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

Biochimica et Biophysica Acta 1834 (2013) 1704–1710

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Hypoxia-inducible haemoglobins of Daphnia pulex and their role in the response to acute and chronic temperature increase☆ Bettina Zeis ⁎, Dörthe Becker, Peter Gerke, Marita Koch, Rüdiger J. Paul Institut für Zoophysiologie, Universität Münster, Schlossplatz 8, 48143 Münster, Germany

a r t i c l e

i n f o

Article history: Received 6 December 2012 Received in revised form 25 January 2013 Accepted 28 January 2013 Available online 4 February 2013 Keywords: Differential isoform expression Oxygen homeostasis Proteomics Stress response Transcriptomics

a b s t r a c t 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 hypoxiainducible Hb isoforms most strongly, resulting in an increase of the haemoglobin mRNA pool by 70% within 8 h. 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. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Individuals of Daphnia pulex are challenged by major oxygen and temperature changes in their habitat, as these poikilothermic zooplankton organisms live in the rather small water bodies of ponds that are characterised by considerable variation in their abiotic conditions [1]. Both, oxygen shortage and high temperatures induce tissue hypoxia, in the latter case due to a mismatch between increasing oxygen demand and lower oxygen solubility in the water. Short-term systemic responses by increased ventilation and perfusion rates (external and internal oxygen transport) can restore the oxygen supply of cells [2]. In response to persisting oxygen or temperature stress, however, the oxygen transport capacity of the hemolymph is raised by haemoglobin (Hb) induction. The Hb of D. pulex consists of di-domain protein subunits of 35 kDa, which carry two oxygen-binding heme groups [3]. Twelve subunits aggregate to the macromolecular multimers [4]. Gene duplications lead to isoform multiplicity [5,6]. Eleven types of extracellular di-domain haemoglobins are present in the D. pulex genome; eight of these are part of a tandem repeat cluster [6]. A hypoxia-induced increase of Hb concentration has been described for Daphnia magna [7,8] and D. pulex [9]. For D. magna, the differential

Abbreviations: Hb, haemoglobin; HIF, hypoxia-inducible factor; HRE, hypoxiaresponsive element ☆ This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. ⁎ Corresponding author. Tel.: +49 251 8323852; fax: +49 251 8323876. E-mail address: [email protected] (B. Zeis). 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.01.036

induction of multiple isoforms [10] was assigned to the presence of hypoxia-responsive elements (HRE) binding the hetero-dimeric transcription factor HIF (hypoxia-inducible factor) in the promoter regions of Hb genes [11]. These regulatory elements are also found upstream of D. pulex Hb genes [12] with analogue roles for differential isoform expression in response to disturbed oxygen homeostasis. This study aims to analyse the induction patterns of the eleven Hb isoforms under hypoxia as well as under moderate and severe temperature stress. As transcriptomic approaches are now possible [6] they can be combined with proteomic data. This way, stress-induced changes on the level of mRNA can be correlated to effects on protein concentration. Expression patterns studied at chronic and acute temperature increases allow to assess the role of potentially HIF-mediated Hb induction for the respective condition. Thus this study provides new insights into the complex mechanisms of adjusting the properties of an oxygen transport protein to environmental changes. 2. Material and methods 2.1. Animals D. pulex LEYDIG originated from a lake near Gräfenhain [13]. Animals were raised in glass beakers with 2 L of Elendt medium [14] under a 16 h:8 h L:D photoperiod and fed with algae (Desmodesmus subspicatus; SAG 53.80, Göttingen, Germany) ad libitum daily. Three-quarter of the culture medium was renewed once a week. Control animals were kept at 20 °C under normoxic conditions (Po2 =20 kPa). Animals acclimated to hypoxia at 20 °C were raised under reduced oxygen concentration

