Bettina Zeis

Acclimatory responses of the Daphnia pulex proteome to environmental changes. II. Chronic exposure to different temperatures (10 and 20°C) mainly affects protein metabolism

BackgroundTemperature affects essentially every aspect of the biology of poikilothermic animals including the energy and mass budgets, activity, growth, and reproduction. While thermal effects in ecologically important groups such as daphnids have been intensively studied at the ecosystem level and at least partly at the organismic level, much less is known about the molecular mechanisms underlying the acclimation to different temperatures. By using 2D gel electrophoresis and mass spectrometry, the present study identified the major elements of the temperature-induced subset of the proteome from differently acclimated Daphnia pulex.ResultsSpecific sets of proteins were found to be differentially expressed in 10°C or 20°C acclimated D. pulex. Most cold-repressed proteins comprised secretory enzymes which are involved in protein digestion (trypsins, chymotrypsins, astacin, carboxypeptidases). The cold-induced sets of proteins included several vitellogenin and actin isoforms (cytoplasmic and muscle-specific), and an AAA+ ATPase. Carbohydrate-modifying enzymes were constitutively expressed or down-regulated in the cold.ConclusionSpecific sets of cold-repressed and cold-induced proteins in D. pulex can be related to changes in the cellular demand for amino acids or to the compensatory control of physiological processes. The increase of proteolytic enzyme concentration and the decrease of vitellogenin, actin and total protein concentration between 10°C and 20°C acclimated animals reflect the increased amino-acids demand and the reduced protein reserves in the animals body. Conversely, the increase of actin concentration in cold-acclimated animals may contribute to a compensatory mechanism which ensures the relative constancy of muscular performance. The sheer number of peptidase genes (serine-peptidase-like: > 200, astacin-like: 36, carboxypeptidase-like: 30) in the D. pulex genome suggests large-scaled gene family expansions that might reflect specific adaptations to the lifestyle of a planktonic filter feeder in a highly variable aquatic environment.

Acclimatory responses of the Daphnia pulex proteome to environmental changes. I. Chronic exposure to hypoxia affects the oxygen transport system and carbohydrate metabolism.

BackgroundFreshwater planktonic crustaceans of the genus Daphnia show a remarkable plasticity to cope with environmental changes in oxygen concentration and temperature. One of the key proteins of adaptive gene control in Daphnia pulex under hypoxia is hemoglobin (Hb), which increases in hemolymph concentration by an order of magnitude and shows an enhanced oxygen affinity due to changes in subunit composition. To explore the full spectrum of adaptive protein expression in response to low-oxygen conditions, two-dimensional gel electrophoresis and mass spectrometry were used to analyze the proteome composition of animals acclimated to normoxia (oxygen partial pressure [P o2]: 20 kPa) and hypoxia (P o2: 3 kPa), respectively.ResultsThe comparative proteome analysis showed an up-regulation of more than 50 protein spots under hypoxia. Identification of a major share of these spots revealed acclimatory changes for Hb, glycolytic enzymes (enolase), and enzymes involved in the degradation of storage and structural carbohydrates (e.g. cellubiohydrolase). Proteolytic enzymes remained constitutively expressed on a high level.ConclusionAcclimatory adjustments of the D. pulex proteome to hypoxia included a strong induction of Hb and carbohydrate-degrading enzymes. The scenario of adaptive protein expression under environmental hypoxia can be interpreted as a process to improve oxygen transport and carbohydrate provision for the maintenance of ATP production, even during short episodes of tissue hypoxia requiring support from anaerobic metabolism.

Differential haemoglobin gene expression in the crustacean Daphnia magna exposed to different oxygen partial pressures.

