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Alternative translation initiation codons for the plastid maturase MatK: unraveling the pseudogene misconception in the Orchidaceae.

Barthet MM, Moukarzel K, Smith KN, Patel J, Hilu KW - BMC Evol. Biol. (2015)

Bottom Line: We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods.We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history.These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Coastal Carolina University, Conway, SC, 29526, USA. mbarthet@coastal.edu.

ABSTRACT

Background: The plastid maturase MatK has been implicated as a possible model for the evolutionary "missing link" between prokaryotic and eukaryotic splicing machinery. This evolutionary implication has sparked investigations concerning the function of this unusual maturase. Intron targets of MatK activity suggest that this is an essential enzyme for plastid function. The matK gene, however, is described as a pseudogene in many photosynthetic orchid species due to presence of premature stop codons in translations, and its high rate of nucleotide and amino acid substitution.

Results: Sequence analysis of the matK gene from orchids identified an out-of-frame alternative AUG initiation codon upstream from the consensus initiation codon used for translation in other angiosperms. We demonstrate translation from the alternative initiation codon generates a conserved MatK reading frame. We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods. We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history.

Conclusion: These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.

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Related in: MedlinePlus

Immunoblot detection of MatK from orchid species. Orchid protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane. MatK protein was detected using anti-MatK antibody as described in Barthet and Hilu [12]. Orchid species analysed represent two different subfamilies of Orchidaceae (Orchidoideae: Spiranthes vernalis, S. cernua, S. sinensis, Caladenia catenata and Cryptostylis erecta and Epidendroideae: Phaius tancarvilleae) and are representative of orchids that require the alternative initiation codon for full-length MatK translation (Figure 2A and B). All immunoblots were repeated twice to verify results. (a) Immunoblot detection of MatK from 50 μg of total protein from Spiranthes vernalis, S. cernua and Phaius tancarvilleae. N = 3 biological replicates. Mass standard = PageRuler Prestained Protein Ladder (Thermo Scientific). Ponceau S stain of RbcS shown as loading control. (b) Immunoblot detection of MatK from 75 μg of total protein from Caladenia catenata, Cryptostylis erecta and Spiranthes sinensis. N = 1 biological replicate due to tissue limitations. Oryza sativa (rice) was used as a control for detection. Mass standard = 6–185 kDa Protein Ladder (NEB). Ponceau S stain of RbcS shown as loading control. All immunoblots were repeated twice to verify results. (c) Alignment of MatK peptide region used for antibody generation to orchid species examined in this study
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Fig4: Immunoblot detection of MatK from orchid species. Orchid protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane. MatK protein was detected using anti-MatK antibody as described in Barthet and Hilu [12]. Orchid species analysed represent two different subfamilies of Orchidaceae (Orchidoideae: Spiranthes vernalis, S. cernua, S. sinensis, Caladenia catenata and Cryptostylis erecta and Epidendroideae: Phaius tancarvilleae) and are representative of orchids that require the alternative initiation codon for full-length MatK translation (Figure 2A and B). All immunoblots were repeated twice to verify results. (a) Immunoblot detection of MatK from 50 μg of total protein from Spiranthes vernalis, S. cernua and Phaius tancarvilleae. N = 3 biological replicates. Mass standard = PageRuler Prestained Protein Ladder (Thermo Scientific). Ponceau S stain of RbcS shown as loading control. (b) Immunoblot detection of MatK from 75 μg of total protein from Caladenia catenata, Cryptostylis erecta and Spiranthes sinensis. N = 1 biological replicate due to tissue limitations. Oryza sativa (rice) was used as a control for detection. Mass standard = 6–185 kDa Protein Ladder (NEB). Ponceau S stain of RbcS shown as loading control. All immunoblots were repeated twice to verify results. (c) Alignment of MatK peptide region used for antibody generation to orchid species examined in this study

Mentions: Protein expression of MatK was examined in five orchid species noted to contain matK as a pseudogene: Caladenia catenata, Cryptostylis erecta, Spiranthes vernalis [39], S. cernua [40] and S. sinensis [41]. This designation is due to the emergence of a premature stop codon in translations from the cic (Fig.1b). When translation was based on the aic, a full-length MatK reading frame was evident for these same species with the exception of C. catenata which is addressed later (Fig. 2a and b). In addition to these five species, we also examined MatK expression in the orchid Phaius tancarvilleae. The matK gene is noted as a pseudogene in one accession [GenBank: EF079306] but as a functional gene in a different accession [GenBank: KF673844] requiring the aic for full-length translation in the latter accession (Fig. 2a and b). An immune-reactive band ranging from 55 to 65 kDa in mass was observed from Western blots of resolved total protein using an anti-MatK antibody [12] from all six orchid species (Fig. 4a and b). The expected molecular mass of MatK from these orchid taxa is approximately 62 kDa based on amino acid sequence.Fig. 4


Alternative translation initiation codons for the plastid maturase MatK: unraveling the pseudogene misconception in the Orchidaceae.

Barthet MM, Moukarzel K, Smith KN, Patel J, Hilu KW - BMC Evol. Biol. (2015)

Immunoblot detection of MatK from orchid species. Orchid protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane. MatK protein was detected using anti-MatK antibody as described in Barthet and Hilu [12]. Orchid species analysed represent two different subfamilies of Orchidaceae (Orchidoideae: Spiranthes vernalis, S. cernua, S. sinensis, Caladenia catenata and Cryptostylis erecta and Epidendroideae: Phaius tancarvilleae) and are representative of orchids that require the alternative initiation codon for full-length MatK translation (Figure 2A and B). All immunoblots were repeated twice to verify results. (a) Immunoblot detection of MatK from 50 μg of total protein from Spiranthes vernalis, S. cernua and Phaius tancarvilleae. N = 3 biological replicates. Mass standard = PageRuler Prestained Protein Ladder (Thermo Scientific). Ponceau S stain of RbcS shown as loading control. (b) Immunoblot detection of MatK from 75 μg of total protein from Caladenia catenata, Cryptostylis erecta and Spiranthes sinensis. N = 1 biological replicate due to tissue limitations. Oryza sativa (rice) was used as a control for detection. Mass standard = 6–185 kDa Protein Ladder (NEB). Ponceau S stain of RbcS shown as loading control. All immunoblots were repeated twice to verify results. (c) Alignment of MatK peptide region used for antibody generation to orchid species examined in this study
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Immunoblot detection of MatK from orchid species. Orchid protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane. MatK protein was detected using anti-MatK antibody as described in Barthet and Hilu [12]. Orchid species analysed represent two different subfamilies of Orchidaceae (Orchidoideae: Spiranthes vernalis, S. cernua, S. sinensis, Caladenia catenata and Cryptostylis erecta and Epidendroideae: Phaius tancarvilleae) and are representative of orchids that require the alternative initiation codon for full-length MatK translation (Figure 2A and B). All immunoblots were repeated twice to verify results. (a) Immunoblot detection of MatK from 50 μg of total protein from Spiranthes vernalis, S. cernua and Phaius tancarvilleae. N = 3 biological replicates. Mass standard = PageRuler Prestained Protein Ladder (Thermo Scientific). Ponceau S stain of RbcS shown as loading control. (b) Immunoblot detection of MatK from 75 μg of total protein from Caladenia catenata, Cryptostylis erecta and Spiranthes sinensis. N = 1 biological replicate due to tissue limitations. Oryza sativa (rice) was used as a control for detection. Mass standard = 6–185 kDa Protein Ladder (NEB). Ponceau S stain of RbcS shown as loading control. All immunoblots were repeated twice to verify results. (c) Alignment of MatK peptide region used for antibody generation to orchid species examined in this study
Mentions: Protein expression of MatK was examined in five orchid species noted to contain matK as a pseudogene: Caladenia catenata, Cryptostylis erecta, Spiranthes vernalis [39], S. cernua [40] and S. sinensis [41]. This designation is due to the emergence of a premature stop codon in translations from the cic (Fig.1b). When translation was based on the aic, a full-length MatK reading frame was evident for these same species with the exception of C. catenata which is addressed later (Fig. 2a and b). In addition to these five species, we also examined MatK expression in the orchid Phaius tancarvilleae. The matK gene is noted as a pseudogene in one accession [GenBank: EF079306] but as a functional gene in a different accession [GenBank: KF673844] requiring the aic for full-length translation in the latter accession (Fig. 2a and b). An immune-reactive band ranging from 55 to 65 kDa in mass was observed from Western blots of resolved total protein using an anti-MatK antibody [12] from all six orchid species (Fig. 4a and b). The expected molecular mass of MatK from these orchid taxa is approximately 62 kDa based on amino acid sequence.Fig. 4

Bottom Line: We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods.We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history.These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Coastal Carolina University, Conway, SC, 29526, USA. mbarthet@coastal.edu.

ABSTRACT

Background: The plastid maturase MatK has been implicated as a possible model for the evolutionary "missing link" between prokaryotic and eukaryotic splicing machinery. This evolutionary implication has sparked investigations concerning the function of this unusual maturase. Intron targets of MatK activity suggest that this is an essential enzyme for plastid function. The matK gene, however, is described as a pseudogene in many photosynthetic orchid species due to presence of premature stop codons in translations, and its high rate of nucleotide and amino acid substitution.

Results: Sequence analysis of the matK gene from orchids identified an out-of-frame alternative AUG initiation codon upstream from the consensus initiation codon used for translation in other angiosperms. We demonstrate translation from the alternative initiation codon generates a conserved MatK reading frame. We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods. We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history.

Conclusion: These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.

Show MeSH
Related in: MedlinePlus