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.     

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.     

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.     

Cadmium disrupts apoplastic ascorbate redox status in barley root tips

Using a short-term Cd treatment (5–30 min), we analysed the effect of Cd on apoplastic ascorbate redox status and their regeneration during the recovery period in barley root tips. Root growth inhibition induced by 15 μM Cd was detectable after 5 min of exposure and increased in a time-dependent manner up to 15 min of exposure. High 30 μM Cd concentration completely inhibited root growth during the first 6 h after short-term treatment. In parallel with Cd-induced root growth inhibition, a rapid decrease of apoplastic ascorbate dehydroascorbate ratio was observed immediately after short-term treatments. During the recovery from 15 μM Cd short-term treatment, apoplastic ascorbate was rapidly regenerated to the control level in the first root segment containing meristem and elongation zone. In contrast to 15 μM Cd treatment, in 30 μM Cd-treated roots apoplastic ascorbate level was sustained at a significantly lower level compared to control roots. We confirmed that a decrease of apoplastic ascorbate/dehydroascorbate ratio in the elongation zone was associated with root growth inhibition or arrest.     

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.     

Exogenous auxin affects ascorbate metabolism in roots of tomato seedlings

Ascorbate levels and redox states, as well as the activities of the enzymes of ascorbate metabolism, were analyzed in roots of tomato seedlings during the culture on a medium supplemented with auxin and compared to the control cultured on an auxin-free medium. Biochemical parameters were determined separately in the distal part of the root where the inhibitory effect of auxin on root elongation growth is observed and in the proximal half on the organ which reacts to auxin treatment with increased lateral root proliferation. ASC peroxidase activity was found to be stimulated by auxin treatment in the lateral-root forming part of the root. This effect was not observed in the distal part of the organ. On the other hand, ASC oxidase activity was raised by auxin exclusively in the distal part of the root. An inhibitory effect of auxin supplementation to the medium on ASC—reducing enzymes was observed. The dehydroascorbate reductase activity was found to be inhibited by auxin only in the proximal part, while the activity of monodehydroascorbate reductase in both, the proximal and distal parts of the root. Ascorbate content increased in roots during culture irrespective of the presence of auxin. However, auxin treatment resulted in higher DHA levels and more significant participation of DHA in the total ascorbate pool when compared to the control grown on the auxin-free medium. Similar to auxin, adding DHA to the culture medium stimulated lateral root formation and inhibited primary root elongation. In contrast to DHA, ASC treatment affected significantly neither lateral root formation nor primary root growth and partly reversed the stimulatory effect of IAA on root formation and the inhibitory effect on root elongation. These results suggest that auxin induced changes in ascorbate metabolism may be involved in developmental reactions in tomato roots.     

Apoplastic ascorbate metabolism and its role in the regulation of cell signalling

The apoplast has crucial functions in plant biology. It comprises all the compartments beyond the plasmalemma, including the cell wall. As the reservoir of information on the biotic and abiotic environment surrounding the cell and a major conduit of information between cells, the apoplast has functions in stress perception and the subsequent appropriate control of growth and defence. The oxidative burst phenomenon, caused by environmental challenges and pathogen attack in particular, oxidises the apoplast. Ascorbic acid (AA), the major and probably the only antioxidant buffer in the apoplast, becomes oxidised in these conditions. The apoplastic enzyme ascorbate oxidase (AO) also regulates the reduction/oxidation (redox) state of the apoplastic ascorbate pool. We propose that a key function of the oxidative burst and of AO is to modify the apoplastic redox state in such a way as to modify receptor activity and signal transduction to regulate defence and growth.     

