Photoinactivation of the detoxifying enzyme nitrophenol reductase from Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus E1F1 detoxifies 2,4-dinitrophenol by inducing an NAD(P)H-dependent iron flavoprotein that reduces this compound to the less toxic end product 2-amino-4-nitrophenol. This nitrophenol reductase was stable in crude extracts containing carotenes, but it became rapidly inactivated when purified protein was exposed to intense white light or moderate blue light intensities, especially in the presence of exogenous flavins. Red light irradiation had no effect on nitrophenol reductase activity. Photoinactivation of the enzyme was irreversible and increased under anoxic conditions. This photoinactivation was prevented by reductants such as NAD(P)H and EDTA and by the excited flavin quencher iodide. Addition of superoxide dismutase, catalase, tryptophan or histidine did not affect photoinactivation of nitrophenol reductase, thus excluding these reactive dioxygen species as the inactivating agent. Substantial protection by 2,4-dinitrophenol also took place when the enzyme was irradiated at a wavelength coinciding with one of the absorption peaks of this compound (365nm). These results suggest that the lability of nitrophenol reductase was due to the absorption of blue light by the flavin prosthetic group, thus producing an excited flavin that might irreversibly oxidize some functional group(s) necessary for enzyme catalysis. Nitrophenol reductase may be preserved in vivo from blue light photoinactivation by the high content of carotenes and excess of reducing equivalents in phototrophic growing cells.Abbreviations 2,4-DNP 2,4-dinitrophenol - ANP 2-amino-4-nitrophenol - EDTA ethylenediamine tetraacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid - NPR nitrophenol reductase     

Photoprotective and antioxidant responses to light spectrum and intensity variations in the coastal diatom Skeletonema marinoi

Photosynthesis is known to produce reactive oxygen species together with the transformation of light into biochemical energy. To fill the gap of the knowledge on the protective antioxidant network of microalgae, a series of experiments to explore the role of spectral composition and intensity of light in the modulation of the photodefence mechanisms developed by the coastal diatom Skeletonema marinoi were performed. The modulation of the total phenolic content, ascorbic acid and the enzymes glutathione reductase, catalase, ascorbate peroxidase and superoxide dismutase together with xanthophyll cycle and non‐photochemical quenching in response to variations in the light environment were analysed. Most of the enzymes' activity was promptly affected by the red light. Yet, the monochromatic high intensity blue light enhanced the synthesis of total phenolic content and ascorbic acid in parallel to the xanthophyll cycle activity. This study reveals the dual effects of spectral composition and intensity of light on the modulation of photoprotective mechanisms. Diatoms developed a complementary and/or alternative tuning processes to cope with the variable light environment they experience in the water column. They also provided valuable insights into light manipulation regimes for diatom cultivation that will help to maximize production of bioactive molecules.     

Induction mechanism of adventitious root from leaf explants of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> as affected by auxin and light quality

An efficient protocol for adventitious root induction from leaf explants of Morinda citrifolia treated with different concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) was established in relation to physiological process changes during adventitious root induction under different light sources (fluorescent, red, blue, red + blue, and far-red). Among the different concentrations of IBA and NAA, 1.0 mg l⁻¹ IBA was proven as the best auxin source for adventitious root induction under fluorescent light. Higher concentrations of IBA and NAA trigger callus formation in both light and dark conditions. Maximum numbers of adventitious roots were induced under red light (26) followed by blue light (22) and the lowest under far-red light (6). In contrast, numerous callus formations were induced by red + blue followed by red and blue, while the highest root length (1.66 cm) with negligible callusing was observed under fluorescent light. Catalase and guaicacol peroxidase activities were highest under red light followed by fluorescent light and the lowest under red + blue light, but superoxide dismutase activity was not significantly influenced by different light sources. Ascorbate peroxidase played an important role in detoxification of the harmful effects of hydrogen peroxide (H₂O₂). Under fluorescent light, significantly lower accumulation of H₂O₂ was observed. Accumulation of H₂O₂ in the induced root under different light showed a positive correlation with peroxidation of lipids and was observed higher under far-red followed by red + blue and blue light.     

