Effects of ozone on apoplast/cytoplasm partitioning of ascorbic acid in snap bean

Apoplast/cytoplasm partitioning of ascorbic acid (AA) was examined in four genotypes of snap bean ( Phaseolus vulgaris L.) known to differ in ozone sensitivity. Plants were grown in pots under field conditions using open-top chambers to establish charcoal-filtered (CF) air (36 nmol mol⁻¹ ozone) or elevated ozone (77 nmol mol⁻¹ ozone) treatments. AA in fully expanded leaves of 36-day-old plants was separated into apoplast and cytoplasm fractions by vacuum infiltration methods using glucose 6-phosphate as a marker for cytoplasm contamination. Apoplast ascorbate levels ranged from 30 to 150 nmol g⁻¹ fresh weight. Ozone-sensitive genotypes partitioned 1–2% of total AA into the apoplast under CF conditions and up to 7% following a 7-day ozone exposure. In contrast, an ozone-tolerant genotype partitioned 3–4% of total leaf AA into the leaf apoplast in both CF and ozone-treated plants. The results suggest that genetic background and ozone stress are factors that affect AA levels in the extracellular space. For all genotypes, the fraction of AA in the oxidized form was higher in the apoplast compared to the cytoplasm, indicative of a more oxidizing environment within the cell wall.     

Role of leaf apoplast in silicon-mediated manganese tolerance of Cucumis sativus L.

Silicon (Si) supplied as sodium silicate (1·8 mm ) clearly decreased symptoms of manganese (Mn) toxicity in Cucumis sativus L. (cv. Chinesische Schlange) grown in nutrient solution with low to elevated Mn concentrations (0·5–1000 µm ). Despite approximately the same total Mn content in the leaves, plants not treated with Si had higher Mn concentrations in the intercellular washing fluid (IWF) compared with plants treated with Si, especially in the BaCl₂‐ and DTPA‐exchangeable fraction of the leaf apoplast. The Mn concentration of the IWF correlated positively with the severity of Mn‐toxicity symptoms and negatively with the Si supply. Furthermore, in Si‐treated plants less Mn was located in the symplast (< 10%) and more Mn was bound to the cell wall (> 90%) compared with non‐Si‐treated plants (about 50% in each compartment). Manganese present in Si‐treated plants is therefore less available and for this reason less toxic than in plants not treated with Si. It is concluded that Si‐mediated tolerance of Mn in C. sativus is a consequence of stronger binding of Mn to cell walls and a lowering of Mn concentration within the symplast. These results support the role of Si as an important beneficial element in plant nutrition.     

Redox state of ascorbic acid in the apoplast of stems of Kalanchoë daigremontiana

The aqueous phase of cell walls in stems of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie (apoplast) contained ascorbic acid (AA) and dehydroascorbic acid (DHA). Ratios of AA/(AA + DHA) were 0.31 ± 0.12 (SD, n = 4), whereas those of whole stems (tissues plus apoplast) were >0.9. The amounts of (AA + DHA) in the stems were 1970 ± 190 (SD, n = 4) nmol g⁻¹ fresh weight and those in the apoplast were 14 ± 2 (SD, n = 4) nmol g⁻¹ fresh weight of stems. Ratios of AA/(AA + DHA) differed in different tissues of the stems. The ratios of AA/(AA + DHA) of apoplast plus symplast were in the following order: pith ⋍ epidermis plus cortex > vascular bundle system, and those of apoplast were: pith > epidermis plus cortex > vascular bundle system. Ratios of AA/(AA + DHA) in the apoplast of the different tissues decreased to about 1/3 of the original values after wounding, while the amounts of (AA + DHA) remained largely unaffected. In contrast, soluble apoplastic peroxidase activities increased 30- to 70-fold on wounding. Hydrogen peroxide infiltrated into stems caused a rapid oxidation of AA. Coniferyl alcohol was oxidized by peroxidase in intercellular washing fluid and by cell wall-bound peroxidase. The oxidation of coniferyl alcohol by peroxidase in intercellular washing fluid was completely inhibited as long as AA was present in reaction mixtures. The oxidation of the coniferyl alcohol by cell wall-bound peroxidase was partially inihibited by AA and the degree of inhibition was dependent upon the concentration of AA. The possible functions of AA in the apoplast are discussed in relation to the control of peroxidase-dependent oxidation of phenolics.     