Differential distribution of ascorbic acid,peroxidase activity,and hydrogen peroxide along the root axis in Allium cepa L. and its possible relationship with cell growth and differentiation

Summary.  In this paper we show an asymmetrical distribution of apoplastic and symplastic ascorbic acid content, peroxidase activities and hydrogen peroxide along the root axis in Allium cepa L. For most of these metabolites, a marked gradient from the root apex to the onion base was observed and was different for apoplastic and symplastic compartments. In total homogenates, ascorbic acid content was higher in the zones closer to the apex and decreased towards the root base. However, an opposite pattern was observed in the apoplastic fraction. Peroxidase activities with guaiacol, ferulic acid, ascorbic acid, and coniferyl alcohol were also different depending on the evaluated zone and the fraction used (apoplastic or symplastic). In general, each activity had a specific and unique pattern. Immunodetection of peroxidase proteins in Western blots using anti-horseradish peroxidase and anti-ascorbate peroxidase antibodies revealed different bands at the different zones of the root. Hydrogen peroxide was detected by electron microscopy and was mainly found in cell walls of epidermis (or rhizodermis), meristem, and elongating cells. The number of cell walls showing hydrogen peroxide decreased dramatically towards the root base. The results suggest that the different zones of the root show specific requirements for ascorbic acid and hydrogen peroxide. Also, each fragment of the root seems to express specific peroxidase proteins. Different processes that take place at every part of the root, as cell proliferation and elongation near the root apex and gradual lignification and differentiation towards the root base are the key to explain the results. Received May 10, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Edificio C-6, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Role of apoplastic ascorbate and hydrogen peroxide in the control of cell growth in pine hypocotyls

The growth cessation of plant axis has been related with the formation of diphenyl bridges among the pectic components of the cell wall caused by the action of apoplastic peroxidases using hydrogen peroxide as electron acceptor. The formation of diphenyl bridges is prevented by the presence of ascorbate in the apoplastic fluid which acts as a hydrogen peroxide scavenger. The current work focuses on the role of the apoplastic ascorbate and hydrogen peroxide in the cell growth. The addition of hydrogen peroxide caused an inhibition of the auxin-induced growth as well as a significant decrease in the cell wall creep induced by acid-pH solutions. The hydrogen peroxide content in apoplastic fluid increased with the hypocotyl age and along the hypocotyl axis of 10-day-old pine seedlings, as the growth capacity decreased. On the other hand, the ascorbate content in the apoplastic fluid decreased with the hypocotyl age and along the hypocotyl axis of 10-day-old seedlings. A very significant correlation between the hydrogen peroxide apoplastic level and the growth rate as well as between the ascorbate/hydrogen peroxide molar ratio and the growth rate of hypocotyls have been found suggesting that the redox state is the main factor controlling the cell wall stiffening mechanism and thus growth in pine hypocotyls.     

Stimulation of onion root elongation by ascorbate and ascorbate free radical inAllium cepa L.

Summary We report that ascorbate free radical stimulates onion root growth at 15 °C and 25 °C. The fully reduced form, ascorbate, also stimulates root elongation if culture conditions allow its oxidation. When ascorbate oxidation was inhibited, no stimulation of root growth was found. The effect of the fully oxidized form, dehydroascorbate, was inhibitory. We show also that ascorbate free radical generated by ascorbate oxidation, is reduced back probably by a transplasmalemma reductase. These results are discussed on the basis of an activation of a transplasma membrane redox system likely involved in processes related to cell growth.Abbreviations AFR ascorbate free radical - ASC ascorbate - DHA dehydroascorbate     

Differences in Cowpea Root Growth Triggered by Salinity and Dehydration are Associated with Oxidative Modulation Involving Types I and III Peroxidases and Apoplastic Ascorbate

