Requirement for direct association of ammonia-excreting Anabaena variabilis mutant (SA-1) with roots for maximal growth and yield of wheat

Direct association between wheat roots and an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis, strain SA-1, was required for maximal enhancement of growth of wheat plants in nitrogen (N)-free, hydroponic medium. Over 85% of the cyanobacterial mutant SA-1 inoculated to the roots were adsorbed under non-saturating conditions. The adsorption process of SA-1 to wheat roots was biphasic: an initial rapid adsorption was followed by a slow phase with about 10% of the initial adsorption rate. The maximal adsorption rate of filaments observed was 1.6 mg dry wt. SA-1 adsorbed·plant^–1·h^–1. Bypassing CO₂ fixation and sugar formation, the ¹⁴C label from [¹⁴C]sucrose was directly applied to leaf blades to study sugar translocation. The ¹⁴C label from this treatment appeared in the wheat culture medium within an hour. Nitrate-grown plants excreted about 30% of the ¹⁴C label into the medium, compared to only 10% excreted by wheat/Anabaena co-cultures. SA-1 assimilated 27% of all ¹⁴C translocated from [U-¹⁴C]sucrose applied to wheat leaves, and ¹⁴C label from this treatment was recovered from strain SA-1 after 30 min. Roots and cyanobacteria accounted for 51% of all radioactive label recovered in the plants co-cultured with SA-1 vs 20% for nitrate-grown plants. We studied the activity of -fructosidase (invertase) in wheat of variety Yecora rojo. Roots from nitrate-grown wheat plants produced high levels of invertase activity, which converted over 85% of 3 mm sucrose into glucose and fructose in 24 h. The rate of sucrose disappearance in the medium of co-cultures using A. variabilis SA-1, was 70% of that of nitrate-grown plants, but the levels of glucose and fructose in these cultures were always very low during sucrose conversion, suggesting hexose assimilation. To study the role of diffusible metabolites, a dialysis membrane was employed to separate the ammonia-excreting SA-1 from the wheat roots. Containing SA-1 in a dialysis bag away from direct root contact, severely limited leaf growth to less than one-third of the growth rate of nitrate control cultures. Ammonia produced by mutant SA-1 in dialysis bag cultures was excreted into the medium at 0.4 mm vs 1.2 mm in free-living cultures, but ammonia was not detectable in co-cultures with or without the dialysis bag containing the mutant. The nitrogenase activity derepressed in the mutant and responsible for ammonia excretion was always higher in the association co-cultures than in either free cells or in dialysis-bag cultures. The nitrogenase activity of strain SA-1 was highest (200 mol ethylene formed·mg^–1 Chl·h^–1) when the cyanobacterium was associated with the root tips. Dialysis membrane separation of plant and cyanobacterium severely inhibited growth of wheat during a complete growth cycle of 2 months. Total biomass and grain yield were very similar for control cultures without inorganic N or SA-1, and for diffusion cultures containing SA-1, kept in a dialysis bag around the roots. Total biomass of the association co-culture attained 75% of the biomass of the nitrate-grown control. It is proposed that wheat roots supplied fructose derived from sucrose for growth and nitrogen fixation of SA-1 in the light, and that ammonia excreted by SA-1 was utilized by the wheat plant for its own growth. Correspondence to: H. Spiller     

Photosynthetic production of hydrogen peroxide by Ulva rigida C. Ag. (Chlorophyta)

Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H₂O₂ was light dependent with a production of 1.2 mol·g FW^–1·h^–1 in sea water (pH 8.2) at an irradiance of 700 mol photons m^–2·s^–1. The excretion was also pH dependent: in pH 6.5 the production was not detectable (< 5 nmol·g FW^–1·h^–1) but at pH 9.0 the production was 5.0 mol·g FW^–1·h^–1. The production of H₂O₂ was totally inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). The ability of U. rigida growing in tanks (7501) under a natural light regime to excrete H₂O₂ was checked and found to be seven times higher at 08.00 hours than other times of the day. The H₂O₂ concentration in the cultivation tank (density: 2 g FW·l^–1) reached the highest value (3 M) at 11.00 hours. Photosynthesis was not influenced by H₂O₂ formation. The H₂O₂ is suggested to come from the Mehler reaction (pseudocyclic photophosphorylation). With an oxygen evolution of 120 mmol·g FW^–1·h^–1 at pH 8.2 and 90 mmol·g FW^–1·h^–1 at pH 9.0, 0.5% and 2.7% of the electrons were used for extracellular H₂O₂ production. The H₂O₂ production is sufficiently high to be of physiological and ecological significance, and is suggested to be a part of the defence against epi and endophytes.Abbreviations ACL artificial, continuous light - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - GNL greenhouse - LDC Luminol-dependent chemiluminescence - SOD Superoxide dismutase This investigation was supported by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Hierta-Retzius Foundation, Marianne and Marcus Wallenberg Foundation, the Swedish Environmental Protection Board, and CICYT Spain.     

The oxygen uptake-power regression in cyclists and untrained men: implications for the accumulated oxygen deficit

The regression of oxygen uptake (O₂) on power output and the O₂ demand predicted for suprapeak oxygen uptake (O_2peak) exercise (power output = 432 W) were compared in ten male cyclists [C, mean O_2peak = 67.9 (SD 4.2) ml · kg^–1 · min^–1] and nine active, yet untrained men [UT, mean O_2peak = 54.1 (SD 6.5) ml · kg^–1 · min^–1]. The O₂-power regression was determined using a continuous incremental cycle test (CON4), performed twice, which comprised several 4-min exercise periods progressing in intensity from approximately 40%–85% O_2peak. Minute ventilation (_E), heart rate (HR), respiratory exchange ratio (R), blood lactate concentration ([1a^–]_b) and rectal temperature (T _re) were measured at rest and during CON4. The slope of the O₂-power regression was greater (P 0.05) in C [12.4 (SD 0.7) ml · min^–1. W^–1] compared to UT [11.7 (SD 0.4) ml · min^–1 W^–1]; as a result, the O₂ demand (at 432 W) was also higher (P 0.05) in C [5.97 (SD 0.23) l · min^–1] than UT [5.70 (SD 0.15) 1 · min^–1]. ExerciseR and [la^–]_b were lower (P 0.05) in C .in comparison to UT at all power outputs, whereas _E and HR were relatively lower (P 0.05) in C at power outputs approximating 180 W, 220 W and 270 W. Differences in fat metabolism estimated over the first three power outputs accounted for approximately 19% of the difference in O₂-power slopes between the groups and up to 46% of the difference in O₂ at a given intensity. Although the O₂-power regressions were linear for C [r = 0.997 (SD 0.001)] and UT [r = 0.997 (SD 0.001)], the O₂-power slope was higher at power outputs at or above the lactate threshold (13.2 ml · min^–1 · W^–1 than at lower intensities (11.6 ml · min^–1 · W^–1) in C, an effect which was less profound in UT. As a result, the exclusion of O₂ at the highest power outputs completely abolished the difference in O₂-power slopes between C and UT. Thus, the relatively higher O₂ during incremental exercise in C can be almost entirely attributed to the higher O₂ cost of cycling at higher power outputs. In addition, the presence of non-linear responses in O₂ at higher intensities also confirms the invalidity of describing the O₂ response across a wide range of power outputs using a linear function, and challenges the validity of predicting the O₂ demand of more intense exercise by a linear extrapolation of this same function.     