Photoinactivation of catalase at low temperature and its relevance to photosynthetic and peroxide metabolism in leaves

Abstract In green as well as in etiolated leaves of rye (Secale cereale L. ev. ‘Halo’), exposed to strong light at low temperature (0.4°C) catalase was inactivated. Other heme-containing enzymes (peroxidases) and various enzymes of photosynthetic, photorespiratory or peroxide metabolism were not photoinactivated. After returning plants from a low to a physiological temperature (22°C), catalase activity recovered within 12 h through new synthesis. The leaf contents of H₂O₂ and organic peroxides were not affected by the photoinactivation of catalse. The content of malondialdehyde generally increased after exposure to a higher light intensity. High-light-induced increases of ascorbate, and particularly of glutathione, were more marked in catalase-deficient than in normal leaves. Photoinactivation of catalase was accompanied by severe inhibition of photosynthesis. Photoinhibition of photosynthesis was not related to the lack of catalase because photosynthesis was not impaired when catalase activity was kept low by growing the plants under non-photorespiratory conditions. Photoinhibition appeared to result from photodamage in primary photochemistry of photosystem II, as indicated by a decrease of the maximal variable fluorescence. Photoinhibition of photosynthesis and of catalase have in common that in both instances proteins are involved that are continuously inactivated in light and, therefore, particularly sensitive to stress conditions that prevent their replacement by repair synthesis.     

Change in the content of salicylic acid and activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the influence of Azospirilium lectins

The time course of changes in the endogenous content of salicylic acid, the ratio between the acid’s free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum (A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3) is investigated. Differences in plant response to the action of the lectins from these two strains are established. On the basis of the obtained data, a model is proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in the accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and formation of induced resistance.     

Glycollate metabolism of wheat and rice leaves during senescence and under the influence of growth regulators

The glycollate metabolism of wheat (Triticum vulgare Vill. cv. Sonalika) and rice (Oryza sativa L. ev. Jaya) leaves was studied during senescence by estimating the endogenous levels of glycollate and hydrogen peroxide (H₂O₂) and the activities of glycollate oxidase and catalase. In comparison with light incubation the incubation of excised leaves in the dark caused a decline in the glycollate content and in the activities of glycollate oxidase and catalase, and an increase in the H₂O₂ content, more marked in the leaves of rice than in the leaves of wheat. Glycollate oxidase activity gradually decreased with incubation time, and glycollate metabolism decreased during senescence. The glycollate oxidase in particular and glycollate metabolism of rice were more sensitive to incubation time than those of wheat. Kinetin increased the glycollate oxidase activity and glycollate metabolism during senescence, while ethrel (2-chloroethylpho-sphonic acid) and ABA (abscisic acid) reduced these activities in both plant species.     

Alleviation of NaCl Stress by Pretreatment with Phytohormones in Vigna radiata

Efficiency of pretreatment as foliar spray of indole-3-acetic acid, gibberellic acid and kinetin, each ranging from 0.1 to 10.0 μM concentration, in restoring the metabolic alterations imposed by NaCl salinity was investigated in Vigna radiata (L.) Wilczek. Glycolate oxidase, superoxide dismutase, catalase and peroxidase activities increased under stress in leaves and roots also. Malondialdehyde content and total peroxide content also increased under stress. All the three hormones used were able to overcome to variable extents the adverse effects of stress imposed by NaCl to these parameters. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.     

Serratia marcescens SYBC08 catalase isolated from sludge containing hydrogen peroxide shows increased catalase production by regulation of carbon metabolism