B. Zeis et al. / Biochimica et Biophysica Acta 1834 (2013) 1704–1710

(Po2 = 2 kPa) for at least four weeks. Animals from long-termacclimation at a slightly elevated temperature (24 °C, Po2 = 20 kPa) were considered as chronically exposed to moderate heat stress. At 30 °C, this clonal line of D. pulex did not reproduce but survived for several days. Thus, this temperature was considered to cause severe temperature stress and was used for experiments of acute heat exposure, as maximal differential expression can be expected for substantial but sub-lethal changes of the stressor. Animals were rapidly transferred from control conditions (20 °C) to 20 °C (control) and 30 °C (both at normoxia), and samples were taken after 2, 4 and 8 h. Only females of 2–3 mm mostly carrying parthenogenetic eggs and embryos were used for experiments. 2.2. Transcriptomics RNA was prepared from 50 animals of the respective treatment (long-term culture at 20 °C and 24 °C, transfer to 20 °C (control) and 30 °C for 2, 4 and 8 h, four replicates for each condition) shockfrozen in liquid nitrogen after removing adhering water with a tissue paper. To assess patterns of gene expression, D. pulex multiplex long-oligonucleotide microarrays were used [6]. Total RNA was extracted using RNAiso-G + reagent (Segenetic, Borken, Germany), and purified using the Qiagen (Venlo, Netherlands) RNeasy protocol with on-column DNase treatment [15]. Experiments were conducted with four biological replicates resulting in two pairs of swapped dye combinations, using either Cy3- or Cy5-coupled nonamer primers (NimbleGen Dual-Color Labeling Kit, Roche NimbleGen Inc., Madison, WI, USA). For cDNA samples of each test vs control analysis pair, fluorescence intensity was detected (NimbleGen MS 200 Microarray Scanner, Roche NimbleGen Inc., Madison/WI, USA). Genes printed on the array refer to the D. pulex genome assembly v1.1 (http:// wfleabase.org/genome/Daphnia_pulex/). Microarray data were imported into an in-house analysis pipeline using bioconductor for normalisation and analysis [16]. All probes were quantile-normalised across arrays, samples and replicates and the median signal of probes representing genes is given in fluorescence intensities in arbitrary units. For this study, only the genes for the eleven di-domain haemoglobins [6], each represented by three independent oligonucleotides, were selected for analysis. 2.3. Proteomics The hemolymph of 5–20 animals was collected individually and pooled (for details see [12]). Hb concentration was determined from spectra using the extinction coefficient of 14.8 L mmol−1 cm–1 at 576 nm [17]. 2D gels were performed as described elsewhere [12] and stained with SyproRuby® (Bio-Rad, Munich, Germany). Spots were cut out of the gels and proteins were identified by mass spectrometry using the D. pulex genome assembly v1.1 (http://wfleabase.org/genome/ Daphnia_pulex/) [12]. Spot intensity indicating protein amount was quantified by densitometric analysis using MATLAB (for details see [12]). For one spot with spectrometric evidence for both, Hb01 and Hb02, equal contributions of both isoforms were assumed, and 50% of the calculated concentration was assigned to each isoform. The relative contribution (in percent) was calculated for each isoform. As total Hb concentrations were measured (s.a.), the absolute amount in the hemolymph (in μmol heme/L) was calculated for each subunit. 2.4. Data processing For the eleven Hb isoforms of D. pulex, the quantification of transcript and protein levels was the basis to calculate induction factors, i.e. the ratio of the amounts under stress versus control conditions. This calculation was not applicable in case of subunits missing in one condition. In addition, the relative percentage share of the respective contribution to the mRNA or protein pool was determined. The statistical significance of differences to control conditions (20 °C, normoxia)