Effect of long-term photoinhibition on growth and photosynthesis of cold-hardened spring and winter wheat

The effect of repeated exposure to high light (1200 mol · m^–2 · s^–1 photosynthetic photon flux density, PPFD) at 5° C was examined in attached leaves of cold-grown spring (cv. Katepwa) and winter (cv. Kharkov) wheat (Triticum aestivum L.) over an eight-week period. Under these conditions, Kharkov winter wheat exhibited a daily reduction of 24% in F_V/F_M (the ratio of variable to maximal fluorescence in the dark-adapted state), in contrast to 41% for cold-grown Katepwa spring wheat. Both cultivars were able to recover from this daily suppression of F_V/F_M such that the leaves exhibited an average morning F_V/F_M of 0.651 ± 0.004. Fluorescence measurements made under steady-state conditions as a function of irradiance from 60 to 2000 mol · m^–2 · s^–1 indicated that the yield of photosystem II (PSII) electron transport under light-saturating conditions was the same for photoinhibited and control cold-grown plants, regardless of cultivar. Repeated daily exposure to high light at low temperature did not increase resistance to short-term photoinhibition, although zeaxanthin levels increased by three- to fourfold. In addition, both cultivars increased the rate of dry-matter accumulation, relative to control plants maintained at 5° C and 250 mol · m^–2 · s^–1 PPFD (10% and 28% for Katepwa and Kharkov, respectively), despite exhibiting suppressed f_v/f_m and reduced photon yields for O₂ evolution following daily high-light treatments. Thus, although photosynthetic efficiency is suppressed by a longterm, photoinhibitory treatment, light-saturated rates of photosynthesis are sufficiently high during the high-light treatment to offset any reduction in photochemical efficiency of PSII. We suggest that in these cold-tolerant plants, photoinhibition of PSII may represent a longterm, stable, down-regulation of photochemistry to match the overall photosynthetic demand for ATP and reducing equivalents.Abbreviations and Symbols Chl chlorophyll - HL high light - PPFD photosynthetic photon flux density - F_O minimum fluorescence in the dark-adapted state - F_M maximum fluorescence in the dark-adapted state - F_V maximum variable fluorescence in the dark-adapted state (F_M-F_O) - F_V/F_V photosynthetic efficiency of the dark-adapted state - f_V/f_M photosynthetic efficiency of the light-adapted steady state - q_P photochemical quenching parameter - q_N non-photochemical quenching parameter - _e yield of electron transport and equals q_P · f_V/f_M - 1-q_O F_O quenching parameter - _app apparent photon yield. The assistance of Amy So is gratefully acknowledged. This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERCC) Operating Grant to N.P.A.H. G.Ö. was supported by an NSERCC International Exchange Award and the Swedish Natural Sciences Research Council.     

Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves

Exchange of CO₂ and O₂ and chlorophyll fluorescence were measured in the presence of 360 1 · 1^–1 CO₂ in nitrogen in Helianthus annuss L. leaves which had been preconditioned in the dark or at a photon flux density (PFD) of 24 mol · m^–2 · s^–1 either in 21 or 0% O₂. An initial light-dependent O₂ outburst of 6 mol · m^–2 was measured after aerobic dark incubation. It was attributed to the reduction of electron carriers, predominantly plastoquinone. The maximum initial rate of O₂ evolution at PFD 8000 mol · m^–2 · s^–1 was 170 mol · m^–2 · s^–2 or about four times the steady CO₂-and light-saturated rate of photosynthesis. Fluorescence measurements showed that the rate was still acceptor-limited. Fast O₂ evolution ceased after electron carriers were reduced in the dark-adapted leaf, but continued for a short time at the lower rate of 62 mol · m^–2 · s^–1 in the light-adapted leaf. The data are interpreted to show that enzymes involved in 3-phosphoglycerate reduction are dark-inhibited, but were fully active in low light. In a dark-adapted leaf, respiratory CO₂ evolution continued under nitrogen; it was partially inhibited by illumination. Prolonged exposure of a leaf to anaerobic conditions caused reducing equivalents to accumulate. This was shown by a slowly increasing chlorophyll fluorescence yield which indicated the reduction of the PSII acceptor Q_A in the dark. When the leaf was illuminated, no O₂ evolution was detected from short light pulses, although transient O₂ production was appreciable during longer light pulses. This indicates that an electron donor (pool size about 2–3 e/PSII reaction center) became reduced in the dark and the first photons were used to oxidise this donor instead of water.Abbreviations Chl chlorophyll - CRC carbon reduction cycle - GAPDH NADP-glyceraldehyde-phosphate dehydrogenase - PFD photon flux density - PGA 3-phosphoglycerate - RuBP ribulose bisphosphate - TCA tricarboxylic acid cycle To whom correspondence should be addressedThis work received support by the Estonian Academy of Sciences, the Gottfried-Wilhelm-Leibniz Program of the Deutsche For-schungsgemeinschaft and the Sonderforschungsbereich 251 of the University of Würzburg.     

