The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.     

Effects of inhibitors on the enzyme system producing C6-aldehydes from C18-unsaturated fatty acids in chloroplasts of Japanese silver (Farfugium japonicum) leaves

When chloroplasts isolated from Farfugium japonicum (Japanesesilver) leaves were used as an enzyme source, the activity ofthe enzyme system producing C₆-aldehydes (cis-3-hexenal andn-hexanal) from C₁₈-unsaturated fatty acids (linolenic and linoleicacids) decreased upon treatment with LAHase from potato. Thisenzyme system could not be separated from chlorophylls and lipidsby detergent treatment and was not affected by light illumination,CCCP or DCMU. The activity of the enzyme system was inhibitedby MB and NTB used as a redox reagent, SKF 525-A as an oxidaseinhibitor and DABCO as a quencher of singlet oxygen, but notby DCIP, PMS and SOD. These data suggest that; i) interactionof the enzyme system with lipids is required for maximal enzymeactivity, ii) this enzyme system may involve electron mediator(s),and iii) singlet oxygen takes part in the enzyme reaction. (Received October 28, 1977; )     

On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12

The possible mutagenic effects induced by singlet oxygen, which is formed during UVA irradiation of bacterial cells pretreated with 8-methoxypsoralen (8-MOP), were investigated. As genetic endpoint, back mutation from arg₅₆^? to arg⁺ was assayed in strain Escherichia coli K-12/343/113/uvrB; this system, in preliminary experiments, was rather sensitive to 8-MOP-induced photodynamic effects. To assess the involvement of singlet oxygen (¹O₂) in the mutation induction process, 2 tests were applied, namely, comparative mutation induction in D₂O and in H₂O media (pH 7.0) and quenching of ¹O₂ with 1,4-diazabicyclo[2.2.2]octane (DABCO).When photodynamy was performed with the indicator cells suspended in D₂O buffer, the mutagenic effect was substantially higher than that obtained with cells suspended in H₂O buffer; this increase was even more pronounced when the incubation mixtures were thoroughly oxygenated before irradiation. D₂O itself was not mutagenic under the present experimental conditions. Addition of DABCO in concentrations of 0.1–10 mM to the irradiation mixtures effectively reduced the number of 8-MOP-induced mutant yields by about 40%. DABCO itself had no effect on cell viability or on spontaneous mutation frequency under our experimental conditions.From these 2 sets of results, and from the preliminary findings that the photomutagenic effect of 8-MOP is higher in the uvrB derivative than in the corresponding excision-repair-proficient parent strain, which is in concordance with previous observations in other E coli strains, it can be concluded that ¹O₂ generated upon UVA irradiation of 8-MOP solutions is probably responsible for part of the observed genetic effects.     

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium

Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa₃ as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa₃ and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.     

Catalysis of singlet oxygen production in the reaction of hydrogen peroxide and hypochlorous acid by 1,4-diazabicyclo[2.2.2]octane (DABCO)

The kinetics of the singlet oxygen production in the hydrogen peroxide plus hypochlorous acid reaction were studied by measuring the time course of the singlet oxygen emission at 1268 nm. The addition of 1,4-diazabicyclo[2.2.2]octane (DABCO) increased the peak intensity of the chemiluminescence, but decreased its duration. The increased rate of singlet oxygen production likely accounts for the enhancement of singlet oxygen dimol emission reported in 1976 by Deneke and Krinsky (J. Am. Chem. Soc. 98, 3041-3042). This phenomenon was not seen when singlet oxygen was generated with the reaction of hypobromous acid and hydrogen peroxide. Thus, the enhancement of red chemiluminescence by DABCO should not be regarded as a general test for the production of singlet oxygen in complex biochemical systems.     

Reactive oxygen intermediates modulate nitric oxide signaling in the plant hypersensitive disease-resistance response

The mechanisms involved in plant defense show several similar characteristics with the innate immune systems of vertebrates and invertebrates. In animals, nitric oxide (NO) cooperates with reactive oxygen intermediates (ROI) to kill tumor cells and is also required for macrophage killing of bacteria. Such cytotoxic events occur because unregulated levels of NO determine its diffusion-limited reaction with O₂^– generating peroxynitrite (ONOO^–), a mediator of cellular injury in many biological systems. In soybean suspension cells, unregulated NO production during the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate the hypersensitive cell death, which is triggered only by fine tuning the NO/ROI ratio. Furthermore, that hypersensitive cell death is activated following interaction of NO with H₂O₂, rather than O₂^–. Increasing O₂^– levels reduces NO-derived toxicity, and the addition of ONOO^– to soybean suspensions does not affect cell viability. Consistently with the fact that ONOO^– is not an essential mediator of NO/RO-induced cell death, during the HR superoxide dismutase (SOD) accelerates O₂^– dismutation to H₂O₂ and therefore minimizes the loss of NO by reaction with O₂^– and triggers hypersensitive cell death through the NO/H₂O₂ synergism. Consequently, the rates of production and dismutation of O₂^– generated during the oxidative burst play a crucial role in modulating NO signaling through the cell death pathway, which proceeds through mechanisms different from those commonly observed in animals.     

Cellular toxicity of elsinochrome phytotoxins produced by the pathogenic fungus, Elsino? fawcettii causing citrus scab.

Elsinochromes are the red/orange pigments produced by many Elsino? fungal species and are structurally similar to the phytotoxin, cercosporin. Here, pigments were extracted from cultures of a citrus pathogen, Elsino? fawcettii and tested for cellular toxicity. On irradiation with light, elsinochromes rapidly killed suspension cultured citrus and tobacco cells. The toxicity was decreased by adding the singlet oxygen ((1)O(2)) quenchers (bixin (carotenoid carboxylic acid), DABCO (1, 4-diazabicyco octane), ascorbate or reduced glutathione). Application of elsinochromes onto rough lemon leaves resulted in necrotic lesions, whereas lesion development was inhibited by the addition of bixin, DABCO or ascorbate, but not a-tocopherol. Incubation of rough lemon leaf discs with elsinochromes in the light induced a steady increase of electrolyte leakage. Compared with two photosensitizing compounds, hematoporphyrin and cercosporin, the accumulation of (1)O(2) induced by elsinochromes after irradiation was indicated by successful detection of the cholesterol oxidation product, 5a-hydroperoxide. Addition of a potent quencher, beta-carotene prevented 5alpha-hydroperoxide production. Elsinochromes generated superoxide ions (O(2)(*-)), whereas accumulation of O(2)(*-)was blocked by addition of the superoxide dismutase, a scavenger of O(2)(*-), but not the (1)O(2)-quencher, DABCO. Our study indicated that elsinochromes are functioning as photosensitizing compounds that produce (1)O(2)and O(2)(*-), and exert toxicity to plant cells.     

Ionic homeostasis disturbance is involved in tomato cell death induced by NaCl and salicylic acid

The ability of salicylic acid and NaCl to induce programmed cell death by disturbing ionic homeostasis was investigated using tomato suspension culture cells. NaCl (300?mM) and salicylic acid (1?mM) inhibited cell growth and caused cell death within 1?wk of exposure. Treatment with NaCl increased the production of reactive oxygen species and the permeability of plasma membrane, but it also led to a reduction in the pH of the culture medium and resulted in a disturbance in ionic homeostasis of the cells. Salicylic acid-induced cell death in tomato suspension culture was also accompanied by production of reactive oxygen species and increases in both electrolyte leakage and pH of the culture media. However, reactive oxygen species production was not significantly different in cultures treated with a lethal salicylic acid concentration and 100?mM NaCl, in which most of the cells survived. A decrease in the K⁺/Na⁺ ratio was observed only in those cell cultures in which the salicylic acid treatment induced the death of cells. These results suggest that the decrease of the intracellular K⁺ concentration and K⁺/Na⁺ ratio is a common phenomenon in triggering programmed cell death by lethal concentrations of salicylic acid and NaCl.     

Effects of scavengers for active oxygen species on cell death by cryptogein

The hypersensitive reaction is a type of programmed cell death in plants. Cryptogein is a proteinaceous elicitor secreted from Phythophthora cryptogea. In one current model, active oxygen species (AOS) trigger programmed cell death in plants. In this study, we examined a variety of AOS scavengers to elucidate the function of AOS in the death program. Most of these AOS scavengers, including tiron, a scavenger for superoxide radical, catalase for hydrogen peroxide, and hydroquinone, sodium ascorbate and propyl gallate for free radicals, almost completely removed extracellular AOS. However, none of the reagents completely blocked the cell death process. Other reagents, such as histidine and dimethylfuran, scavengers for singlet oxygen, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, showed significant toxicity in BY-2 cells. These results indicate that AOS produced in the extracellular space do not play a role in hypersensitive cell death.     

Different responses of two tobacco cultivars and their cell suspension cultures to quercinin,a novel elicitin from Phytophthora quercina

In this study we describe the response of two tobacco cultivars (Nicotiana tabacum L. cv. Bel B and Bel W3) and their cell suspension cultures to quercinin, a novel elicitin produced by the oak pathogen Phytophthora quercina. N-terminal sequencing of the purified protein proved that it belongs to the basic β-elicitins with threonine on position 13. Both tobacco leaves and cells of the cultivar Bel W3 showed hypersensitive cell death after quercinin treatment. Leaves of Bel B also developed quercinin-induced necrosis but higher concentrations of quercinin were necessary as compared to Bel W3. Also Bel B cells showed cell death induction only at the highest quercinin concentration (20 nM). In cell suspension experiments we also measured the quercinin-induced oxidative burst, which occurred in both cultivars. H₂O₂ production in Bel B increased with increasing quercinin concentration and was inhibited only at the highest elicitin concentration (20 nM) whereas the oxidative burst in Bel W3 was completely abolished by 5 nM quercinin. Furthermore we demonstrated that neither H₂O₂ nor superoxide were responsible for cell death induction since neither the inhibitor diphenyleneiodonium (DPI) nor the enzymes catalase (CAT) and superoxide dismutase (SOD) influenced the hypersensitive reaction (HR) in Bel W3 cells. Due to the different response of Bel W3 and Bel B towards the P. quercina elicitin, our system represents an interesting tool to elucidate signaling pathways in tobacco leading to hypersensitive cell death.     

Production of L-DOPA by cell suspension cultures of Mucuna pruriens

The endogenous synthesis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) by cell suspension cultures of Mucuna pruriens was found to be influenced by several environmental parameters. The nature of the nitrogen source as well as the concentration of nitrogen containing salts, sucrose and phosphate in the culture medium were found to affect the biosynthesis of L-DOPA. Addition of 2, 4-dichlorophenoxyacetic acid to the medium suppressed L-DOPA production; continuous illumination of the cultures had a strong beneficial effect on L-DOPA production. L-DOPA was accumulated intracellularly by the cell suspension cultures. These observations further demonstrate that for certain products of plant cell suspensions product synthesis can be manipulated by a proper selection of specified nutrients.     

Involvement of phospholipase A2 in the release of silymarin to the culture medium of Silybum marianum cell suspensions

In suspension cell cultures of Silybum marianum, methyl jasmonate (MJ) stimulated the accumulation and release of silymarin (Sm) to the culture medium. This study shows that phospholipase A2 (PLA2) plays a role in the release of Sm in elicited cultures. PLA2 activity increased in cell suspensions treated with MJ. Addition of aristolochic acid (AA) or bromoenol lactone (BEL) compounds that inhibit PLA2 activity impeded silymarin release. The addition of linoleic or linolenic acid reversed the inhibitory action of AA. Fatty acids (FAs) stimulated Sm release when added alone to control cultures. By contrast, oleic acid and saturated FA were ineffective in emulating MJ action.     

Monitoring of haploid maize cell suspension culture conditions in bioreactors

Maize (Zea mays L.) haploid cells were cultivated in a 1500 ml aerated and stirred batch bioreactor using modified BM medium. Cell growth was highly affected by pH and dissolved oxygen, and we observed two fairly distinct growth phases. During the first two days after inoculation at pH 5.8, oxygen consumption was high and the cells lowered the pH to a value around 4.3. After this period the pH stabilized at 4.5 and the dissolved oxygen reached a steady level. Decreasing dissolved oxygen concentration leads to lower growth rate and to higher pH. Both events mean stress conditions for the cell culture and probably result in increased genetic variability, and the loss of regeneration capacity. The stress condition during the adaptation phase can be eliminated by decreasing the pH of the medium to 4.7 before inoculation and by keeping dissolved oxygen above 40%. These conditions provide prolonged exponential growth dynamics and the cell suspensions could be the basis of large scale cultures also.Abbreviations 2,4-d 2,4-dichlorophenoxyacetitc acid - NAA naphthalene acetic acid     

Modeling of the Competitive Growth of Listeria monocytogenes and Lactococcus lactis in Vegetable Broth

Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods.     

Vanillin as an antioxidant in rat liver mitochondria: Inhibition of protein oxidation and lipid peroxidation induced by photosensitization

Using rat liver mitochondria, as model systems, we have examined the ability of the natural compound and the food-flavoring agent, vanillin to protect membranes against oxidative damage induced by photosensitization at concentrations normally used in food preparations. Vanillin, at a concentration of 2.5 mmol/L, has afforded significant protection against protein oxidation and lipid peroxidation in hepatic mitochondria induced by photosensitization with methylene blue plus light. The effect observed was both time- and concentration-dependent. The inhibitory effect is similar to ascorbic acid and the singlet oxygen quencher, diazabicyclo[2.2.2]octane (DABCO) but less effective than sodium azide and glutathione. Examination of possible mechanisms responsible for the observed protection, showed that vanillin has a significant ability to quench singlet oxygen (¹O₂), a reactive species responsible for damage induced during photosensitization by Type II mechanism. Hence, this flavoring compound, due to its antioxidant ability, may have potential to prevent oxidative damage to membranes in mammalian tissues and thereby the ensuing diseased states.     

Influence of neopterin on the generation of reactive oxygen species in human neutrophils

Neopterin is synthesized by human monocyte-derived macrophages primarily upon stimulation with the cytokine interferon-gamma. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earl's solution (pH=7.4) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D-mannitol, L-histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol-dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduced neopterin-induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)-independent pathways.     

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.     

Defence gene expression in soybean is linked to the status of the cell death program

Soybean cell cultures (cv. Williams 82) respond to Pseudomonas syringae bacteria expressing the avirulence gene AvrA with a hypersensitive reaction, a programmed cell death (PCD) of plant cells to pathogen attack. This PCD is under control of salicylic acid (SA) via an unknown mechanism. In the presence of low concentrations of SA, the cells undergo a very rapid cell death, which needs only half of the time required for the normal hypersensitive reaction (HR). Northern blot studies for defence-related genes show that the expression of many of these genes is tightly linked to the status of the cell death program rather than to pathogen-derived elicitors. Thus the expression is much faster in the SA-accelerated PCD than in the normal hypersensitive reaction. In contrast, other pathogen-responsive genes are induced independently of the speed of PCD, indicating a divergent signalling mechanism. The production of reactive oxygen species during the oxidative burst of bacteria-inoculated soybean cells is slightly enhanced in the presence of SA but occurs at the same time as in untreated cells, suggesting that SA exhibits the control of the PCD downstream of the oxidative burst. Consistent with these findings a HR-specific marker gene is neither directly induced by H₂O₂ or SA. However, this gene shows a high expression in the regular HR and is induced much faster in the SA-accelerated PCD.     

Thaxtomin A induces programmed cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.     

Bioreactor culture of heterotrophic sandalwood (Santalum album L.) cell suspensions utilizing a cell-lift impeller

Summary Growth and phenolic production by two heterotrophic suspension cultures (SW-1 and SW-2) of sandalwood cultivated in a 2.5 L bioreactor were investigated. Cultures of SW-1 cell suspensions resulted in a maximum phenolic content of 32.5 mg L^–1 compared to 12.5 mg L^–1 produced by SW-2 cell suspensions. Fresh weight doubling time (Td) was 5.8 days and the specific growth rate () was 0.12 d^–1 during exponential growth for both cell lines. The pH of the culture medium decreased from 5.5 to 3.5 during the exponential growth phase of SW-1 and SW-2 cell suspensions. The dissolved oxygen content also dropped steadily during culture and remained at 40% throughout exponential growth phase. These results should provide a basis for developing sandalwood cell cultures for bioproduction of useful compounds.Abbreviations 2,4-D: 2–4 dichlorophenoxyacetic acid - BA N⁶-benzyladenine - Eh Medium oxidation-reduction potential - K_La Oxygen transfer coefficient - MS Murashige and Skoog (1962) basal medium - SW-1 and SW-2 Sandalwood suspension lines