Ocean Acidification Alters the Photosynthetic Responses of a Coccolithophorid to Fluctuating Ultraviolet and Visible Radiation

Mixing of seawater subjects phytoplankton to fluctuations in photosynthetically active radiation (400–700 nm) and ultraviolet radiation (UVR; 280–400 nm). These irradiance fluctuations are now superimposed upon ocean acidification and thinning of the upper mixing layer through stratification, which alters mixing regimes. Therefore, we examined the photosynthetic carbon fixation and photochemical performance of a coccolithophore, Gephyrocapsa oceanica, grown under high, future (1,000 μatm) and low, current (390 μatm) CO₂ levels, under regimes of fluctuating irradiances with or without UVR. Under both CO₂ levels, fluctuating irradiances, as compared with constant irradiance, led to lower nonphotochemical quenching and less UVR-induced inhibition of carbon fixation and photosystem II electron transport. The cells grown under high CO₂ showed a lower photosynthetic carbon fixation rate but lower nonphotochemical quenching and less ultraviolet B (280–315 nm)-induced inhibition. Ultraviolet A (315–400 nm) led to less enhancement of the photosynthetic carbon fixation in the high-CO₂-grown cells under fluctuating irradiance. Our data suggest that ocean acidification and fast mixing or fluctuation of solar radiation will act synergistically to lower carbon fixation by G. oceanica, although ocean acidification may decrease ultraviolet B-related photochemical inhibition.The oceans absorb about 25 million tons of CO₂ per day from the atmosphere (Sabine et al., 2004), leading to the acidification of seawater in surface oceans. The pH of oceanic surface seawater will decline by 0.3 to 0.4 units, reflecting a 100% to 150% increase in [H⁺], by the year 2100 under “a fossil-fuel intensive” emission scenario (Houghton, 2001). This ocean acidification and the associated chemical changes may bring about critical ecological and social consequences (Turley et al., 2010).Coccolithophores, as a key group of oceanic primary producers, with coccolith scales made of CaCO₃, are important to global carbon cycles (Riebesell and Tortell, 2011). Ocean acidification generally decreases calcification by coccolithophores (Riebesell et al., 2000; Zondervan et al., 2002; Delille et al., 2005; Beaufort et al., 2011) and other calcifying algae (Gao and Zheng, 2010; Sinutok et al., 2011), with responses differing across species or different environmental conditions (Langer et al., 2006, 2009; Iglesias-Rodriguez et al., 2008; Doney et al., 2009; Shi et al., 2009). Algal calcification, in turn, influences the impacts of solar ultraviolet radiation (UVR; 280–400 nm) on the algae’s photophysiology (Gao et al., 2009; Gao and Zheng, 2010; Guan and Gao, 2010).Although the Montreal Protocol has resulted in a slowing of ozone depletion, ultraviolet B (UVB) irradiance (280–315 nm) reaching northern temperate regions increased 10% between 1983 and 2003 (Josefsson, 2006), and a recent observation found an ozone hole above the Arctic (Manney et al., 2011), reflecting ongoing impacts of climate change on ozone depletion. Biologically significant levels of UVR reach as deep as 80 m in pelagic oceans (Smith et al., 1992). In coastal waters or areas with high productivity, UVB irradiance usually penetrates only a few meters due to the attenuation caused by suspended particles and dissolved organic matter (Hargreaves, 2003; Tedetti and Sempéré, 2006). Ultraviolet A (UVA) and photosynthetically active radiation (PAR) are also attenuated but penetrate to much deeper depths due to their wavelength properties and intensities. UVA and UVB can both act synergistically with ocean acidification to inhibit algal photosynthetic performance (Gao et al., 2009), and the inhibition caused by UVB could be about 2.5 times that caused by UVA (Gao and Zheng, 2010); however, an antagonistic effect of UVB and ocean acidification was also found in a diatom (Li et al., 2012).In parallel, global warming due to increased atmospheric CO₂ concentration causes ocean warming, which results in a decrease in the depth of the upper mixing layer (UML; Sarmiento et al., 2004). Such stratification increases integrated exposures of phytoplankton cells within the UML to solar UVR and visible radiation and decreases the upward transport of nutrients from deeper water layers, influencing phytoplankton photophysiology (Beardall et al., 2009; Gao et al., 2012a). Fluctuations of both solar PAR and UVR within the UML affect phytoplankton photosynthetic activity and carbon fixation (Helbling et al., 2003; Villafañe et al., 2007; Guan and Gao, 2008; Dimier et al., 2009). Mixing depths and/or mixing rates in the upper oceans also change in response to increased stratification and/or wind speed due to global climate change (Sarmiento et al., 2004; Boyd et al., 2010).Phytoplankton responses to fluctuating solar radiation vary, particularly if considered in combination with other environmental factors, due to antagonistic or synergistic interactions. Fluctuation of solar radiation on cloudy days led to higher primary production in the presence of UVA (315–400 nm) as compared with the presence of UVA on sunny days (Gao et al., 2007). Algal acclimation to fluctuating irradiance can lead to differences in growth rates and cellular pigment content compared with the cells acclimated to constant irradiance (Van de Poll et al., 2007, 2010). On the other hand, the mixing rate in the UML is strongly controlled by wind (Denman and Gargett, 1983; MacIntyre, 1993), which may increase due to global warming (Toggweiler and Russell, 2008). Therefore, changes in mixing rate and stratification may interact with ocean acidification to affect the photophysiology of phytoplankton. Nevertheless, to our knowledge, nothing has yet been documented on the combined impacts of the fluctuation of PAR or UVR and ocean acidification on the photosynthetic performance of coccolithophores.Under this scenario, we expect that the photosynthesis of coccolithophores will respond differentially to fluctuating PAR, with or without UVR, when grown under ocean acidification conditions, since the balance of high PAR- or UVR-induced damage and the counteracting repair could differ under elevated CO₂ or acidity. To test these interactions, we grew Gephyrocapsa oceanica, which is widely distributed in temperate and tropical waters (Okabe, 1997), under current and ocean acidification conditions and examined its photochemical activity and photosynthesis under different combinations of fluctuating PAR and UVR.     

Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

Chloroplast Integrity and ATP-Dependent CO(2) Fixation in Spinacia oleracea

Washed whole chloroplasts of Spinacia oleracea isolated and assayed in a tris (hydroxymethyl aminomethane)-HCl buffered sucrose solution exhibited low dark CO₂ fixing activity, whereas washed whole chloroplasts isolated in the same buffer but assayed in that buffer without sucrose exhibited much greater dark CO₂ fixing activity. The lowered activity could be attributed to the impermeability of the chloroplast membrane to ribose-5-phosphate or adenosine triphosphate. The preservation of the integrity of the chloroplast membrane, as reflected by its impermeability to either or both of the abovementioned compounds, was measured by the fixation of ¹⁴CO₂ into acid-stable products in the presence of ribose-5-phosphate and adenosine triphosphate by the whole chloroplast as compared with fixation by the chloroplast extract. An effect (i.e., apparent resistance to the passage of ribose-5-phosphate or adenosine-5-triphosphate into the chloroplast) similar to, but less pronounced than, that produced by the presence of sucrose in the isolation medium was observed upon the addition of MnCl₂ or CaCl₂ to the buffered sucrose isolation medium. The addition of KCl enhanced slightly the effect produced by addition of sucrose alone to the isolation medium. The presence of MgCl₂ in the isolation medium, however, either caused the chloroplasts to become leaky or more fragile since more of the activity of the carboxylative phase enzymes appeared in the cytoplasm. When a mixture of all of the metal ions was added to the buffered sucrose suspending medium, the chloroplasts exhibited the same response observed with MgCl₂ alone. The addition of ethylene diaminetetraacetate or dithiothreitol appeared to alter the permeability of the chloroplast membrane nonspecifically when the assay was conducted in the absence of sucrose. Specific activities (μmoles CO₂ fixed/mg chlorophyll × hr) as high as 329.6 have been observed for dark fixation by chloroplasts. The phosphoenolpyruvate carboxylase activity in the chloroplasts was only one-seventh that of ribulose diphosphate carboxylase. The phosphoenolpyruvate carboxylase activity in the cytoplasm was 5 times that of the chloroplasts.     

