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.

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.

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-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 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.

Evaluating the late career nurse initiative: a cross-sectional survey of senior nurses in Ontario.

AIM: This study evaluated the impact of the late career nurse initiative on nurse perceptions of their work environment, workplace burnout, job satisfaction, organisational commitment and intention to remain. BACKGROUND: The Ontario Ministry of Health and Long-Term Care introduced the late career nurse initiative with the goal of improving the retention of front-line nurses aged 55 and over by implementing a 0.20 full-time equivalent reduction of physically or psychologically demanding duties, enabling nurses to engage in special projects for the improvement of their organisations and patient care. METHODS: A sample of 902 nurses aged 55 and over from acute and long-term care facilities were surveyed using valid and reliable questionnaires. RESULTS: Nurses who had participated in the initiative did not differ significantly from those who had not in terms of workplace burnout, job satisfaction, length of service or intention to remain within their current organisation. The late career nurse initiative participants reported significantly higher perceptions of managers' ability, leadership and support and their level of participation in hospital affairs. CONCLUSION: The late career nurse initiative was associated with perceived differences in nurses' work environment but not outcomes. IMPLICATIONS FOR NURSING MANAGEMENT: Leaders need to pay attention to how late career nurses are selected and matched to organisational projects.

Glutaredoxin GRXS13 plays a key role in protection against photooxidative stress in Arabidopsis.

Glutaredoxins (GRXs) belong to the antioxidant and signalling network involved in the cellular response to oxidative stress in bacterial and eukaryotic cells. In spite of the high number of GRX genes in plant genomes, the biological functions and physiological roles of most of them remain unknown. Here the functional characterization of the Arabidopsis GRXS13 gene (At1g03850), that codes for two CC-type GRX isoforms, is reported. The transcript variant coding for the GRXS13.2 isoform is predominantly expressed under basal conditions and is the isoform that is induced by photooxidative stress. Transgenic lines where the GRXS13 gene has been knocked down show increased basal levels of superoxide radicals and reduced plant growth. These lines also display reduced tolerance to methyl viologen (MeV) and high light (HL) treatments, both conditions of photooxidative stress characterized by increased production of superoxide ions. Consistently, lines overexpressing the GRXS13.2 variant show reduced MeV- and HL-induced damage. Alterations in GRXS13 expression also affect superoxide levels and the ascorbate/dehydroascorbate ratio after HL-induced stress. These results indicate that GRXS13 gene expression is critical for limiting basal and photooxidative stress-induced reactive oxygen species (ROS) production. Together, these results place GRXS13.2 as a member of the ROS-scavenging/antioxidant network that shows a particularly low functional redundancy in the Arabidopsis GRX family.