The aim of this work was to investigate the balance between the activities of ascorbate peroxidase (APX) and phenol peroxidases (POD) and cowpea root growth in response to dehydration and salt stress. Root growth and indicators of oxidative response were markedly changed in response to salinity and dehydration. Salt treatment strongly inhibited root elongation, which was associated with an increase in lignin content and a significant decrease in the concentrations of apoplastic hydrogen peroxide (H₂O₂) and ascorbate. In conditions of extreme salinity, cytosol–APX activity was significantly decreased. In contrast, cell-wall POD activity was greatly increased, whereas lipid peroxidation was unchanged. These results indicate that POD could be involved in both H₂O₂ scavenging and the inhibition of root elongation under high salinity. In contrast, dehydration stimulated primary root elongation and increased lipid peroxidation and apoplastic ascorbate content, but it did not change APX and POD activities or H₂O₂ concentration. When cowpea roots were subjected to salinity followed by dehydration, the water and pressure potentials were decreased, and lipid peroxidation was markedly increased, highlighting the additive nature of the inhibitory effects caused by salt and dehydration. The proline concentration was markedly increased by dehydration alone, as well as by salt followed by dehydration, suggesting a possible role for proline in osmotic adjustment. Salinity and dehydration induce contrasting responses in the growth and morphology of cowpea roots. These effects are associated with different types of oxidative modulation involving cytosolic-APX and cell-wall POD activities and apoplast H₂O₂ and ascorbate levels.     

A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit

The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild‐type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose : sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.     

Transport and action of ascorbate at the plant plasma membrane

The plasmalemma is both a bridge and a barrier between the cytoplasm and the outside world. It is a dynamic interface that perceives and transmits information concerning changes in the environment to the nucleus to modify gene expression. In plants, ascorbate is an essential part of this dialogue. The concentration and ratio of reduced to oxidized ascorbate in the apoplast, for example, possibly modulates cell division and growth. The leaf apoplast contains millimolar amounts of ascorbate that protect the plasmalemma against oxidative damage. The apoplastic ascorbate-dehydroascorbate redox couple is linked to the cytoplasmic ascorbate-dehydroascorbate redox couple by specific transporters for either or both metabolites. Although evidence about the mechanisms driving ascorbate or dehydroascorbate transport remains inconclusive, these carrier proteins potentially regulate the level and redox status of ascorbate in the apoplast. The redox coupling between compartments facilitated by these transport systems allows coordinated control of key physiological responses to environmental cues.     

Short- and long-term effects of dehydroascorbate in Lupinus albus and Allium cepa roots

Administration of 1 mM dehydroascorbate (DHA) results in a rapid and large increase in cellular ascorbate (AA) content in both Lupinus albus L. and Allium cepa L. root tips. Uptake of DHA from the medium occurs at a high rate within 10-12 h of incubation, and is slowed down thereafter. In the first few h, DHA reduction to AA is apparently correlated to GSH depletion and slightly higher DHA reductase activity. DHA incubation also seems to induce new GSH synthesis. Longer DHA incubation (24 h) affects root growth by inhibiting cell proliferation. At this stage, an apparently generalised oxidation of SH-containing proteins is observed in DHA-treated roots. Treatment with 1 mM L-galactono-gamma-lactone, the last precursor of AA biosynthesis, results in an increase in AA content similar to that obtained with DHA, but stimulates growth and affects the redox state of SH-containing proteins in the opposite way. A possible multi-step mechanism of DHA reduction/removal is suggested and the hypothesis that DHA inhibits cell cycle progression by affecting the redox state of SH-containing proteins is discussed.     

Impacts of ozone on Plantago major: apoplastic and symplastic antioxidant status

The aim of this work was to examine the correspondence between apoplastic/symplastic antioxidant status and previously reported plant age-related shifts in the ozone (O₃) resistance of Plantago major L. Seed-grown plants were fumigated in duplicate controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 70 nmol mol⁻¹ O₃ for 7 h d⁻¹ over a 42 d period. Measurements of stomatal conductance and antioxidants were made after 14, 28 and 42 d fumigation, on leaves at an equivalent stage of development (youngest fully expanded leaf, measured c . 9 d after emergence). Ozone exposure resulted in a similar decline in stomatal conductance across plant ages, indicating that increases in O₃ resistance with plant age were mediated through changes in the tolerance of leaf tissue rather than enhanced pollutant exclusion. Leaf apoplastic washing fluid was found to contain 'unspecific' peroxidase, ascorbate peroxidase, superoxide dismutase and ascorbate, but not glutathione and the enzymes required to facilitate the regeneration of ascorbate from its oxidized forms. A weak induction in the activity of certain symplastic antioxidants was found after 14 d O₃ fumigation, despite a lack of visible symptoms of injury, but shifts in symplastic antioxidant enzyme activity were not consistent with previously observed increases in resistance to O₃ with plant age. By contrast, changes in 'unspecific' peroxidase activity and in the small pool of ascorbate in the leaf apoplast were found to accompany age-related shifts in O₃ resistance. It is concluded that constituents of the leaf apoplast may constitute a potentially important front line defence against O₃.     

Endogenous ascorbate restrains apoplastic peroxidase activity during sunflower leaf development

Several apoplastic enzymes have been implicated in the control of elongation growth of plant cells. Among them, peroxidases contribute to both loosening and stiffening of the cell wall. They appear to be regulated by various mechanisms, including the action of extracellular inhibitors. To obtain evidence of the role of the enzyme–inhibitor interaction during leaf development, the intercellular washing fluids from Helianthus annuus leaves of different ages were isolated using standard methods of vacuum infiltration and centrifugation. Peroxidase activities, assessed using tetramethylbenzidine as substrate, increased during leaf development, reaching a maximum value after the leaves were fully expanded. An inhibitor, chemically characterised as ascorbate, co‐localised with the enzyme in the apoplast. Moreover, there was a strong negative correlation between the action of peroxidase and the micromolar concentration of ascorbate in the apoplastic fluid. The results show that in growing leaves, the in planta ascorbate concentration is able to restrain peroxidase enzyme activity. Then, at the time of growth cessation, the loss of extracellular ascorbate relieves the inhibition on this enzyme that contributes to wall fixation.     

Nitric oxide increases the enzymatic activity of three ascorbate peroxidase isoforms in soybean root nodules

Ascorbate peroxidase is one of the major enzymes regulating the levels of H₂O₂ in plants and plays a crucial role in maintaining root nodule redox status. We used fully developed and mature nitrogen fixing root nodules from soybean plants to analyze the effect of exogenously applied nitric oxide, generated from the nitric oxide donor 2,2′-(hydroxynitrosohydrazono)bis-ethanimine, on the enzymatic activity of soybean root nodule ascorbate peroxidase. Nitric oxide caused an increase in the total enzymatic activity of ascorbate peroxidase. The nitric oxide-induced changes in ascorbate peroxidase enzymatic activity were coupled to altered nodule H₂O₂ content. Further analysis of ascorbate peroxidase enzymatic activity identified three ascorbate peroxidase isoforms for which augmented enzymatic activity occurred in response to nitric oxide. Our results demonstrate that nitric oxide regulates soybean root nodule ascorbate peroxidase activity. We propose a role of nitric oxide in regulating ascorbate-dependent redox status in soybean root nodule tissue.Key words: antioxidant enzymes, ascorbate peroxidase, nitric oxide, oxidative stress, reactive oxygen species, redox homeostasis, soybean root nodules     

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.     

菜豆茎韧皮部卸出的细胞途径以及激素的促进作用(英文)

通过缩小叶面积和去茎尖改变源库比率,以调节韧皮部卸出的途径,证明了韧皮部卸出的共质体与质外体途径的季节变化,和由对氯高汞苯磺酸所诱发的从质外体向共质体途径的转变,是与光合产物的输入有关。缩小叶面积而降低源库比率,能增加夏季生长植株茎韧皮部的质外体卸出,但对冬季生长植株无影响。去尖而增加源库比率,则促进共质体卸出。赤霉酸和激动素能促进共质体的横向转运,但对质外体转运无作用。当质外体为主要运输途径时,赤霉酸和激动素开启共质体途径。赤霉酸和激动素刺激光合产物,通过共质体从筛管一伴胞复合体向韧皮部薄壁纽胞输送,并可能在韧皮部薄壁细胞被动扩散到自由空间。由此可进一步说明蔗糖在激素处理部位自由空间的增加。