A high‐catalase‐producing strain, which was isolated from sludge containing hydrogen peroxide, was identified as Serratia marcescens SYBC08 by 16S rDNA sequence analysis. Serratia spp. was reported as non‐spore‐forming bacterium (except S. marcescens spp. sakuensis), but in our study electron microscopic observation revealed that the strain did produce spores. The content of the main fatty acid C_16:0 (14.8%) was significantly different from that of S. marcescens spp. sakuensis (33.2%) and S. marcescens spp. marcescens DSM 30121^T (34.8%), and the biochemical characteristics were not identical to those of S. marcescens spp. sakuensis. We speculate that the relatively high catalase activity and the spore structures may enable the strain to survive in a hydrogen peroxide environment. The most suitable carbon and nitrogen sources for the catalase production by S. marcescens SYBC08 were citric acid and corn steep liquor powder. A strategy of carbon metabolism regulation to enhance the catalase production was exploited. In the 7‐L fermenter, catalase production (20 353 U/mL) obtained in the presence of glucose and citric acid was 1.68‐ and 1.31‐fold higher than that obtained in the presence of glucose or citric acid, at equimolar carbon concentration. This production yield was much higher than that of many catalase‐producing strains, but only slightly lower than the production by Micrococcus luteus (34 601 U/mL). The results suggest that the new spore‐forming S. marcescens SYBC08 is a potential candidate for the production of catalase.     

Salt Stress Injury Induces Oxidative Alterations and Antioxidative Defence in the Roots of Lemna minor

Lemna minor L. roots were treated with different concentrations of NaCl. Lipid peroxidation was investigated histochemically and biochemically. At higher NaCl concentrations an increase in staining was observed in the root apices as compared to control for lipid peroxidation and loss of membrane integrity as well as an increase in contents of thiobarbituric acid reactive substance and peroxide. Both the non-enzymic antioxidants, ascorbate and glutathione increased with the NaCl concentration in the roots. Whereas an increase in superoxide dismutase, guaiacol peroxidase, and glutathione reductase activities were marked, catalase activity decreased in the roots under NaCl stress. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mitochondrial activity in illuminated leaves of chlorophyll-deficient mutant rice (<Emphasis Type="Italic">OsCHLH</Emphasis>) seedlings

The rice CHLH gene encodes the Mg²⁺-chelatase H subunit, which is involved in chlorophyll biosynthesis. Growth of the chlorophyll-deficient oschlh mutant is supported by mitochondrial activity. In this study, we investigated the activity of mitochondrial respiration in the illuminated leaves during oschlh seedling development. Growth of mutant plants was enhanced in the presence of 3% sucrose, which may be used by mitochondria to meet cellular energy requirements. ATP content in these mutants was, however, significantly lowered in light conditions. Low cytosolic levels of NADH in illuminated oschlh mutant leaves further indicated the inhibition of mitochondrial metabolism. This down-regulation was particularly evident for oxidative stress-responsive genes in the mutant under light conditions. Hydrogen peroxide levels were higher in oschlh mutant leaves than in wild-type leaves; this increase was largely caused by the impairment of the expression of the antioxidant genes, such as OsAPX1, OsRAC1, and OsAOXc in knockout plants. Moreover, treatment of mesophyll protoplasts with ascorbic acid or catalase recovered ATP content in the mutants. Taken together, these results suggest that the light-mediated inhibition of mitochondrial activity leads to stunted growth of CHLH rice seedlings.     

Activity of Enzymes Related to H2O2 Generation and Metabolism in Leaf Apoplastic Fraction of Tomato Leaves Infected with Botrytis cinerea

Hydrogen peroxide generation rates of uninfected and infected leaves of two tomato (Lycopersicon esculentum) cultivars showing differential susceptibility to Botrytis cinerea were determined. The superoxide anion, hydroxyl radical, ascorbate contents and changes in NADH peroxidase, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities in the apoplast fraction were analysed. Infected leaves had an increased hydrogen peroxide level. It was greater and generally occurred earlier in plants of the less susceptible cv. Perkoz than in those of the more susceptible cv. Corindo. Induction of nitrotetrazolium blue reducing activity and SOD levels in apoplast were higher in cv. Perkoz 24 h after inoculation. In the controls, NADH peroxidase activity in apoplast was higher in the more susceptible cv. Corindo, but after infection it increased faster and to a higher level in the less susceptible cv. Perkoz. NADH oxidation was inhibited by only 15% by a specific inhibitor DPI (diphenylene‐iodonium) but was completely inhibited by KCN and NaN₃. Similar increases in APX activity after 48 h and a small increase in catalase activities were observed in both cultivars soon after infection. These results indicate that resistance of tomato plants to infection by the necrotrophic fungus B. cinerea may result from early stimulation of hydrogen peroxide and superoxide radical generations by NADH peroxidase and SOD in apoplastic space, and they confirm the important role of their enhanced production in apoplastic spaces of plants.     

