Oligo-Carrageenan Kappa Increases Expression of Genes Encoding Proteins Involved in Photosynthesis, C, N, and S Assimilation, and Growth in Arabidopsis thaliana.

To analyze the effect of oligo-carrageenan (OC) kappa in the stimulation of growth in Arabidopsis thaliana, plants were sprayed on leaves with an aqueous solution of OC kappa at 1 mg mL-1, 5 times every 2 days and cultivated for 5 or 15 additional days. Plants treated with OC kappa showed an increase in rosette diameter, fresh and dry weight, and primary root length. Plants treated with OC kappa once and cultivated for 0 to 24 h after treatment were subjected to transcriptomic analyses to identify differentially expressed genes, mainly at 12 h after treatment. Transcripts encoding proteins involved in growth and development and photosynthesis were upregulated as well as enzymes involved in primary metabolism. In addition, plants treated with OC kappa once and cultivated for 0 to 96 h showed increased levels of transcripts encoding enzymes involved in C, N, and S assimilation at 6 and 12 h after treatment that remain increased until 96 h. Therefore, OC kappa increased the expression of genes encoding proteins involved in photosynthesis, C, N, and S assimilation, and growth in A. thaliana.

Transcriptomic analyses reveal increased expression of dioxygenases, monooxygenases, and other metabolizing enzymes involved in anthracene degradation in the marine alga Ulva lactuca.

To analyze the mechanisms involved in anthracene (ANT) degradation in the marine alga Ulva lactuca, total RNA was obtained from the alga cultivated without ANT and with 5 µM of ANT for 24 h, and transcriptomic analyses were performed. A de novo transcriptome was assembled, transcripts differentially expressed were selected, and those overexpressed were identified. Overexpressed transcripts potentially involved in ANT degradation were: one aromatic ring dioxygenase, three 2-oxoglutarate Fe (II) dioxygenases (2-OGDOs), and three dienelactone hydrolases that may account for anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid production (pathway 1). In addition, two flavin adenine dinucleotide (FAD)-dependent monooxygenases, four cytP450 monooxygenases, two epoxide hydrolase, one hydroxyphenylpyruvic acid dioxygenase (HPPDO), and two homogentisic acid dioxygenases (HGDOs) were identified that may also participate in ANT degradation (pathway 2). Moreover, an alkane monooxygenase (alkB), two alcohol dehydrogenases, and three aldehyde dehydrogenases were identified, which may participate in linear hydrocarbon degradation (pathway 3). Furthermore, the level of transcripts encoding some of mentioned enzymes were quantified by qRT-PCR are in the alga cultivated with 5 µM of ANT for 0-48 h, and those more increased were 2-OGDO, HGDO, and alkB monooxygenase. Thus, at least three pathways for ANT and linear hydrocarbons degradation may be existed in U. lactuca. In addition, ANT metabolites were analyzed by gas chromatography and mass spectrometry (GC-MS), allowing the identification of anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid, thus validating the pathway 1.

The Genome of the Marine Alga Ulva compressa (Chlorophyta) Reveals Protein-Coding Genes with Similarity to Plants and Green Microalgae, but Also to Animal, Bacterial, and Fungal Genes.

The genome of the marine alga Ulva compressa was assembled using long and short reads. The genome assembly was 80.8 Mb in size and encoded 19,207 protein-coding genes. Several genes encoding antioxidant enzymes and a few genes encoding enzymes that synthesize ascorbate and glutathione were identified, showing similarity to plant and bacterial enzymes. Additionally, several genes encoding signal transduction protein kinases, such as MAPKs, CDPKS, CBLPKs, and CaMKs, were also detected, showing similarity to plants, green microalgae, and bacterial proteins. Regulatory transcription factors, such as ethylene- and ABA-responsive factors, MYB, WRKY, and HSTF, were also present and showed similarity to plant and green microalgae transcription factors. Genes encoding enzymes that synthesize ACC and ABA-aldehyde were also identified, but oxidases that synthesize ethylene and ABA, as well as enzymes that synthesize other plant hormones, were absent. Interestingly, genes involved in plant cell wall synthesis and proteins related to animal extracellular matrix were also detected. Genes encoding cyclins and CDKs were also found, and CDKs showed similarity to animal and fungal CDKs. Few genes encoding voltage-dependent calcium channels and ionotropic glutamate receptors were identified as showing similarity to animal channels. Genes encoding Transient Receptor Potential (TRP) channels were not identified, even though TRPs have been experimentally detected, indicating that the genome is not yet complete. Thus, protein-coding genes present in the genome of U. compressa showed similarity to plant and green microalgae, but also to animal, bacterial, and fungal genes.