Ascorbate in the leaf apoplast is a factor mediating ozone resistance in Plantago major

The role of ascorbate in mediating ozone resistance was examined in Plantago major L. Seedlings of eleven populations which exhibited differential resistance to ozone were fumigated in controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 15 nmol·mol^–1 ozone overnight rising to a maximum between 12:00–16:00 hours of 75 nmol·mol^–1 for 14 d. Measurements of ascorbate content were made on apoplastic and symplastic extracts. Populations differed in their constitutive level of ascorbate in youngest fully expanded leaves, and regression analysis revealed a significant correlation between ascorbate content in ozone-treated leaves and the ozone resistance of the populations. The relationship was stronger using apoplastic ascorbate levels than with corresponding symplastic measurements. The ascorbate content of the youngest fully expanded leaf of an ozone sensitive population was increased by foliar application of ascorbate. No significant difference in stomatal conductance was found between control and ascorbate-treated plants. Following spraying, plants were fumigated with 400 nmol·mol^–1 ozone for 7 h. In control plants, ozone exposure resulted in extensive visible leaf damage (20–70 % at the end of the fumigation period) and decreased rates of CO₂ assimilation (–57 %). However, ascorbate treatment prevented the appearance of visible injury, and ameliorated the decline in photosynthesis induced by ozone (–26 %). Modelled data estimating the extent of protection afforded by apoplastic ascorbate against ozone supported the experimental observations. The results suggested that although apoplastic ascorbate plays an important role, other factors must also contribute to the mediation of ozone resistance in P. major.     

The decline in ascorbic acid content is associated with cadmium toxicity of rice seedlings

Cadmium (Cd) toxicity of rice (Oryza sativa L. cv. Taichung Native 1) seedlings was evaluated by the decrease in chlorophyll content and the increase in malondialdehyde (MDA) in the second leaves of rice seedlings. CdCl₂ (5 μM) treatment was accompanied by a decrease in the contents of ascorbic acid (AsA) and AsA + dehydroascorbate (DHA) and in the ratios of AsA/DHA in leaves. However, CdCl₂ treatment resulted in an increase in DHA content in leaves. Moreover, the decrease in AsA content was prior to the occurrence of chlorosis and associated with the increase in MDA content in the leaves of seedlings treated with Cd. Pretreatment with 0.5 mM AsA or l-galactono-1,4-lactone (GalL), the biosynthetic precursor of AsA, for 6 h resulted in an increase in the contents of AsA and reduced glutathione (GSH), the ratios of AsA/DHA and GSH/oxidized glutathione, and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in the leaves of rice seedlings. Quantitative RT-PCR was applied to quantify the mRNA levels for OsAPX and OsGR genes from rice leaves to examine the effect of AsA or GalL pretreatment on the expression of OsAPX and OsGR genes in rice leaves. The expression of OsAPX2, OsAPX3, OsAPX4, OsAPX5, OsAPX6, OsAPX7, and OsGR1 was increased by AsA or GalL pretreatment. Rice seedlings pretreated with AsA or GalL were observed to reduce the subsequent Cd-induced toxicity. Our results suggest that AsA content may play a role in regulating Cd toxicity of rice seedlings.     

The role of hydrogen peroxide-producing and hydrogen peroxide-consuming peroxidases in the leaf apoplast of cowpea in manganese tolerance

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.     

Influence of pesticide treatments on the dynamics of whiteflies and associated parasitoids in snap bean fields

To determine the influence of pesticidetreatments on the population dynamics of thewhiteflies Trialeurodes vaporariorum(Westwood) and Bemisia tabaci Gennadius (Homoptera: Aleyrodidae)and their naturally occurring parasitoids, weperformed field experiments on insecticidesprayed and unsprayed fields during a croppingseason of snap bean in Pradera, Valle delCauca, Colombia. Substantially largerpopulations of whitefly nymphs occurred in theunsprayed field than in the sprayed field. Parasitoids were more frequent in unsprayedthan in sprayed fields with Encarsianigricephala Dozier being more prevalent thanAmitus fuscipennis MacGown & Nebeker. Insprayed fields the nymphs parasitized by E. nigricephala exceeded the unparasitizedwhitefly nymphs at the end of the croppingseason. Our results suggest that while undercurrent agricultural practices whiteflies onsnap beans cannot be exclusively controlled bynaturally occurring parasitoids, parasitoidsmay be integrated with chemical control inorder to reduce crop damage.     

Low leaf hydraulic conductance associated with drought tolerance in soybean

Lack of water is the most serious environmental constraint on agricultural production. More efficient use of water resources is a key solution for increased plant productivity in water-deficit environments. We examined the hydraulic characteristics of a 'slow wilting' phenotype in soybean ( Glycine max Merr.), PI 416937, which has been shown to have relatively constant transpiration rates above a threshold atmospheric vapor pressure deficit (VPD). The VPD response of PI 416937 was confirmed. Three experiments are reported to examine the hypothesis that the VPD response was a result of low hydraulic conductance in leaves as compared to two other soybean genotypes. Results are reported from experiments to measure transpiration response to VPD when xylem water potential was maintained at zero, leaf rehydration response and leaf carbon assimilation response to petiole cutting. Major interspecific differences in leaf hydraulic properties were observed. The observed low leaf hydraulic conductance in PI 416937 is consistent with an increased water use efficiency, and an increased water conservation by limiting transpiration rates under high evaporative conditions but allowing normal gas exchange rates under more moderate evaporative conditions.     

Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress

Quantitative trait loci (QTL) mapping is a step towards the identification of factors regulating traits such as fruit ascorbic acid content. A previously identified QTL controlling variations in tomato fruit ascorbic acid has been fine mapped and reveals that the QTL has a polygenic and epistatic architecture. A monodehydroascorbate reductase (MDHAR) allele is a candidate for a proportion of the increase in fruit ascorbic acid content. The MDHAR enzyme is active in different stages of fruit ripening, shows increased activity in the introgression lines containing the wild-type ( Solanum pennellii ) allele, and responds to chilling injury in tomato along with the reduced/oxidized ascorbate ratio. Low temperature storage of different tomato introgression lines with all or part of the QTL for ascorbic acid and with or without the wild MDHAR allele shows that enzyme activity explains 84% of the variation in the reduced ascorbic acid levels of tomato fruit following storage at 4 °C, compared with 38% at harvest under non-stress conditions. A role is indicated for MDHAR in the maintenance of ascorbate levels in fruit under stress conditions. Furthermore, an increased fruit MDHAR activity and a lower oxidation level of the fruit ascorbate pool are correlated with decreased loss of firmness because of chilling injury.     

Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate

 This paper presents a mathematical model which enables the semi-quantification of ozone (O₃) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments – based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O₃ reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O₃ taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O₃ in the cell wall. Received: 13 May 1999 / Accepted: 5 August 1999     

Cultivar specific variations in morphological and biochemical characteristics of mung bean due to foliar spray of ascorbic acid under elevated ozone

Effect of exogenous application of ascorbic acid (Asc) solution was examined on the growth, photosynthetic pigments, biochemical and yield characteristics of mung bean cultivars against ozone (O₃). Experiment was performed on six mung bean cultivars in open top chambers under field conditions and Asc was applied as foliar spray prior to the exposure of ambient (AO+) and elevated (EO+) levels of O₃. Application of Asc showed increment in growth attributes as compared to plants not provided Asc (AO). However, O₃ induced the production of reactive oxygen species led to membrane damage. Reductions were depicted in lipid peroxidation, solute leakage and foliar injury % in AO+ and EO+ as compared to plants not provided with Asc. Photosynthetic pigments and ascorbic acid content along with activities of anti-oxidative enzymes (APX, CAT, GR and SOD) showed increments in AO+ and EO+ with cultivar-specific variations. Cultivars HUM-1 and HUM-2 restored yield with Asc application while less response was observed in other test cultivars. Quality of the seeds was also affected by Asc treatment in the plants exposed to ambient and elevated levels of O₃. Therefore, exogenous application of Asc promotes plant’s performance by providing protection against O₃ induced oxidative stress and may be used in screening of the mung bean cultivars against O₃ phytotoxicity.     

Aging is associated with elevated muscle triglyceride content and increased insulin-stimulated fatty acid uptake

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under hyperglycemic and hyperinsulinemic conditions. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [1-14C]palmitate, and [3-3H]glucose. Glucose uptake and palmitate delivery were similar among age groups. Palmitate uptake and oxidation as well as muscle protein concentration of fatty acid translocase (FAT/CD36) and plasma membrane fatty acid-binding protein (FABPPM) were significantly increased (P < or = 0.05) in 24- vs. 5- and 15-mo-old animals. Compared with 5- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 72-145% in red and 112-129% in white muscles of 24-mo-old animals. Palmitate uptake was associated with total preperfusion TG concentration (r2 = 0.27, P < 0.05) and total TG synthesis rate (r2 = 0.68, P < 0.05). These results indicate that, under insulin-stimulated conditions, FA uptake is significantly increased in old animals, which is associated with increased rates of TG synthesis and may contribute to the accumulation of TG in muscle of old animals.     

