Biochemical Characterization of a Novel Thermostable Ulvan Lyase from Tamlana fucoidanivorans CW2-9.

Ulvan is a complex sulfated polysaccharide extracted from Ulva, and ulvan lyases can degrade ulvan through a ß-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The Km and Vmax of recombinant EPL15085 toward ulvan are 0.80 mg·mL-1 and 11.22 µmol·min -1 mg-1·mL-1, respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.

Anthracene induces oxidative stress and activation of antioxidant and detoxification enzymes in Ulva lactuca (Chlorophyta).

In order to analyze whether the marine macroalga Ulva lactuca can absorb and metabolize anthracene (ANT), the alga was cultivated with 5 µM ANT for 0-72 h, and the level of ANT was detected in the culture medium, and in the alga. The level of ANT rapidly decreased in the culture medium reaching a minimal level at 6 h, and rapidly increased in the alga reaching a maximal level at 12 h and then decreased to reach a minimal level at 48 h of culture. In addition, ANT induced an increase in hydrogen peroxide that remained until 72 h and a higher increase in superoxide anions that reach a maximal level at 24 h and remained unchanged until 72 h, indicating that ANT induced an oxidative stress condition. ANT induced an increase in lipoperoxides that reached a maximal level at 24 h and decreased at 48 h indicating that oxidative stress caused membrane damage. The activity of antioxidant enzymes SOD, CAT, AP, GR and GP increased in the alga treated with ANT whereas DHAR remained unchanged. The level of transcripts encoding these antioxidant enzymes increased and those encoding DHAR did not change. Inhibitors of monooxygenases, dioxygenases, polyphenol oxidases, glutathione-S-transferases and sulfotransferases induced an increase in the level of ANT in the alga cultivated for 24 h. These results strongly suggest that ANT is rapidly absorbed and metabolized in U. lactuca and the latter involves Phase I and II metabolizing enzymes.

Molecular cloning and transcriptional regulation of two γ-carbonic anhydrase genes in the green macroalga Ulva prolifera.

Ulva prolifera O.F. Müller (Ulvophyceae, Chlorophyta) is well known as a typical green-tide forming macroalga which has caused the world's largest macroalgal blooms in the Yellow Sea of China. In this study, two full-length γ-carbonic anhydrase (γ-CA) genes (UpγCA1 and UpγCA2) were cloned from U. prolifera. UpγCA1 has three conserved histidine residues, which act as an active site for binding a zinc metal ion. In UpγCA2, two of the three histidine residues were replaced by serine and arginine, respectively. The two γ-CA genes are clustered together with other γ-CAs in Chlorophyta with strong support value (100% bootstrap) in maximum likelihood (ML) phylogenetic tree. Quantitative real-time PCR (qRT-PCR) analysis showed that stressful environmental conditions markedly inhibited transcription levels of these two γ-CA genes. Low pH value (pH 7.5) significantly increased transcription level of UpγCA2 not UpγCA1 at 12 h, whereas high pH value (pH 8.5) significantly inhibited the transcription of these two γ-CA genes at 6 h. These findings enhanced our understanding on transcriptional regulation of γ-CA genes in response to environmental factors in U. prolifera.

Benzopyrene induces oxidative stress and increases expression and activities of antioxidant enzymes, and CYP450 and GST metabolizing enzymes in Ulva lactuca (Chlorophyta).

MAIN CONCLUSION: Benzopyrene is rapidly incorporated and metabolized, and induces oxidative stress and activation of antioxidant enzymes, and CYP450 and GST metabolizing enzymes in Ulva lactuca. To analyze absorption and metabolism of benzo[a]pyrene (BaP) in Ulva lactuca, the alga was cultivated with 5 µM of BaP for 72 h. In the culture medium, BaP level rapidly decreased reaching a minimal level at 12 h and, in the alga, BaP level increased until 6 h, remained stable until 24 h, and decreased until 72 h indicating that BaP is being metabolized in U. lactuca. In addition, BaP induced an initial increase in hydrogen peroxide decreasing until 24 h, superoxide anions level that remained high until 72 h, and lipoperoxides that initially increased and decreased until 72 h, showing that BaP induced oxidative stress. Activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (AP), glutathione reductase (GR) and glutathione peroxidase (GP) were increased, whereas dehydroascorbate reductase (DHAR) activity was unchanged. The level of transcripts encoding these antioxidant enzymes was increased, but transcripts encoding DHAR remained unchanged. Interestingly, the activity of glutathione-S-transferase (GST) was also increased, and inhibitors of cytochrome P450 (CYP450) and GST activities enhanced the level of BaP in algal tissue, suggesting that these enzymes participate in BaP metabolism.