Influence of Light of Different Spectral Compositions on Growth and Metabolism of Citrus Seedlings

Sour orange (Citrus aurantium L.) seedlings grown for six months under covers transmitting light of different spectral composition, were compared with others grown under a white cover (control) and outside in full daylight. The intensity of transmitted light was equalized under all covers and attained only 20% of full daylight. Seedlings grown in daylight were shorter, had more internodes, smaller leaves, less chlorophyll and more ascorbic acid than the others. Blue + far-red covers (no transmission between 560–700 nm) enhanced seedling length, the protein and chlorophyll content and peroxidase activity of leaves. When also the wave-range above 700 nm was cut out (blue) seedlings were the shortest, and leaves had very high protein and chlorophyll content, but much less ascorbic acid and lower peroxidase activity. Red + far-red covers (no transmission below 500 nm) enhanced seedling length more than blue + far-red; leaves contained as much protein as control, but had relatively high chlorophyll and peroxidase activity. Ascorbic acid was as low as in blue light.     

Growth-Promoting Effects of Fatty Acids on Excised Wheat Roots and Oat Coleoptiles

The growth action of some fatty acids and alcohols with carbon number from 1 to 4 was tested on excised wheat roots in aseptic cultures. Growth, cell length, and dry weight were measured after seven days. The tested substances were: Formic acid, acetic acid, propionic acid, n-Butyric acid, isobutyric acid, acelaldehyde, propionaldohyde, ethanol, n-propanol, isopropanol, n-butanoL and isobutanol. The primary alcohols and all of the acids, except formic acid, promoted the cell elongation and the total growth, but the meristematic activity was inhibited in the higher concentrations. The concentrations with maximal growth-promoting activity were 10^?2M to 10^?3M for the alcohols and 10^?4M for the acids. Propionic acid was applied in darkness, red light, and white light and proved to increase the cell length from 215 μ, 180 μ, and 120 μ up to 240 μ in all three treatments. The growth rate was not affected, but the duration of the cell elongation was extended. The presence of iron proved to be necessary for the stimulation. The chlorophyll content in the light grown roots was relatively unaffected when the cell length was increased and the dry weight was not increased as long the cell number was normal. The growth of Avena coleoptile segments was slightly promoted by propionic acid in the presence of IAA.     

不同光质对辣椒叶色黄化突变体光合生理特性的影响研究

以辣椒叶色黄化突变体yl1及其野生型6421为试材,用白光、蓝光、红光、绿光、紫光、黄光和远红光不同光质进行处理,考察其表型、生理及光合特性的变化特征,探究光质对黄叶辣椒植株生长发育的影响。结果显示：(1)蓝光与红光对辣椒幼苗的生长有促进作用,黄光和远红光则会显著抑制幼苗生长,6421生长受不同光质的抑制影响比yl1更大。(2)两个辣椒材料光合色素含量在不同光质下均不同程度降低;6421叶绿素总含量和类胡萝卜素含量在不同光质下均高于yl1,yl1和6421叶片的光合色素含量分别在紫光和黄光下最低。(3)蓝光和绿光能显著提高yl1的净光合速率(P_n),而不同光质处理均显著降低了6421的P_n。(4)紫光处理使yl1的PSⅡ潜在活性(F_v/F_o)和最大光化学效率(F_v/F_m)值均显著降低且显著低于6421,但升高了光化学猝灭系数(qP)和非光化学猝灭系数(NPQ)。(5)蓝光、红光和绿光均能提高辣椒的超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(...     

Induction of catalase in Escherichia coli by ascorbic acid involves hydrogen peroxide

Ascorbic acid at concentrations between 0.57 and 5.7 mM in aerated medium caused an eight fold increase in catalase activity in Escherchia coli. The hydrogen peroxide concentrations resulting from ascorbate oxidation were between 20 and 120 μM and hydrogen peroxide by itself caused a similar increase in catalase levels in both aerobic and anaerobic media. Three catalase activity bands visualized on polyacrylamide gels were increased. Chloramphenicol which inhibits protein synthesis, anaerobic medium and EDTA, which prevent ascorbate oxidation, and exogenous catalase, which removes hydrogen peroxide from the medium, all prevented the increase in catalase in response to ascorbate. Superoxide dismutase activity was not affected by ascorbate.     

