Leland S. Jahnke

Long-term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta.

Tolerance to salinity stress in higher plants correlates to levels of antioxidant enzymes and/or substrates. Do hyperosmotic and hypoosmotic stress induce antioxidant responses in salt tolerant algae, and if so, are these responses the same for both excess and minimal salinity? To answer these questions, cultures of the marine alga Dunaliella tertiolecta (Chlorophyta) were grown in seven salinities covering a 60-fold range from 0.05 to 3.0 mol/L NaCl. Long-term effects of salinity on growth and antioxidant parameters were determined. Growth rates were reduced at the salinity extremes (0.05 mol/L NaCl and 3 mol/L NaCl) indicating the cultures were stressed. The levels of six antioxidant enzymes and three antioxidant substrates were quantified at these growth salinities. Compared to growth at optimum salinities (i.e. 0.2-0.5 mol/L NaCl), high salinities produced a 260% increase in monodehydroascorbate reductase, a doubling of ascorbate peroxidase activity and a three-fold increase in the rate of dark respiration. Cells acclimated to low growth salinities (hyposaline stress, i.e. < 0.2 mol/L NaCl) showed major increases in glutathione and alpha-tocopherol coupled with decreases in Fv/Fm ratios and in total and reduced ascorbate compared to moderate and high external salinities. Cell volumes remained unchanged, except at the lowest salinity where they doubled. Catalase, superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities were not altered by extreme salinities. The involvement of oxidative stress at both salinity extremes is implied by the alterations in antioxidant enzymes and substrates, but the specific changes are very different between hypo and hypersaline stresses.

Massive carotenoid accumulation in Dunaliella bardawil induced by ultraviolet-A radiation

Abstract The effect of supplementing visible light (i.e., wavelengths in the range 400–700 nm) with ultraviolet-A radiation (320–400 nm) during the growth of Dunaliella bardawil has been investigated. This study focuses on the accumulation of carotenoids. Previous investigations have found that large increases in carotenoids are induced by high-intensity photosynthetically active radiation (PAR) and by various environmental stresses (e.g., high salinity, nutrient deficiencies or low temperature), which significantly reduce growth rates. This study has found that UV-A radiation causes massive increases in carotenoid levels without slowing the growth of D. bardawil in culture. By supplementing various levels of PAR with UV-A radiation (38 μmol m −2 s −1 ) during growth, carotenoid:chlorophyll ratios increase by 80–310%. Per unit protein, UV-A produces a 0–35% decrease in chlorophyll coupled with a doubling of total carotenoids. These large increases in carotenoids are induced by UV-A at all salinities (0.5 to 3.0 M), and at all visible photosynthetic photon flux densities (30–1500 μmol m −2 s −1 ) tested. Induced carotenoid levels are proportional to the intensity of UV-A when growth PAR is held constant. Blue light (200 μmol m −2 s −1 ) is no more effective than white light in carotenoid induction, indicating that this is not a shared blue-light/near-UV effect. UV-B (290–320 nm) is also ineffective in inducing carotenoid increases during growth.

Instantaneous and Developmental Effects of Low Temperature on the Catalytic Properties of Antioxidant Enzymes in Two Zea Species

When Zea mays cv. LG11 plants were grown 14˚C(close to the lower thermal limit for leaf expansion), three of the five enzymes of the active oxygen scavenging cycle (Halliwell-Asada Pathway) showed changes in total leaf activity compared with growth at 25˚C. Two of theseenzymes, ascorbate peroxidase (APX) and glutathione reductase (GTR), were selected for further investigation. The effects of assay temperature on threekinetic parameters (Vmax,Km,Vmax/Km) were determined inextracts from Z. mays and compared with extracts fromits low temperature tolerant relative, Z. diploperennisIltis, Doebley & Guzman. The kinetic power(Vmax/Km) was determinedbecause the Km alone may not be a useful predictor of anenzyme’s effectiveness in situ. The decrease inthe kinetic power of APX on lowering the temperature to 5oC was muchsmaller in Z. diploperennis thaninZ. mays. This suggests that theZ. diploperennis APX is better able to removeH2O2 at severely reducedtemperatures than is APX from Z. mays.Z. diploperennis had a twofold greater ascorbate poolthan the chilling-susceptible Z. mays. Only minordifferences were seen in the kinetic properties of GTR and the size of theglutathione pool between the genotypes.