Role of C4 carbon fixation in Ulva prolifera, the macroalga responsible for the world's largest green tides.

Most marine algae preferentially assimilate CO2 via the Calvin-Benson Cycle (C3) and catalyze HCO3- dehydration via carbonic anhydrase (CA) as a CO2-compensatory mechanism, but certain species utilize the Hatch-Slack Cycle (C4) to enhance photosynthesis. The occurrence and importance of the C4 pathway remains uncertain, however. Here, we demonstrate that carbon fixation in Ulva prolifera, a species responsible for massive green tides, involves a combination of C3 and C4 pathways, and a CA-supported HCO3- mechanism. Analysis of CA and key C3 and C4 enzymes, and subsequent analysis of δ13C photosynthetic products showed that the species assimilates CO2 predominately via the C3 pathway, uses HCO3- via the CA mechanism at low CO2 levels, and takes advantage of high irradiance using the C4 pathway. This active and multi-faceted carbon acquisition strategy is advantageous for the formation of massive blooms, as thick floating mats are subject to intense surface irradiance and CO2 limitation.

Physiological and metabolic responses to hypersalinity reveal interpopulation tolerance in the green macroalga Ulva compressa with different pollution histories.

There is scarce investigation addressing interpopulation tolerance responses to address the influence of a history of chronic stress exposure, as that occurring in polluted environments, in photoautotrophs. We evaluated ecophysiological (photosynthetic activity) and metabolic (oxidative stress and damage) responses of two populations of green macroalga Ulva compressa from polluted (Ventanas) and non-polluted (Cachagua) localions of central Chile, and exposed to controlled hypersalinity conditions of 32 (control), 42, 62 and 82 psu (practical salinity units) for 6 h, 48 h and 6 d. Both primary production (ETRmax) and photosynthetic efficiency (αETR) were generally higher in the population from Cachagua compared to Ventanas at all times and salinities. Moreover, at most experimental times and salinities the population from Ventanas had greater levels of H2O2 and lipid peroxidation that individuals from Cachagua. Total ascorbate was higher in the population of Cachagua than Ventanas at 42 and 82 psu after 6 and 48 h, respectively, while at 6 d concentrations were similar between both populations at all salinities. Total glutathione was greater in both populations after 6 h at all salinities, but at 48 h its concentrations were higher only in the population from Cachagua, a trend that was maintained at 6 d under 82 psu only. Reduced and oxidized ascorbate (ASC and DHA, respectively) and glutathione (GSH and GSSG, respectively) demonstrated similar patterns between U. compressa populations, with an increase oxidation with greater salinities but efficient recycling to maintain sufficient batch of ASC and GSH. When assessing the expression of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR), while the population of Ventanas displayed a general trend of upregulation with increasing salinities along the experiments, U. compressa from Cachagua revealed patterns of downregulation. Results demonstrated that although both populations were still viable after the applied hypersalinities during all experimental times, biological performance was usually more affected in the population from the Ventanas than Cachagua, likely due to a depressed baseline metabolism after a long history of exposition to environmental pollution.

Partial Characterization of Protease Inhibitors of Ulva ohnoi and Their Effect on Digestive Proteases of Marine Fish.

This piece of research evaluates the presence of protease inhibitors in the macroalga Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver-Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.

Ulvan lyase assisted structural characterization of ulvan from Ulva pertusa and its antiviral activity against vesicular stomatitis virus.

Marine green algae are valuable sources of diverse health-promoting bioactive components. Ulvan is suitable for biological applications due to its unique structure and numerous bioactivities. Here, the complex structure of ulvan from Ulva pertusa was analyzed using specific ulvan lyase degradation, MS, and NMR detection. Its structure mainly consists of →4)-ß-d-GlcA-(1 â†’ 4)-α-l-Rha3S-(1 â†’ and →4)-ß-d-Xyl-(1 â†’ 4)-α-l-Rha3S-(1 â†’ repeating units. Small amounts of →4)-α-l-IdoA-(1 â†’ 4)-α-l-Rha3S-(1 â†’ unit also exist. In addition, a minor number of branches, a single GlcA, and a long branch containing GlcA-Glc were linked to Rha3S. The antiviral activity of the ulvan and its degraded fragments were further investigated. Ulvan (1068.2 kDa) and ulvan-F1 (38.5 kDa) with relatively high molecular weight showed potency of inhibiting the infection and replication of vesicular stomatitis virus (VSV) at 100 µg/mL, the inhibition rate of VSV replication was 40.75% and 40.13%, respectively. These results indicated that ulvan has potential as a functional agent.

