fe deficiency were apparent in leaves. Root tips of Pb-treated plants, however, had increased Fe(III)-chelate reductase activities.

N2 Fixation, Carbon Metabolism, and Oxidative Damage in Nodules of Dark-Stressed Common Bean Plants

Common beans (Phaseolus vulgaris L.) were exposed to continuous darkness to induce nodule senescence, and several nodule parameters were investigated to identify factors that may be involved in the initial loss of N2 fixation. After only 1 d of darkness, total root respiration decreased by 76% and in vivo nitrogenase (N2ase) activity decreased by 95%. This decline coincided with the almost complete depletion (97%) of sucrose and fructose in nodules. At this stage, the O2 concentration in the infected zone increased to 1%, which may be sufficient to inactivate N2ase; however, key enzymes of carbon and nitrogen metabolism were still active. After 2 d of dark stress there was a significant decrease in the level of N2ase proteins and in the activities of enzymes involved in carbon and nitrogen assimilation. However, the general collapse of nodule metabolism occurred only after 4 d of stress, with a large decline in leghemoglobin and antioxidants. At this final senescent stage, there was an accumulation of oxidatively modified proteins. This oxidative stress may have originated from the decrease in antioxidant defenses and from the Fe-catalyzed generation of activated oxygen due to the increased availability of catalytic Fe and O2 in the infected region.

Metabolic responses in iron deficient tomato plants.

The effects of Fe deficiency on different metabolic processes were characterized in roots, xylem sap and leaves of tomato. The total organic acid pool increased significantly with Fe deficiency in xylem sap and leaves of tomato plants, whereas it did not change in roots. However, the composition of the pool changed with Fe deficiency, with major increases in citrate concentrations in roots (20-fold), leaves (2-fold) and xylem sap (17-fold). The activity of phosphoenolpyruvate carboxylase, an enzyme leading to anaplerotic C fixation, increased 10-fold in root tip extracts with Fe deficiency, whereas no change was observed in leaf extracts. The activities of the organic acid synthesis-related enzymes malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, fumarase and aconitase, as well as those of the enzymes lactate dehydrogenase and pyruvate carboxylase, increased with Fe deficiency in root extracts, whereas only citrate synthase increased significantly with Fe deficiency in leaf extracts. These results suggest that the enhanced C fixation capacity in Fe-deficient tomato roots may result in producing citrate that could be used for Fe xylem transport. Total pyridine nucleotide pools did not change significantly with Fe deficiency in roots or leaves, although NAD(P)H/NAD(P) ratios were lower in Fe-deficient roots than in controls. Rates of O(2) consumption were similar in Fe-deficient and Fe-sufficient roots, but the capacity of the alternative oxidase pathway was decreased by Fe deficiency. Also, increases in Fe reductase activity with Fe deficiency were only 2-fold higher when measured in tomato root tips. These values are significantly lower than those found in other plant species, where Fe deficiency leads to larger increases in organic acid synthesis-related enzyme activities and flavin accumulation. These data support the hypothesis that the extent of activation of different metabolic pathways, including carbon fixation via PEPC, organic acid synthesis-related enzymes and oxygen consumption is different among species, and this could modulate the different levels of efficiency in Strategy I plants.

Involvement of Activated Oxygen in Nitrate-Induced Senescence of Pea Root Nodules.

The effect of short-term nitrate application (10 mM, 0–4 d) on nitrogenase (N2ase) activity, antioxidant defenses, and related parameters was investigated in pea (Pisum sativum L. cv Frilene) nodules. The response of nodules to nitrate comprised two stages. In the first stage (0–2 d), there were major decreases in N2ase activity and N2ase-linked respiration and concomitant increases in carbon cost of N2ase and oxygen diffusion resistance of nodules. There was no apparent oxidative damage, and the decline in N2ase activity was, to a certain extent, reversible. The second stage (>2 d) was typical of a senescent, essentially irreversible process. It was characterized by moderate increases in oxidized proteins and catalytic Fe and by major decreases in antioxidant enzymes and metabolites. The restriction in oxygen supply to bacteroids may explain the initial decline in N2ase activity. The decrease in antioxidant protection is not involved in this process and is not specifically caused by nitrate, since it also occurs with drought stress. However, comparison of nitrate- and drought-induced senescence shows an important difference: there is no lipid degradation or lipid peroxide accumulation with nitrate, indicating that lipid peroxidation is not necessarily involved in nodule senescence.