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was tested using ANOVA, considering p values below 0.05 as indicators of significant changes. 3. Results Significant Hb induction was detected under hypoxia as well as during chronic exposure to moderate heat stress and acute exposure to severe heat stress. On the protein level, Hb subunit composition was dominated by the isoforms Hb01-03, with small contributions of Hb07, Hb04 and Hb08 at normoxia and 20 °C (Fig. 1A). Hb10 was absent at normoxia, but strongly induced under hypoxia. The isoforms Hb05, Hb06, Hb09 and Hb11 were not detected in 2D gels. When oxygen concentration was lowered to about 10% of normoxic conditions, all detected subunits increased in concentration, but to different extents. Under hypoxia, Hb03, Hb07, Hb04, Hb10 and Hb08 concentrations were most prominent (Fig. 1B). Consequently, the highest induction factors were calculated for these isoforms, ranging from 13.1 to 63.4 (Fig. 1C, Table 1). The strong induction of Hb10 could not be quantified by an induction factor, as this subunit was not detected under control conditions (20 °C, normoxia). Nevertheless, it is considered a hypoxia-inducible isoform, as its concentration increased from below detection level to 144 μmol/L, an increase in concentration, which is about 4 times higher than for Hb01 and Hb02. The constitutive subunits Hb01 and Hb02 increased only about twofold under hypoxia (induction factors 2.5 and 2.2, respectively). Thus, the increase in overall Hb concentration in the animals' hemolymph by a factor of 14 was mainly due to the strongly hypoxia-inducible isoforms (Fig. 1D). In animals kept permanently at 24 °C, eight gene products were detected in significant quantities on the transcript level, whereas the mRNA of Hb06, Hb11, and Hb10 was found only in minor amounts (Fig. 2). Comparing results from animals raised at 20 °C and 24 °C (Fig. 2A and B), we found that the overall Hb mRNA quantity remained almost unchanged (Fig. 2D). The contribution of Hb02 increased to the highest extent, which was also reflected by an induction factor of 1.5 (Fig. 2C). For the isoforms Hb01 and Hb10, transcript levels also increased slightly. The transcripts of several other isoforms were down-regulated. Despite these minor changes on the transcript level, the overall Hb concentration in the hemolymph increased by a factor of 1.8 compared to the 20 °C control value (Fig. 2H). The largest rise in protein concentration was detected for the isoform Hb02, and both subunits Hb01 and Hb02 still accounted for the dominant part of the Hb suite as determined at 20 °C. The induction factors calculated for the subunits Hb04, Hb07, and Hb08 were higher than for the constitutive isoforms (Fig. 2G), but they were much lower than in case of hypoxic induction (Fig. 1C). The elevated temperature caused a maximal increase of 10 μmol/L of these hypoxia-responsive isoforms. Thus absolute protein amounts of these isoforms remained low compared to amounts at hypoxia as well as in comparison to the dominating isoforms Hb01, Hb02 and Hb03 at 24 °C. Accordingly, chronic exposure to moderately elevated temperature led to higher levels of the Hb isoforms that had been dominant already under control conditions. Animals exposed to acute heat stress (30 °C) responded with an induction of the Hb mRNA pool by a factor of 1.3 after 2 h, 1.5 after 4 h and 1.7 after 8 h (Fig. 3, Table 1). Again, the signal of Hb06 and Hb11 mRNA was very low. A significant induction was observed for several subunits, both constitutive and hypoxia-inducible isoforms. The highest induction factor was observed for Hb10 with a value of 1.9 after 8 h. The constitutive subunits Hb01, Hb02 and Hb03 showed the highest contribution to overall Hb mRNA at all incubation times analysed (Fig. 3A), with induction factors of 1.8 and 1.7 (Table 1). Hb04, Hb05, and Hb07 also showed high amounts of transcripts, whereas Hb08, Hb09, and Hb10 were less prominent (Fig. 3). Effects of hypoxia or temperature increase were also quantified concerning the relative contribution of isoforms to the pool of mRNA and proteins. Under hypoxia, a major shift in subunit composition

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protein quantity (µmol/L)

1706

60

Table 1 Induction of Daphnia pulex haemoglobin isoforms. For each subunit type the number of hypoxia-responsive elements in the upstream region of the respective gene is given. Induction factors are calculated on the protein level for chronic hypoxia and to 24 °C, and on the transcript level for chronic exposure to 24 °C and acute heat stress (30 °C for 8 h) in relation to values under control conditions (20 °C, normoxia). Bold characters indicate significant changes. n.f.: not found, n.a. not applicable, protein induction factor cannot be calculated, value missing at control conditions. (n = 3 for protein, n = 4 for mRNA).

normoxia (20 kPa)

A

50 40 30 20 10

Isoform

HRE

Hb01 Hb02 Hb03 Hb04 Hb05 Hb06 Hb07 Hb08 Hb09 Hb10 Hb11 Total Hb

3 3 1 5 4 1 3 2 0 8 1 31

protein quantity (µmol/L)

0 250

hypoxia (2 kPa)

* *

200

*

150

*

100

* *

50 0 8

C

256

6

64

4

16

2

4

induction factor

induction (log2)

