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Directed evolution and in silico analysis of reaction centre proteins reveal molecular signatures of photosynthesis adaptation to radiation pressure.

Rea G, Lambreva M, Polticelli F, Bertalan I, Antonacci A, Pastorelli S, Damasso M, Johanningmeier U, Giardi MT - PLoS ONE (2011)

Bottom Line: The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids.A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed.Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Crystallography, National Research Council, Monterotondo, Italy. giuseppina.rea@mlib.ic.cnr.it

ABSTRACT
Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earth's atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues more sensitive to oxidative damage in the eukaryotic/cyanobacterial proteins compared to their bacterial orthologs. These results led us to hypothesize an archaean atmosphere less challenging in terms of ionizing radiation than the present one.

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Comparison of the aminoacidic composition of R. spheroides L and C. reinhardtii D1 protein homologues.A) Comparison of the amino acid composition of R. spheroides L protein homologues (light gray columns), eukaryotic C. reinhardtii D1 protein homologues (white columns) and cyanobacterial C. reinhardtii D1 protein homologues (dark gray columns). Black bars indicate standard deviation values. B) Comparison of the amino acid composition of the QB binding region of R. spheroides L protein homologues (light gray columns) and C. reinhardtii D1 protein homologues (white columns). Black bars indicate standard deviation values. The asterisks indicate statistically significant differences between the Bacteria and Eukarya at p = 0.001.
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pone-0016216-g006: Comparison of the aminoacidic composition of R. spheroides L and C. reinhardtii D1 protein homologues.A) Comparison of the amino acid composition of R. spheroides L protein homologues (light gray columns), eukaryotic C. reinhardtii D1 protein homologues (white columns) and cyanobacterial C. reinhardtii D1 protein homologues (dark gray columns). Black bars indicate standard deviation values. B) Comparison of the amino acid composition of the QB binding region of R. spheroides L protein homologues (light gray columns) and C. reinhardtii D1 protein homologues (white columns). Black bars indicate standard deviation values. The asterisks indicate statistically significant differences between the Bacteria and Eukarya at p = 0.001.

Mentions: The observation that the strains selected under ionizing radiation displayed amino acid changes consistent with tolerance to radiation-induced oxidative damage prompted a comparative study of the D1 amino acid composition. Bacterial, cyanobacterial and eukaryotic L/D1 proteins were analyzed to uncover the molecular signatures of the PSII complex adaptation to the radiation-induced damage occurring during the evolution of photosynthesis. Using R. sphaeroides L and C. reinhardtii D1 proteins as baits, homologues were retrieved from the NCBI sequence database and their amino acid composition was determined and compared (Fig. 6A). Although these proteins are highly conserved, analysis of the data reveals that the content of aliphatic and aromatic residues is significantly higher in Bacteria. In fact, aliphatic and aromatic residues represent almost 70% of the total residues in bacterial RC L proteins, while in eukarya D1 proteins the same residues represent less than 60% of the total. In particular, the percentage of Trp residues in bacterial L proteins is almost twice than that in eukaryotic D1 proteins, while smaller but still significant differences were observed in other residues (such as Gly and Leu) highly prone to damage by reactive oxygen species. Not surprisingly, the amino acid composition of cyanobacterial D1 proteins, in line with the evolutionary conservation of D1 proteins, is practically identical to that of the eukaryotic ones (Fig. 6A).


Directed evolution and in silico analysis of reaction centre proteins reveal molecular signatures of photosynthesis adaptation to radiation pressure.

Rea G, Lambreva M, Polticelli F, Bertalan I, Antonacci A, Pastorelli S, Damasso M, Johanningmeier U, Giardi MT - PLoS ONE (2011)

Comparison of the aminoacidic composition of R. spheroides L and C. reinhardtii D1 protein homologues.A) Comparison of the amino acid composition of R. spheroides L protein homologues (light gray columns), eukaryotic C. reinhardtii D1 protein homologues (white columns) and cyanobacterial C. reinhardtii D1 protein homologues (dark gray columns). Black bars indicate standard deviation values. B) Comparison of the amino acid composition of the QB binding region of R. spheroides L protein homologues (light gray columns) and C. reinhardtii D1 protein homologues (white columns). Black bars indicate standard deviation values. The asterisks indicate statistically significant differences between the Bacteria and Eukarya at p = 0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020971&req=5

pone-0016216-g006: Comparison of the aminoacidic composition of R. spheroides L and C. reinhardtii D1 protein homologues.A) Comparison of the amino acid composition of R. spheroides L protein homologues (light gray columns), eukaryotic C. reinhardtii D1 protein homologues (white columns) and cyanobacterial C. reinhardtii D1 protein homologues (dark gray columns). Black bars indicate standard deviation values. B) Comparison of the amino acid composition of the QB binding region of R. spheroides L protein homologues (light gray columns) and C. reinhardtii D1 protein homologues (white columns). Black bars indicate standard deviation values. The asterisks indicate statistically significant differences between the Bacteria and Eukarya at p = 0.001.
Mentions: The observation that the strains selected under ionizing radiation displayed amino acid changes consistent with tolerance to radiation-induced oxidative damage prompted a comparative study of the D1 amino acid composition. Bacterial, cyanobacterial and eukaryotic L/D1 proteins were analyzed to uncover the molecular signatures of the PSII complex adaptation to the radiation-induced damage occurring during the evolution of photosynthesis. Using R. sphaeroides L and C. reinhardtii D1 proteins as baits, homologues were retrieved from the NCBI sequence database and their amino acid composition was determined and compared (Fig. 6A). Although these proteins are highly conserved, analysis of the data reveals that the content of aliphatic and aromatic residues is significantly higher in Bacteria. In fact, aliphatic and aromatic residues represent almost 70% of the total residues in bacterial RC L proteins, while in eukarya D1 proteins the same residues represent less than 60% of the total. In particular, the percentage of Trp residues in bacterial L proteins is almost twice than that in eukaryotic D1 proteins, while smaller but still significant differences were observed in other residues (such as Gly and Leu) highly prone to damage by reactive oxygen species. Not surprisingly, the amino acid composition of cyanobacterial D1 proteins, in line with the evolutionary conservation of D1 proteins, is practically identical to that of the eukaryotic ones (Fig. 6A).

Bottom Line: The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids.A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed.Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Crystallography, National Research Council, Monterotondo, Italy. giuseppina.rea@mlib.ic.cnr.it

ABSTRACT
Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earth's atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues more sensitive to oxidative damage in the eukaryotic/cyanobacterial proteins compared to their bacterial orthologs. These results led us to hypothesize an archaean atmosphere less challenging in terms of ionizing radiation than the present one.

Show MeSH
Related in: MedlinePlus