Abstract The quantity and quality of the haemoglobin (Hb) of Daphnia magna is related to oxygen partial pressure in the water. Both the dynamics of hypoxia-induced Hb gene transcription, as well as Hb properties in animals incubated long-term at hyperoxia, normoxia and hypoxia, were investigated. Examination of Hb gene (dhb1-dhb3) transcription showed the expression of dhb2 and especially dhb3 to increase markedly approximately one hour after the onset of hypoxia, whereas dhb1 was expressed more or less constitutively. At an incubation close to anoxia, an onset of dhb3 transcription was found already after two minutes. In longterm incubated animals, concentration and oxygen affinity of Hb were lower at higher oxygen partial pressures. With decreasing oxygen availability, the subunit composition of Hb macromolecules changed. The share of the dhb2-encoded subunit, DHbF, increased already during moderate hypoxia. The increase of dhb3 mRNA (encoding DHbC) may be related to a transient increase of DHbC in the first days of hypoxia and/or to an additional coding of dhb3 for DHbD. The rise of DHbD, and particularly DHbA, only at severe hypoxia coincided with the increase of Hb oxygen affinity. The dhb1-encoded subunits DHbB and DHbE showed either a relatively moderate increase or even a decrease in concentration at hypoxia. In small animals with restricted homeostasis capabilities such as Daphnia, adaptation of the protein equipment seems to be a more effective strategy than allosteric modulator control.

The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties

The process of oxygen-dependent hemoglobin induction in Daphnia magna was studied over an 11-day period of hypoxia (ambient oxygen partial pressure: 3 kPa). Along with the increase of hemoglobin concentration in the hemolymph, hemoglobin became the dominant protein fraction in gel filtration experiments using extracts of whole animals. The size of the native aggregates was constant. However, subunit composition depended on the duration of hypoxia: the pattern of predominantly expressed subunits under hypoxia deviated from that of normoxic individuals. The varying degree of hypoxic induction for different hemoglobin subunits was confirmed by autoradiography. Along with changes in hemoglobin subunit composition, oxygen affinity of the respiratory protein increased. The dynamics of the hemoglobin induction process was analysed. Newly synthesized hemoglobin can be detected within 18 h after the onset of hypoxia. A marked increase in hemoglobin concentration is evident from the third day of hypoxia, and a steady state of hemoglobin concentration is reached within 11 days. The changes of hemoglobin subunit expression in response to hypoxia form the structural basis for the observed adjustments of hemoglobin function leading to enhanced oxygen transport at low ambient oxygen concentrations.

Acute changes in temperature or oxygen availability induce ROS fluctuations in Daphnia magna linked with fluctuations of reduced and oxidized glutathione, catalase activity and gene (haemoglobin) expression.

Background information. ROS (reactive oxygen species) as well as components of the antioxidant redox systems may act as signals. To link acute environmental change with gene expression, changes in ROS and GSH/GSSG (reduced/oxidized glutathione) level were measured upon acute changes in temperature or oxygen availability in the aquatic key species Daphnia magna together with HIF‐1 (hypoxia‐inducible factor 1)‐mediated Hb (haemoglobin) expression.

Adaptive haemoglobin gene control in Daphnia pulex at different oxygen and temperature conditions.

Hypoxia-induced haemoglobin (Hb) expression is a central regulatory mechanism in Daphnia in response to environmental hypoxia or warm temperatures. Changes in Hb concentration as well as Hb subunit composition, which modulate Hb oxygen affinity, guarantee the oxygen supply of tissues under these environmental conditions. Based on the sequenced D. pulex genome, Hb genes were related to the properties of haemolymph Hb, which included its concentration and oxygen affinity (both measured by spectrophotometry) as well as the Hb subunit composition (determined by 2-D gel electrophoresis and ESI-MS analysis). Permanent cultures of D. pulex acclimated to different oxygen conditions (normoxia and hypoxia) and temperatures (10°C, 20°C, and 24°C), showed characteristic changes in Hb concentration, subunit composition and oxygen affinity. Several subunits (Hb4, Hb7, Hb8, and Hb10) were obviously responsible for changes in oxygen affinity including those, which carry a number of hypoxia-responsive elements (HREs) upstream of the respective gene (hb4 and hb10). Analysing the effects of different oxygen- or temperature-acclimations on Hb subunit expression in D. pulex and D. magna on a common basis (Hb concentration or oxygen affinity) revealed a general pattern of oxygen and temperature effects on Hb, which implies that Hb quantity and quality are mostly influenced by the degree of tissue hypoxia. Differences between both species in the onset of hypoxia-induced differential Hb expression and Hb oxygen affinity, which are probably related to different HRE patterns and functionally important differences in the amino acid sequence of only a few subunits, cause a reduced ability of D. pulex to adjust Hb function to temperature changes in comparison to D. magna.