Isolation and Characterization of Copper- and Zinc- Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta).

In this work, transcripts encoding three metallothioneins from Ulva compressa (UcMTs) were amplified: The 5'and 3' UTRs by RACE-PCR, and the open reading frames (ORFs) by PCR. Transcripts encoding UcMT1.1 (Crassostrea-like), UcMT2 (Mytilus-like), and UcMT3 (Dreissena-like) showed a 5'UTR of 61, 71, and 65 nucleotides and a 3'UTR of 418, 235, and 193 nucleotides, respectively. UcMT1.1 ORF encodes a protein of 81 amino acids (MW 8.2 KDa) with 25 cysteines (29.4%), arranged as three motifs CC and nine motifs CXC; UcMT2 ORF encode a protein of 90 amino acids (9.05 kDa) with 27 cysteines (30%), arranged as three motifs CC, nine motifs CXC, and one motif CXXC; UcMT3 encode a protein of 139 amino acids (13.4 kDa) with 34 cysteines (24%), arranged as seven motifs CC and seven motifs CXC. UcMT1 and UcMT2 were more similar among each other, showing 60% similarity in amino acids; UcMT3 showed only 31% similarity with UcMT1 and UcMT2. In addition, UcMTs displayed structural similarity with MTs of marine invertebrates MTs and the terrestrial invertebrate Caenorhabtidis elegans MTs, but not with MTs from red or brown macroalgae. The ORFs fused with GST were expressed in bacteria allowing copper accumulation, mainly in MT1 and MT2, and zinc, in the case of the three MTs. Thus, the three MTs allowed copper and zinc accumulation in vivo. UcMTs may play a role in copper and zinc accumulation in U. compressa.

Structural and enzymatic analysis of the cytochrome b5 reductase domain of Ulva prolifera nitrate reductase.

Rapid accumulations of unattached green macroalgae, referred to as blooms, constitute ecological disasters and occur in many coastal regions. Ulva are a major cause of blooms, owing to their high nitrogen utilization capacity, which requires nitrate reductase (NR) activity; however, molecular characterization of Ulva NR remains lacking. Herein we determined the crystal structure and performed an enzymatic analysis of the cytochrome b5 reductase domain of Ulva prolifera NR (UpCbRNR). The structural analysis revealed an N-terminal FAD-binding domain primarily consisting of six antiparallel ß strands, a C-terminal NADH-binding domain forming a Rossmann fold, and a three ß-stranded linker region connecting these two domains. The FAD cofactor was located in the cleft between the two domains and interacted primarily with the FAD-binding domain. UpCbRNR shares similarities in overall structure and cofactor interactions with homologs, and its catalytic ability is comparable to that of higher plant CbRNRs. Structure and sequence comparisons of homologs revealed two regions of sequence length variation potentially useful for phylogenetic analysis: one in the FAD-binding domain, specific to U. prolifera, and another in the linker region that may be used to differentiate between plant, fungi, and animal homologs. Our data will facilitate molecular-level understanding of nitrate assimilation in Ulva.

Efficient renaturation of inclusion body proteins denatured by SDS.

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins.

Copper-induced overexpression of genes encoding antioxidant system enzymes and metallothioneins involve the activation of CaMs, CDPKs and MEK1/2 in the marine alga Ulva compressa.

