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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.


Expression of SLS1 and SLS2 in C. roseus organs. SLS1 (light gray bars) and SLS2 (dark gray bars) transcript levels were determined by real-time RT-PCR analyses performed on total RNA extracted from C. roseus organs and transcript copy numbers were normalized using CrRPS9. R, Roots; S, stems; YL, young leaves; ML, mature leaves; FB, flower buds; Fl, flowers; Fr, fruits
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Fig5: Expression of SLS1 and SLS2 in C. roseus organs. SLS1 (light gray bars) and SLS2 (dark gray bars) transcript levels were determined by real-time RT-PCR analyses performed on total RNA extracted from C. roseus organs and transcript copy numbers were normalized using CrRPS9. R, Roots; S, stems; YL, young leaves; ML, mature leaves; FB, flower buds; Fl, flowers; Fr, fruits

Mentions: Besides T16H and IS, SLS corresponds to the third type of enzymes from the MIA biosynthetic pathway displaying more than one isoform [22, 60]. While T16H1 and T16H2 have distinct organ specific-roles in MIA biosynthesis, IS4 and IS5 display somewhat redundant functions. To gain insight into the respective involvement of the two SLS isoforms, SLS1 and SLS2 gene expression was measured in the main C. roseus organs (Fig. 5). SLS2 expression was detected in all the tested organs and reached maxima in those directly associated with MIA biosynthesis including roots, flower buds and leaves. By contrast, SLS1 transcripts were barely detectable in all organs except in roots where expression was three-fold lower than SLS2. It is interesting to note that SLS1 was initially characterized from a cell suspension culture cDNA library [15]. Therefore, these results suggest that SLS1 and SLS2 can contribute concomitantly to secologanin biosynthesis in roots while SLS2 can be the prominent isoform of secologanin biosynthesis in the aerial parts of the plant.Fig. 5


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)

Expression of SLS1 and SLS2 in C. roseus organs. SLS1 (light gray bars) and SLS2 (dark gray bars) transcript levels were determined by real-time RT-PCR analyses performed on total RNA extracted from C. roseus organs and transcript copy numbers were normalized using CrRPS9. R, Roots; S, stems; YL, young leaves; ML, mature leaves; FB, flower buds; Fl, flowers; Fr, fruits
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Expression of SLS1 and SLS2 in C. roseus organs. SLS1 (light gray bars) and SLS2 (dark gray bars) transcript levels were determined by real-time RT-PCR analyses performed on total RNA extracted from C. roseus organs and transcript copy numbers were normalized using CrRPS9. R, Roots; S, stems; YL, young leaves; ML, mature leaves; FB, flower buds; Fl, flowers; Fr, fruits
Mentions: Besides T16H and IS, SLS corresponds to the third type of enzymes from the MIA biosynthetic pathway displaying more than one isoform [22, 60]. While T16H1 and T16H2 have distinct organ specific-roles in MIA biosynthesis, IS4 and IS5 display somewhat redundant functions. To gain insight into the respective involvement of the two SLS isoforms, SLS1 and SLS2 gene expression was measured in the main C. roseus organs (Fig. 5). SLS2 expression was detected in all the tested organs and reached maxima in those directly associated with MIA biosynthesis including roots, flower buds and leaves. By contrast, SLS1 transcripts were barely detectable in all organs except in roots where expression was three-fold lower than SLS2. It is interesting to note that SLS1 was initially characterized from a cell suspension culture cDNA library [15]. Therefore, these results suggest that SLS1 and SLS2 can contribute concomitantly to secologanin biosynthesis in roots while SLS2 can be the prominent isoform of secologanin biosynthesis in the aerial parts of the plant.Fig. 5

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.