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Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane.

Day PM, Potter D, Inoue K - Front Plant Sci (2014)

Bottom Line: Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent.The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80.Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, University of California at Davis Davis, CA, USA.

ABSTRACT
Translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75) is the core component of the chloroplast protein import machinery. It belongs to the Omp85 family whose members exist in various Gram-negative bacteria, mitochondria, and chloroplasts of eukaryotes. Chloroplasts of Viridiplantae contain another Omp85 homolog called outer envelope protein 80 (OEP80), whose exact function is unknown. In addition, the Arabidopsis thaliana genome encodes truncated forms of Toc75 and OEP80. Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent. The bipartite targeting sequence-dependent sorting of Toc75 has been demonstrated but the targeting mechanisms of other chloroplast Omp85 homologs remain largely unexplored. This study was aimed to address these unresolved issues in order to further our understanding of chloroplast evolution. Sequence alignments and recently determined structures of bacterial Omp85 homologs were used to predict structures of chloroplast Omp85 homologs. The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80. Phylogenetic analyses using Omp85 homologs from various cyanobacteria and chloroplasts provided strong support for the grouping of Toc75 and OEP80 sister to cyanoOmp85. However, this support was diminished when the analysis included Omp85 homologs from other bacteria and mitochondria. Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic trees of Omp85 homologs. Sequences of chloroplast Omp85 homologs (Toc75, OEP80, and OEP80tr), cyanoOmp85, and outgroups from other bacteria and mitochondria were aligned with MAFFT. Phylogenetic inference was done using Bayesian inference. Each tree shown is the 50% majority-rule consensus tree generated for the 3002 trees produced by two runs of two million generations each, sampled every 1000 generations with a 25% burn in. (A) Consensus tree using an alignment of amino acid sequences corresponding to residues 438 to 818 of A. thaliana Toc75 with poorly aligned regions removed by Gblocks. Numbers at nodes represent the proportion of trees in which a clade appeared, interpreted as the posterior probability (PP) of that clade. Discrepancies between our trees and the generally excepted plant species relationships are indicated as lower-case roman numerals (i–iii). (B) Consensus tree using an alignment of amino acid sequences corresponding to residues 648–818 of A. thaliana Toc75 with no interior section removed. (C) Consensus tree using an alignment of amino acid sequences excluding outgroup sequences corresponding to residues 438 to 818 of A. thaliana Toc75. The tree showed the chloroplast Omp85 homologs (*) nesting within cyanobacteria (#).
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Figure 3: Phylogenetic trees of Omp85 homologs. Sequences of chloroplast Omp85 homologs (Toc75, OEP80, and OEP80tr), cyanoOmp85, and outgroups from other bacteria and mitochondria were aligned with MAFFT. Phylogenetic inference was done using Bayesian inference. Each tree shown is the 50% majority-rule consensus tree generated for the 3002 trees produced by two runs of two million generations each, sampled every 1000 generations with a 25% burn in. (A) Consensus tree using an alignment of amino acid sequences corresponding to residues 438 to 818 of A. thaliana Toc75 with poorly aligned regions removed by Gblocks. Numbers at nodes represent the proportion of trees in which a clade appeared, interpreted as the posterior probability (PP) of that clade. Discrepancies between our trees and the generally excepted plant species relationships are indicated as lower-case roman numerals (i–iii). (B) Consensus tree using an alignment of amino acid sequences corresponding to residues 648–818 of A. thaliana Toc75 with no interior section removed. (C) Consensus tree using an alignment of amino acid sequences excluding outgroup sequences corresponding to residues 438 to 818 of A. thaliana Toc75. The tree showed the chloroplast Omp85 homologs (*) nesting within cyanobacteria (#).

Mentions: We first used the alignment of sequences corresponding to residues 438–818 of A. thaliana Toc75. This region includes P3-β3 and the entire transmembrane β-barrel, which was shown to be conserved well among various homologs (Reumann et al., 1999). To increase the reliability of the analysis, we removed ambiguously aligned regions using the Gblocks server (Castresana, 2000). Bayesian inference using this alignment provided strong support for a monophyletic relationship of the chloroplast Omp85 homologs and cyanoOmp85 (PP = 0.908). The analysis grouped OEP80 with cyanoOmp85 although the support for this grouping was low (PP = 0.561) (Figure 3A). The Toc75 orthologs from the green lineages, but not those from red algae, were placed sister to this clade although support for this relationship was also low (PP = 0.676). Similar to the previous report, we were able to identify only one chloroplast Omp85 homolog, which belongs to the Toc75 group, from each of the two Rhodophyta species (Topel et al., 2012) (Figure 3A). The tree topology was not entirely consistent with the established organismal classification. Within the Toc75 group, five sequences from the chlorophyte lineage were separated into three distinct groups [Figure 3A, indicated by (i)], and the sequence from the basal angiosperm Amborella trichopoda (Amborella Genome, 2013) was grouped with those from eudicots rather than being sister to the rest of the angiosperms [Figure 3A, indicated by (ii)]. In addition, sequences from the moss (Physicomitrella patens) and liverwort (Marchantia polymorpha) were grouped together as a clade sister to all vascular plant sequences [Figure 3A, indicated by (iii)]. In the case of the OEP80 clade, the location of the A. trichopoda sequence was consistent with the organismal classification. However, those of the algal sequences were not; instead they formed two groups. Interestingly, sequences from the moss, liverwort, a lycophyte (Selaginella moellendorffii), a streptophyte green alga (Nitella mirabilis) and three gymnosperms [white spruce (Picea glauca), loblolly pine (Pinus taeda), and Douglas fir (Pseudotsuga menziesii)] formed a clade sister to angiosperm non-truncated OEP80 sequences [Figure 3A, indicated by (iii)]. This result does not appear to follow the generally accepted relationships where (a) gymnosperms are grouped with angiosperms forming the seed plants, (b) lycophytes are grouped within vascular plants, (c) mosses are more closely related to vascular plants than to liverworts, and (d) land plants form a monophyletic group (Bowman, 2013). This may indicate large changes within the angiosperms and the absence of the species representing the intermediate state.


Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane.

Day PM, Potter D, Inoue K - Front Plant Sci (2014)

Phylogenetic trees of Omp85 homologs. Sequences of chloroplast Omp85 homologs (Toc75, OEP80, and OEP80tr), cyanoOmp85, and outgroups from other bacteria and mitochondria were aligned with MAFFT. Phylogenetic inference was done using Bayesian inference. Each tree shown is the 50% majority-rule consensus tree generated for the 3002 trees produced by two runs of two million generations each, sampled every 1000 generations with a 25% burn in. (A) Consensus tree using an alignment of amino acid sequences corresponding to residues 438 to 818 of A. thaliana Toc75 with poorly aligned regions removed by Gblocks. Numbers at nodes represent the proportion of trees in which a clade appeared, interpreted as the posterior probability (PP) of that clade. Discrepancies between our trees and the generally excepted plant species relationships are indicated as lower-case roman numerals (i–iii). (B) Consensus tree using an alignment of amino acid sequences corresponding to residues 648–818 of A. thaliana Toc75 with no interior section removed. (C) Consensus tree using an alignment of amino acid sequences excluding outgroup sequences corresponding to residues 438 to 818 of A. thaliana Toc75. The tree showed the chloroplast Omp85 homologs (*) nesting within cyanobacteria (#).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4195282&req=5