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

Modulation of fatty acid-binding capacity of heart fatty acid-binding protein by oxygen — derived free radicals

Summary In this study, we examined the effects of exposure of heart fatty acid-binding protein (h-FABP) to chemically generated O₂ ^– or OH · with respect to its oleate binding and to its electrophoretic properties. Purified rat h-FABP at 40 M scavenged as much as 30% O₂ ^– and 85% OH ·. On the other hand, when 2 nmol (4 M) FABP were exposed to free radicals, the maximum oleate binding capacity as measured by Scatchard analysis was reduced only by 14% and 27% for O₂ ^– and OH ·, respectively. The electrophoretic pattern of free radical-exposed FABP was not markedly different when examined either by the non-denaturing or by denaturing PAGE, suggesting the absence of any degradation or aggregation of FABP by O₂ ^– or OH ·. It is hypothesized that O₂ ^– or OH · in physiological concentration may not alter the function of FABP markedly in the ischemic-reperfused myocardium.Abbreviations h-FABP Heart Fatty Acid Binding Protein - NEFA Non-Esterified Fatty Acids - O₂ ^– Superoxide anions - OH· hydroxyl radicals - OCI hypohalite radicals - H₂O₂ hydrogen peroxide - HPLC High Pressure Liquid Chromatography     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Foraminifera and meiofauna on an intertidal mudflat,Cornwall, England: Populations; respiration and secondary production; and energy budget

A rich and varied meiofauna inhabits a Cornish mudflat near the mouth of the Tamar River in southwestern England. Population densities range from 117 to 943 individuals · g^–1 (wet) sediment (1.4–11.4 × 10⁶ individuals · m^–2), with foraminifera, harpacticoid copepods and nematodes appearing in nearly equal numbers and comprising most of the meiofauna. Seasonally, meiofaunal numbers rise and fall with solar radiation and vary inversely with river discharge. Two species, the atestate allogromiid A and the calcareous Haynesina germanica (Ehrenberg), far outnumber other foraminifera; their population densities and growth rates reach maxima in spring and summer.Monthly rates of sediment respiration are locally variable, but clearly increase from winter (4.13 ml O₂ · m^–2 · h^–1 in December) to spring (38.87 ml O₂ · m^–2 · h^–1 in April). Experiments and calculations ascribe approximately 30% of this total to the meiofauna (including microfauna and microflora), 50% to bacteria and less than 20% to chemical oxidation. A tentative energy budget for the mudflat suggests that secondary production by meiofauna is small as compared with coastal environments elsewhere, and that meiofaunal production (426 Kcal · m^–2 · y^–1) is nearly twice meiofaunal respiration (252 Kcal · m^–2 · yr^–1).     

Maximal oxygen uptake, maximal voluntary isometric contraction and physical activity in young Danish adults

In a randomly selected sample of 88 men and 115 women, aged 23–27 years from Denmark, maximal oxygen uptake ( O_2max), maximal voluntary isometric contraction (MVC) in four muscle groups and physical activity were studied. The O_2max was 48.0 ml · min^–1 kg^–1 and 39.6 ml · min^–1 · kg^–1 for the men and the women, respectively. The MVC was 10% lower than in a comparable group of Danes of the same age and height studied 35 years ago. Only in men was sports activity directly related to O_2max (ml · min^–1 · kg^–1; r=0.31, P<0.01). The MVC of the knee extensors was related to O_2max in the men (r=0.31, P<0.01), but there was no relationship between the other measurements of MVC and O_2max. In the women O_2max (ml · min^–1 · kg^–1) was only related to body size, i.e. body mass index, percentage body fat and body mass [(r= –0.47, –0.48 (both P<0.001) and –0.34. (P<0.01), respectively)]. There were differences in O_2max in the men, according to education and occupation. Blue collar workers and subjects attending vocational or trade schools in 1983 had lower O_2max and more of them were physically inactive. In the women differences were also found, but there was no clear pattern among the groups. More of the women participated regularly in sports activity, but more of the men were very active compared to the women.     

