Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.     

Cytosolic ascorbate peroxidase in Gymnosperms

ABSTRACT

Sixteen species of Gymnosperms have been screened for cytosolic ascorbate peroxidase by means of native polyacrylamide gel electrophoresis. This analysis shows that a single form of the enzyme is the most common situation. The enzyme reveals a similar electrophoretic mobility in species belonging to the same genus and sometimes to different genera. In some Pinaceae, two bands of activity were observed. The presence in the archaic spermatophyte Ginkgo biloba, as well as in the more advanced monocotyledons, of three isoforms of ascorbate peroxidase, might suggest that three different cytosolic ascorbate peroxidase genes were already present in this archaic species.     

Distribution of cytosolic ascorbate peroxidase in Angiosperms

Abstract

Over 80 Angiosperms have been screened for cytosolic ascorbate peroxidase by means of native-polyacrylamide gel electrophoresis techniques. The results of our analysis show that the presence of a single cytosolic ascorbate peroxidase form is the most common case in the Angiosperms investigated when seedlings or young tissues are analized. This is a conserved character in orders of the Dicots. Two electrophoretic distinct AApx forms have been identified in Magnoliales, the ancestor group from which both Monocots and Dicots were originated and in few other orders. A notable increase in the isoenzyme number is observed in the advanced Monocots (Poales), thus suggesting the existence of an evolutionary trend leading in Monocots to an apparently progressive rise in the ascorbate-dependent enzymatic scavenging of hydrogen peroxide.     

Conjugation of benzo[a]pyrene metabolites by freshwater green alga Selenastrum capricornutum

Benzo[a]pyrene (BaP) undergoes metabolic transformation in mammals via oxidative, hydrolytic, and conjugative processes; however, little is known concerning BaP conjugation in freshwater algae. It has been shown in this laboratory that BaP is metabolized by Selenastrum capricornutum via a dioxygenase pathway. This study describes the conjugation of BaP metabolites by a green alga, Selenastrum capricornutum. Cultures were exposed to 1160 micrograms/l [14C]BaP for 4 days at 23 degrees C under gold fluorescent lights on a diurnal cycle of 16 h light, 8 h dark. Of the total metabolites in the algal culture, 89% were present in media. BaP and non-conjugated metabolites were separated from conjugated metabolites by chromatography on neutral alumina columns using solvents of increasing polarity. Seventy-one percent of the BaP metabolites were conjugates of which 12.2%, 12.0% and 12.4% were sulfate ester and alpha- and beta-glucose conjugates, respectively. Conjugates that coeluted with sulfate esters were hydrolyzed with arylsulfatase, alpha- or beta-glucosidase; high performance liquid chromatography (HPLC) analysis indicated that the major product of each enzymatic hydrolysis was the 4,5-dihydrodiol (87.2, 69 and 53%, respectively). Eighty-six percent of the conjugates were acid labile following incubation for 2 h in 4 N HCl at 37 degrees C. To our knowledge this is the first demonstration of the metabolism of a polynuclear aromatic hydrocarbon by a freshwater green alga through a dioxygenase pathway and subsequent conjugation and excretion.     

Phosphate uptake by the yeast, Rhodotorula rubra, and the green alga, Selenastrum capricornutum Printz, after phosphate additions to steady-state continuous cultures

Abstract We examined phosphate (P_i) uptake by two well-characterized microorganisms: a green alga ( Selenastrum capricornutum ) and a heterotrophic yeast ( Rhodotorula rubra ). Phosphate uptake was measured in dual- and single-species continuous cultures after perturbation of a phosphorus (P)-limited steady-state culture by additions of varying concentrations of P_i. We found that, under these conditions, both organisms had very high transport rates for P_i. The yeast was able to attain higher internal P concentrations than predicted from either steady-state or from P-starved batch culture data. Because the yeast was able to sequester and store P_i more efficiently than the alga under dilute P_i continuous culture conditions, co-existence of the two organisms was ultimately controlled by the concentration of carbon available for growth of the yeast.     