Transcriptomic analyses were performed in the green macroalga Ulva compressa cultivated with 10µM copper for 24h. Nucleotide sequences encoding antioxidant enzymes, ascorbate peroxidase (ap), dehydroascorbate reductase (dhar) and glutathione reductase (gr), enzymes involved in ascorbate (ASC) synthesis l-galactose dehydrogenase (l-gdh) and l-galactono lactone dehydrogenase (l-gldh), in glutathione (GSH) synthesis, γ-glutamate-cysteine ligase (γ-gcl) and glutathione synthase (gs), and metal-chelating proteins metallothioneins (mt) were identified. Amino acid sequences encoded by transcripts identified in U. compressa corresponding to antioxidant system enzymes showed homology mainly to plant and green alga enzymes but those corresponding to MTs displayed homology to animal and plant MTs. Level of transcripts encoding the latter proteins were quantified in the alga cultivated with 10µM copper for 0-12 days. Transcripts encoding enzymes of the antioxidant system increased with maximal levels at day 7, 9 or 12, and for MTs at day 3, 7 or 12. In addition, the involvement of calmodulins (CaMs), calcium-dependent protein kinases (CDPKs), and the mitogen-activated protein kinase kinase (MEK1/2) in the increase of the level of the latter transcripts was analyzed using inhibitors. Transcript levels decreased with inhibitors of CaMs, CDPKs and MEK1/2. Thus, copper induces overexpression of genes encoding antioxidant enzymes, enzymes involved in ASC and GSH syntheses and MTs. The increase in transcript levels may involve the activation of CaMs, CDPKs and MEK1/2 in U. compressa.

A luciferase-based method for assay of 5'-adenylylsulfate reductase.

A luciferase-based method was developed for measurement of 5'-adenylylsulfate (APS) reductase (APR), an enzyme of the reductive sulfate assimilation pathway in prokaryotes and plants. APR catalyzes the two-electron reduction of APS and forms sulfite and adenosine 5'-monophospahate (AMP). The luciferase-based assay measures AMP production using an enzyme-coupled system that generates luminescence. The method is shown to provide an accurate measurement of APR kinetic properties and can be used for both endpoint and continuous assays. APR activity can be measured from pure enzyme preparations as well as from crude protein extracts of tissues. In addition, the assay is ideally suited to high-throughput sample analysis of APR activity in a microtiter dish format. The method adds new capability to the study of the biochemistry and physiology of APR.

Nitrate and phosphate regimes induced lipidomic and biochemical changes in the intertidal macroalga Ulva lactuca (Ulvophyceae, Chlorophyta).

This study was carried out in order to understand the lipid and biochemical alterations resulting from different nutritional regimes of nitrate and phosphate in Ulva lactuca. The algal thalli cultured in artificial seawater (ASW) showed higher levels of carbohydrates and non-polar lipids and increased phosphatase activities, accompanied by degradation of polar lipids, proteins and pigments. Further, higher levels of lipid hydroperoxides indicated reative oxygen species (ROS)-mediated non-enzymatic lipid peroxidation due to nutritional limitation-induced oxidative stress. Those thalli cultured in ASW supplemented with nitrate showed responses corresponding to nitrate addition, such as an increase in pigments, monogalactosyldiacylglycerols, polyunsaturated fatty acids and nitrate reductase. In addition, these thalli showed partial induction of phosphatases, low phospholipids, and high sulfolipid and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS) due to phosphate limitation. Similarly, algal thalli cultured in ASW supplemented with phosphate showed down-regulation of phosphatases, an increase in phospholipids due to availability of phosphate as well as a decrease in nitrate reductase, pigment, monogalactosyldiacylglycerols and polyunsaturated fatty acids due to nitrate limitation. On the other hand, algal thalli cultured in ASW supplemented with both nitrate and phosphate showed recovery of lost pigments and proteins, a high monogalactosyldiacylglycerol/digalactosyldiacylglycerol ratio, high unsaturation and high oxylipin levels (both C18 and C20). Further, the accumulation of indole-3-acetic acid in nutrient-limited thalli and of kinetin and kinetin riboside in nutrient-supplemented thalli indicated their antagonistic roles under nutrient stress. Thus, U. lactuca copes with nitrate and phosphate nutritional stress by altering the metabolic pathways involved in lipid biosynthesis including a shift in lipid classes, fatty acids, oxylipins and indole-3-acetic acid/kinetin cross-talk.

Metal accumulation and oxidative stress responses in Ulva spp. in the presence of nocturnal pulses of metals from sediment: a field transplantation experiment under eutrophic conditions.