Ulva compressa from Copper-Polluted Sites Exhibits Intracellular Copper Accumulation, Increased Expression of Metallothioneins and Copper-Containing Nanoparticles in Chloroplasts.

In order to analyze the mechanisms involved in copper accumulation in Ulva compressa, algae were collected at control sites of central and northern Chile, and at two copper-polluted sites of northern Chile. The level of intracellular copper, reduced glutathione (GSH), phytochelatins (PCs), PC2 and PC4, and transcripts encoding metallothioneins (MTs) of U. compressa, UcMT1, UcMT2 and UcMT3, were determined. Algae of control sites contained around 20 µg of copper g-1 of dry tissue (DT) whereas algae of copper-polluted sites contained 260 and 272 µg of copper g-1 of DT. Algae of control sites and copper-polluted sites did not show detectable amounts of GSH, the level of PC2 did not change among sites whereas PC4 was increased in one of the copper-polluted sites. The level of transcripts of UcMT1 and UcMT2 were increased in algae of copper-polluted sites, but the level of UcMT3 did not change. Algae of a control site and a copper-polluted site were visualized by transmission electron microscopy (TEM) and the existence of copper in electrodense particles was analyzed using energy dispersive x-ray spectroscopy (EDXS). Algae of copper-polluted sites showed electrodense nanoparticles containing copper in the chloroplasts, whereas algae of control sites did not. Algae of a control site, Cachagua, were cultivated without copper (control) and with 10 µM copper for 5 days and they were analyzed by TEM-EDXS. Algae cultivated with copper showed copper-containing nanoparticles in the chloroplast whereas control algae did not. Thus, U. compressa from copper-polluted sites exhibits intracellular copper accumulation, an increase in the level of PC4 and expression of UcMTs, and the accumulation of copper-containing particles in chloroplasts.

Cadmium Accumulation Involves Synthesis of Glutathione and Phytochelatins, and Activation of CDPK, CaMK, CBLPK, and MAPK Signaling Pathways in Ulva compressa.

In order to analyze the effect of cadmium in Ulva compressa (Chlorophyta), the alga was cultivated with 10, 25, and 50 µM of cadmium for 7 days, and the level of intracellular cadmium was determined. Intracellular cadmium showed an increase on day 1, no change until day 5, and an increase on day 7. Then, the alga was cultivated with 10 µM for 7 days, and the level of hydrogen peroxide, superoxide anions, and lipoperoxides; activities of antioxidant enzymes ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR), and glutathione reductase (GR); the level of glutathione (GSH) and ascorbate (ASC); and the level of phytochelatins (PCs) and transcripts encoding metallothioneins (UcMTs) levels were determined. The level of hydrogen peroxide increased at 2 and 12 h, superoxide anions on day 1, and lipoperoxides on days 3 to 5. The activities of AP and GR were increased, but not the DHAR activity. The level of GSH increased on day 1, decreased on day 3, and increased again on day 5, whereas ASC slightly increased on days 3 and 7, and activities of enzymes involved in GSH and ASC synthesis were increased on days 3 to 7. The level of PC2 and PC4 decreased on day 3 but increased again on day 5. The level of transcripts encoding UcMT1 and UcMT2 increased on days 3 to 5, mainly that of UcMT2. Thus, cadmium accumulation induced an oxidative stress condition that was mitigated by the activation of antioxidant enzymes and synthesis of GSH and ASC. Then, the alga cultivated with inhibitors of calcium-dependent protein kinases (CDPKs), calmodulin-dependent protein kinases (CaMKs), calcineurin B-like protein kinases (CBLPKs), and MAPKs and 10 µM of cadmium for 5 days showed a decrease in intracellular cadmium and in the level of GSH and PCs, with the four inhibitors, and in the level of transcripts encoding UcMTs, with two inhibitors. Thus, CDPKs, CaMK, CBLPKS, and MAPKs are involved in cadmium accumulation and GSH and PC synthesis, and GSH and PCs and/or UcMTs may participate in cadmium accumulation.