Hypoxia-inducible haemoglobins of Daphnia pulex and their role in the response to acute and chronic temperature increase☆

Daphnia pulex is challenged by severe oxygen and temperature changes in its habitat. In response to hypoxia, the equipment of oxygen transport proteins is adjusted in quantity and quality by differential expression of haemoglobin isoforms. This study focuses on the response of 20°C acclimated animals to elevated temperature using transcriptomic and proteomic approaches. Acute temperature stress (30°C) induced the hypoxia-inducible Hb isoforms most strongly, resulting in an increase of the haemoglobin mRNA pool by 70% within 8h. Long-term-acclimation to moderately elevated temperature (24°C) only evoked minor changes of the Hb mRNA suite. Nevertheless, the concentration of the hemolymph pool of haemoglobin was elevated by 80%. In this case, the constitutive Hb isoforms showed the strongest increase, with Hb01 and Hb02 contributing by 64% to the total amount of respiratory protein. The regulation patterns upon acute temperature stress likely reflect temperature-induced tissue hypoxia, whereas in case of persisting exposure to moderately elevated temperature, acclimation processes enabled the successful return to oxygen homeostasis. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Macromolecular isoforms of Daphnia magna haemoglobin.

Abstract The haemoglobin (Hb) of Daphnia magna acclimated to different oxygen conditions was sampled, and in its natively assembled state it was separated by chromatofocusing. The Hb isoforms were analysed for their subunit composition under denaturating conditions by two-dimensional gel electrophoresis. The Hb system is suggested to consist of three predominant Hb aggregates, which are characterised by a specific subunit composition and synthesised in response to different ambient oxygen conditions. In normoxia, a dominant Hb aggregate (DmHbI) with a pI of 4.4–4.6 was composed of subunits B, C, E, F and G. In severe hypoxia, a different dominant Hb isoform (DmHbIII) with a pI of 5.7–5.9 was composed of subunits A, B, C, D, E and F. Further analyses in moderate hypoxia provided evidence for a third Hb isoform (DmHbII) composed of subunits B, C, D, E and F. Sequence alignment and homology modelling of the tertiary structure of the D. magna Hb domains 1 and 2 revealed functionally relevant substitutions of amino acid residues at positions B10, E7 and E11, which determine the functional properties of D. magna haemoglobin in terms of haem contact, oxygen binding and affinity. Both domains are predicted to possess the common haemoglobin fold, but helices C and D are not properly formed, and helix G is interrupted by a short coil.

Reactive oxygen species (ROS) and the heat stress response of Daphnia pulex: ROS‐mediated activation of hypoxia‐inducible factor 1 (HIF‐1) and heat shock factor 1 (HSF‐1) and the clustered expression of stress genes

Heat stress in ectotherms involves direct (e.g. protein damage) and/or indirect effects (temperature‐induced hypoxia and ROS formation), which cause activation of the transcription factors (TF) heat shock factor 1 (HSF‐1) and/or hypoxia‐inducible factor 1 (HIF‐1). The present study focused on the links between stress (ROS) signals, nuclear (n) and cytoplasmic (c) HSF‐1/HIF‐1 levels, and stress gene expression on mRNA and protein levels (e.g. heat‐shock protein 90, HSP90) upon acute heat and ROS (H2O2) stress.

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh‐water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia‐incubated, hemoglobin‐rich and normoxia‐incubated, hemoglobin‐poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 ± 0.1 S, and its molecular mass was 583 kDa (hemoglobin‐rich animals) determined by AUC and 590.4 ± 11.1 kDa (hemoglobin‐rich animals) and 597.5 ± 49 kDa (hemoglobin‐poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin‐rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 ± 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O‐linked glycosylation in the pre‐A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three‐dimensional reconstructions via single‐particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.