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta)

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by ‘selection’ to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.     

Temperature Dependent Effects of Elevated CO2 on Shell Composition and Mechanical Properties of Hydroides elegans: Insights from a Multiple Stressor Experiment

The majority of marine benthic invertebrates protect themselves from predators by producing calcareous tubes or shells that have remarkable mechanical strength. An elevation of CO₂ or a decrease in pH in the environment can reduce intracellular pH at the site of calcification and thus interfere with animal’s ability to accrete CaCO₃. In nature, decreased pH in combination with stressors associated with climate change may result in the animal producing severely damaged and mechanically weak tubes. This study investigated how the interaction of environmental drivers affects production of calcareous tubes by the serpulid tubeworm, Hydroides elegans. In a factorial manipulative experiment, we analyzed the effects of pH (8.1 and 7.8), salinity (34 and 27‰), and temperature (23°C and 29°C) on the biomineral composition, ultrastructure and mechanical properties of the tubes. At an elevated temperature of 29°C, the tube calcite/aragonite ratio and Mg/Ca ratio were both increased, the Sr/Ca ratio was decreased, and the amorphous CaCO₃ content was reduced. Notably, at elevated temperature with decreased pH and reduced salinity, the constructed tubes had a more compact ultrastructure with enhanced hardness and elasticity compared to decreased pH at ambient temperature. Thus, elevated temperature rescued the decreased pH-induced tube impairments. This indicates that tubeworms are likely to thrive in early subtropical summer climate. In the context of climate change, tubeworms could be resilient to the projected near-future decreased pH or salinity as long as surface seawater temperature rise at least by 4°C.     

Giant Clams and Rising CO2: Light May Ameliorate Effects of Ocean Acidification on a Solar-Powered Animal

Global climate change and ocean acidification pose a serious threat to marine life. Marine invertebrates are particularly susceptible to ocean acidification, especially highly calcareous taxa such as molluscs, echinoderms and corals. The largest of all bivalve molluscs, giant clams, are already threatened by a variety of local pressures, including overharvesting, and are in decline worldwide. Several giant clam species are listed as ‘Vulnerable’ on the IUCN Red List of Threatened Species and now climate change and ocean acidification pose an additional threat to their conservation. Unlike most other molluscs, giant clams are ‘solar-powered’ animals containing photosynthetic algal symbionts suggesting that light could influence the effects of ocean acidification on these vulnerable animals. In this study, juvenile fluted giant clams Tridacna squamosa were exposed to three levels of carbon dioxide (CO₂) (control ~400, mid ~650 and high ~950 μatm) and light (photosynthetically active radiation 35, 65 and 304 μmol photons m^-2 s^-1). Elevated CO₂ projected for the end of this century (~650 and ~950 μatm) reduced giant clam survival and growth at mid-light levels. However, effects of CO₂ on survival were absent at high-light, with 100% survival across all CO₂ levels. Effects of CO₂ on growth of surviving clams were lessened, but not removed, at high-light levels. Shell growth and total animal mass gain were still reduced at high-CO₂. This study demonstrates the potential for light to alleviate effects of ocean acidification on survival and growth in a threatened calcareous marine invertebrate. Managing water quality (e.g. turbidity and sedimentation) in coastal areas to maintain water clarity may help ameliorate some negative effects of ocean acidification on giant clams and potentially other solar-powered calcifiers, such as hard corals.     

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): Role of bacterial extracellular polymers

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV‐irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV‐dosage. Since UV energy crosslinks both intra‐ and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.     

Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): role of bacterial extracellular polymers

Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films. Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H. elegans was investigated. Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e. larvae slowed down, secreted a mucous thread and crawled over the surface. This response is typical of larvae that encounter an attractive bacterial film. In contrast, bioassays with exopolymers in association with UV-irradiated, metabolically inactive bacterial films evoked complete settlement. However, the percentage of responding larvae was negatively correlated with the magnitude of UV-dosage. Since UV energy crosslinks both intra- and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity. Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect.     

