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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.


Time course analyses of the reaction catalyzed by SLS1 and SLS2. Proteins extracts from yeast cells expressing either SLS1 or SLS2 were incubated at 30 °C with loganin or secologanin. Formation of the resulting products was monitored by LC-MS analysis. Black triangles, loganin; dark grey squares, secologanin; light circles, secoxyloganin
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Fig3: Time course analyses of the reaction catalyzed by SLS1 and SLS2. Proteins extracts from yeast cells expressing either SLS1 or SLS2 were incubated at 30 °C with loganin or secologanin. Formation of the resulting products was monitored by LC-MS analysis. Black triangles, loganin; dark grey squares, secologanin; light circles, secoxyloganin

Mentions: Interestingly, we noted that both SLS1 and SLS2 also convert loganin into a more polar compound identified as secoxyloganin according to UV and MS spectra and a comparison with a pure authentic standard (Fig. 2; Additional file 2: Figure S2). By contrast, this product was not produced using the empty vector crude extract suggesting that it results from a reaction catalyzed by SLS. For SLS1 and SLS2, time course reactions showed a decrease of the loganin content accompanied by the formation of both secologanin and secoxyloganin (Fig. 3). Since secoxyloganin corresponds to the acidic form of secologanin, it may result from the oxidation of the aldehyde function of secologanin. Therefore, we tested the capacity of both SLS1 and SLS2 to convert secologanin into secoxyloganin. While no formation of secoxyloganin was monitored by incubating secologanin with the empty vector crude extract, both SLS1 and SLS2 directly produce secoxyloganin from secologanin in a stoichiometric manner at least during the early times of the reaction (Fig. 2, Fig. 3). As a consequence, these results suggest that SLS1 and SLS2 not only catalyze the oxidative ring cleavage of loganin to produce secologanin but also perform the oxidation of secologanin into secoxyloganin. Besides G10H and IO, SLS1 and SLS2 constitute the third type of P450 from the seco-iridoid pathway performing more than one catalytic reaction [8, 11]. Interestingly, the additional reaction catalyzed by SLS1 and SLS2 is similar to the third oxidation performed by IO to generate 7-deoxyloganetic acid, suggesting that regiospecific multi-oxidation is rather common to P450s acting in secoiridoid biosynthesis. The occurrence of sequential oxidations has been reported for several P450s [57] but the dissociation of intermediates is still a question of debate since it ranges from an absence of dissociation for P450 11B2 [58] to a dissociation of 85 % for P450 2C11 [59]. In the absence of pulse-chase experiments, we are not able to propose a reaction scheme for both SLS1 and SLS2 concerning secologanin release.Fig. 3


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)

Time course analyses of the reaction catalyzed by SLS1 and SLS2. Proteins extracts from yeast cells expressing either SLS1 or SLS2 were incubated at 30 °C with loganin or secologanin. Formation of the resulting products was monitored by LC-MS analysis. Black triangles, loganin; dark grey squares, secologanin; light circles, secoxyloganin
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Time course analyses of the reaction catalyzed by SLS1 and SLS2. Proteins extracts from yeast cells expressing either SLS1 or SLS2 were incubated at 30 °C with loganin or secologanin. Formation of the resulting products was monitored by LC-MS analysis. Black triangles, loganin; dark grey squares, secologanin; light circles, secoxyloganin
Mentions: Interestingly, we noted that both SLS1 and SLS2 also convert loganin into a more polar compound identified as secoxyloganin according to UV and MS spectra and a comparison with a pure authentic standard (Fig. 2; Additional file 2: Figure S2). By contrast, this product was not produced using the empty vector crude extract suggesting that it results from a reaction catalyzed by SLS. For SLS1 and SLS2, time course reactions showed a decrease of the loganin content accompanied by the formation of both secologanin and secoxyloganin (Fig. 3). Since secoxyloganin corresponds to the acidic form of secologanin, it may result from the oxidation of the aldehyde function of secologanin. Therefore, we tested the capacity of both SLS1 and SLS2 to convert secologanin into secoxyloganin. While no formation of secoxyloganin was monitored by incubating secologanin with the empty vector crude extract, both SLS1 and SLS2 directly produce secoxyloganin from secologanin in a stoichiometric manner at least during the early times of the reaction (Fig. 2, Fig. 3). As a consequence, these results suggest that SLS1 and SLS2 not only catalyze the oxidative ring cleavage of loganin to produce secologanin but also perform the oxidation of secologanin into secoxyloganin. Besides G10H and IO, SLS1 and SLS2 constitute the third type of P450 from the seco-iridoid pathway performing more than one catalytic reaction [8, 11]. Interestingly, the additional reaction catalyzed by SLS1 and SLS2 is similar to the third oxidation performed by IO to generate 7-deoxyloganetic acid, suggesting that regiospecific multi-oxidation is rather common to P450s acting in secoiridoid biosynthesis. The occurrence of sequential oxidations has been reported for several P450s [57] but the dissociation of intermediates is still a question of debate since it ranges from an absence of dissociation for P450 11B2 [58] to a dissociation of 85 % for P450 2C11 [59]. In the absence of pulse-chase experiments, we are not able to propose a reaction scheme for both SLS1 and SLS2 concerning secologanin release.Fig. 3

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.