Phenotypic diversity and relationships of fruit quality traits in peach and nectarine (Prunus persica (L.) Batsch) breeding progenies

Agronomic and fruit quality traits were evaluated and compared for three consecutive years on 1,111 seedlings coming from 15 peach and nectarine breeding crosses, grown under a Mediterranean climate. Significant differences among and within the different progenies were found for most of the traits analyzed. The breeding population segregated for several Mendelian characters such as peach or nectarine fruit, round or flat fruit, yellow or white flesh and freestone or clingstone. In addition, aborting fruit and flat fruit trees were found in our progeny, and our data seem to support multi-allelic control of both flat shape and aborting fruit. The variation within the progenies of some traits such as blooming and harvesting date, yield, fruit weight and SSC was continuous, suggesting a polygenic inheritance. Relationships between qualitative pomological traits and these agronomic and fruit quality parameters were also found. Valuable correlations among agronomic and fruit quality parameters were found, although coefficients of variation depending on the progeny should be considered. In addition, principal component analysis (PCA) revealed several relationships among quality traits in the evaluated progenies. Based on this evaluation, 26 outstanding genotypes were pre-selected from the initial breeding population for further studies.

Induction of in vivo root ferric chelate reductase activity in fruit tree rootstock

Abstract A method has been developed to consistently induce increases in root ferric chelate reductase activity in the fruit tree rootstock GF 677 (Prunus amygdalopersica) grown under iron (Fe) deficiency. Clonal GF 677 plants were grown hydroponically in a growth chamber with 0 or 90 μM Fe(III)‐EDTA. Root ferric chelate reductase activity was measured in vivo using BPDS. Plants grown without Fe developed visible symptoms of chlorosis and had lower root ferric chelate reductase activities than those grown with Fe. Root ferric chelate reductase activities were 0.1–1.9 and 0.6–5.3 nmol of Fe reduced per gram of fresh mass and minute, respectively, in Fe‐deficient and sufficient plants. However, when plants grown without Fe for several days were resupplied with 180 μM of Fe(III)‐EDTA, FC‐R activities increased within 1 day. The FC‐R values after Fe resupply were 20‐fold higher than those found in Fe‐deficient plants and 5‐fold higher than those found in the Fe‐sufficient controls. After three days of the Fe treatments the FC‐R activities had decreased again to the control values. The reduction of Fe was localized at the subapical root zone. In the conditions used we have found no decreases of the nutrient solution pH values, indicating that this type of response is not strong enough to be detected in peach tree rootstocks. Also, no major changes in root morphology have been found in response to Fe deficiency. This ferric chelate reductase induction protocol may be used in screening assays to select rootstock genotypes tolerant to Fe chlorosis.

A New Technique for Screening Iron-Efficient Genotypes in Peach Rootstocks: Elicitation of Root Ferric Chelate Reductase by Manipulation of External Iron Concentrations

Abstract The possibility of using the root ferric chelate reductase enzymatic activity as a tool for iron (Fe) efficient rootstock screening in breeding programs has been investigated. Ferric chelate reductase activities were estimated spectrophotometrically from the formation of the Fe(II)–bathophenantroline complex by exposing the roots of intact plants to nutrient solution containing Fe(III)-EDTA. Four micro-propagated peach rootstocks, GF-677, Adesoto (syn. Puebla de Soto), Barrier and Cadaman, known to differ in tolerance to Fe deficiency under field conditions, were grown in hydroponic cultures containing 90 µM Fe(III)-EDTA. After 9 days, plants were grown without any Fe in the nutrient solution for 4 more days. Then, some plants were re-supplied with 45 and 180 µM of Fe(III)-EDTA for 1, 2, or 3 days. A transient increase in root ferric chelate reductase activity was elicited by Fe resupply in GF-677 and Adesoto, two Fe-efficient genotypes, whereas no induction was found in other two genotypes, Barrier and Cadaman, which are known to be Fe-inefficient. Leaf Fe concentrations after resupply were higher in the Fe-efficient genotypes than in the Fe-inefficient ones. These results indicate that the capacity of the roots to reduce ferric compounds could be used as a screening technique for Fe efficiency, provided it is elicited by manipulation of the external Fe levels. This protocol could be easier to use for commercial breeders looking for Fe-efficient rootstock genotypes.

The use of RAPD markers for identification of cultivated grapevine (Vitis vinifera L.)

Abstract Polymorphism among 31 grapevine accessions was studied utilizing random amplified polymorphic DNA (RAPD) markers. Fourteen decamer oligonucleotide primers were tested. All experiments were repeated three times. Primers could be grouped according to the polymorphism obtained as well as the constancy of results. Choosing the right primers, all varieties and some of the clones could be discriminated using reliable bands. Synonymies were confirmed, and a case of homonymy was detected. The use of RAPD markers as a technique for identification of grapevine varieties and clones can be encouraged.