Changes in activity of antioxidative enzymes in wheat (Triticum aestivum) seedlings under cold acclimation

Activated oxygen species such as superoxide radicals, singlet oxygen, hydrogen peroxide and hydroxyl radicals can be produced in plants exposed to low, non-freezing, non-injurious temperatures. To prevent or alleviate oxidative injury, plants have evolved several mechanisms which include scavenging by natural antioxidants and enzymatic antioxidant systems such as superoxide dismutases, catalase and peroxidases. Although overproduction of hydrogen peroxide and increased tolerance to oxidative stress can be induced in wheat by low-temperature treatments, data concerning changes in the enzymatic antioxidant systems are almost absent. With the aim to provide this information, antioxidant enzyme (superoxide dismutases, catalase and peroxidases) activities were analysed in leaves and roots of Triticum aestivum cvs Brasilia (frost resistant in field) and Eridano (less frost resistant in field) seedlings grown at day/night temperatures of 24/22°C (control treatment) and 12/5°C (low-temperature treatment). Our data showed that superoxide dismutase activities were unaffected by low-temperature treatment both in leaves and roots. Catalase activity in leaves and roots was decreased in 12/5°C-grown seedlings, but Brasilia maintained higher catalase activity than Eridano. Differences were also observed in guaiacol peroxidase activities between control and acclimated seedlings: Higher guaiacol peroxidase activities were found in the leaves of 12/5°C-grown seedlings while in roots these activities were lower. Moreover, Brasilia guaiacol peroxidase activities were higher than Eridano. Superoxide dismutase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by low-temperature treatment. Changes in the activities of antioxidant enzymes induced by cold acclimation support the hypothesis that a frost-resistant wheat cultivar, in comparison with a less frost-resistant one, maintains a better defence against activated oxygen species during low-temperature treatment.     

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.     

Catalase production influences germination,stress tolerance and virulence of Lecanicillium muscarium conidia

Catalases are the most important enzymatic systems used to degrade hydrogen peroxide (H₂O₂) into water and oxygen, thereby lowering intracellular hydrogen peroxide levels. Entomopathogenic fungi display increased catalase activity during germination and growth, which is necessary to counteract the hyperoxidant state produced by oxidative metabolism. We studied the influence of five different hydrocarbons on catalase production by Lecanicillium muscarium to determine the importance of catalase induction in fungal germination, stress tolerance and virulence. Conidia produced by colonies grown on different hydrocarbons showed higher rates of catalase activity compared to the control and the catalase activity of conidia produced on n-octacosane was three times higher than the activity of the control. This increase in catalase activity was accompanied by a higher level of resistance to exogenous hydrogen peroxide and a reduction in the germination time. Our study has helped to identify that increased catalase activity improves the germination and tolerance to different antioxidant stress response of L. muscarium.     

Responses of antioxidant defense system of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don. to paclobutrazol treatment under salinity

The effect of paclobutrazol, a plant growth regulator, on antioxidant defense system was investigated in Catharanthus roseus (L.) G. Don. plants subjected to NaCl stress. The growth parameters were significantly reduced under 80 mM NaCl treatment; however, this growth inhibition was less in paclobutrazol-treated (15 mg l⁻¹ plant⁻¹) plants. The non-enzymatic antioxidants ascorbic acid and reduced glutathione were affected under NaCl stress and they increased significantly under paclobutrazol treatment when compared to NaCl treated as well as control plants (P ≤ 0.05). The activity of antioxidant enzyme ascorbate peroxidase showed a significant enhancement under salinity stress. The catalase activity decreased in roots of NaCl-treated plants, but recovered with paclobutrazol treatment. The results suggested that paclobutrazol have significant role in contributing salt stress tolerance of C. roseus by improving the components of antioxidant defense system.