黄芩过氧化物酶同工酶电泳和抗坏血酸过氧化物酶活性分析

对黄芩（Scutellaria baicalensis Georgi)二倍体和同源四倍体过氧化物酶进行了同工酶电泳分析及抗坏血酸过氧化物酶活性测定。结果表明,黄芩二倍体与同源四倍体过氧化物酶同工酶酶谱一致,但后者的着色程度大于前者,二年生黄芩叶子在快速区Rf为0．494和0．512处出现新的谱带,不同发育阶段和不同组织器官的谱带存在明显差异;根和叶中谱带数最多,花其次,种子最少;试管苗谱带数先减少后增加,并在整个培养过程中出现特征性谱带C。各组织器官抗坏血酸过氧化物酶总活力差异明显,顶芽最高,叶子次之,花最低。黄芩一年生和二年生各个多倍体株系叶子抗坏血酸过氧酶总活力均高于二倍体叶子抗坏血酸过氧化酶总活力。试管苗生长过程中抗坏血酶过氧化物酶活活力的变化与生长趋势一致,表明该酶与植株生长发育紧密相关。     

Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E. coli

Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products. Received: 10 May 1997 / Accepted: 19 June 1997     

Peroxidase from the green alga Enteromorpha linza

An enzyme displaying peroxidase activity has been extracted and purified 27-fold from the green alga Enteromorpha linza. The partially purified pre     

Identification of the 11,12-dihydro-11,12-dihydroxybenzo(a)pyrene as a major metabolite produced by the green alga, Selenastrum capricornutum

Benzo(a)pyrene metabolites were isolated after incubation of [14C]-benzo(a)pyrene with the green alga, Selenastrum capricornutum. A significant amount of radioactivity chromatographed in the dihydrodiol region which did not coelute with any of the previously identified dihydrodiol metabolites isolated from this system. Following characterization by mass spectrometry, fluorescence spectroscopy, and high pressure liquid chromatography, this metabolite was identified as the cis-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene. This metabolite has not been identified previously as a metabolite formed in a plant system.     

Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress

To evaluate the physiological importance of thylakoid membrane-bound ascorbate peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.     

Metabolism of benzo(a)pyrene by a dioxygenase enzyme system of the freshwater green alga Selenastrum capricornutum

The green alga Selenastrum capricornutum was incubated with benzo(a)-pyrene under an atmosphere of 20% (18)O2: 80% N2. The cis-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene, cis-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and cis-4,5-dihydro-4,5-dihydroxybenzo(a)pyrene, were isolated by HPLC and analyzed by mass spectrometry. The metabolites produced molecular ions at m/z 290 and 286. Elemental analysis of the ion at m/z 290 gave an elemental composition of C20H14(18)O2 with 13% (18)O2 incorporation. The results indicate that S. capricornutum produces cis vicinal dihydrodiols from molecular oxygen via a dioxygenase enzyme pathway. The dioxygenase enzymes are characteristic of the bacterial metabolic pathway and unlike those of eukaryotic organisms which involve monooxygenase enzymes.     

Photosynthetic and population growth response of the test alga Selenastrum capricornutum Printz to zinc, cadmium and suspended sediment elutriates

Short-term ¹⁴C-fixation (4 h) Selenastrum capricornutum algal toxicity tests were conducted with Cd (n=8), Zn (n=9) and suspended sediment aqueous elutriates (n=28) and the results were compared to those obtained in a 48 h population growth test. In order to provide more realistic experimental conditions, toxicity tests were carried out in prefiltered nutrient-spiked Lake Geneva water. The population growth inhibition test was significantly more sensitive than the¹⁴ C-fixation test for Cd (median EC50-4h and EC50-48h values of 600 and 118 μg L^-1, respectively) whereas no significant difference was measured for Zn toxicity (median EC50-4h and EC50-48h values of 97 and 96 μg L^-1, respectively). With suspended sediment aqueous elutriates, the relative sensitivity of the two different end points is sample dependent, with ratios of the EC25 for the¹⁴ C-fixation: population growth test ranging from <0.26 to >53.3. Elutriate toxicity shows no apparent relationship between the acute and chronic test, indicating that population growth inhibition cannot be derived directly or predicted from¹⁴ C-fixation. Both tests with their specific advantages and limitations provide valuable complementary information to measure the impact of single toxicants or complex mixtures on aquatic plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genome-wide analysis of core cell cycle genes in the unicellular green alga Ostreococcus tauri