Reversible anti-settlement activity against Amphibalanus (=Balanus) amphitrite,Bugula neritina,and Hydroides elegans by a nontoxic pharmaceutical compound,mizolastine

Mizolastine, an antihistamine pharmaceutical, was found to significantly inhibit larval settlement of the barnacle Amphibalanus (=Balanus) amphitrite, the bryozoan Bugula neritina, and the polychaete Hydroides elegans with EC₅₀ values of 4.2, 11.2, and 4.1 µg ml^?1, respectively. No toxicity against the larvae of these three species was observed at the concentration range tested during incubations with mizolastine. To determine whether the anti-settlement activity of mizolastine is reversible, recovery bioassays using these three species were conducted. More than 70% of the larvae that had been exposed for 4 h to mizolastine at concentrations four-fold greater than their respective EC₅₀ values completed normal metamorphosis. The results of the recovery bioassay provide evidence that the anti-settlement effect of mizolastine is reversible in addition to being nontoxic. The anti-settlement activities of several intermediates of the synthesis process of mizolastine were also examined. One of the intermediates, 2-chloro-1-(4-fluorobenzyl)-1H-benzo[d]imidazole, inhibited larval settlement and metamorphosis with low toxicity. These results may improve the understanding of the key functional group responsible for the anti-settlement activity of mizolastine.     

Flux of SO(2) into Leaf Cells and Cellular Acidification by SO(2)

A comparison of fluxes of SO₂ from the atmosphere into leaves with fluxes across biomembranes revealed that, apart from the cuticle, the main barrier to SO₂ entry into leaves are the stomates. SO₂ fluxes into leaves can be calculated with an accuracy sufficient for many purposes on the assumption that the intracellular SO₂ concentration is zero. SO₂ entering green leaf cells is trapped in the cytoplasm. In the light, the products formed in its reaction with water are processed particularly in the chloroplasts. Flux of SO₂ to the acidic central vacuole of leaf cells is insignificant. Intracellular acidification of barley mesophyll protoplasts by SO₂ was measured by the uptake of ¹⁴C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. The measured acidification was similar to the acidification calculated from known buffer capacities and the rate of SO₂ influx when the H⁺/SO₂ ratio was assumed to be 2. A comparison of photosynthesis inhibition by SO₂ with calculated acidification revealed different mechanisms of inhibition at low and at high concentrations of SO₂. At very low concentrations, inhibition by SO₂ was even smaller than expected from calculated acidification. The data suggest that, if acidification cannot be compensated by pH-stabilizing cellular mechanisms, it is a main factor of SO₂ toxicity at low SO₂ levels. At high levels of SO₂, anion toxicity and/or radical formation during oxidation of SO₂ to sulfate may play a large role in inhibition.     

Cyclic AMP concentration and protein kinase A (PKA) gene expression at different developmental stages of the polychaete Hydroides elegans

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) showed inductive effect on larval settlement of the polychaete Hydroides elegans. It has been suggested that IBMX triggers larval settlement by elevating the cellular adenosine 3',5'-cyclic monophosphate (cAMP) level in this species. To test this hypothesis, we first examined cAMP-level changes in both the competent (CL) and attached larvae (AL) and then characterized the cAMP-dependent protein kinase in H. elegans, which is the major mediator of cAMP action. Tissue extracts of the larvae were assayed for cAMP by enzyme immunoassay; the results showed that IBMX increased cAMP production up to approximately two-folds in the CL. However, there was no significant difference in the cAMP concentration between the CL and AL that were not treated with IBMX. The catalytic subunit of protein kinase A gene from H. elegans (designated HePKAc) was cloned, and its expression in different developmental stages of H. elegans was examined using quantitative real-time polymerase chain reaction. The gene expression level in the pre-competent trochophore larvae was the lowest, increased in the CL, reached the highest in the larvae undergoing normal and IBMX-induced metamorphosis, and then decreased in the adult stage. In situ hybridization results showed that HePKAc expressed mainly around eye regions and along body fragments of the CL and AL. Our results indicated that the IBMX-induced cAMP changes and the cAMP-dependent protein kinase gene may mediate larval development and settlement of H. elegans.     