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.

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.

Antarctic intertidal macroalgae under predicted increased temperatures mediated by global climate change: Would they cope?

The Antarctic Peninsula is one of the regions to be most affected by increase in sea surface temperatures (SSTs) mediated by Global Climate Change; indeed, most negative predictions imply an up to 6 °C increment by the end of the XXI century. Temperature is one of the most important factors mediating diversity and distribution of macroalgae, although there is still no consensus as to the likely effects of higher SSTs, especially for polar seaweeds. Some available information suggests that potential strategies to withstand future increases in SSTs will be founded upon the glutathione-ascorbate cycle and the induction of chaperone-functioning heat shock proteins (HSPs); however, their eventual role, even for general stress responses, is unclear. The intertidal green, brown and red macroalgae species Monostroma hariotii, Adenocystis utricularis and Pyropia endiviifolia, respectively, from King George Island, Antarctic Peninsula, were exposed to 2 °C (control) and 8 °C (climate change scenario) for up to 5 days (d). Photosynthetic activity (αETR and ETRmax, and EkETR), photoinhibition (Fv/Fm) and photoprotection processes (αNPQ, NPQmax, and EkNPQ) provided no evidence of negative ecophysiological effects. There were moderate increases in H2O2 production and levels of lipid peroxidation with temperature, results supported by stable levels of total glutathione and ascorbate pools, with mostly higher levels of reduced ascorbate and glutathione than oxidized forms in all species. Transcripts of P. endiviifolia indicated a general upregulation of all antioxidant enzymes and HSPs genes studied under warmer temperature, although with different levels of activation with time. This pioneering investigation exploring different levels of biological organization, suggested that Antarctic intertidal macroalgae may be able to withstand future rise in SSTs, probably slightly altering their latitudinal distribution and/or range of thermal tolerance, by exhibiting robust glutathione-ascorbate production and recycling, as well as the induction of associated antioxidant enzymatic machinery and the syntheses of HSPs.

Copper-Induced Activation of MAPKs, CDPKs and CaMKs Triggers Activation of Hexokinase and Inhibition of Pyruvate Kinase Leading to Increased Synthesis of ASC, GSH and NADPH in Ulva compressa.

In order to analyze whether copper induces activation of CaMK, CDPK and/or MAPK signaling pathways leading to carbon flux reprogramming and to the synthesis of ascorbate (ASC), glutathione (GSH) and NADPH in order to buffer copper-induced oxidative stress, U. compressa was initially cultivated with 10 µM copper for 0 to 10 days. The activities of hexokinase (HK), pyruvate kinase (PK), L-galactone 1,4 lactone dehydrogenase (L-GLDH) and glucose 6-P dehydrogenase (G6PDH) were analyzed. HK activity was increased whereas PK was inhibited, and L-GLDH and G6PDH activities were increased indicating a copper-induced modulation of glycolysis leading to carbon flux reprogramming. Then, the alga was cultivated with an inhibitor of CaMs and CaMKs, CDPKs and MAPKs, and with 10 µM of copper for 5 days and the activities of HK, PK, L-GLDH, G6PDH and glutathione synthase (GS), the levels of ASC/DHA, GSG/GSSG and NADPH/NADP, the levels of superoxide anions (SA) and hydrogen peroxide (HP) and the integrity of plasma membrane were determined. The activation of HK was dependent on MAPKs, the inhibition of PK on CDPKs/MAPKs, the activation of L-GLDH on MAPKs, the activation GS on CDPKs/MAPKs, and the activation of G6PDH on MAPKs. Increases in the level of ASC/DHA were dependent on activation of CaMKs/CDPKs/MAPKs, those of GSG/GSSG on MAPKs and those NADPH/NADP on CaMKs/CDPKs/MAPKs. The accumulation of superoxide anions and hydrogen peroxide and the integrity of plasma membrane were dependent on CaMKs/CDPKs/MAPKs. Thus, copper induced the activation of MAPKs, CDPKs and CaMKs leading to the modulation of glycolysis and carbon flux reprogramming which trigger an increase in ASC, GSH and NADPH syntheses and the activation of antioxidant enzymes in order to buffer copper-induced oxidative stress in U. compressa.

Mechanisms of Copper Tolerance, Accumulation, and Detoxification in the Marine Macroalga Ulva compressa (Chlorophyta): 20 Years of Research.

Copper induces an oxidative stress condition in the marine alga Ulva compressa that is due to the production of superoxide anions and hydrogen peroxide, mainly in organelles. The increase in hydrogen peroxide is accompanied by increases in intracellular calcium and nitric oxide, and there is a crosstalk among these signals. The increase in intracellular calcium activates signaling pathways involving Calmodulin-dependent Protein Kinases (CaMKs) and Calcium-Dependent Protein Kinases (CDPKs), leading to activation of gene expression of antioxidant enzymes and enzymes involved in ascorbate (ASC) and glutathione (GSH) synthesis. It was recently shown that copper also activates Mitogen-Activated Protein Kinases (MAPKs) that participate in the increase in the expression of antioxidant enzymes. The increase in gene expression leads to enhanced activities of antioxidant enzymes and to enhanced levels of ASC and GSH. In addition, copper induces an increase in photosynthesis leading to an increase in the leve of Nicotinamide Adenine Dinucleotide Phosphate (NADPH). Copper also induces an increase in activities of enzymes involved in C, N, and S assimilation, allowing the replacement of proteins damaged by oxidative stress. The accumulation of copper in acute exposure involved increases in GSH, phytochelatins (PCs), and metallothioneins (MTs) whereas the accumulation of copper in chronic exposure involved only MTs. Acute and chronic copper exposure induced the accumulation of copper-containing particles in chloroplasts. On the other hand, copper is extruded from the alga with an equimolar amount of GSH. Thus, the increases in activities of antioxidant enzymes, in ASC, GSH, and NADPH levels, and in C, N, and S assimilation, the accumulation of copper-containing particles in chloroplasts, and the extrusion of copper ions from the alga constitute essential mechanisms that participate in the buffering of copper-induced oxidative stress in U. compressa.

Copper-induced concomitant increases in photosynthesis, respiration, and C, N and S assimilation revealed by transcriptomic analyses in Ulva compressa (Chlorophyta).

BACKGROUND: The marine alga Ulva compressa is the dominant species in copper-polluted coastal areas in northern Chile. It has been shown that the alga tolerates micromolar concentrations of copper and accumulates copper at the intracellular level. Transcriptomic analyses were performed using total RNA of the alga cultivated with 10 µ M copper for 0, 1, 3 and 5 days using RNA-seq in order to identify processes involved in copper tolerance. RESULTS: The levels of transcripts encoding proteins belonging to Light Harvesting Complex II (LHCII), photosystem II (PSII), cytochrome b6f, PSI, LHCI, ATP synthase and proteins involved in repair of PSII and protection of PSI were increased in the alga cultivated with copper. In addition, the level of transcripts encoding proteins of mitochondrial electron transport chain, ATP synthase, and enzymes involved in C, N and S assimilation were also enhanced. The higher percentages of increase in the level of transcripts were mainly observed at days 3 and 5. In contrast, transcripts involved protein synthesis and degradation, signal transduction, and replication and DNA repair, were decreased. In addition, net photosynthesis and respiration increased in the alga cultivated with copper, mainly at days 1 to 3. Furthermore, the activities of enzymes involved in C, N and S assimilation, rubisco, glutamine synthase and cysteine synthase, respectively, were also increased, mainly at days 1 and 3. CONCLUSIONS: The marine alga U. compressa tolerates copper excess through a concomitant increase in expression of proteins involved in photosynthesis, respiration, and C, N and S assimilation, which represents an exceptional mechanism of copper tolerance.

