Zonal changes in ascorbate and hydrogen peroxide contents,peroxidase, and ascorbate-related enzyme activities in onion roots

Onion (Allium cepa) roots growing hydroponically show differential zonal values for intra- (symplastic) and extra- (apoplastic) cellular ascorbate (ASC) and dehydroascorbate (DHA) contents and for related enzyme activities. In whole roots, ASC and DHA concentrations were higher in root apex and meristem and gradually decreased toward the root base. Guaiacol peroxidase, ASC peroxidase, monodehydroascorbate oxidoreductase, DHA reductase, catalase, and glutathione reductase activities showed differential activity patterns depending on the zone of the root and their apoplastic or symplastic origin. An in vivo staining of peroxidase activity also revealed a specific distribution pattern along the root axis. Using electron microscopy, hydrogen peroxide was found at different locations depending on the root zone but was mainly located in cell walls from epidermal and meristematic cells and in cells undergoing lignification. A balanced control of all of these molecules seems to exist along the root axis and may be directly related to the mechanisms in which the ASC system is involved, as cell division and elongation. The role of ASC on growth and development in relation to its presence at the different zones of the root is discussed.     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Changes in intracellular and apoplastic peroxidase activity, ascorbate redox status, and root elongation induced by enhanced ascorbate content in Allium cepa L

Onions (Allium cepa L.) treated with external ascorbic acid or with the immediate precursor of its synthesis L-galactono-gamma-lactone show a stimulated elongation rate of the roots and an increase in the number of new radicles appearing at the bulb base. Treatment with both molecules resulted in an enhanced accumulation of ascorbate and dehydroascorbate along the root axis, but the distribution of these redox forms was not uniform along the root, as detected in intracellular (symplastic) and extracellular (apoplastic) compartments. Thus, those radicular zones metabolically more active, such as the meristem and the elongation zone, accumulated the highest amount of both redox forms of ascorbate. On the other hand, ascorbate and L-galactono-gamma-lactone also stimulated cytosolic glucose-6-phosphate dehydrogenase activity and inhibited peroxidase activity as deduced from in vivo and in vitro experiments. Differences were also found when comparing apoplastic and symplastic activities. These results are compatible with the idea of an ascorbate-mediated stimulation of root growth by inhibiting cell wall stiffening and increasing root metabolism.     

Changes in Growth Pattern,Enzymatic Activities Related to Ascorbate Metabolism,and Hydrogen Peroxide in Onion Roots Growing Under Experimentally Increased Ascorbate Content

Onion (Allium cepa L.) roots treated with external ascorbate or with the immediate precursor of its synthesis, L-galactono-γ-lactone, increased root development measured as an increase in fresh and dry weights after 48-h treatments compared to controls. Also, treatments induced changes in extracellular (apoplastic) and cytosolic (symplastic) enzyme activities related to ascorbate metabolism and antioxidant protection, such as ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and catalase. Finally, we have found that both chemicals induced increased content of hydrogen peroxide in well-differentiated zones of the root, and local increases in meristematic and elongation zones were detected by cytochemistry as well. The results are discussed on the basis of changes in the root growth rate and other physiologic processes mediated by ascorbate in higher plants.     

Inhibition of neutral Mg2+-dependent sphingomyelinase by ubiquinol-mediated plasma membrane electron transport

Summary.  Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, has emerged as an important lipid signaling molecule. Previous work carried out by our group has documented that plasma membrane Mg²⁺-dependent neutral sphingomyelinase can be effectively inhibited by exogenous ubiquinol. In this work, we have tested whether or not plasma-membrane-associated electron transport can also achieve this inhibition through endogenous ubiquinol. Our results have shown that Mg²⁺-dependent neutral sphingomyelinase in isolated plasma membranes was inhibited by NAD(P)H under conditions where ubiquinone is reduced to ubiquinol. This inhibition was potentiated in the presence of an extra amount of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2). Depletion of plasma membranes from lipophilic antioxidants by solvent extraction abolished the inhibition by reduced pyridine nucleotides without affecting the sensitivity of the neutral sphingomyelinase to exogenous ubiquinol. Reconstitution of plasma membranes with ubiquinone restored the ability of NAD(P)H to inhibit the enzyme. Our results support that the reduction of endogenous ubiquinone to ubiquinol by NAD(P)H-driven electron transport may regulate the activity of the plasma membrane neutral sphingomyelinase. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Department of Biomedical Engineering, School of Medicine, University of Baltimore, Maryland, U.S.A. RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio C-6, Campus Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Effect of dietary coenzyme Q and fatty acids on the antioxidant status of rat tissues