Figure 3: Phylogenetic trees of Omp85 homologs. Sequences of chloroplast Omp85 homologs (Toc75, OEP80, and OEP80tr), cyanoOmp85, and outgroups from other bacteria and mitochondria were aligned with MAFFT. Phylogenetic inference was done using Bayesian inference. Each tree shown is the 50% majority-rule consensus tree generated for the 3002 trees produced by two runs of two million generations each, sampled every 1000 generations with a 25% burn in. (A) Consensus tree using an alignment of amino acid sequences corresponding to residues 438 to 818 of A. thaliana Toc75 with poorly aligned regions removed by Gblocks. Numbers at nodes represent the proportion of trees in which a clade appeared, interpreted as the posterior probability (PP) of that clade. Discrepancies between our trees and the generally excepted plant species relationships are indicated as lower-case roman numerals (i–iii). (B) Consensus tree using an alignment of amino acid sequences corresponding to residues 648–818 of A. thaliana Toc75 with no interior section removed. (C) Consensus tree using an alignment of amino acid sequences excluding outgroup sequences corresponding to residues 438 to 818 of A. thaliana Toc75. The tree showed the chloroplast Omp85 homologs (*) nesting within cyanobacteria (#).
Mentions: We first used the alignment of sequences corresponding to residues 438–818 of A. thaliana Toc75. This region includes P3-β3 and the entire transmembrane β-barrel, which was shown to be conserved well among various homologs (Reumann et al., 1999). To increase the reliability of the analysis, we removed ambiguously aligned regions using the Gblocks server (Castresana, 2000). Bayesian inference using this alignment provided strong support for a monophyletic relationship of the chloroplast Omp85 homologs and cyanoOmp85 (PP = 0.908). The analysis grouped OEP80 with cyanoOmp85 although the support for this grouping was low (PP = 0.561) (Figure 3A). The Toc75 orthologs from the green lineages, but not those from red algae, were placed sister to this clade although support for this relationship was also low (PP = 0.676). Similar to the previous report, we were able to identify only one chloroplast Omp85 homolog, which belongs to the Toc75 group, from each of the two Rhodophyta species (Topel et al., 2012) (Figure 3A). The tree topology was not entirely consistent with the established organismal classification. Within the Toc75 group, five sequences from the chlorophyte lineage were separated into three distinct groups [Figure 3A, indicated by (i)], and the sequence from the basal angiosperm Amborella trichopoda (Amborella Genome, 2013) was grouped with those from eudicots rather than being sister to the rest of the angiosperms [Figure 3A, indicated by (ii)]. In addition, sequences from the moss (Physicomitrella patens) and liverwort (Marchantia polymorpha) were grouped together as a clade sister to all vascular plant sequences [Figure 3A, indicated by (iii)]. In the case of the OEP80 clade, the location of the A. trichopoda sequence was consistent with the organismal classification. However, those of the algal sequences were not; instead they formed two groups. Interestingly, sequences from the moss, liverwort, a lycophyte (Selaginella moellendorffii), a streptophyte green alga (Nitella mirabilis) and three gymnosperms [white spruce (Picea glauca), loblolly pine (Pinus taeda), and Douglas fir (Pseudotsuga menziesii)] formed a clade sister to angiosperm non-truncated OEP80 sequences [Figure 3A, indicated by (iii)]. This result does not appear to follow the generally accepted relationships where (a) gymnosperms are grouped with angiosperms forming the seed plants, (b) lycophytes are grouped within vascular plants, (c) mosses are more closely related to vascular plants than to liverworts, and (d) land plants form a monophyletic group (Bowman, 2013). This may indicate large changes within the angiosperms and the absence of the species representing the intermediate state.

Bottom Line: Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent.The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80.Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, University of California at Davis Davis, CA, USA.

ABSTRACT
Translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75) is the core component of the chloroplast protein import machinery. It belongs to the Omp85 family whose members exist in various Gram-negative bacteria, mitochondria, and chloroplasts of eukaryotes. Chloroplasts of Viridiplantae contain another Omp85 homolog called outer envelope protein 80 (OEP80), whose exact function is unknown. In addition, the Arabidopsis thaliana genome encodes truncated forms of Toc75 and OEP80. Multiple studies have shown a common origin of the Omp85 homologs of cyanobacteria and chloroplasts but their results about evolutionary relationships among cyanobacterial Omp85 (cyanoOmp85), Toc75, and OEP80 are inconsistent. The bipartite targeting sequence-dependent sorting of Toc75 has been demonstrated but the targeting mechanisms of other chloroplast Omp85 homologs remain largely unexplored. This study was aimed to address these unresolved issues in order to further our understanding of chloroplast evolution. Sequence alignments and recently determined structures of bacterial Omp85 homologs were used to predict structures of chloroplast Omp85 homologs. The results enabled us to identify amino acid residues that may indicate functional divergence of Toc75 from cyanoOmp85 and OEP80. Phylogenetic analyses using Omp85 homologs from various cyanobacteria and chloroplasts provided strong support for the grouping of Toc75 and OEP80 sister to cyanoOmp85. However, this support was diminished when the analysis included Omp85 homologs from other bacteria and mitochondria. Finally, results of import assays using isolated chloroplasts support outer membrane localization of OEP80tr and indicate that OEP80 may carry a cleavable targeting sequence.

No MeSH data available.


Related in: MedlinePlus