*

B

1

0 1

2

3

4

5

6

7

8

9 10 11

Chronic exposure to hypoxia

Chronic exposure to 24 °C

Chronic exposure to 24 °C

8 h acute heat stress (30 °C)

Protein

Protein

mRNA

mRNA

2.5 2.2 13.1 52.6 n.f. n.f. 56.8 63.4 n.f. n.a. n.f. 14.0

1.7 2.1 1.5 3.4 n.f. n.f. 2.9 2.4 n.f. n.f. n.f. 1.8

1.1 1.5 1.0 0.9 1.0 1.0 1.1 0.7 0.5 1.1 0.7 1.0

1.8 1.8 1.7 1.7 1.7 1.2 1.7 1.5 1.1 1.9 1.8 1.7

conditions (20 °C), corresponding to a rather uniform increase in absolute isoform concentrations (Fig. 3). Concerning the relative contributions to protein amounts, there were only minor changes between 20 and 24 °C long-term-exposures. The largest rise was detected for Hb02, matching its increase observed on the transcript level (Fig. 4). 4. Discussion

Hb isoforms protein quantity (µmol/L)

1200

D

Hb10 Hb08 Hb07 Hb04 Hb03 Hb02 Hb01

1000 800 600 400 200 0

normoxia (20 kPa O2)

hypoxia (2 kPa O2)

Fig. 1. Hypoxic induction of Daphnia pulex haemoglobin. The concentration of isoforms detected in 2D gels under normoxic (A) or hypoxic conditions (B) (20 kPa or 2 kPa O2 at 20 °C) was calculated from 2D gels of Hb solutions of known concentrations. Please note the different scales on the y-axes. The induction factor (C) and the contribution of each isoform to the total amount of haemoglobin (D) were calculated from these data. Asterisks indicate significant changes in relation to control conditions (20 °C, normoxia) (n=3).

occurred on the protein level (Fig. 4B). While the isoforms Hb01 and Hb02 dominated under normoxia at both temperatures, they only contributed to about 5% under hypoxia. At low oxygen conditions, the protein subunits Hb03 and Hb07 were most prominent, followed by Hb04, Hb10, and Hb08. Thus, hypoxic induction evoked major changes in the isoform pattern on the protein level. Data on Hb gene transcripts from hypoxic animals are still lacking. The changes resulting from longterm-incubation at 24 °C were small for most Hb types on the transcript level. Hb02 raised its relative share most strongly, whereas Hb04 and Hb08 contributed less to the Hb mRNA pool at 24 °C. Thus, permanently elevated temperature caused the share of the isoforms Hb01 and Hb02 to increase, which were already the most prominent isoforms under control conditions. Upon acute heat stress, the relative share of Hb mRNAs changed only to a minor extent compared to acclimation

Functional isoform multiplicity [18] allows for adjustments in response to altered environmental conditions. In Daphnia, duplication events leading to extensions of gene families were observed to a larger extent than in other organisms, which may be one decisive feature to the success of these animals in variable habitats (described as “the eco-responsive genome” of D. pulex [6]. The presence of genes coding for several isoforms subjected to changes within the coding sequences during evolution may provide the basis for alterations of functional characteristics, whereas changes of intergenic regions upstream of the coding sequences may affect regulatory processes, altering the expression pattern of the isoform suite. Evidence for both processes can be found in case of Daphnia haemoglobin. 4.1. Adjustment of functional properties The affinity of D. pulex Hb for its ligand oxygen is affected by hypoxic acclimation. In animals raised at 20 °C and normoxia, the oxygen partial pressure necessary for half-maximal loading of Hb O2-binding sites (P50) is 1.99 kPa. For Hb from hypoxia-acclimated animals, a P50 of 0.17 kPa was determined, indicating a major increase in oxygen affinity [12]. Haemoglobin of animals kept at 24 °C and normoxia shows a P50 of 1.46 kPa, thus, the affinity is increased also at elevated temperatures, but the change is less severe than in case of hypoxia-acclimation [12]. These functional adjustments are correlated to changes in expression patterns of Hb subunits. The constitutive subunits most prominent in the hemolymph at 20 °C and normoxia are Hb01 and Hb02, whereas hypoxic induction leads to a Hb pool that is dominated by isoforms Hb03, Hb07, Hb10, Hb04, and Hb08, respectively (Fig. 1). Although oxygen affinity has not been studied for individual subunits yet, it must be assumed that the changes in affinity result from enhanced amounts of the hypoxia-induced subunits. In animals raised at 24 °C, the amount of subunits that are expected to have higher oxygen affinity increases only slightly, which is in line with a moderate change in affinity in this case.