Summary.  Wistar rats were fed with different diets with or without supplement coenzyme Q₁₀ (CoQ₁₀) and with oil of different sources (sunflower or virgin olive oil) for six or twelve months. Ubiquinone contents (CoQ₉ and CoQ₁₀) were quantified in homogenates of livers and brains from rats fed with the four diets. In the brain, younger rats showed a 3-fold higher amount of ubiquinone than older ones for all diets. In the liver, however, CoQ₁₀ supplementation increased the amount of CoQ₉ and CoQ₁₀ in both total homogenates and plasma membranes. Rats fed with sunflower oil as fat source showed higher amounts of ubiquinone content than those fed with olive oil, in total liver homogenates, but the total ubiquinone content in plasma membranes was similar with both fat sources. Older rats showed a higher amount of ubiquinone after diets supplemented with CoQ₁₀. Two ubiquinone-dependent antioxidant enzyme activities were measured. NADH-ferricyanide reductase activity in hepatocyte plasma membranes was unaltered by ubiquinone accumulation, but this activity increased slightly with age. Both cytosolic and membrane-bound dicumarol-sensitive NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase, EC 1.6.99.2) activities were decreased by diets supplemented with CoQ₁₀. Animals fed with olive oil presented lower DT-diaphorase activity than those fed with sunflower oil, suggesting that the CoQ₁₀ antioxidant protection is strengthened by olive oil as fat source. Received May 22, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba Edificio Severo Ochoa, Campus de Rabanales, 14071 Córdoba, Spain.     

Exogenous application of salicylic acid alleviates cadmium toxicity and reduces hydrogen peroxide accumulation in root apoplasts of Phaseolus aureus and Vicia sativa

We examined ameliorative effects of salicylic acid (SA) on two cadmium (Cd)-stressed legume crops with different Cd tolerances, viz. Phaseolus aureus (Cd sensitive) and Vicia sativa (Cd tolerant). Cd at 50 μM significantly increased the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^·−) in root apoplasts of P. aureus and V. sativa. When comparing the two species, we determined that Cd-induced production of H₂O₂ and O₂^·− was more pronounced in P. aureus root apoplasts than in V. sativa root apoplasts. V. sativa had higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) than P. aureus in root symplasts and apoplasts. Seed-soaking pretreatment with 100 μM SA decreased Cd-induced production of H₂O₂ and O₂^·− in apoplasts of both species, and increased activities of symplastic and apoplastic SOD, symplastic APX, and apoplastic CAT under Cd stress. Hence, SA-induced Cd tolerances in P. aureus and V. sativa are likely associated with increases in symplastic and apoplastic antioxidant enzyme activities.     

Synoptic and meteorological characterisation of olive pollen transport in Córdoba province (south-western Spain)

The main goal of the present study was to provide a detailed analysis of olive pollen transport dynamics in the province of Córdoba (south-western Spain) by applying back-trajectory analysis. Pollen data from 2006 and 2007 were analysed at four monitoring sites: Córdoba city in the centre of the province, Baena and Priego de Córdoba located in the south, and El Cabril reserve (Hornachuelos Natural Park) in the north. Particular attention was paid to nine episodes of high pollen counts. Synoptic surface maps were used, and kinematic back-trajectories (3D) were computed using the hybrid single particle Lagrangian integrated trajectory model (HYSPLIT) at 500 m above ground level, run with a time-step of 6 h over a period of 36 h. Findings were analysed in conjunction with daily and bi-hourly airborne pollen data, field phenological data and hourly surface meteorological data recorded at nearby stations: temperature, relative humidity, rainfall, wind direction and wind speed. The results identified two pollen source areas over the Córdoba province, the largest one located in the south, affecting Baena, Priego de Córdoba and Córdoba city, and one smaller located in the west, which determines mainly the pollen cycle over the north of the province, El Cabril. In addition, two air mass circulations were found, one coming from the south and crossing the main olive pollen sources very close to the surface and being frequently associated with higher pollen counts, and the other coming from the west and, in the episodes investigated, influencing mainly the north of the province.     

