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.     

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.     

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.     

Role of Apoplastic and Cell-Wall Peroxidases on the Stimulation of Root Elongation by Ascorbate

Elongation of onion (Allium cepa L.) roots was highly stimulated by ascorbate (ASC) and its natural precursor I-galactone-[gamma]-lactone (GL). When incubation media were supplemented with lycorine (Lyc), an inhibitor of the ASC biosynthesis, root growth was negligible even in the presence of ASC or GL. ASC completely inhibited in vitro guaiacol peroxidase activities that were isolated from both the apoplast and the cell wall. However, ferulic-acid-dependent peroxidase from the cell wall was partially inhibited by ASC, whereas ferulic acid peroxidase activity from the apoplastic fluid was completely inhibited by ASC as long as ASC was present in the assay medium. ASC content in cells was increased by preincubations with ASC or GL, whereas Lyc reduced it. On the other hand, ASC or GL treatments decreased both apoplast and cell-wall-bound peroxidase activities, whereas Lyc had a slight stimulating effect. These results are discussed on the basis of a possible control of root elongation by ASC via its action on peroxidases that are involved in the regulation of cell-wall extensibility.     

Effects of ambient oxygen and of fixed nitrogen on concentrations of glutathione, ascrobate, and associated enzymes in soybean root nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle for defense against activated forms of oxygen. Nodulated roots of hydroponically grown soybean plants were exposed to atmospheres containing 2, 21, 50, or alternating 21 and 50 kilopascals of O₂. The activities of ascorbate (ASC) peroxidase, monodehydroascorbate (MDHA) reductase, dehydroascorbate (DHA) reductase, and glutathione (GSSG) reductase were higher in nodules exposed to high pO₂. Nodule contents of ascorbate and reduced glutathione were also greater in the high pO₂ treatments. Treatment of nodulated plants with fixed nitrogen (urea) led to concomitant decreases in acetylene reduction activity, in leghemoglobin content, and in activities of ASC peroxidase, DHA reductase, and GSSG reductase. Activity of MDHA reductase and glutathione concentrations in nodules were not affected by treatment with urea. The enzymes of the ascorbate-glutathione cycle were also detected in uninfected soybean roots, although at levels substantially below those in nodules. These observations indicate that the ascorbate-glutathione cycle can adjust to varying physiological conditions in nodules and that there is a key link between N₂ fixation and defenses against activated forms of oxygen.     

The Effects of Ascorbate on Root Regeneration in Seedling Cuttings of Tomato

The changes in ascorbate (ASC) and dehydroascorbate (DHA) levels and the activities of ascorbate metabolising enzymes were examined during adventitious root formation in cuttings of tomato (Lycopersicon esculentum Mill. cv. Paw) seedlings. The effects of ASC, DHA and the immediate ascorbate precursor – galactono-γ-lactone (GalL) supplemented to the culture medium on the rooting response, ascorbate content and the activities of the ASC-metabolising enzymes were also investigated. The cuttings treated with abovementioned compounds formed more roots then control plants. However, in contrast to the number of regenerated organs, the elongation of newly formed roots was markedly inhibited. Treatment with auxin (IAA) resulted in a similar phenotype. The inhibitor of auxin polar transport-TIBA (2,3,5-triiodobenzoic acid) effectively blocked rooting. The inhibitory effect of TIBA was reversed by auxin and ASC treatments, while DHA and GalL were ineffective. Both auxin and ASC stimulated cell divisions in an area of pericycle layer of TIBA-treated rooting zones, that enabled cuttings to form roots in the presence of the inhibitor of auxin polar transport. It has been found that the first stages of rooting, preceding the emergence of roots, are accompanied by an increase in endogenous content of ASC with a peak in the 3rd day of rooting. Subsequent stages, when elongation of newly formed roots occurs, are characterised by low level of ASC. The activities of the ascorbate peroxidase (APX), ascorbate oxidase (AOX), ascorbate free radical reductase (AFRR) and dehydroascorbate reductase (DHR) increased in the first 3 days of root formation. The initial period of rooting was also accompanied by the increase of the hydrogen peroxide content and the activities of catalase (CAT) and guaiacol peroxidase (GPX) in the rooting zones. IAA, ASC, DHA as well as Gal stimulated the APX activity, however the rise of the enzyme's activity induced by ASC, DHA and Gal was reversed by TIBA, which was found to inhibit APX. Only exogenous IAA was able to maintain the high level of APX activity in the TIBA-treated cuttings. AOX was strongly affected by ASC and GalL – treatments, its activity increased in the cuttings grown on the media containing ASC in the absence as well as in the presence of TIBA. On the other hand, GalL-dependent stimulation of its activity was suppressed if TIBA was present in a rooting medium.     

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.     

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.     

Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on changes of the activities of major antioxidant enzymes, superoxide anion accumulation (O₂ ⁻), ascorbate, total glutathione (TG), malondialdehyde (MDA) content and ginsenoside accumulation were investigated in ginseng roots (Panax ginseng L.) in 4 l (working volume) air lift bioreactors. Single treatment of 200 μM MJ and SA to P. ginseng roots enhanced ginsenoside accumulation compared to the control and harvested 3, 5, 7 and 9 days after treatment. MJ and SA treatment induced an oxidative stress in P. ginseng roots, as shown by an increase in lipid peroxidation due to rise in O₂ ⁻ accumulation. Activity of superoxide dismutase (SOD) was inhibited in MJ-treated roots, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), SOD, guaiacol peroxidase (G-POD), glutathione peroxidase (GPx) and glutathione reductase (GR) were induced in SA-treated roots. A strong decrease in the activity of catalase (CAT) was obtained in both MJ- and SA-treated roots. Activities of ascorbate peroxidase (APX) and glutathione S transferase (GST) were higher in MJ than SA while the contents of reduced ascorbate (ASC), redox state (ASC/(ASC+DHA)) and TG were higher in SA- than MJ-treated roots while oxidized ascorbate (DHA) decreased in both cases. The result of these analyses suggests that roots are better protected against the O₂ ⁻ stress, thus mitigating MJ and SA stress. The information obtained in this work is useful for efficient large-scale production of ginsenoside by plant-root cultures.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

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.     

Zinc-alleviating effects on iron-induced phytotoxicity in roots of <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The mechanisms of growth inhibition and antioxidative response were investigated in wheat roots exposed to 300 μM iron together with different zinc concentrations (0, 50, and 250 μM). All Zn concentrations decreased Fe content but increased Zn content in the roots and leaves of Fe-treated seedlings. Compared with Fe stress alone, 50 or 250 μM Zn + Fe treatment stimulated root growth, and increased cell viability but decreased malondialdehyde content, which were correlated with the decreases of total and apoplastic hydrogen peroxide and superoxide anion radical (O₂ ^·?) content along with apoplastic hydroxyl radical content. Generation of O₂ ^·? in response to 10 μM diphenylene iodonium suggested that NADPH oxidase activity was lower in Zn + Fe-treated roots than in other roots. In addition, cell wallbound peroxidase, diamine oxidase, and polyamine oxidase in Fe-treated roots were insensitive to Zn addition. Further study showed the stimulation of total superoxide dismutase and glutathione reductase (GR) activities as well as apoplastic catalase, ascorbate peroxidase, and GR in Zn + Fe-stressed roots in comparison with Fe-alone-treated ones. Taken together, Zn could alleviate iron-inhibitory effect on root growth, which might be associated with the decrease of lipid peroxidation, the increase of cell viability and the reductions of reactive oxygen species generation.     

Lead accumulation and its translocation barriers in roots of Allium cepa L.—autoradiographic and ultrastructural studies

Abstract Lead migrating through the tissues of Allium cepa L. was found, by electron microscopy, autoradiography and other methods, to encounter at least three barriers to penetration. The layers of protoderm and hypodermic meristematic cells in the root meristematic zone and the layer of endodermis in the mature root zone were barriers to apoplastic transport. The central zone was a barrier to apoplastic and symplastic transport. It comprises the quiescent centre in the root meristem and the central part of the root cap. The cells of the deepest ground meristematic tissue layers seemed to act as a barrier, which keeps lead away from the procambium. Lead accumulated in roots but it was not uniformly distributed between their various tissues. The largest amount of lead accumulated both in ground meristematic and cortex tissues.     

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.     

Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L

To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-gamma-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis.     

Antioxidant Systems and O2.−/H2O2 Production in the Apoplast of Pea Leaves. Its Relation with Salt-Induced Necrotic Lesions in Minor Veins

The present work describes, for the first time, the changes that take place in the leaf apoplastic antioxidant defenses in response to NaCl stress in two pea (Pisum sativum) cultivars (cv Lincoln and cv Puget) showing different degrees of sensitivity to high NaCl concentrations. The results showed that only superoxide dismutase, and probably dehydroascorbate reductase (DHAR), were present in the leaf apoplastic space, whereas ascorbate (ASC) peroxidase, monodehydroascorbate reductase (MDHAR), and glutathione (GSH) reductase (GR) seemed to be absent. Both ASC and GSH were detected in the leaf apoplastic space and although their absolute levels did not change in response to salt stress, the ASC/dehydroascorbate and GSH to GSH oxidized form ratios decreased progressively with the severity of the stress. Apoplastic superoxide dismutase activity was induced in NaCl-treated pea cv Puget but decreased in NaCl-treated pea cv Lincoln. An increase in DHAR and GR and a decrease in ASC peroxidase, MDHAR, ASC, and GSH levels was observed in the symplast from NaCl-treated pea cv Lincoln, whereas in pea cv Puget an increase in DHAR, GR, and MDHAR occurred. The results suggest a strong interaction between both cell compartments in the control of the apoplastic ASC content in pea leaves. However, this anti-oxidative response does not seem to be sufficient to remove the harmful effects of high salinity. This finding is more evident in pea cv Lincoln, which is characterized by a greater inhibition of the growth response and by a higher rise in the apoplastic hydrogen peroxide content, O(2)(.-) production and thiobarbituric acid-reactive substances, and CO protein levels. This NaCl-induced oxidative stress in the apoplasts might be related to the appearance of highly localized O(2)(.-)/H(2)O(2)-induced necrotic lesions in the minor veins in NaCl-treated pea plants. It is possible that both the different anti-oxidative capacity and the NaCl-induced response in the apoplast and in the symplast from pea cv Puget in comparison with pea cv Lincoln contributes to a better protection of pea cv Puget against salt stress.     

Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules

Lucifer Yellow (LYCH) and carboxyfluorescein (CF) served in Medicago truncatula roots and root nodules as the markers of apoplastic and symplastic transport, respectively. The aim of this study was to understand better the water and photoassimilate translocation pathways to and within nodules. The present study shows that in damaged roots LYCH moves apoplastically through the vascular elements but it was not detected within the nodule vascular bundles. In intact roots, the outer cortex was strongly labeled but the dye was not present in the interior of intact root nodules. The inwards movement of LYCH was halted in the endodermis. When the dye was introduced into a damaged nodule by infiltration, it spread only in the cell walls and the intercellular spaces up to the inner cortex. Our research showed that in addition to the outer cortex, the inner tissue containing bacteroid-infected cells is also an apoplastic domain. Our results are consistent with the hypothesis that nodules do not receive water from the xylem but get it and photoassimilates from phloem. A comparison between using LYCH and LYCH followed by glutaraldehyde fixation indicates that glutaraldehyde is responsible for fluorescence of some organelles within root nodule cells. The influence of the fixation on nodule fluorescence has not been reported before but must be taken into consideration to avoid errors. An attempt was made to follow carboxyfluorescein (6(5) CF) translocation from leaflets into roots and root nodules. In root nodules, CF was present in all or a couple of vascular bundles (VB), vascular endodermis and some adjacent cells. The leakage of CF from the VBs was observed, which suggests symplastic continuity between the VBs and the nodule parenchyma. The lack of CF in inner tissue was observed. Therefore, photoassimilate entry to the infected region of nodule must involve an apoplastic pathway.     

Effect of carbon dioxide on antioxidant enzymes and ginsenoside production in root suspension cultures of Panax ginseng

The aim of this study was to investigate the effect of CO₂ at various concentrations (1, 2.5 and 5%) on antioxidant enzymes and ginsenoside accumulation in Panax ginseng roots in 5 l airlift bioreactors (working volume 4 l). One and 2.5% CO₂ was beneficial for root biomass accumulation, but 5% CO₂ decreased the biomass. Ginsenoside concentration decreased with increasing concentration of CO₂. No significant difference was observed in the malondialdehyde (MDA) content and lipoxygenase (LOX) activity between respective controls and CO₂ treated roots. Antioxidant enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), catalase (CAT), guaiacol peroxidase (G-POD) including reduced ascorbate and total glutathione were induced in CO₂ exposed roots which emphasized the protective role of antioxidants against CO₂ induced stress. Superoxide dismutase activity (SOD) which was induced after 15 days was significantly inhibited after 45 days. Glutathione-S-transferase (GST) and glutathione peroxidase (GPX) activities also increased when the roots were subjected to 1 and 2.5% CO₂ compared to the respective controls but not at 5%. A higher reduced ascorbate to oxidized (ASC/DHA) ratio in CO₂ treated root indicates the plant's ability to tolerate CO₂ stress. These observations suggest that an increase in antioxidant enzymes may affect a defense response to the cellular damage induced by CO₂. Probably, this increase could not stop the deleterious effects of CO₂ concentration on ginsenoside concentration, but reduced stress severity and thereby allowing root growth to occur.     

Calcium and L-histidine effects on ascorbate-glutathione cycle components under nickel-induced oxidative stress in tomato plants

The effects of NiSO₄, calcium, and L-histidine (His) on the components of ascorbate-glutathione cycle, antioxidant enzymes and lipid peroxidation in a tomato cultivar Early Urbana Y was investigated. The activities of enzymes including catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), lipoxygenase (LOX), and phenylalanine ammonia lyase (PAL) were measured. In addition, the content of H₂O₂, ascorbate (ASC), dehydroascorbate (DHA), reduced glutathione (GSH), chlorophyll (Chl) a+b, carotenoids, proteins, malondialdehyde (MDA), membrane aldehydes, and electrolyte leakage (EL) were determined. Results suggest that the excess of Ni increased the content of H₂O₂, MDA, membrane aldehydes and proteins in roots as well as GPX, LOX, APX activities, and EL in leaves, whereas Ca and His ameliorated these effects. Moreover, decreasing leaf GSH and DHA content and GR activity were observed under the Ni stress, but these parameters were raised by Ca plus His treatment. However, no improvement in leaf protein, ASC, root GSH content, and activities of PAL and CAT were observed by using Ca or His under Ni stress.