B. Zeis et al. / Biochimica et Biophysica Acta 1834 (2013) 1704–1710

40000

A

20°C

60

1707

E

20°C

50 40 30

20000

protein quantity (µmol/L)

mRNA quantity (relative units)

30000

10000 0 40000

*

B

24°C

30000

20 10 0 60 50

*

F *

40

*

30

20000

24°C

20 10000

0

0

C

4

0

1

-1

-2 1

2

3

4

5

6

7

8

induction (log2)

2

G

4

1

2

0

1

0.5

-1

0.25

-2

0.5

0.25

1

9 10 11

2

D

160000

Hb11 Hb10 Hb09 Hb08 Hb07 Hb06 Hb05 Hb04 Hb03 Hb02 Hb01

120000 80000 40000

3

4

5

6

7

8

9 10 11

Hb isoforms 160

protein quantity (µmol/L)

mRNA quantity (relative units)

Hb isoforms 200000

induction factor

1

2

induction factor

induction (log2)

2

*

10

140

H

120

Hb11 Hb10 Hb09 Hb08 Hb07 Hb06 Hb05 Hb04 Hb03 Hb02 Hb01

100 80 60 40 20 0

0 20

24

acclimation temperature (°C)

20

24

acclimation temperature (°C)

Fig. 2. Effect of chronic heat stress on Daphnia pulex haemoglobin on the transcript and protein levels. Animals were permanently kept under control conditions (20 °C) or at elevated temperature (24 °C) under normoxic conditions. (A and B) mRNA quantity was calculated from fluorescence intensity (given in arbitrary units) for all Hb genes. (E and F) Protein quantity was calculated from 2D gels of Hb solutions of specific concentration. (C and G) Induction factors were determined for each isoform, and the contribution of each isoform to the total amount of haemoglobin mRNA (D) or protein (H) was specified. Asterisks indicate significant changes in relation to control conditions (20 °C, normoxia) (n = 3 for protein, n = 4 for mRNA).

4.2. Regulation of expression patterns As a prerequisite for the observed differential isoform expression, the regulatory elements upstream of the Hb genes must show a deviating inventory of transcription factor binding sites. Indeed, hypoxia-responsive elements (HREs) as binding sites for the hypoxia-inducible factor (HIF) are present in the intergenic regions of the Hb gene cluster in D. magna and D. pulex [6,11,12]. Moreover, the amount and position of these DNA motifs are specific for each isoform, its number ranging from zero (Hb09) to eight (Hb10) in D. pulex (Table 1). Unfortunately, for both isoforms, an induction factor could not be calculated for the protein amounts, as Hb09 was not found on the protein level at all, and Hb10 was not detected in animals raised under normoxic conditions. But a

very high induction was observed for this latter subunit, as it increased from below detection level to 144 μmol/L (15% of the Hb pool) in response to hypoxia. A correlation of the number of HREs and hypoxic induction was also absent for other isoforms. For instance, a strong induction was observed for Hb07 with three HIF binding sites (induction factor of 56.8), which is identical to the number of HREs in case of Hb01 and Hb02, the constitutive isoforms that respond to hypoxia only by a 2.5- and 2.2-fold increase (Table 1). Moreover, the highest hypoxiainduced increase was found for Hb08 with two HREs in the upstream region. It seems that isoforms of genes, which are silent or expressed only in minor amounts under normoxic conditions, have the largest induction potential, whereas isoforms already transcribed under normoxic conditions show less potential for HIF induction, which might be due

B. Zeis et al. / Biochimica et Biophysica Acta 1834 (2013) 1704–1710

45000

A

Hb01 Hb02

*

Hb03 Hb04

30000

Hb05 Hb06 Hb07 Hb08 Hb09

15000

Hb10 Hb11

0

0

2

4

6

8

10

time (h) 2

B

8 h 30°C vs 20°C

1

2

3

4

5

6

7

8

9 10 11

0.5

mRNA quantity (relative units)