Changes in the tobacco leaf apoplast proteome in response to salt stress

The apoplast of plant cells is a dynamic compartment involved in many processes, including maintenance of tissue shape, development, nutrition, signalling, detoxification and defence. In this work we used Nicotiana tabacum plants as a model to investigate changes in the soluble apoplast composition induced in response to salt stress. Apoplastic fluid was extracted from leaves of control plants and plants exposed to salt stress, using a vacuum infiltration procedure. Two-dimension electrophoretic analyses revealed about 150 polypeptide spots in the pH range of 3.0 to 10.0, in independent protein extracts, with a high level of reproducibility between the two sample sets. Quantitative evaluation and statistical analyses of the resolved spots in treated and untreated samples revealed 20 polypeptides whose abundance changed in response to salt stress. Mass spectroscopic peptide separation and sequencing was used to identify polypeptides affected by salt stress. While the levels of some proteins were reduced by salt-treatment, an enhanced accumulation of protein species known to be induced by biotic and abiotic stresses was observed. In particular, two chitinases and a germin-like protein increased significantly and two lipid transfer proteins were expressed entirely de novo. Some apoplastic polypeptides, involved in cell wall modifications during plant development, remained largely unchanged. The significance of these components is discussed in the context of stress responses in plants.     

Ozone concentration in leaf intercellular air spaces is close to zero

Transpiration and ozone uptake rates were measured simultaneously in sunflower leaves at different stomatal openings and various ozone concentrations. Ozone uptake rates were proportional to the ozone concentration up to 1500 nanoliters per liter. The leaf gas phase diffusion resistance (stomatal plus boundary layer) to water vapor was calculated and converted to the resistance to ozone multiplying it by the theoretical ratio of diffusion coefficients for water vapor and ozone in air (1.67). The ozone concentration in intercellular air spaces calculated from the ozone uptake rate and diffusion resistance to ozone scattered around zero. The ozone concentration in intercellular air spaces was measured directly by supplying ozone to the leaf from one side and measuring the equilibrium concentration above the other side, and it was found to be zero. The total leaf resistance to ozone was proportional to the gas phase resistance to water vapor with a coefficient of 1.68. It is concluded that ozone enters the leaf by diffusion through the stomata, and is rapidly decomposed in cell walls and plasmalemma.     

Coronary endothelial dysfunction is not rapidly reversible with ascorbic acid

In humans with cardiovascular risk factors, increased vascular production of superoxide anion may contribute to endothelial dysfunction by its reacting with nitric oxide and reducing its biological activity. High concentrations of ascorbic acid scavenge superoxide anion and restore normal endothelium-dependent vasodilation in humans with cardiovascular risk factors. To investigate the contribution of increased superoxide anion to endothelial dysfunction in atherosclerotic coronary arteries, we examined the effect of sequential infusions of ascorbic acid (final concentration 0.1, 1.0, and 10 mmol/L) or placebo on coronary endothelial function in 26 subjects referred for cardiac catheterization to evaluate coronary artery disease. Coronary vasomotor function was evaluated using intracoronary agonist infusion, quantitative angiography, and intracoronary Doppler measurements. At baseline, endothelium-dependent vasodilation of epicardial arteries and coronary microvessels was impaired to an equivalent extent in the ascorbic acid and placebo groups. Sequential ascorbic acid infusions had no effect on the acetylcholine-induced change in coronary artery diameter (-11+/-8, -12+/-10, and -9+/-9%) compared with the effect of placebo (-14+/-13, -16+/-10, and -13+/-9%) infusions (p=0.98). Similarly, the changes in coronary blood flow during acetylcholine infusions were equivalent during ascorbic acid (51+/-44, 67+/-66, and 62+/-52%) and placebo (61+/-104, 55+/-93, and 50+/-69%) infusions (p=0.63). Ascorbic acid also had no effect on the dilator response to intracoronary nitroglycerin (p=0.19). These data argue against an important role for superoxide-mediated "inactivation" of nitric oxide or another rapidly reversible form of oxidative stress as a mechanism of coronary endothelial dysfunction in patients with coronary atherosclerosis.     

Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea

Excess manganese (Mn) supply causes formation of visible brown depositions in the cell walls of leaves of cowpea (Vigna unguiculata), which consist of oxidized Mn and oxidized phenols. Because oxidation of Mn and phenolic compounds in the leaf apoplast was proposed to be catalyzed by apoplastic peroxidases (PODs), induction of these enzymes by Mn excess was investigated. POD activity increased upon prolonged Mn treatment in the leaf tissue. Simultaneously, a significant increase in the concentration of soluble apoplastic proteins in "apoplastic washing fluid" was observed. The identity of the released proteins was systematically characterized by analysis of the apoplast proteome using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. Some of the identified proteins exhibit sequence identity to acidic PODs from other plants. Several other proteins show homologies to pathogenesis-related proteins, e.g. glucanase, chitinase, and thaumatin-like proteins. Because pathogenesis-related-like proteins are known to be induced by various other abiotic and biotic stresses, a specific physiological role of these proteins in response to excess Mn supply remains to be established. The specific role of apoplastic PODs in the response of plants to Mn stress is discussed.