The cell cycle has been extensively studied in various organisms, and the recent access to an overwhelming amount of genomic data has given birth to a new integrated approach called comparative genomics. Comparing the cell cycle across species shows that its regulation is evolutionarily conserved; the best-known example is the pivotal role of cyclin-dependent kinases in all the eukaryotic lineages hitherto investigated. Interestingly, the molecular network associated with the activity of the CDK-cyclin complexes is also evolutionarily conserved, thus, defining a core cell cycle set of genes together with lineage-specific adaptations. In this paper, we describe the core cell cycle genes of Ostreococcus tauri, the smallest free-living eukaryotic cell having a minimal cellular organization with a nucleus, a single chloroplast, and only one mitochondrion. This unicellular marine green alga, which has diverged at the base of the green lineage, shows the minimal yet complete set of core cell cycle genes described to date. It has only one homolog of CDKA, CDKB, CDKD, cyclin A, cyclin B, cyclin D, cyclin H, Cks, Rb, E2F, DP, DEL, Cdc25, and Wee1. We have also added the APC and SCF E3 ligases to the core cell cycle gene set. We discuss the potential of genome-wide analysis in the identification of divergent orthologs of cell cycle genes in different lineages by mining the genomes of evolutionarily important and strategic organisms.     

Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis

This paper reports for the first time the transient expression of areporter gene, LacZ, in the unicellular green alga Haematococcuspluvialis. By employing the micro-particle bombardment method,motilecells in the exponential phase showed transient expression oflacZ. This was detected in bombarded motile cells undertherupture-disc pressures of 3103 KPa and 4137 KPa.Transient expression of LacZ gene could not be observed in non-motile cells ofthis alga under the same transformation condition. No LacZ background was foundin either the motile cells or the non-motile cells. The study suggests apromising potential of the SV40 promoter and the lacZreporter gene in genetic engineering of unicellular green algae.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Variation in cell composition and utilization of N and P for growth of Selenastrum capricornutum

Cell material from different phases of growth for the green alga Selenastrum capricornutum Printz was analysed for major elements and chlorophyll a content. The ranges in percentage of the dry weight were 50–80%, 4.4–8.8%, 0.24–3.0%, and 0.20–3.23% for C, N, P and chlorophyll a, respectively. The dry weight per cell varied 2.3 times between different growth phases. A N/P-quotient of about 10 (by weight) was found to be a critical level for Selenastrum when these elements were the most limiting nutrients for growth. 31 different organic N-compounds were tested as single N sources in growth experiments with Selenastrum. At least 19 of them could be utilized to varying extent for algal growth. Three organic P-compounds of six tested, were utilized for algal growth. Yield coefficients (mm³ cell volume/mg limiting nutrient element) for Selenastrum were determined with NO₃, -N (y_N= 134). NH₄-N (Y_N= 111) and PO₄-P (Y_P= 2580).     

Interactions between inorganic phosphate (Pi) assimilation,photosynthesis and respiration in the Pi-limited green alga Selenastrum minutum*

Inorganic phosphate (P_i)-limited chemostat cultures of the green alga Selenastrum minutum were employed to investigate interactions between P_i assimilation, respiration and photosynthetic processes. Changes in net and gross gas exchange rates indicated that O₂ evolution decreases during photosynthetic P_i assimilation. Room temperature and 77K Chi a fluorescence measurements revealed that this photosynthetic suppression is correlated with a transition from state 1 to state 2. Substantial photosynthetic P_i uptake rates occur in the presence of DCMU and KCN. Additionally, the cellular ratio of ATP:NADPH increases following P_i enrichment, suggesting that the ratio of cyclic to linear electron flow is enhanced in response to the high energy requirements of P_i uptake. Net starch degradation was observed during photosynthetic P_i assimilation and the cellular pool size of 3-phosphoglycerate increased; however, gross gas exchange parameters and cellular metabolite pool sizes indicated that mitochondrial respiration plays a smaller role during P_i assimilation in the light than it does in the dark. These observations were used to formulate a model depicting possible interactions between photosynthetic electron flow, photosynthetic and respiratory carbon metabolism and metabolite exchange between the chloroplast, cytosol and mitochondrion during photosynthetic P_i assimilation.