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.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms.

Following the physiological complementary/parallel Celis-Plá et al., by inhibiting extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and cytokinin specific binding protein (p38), we assessed the role of the mitogen-activated protein kinases (MAPK) pathway in detoxification responses mediated by chronic copper (10 µM) in U. compressa. Parameters were taken at 6, 24, and 48 h, and 6 days (d). H2O2 and lipid peroxidation under copper and inhibition of ERK, JNK, or p38 alone increased but recovered by the sixth day. By blocking two or more MAPKs under copper, H2O2 and lipid peroxidation decayed even below controls. Inhibition of more than one MAPK (at 6 d) caused a decrease in total glutathione (reduced glutathione (GSH) + oxidised glutathione (GSSG)) and ascorbate (reduced ascorbate (ASC) + dehydroascorbate (DHA)), although in the latter it did not occur when the whole MAPK was blocked. Catalase (CAT), superoxide dismutase (SOD), thioredoxin (TRX) ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione synthase (GS), were downregulated when blocking more than one MAPK pathway. When one MAPK pathway was blocked under copper, a recovery and even enhancement of detoxification mechanisms was observed, likely due to crosstalk within the MAPKs and/or other signalling processes. In contrast, when more than one MAPK pathway were blocked under copper, impairment of detoxification defences occurred, demonstrating that MAPKs were key signalling mechanisms for detoxification in macroalgae.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion/Extrusion Mechanisms and Photosynthesis.

There is currently no information regarding the role that whole mitogen activated protein kinase (MAPK) pathways play in counteracting environmental stress in photosynthetic organisms. To address this gap, we exposed Ulva compressa to chronic levels of copper (10 µM) specific inhibitors of Extracellular Signal Regulated Kinases (ERK), c-Jun N-terminal Kinases (JNK), and Cytokinin Specific Binding Protein (p38) MAPKs alone or in combination. Intracellular copper accumulation and photosynthetic activity (in vivo chlorophyll a fluorescence) were measured after 6 h, 24 h, 48 h, and 6 days of exposure. By day 6, when one (except JNK) or more of the MAPK pathways were inhibited under copper stress, there was a decrease in copper accumulation compared with algae exposed to copper alone. When at least two MAPKs were blocked, there was a decrease in photosynthetic activity expressed in lower productivity (ETRmax), efficiency (αETR), and saturation of irradiance (EkETR), accompanied by higher non-photochemical quenching (NPQmax), compared to both the control and copper-only treatments. In terms of accumulation, once the MAPK pathways were partially or completely blocked under copper, there was crosstalk between these and other signaling mechanisms to enhance metal extrusion/exclusion from cells. Crosstalk occurred among MAPK pathways to maintain photosynthesis homeostasis, demonstrating the importance of the signaling pathways for physiological performance. This study is complemented by a parallel/complementary article Rodríguez-Rojas et al. on the role of MAPKs in copper-detoxification.

Oligo-carrageenan kappa increases glucose, trehalose and TOR-P and subsequently stimulates the expression of genes involved in photosynthesis, and basal and secondary metabolisms in Eucalyptus globulus.

