RESUMEN
AIM: Experimental simulation of near-future ocean acidification (OA) has been demonstrated to affect growth and development of echinoderm larval stages through energy allocation towards ion and pH compensatory processes. To date, it remains largely unknown how major pH regulatory systems and their energetics are affected by trans-generational exposure to near-future acidification levels. METHODS: Here, we used the common sea star Asterias rubens in a reciprocal transplant experiment comprising different combinations of OA scenarios, to study trans-generational plasticity using morphological and physiological endpoints. RESULTS: Acclimation of adults to pHT 7.2 (pCO2 3500 µatm) led to reductions in feeding rates, gonad weight and fecundity. No effects were evident at moderate acidification levels (pHT 7.4; pCO2 2000 µatm). Parental pre-acclimation to pHT 7.2 for 85 days reduced developmental rates even when larvae were raised under moderate and high pH conditions, whereas pre-acclimation to pHT 7.4 did not alter offspring performance. Microelectrode measurements and pharmacological inhibitor studies carried out on larval stages demonstrated that maintenance of alkaline gastric pH represents a substantial energy sink under acidified conditions that may contribute up to 30% to the total energy budget. CONCLUSION: Parental pre-acclimation to acidification levels that are beyond the pH that is encountered by this population in its natural habitat (eg, pHT 7.2) negatively affected larval size and development, potentially through reduced energy transfer. Maintenance of alkaline gastric pH and reductions in maternal energy reserves probably constitute the main factors for a reduced juvenile recruitment of this marine keystone species under simulated OA.
Asunto(s)
Aclimatación/fisiología , Asterias/fisiología , Tracto Gastrointestinal/fisiología , Homeostasis/fisiología , Agua de Mar/química , Animales , Cambio Climático , Tracto Gastrointestinal/química , Humanos , Concentración de Iones de Hidrógeno , LarvaRESUMEN
Anthropogenic CO(2) emission will lead to an increase in seawater pCO(2) of up to 80-100 Pa (800-1000 µatm) within this century and to an acidification of the oceans. Green sea urchins (Strongylocentrotus droebachiensis) occurring in Kattegat experience seasonal hypercapnic and hypoxic conditions already today. Thus, anthropogenic CO(2) emissions will add up to existing values and will lead to even higher pCO(2) values >200 Pa (>2000 µatm). To estimate the green sea urchins' potential to acclimate to acidified seawater, we calculated an energy budget and determined the extracellular acid base status of adult S. droebachiensis exposed to moderately (102-145 Pa, 1007-1431 µatm) and highly (284-385 Pa, 2800-3800 µatm) elevated seawater pCO(2) for 10 and 45 days. A 45-day exposure to elevated pCO(2) resulted in a shift in energy budgets, leading to reduced somatic and reproductive growth. Metabolic rates were not significantly affected, but ammonium excretion increased in response to elevated pCO(2). This led to decreased O:N ratios. These findings suggest that protein metabolism is possibly enhanced under elevated pCO(2) in order to support ion homeostasis by increasing net acid extrusion. The perivisceral coelomic fluid acid-base status revealed that S. droebachiensis is able to fully (intermediate pCO(2)) or partially (high pCO(2)) compensate extracellular pH (pH(e)) changes by accumulation of bicarbonate (maximum increases 2.5mM), albeit at a slower rate than typically observed in other taxa (10-day duration for full pH(e) compensation). At intermediate pCO(2), sea urchins were able to maintain fully compensated pH(e) for 45 days. Sea urchins from the higher pCO(2) treatment could be divided into two groups following medium-term acclimation: one group of experimental animals (29%) contained remnants of food in their digestive system and maintained partially compensated pH(e) (+2.3mM HCO(3)(-)), while the other group (71%) exhibited an empty digestive system and a severe metabolic acidosis (-0.5 pH units, -2.4mM HCO(3)(-)). There was no difference in mortality between the three pCO(2) treatments. The results of this study suggest that S. droebachiensis occurring in the Kattegat might be pre-adapted to hypercapnia due to natural variability in pCO(2) in its habitat. We show for the first time that some echinoderm species can actively compensate extracellular pH. Seawater pCO(2) values of >200 Pa, which will occur in the Kattegat within this century during seasonal hypoxic events, can possibly only be endured for a short time period of a few weeks. Increases in anthropogenic CO(2) emissions and leakages from potential sub-seabed CO(2) storage (CCS) sites thus impose a threat to the ecologically and economically important species S. droebachiensis.