In aquatic systems under eutrophic conditions, remobilization of metals from sediment to the overlying water may occur. Consequently, adaptive responses of local organisms could result from the accumulation of metals intermittently released from the sediment. In summer 2007, a field transplantation experiment was performed in the Óbidos lagoon (Portugal) with Ulva spp. comprising three short-term exposures (between 15:30-23:30; 23:30-07:30; 07:30-15:30) during a 24-h period. In each period, Ulva spp. was collected at a reference site located in the lower lagoon (LL) and transplanted to a eutrophic site located at the Barrosa branch (BB), characterized by moderate metal contamination. For comparison purposes, macroalgae samples were simultaneously exposed at LL under the same conditions. Both sites were surveyed in short-time scales (2-4 h) for the analysis of the variability of physical-chemical parameters in the water and metal levels in suspended particulate matter. The ratios to Al of particulate Mn, Fe, Cu and Pb increased during the period of lower water oxygenation at the eutrophic site, reaching 751 × 10⁻4, 0.67, 12 × 10⁻4, 9.9 × 10⁻4, respectively, confirming the release of metals from the sediment to water during the night. At the reference site, dissolved oxygen oscillated around 100%, Mn/Al ratios were considerably lower (81 × 10⁻4-301 × 10⁻4) compared to BB (234 × 10⁻4-790 × 10⁻4), and no increases of metal/Al ratios were found during the night. In general, algae uptake of Mn, Cu, Fe, Pb and Cd was significantly higher at the eutrophic site compared to the reference site. The results confirmed the potential of Ulva spp. as bioindicator of metal contamination and its capability to respond within short periods. An induction of SOD, an inhibition of CAT and the increase of LPO were recorded in Ulva spp. exposed at BB (between 23:30 and 7:30) probably as a response to the higher incorporation of Mn, Fe and Pb in combination with the lack of dissolved oxygen in the water. Current findings emphasize the importance of assessing, in eutrophic systems, the relationship between the variability of chemical conditions and its repercussions on autochthonous organisms over day-night cycles.

Activities of principal photosynthetic enzymes in green macroalga Ulva linza: functional implication of C4 pathway in CO2 assimilation.

The green-tide-forming macroalga Ulva linza was profiled by transcriptome sequencing to ascertain whether the alga carries both C3 and C4 photosynthesis genes. The key enzymes involved in C4 metabolism including pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK) were found. When measured under normal and different stress conditions, expression of rbcL was higher under normal conditions and lower under the adverse conditions, whereas that of PPDK was higher under some adverse conditions, namely desiccation, high salinity, and low salinity. Both ribulose-1, 5-biphosphate carboxylase (RuBPCase) and PPDK were found to play a role in carbon fixation, with significantly higher PPDK activity across the stress conditions. These results suggest that elevated PPDK activity alters carbon metabolism in U. linza leading to partial operation of the C4 carbon metabolism, a pathway that, under stress conditions, probably contributes to the hardy character of U. linza and thus to its wide distribution.

Accumulation of selenium in Ulva sp. and effects on morphology, ultrastructure and antioxidant enzymes and metabolites.

The impact of selenium (Se) on Ulva sp., a green macroalga naturally growing in the Venice Lagoon, was investigated. The alga was provided for 10 days with concentrations of selenate (Na(2)SeO(4)) ranging from 0 to 100 µM. Se accumulation in the algal biomass was linearly related to the selenate dose and this relationship was not affected by the high sulfate concentration measured in the seawater. The amount of Se measured in the alga was always relatively low and not hazardous to algal consumers. However, Se induced the formation of hydrogen peroxide (H(2)O(2)) in Ulva sp. and, as a result, the activity of antioxidant enzymes (superoxide dismutase, SOD, and catalase, CAT) and the amount of antioxidant metabolites (phenols, flavonoids and carotenoids) increased, even when selenate was supplied to the macroalga at low concentration (2.5 µM). This indicated that different components of the antioxidant defence system played a pivotal role in overcoming oxidative damage by Se in the macroalga, and explained the lack of morphological and ultrastructural alterations in Ulva sp. exposed to selenate.

The organophosphate insecticide Coumaphos induces oxidative stress and increases antioxidant and detoxification defences in the green macroalgae Ulva pertusa.