BACKGROUND: It has been previously shown that oligo-carrageenan (OC) kappa increases growth, photosynthesis and activities of enzymes involved in basal and secondary metabolisms in Eucalyptus globulus. However, it is not known whether OC kappa may induce the activation of TOR pathway and the increase in expression of genes encoding proteins involved in photosynthesis and enzymes of basal and secondary metabolisms. RESULTS: E. globulus trees were sprayed on leaves with water (control) or with OC kappa 1 mg mL- 1, once a week, four times in total, and cultivated for 17 additional weeks (21 weeks in total). Treated trees showed a higher level of net photosynthesis than controls, beginning at week 3, a higher height, beginning at week 9, and those differences remained until week 21. In addition, treated trees showed an increase in the level of glucose beginning at week 1, trehalose at weeks 1-3, and in TOR-P level at week 1-2. On the other hand, transcripts encoding proteins involved in photosynthesis, and enzymes involved in glucose accumulation, C, N and S assimilation, and synthesis of secondary metabolites began at weeks 3-4 and with additional peaks at weeks 5-6, 8-11,13-14 and 17-19. Thus, OC kappa induced initial increases in glucose, trehalose and TOR-P levels that were followed by oscillatory increases in the level of transcripts coding for proteins involved in photosynthesis, and in basal and secondary metabolisms suggesting that initial increases in glucose, trehalose and TOR-P may trigger activation of gene expression. CONCLUSIONS: The stimulation of growth induced by OC kappa in E. globulus trees is due, at least in part, to activation of TOR pathway and the increase in expression of genes encoding proteins involved in photosynthesis and enzymes of basal metabolism.

Copper excess detoxification is mediated by a coordinated and complementary induction of glutathione, phytochelatins and metallothioneins in the green seaweed Ulva compressa.

In order to analyze the involvement of intracellular thiol-chelators in the accumulation and detoxification of copper, the marine alga Ulva compressa was cultivated with increasing concentrations of copper such as 2.5, 5, 7.5 and 10 µM for up to 12 d, and the amount of intracellular copper, glutathione (GSH), phytochelatins (PCs) and transcripts encoding three metallothioneins (MTs) were determined. Over this exposure period and concentration range there was a linear correlation between intracellular copper and the copper concentration in the culture medium. Increases in GSH concentrations occurred mainly between days 1 and 3 and at lower concentrations of copper (2.5 and 5 µM). The level of PCs, and particularly PC2, increased from day 1 of exposure mainly at higher concentrations of copper (7.5 and 10 µM). The levels of transcripts encoding MT7 increased at day 3, whereas those of MT3 and MT6 increased between days 9-12, mainly at higher concentrations of copper. Thus in U. compressa, the initial responses to increasing intracellular copper concentrations are increases in GSH and PCs that are followed by higher levels of MTs expression, suggesting that thiol-containing peptides and proteins may participate in copper accumulation and detoxification responding in a coordinated and complementary manner. In addition, the alga was cultivated with 10 µM copper for 5 d and transferred to synthetic seawater with no copper and cultivated for 3 d. The release of copper from cells to culture medium was observed and accompanied by a similar nanomolar amount of GSH; no PCs or small proteins were detected. These results could suggest that a component of the detoxification mechanism also involves the release of copper and GSH to the extracellular medium.

Copper-induced increased expression of genes involved in photosynthesis, carotenoid synthesis and C assimilation in the marine alga Ulva compressa.

BACKGROUND: The marine alga Ulva compressa is the dominant species in coastal areas receiving effluents from copper mines. The alga can accumulate high amounts of copper and possesses a strong antioxidant system. Here, we performed short-term transcriptomic analyses using total RNA of the alga cultivated with 10 µM of copper for 0, 3, 6, 12 and 24 h by RNA-seq. RESULTS: De novo transcriptomes were assembled using the Trinity software, putative proteins were annotated and classified using Blast2GO. Differentially expressed transcripts were identified using edgeR. Transcript levels were compared by paired times 0 vs 3, 0 vs 6, 0 vs 12 and 0 vs 24 h at an FDR < 0.01 and Log2 Fold Change > 2. Up-regulated transcripts encode proteins belonging to photosystem II (PSII), Light Harvesting II Complex (LHCII), PSI and LHCI, proteins involved in assembly and repair of PSII, and assembly and protection of PSI. In addition, transcripts encoding enzymes leading to ß-carotene synthesis and enzymes belonging to the Calvin-Benson cycle were also increased. We further analyzed photosynthesis and carotenoid levels in the alga cultivated with 10 µM of copper for 0 to 24 h. Photosynthesis was increased from 3 to 24 h as well as the level of total carotenoids. The increase in transcripts encoding enzymes of the Calvin-Benson cycle suggests that C assimilation may also be increased. CONCLUSIONS: Thus, U. compressa displays a short-term response to copper stress enhancing the expression of genes encoding proteins involved in photosynthesis, enzymes involved carotenoids synthesis, as well as those belonging to the Calvin-Benson cycle, which may result in an increase in C assimilation.