Hb isoforms 300000

A

normoxia, 20°C acclimation to normoxia, 24°C acute heat stress, 8 h at normoxia, 30°C

15

10

5

0

protein, relative contribution (%)

1

0

-1

20

40

induction factor

induction (log2)

1

mRNA, relative contribution (%)

mRNA quantity (relative units)

1708

C

B

normoxia, 20°C hypoxia, 20°C normoxia, 24°C

30

20

10

0 1

250000

Hb11 Hb10 Hb09 Hb08 Hb07 Hb06 Hb05 Hb04 Hb03 Hb02 Hb01

200000 150000 100000 50000 0 0

2

4

6

8

10

2

3

4

5

6

7

8

9

10

11

Hb isoforms Fig. 4. Effect of hypoxia, chronic or acute heat stress on the relative contribution of Daphnia pulex haemoglobin isoforms on the transcript and protein levels. (A) The share (in percent) of each mRNA subtype was calculated for control conditions (20 °C, 20 kPa, black columns), permanently elevated temperature (24 °C, 20 kPa, dark grey columns) and after 8 h of severe heat stress (30 °C, 20 kPa, grey columns). (B) The share (in percent) of each protein isoform was calculated for control conditions (20 °C, 20 kPa, black columns), hypoxia (2 kPa, 20 °C, grey columns) and permanently elevated temperature (24 °C, 20 kPa, dark grey columns) (n=3 for protein, n=4 for mRNA).

time (h) Fig. 3. Effect of acute heat stress on Daphnia pulex haemoglobin on the transcript level. Animals were transferred from control conditions (20 °C, t=0) to 30 °C at nomoxic conditions and mRNA was quantified after 2, 4 and 8 h. mRNA quantity was calculated from fluorescence intensity (given in arbitrary units) for all Hb genes (A). The respective induction factor was determined (B). The contribution of each isoform to the total amount of haemoglobin mRNA was calculated (C). Asterisk indicates significant changes in relation to control conditions (20 °C, normoxia) for six isoforms (bar) (n=4).

to interfering effects of other regulating elements [19]. Thus, the role of HIF binding, the position of binding sites and possible interactions with other transcription factors [11,19] still have to be elucidated.

underlining the close relation of heat stress and cellular oxygen shortage. HIF concentration is directly related to oxygen concentration [21], moreover, the continuous HIF degradation under normoxia is disturbed by ROS and changes in the cellular redox state [22], parameters which are also affected by elevated temperature in Daphnia [23]. As a result of HIF-based Hb induction following acute temperature stress, oxygen transport capacity is enhanced, restoring the oxygen supply of the cells. At temperatures below critical thresholds, Hb induction in Daphnia is an example of a stressor-specific homeostasis response [24]. This second line of stress defense results in restored cellular oxygen conditions. Once the enhanced oxygen demand of the cells is sufficiently supplied, HIF levels are decreased again.

4.3. Acute heat stress resembles hypoxia 4.4. Temperature acclimation restores oxygen homeostasis When the animals were challenged by acute heat stress (transfer from 20 °C to 30 °C at normoxia), significant induction of Hb transcripts was observed (Fig. 3). The mRNAs for the constitutive subunits (Hb01, Hb02 and Hb03) increased as well as the hypoxia-inducible subunits; Hb10, Hb04 and Hb07 were affected to the largest extent (induction factors of 1.9 or 1.7 within 8 h, Fig. 3, Table 1). Thus, the isoforms most responsive to hypoxia are induced upon exposure to severe heat as well, indicating disturbed oxygen supply to the cells at 30 °C. Temperature affected HIF levels also in another invertebrate species: Caenorhabditis elegans HIF loss-of-function mutants lost the ability to acclimate to elevated temperatures, whereas animals overexpressing HIF gained heat tolerance [20]. Even a cross tolerance of heat-acclimated animals towards hypoxia was described [20],

