Comparison of the chloroplast peroxidase system in the chlorophyte Chlamydomonas reinhardtii, the bryophyte Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana

Abstract

Background: Oxygenic photosynthesis is accompanied by the formation of reactive oxygen species (ROS), which damage proteins, lipids, DNA and finally limit plant yield. The enzymes of the chloroplast antioxidant system are exclusively nuclear encoded. During evolution, plastid and mitochondrial genes were post-endosymbiotically transferred to the nucleus, adapted for eukaryotic gene expression and post-translational protein targeting and supplemented with genes of eukaryotic origin.

Results: Here, the genomes of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii and the seed plant Arabidopsis thaliana were screened for ORFs encoding chloroplast peroxidases. The identified genes were compared for their amino acid sequence similarities and gene structures. Stromal and thylakoid-bound ascorbate peroxidases (APx) share common splice sites demonstrating that they evolved from a common ancestral gene. In contrast to most cormophytes, our results predict that chloroplast APx activity is restricted to the stroma in Chlamydomonas and to thylakoids in Physcomitrella. The moss gene is of retrotransposonal origin.The exon-intron-structures of 2CP genes differ between chlorophytes and streptophytes indicating an independent evolution. According to amino acid sequence characteristics only the A-isoform of Chlamydomonas 2CP may be functionally equivalent to streptophyte 2CP, while the weakly expressed B- and C-isoforms show chlorophyte specific surfaces and amino acid sequence characteristics. The amino acid sequences of chloroplast PrxII are widely conserved between the investigated species. In the analyzed streptophytes, the genes are unspliced, but accumulated four introns in Chlamydomonas. A conserved splice site indicates also a common origin of chlorobiont PrxQ.The similarity of splice sites also demonstrates that streptophyte glutathione peroxidases (GPx) are of common origin. Besides a less related cysteine-type GPx, Chlamydomonas encodes two selenocysteine-type GPx. The latter were lost prior or during streptophyte evolution.

Conclusion: Throughout plant evolution, there was a strong selective pressure on maintaining the activity of all three investigated types of peroxidases in chloroplasts. APx evolved from a gene, which dates back to times before differentiation of chlorobionts into chlorophytes and streptophytes, while Prx and presumably also GPx gene patterns may have evolved independently in the streptophyte and chlorophyte branches.

Figure 1

Comparison of ascorbate peroxidase amino acid sequences. A: Amino acid sequence alignment of ascorbate peroxidases (APx) from Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). The label "H₂O₂" marks the H₂O₂-binding site, "C" the amino acids involved in formation of the catalytic site, "P" the proximal and "D" the distal cation binding site and "H" the heme binding amino acids. B: Phylogramme of APx proteins. The proteins depicted in Fig. 1A are marked in red. They are compared to all putative full-length organellar APx listed in PeroxiBase and a selection of extra-organellar APx. The tree was calculated based on the neighborhood joining algorithm. Additional in PeroxiBase predicted, but not in Fig. 1A listed APx from Arabidopsis thaliana, Physcomitrella patens and Chlamydomonas reinhardtii are labeled in green. For all PeroxiBase-data the data base IDs are presented in the labels. The numbers represent bootstrap values for the branches as calculated based on 500 bootstraps. Maximum parsimony and minimum evolution trees are shown in the additional files 1 and 2.

Figure 2

Relative exon and intron lengths of chloroplast APx in Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). tAPx are shown in red, sAPX in black. The vertical lines connect corresponding splice sites. The numbers represent positions of corresponding amino acids in the alignment shown in fig. 1A and the relative splice sites within the corresponding codon.

Figure 3

PCR amplification of full-length genomic DNA (gDNA) and cDNA fragments encoding the predicted stromal and thylakoid-bound ascorbate peroxidases in Chlamydomonas reinhardtii, Physomitrella patens and Selaginella moellendorffii proving the predicted cDNA lengths.

Figure 4

Superimposition of AtsAPx (yellow) and CrsAPxA (green) structures. The two loops formed by the insertions aa²⁴⁴-aa²⁴⁸ (EVaLo I) and aa³⁰³-aa³²¹ (EVaLO II) are shown in red.

Figure 5

Comparison of 2-Cys peroxiredoxin amino acid sequences. A: Amino acid sequence alignment of 2-Cys peroxiredoxins (2CP) from Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). The peroxidatic and resolving cysteine residues are labeled with "*", "T" indicates the amino acid residues involved in decamer formation and "C" the residues forming the catalytic site. B: Phylogramme of the 2CP sequences shown in Fig. 5A (red) and additional 2CP from chlorobionts and cyanobacteria as listed in PeroxiBase [96]. The tree was calculated based on the neighborhood joining algorithm. For all PeroxiBase-data the data base IDs are presented in the labels. The numbers represent bootstrap values. Maximum parsimony and minimum evolution trees are shown in the additional files 3 and 4.