Copper-induced activation of TRPs and VDCCs triggers a calcium signature response regulating gene expression in Ectocarpus siliculosus.

In certain multicellular photoautotrophs, such as plants and green macroalgae, it has been demonstrated that calcium signaling importantly mediates tolerance to copper excess. However, there is no information in brown macroalgae, which are phylogenetically distant from green algae and plants. We have previously shown that chronic copper levels (2.5 µM) activate transient receptor potential (TRP) channels in the model brown macroalga Ectocarpus siliculosus, allowing extracellular calcium entry at 13, 29, 39 and 51 min. Here, we showed that intracellular calcium increases also occurred at 3 and 5 h of exposure; these increases were inhibited by antagonists of voltage-dependent calcium channels (VDCCs); a chelating agent of extracellular calcium; an antagonist of endoplasmic reticulum (ER) ATPase; and antagonists of cADPR-, NAADP- and IP3-dependent calcium channels. Thus, copper activates VDCCs allowing extracellular calcium entry and intracellular calcium release from the ER via cADPR-, IP3- and NAADP-dependent channels. Furthermore, the level of transcripts encoding a phytochelatin synthase (PS) and a metallothionein (MT) were analyzed in the alga exposed to 2.5 µM copper from 3 to 24 h. The level of ps and mt transcripts increased until 24 h and these increases were inhibited by antagonists of calmodulins (CaMs), calcineurin B-like proteins (CBLs) and calcium-dependent protein kinases (CDPKs). Finally, activation of VDCC was inhibited by a mixture of TRP antagonists and by inhibitors of protein kinases. Thus, copper-mediated activation of TRPs triggers VDCCs via protein kinases, allowing extracellular calcium entry and intracellular calcium release from ER that, in turn, activate CaMs, CBLs and CDPKs increasing expression of PS and MT encoding genes in E. siliculosus.

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of antioxidant enzymes in Ectocarpus siliculosus.

The existence of functional Transient Receptor Potential (TRP) channels was analyzed in Ectocarpus siliculosus using agonists of human TRPs and specific antagonists of TRPA1, TRPC5, TRPM8 and TRPV; intracellular calcium was detected for 60 min. Increases in intracellular calcium were observed at 13, 29, 39 and 50-52 min, which appeared to be mediated by the activation of TRPM8/V1 at 13 min, TRPV1 at 29 min, TRPA1/V1 at 39 min and TRPA1/C5 at 50-52 min. In addition, intracellular calcium increases appear to be due to extracellular calcium entry, not requiring protein kinase activation. On the other hand, 2.5 µM copper exposure induced increased intracellular calcium at 13, 29, 39 and 51 min, likely due to the activation of a TRPA1/V1 at 13 min, TRPA1/C5/M8 at 29 min, TRPC5/M8 at 39 min, and a TRPC5/V1 at 51 min. The increases in intracellular calcium induced by copper were due to extracellular calcium entry and required protein kinase activation. Furthermore, from 3 to 24 h, copper exposure induced an increase in the level of transcripts encoding antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and peroxiredoxin. The described upregulation decreased with inhibitors of CaMK, PKA, PKC, PKG and CBLPK, as well as with a mixture of TRP inhibitors. Thus, copper induces the activation of TRP channels allowing extracellular calcium entry as well as the activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of genes encoding antioxidant enzymes in E. siliculosus.

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.