Asunto(s)
Dióxido de Carbono/toxicidad , Agua de Mar/química , Strongylocentrotus/fisiología , Contaminantes Químicos del Agua/toxicidad , Aclimatación , Equilibrio Ácido-Base , Animales , Concentración de Iones de HidrógenoRESUMEN
Extensive use of fossil fuels is leading to increasing CO(2) concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO(2). As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO(2) 41 Pa e.g. 399 µatm) and CO(2) acidified seawater with pH of 7.7 (pCO(2) 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10% reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (two-way ANOSIM: Global R=1) while acidification effects were less pronounced (Global R=0.518). Significant differences in gene expression patterns (ANOSIM R=0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO(2) treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO(2) effect. We found an up regulation of metabolic genes (between 10%and 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23% and 36% in msp130, SM30B, and SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na(+)/K(+)-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.
Asunto(s)
Dióxido de Carbono/toxicidad , Ácido Carbónico/química , Contaminación Ambiental , Perfilación de la Expresión Génica , Erizos de Mar/crecimiento & desarrollo , Erizos de Mar/genética , Animales , Dióxido de Carbono/química , Humanos , Concentración de Iones de Hidrógeno , Larva/crecimiento & desarrollo , Agua de Mar/química , Regulación hacia ArribaRESUMEN
Anthropogenic CO(2) emissions are acidifying the world's oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO(2) (129 Pa, 1271 µatm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of +100% under elevated pCO(2), while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO(2) spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters.
Asunto(s)
Dióxido de Carbono/toxicidad , Ácido Carbónico/química , Contaminación Ambiental , Larva/crecimiento & desarrollo , Larva/metabolismo , Erizos de Mar/crecimiento & desarrollo , Erizos de Mar/metabolismo , Animales , Dióxido de Carbono/química , Humanos , Concentración de Iones de Hidrógeno , Larva/anatomía & histología , Erizos de Mar/anatomía & histología , Agua de Mar/químicaRESUMEN
Possible target proteins of chloroplast thioredoxin (Trx) have been investigated in the stroma lysate of spinach chloroplasts. For that purpose, we immobilized a mutant of m-type Trx in which an internal cysteine at the active site was substituted with serine, on cyanogen bromide-activated resin. By using this resin, the target proteins in chloroplast were efficiently acquired when they formed the mixed-disulfide intermediates with the immobilized Trxs. We could acquire Rubisco activase (45 kDa) and 2-Cys-type peroxiredoxin (Prx), which were recently identified as targets of chloroplast Trxs. Glyceraldehyde-3-phosphate dehydrogenase and sedoheputulose 1,7-bisphosphatase, well-known thiol enzymes in the Calvin cycle, also were recognized among the collected proteins, suggesting the method is applicable for our purpose. Furthermore, four proteins were identified from a homology search of the NH(2)-terminal sequence of the acquired proteins: glutamine synthetase, a protein homologous to chloroplast cyclophilin, a homolog of Prx-Q, and the Rubisco small subunit. The Trx susceptibilities of the recombinant cyclophilin and Prx-Q of Arabidopsis thaliana were then examined. The method developed in the present study is thus applicable to investigate the various redox networks via Trxs and the related enzymes in the cell.