A multi-level approach using Gambusia affinis as a bioindicator of environmental pollution in the middle-lower basin of Suquía River

The Suquía River middle-lower basin (Córdoba, Argentina) is subjected to a strong anthropogenic impact because it receives pollutants from different sources. The main goal of this study was to evaluate the introduced fish species Gambusia affinis as a bioindicator of environmental pollution in the middle-lower basin of the Suquía River. The assessment was performed by measuring biomarkers at different levels of biological organization, at two sampling sites (before and after Córdoba city), and at dry and wet seasons. Water quality evaluation was made through a water-quality index (based on physico-chemical parameters), heavy-metals and pesticides concentrations in water. The water quality varied between sampling sites, showing the most degraded conditions downstream Córdoba city. The same pattern of variation was detected in the biomarkers studied, mainly in: gill and liver histopathological indexes, copulatory organ (gonopodium) morphology and vitellogenin expression in males and females. The present study characterized the environmental conditions in the middle-lower basin of the Suquía River and revealed the low freshwater quality at the most polluted site. Although G. affinis is an introduced species, it could be considered a good sentinel of water resource quality of invaded Neotropical basins. Our results demonstrated the importance of addressing the environmental quality monitoring through an integrated analysis of water quality parameters together with histological, morphological and molecular parameters. Thus, our study provides a good model for application in other basins of South America.     

Comparison of airborne herb pollen types in Córdoba (Southwestern Spain) and Poznan (Western Poland)

This study sought to compare airborne pollen counts for a number of common herbaceous species (Plantago, Chenopodiaceae–Amaranthaceae, Rumex, and Urticaceae) in two cities with differing weather conditions, Córdoba (Southwestern Spain) and Poznan (Western Poland). Pollen seasons for these species were studied from 1995 to 2005. Aerobiological sampling was performed using a Hirst type 7-day spore trap, in accordance with the procedure developed by the European Aerobiology Network. A Spearman correlation test was used to test for correlations between meteorological parameters and daily airborne pollen counts. The Spearman correlation test and the Wilcoxon signed ranks test were also used to compare mean daily pollen counts for the two study sites. In Córdoba, the pollen season generally started around two months earlier than in Poznan, and also lasted longer. These findings were attributed to the presence of a larger number of species in Córdoba, with overlapping pollen seasons, and also to more favorable weather conditions. Trends in pollen season start dates were fairly stable over the study period, with a slight tendency to delayed onset in Córdoba and a modest advance in start date in Poznan. The pollen season end date also remained reasonably stable over the study, with only a slight tendency for the season to end earlier in Córdoba and later in Poznan. A clear trend towards declining annual pollen counts was recorded over the study period for all pollen types in both cities.     

Assessing temperature and humidity conditions for dairy cattle in Córdoba,Argentina

Temperature and humidity conditions affect livestock production in Central Argentina. This study evaluates the risk of thermal stress affecting dairy production. The temperature-humidity index (THI) was used to analyze the regional and seasonal effects of temperature and humidity. Statistically, the THI was found to be normally distributed. The probability of occurrence of a daily THI higher than 72 was 40% for Río Cuarto during January. Regional variability of THI indicates a low risk of harmful extreme thermal stress conditions. The probability of THI being 78 or above ranges between 4% and 10% for the main dairy region of Córdoba during January. Also, in January and February, dairy production losses between 3 and 4 l cow(-1) day(-1) could be expected with a frequency of 5% in Río Cuarto and 15% in Villa de María de Río Seco.     