Temperature insensitive O2 in blood of the tree frogChiromantis petersi

Summary Respiratory gas exchange and blood respiratory properties have been studied in the East-African tree frogChiromantis petersi. This frog is unusually xerophilous, occupies dry habitats and prefers body temperatures near 40°C and direct solar exposure. Total O₂ uptake was low at 81 l O₂·g^–1·h^–1±19.0 (SD) at 25°C increasing to 253.5 l O₂·g^–1·h^–1±94.8 (SD) at 40°C giving aQ ₁₀ value of 2.1. Skin O₂ uptake at 25°C was 38.5% of total. The gas exchange ratio was 0.71 for whole body gas exchange, 0.61 for the lungs and 1.02 for the skin at 25°C.Blood O₂ affinity was low with aP ₅₀ of 47.5 mmHg at 25°C and pH 7.65. Then _H-value at 25°C increased from 2.7 aroundP ₅₀ to 5.0 at O₂ saturations exceeding 70–80%. Surprisingly, blood O₂ affinity was nearly insensitive to temperature expressed by a H value of ±1.0 kcal·mole between 25 and 40°C.The adaptive significance of the low O₂ affinity, the increase ofn _H with O₂ saturation and the temperature insensitive O₂-Hb binding is discussed in relation to the high and fluctuating body temperatures ofChiromantis.     

Zingerone [4-(4-hydroxy-3-methoxyphenyl)-2-butanone] Prevents 6-Hydroxydopamine-induced Dopamine Depression in Mouse Striatum and Increases Superoxide Scavenging Activity in Serum

As superoxide (·O₂^–) and hydroxyl radical (·OH) have been implicated in pathogenesis of Parkinsons disease, free radical scavenging, antioxidant, and neuroprotective agents have attracted attention as ways to prevent progression. We examined effects of zingerone, an alkaloid extracted from ginger root, on 6-hydroxydopamine (6-OHDA)-induced dopamine (DA) reduction in mouse striatum. Zingerone administration 1 h before and for 6 more days following one intracerebroventricular 6-OHDA injection prevented reductions of striatal DA and its metabolites, and increased serum ·O₂^– scavenging activity. Zingerone did not change activities of catalase or glutathione peroxidase in striatum or serum, or ·O₂^– scavenging activity in striatum. Treatment with diethyldithiocarbamate, SOD inhibitor, abolished the protective effect of zingerone against 6-OHDA-induced DA reduction. In vitro, zingerone scavenged ·O₂^– and ·OH and suppressed lipid peroxidation only weakly. Thus, direct antioxidant effects may be a minor component of its putative neuroprotective effect; instead, zingerone acted mainly by increasing systemic superoxide dismutase activity. Effects of zingerone treatment in this model suggest possible value in treatment of Parkinsons disease.     

Oxygen transfer properties and dimensions of red blood cells in high-altitude camelids,dromedary camel and goat

