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First published online May 24, 2005
Journal of Experimental Biology 208, 2157-2165 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01571
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Symbiotic Hydra express a plant-like peroxidase gene during oogenesis

Matthias Habetha and Thomas C. G. Bosch*

Zoological Institute, Christian-Albrechts-University Kiel, Olshausenstrasse 40, 24098 Kiel, Germany



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  		Fig. 1. (A) Schematic diagram of the Hydra viridis HvAPX1 mRNA depicting the region coding for the plant peroxidase domain and other putative structural elements. UTR, untranslated region; SigP, ER signal peptide. (B) Multiple amino acid sequence comparison of the plant peroxidase domain (amino acids 48-229) of HvAPX1 with the plant ascorbate peroxidases from Zantedischia aethiopica, AAC085761, (Magnoliaceae), Euglena gracilis, BAC054841 (Euglenida), Galdieria partita, BAC411991 (Rhodophyceae). NCBI BLAST search ranked these plant peroxidases highest in amino acid scores. Additionally ascorbate peroxidase from symbiotic Chlorella, which is in the aligned region identical to free living Chlorella 211-11b is shown. Comparison was carried out with ClustalW under standard parameter settings. Similarities between HvAPX1 and the other sequences are shaded. Residues that are conserved between Hydra and in at least three other sequences are shown in white capitals on a black background. Residues conserved between Hydra and two or one of the other sequences are shaded dark or light grey respectively. The number of conserved residues between HvAPX1 and the aligned sequences are as follows: Hydra/Zantedischia=74 (41%), the expect as given by NCBI BLAST analysis is 2e-09; Hydra/Euglena=73 (41%), expect=1e-08; Hydra/Galdieria=70 (39%), expect=1e-08; Hydra/Chlorella=65 (36%), expect=0.83. Five amino acids implicated in the redox activity of plant ascorbate peroxidases are located within the plant peroxidase domain of HvAPX1 (indicated by the letters R and H and one asterisk above the aligned blocks). Seven boxes (I-VII) of high similarity between HvAPX1 and all ascorbate peroxidases aligned by NCBI BLASTp are framed in grey.

 


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  		Fig. 2. Unrooted distance tree, depicting the relationships between Hydra viridis ascorbate peroxidase 1 and ascorbate peroxidase sequences from different protozoans including Trypanosoma cruzi (CAD300231), free living Chlorella vulgaris 211-11b, and symbiotic Chlorella from Hydra viridis strain A99. Related catalase sequences are from Danio rerio (Q9PT92), Homo sapiens (P04040), Drosophila melanogaster (NP_536731.1) and `putative' peroxidases PPOD1 (AAK564451) and PPOD2 (AAK564461) from Hydra vulgaris. Numbers on branches indicate bootstrap values.

 


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  		Fig. 3. Expression of HvAPX1 in symbiotic and aposymbiotic polyps. (A) RT-PCR. The quantity of the cDNA template was equalized using Hydra viridis actin specific primers. (B) Northern blot hybridisation of 8 µg total RNA from asexual, testes-bearing and hermaphroditic polyps as well as 4 µg of total RNA from symbiotic Chlorella with radiolabelled HvAPX1 revealing a transcript of about 1300 bases. Lower panel shows the amount of ribosomal RNA within the blotted samples. s, symbiotic polyps; a, aposymbiotic polyps; Chl, symbiotic Chlorella.

 


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  		Fig. 4. Spatiotemporal expression of HvAPX1 and ascorbate-dependent peroxidase activity in hermaphroditic H. viridis. (A-C) Whole-mount in situ hybridisation. (D,E) Cellular in situ hybridisation. (D) Early oocyte; (E) cells from the ovary region showing HvAPX1 expression in one of the interstitial cells. (F-I) Peroxidase activity in the absence (F) or presence of ascorbate as competing substrate at 1 mmol l-1 (G), 500 µmol l-1 (H), and 200 µmol l-1 (I). Scale bars: 100 µm (D) and 5 µm (E).

 


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  		Fig. 5. HvAPX1 in contrast to plant ascorbate peroxidases lacks introns. RT PCR (lanes 1,3,5) and genomic PCR amplification (lanes 2,4,6) of Chlorella vulgaris 211-11b and Hydra viridis ascorbate peroxidase. PCR was carried out with one set of primers against the Chlorella APX peroxidase domain (lanes 1,2), another set against the homologous region of HvAPX1 (lanes 3,4), and a third pair of primers flanking the complete open reading frame of HvAPX1 (lanes 5,6). PCR with primers flanking the peroxidase domain resulted in fragment sizes of 527 bp (lane 1) from Chlorella cDNA and of about 1000 bp from Chlorella genomic DNA as well as 523 bp (lane 3) and 1123 bp (lane 5) from Hydra cDNA and equally sized genomic fragments (lanes 4,6). M, size markers.

 

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© The Company of Biologists Ltd 2005