Hypodermosis in the red deer Cervus elaphus in Cordoba, Spain

The prevalence of red deer hypodermosis and the life cycle of Hypodermia diana Brauer 1858 in three provinces of the south of Spain, Córdoba, Jaen and Ciudad Real, were studied by inspecting 254, thirty-eight and thirty-five deer from each province respectively. The prevalence of infestations was: Córdoba, 87.75%; Jaen, 92.10%, Ciudad Real, 91.42%. From this we deduced an overall prevalence of 88.67%, comprising 88.23% in male deer and 89.96% in female deer. The intensity of the parasitism in 213 of the 245 animals from Córdoba varied between one warble per animal to more than 100 in 28 of the inspected animals. The chronology of the life cycle during the hunting period (October to the end of February) was investigated.     

Changes in ascorbic acid levels in apoplastic fluid during growth of pine hypocotyls. Effect on peroxidase activities associated with cell walls

The apoplastic fluid of pine ( Pinus pinaster Aiton) hypocotyls contains ascorbic acid (AA) and dehydroascorbic acid (DHA). The amounts of ascorbic and dehydroascorbic acids were in the nmol (g fresh weight)⁻¹ range and decreased with the hypocotyl age as well as along the hypocotyl axis. The ratio AA/(AA+DHA) also decreased with the hypocotyl age and along the hypocotyl. Both ascorbic oxidase and peroxidase activity against ascorbic acid showed very low activity not only in the apoplastic fluid but also in the fractions ionically and covalently bound to the cell walls. However, the peroxidase activity in the three abovementioned fractions was strongly increased in the presence of ferulic acid. That stimulation effect increased with the hypocotyl age and from the apical towards the basal region of the hypocotyls of 10-day-old seedlings. Furthermore, the oxidation of ferulic acid by apoplastic and ionically- and covalently-bound peroxidases was inhibited by ascorbic acid as long as ascorbate was available. A regulatory role of apoplastic ascorbic acid levels in the formation of dehydrodiferulic bridges between wall polysaccharides catalysed by cell wall peroxidases and thus in the cell wall stiffening during plant growth is proposed.     

New Patagonian baurusuchids (Crocodylomorpha; Notosuchia) from the Bajo de la Carpa Formation (Upper Cretaceous; Neuquén,Argentina): New evidences of the early sebecosuchian diversification in Gondwana

The Bajo de la Carpa Formation (Santonian) of the Neuquén basin (southwestern Argentina) has one of the most complete crocodyliform records of South America, together with the Adamantina and Marília formations of Brazil. In this contribution we report two fragmentary individuals from two different localities of the Bajo de la Carpa Formation: a middle-sized individual (MLP 26-IV-30-2), represented by postcranial remains from the Boca del Sapo locality and a large-bodied individual (MACN Pv-RN 1150), consisting only of snout elements from Paso Córdoba locality. Despite the remains of both specimens are fragmentary, they display anatomical characters of a specialized notosuchian carnivore clade: Baurusuchidae. Our phylogenetic analysis recovers these new individuals as non-baurusuchine baurusuchids, although they take multiple phylogenetic positions on the different most parsimonious trees. These new remains highlight the relevance of the Bajo de la Carpa Formation for understanding the rise of notosuchians during the Late Cretaceous.     

Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.     

Net sodium fluxes change significantly at anatomically distinct root zones of rice (Oryza sativa L.) seedlings

Casparian bands of endodermis and exodermis play crucial roles in blocking apoplastic movement of ions and water into the stele of roots through the cortex. These apoplastic barriers differ considerably in structure and function along the developing root. The present study assessed net Na⁺ fluxes in anatomically distinct root zones of rice seedlings and analyzed parts of individual roots showing different Na⁺ uptake. The results indicated that anatomically distinct root zones contributed differently to the overall uptake of Na⁺. The average Na⁺ uptake in root zones in which Casparian bands of the endo- and exo-dermis were interrupted by initiating lateral root primordia (root zone III) was significantly greater than that at the root apex, where Casparian bands were not yet formed (root zone I), or in the region where endo- and exo-dermis with Casparian bands were well developed (root zone II). The measurement of net Na⁺ fluxes using a non-invasive scanning ion-selective electrode technique (SIET) demonstrated that net Na⁺ flux varied significantly in different positions along developing rice roots, and a net Na⁺ influx was obvious at the base of young lateral root primordia. Since sodium fluxes changed significantly along developing roots of rice seedlings, we suggest that the significantly distinct net Na⁺ flux profile may be attributed to different apoplastic permeability due to lateral root primordia development for non-selective apoplastic bypass of ions along the apoplast.     