In D. pulex exposed to 30 °C, however, a successful return to cellular homeostasis is evidently impossible, as animals do not grow or reproduce under these conditions and die after several days. But persisting exposure to moderately elevated temperatures (24 °C) apparently enables complete return to cellular homeostasis, due to adjusted oxygen transport capacity. Oxygen consumption at 24 °C increased by 53% compared to 20 °C in D. pulex (Zeis and Paul, unpublished data). The long-term process of acclimation results in higher amounts of the constitutive subunits (Hb01, Hb02), as a result, the oxygen transport matches the increased demand. The low amount of hypoxia-inducible isoforms under these conditions indicates HIF levels similar to control conditions and suggests that oxygen

B. Zeis et al. / Biochimica et Biophysica Acta 1834 (2013) 1704–1710

shortage occurred only transiently. Acclimation to moderately elevated temperatures thus affects Hb isoforms different from those responding to acute heat stress.

4.5. Correlation of mRNA and protein Comparing the adjustments in response to temperature increase for both, transcripts and proteins, we found that the most prominent isoforms match on both levels. The constitutive subunits Hb01 and Hb02 are present in high concentrations and show similar temperatureinduced increases for mRNA and protein (Fig. 2). Isoforms Hb06 and Hb11 are absent or present in minor amounts at 20 °C and 24 °C both on the messenger and protein level. For Hb06 and Hb09, N-terminal signal peptides are lacking, suggesting intracellular location; accordingly, their products would not be detected in the hemolymph analysed in this study. Striking differences were obvious, however, concerning the other isoforms. For subunit Hb05, which carries a signal peptide for extracellular location, the protein was not detected despite high levels of mRNA. This contrast between transcript and protein level remains unsolved. Similarly, the subunits Hb04, Hb07 and Hb08 were present in low amounts on the protein level, although their mRNA amount is in a comparable range as that of the dominant isoforms. For these isoforms, a strong induction on the protein level occurred despite almost unchanged amount of mRNA during temperature acclimation. Similarly, overall Hb concentration increased by 80%, although mRNA amounts only increased by 2%. Thus, the effect of temperature on the translation of Hb transcripts into protein showed specific differences between isoforms. Isoform-specific stability of transcripts or proteins might contribute to the observed variations of temperature effects. Although there is a high level of similarity between Hb isoforms, (52–80% for nucleotides, 53–95% for proteins), differences in turnover rates of transcripts or proteins seem possible. Generally, acceleration of metabolic processes at elevated temperatures may cause faster protein synthesis rates from only slightly increased mRNA amounts. A concomitant reduction of protein degradation could result in higher steady-state concentrations of proteins. Proteases are indeed down-regulated in response to acute temperature stress (Becker et al., unpublished results), but they increased upon long-term acclimation to elevated temperatures [25] (Becker et al., unpublished results). Consequently, the reasons for deviating changes on both levels require further investigations. In summary, the attempt to compare transcript and protein amounts in this study provides another example about poor correlation between both levels, as has been described earlier [26], in particular with respect to regulatory processes involving HIF [27].

4.6. Conclusions D. pulex adjusted its equipment of oxygen transport proteins to environmental challenges linked to reduced oxygen supply. The response to acute temperature stress (30 °C) involved predominantly the hypoxia-inducible Hb isoforms, resulting in an increase of the Hb mRNA pool by 70% within 8 h. Long-term acclimation to moderately elevated temperature (24 °C) resulted only in slight shifts in the contribution of isoforms to the Hb mRNA suite, leaving the total amount of Hb transcripts almost unchanged. Nevertheless, the Hb concentration in the hemolymph was increased by 80%. In this case, the constitutive Hb isoforms were most strongly affected, with Hb01 and Hb02 adding to 64% of the total amount of respiratory protein. The regulation patterns upon acute temperature increase reflects temperature-induced tissue hypoxia, whereas in case of persisting exposure to moderately elevated temperature, acclimation processes enable the successful return to oxygen homeostasis.

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Acknowledgements The authors want to thank Ulrike Gigengack and Ina Buchen for the help in raising the animals. We gratefully acknowledge the contributions of John K. Colbourne, Jacqueline A. Lopez and Craig Jackson in performing and evaluating the transcriptomic analyses. Our work benefits from and contributes to the Daphnia Genomics Consortium http://daphnia.cgb.indiana.edu.

Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbapap.2013.01.036.

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