Photooxidative reactions in chloroplast thylakoids. Evidence for a Fenton-type reaction promoted by superoxide or ascorbate.

A methyl viologen (MV)* mediated Mehler reaction was studied using Type C and D chloroplasts (thylakoids) from spinach. The extent of photooxidative reactions were measured as (a) rate of ethylene formation from methional oxidation indicating the production of oxygen radicals, and (b) rate of malondialdehyde (MDA) formation as a measure of lipid peroxidation. Without added ascorbate, 1 μM FerricEDTA increased ethylene formation by greater than 4-fold, but had no effect on MDA production. Ascorbate (1 mM) produced a tripling of ethylene while it reduced MDA formation in the presence of iron. Radical scavengers diethyldithiocarbamate (DDTC), formate, 1,4-diazabicyclo (2.2.2octane) (DABCO), inhibited ethylene formation. Using 0,4 M mannitol to scavenge hydroxyl radicals, the rates of ethylene formation were reduced 40 to 60% with or without 1 μM Fe(III) EDTA. The strong oxidant(s) not scavenged by mannitol are hypothesized to be either alkoxyl radicals from lipid peroxidation, or ‘site specific’ formation of hydroxyl radicals in a lipophillic environment not exposed to mannitol. Singlet oxygen does not appear to be a significant factor in this system. Catalase strongly inhibited both ethylene and MDA synthesis under all conditions; 1 mM ascorbate did not reverse this inhibition. However, the strong superoxide dismutase (SOD) inhibition of ethylene and MDA formation was completely reversed by 1 mM ascorbate. This suggests that superoxide was functioning as an iron reducing agent and that in its absence, ascorbate was similarly promoting oxidations. Therefore, these oxidative processes were dependent on the presence of H2O2 and a reducing agent, suggesting the involvement of a Fenton-type reaction.

Carbonic anhydrase, carbondioxide levels and growth of Nitrosomonas

The ammonia oxidizing bacterium Nitrosomonas europaea was grown either (a) with added bicarbonate in the absence of added CO2 (bubbled through the culture), (b) with added bicarbonate plus low added CO2 (0.03% v/v), or (c) without added bicarbonate with high added CO2 (1% v/v). Cell doubling times of 12 h were observed in 1% cultures; doubling times of 2 to 3-fold longer wre found with 0.03% CO2 and/or bicarbonate grown cultures. The specific activity of carbonic anhydrase was 40–80% lower in cultures grown on 1% CO2. These results are compared with those in heterotrophic and photosynthetic microorganisms.

Removing UV-A and UV-C Radiation from UV-B Fluorescent Lamp Emissions. Differences in the Inhibition of Photosynthesis in the Marine Alga Dunaliella tertiolecta Using Chromate Versus Cellulose Acetate-Polyester Filters¶

Ultraviolet‐B (UV‐B; 280‐320 nm)‐emitting lamps unavoidably emit ultraviolet‐A (UV‐A; 320‐400 nm) and ultraviolet‐C (UV‐C; < 280 nm) radiation. Short‐wavelength‐blocking filters are generally used to limit the wave bands of UV under investigation. The widespread use of such filters means that all exposures to UV‐B radiation will have a significant UV‐A component. Therefore, the physiological effects unique to UV‐B exposure are difficult to clearly isolate. This study presents a method to remove the UV‐A and UV‐C “contamination” using a liquid potassium chromate (K2CrO4) filter, thus allowing more direct assessment of the effects of UV‐B exposure. Cultures of the green marine alga Dunaliella tertiolecta were grown in the absence of UV radiation. Sunlamps supplied the UV radiation for a 24 h exposure (solar radiation was not used in this study). The UV radiation was filtered either by the standard method (i.e. cellulose acetate (CA) with polyester = Mylar controls) or by a liquid filter of potassium chromate. Photosynthetic responses were compared. Major decreases in the ratio of variable to maximal fluorescence in dark‐adapted cells and photosynthetic capacity were observed in CA‐filtered cultures, whereas no change was observed in cells exposed to the same UV‐B flux with the UV‐A removed by K2CrO4. The use of a CA filter with a Mylar control does not link results unequivocally to UV‐B radiation. Such results should be interpreted with caution.

Evidence for Bicarbonate Active Transport in Elodea Nuttallii

In aquatic wastewater treatment systems, submerged aquatic macrophytes can be exposed to high oxygen and low CT (see abbreviations below) concentrations which are detrimentally conducive to photorespiration. Many macrophytes adapt to such conditions by entering a low photorespiratory state which involves a decrease in the CO2 compensation point (Γ) and consequently an increase In the affinity (Km) for CO2. Two seChapaute Inducible enzyme mechanisms are utilized (1,2). Both utilize HCO2 -, which can be the only available inorganic carbon species, and both promote carbcxylatlon over oxygenation at RUBISCO. Hydrilla synthesizes PEPcase which fixes HCO3 - into C4 acids which can then be decarboxylated as a CO2 source for RUBISCO (2). Conversely, Myriophyllum synthesizes CA which is thought to be involved in a HCO3 - active transport system (1). We have grown Elodea nuttallii in aquatic treatment systems; the macrophyte exhibits low photorespiratory and HCO33- utilization characteristics (3)Γ=44 μL L-1, Km [C2]=98μ M, RUBISCO/PEPcase=6.6, CA=14 EU mg Chl-1, acid and alkaline banding, and 1:1 HCO3 - utilization: OH- production stolchlometries. The purpose of this study was to determine if E. nuttallii employs a HCO3 - active transport system as part of its adaptive response to photorespiratory conditions. The data suggests the E. nuttallii possesses a HOO3 - proton symport to accumulate CT.

Inhibition of the photosynthetic electron transport of isolated thylakoids by hemolyzed rabbit sera. Evidence for the potential involvement of parallel electron transport in photosystem I Mehler reactions.

The inhibition patterns of rabbit sera (RS1 & RS2) from two different rabbits on the photosynthetic electron transport of isolated spinach thylakoids were studied. Fifty μl of RSI were required for 100% inhibition of a H2O → MV/O2 reaction, while only 10 μl of a 1:10 dilution of RS2 were needed for 100% inhibition. The RS2 serum was greatly hemolyzed. The γ-globulin fraction from purified rabbit serum (RS1) did not inhibit photosynthetic electron transport, indicating that the antibody fraction of the rabbit serum does not contain the inhibitor. It appears that the inhibitor is from the hemolyzed red blood cells. Rabbit sera added prior to chloroplast illumination caused no inhibition, while addition of rabbit sera during illumination inhibited a H2O → MV/O2 reaction within 1–3s. Aminotriazole, a catalase inhibitor, did not affect the efficacy of the rabbit sera indicating that the unknown rabbit serum inhibitor is not catalase. Various Hill reactions were employed to determine the site of inhibition. Rabbit sera inhibited the following reactions: DHQ/DCMU → MV/O2, DAD/Asc/DBMIB → MV/O2, and DCIP/Asc/DBMIB → MV/O2. Rabbit sera did not inhibit a H2O → DADox reaction indicating that inhibition is on the reducing side of PSI. However, a H2O → Fd/NADP+ reaction was not inhibited by rabbit sera. NADP did not interfere with the ability of RS2 to inhibit a MV-mediated Mehler reaction. In simultaneously measured assays of Fd-mediated O2 and NADP+ reductions, RS2 serum inhibited the reduction of O2 by ferredoxin without inhibiting the reduction of NADP+. These results indicate the potential involvement of parallel (branched) electron transport of the reducing side of PSI in the reduction of oxygen.

Indirect Effects of α-Hydroxypyridinemethanesulfonate and Cyanide on Photosynthesis and Glycolate Excretion in Chlorella

Summary The effects of two inhibitors of photosynthetic carbon metabolism (HPMS and cyanide) were studied in Chlorella pyrenoidosa . Cultures were grown either with air or high CO 2 (5% CO 2 in air). The effects of these two inhibitors on glycolate excretion and 14 CO 2 fixation were determined and compared with the effects of the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZA). Air grown Chlorella had high carbonic anhydrase activities and released small quantities of glycolate when incubated with 100% O 2 with saturating light intensities. Identical cultures treated with 10 mM HPMS or 100 μM NaCN doubled the rate of glycolate excretion while reducing carbon-fixation by 31 and 40% respectively. Saturating concentrations (≥ 0.5 mM) of EZA caused a greater than five-fold increase in glycolate excretion and a 73% inhibition of carbon fixation. HPMS when used with EZA further increased glycolate excretion compared to HPMS alone. Saturating concentrations of EZA with 100 μM cyanide caused a 60% increase in glycolate excretion compared with cyanide alone. Cultures grown with high CO 2 responded to the inhibitors very differently. They had no detectable carbonic anhydrase activity and excreted glycolate at more than five times the rate of the air grown cells with no inhibitor treatment. The addition of 10 mM HPMS either with or without EZA produced only relatively small changes in both glycolate excretion and carbon-fixation. Saturating levels of EZA caused only a 34% increase in measured glycolate, and HPMS with EZA showed a 24% increase compared with the control. Cyanide inhibited glycolate synthesis in the high CO 2 -grown cells by 27% and by 19% when tested with EZA; cyanide inhibited carbon-fixation by 29% alone and by 37% when used with EZA. Consistent with the low levels of CA in these high CO 2 grown cells is the small effect that EZA has on carbon-fixation and glycolate excretion either by itself or with HPMS or with cyanide. The inhibition of glycolate dehydrogenase by HPMS or cyanide does not appear to cause significant changes in glycolate excretion or carbon- fixation. The activity of glycolate dehydrogenase is low and is not altered by the growth conditions, as is carbonic anhydrase. It is concluded that both HPMS and cyanide do significantly alter photosynthetic carbon metabolism in air-grown Chlorella pyrenoidosa by inhibition of carbonic anhydrase. Cells grown with high CO 2 do not show these effects because very little CA activity occurs in such cells.

A new filter that accurately mimics the solar UV-B spectrum using standard UV lamps: the photochemical properties, stabilization and use of the urate anion liquid filter.

The physiological effects unique to solar ultraviolet (UV)-B exposure (280-315 nm) are difficult to accurately replicate in the laboratory. This study evaluates the effectiveness of the sodium urate anion in a liquid filter that yields a spectrum nearly indistinguishable from the solar UV-B spectrum while filtering the emissions of widely used UV-B lamps. The photochemical properties and stability of this filter are examined and weighed against a typical spectrum of ground-level solar UV-B radiation. To test the effectiveness of this filter, light-saturated photosynthetic oxygen evolution rates were measured following exposure to UV-B filtered either by this urate filter or the widely used cellulose acetate (CA) filter. The ubiquitous marine Chlorophyte alga Dunaliella tertiolecta was tested under identical UV-B flux densities coupled with ecologically realistic fluxes of UV-A and visible radiation for 6 and 12 h exposures. These results indicate that the urate-filtered UV-B radiation yields minor photosynthetic inhibition when compared with exposures lacking in UV-B. This is in agreement with published experiments using solar radiation. In sharp contrast, radiation filtered by CA filters produced large inhibition of photosynthesis.