Target sites of aluminum phytotoxicity

The primary phytotoxic effect of aluminum (Al) is confined to the root apex. It is a matter of debate whether the primary injury of Al toxicity is apoplastic or symplastic. This review paper summarizes our current understanding of the spatial and metabolic sites of Al phytotoxicity. At tissue level, the meristematic, distal transition, and apical elongation zones of the root apex are most sensitive to Al. At cellular and molecular level, many cell components are implicated in Al toxicity including DNA in nucleus, numerous cytoplastic compounds, the plasma membrane, and the cell wall. Although it is difficult to distinguish the primary targets from the secondary effects so far, understanding of the target sites of Al toxicity is helpful for elucidating the mechanisms by which Al exerts its deleterious effects on root growth.This work was partly supported by fund from the Huoyingdong Foundation, Education Ministry of China and Natural Science Foundation of China (Contact No. 30170548).     

Symplastic and apoplastic uptake and root to shoot translocation of nickel in wheat as affected by exogenous amino acids

This study investigated the effect of exogenous amino acids on apoplastic and symplastic uptake and root to shoot translocation of nickel (Ni) in two wheat cultivars. Seedlings of a bread (Triticum aestivum cv. Back Cross) and a durum wheat cultivar (T. durum cv. Durum) were grown in a modified Johnson nutrient solution and exposed to two levels (50 and 100 μM) of histidine, glycine, and glutamine. Application of amino acids resulted in increasing symplastic to apoplastic Ni ratio in roots of both wheat cultivars, although glutamine and glycine were more effective than histidine under our experimental conditions. The amino acid used in the present study generally increased the relative transport of Ni from the roots to shoots in both wheat cultivars. Higher amounts of Ni were translocated to wheat shoots in the presence of histidine than the other amino acids studied, which indicated that histidine was more effective in translocation of Ni from roots to shoots. Amino acids used in the present study largely increased root symplastic Ni, but shoot Ni accumulation was much lower than the total Ni accumulation in roots, indicating a large proportion of Ni was retained or immobilized in wheat roots (either in the apoplastic or symplastic space), with only a very small fraction of Ni being translocated from the root to the shoot. According to the results, glutamine and glycine were more effective than histidine in enhancing the symplastic to apoplastic Ni ratio in the roots, while more Ni was translocated from the roots to the shoots in the presence of histidine.     

Distribution of a 9.1-kb insertion-deletion polymorphism among native and admixed populations from Argentina

We studied five population samples from Argentina, four drawn from Native American groups of the northeast region (Wichí, Pilagá, Toba, and Mbyá-Guaraní) and one from two small villages of the Córdoba province. In this study we report genotypes and allele frequencies of the 9.1-kb insertion-deletion polymorphism located on chromosome 22. The frequency of the deletion allele ranges from 0.276 in the Mbyá-Guaraní to 0.470 in the Pilagá. The coefficient of population differentiation is fairly low (F(ST) = 0.013), does not reflect any geographic or linguistic pattern, and seems to be more related to stochastic processes than to directional forces.     

Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humour

1. Glutathione peroxidase has been demonstrated in cattle, rabbit and guineapig lenses. 2. The enzyme will oxidize GSH either with hydrogen peroxide added at the start of the reaction or with hydrogen peroxide generated enzymically with glucose oxidase. 3. No product other than GSSG was detected. 4. Oxidation of GSH can be coupled with oxidation of malate through the intermediate reaction of glutathione reductase and NADPH₂. 5. Traces of hydrogen peroxide are present in aqueous humour: it is formed when the ascorbic acid of aqueous humour is oxidized. 6. Hydrogen peroxide will diffuse into the explanted intact lens and oxidize the contained GSH. The addition of glucose to the medium together with hydrogen peroxide maintains the concentration of lens GSH. 7. Glutathione peroxidase in lens extracts will couple with the oxidation of ascorbic acid. 8. It is suggested that, as there is only weak catalase activity in lens, glutathione peroxidase may act as one link between the oxygen of the aqueous humour and NADPH₂.