Adrenoceptor Compounds Prevent the Settlement of Marine Invertebrate Larvae: Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) and Hydroides elegans (Polychaeta)

The effects of the neurotransmitter blockers idazoxan and phentolamine on the larval settlement of three marine invertebrate species belonging to three different phyla were investigated by using in vitro concentration-response bioassays. Since neurotransmitters are known to influence metamorphic transitions in invertebrate larvae, neurotransmitter blockers were tested to evaluate their sublethal effects on larvae. The α-adrenergic antagonists idazoxan and phentolamine inhibited settlement of Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) larvae, and larvae of the polychaete Hydroides elegans (Polychaeta) in a concentration- and taxon-dependent manner. At concentrations of 10^?3 M of both agents, larvae of all three species became immobile and subsequently died within 24 h. While cumulative settlement rates were observed after 48 h for B. amphitrite and H. elegans, and after 5 h for B. neritina, > 90% of the larvae that settled did so within 24 h for the first two species and within 1 h for B. neritina. The tendency of the hydrophobic idazoxan and phentolamine to accumulate at solid surfaces most probably contributes to their successful inhibition of larval settlement. This ability makes them particularly attractive as candidates for the development of slow-release carriers in antifouling paints.     

Adrenoceptor compounds prevent the settlement of marine invertebrate larvae: Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) and Hydroides elegans (Polychaeta)

The effects of the neurotransmitter blockers idazoxan and phentolamine on the larval settlement of three marine invertebrate species belonging to three different phyla were investigated by using in vitro concentration-response bioassays. Since neurotransmitters are known to influence metamorphic transitions in invertebrate larvae, neurotransmitter blockers were tested to evaluated their sublethal effects on larvae. The alpha-adrenergic antagonists idazoxan and phentolamine inhibited settlement of Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) larvae, and larvae of the polychaete Hydroides elegans (Polychaeta) in a concentration-and taxon-dependent manner. At concentrations of 10(-3) M of both agents, larvae of all three species became immobile and subsequently died within 24 h. While cumulative settlement rates were observed after 48 h for B. amphitrite and H. elegans, and after 5 h for B. neritina, >90% of the larvae that settled did so within 24 h for the first two species and within 1 h for B. neritina. The tendency of the hydrophobic idazoxan and phentolamine to accumulate at solid surfaces most probably contributes to their successful inhibition of larval settlement. This ability makes them particularly attractive as candidates for the development of slow-release carriers in antifouling paints.     

Effects of Ocean Acidification on Juvenile Red King Crab (Paralithodes camtschaticus) and Tanner Crab (Chionoecetes bairdi) Growth,Condition, Calcification,and Survival

Ocean acidification, a decrease in the pH in marine waters associated with rising atmospheric CO₂ levels, is a serious threat to marine ecosystems. In this paper, we determine the effects of long-term exposure to near-future levels of ocean acidification on the growth, condition, calcification, and survival of juvenile red king crabs, Paralithodes camtschaticus, and Tanner crabs, Chionoecetes bairdi. Juveniles were reared in individual containers for nearly 200 days in flowing control (pH 8.0), pH 7.8, and pH 7.5 seawater at ambient temperatures (range 4.4–11.9 °C). In both species, survival decreased with pH, with 100% mortality of red king crabs occurring after 95 days in pH 7.5 water. Though the morphology of neither species was affected by acidification, both species grew slower in acidified water. At the end of the experiment, calcium concentration was measured in each crab and the dry mass and condition index of each crab were determined. Ocean acidification did not affect the calcium content of red king crab but did decrease the condition index, while it had the opposite effect on Tanner crabs, decreasing calcium content but leaving the condition index unchanged. This suggests that red king crab may be able to maintain calcification rates, but at a high energetic cost. The decrease in survival and growth of each species is likely to have a serious negative effect on their populations in the absence of evolutionary adaptation or acclimatization over the coming decades.     