Summary To estimate the advantage of the small red blood cells (RBC) of high-altitude camelids for O₂ transfer, the kinetics of O₂ uptake into and release from the RBC obtained from llama, vicuña and alpaca were investigated at 37°C with a stopped-flow technique. O₂ transfer conductance of RBC (G) was estimated from the rate of O₂ saturation change and the corresponding O₂ pressure difference between medium and hemoglobin. For comparison, O₂ kinetics for the RBC of a lowaltitude camelid (dromedary camel) and the pygmy goat were determined and previously measured values for human RBC were used. O₂ transfer of RBC was found to be strongly influenced by extracellular diffusion, except with O₂ release into dithionite solutions of sufficiently high concentration (>30 mM). TheG values measured in these standard conditions,G _st (in mmol · min^–1 · Torr^–1 · (ml RBC)^–1) were: high-altitude camelids, 0.58 (averaged for llama, alpaca and vicuña since there were no significant interspecific differences); camel 0.42; goat, 0.42; man, 0.39. The differences can in part be attributed to expected effects of the size and shape of the RBC (volume, surface area, mean thickness), as well as to the intracellular O₂ diffusivity which depends on the concentration of cellular hemoglobin. The highG _st of RBC of highaltitude camelids may be considered to enhance O₂ transfer in lungs and tissues. But the O₂ transfer conductance of blood, , equal toG _st multiplied by hematocrit (in mmol · min^–1 · Torr^–1 · (ml blood)^–1), was only slightly higher as compared to other species: 0.20 (llama, alpaca, vicuña), 0.14 (camel), 0.18 (goat), 0.17 (man).Abbreviations DPG 2,3-diphosphoglycerate - G conductance - Hb hemoglobin - RBC red blood cells - percent saturation of hemoglobin     

Signal role for activation of caspase-3-like protease and burst of superoxide anions during Ce<Superscript>4+</Superscript>-induced apoptosis of cultured <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

The signal events of 1 mM Ce⁴⁺ (Ce(NH₄)₂(NO₃)₆)-induced apoptosis of cultured Taxus cuspidata cells were investigated. The percentage of apoptotic cells increased from 0.82% to 51.32% within 6 days. Caspase-3-like protease activity became notable during the second day of Ce⁴⁺-treatment, and the maximum activity was 5-fold higher than that of control cells at the fourth day. When the experiment system was pretreated with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) at 100 M, caspase-3-like activity resulted in distinct inhibition by 70% and 77.3% after 3 and 4 days of induction. Furthermore, 100 M Ac-DEVD-CHO partially reduced the apoptotic cells by 58.6% and 60.8% at day 4 and 5 respectively. Ce⁴⁺ induced superoxide anions (O₂·–) transient burst, and the first peak appeared at around 3.7–4 h, the second appeared at about 7 h. Both O₂·– burst and cell apoptosis were effectively suppressed by application of diphenyl iodonium (NADPH oxidase inhibitor). Inhibition of O₂·– production attenuated caspase-3-like activation by 49% and 53.6% during day 3 and 4 respectively. In addition, a total of 15 protein spots changed in response to caspase-3-like protease activation were identified by two-dimensional gel electrophoresis. These results suggest that Ce⁴⁺ of 1 mM induces apoptosis in suspension cultures of T. cuspidata through O₂·– burst as well as caspase-3-like protease activation. The burst of O₂·– exerts its activity as an upstream of caspase-3-like activation. Our results also implicate that other signal pathways independent of an O₂·– burst possibly participate in mediating caspase-3-like protease activation.     

Ozone detoxification in the apoplasm and symplasm of spinach,broad bean and beech leaves at ambient and elevated concentrations of ozone in air

Spinach (Spinacia oleracea L.), broad bean (Vicia faba L.) and beech (Fagus sylvatica L.) plants were exposed to ozone at concentrations often measured in air during the summer months (120–300 g·m^–3) and antioxidants were determined in the leaf tissue and in the aqueous phase of the cell wall, the apoplasm. Concentrations of both reduced ascorbate (AA) and its oxidized form, dehydroascorbate (DHA), showed the tendency to increase transiently in the apoplasm of spinach leaves 6–24 h after starting fumigation with ozone. In beech leaves, apoplasmic AA and DHA increased 3–7 d after beginning of treatment. At the very high concentration of 1600 g O₃·m^–3, an increase of apoplasmic AA was already measured after 1 d in beech leaves. Apparently, spinach and beech leaves respond to oxidative stress by increasing AA transport into the apoplasm and by accelerating DHA export. In contrast to these observations, DHA accumulated during 3 d of fumigation with only 120 g O₃·m^–3 in the apoplasm of broad bean leaves, while AA contents did not increase. After termination of fumigation, the extracellular redox state of ascorbate normalized within 1 d. Glutathione could not be detected in the apoplasm of any of the three leaf species. Intracellular AA changed its redox state in response to exposure to elevated concentrations of ozone. After 4–6 weeks of fumigation with 200–300 g O₃·m^–3 an increase of intracellular DHA was measured in beech leaves. At the same time, chlorophyll contents decreased and characteristic symptoms of ozone damage could be observed. However, no significant change in the redox state of apoplasmic ascorbate could be detected in beech leaves. Evidently, detoxification of ozone by apoplasmic AA was insufficient to protect the leaf tissue. Fumigation with a high ozone concentration (1600 g·m^–3) caused an appreciable increase in the cellular contents of the oxidized forms of ascorbate and glutathione in beech leaves. Whereas in spinach leaves intracellular antioxidant contents and redox states were not altered during fumigation with 120–240 g O₃·m^–3, in broad bean leaves the intracellular DHA concentration increased and intracellular ascorbate became more oxidized after fumigation of the plants with 120 g O₃·m^–3. Apparently, broad bean leaves are more sensitive to ozone than beech and spinach leaves.Abbreviations AA ascorbate, reduced form - DHA ascorbate, oxidized form (dehydroascorbate) - FW fresh weight - GSH glutathione, reduced form - GSSG glutathione, oxidized form - IWF intercellular washing fluid - V_air intercellular air space volume of leaves - V_apo apoplasmic water volume of leaves This work was supported within the Sonderforschungsbereich 251 of the University of Würzburg.     

Elektron-Spin-Resonanz-Untersuchungen der Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins

Zusammenfassung Die Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins wurde in flüssiger Phase bei Raumtemperatur mittels ESR-Spektroskopie untersucht. Mit Hilfe eines geeigneten photochemischen Initiationssystems ließen sich in Cyclohexanlösung unter UV-Bestrahlung (235 nm265 nm) genau dieselben freien Radikale des Cholesterins darstellen, die schon früher [9, 7] in röntgenbestrahltem Cholesterinpulver beobachtet worden waren. Bei ausreichendem O₂-Partialdruck (3·10⁴Torr) über der Probenlösung trat das ESR-Spektrum eines Peroxyradikals auf, das mittels der Analyse seiner Reaktionsprodukte (7-Hydroxy-Cholesterin und 7-Keto-Cholesterin) mit dem Cholesteryl-7-peroxyradikal identifiziert wurde. Die Kinetik sowohl der Bildung als auch des Zerfalls des Radikals entsprachen einer Reaktion von 2. Ordnung. Die Geschwindigkeitskonstante für den bimolekularen Zerfall, eine Disproportionierung in Alkohol und Keton unter Abgabe eines Moleküls O₂, wurde bei Raumtemperatur zuk ₂=(1,8 _–0,6 ^+0,9 )·10⁶ sec^–1M^–1·l bestimmt. Ferner wurde gezeigt, daß das Cholesteryl-7-peroxyradikal aus dem freien Radikal Cholesteryl-7 durch Anlagerung eines Moleküls O₂ entsteht. Für die Geschwindigkeitskonstante dieser Reaktion ergab sich eine untere Schranke vonk ₁=0,40·10¹⁰ sec^–1M^–1·l.

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Electron spin resonance investigations on radiation-induced free radicals of cholesterol in liquid phase

Summary The reaction kinetics of radiation-induced free radicals of cholesterol was studied in liquid phase at room temperature by means of e.s.r. spectroscopy on a solution of cholesterol in cyclohexane. Using a convenient photochemical initiation system, just those free radicals of cholesterol could be generated by the filtered u.v. radiation from a Xe high pressure lamp (235 nm265 nm) as were observed already a decade ago by Gordy [9] and by Ehrenberg, Löfroth [7] in X-irradiated cholesterol powder. At sufficiently high O₂-pressures (3·10^–4 Torr) over the sample solution a peroxy radical e.s.r. spectrum arose during u.v. irradiation which was identified by product analysis (7-hydroxy-cholesterol and 7-keto-cholesterol) to be dueto a cholesteryl-7-peroxyradical. The radical'sgeneration and decay kinetics was governed by a second order reaction. The velocity constant for bimolecular decay of the cholesteryl-7-peroxyradical was found to be k₂=(1.8 _–0,6 ^+0,9 )·10⁶sec^–1M^–1·l at room temperature. Furthermore it could be shown that the cholesteryl-7-peroxyradical was built up by the addition of one molecule of O₂ to a cholesteryl-7 free radical. For this reaction a value ofk ₁=0.4·10¹⁰ sec^–1 M^–1·l was estimated as a lower limit of the velocity constant.

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Die Arbeit stellt einen Auszug aus einer Dissertation an der Technischen Hochschule München dar.     

Some effects of long-term ozone fumigations on Norway spruce

Summary Potted cuttings of a 12-year-old, and grafts of an 80-year-old, Norway spruce (Picea abies (L.) Karst.) were subjected to 100 or 300 g O₃·m^–3 for 1215 h (45 h of daylight per week) during the growing season of 1985. At the end of the fumigation the plants did not exhibit any visible signs of injury. Whereas in the fumigation with 100g O₃·m^–3 we did not detect any significant change in gas exchange, 300 g O₃·m^–3 did alter the CO₂ uptake after 27 weeks, and in one clone transpiration was also altered. Stomatal reaction to a change of light suggested sluggishness, but the change was not statistically significant.     

The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO₂ assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO₂ partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO₂ assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO₂, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, k_cat, of 3.5+0.2 mol CO₂·(mol sites)^–1·s^–1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO₂·(mol sites)^–1·^–1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO₂ and O₂, the rate of CO₂ assimilation was measured at 25° C at different intercellular partial pressures of CO₂ and O₂. These measurements were combined with carbon-isotope analysis (¹³C/¹²C) of CO₂ in the air passing over the leaf to estimate the conductance for transfer of CO₂ from the substomatal cavities to the sites of carboxylation (0.3 mol·m^–2·s^–1·bar^–1) and thus the partial pressure of CO₂ at the sites of carboxylation. The calculated Michaelis-Menten constants for CO₂ and O₂ were 259 ±57 bar (8.6±1.9M) and 179 mbar (226 M), respectively, and the effective Michaelis-Menten constant for CO₂ in 200 mbar O₂ was 549 bar (18.3 M). From measurements of the photocompensation point (_* = 38.6 ubar) we estimated Rubisco's relative specificity for CO₂, as opposed to O₂ to be 97.5 in vivo. These values were dependent on the size of the estimated CO₂-transfer conductance.Abbreviations and Symbols A CO₂-assimilation rate - g_w conductance for CO₂ transfer from the substomatal cavities to the sites of carboxylation - K_c, K_o Michaelis-Menten constants for carboxylation, oxygenation of Rubisco - k_cat V_cmax/[active site] - O partial pressure of O₂ at the site of carboxylation - p_c partial pressure of CO₂ at the site of carboxylation - p_i intercellular CO₂ partial pressure - R_d day respiration (non-photorespiratory CO₂ evolution) - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - S_c/o relative specificity factor for Rubisco - SSu small subunit of Rubisco - V_cmax, V_omax maximum rates of Rubisco carboxylation, oxygenation - _* partial pressure of CO₂ in the chloroplast at which photorespiratory CO₂ evolution equals the rate of carboxylation     

Necessity of Superoxide Production for Development of Etiolated Wheat Seedlings

It was found that production of superoxide (O₂ ^{– ·}) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O₂ ^{– ·} production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O₂ ^{– ·} production was clearly observed in the period from two to four days of seedling development, then the rate of O₂ ^{– ·} production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O₂ ^{– ·} production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O₂ ^{– ·} production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O₂ ^{– ·} amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O₂ ^{– ·} controls plant growth by cell elongation at the early stages of seedling development but later O₂ ^{– ·} controls (induces) apoptotic DNA fragmentation and protein disintegration.