Asunto(s)
Cloroplastos/metabolismo , Peroxidasas/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arabidopsis/metabolismo , Sitios de Unión , Clonación Molecular , Ciclofilinas/química , Ciclofilinas/metabolismo , Cisteína , Disulfuros/análisis , Glutamato-Amoníaco Ligasa/química , Glutamato-Amoníaco Ligasa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Peroxidasas/química , Peroxirredoxinas , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Resinas de Plantas , Ribulosa-Bifosfato Carboxilasa/metabolismo , Serina , Spinacia oleracea/metabolismoRESUMEN
Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.
Asunto(s)
Cloroplastos/enzimología , Mutación/genética , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Bacillus/enzimología , Disulfuros/metabolismo , Ditiotreitol/metabolismo , Ditiotreitol/farmacología , Activación Enzimática/efectos de los fármacos , Datos de Secuencia Molecular , Oxidación-Reducción/efectos de los fármacos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Subunidades de Proteína , ATPasas de Translocación de Protón/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Sustancias Reductoras/metabolismo , Sustancias Reductoras/farmacología , Rotación , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacologíaRESUMEN
Methylobacterium dichloromethanicum DM4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase-glutathione S-transferase. A mutant of the dichloromethane-degrading strain M. dichloromethanicum DM4, strain DM4-1445, was obtained by mini-Tn5 transposon mutagenesis that was no longer able to grow with dichloromethane. Dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain. The site of mini-Tn5 insertion in this mutant was located in the polA gene encoding DNA polymerase I, an enzyme with a well-known role in DNA repair. DNA polymerase activity was not detected in cell extracts of the polA mutant. Conjugation of a plasmid containing the intact DNA polymerase I gene into the polA mutant restored growth with dichloromethane, indicating that the polA gene defect was responsible for the observed lack of growth of this mutant with dichloromethane. Viability of the DM4-1445 mutant was strongly reduced upon exposure to both UV light and dichloromethane. The polA'-lacZ transcriptional fusion resulting from mini-Tn5 insertion was constitutively expressed at high levels and induced about twofold after addition of 10 mM dichloromethane. Taken together, these data indicate that DNA polymerase I is essential for growth of M. dichloromethanicum DM4 with dichloromethane and further suggest an important role of the DNA repair machinery in the degradation of halogenated, DNA-alkylating compounds by bacteria.
Asunto(s)
ADN Polimerasa I/metabolismo , Cloruro de Metileno/metabolismo , Methylobacterium/enzimología , Carbono/metabolismo , Clonación Molecular , Medios de Cultivo , ADN Polimerasa I/genética , Elementos Transponibles de ADN , Expresión Génica , Genes Bacterianos , Cloruro de Metileno/farmacología , Methylobacterium/efectos de los fármacos , Methylobacterium/genética , Methylobacterium/crecimiento & desarrollo , Mutagénesis Insercional , Análisis de Secuencia de ADNRESUMEN
A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic). By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex. The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol. No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C. alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.
Asunto(s)
Cloroplastos/química , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Bacillus/química , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Mutagénesis , Oxidación-Reducción , Plásmidos , Unión Proteica , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Temperatura , Tiorredoxinas/farmacología , Factores de TiempoRESUMEN
The activity of the chloroplast H+-ATPase (CFoCF1) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the gamma-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CFoCF1 (CF1) and includes two successive steps, namely the binding of Trx to CF1 and the subsequent reduction or oxidation of CF1. To study this process thoroughly, a new expression system for spinach Trx-f and Trx-m was designed. In the presence of dithiothreitol (DTT) both forms of the expressed Trx could reduce the disulphide bridge on the gamma-subunit of CF1 and thus activate the ATPase. Trx mutants deficient in the internal, or both, cysteines of the active site were designed to study the details of the interaction. The Trx mutant proteins could still activate CF1-ATPase in the presence of DTT and they also increased the apparent affinity of CF1 for DTT. This implies that the binding of Trx to the CF1 gamma-subunit induces a conformational change facilitating the reduction of the disulphide bridge, and partially explains the high efficiency of Trx as a reductant in vivo.