The Monocyte Locomotion Inhibitory Factor (MLIF) Produced by Entamoeba histolytica Alters the Expression of Genes Related to the Wound-Healing Process

The monocyte locomotion inhibitory factor (MLIF) is a pentapeptide produced by Entamoeba histolytica. This factor displays several in vivo and in vitro anti-inflammatory properties, among others, inhibition of monocyte locomotion and the respiratory burst in monocyte and neutrophils. A synthetic peptide had the same selective anti-inflammatory features as the native material. We now evaluated MLIF on the constitutive and induced gene expression in the MRC-5 human fibroblasts cell line. The MLIF affected constitutive expression of 21 genes and induced expression of 75 genes. Some of these genes involved in the inflammatory response and other participated in the remodeling and the wound-healing processes.     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

Quantitation of the O(2)-Dependent, CO(2)-Reversible Component of the Postillumination CO(2) Exchange Transient in Tobacco and Maize Leaves

The postillumination transient of CO₂ exchange and its relation to photorespiration has been examined in leaf discs from tobacco (Nicotiana tabacum) and maize (Zea mays). Studies of the transients observed by infrared gas analysis at 1, 21, and 43% O₂ in an open system were extended using the nonsteady state model described previously (Peterson and Ferrandino 1984 Plant Physiol 76: 976-978). Cumulative CO₂ exchange equivalents (i.e. nanomoles CO₂) versus time were derived from the analyzer responses of individual transients. In tobacco (C₃), subtraction of the time course of cumulative CO₂ exchange under photorespiratory conditions (21 or 43% O₂) from that obtained under nonphotorespiratory conditions (1% O₂) revealed the presence of an O₂-dependent and CO₂-reversible component within the first 60 seconds following darkening. This component was absent in maize (C₄) and at low external O₂:CO₂ ratios (i.e. <100) in tobacco. The size of the component in tobacco increased with net photosynthesis as irradiance was increased and was positively associated with inhibition of net photosynthesis by O₂. This relatively simple and rapid method of analysis of the transient is introduced to eliminate some uncertainties associated with estimation of photorespiration based on the maximal rate of postillumination CO₂ evolution. This method also provides a useful and complementary tool for detecting variation in photorespiration.     

CO2 Saturation and Trophic Shift Induced by Microbial Metabolic Processes in a River-Dominated Ocean Margin (Tropical Shallow Lagoon,Chilika, India)

An effort has been made for the first time in Asia's largest brackish water lagoon, Chilika, to investigate the spatio-temporal variability in primary productivity (PP), bacterial productivity (BP), bacterial abundance (BA), bacterial respiration (BR) and bacterial growth efficiency (BGE) in relation to partial pressure of CO₂ (pCO₂) and CO₂ air–water flux and the resultant trophic switchover. Annually, PP ranged between 24 and 376 µg C L^?1 d^?1 with significantly low values throughout the monsoon (MN), caused by light limitation due to inputs of riverine suspended matter. On the contrary, BP and BR ranged from 11.5 to 186.3 µg C L^?1 d^?1 and from 14.1 to 389.4 µg C L^?1 d^?1, respectively, with exceptionally higher values during MN. A wide spatial and temporal variation in the lagoon trophic status was apparent from BP/PP (0.05–6.4) and PP/BR (0.10–18.2) ratios. The seasonal shift in net pelagic production from autotrophy to heterotrophy due to terrestrial organic matter inputs via rivers, enhanced the bacterial metabolism during the MN, as evident from the high pCO₂ (10,134 µatm) and CO₂ air–water flux (714 mm m^?2 d^?1). Large variability in BGE and BP/PP ratios especially during MN led to high bacteria-mediated carbon fluxes which was evident from significantly high bacterial carbon demand (BCD >100% of PP) during this season. This suggested that the net amount of organic carbon (either dissolved or particulate form) synthesized by primary producers in the lagoon was not sufficient to satisfy the bacterial carbon requirements. Lagoon sustained low to moderate autotrophic–heterotrophic coupling with annual mean BCD of 231% relative to the primary production, which depicted that bacterioplankton are the mainstay of the lagoon biogeochemical cycles and principal players that bring changes in trophic status. Study disclosed that the high CO₂ supersaturation and oxygen undersaturation during MN was attributed to the increased heterotrophic respiration (in excess of PP) fuelled by allochthonous organic matter. On a spatial scale, lagoon sectors such as south sector, central sector and outer channel recorded “net autotrophic,” while the northern sector showed “net heterotrophic” throughout the study period.     

Cuticle Integrity and Biogenic Amine Synthesis in Caenorhabditis elegans Require the Cofactor Tetrahydrobiopterin (BH4)

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions.