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Characterization of a second secologanin synthase isoform producing both secologanin and secoxyloganin allows enhanced de novo assembly of a Catharanthus roseus transcriptome.

Dugé de Bernonville T, Foureau E, Parage C, Lanoue A, Clastre M, Londono MA, Oudin A, Houillé B, Papon N, Besseau S, Glévarec G, Atehortùa L, Giglioli-Guivarc'h N, St-Pierre B, De Luca V, O'Connor SE, Courdavault V - BMC Genomics (2015)

Bottom Line: The new consensus transcriptome allowed a precise estimation of abundance of SLS and T16H isoforms, similar to qPCR measurements.The C. roseus consensus transcriptome can now be used for characterization of new genes of the MIA pathway.Furthermore, additional isoforms of genes encoding distinct MIA biosynthetic enzymes isoforms could be predicted suggesting the existence of a higher level of complexity in the synthesis of MIA, raising the question of the evolutionary events behind what seems like redundancy.

View Article: PubMed Central - PubMed

Affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", UFR Sciences et Techniques, 37200, Tours, France. Bernonvillethomas.duge@univ-tours.fr.

ABSTRACT

Background: Transcriptome sequencing offers a great resource for the study of non-model plants such as Catharanthus roseus, which produces valuable monoterpenoid indole alkaloids (MIAs) via a complex biosynthetic pathway whose characterization is still undergoing. Transcriptome databases dedicated to this plant were recently developed by several consortia to uncover new biosynthetic genes. However, the identification of missing steps in MIA biosynthesis based on these large datasets may be limited by the erroneous assembly of close transcripts and isoforms, even with the multiple available transcriptomes.

Results: Secologanin synthases (SLS) are P450 enzymes that catalyze an unusual ring-opening reaction of loganin in the biosynthesis of the MIA precursor secologanin. We report here the identification and characterization in C. roseus of a new isoform of SLS, SLS2, sharing 97 % nucleotide sequence identity with the previously characterized SLS1. We also discovered that both isoforms further oxidize secologanin into secoxyloganin. SLS2 had however a different expression profile, being the major isoform in aerial organs that constitute the main site of MIA accumulation. Unfortunately, we were unable to find a current C. roseus transcriptome database containing simultaneously well reconstructed sequences of SLS isoforms and accurate expression levels. After a pair of close mRNA encoding tabersonine 16-hydroxylase (T16H1 and T16H2), this is the second example of improperly assembled transcripts from the MIA pathway in the public transcriptome databases. To construct a more complete transcriptome resource for C. roseus, we re-processed previously published transcriptome data by combining new single assemblies. Care was particularly taken during clustering and filtering steps to remove redundant contigs but not transcripts encoding potential isoforms by monitoring quality reconstruction of MIA genes and specific SLS and T16H isoforms. The new consensus transcriptome allowed a precise estimation of abundance of SLS and T16H isoforms, similar to qPCR measurements.

Conclusions: The C. roseus consensus transcriptome can now be used for characterization of new genes of the MIA pathway. Furthermore, additional isoforms of genes encoding distinct MIA biosynthetic enzymes isoforms could be predicted suggesting the existence of a higher level of complexity in the synthesis of MIA, raising the question of the evolutionary events behind what seems like redundancy.

No MeSH data available.


SLS1 and SLS2 catalyze the conversion of loganin in secologanin and secoxyloganin. a LC-MS chromatograms using selected ion monitoring (loganin, mass-to-charge ratio 413; secologanin, mass-to-charge ratio 433; secoxyloganin, mass-to-charge ration 403) of the reaction products of yeast extracts from cell cultures expressing either SLS1 or SLS2 or containing the empty pYeDP60 vector, incubated with loganin or secologanin. b Schematic reaction catalyzed by SLS1 and SLS2 highlighting the aldehyde to acid conversion
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Fig2: SLS1 and SLS2 catalyze the conversion of loganin in secologanin and secoxyloganin. a LC-MS chromatograms using selected ion monitoring (loganin, mass-to-charge ratio 413; secologanin, mass-to-charge ratio 433; secoxyloganin, mass-to-charge ration 403) of the reaction products of yeast extracts from cell cultures expressing either SLS1 or SLS2 or containing the empty pYeDP60 vector, incubated with loganin or secologanin. b Schematic reaction catalyzed by SLS1 and SLS2 highlighting the aldehyde to acid conversion

Mentions: While amplifying the coding sequence of SLS (CYP71A1, Genbank accession number L10081) [14], sequencing of the PCR products revealed the presence of a second putative isoform exhibiting 96 % identity with the original SLS isoform. Interrogation of the C. roseus transcriptomic databases (Medicinal Plant Genomics Resource, CathaCyc/Orcae and Phytometasyn) led to the identification of identical but partial sequences confirming thus the existence of this new SLS sequence that has been recently deposited to Genbank under accession number KF415117. The corresponding P450 also displayed a high level of identity (97 %) with the first SLS isoform (Additional file 1: Figure S1) suggesting that it could also catalyze the oxidative ring cleavage of loganin to produce secologanin. To test this hypothesis. The original and the new putative SLS isoforms were individually expressed in the Saccharomyces WAT11 strain that overexpresses the Arabidopsis NADPH P450 reductase [56]. Crude extracts of galactose-induced yeasts transformed with the pYeDP60 empty control vector or the pYeDP60 expressing each P450 were subsequently incubated with NADPH, H+ and loganin, and analyzed by UPLC-MS (Fig. 2). While no modification of loganin occurred with the empty vector crude extract, a conversion of loganin into secologanin was observed with the crude extract of each enzyme. This established that the putative SLS isoform truly corresponds to a new SLS isoform, named SLS2 as reference to CYP71A1, renamed SLS1.Fig. 2


Characterization of a second secologanin synthase isoform producing both secologanin and secoxyloganin allows enhanced de novo assembly of a Catharanthus roseus transcriptome.

Dugé de Bernonville T, Foureau E, Parage C, Lanoue A, Clastre M, Londono MA, Oudin A, Houillé B, Papon N, Besseau S, Glévarec G, Atehortùa L, Giglioli-Guivarc'h N, St-Pierre B, De Luca V, O'Connor SE, Courdavault V - BMC Genomics (2015)

SLS1 and SLS2 catalyze the conversion of loganin in secologanin and secoxyloganin. a LC-MS chromatograms using selected ion monitoring (loganin, mass-to-charge ratio 413; secologanin, mass-to-charge ratio 433; secoxyloganin, mass-to-charge ration 403) of the reaction products of yeast extracts from cell cultures expressing either SLS1 or SLS2 or containing the empty pYeDP60 vector, incubated with loganin or secologanin. b Schematic reaction catalyzed by SLS1 and SLS2 highlighting the aldehyde to acid conversion
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4541752&req=5

Fig2: SLS1 and SLS2 catalyze the conversion of loganin in secologanin and secoxyloganin. a LC-MS chromatograms using selected ion monitoring (loganin, mass-to-charge ratio 413; secologanin, mass-to-charge ratio 433; secoxyloganin, mass-to-charge ration 403) of the reaction products of yeast extracts from cell cultures expressing either SLS1 or SLS2 or containing the empty pYeDP60 vector, incubated with loganin or secologanin. b Schematic reaction catalyzed by SLS1 and SLS2 highlighting the aldehyde to acid conversion
Mentions: While amplifying the coding sequence of SLS (CYP71A1, Genbank accession number L10081) [14], sequencing of the PCR products revealed the presence of a second putative isoform exhibiting 96 % identity with the original SLS isoform. Interrogation of the C. roseus transcriptomic databases (Medicinal Plant Genomics Resource, CathaCyc/Orcae and Phytometasyn) led to the identification of identical but partial sequences confirming thus the existence of this new SLS sequence that has been recently deposited to Genbank under accession number KF415117. The corresponding P450 also displayed a high level of identity (97 %) with the first SLS isoform (Additional file 1: Figure S1) suggesting that it could also catalyze the oxidative ring cleavage of loganin to produce secologanin. To test this hypothesis. The original and the new putative SLS isoforms were individually expressed in the Saccharomyces WAT11 strain that overexpresses the Arabidopsis NADPH P450 reductase [56]. Crude extracts of galactose-induced yeasts transformed with the pYeDP60 empty control vector or the pYeDP60 expressing each P450 were subsequently incubated with NADPH, H+ and loganin, and analyzed by UPLC-MS (Fig. 2). While no modification of loganin occurred with the empty vector crude extract, a conversion of loganin into secologanin was observed with the crude extract of each enzyme. This established that the putative SLS isoform truly corresponds to a new SLS isoform, named SLS2 as reference to CYP71A1, renamed SLS1.Fig. 2

Bottom Line: The new consensus transcriptome allowed a precise estimation of abundance of SLS and T16H isoforms, similar to qPCR measurements.The C. roseus consensus transcriptome can now be used for characterization of new genes of the MIA pathway.Furthermore, additional isoforms of genes encoding distinct MIA biosynthetic enzymes isoforms could be predicted suggesting the existence of a higher level of complexity in the synthesis of MIA, raising the question of the evolutionary events behind what seems like redundancy.

View Article: PubMed Central - PubMed

Affiliation: Université François-Rabelais de Tours, EA2106 "Biomolécules et Biotechnologies Végétales", UFR Sciences et Techniques, 37200, Tours, France. Bernonvillethomas.duge@univ-tours.fr.

ABSTRACT

Background: Transcriptome sequencing offers a great resource for the study of non-model plants such as Catharanthus roseus, which produces valuable monoterpenoid indole alkaloids (MIAs) via a complex biosynthetic pathway whose characterization is still undergoing. Transcriptome databases dedicated to this plant were recently developed by several consortia to uncover new biosynthetic genes. However, the identification of missing steps in MIA biosynthesis based on these large datasets may be limited by the erroneous assembly of close transcripts and isoforms, even with the multiple available transcriptomes.

Results: Secologanin synthases (SLS) are P450 enzymes that catalyze an unusual ring-opening reaction of loganin in the biosynthesis of the MIA precursor secologanin. We report here the identification and characterization in C. roseus of a new isoform of SLS, SLS2, sharing 97 % nucleotide sequence identity with the previously characterized SLS1. We also discovered that both isoforms further oxidize secologanin into secoxyloganin. SLS2 had however a different expression profile, being the major isoform in aerial organs that constitute the main site of MIA accumulation. Unfortunately, we were unable to find a current C. roseus transcriptome database containing simultaneously well reconstructed sequences of SLS isoforms and accurate expression levels. After a pair of close mRNA encoding tabersonine 16-hydroxylase (T16H1 and T16H2), this is the second example of improperly assembled transcripts from the MIA pathway in the public transcriptome databases. To construct a more complete transcriptome resource for C. roseus, we re-processed previously published transcriptome data by combining new single assemblies. Care was particularly taken during clustering and filtering steps to remove redundant contigs but not transcripts encoding potential isoforms by monitoring quality reconstruction of MIA genes and specific SLS and T16H isoforms. The new consensus transcriptome allowed a precise estimation of abundance of SLS and T16H isoforms, similar to qPCR measurements.

Conclusions: The C. roseus consensus transcriptome can now be used for characterization of new genes of the MIA pathway. Furthermore, additional isoforms of genes encoding distinct MIA biosynthetic enzymes isoforms could be predicted suggesting the existence of a higher level of complexity in the synthesis of MIA, raising the question of the evolutionary events behind what seems like redundancy.

No MeSH data available.