It is well established that many pesticides used in the farming and horticultural industries are harmful to not only the target species they were developed for, but also other organisms. Organophosphates were introduced as a replacement for the organochlorines and are generally considered non-toxic to plants and algae. This study investigated the impact of Coumaphos, a commonly used organophosphate, on the estuarine macrophyte Ulva pertusa. In a seven-day experiment U. pertusa cultures were exposed to four environmentally relevant concentrations of Coumaphos (0.01 mg/L, 0.05 mg/L, 0.1mg/L, 0.5 mg/L), well below the aqueous solubility maximum of the insecticide. The impact of Coumaphos was determined at a cellular level by assessing oxidative damage in the form of protein carbonyl and lipid hydroperoxide levels. Furthermore, non-enzymatic antioxidant levels and changes in the levels of enzymatic antioxidants and the enzyme GST were measured. Concentrations of Coumaphos above 0.01 mg/L caused rapid increases in the levels of protein carbonyls and lipid hydroperoxides peaking after 2-3 days of exposure, followed by a rapid decline in both markers of oxidative stress. Glutathione levels and the activities SOD, CAT, GR, APOX and GST all increased in response to the higher concentrations of Coumaphos tested and remained elevated for the duration of the experiment. These results demonstrate that environmentally relevant levels of the insecticide Coumaphos can cause oxidative damage and increase the antioxidant scavenging capacity, and GST activity in U. pertusa. This could potentially alter resource allocation within this alga, impacting algal growth and development, with possible indirect ecological consequences.

Nitric oxide up-regulates the expression of methionine sulfoxide reductase genes in the intertidal macroalga Ulva fasciata for high light acclimation.

Nitric oxide (NO) has emerged as a fundamental signal molecule involved in the responses of plant to stress. A role for NO in the regulation of methionine sulfoxide reductase (MSR) mRNA expression and high light acclimation was studied in a green macroalga Ulva fasciata Delile. Transfer from darkness to high light (≥1,200 µmol photons m(-2) s(-1)) inhibited photosynthesis and growth but increased NO production and UfMSRA and UfMSRB transcripts. Treatment with an NO scavenger, 2-(4-carboxy- phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), at 1,200 µmol photons m(-2) s(-1) caused a further growth inhibition accompanied by an inhibition of the increase of UfMSRA and UfMSRB transcripts by high light, while treatment with an NO generator, sodium nitroprusside (SNP), alleviated the growth inhibition and enhanced UfMSRA and UfMSRB expression. Exposure to moderate light (300 µmol photons m(-2) s(-1)) conditions also increased UfMSRA and UfMSRB transcripts, which were not affected by cPTIO treatment but were enhanced by SNP treatment. So, NO does not mediate the up-regulation of UfMSR genes by transfer to moderate light possibly as a precautionary mechanism in the sense of increasing light intensities in the daytime. In conclusion, NO production can be induced in U. fasciata upon exposure to high light for up-regulation of UfMSRA and UfMSRB expression but the level of NO production is not sufficient for acquisition of full tolerance to high light stress. Enhanced NO production by an exogenously applied NO generator can effectively trigger the high light acclimation process, including UfMSRA and UfMSRB expression.

Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca: oxidative stress and DNA damage.

The green credentials of ionic liquids (ILs) are being increasingly questioned due to the growing evidence of their toxicity to aquatic ecosystems, although the mechanisms of toxicity are unknown. This study provides insights into the mechanism of toxicity and biological effects of 1-alkyl-3-methylimidazolium bromide [C(n)mim]Br (n = 4 to 16) on the marine macroalga Ulva lactuca. The cell viability of this alga during IL exposure was found to be negatively correlated to the chain length of the alkyl group. The IL ([C(12)mim]Br) exposure triggers the generation of reactive oxygen species (ROS viz. O(2)(•-), H(2)O(2), and OH(•)), damage of the membrane and DNA, and inhibition of antioxidant systems in the alga. The enhanced production of ROS and lipid peroxidation in the alga subjected to LC(50) concentration for 4 days was largely attributed to lipoxygenase (LOX) activity coupled with the induction of two LOX isoforms (~80 kDa and ~55 kDa). Pretreatment of the algal thallus with enzyme inhibitors such as diphenylene iodonium, sodium azide, cantharidin, and oxadiazoloquinoxalin-1-one, prior to [C(12)mim]Br exposure showed the regulation of ROS by the activation of membrane bound NADPH-oxidase and cytochrome oxidase. The IL exposure resulted in the accumulation of n-3 and n-6 fatty acids at 0.5 LC(50) concentration indicating the induction of desaturase enzymes. Furthermore, antioxidant enzyme activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced by 1.3-2.0-fold, while glutathione peroxidase (GSH-Px) diminished, together with a higher regeneration rate of reduced ascorbate and glutathione. The isoforms of antioxidant enzymes, namely, Mn-SOD (~85 kDa), APX (~125 and 45 kDa), and GR (~135 kDa) regulated differentially to IL exposure. The comet assay performed for the first time for seaweeds revealed the significant induction of DNA damage (>50-70% increase in % tail DNA over control) in alga exposed to ≥ LC(50) concentration.