Figure 6

Proportional comparison of the gene structures of 2CP in Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). ESTcovered 2CP genes are shown in green, putatively non-expressed in blue. The vertical lines connect corresponding splice sites. The numbers represent positions of corresponding amino acids in the alignment shown in Fig. 5A and the relative splice sites within the corresponding codon.

Figure 7

Superimposition of At2CPA (yellow) and Cr2CPB (green) monomers. The three amino acid insertion (aa²⁵⁸ - aa²⁶⁰) extends the length of a β-sheet and modifies the protein surface. In the decameric toroid structure (right) the three EVaLo-s modify the inner and outer ring surface (red-green labels in the right figure).

Figure 8

Comparison of peroxiredoxin Q amino acid sequences. A: Amino acid sequence alignment of the here analyzed PrxQ from Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). B: Phylogramme of the PrxQ sequences shown in Fig. 8A (red) and putative full-length PrxQ sequences of chlorobiont and cyanobacterial origin as listed in PeroxiBase [96]. The tree was calculated based on the neighborhood joining algorithm. The numbers represent bootstrap values. Maximum parsimony and minimum evolution trees are shown in the additional files 5 and 6.

Figure 9

Comparison of chloroplast type-II peroxiredoxin amino acid sequences. A: Amino acid sequence alignment of PrxII from Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). B: Phylogramme of the PrxII sequences shown in Fig. 11A (red) and a selection of PrxII full length sequences listed in PeroxiBase [96]. A Chlamydomonas reinhardtii of uncertain location, which is listed in PeroxiBase, but not in our analysis is labeled in green. For all PeroxiBasedata the data base IDs are presented in the labels. The tree was calculated based on the neighborhood joining algorithm. The numbers represent bootstrap values. Maximum parsimony and minimum evolution trees are shown in the additional files 7 and 8.

Figure 10

Gene structures of PrxQ in Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). Expressed PrxQ genes are shown in green, non-expressed in blue. The vertical lines connect corresponding splice sites. The numbers represent positions of corresponding amino acids in the alignment shown in Fig. 8A the relative splice site within the corresponding codon.

Figure 11

PrxII gene structures of Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). Expressed PrxII genes are shown in green. The vertical lines connect corresponding splice sites. The numbers represent position of corresponding amino acids in the alignment shown in Fig. 11 A and the relative splice sites within the corresponding codon.

Figure 12

Superimposition of AtPrxQ (yellow) and SmPrxQA. 1 (pink) and AtPrxQ (yellow) and CrPrxQ (green) in two views. The positions of the three and five flexible elements on the protein surface are numbered in white for better comparison. The peroxidatic and resolving C are labeled in blue.

Figure 13

Superimposition of AtPrxIIE (yellow) and SmPrxII.1 (pink) showing the peroxidatic and resolving C residues in blue and nine flexible loops (labeled with white numbers).

Figure 14

Comparison of glutathione peroxidase amino acid sequences. A: Amino acid sequence alignment of GPx from Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). B: Phylogramme of the GPx sequences shown in Fig. 14A (red) and a selection of plant GPx full length sequences listed in PeroxiBase [96]. For all PeroxiBase-data the data base IDs are presented in the labels. The tree was calculated based on the neighborhood joining algorithm. The numbers represent bootstrap values. Maximum parsimony and minimum evolution trees are shown in the additional files 9 and 10.

Figure 15

GPx gene structures of Arabidopsis thaliana (At), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Chlamydomonas reinhardtii (Cr). Expressed GPx genes are shown in green, putatively non-expressed in blue. The vertical lines connect corresponding splice sites. The numbers represent position of corresponding amino acids in the alignment shown in Fig. 14A and the relative splice sites within the corresponding codon.

Figure 16

PCR amplification of cDNA fragments encoding the predicted glutathione peroxidases in Chlamydomonas reinhardtii, Physomitrella patens and Selaginella moellendorffii. Samples with unspecific bands are labeled with an asterisk.

Figure 17

Superimposition of AtGPx1 (yellow) and (A) AtGPx6 (red), (B) SmGPxB.1 (pink), (C) PpGPxB (light blue) and (D) CrGPxC (green). The catalytic sites are labeled in blue. The arrows point at the helices 1 and 2 for which the structures of the presented proteins differ from the AtGPx1/AtGPx7 structure.