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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.


Related in: MedlinePlus

SLS2 is located to the endoplasmic reticulum. C. roseus cells were transiently transformed with the SLS2-YFP-expressing vector (SLS2-YFP; (a) in combination with the plasmid expressing an ER-CFP marker (“ER”-CFP; (b). Colocalization of the two fluorescence signals appears on the merged images (c). Cell morphology (d) was observed with differential interference contrast (DIC). Bars = 10 μm
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Fig4: SLS2 is located to the endoplasmic reticulum. C. roseus cells were transiently transformed with the SLS2-YFP-expressing vector (SLS2-YFP; (a) in combination with the plasmid expressing an ER-CFP marker (“ER”-CFP; (b). Colocalization of the two fluorescence signals appears on the merged images (c). Cell morphology (d) was observed with differential interference contrast (DIC). Bars = 10 μm

Mentions: While important amounts of secologanin can be measured in the different organs of C. roseus [22] we never detected the presence of secoxyloganin (data not shown), raising the question of the physiological signification of this compound production but also suggesting that secologanin could be release from the SLS catalytic site during the sequential oxidation process. Secoxyloganin is an acidic compound derived from secologanin that is no longer able to be condensed with tryptamine by STR in the vacuole, due to the absence of the aldehyde function. If secoxyloganin formation occurs in vivo, the resulting depletion of the secologanin pool would be deleterious for the subsequent synthesis of MIAs. Although we cannot exclude that additional enzymes might convert secoxyloganin back to secologanin, the subcellular compartmentation of secologanin biosynthesis may also limit secoxyloganin formation in planta. Subcellular localization studies showed that SLS2 is located to the endoplasmic reticulum as previously observed for SLS1 [36] (Fig. 4). Since both SLS1 and SLS2 are anchored to this subcellular compartment and release their product in the cytosol, active transport of secologanin to the vacuolar compartment with high efficiency might allow its import before additional oxidation by SLS1 or SLS2. This would be a direct and interesting consequence of the complex subcellular organization of the MIA biosynthetic pathway regarding regulation of the metabolic flux.Fig. 4


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)

SLS2 is located to the endoplasmic reticulum. C. roseus cells were transiently transformed with the SLS2-YFP-expressing vector (SLS2-YFP; (a) in combination with the plasmid expressing an ER-CFP marker (“ER”-CFP; (b). Colocalization of the two fluorescence signals appears on the merged images (c). Cell morphology (d) was observed with differential interference contrast (DIC). Bars = 10 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: SLS2 is located to the endoplasmic reticulum. C. roseus cells were transiently transformed with the SLS2-YFP-expressing vector (SLS2-YFP; (a) in combination with the plasmid expressing an ER-CFP marker (“ER”-CFP; (b). Colocalization of the two fluorescence signals appears on the merged images (c). Cell morphology (d) was observed with differential interference contrast (DIC). Bars = 10 μm
Mentions: While important amounts of secologanin can be measured in the different organs of C. roseus [22] we never detected the presence of secoxyloganin (data not shown), raising the question of the physiological signification of this compound production but also suggesting that secologanin could be release from the SLS catalytic site during the sequential oxidation process. Secoxyloganin is an acidic compound derived from secologanin that is no longer able to be condensed with tryptamine by STR in the vacuole, due to the absence of the aldehyde function. If secoxyloganin formation occurs in vivo, the resulting depletion of the secologanin pool would be deleterious for the subsequent synthesis of MIAs. Although we cannot exclude that additional enzymes might convert secoxyloganin back to secologanin, the subcellular compartmentation of secologanin biosynthesis may also limit secoxyloganin formation in planta. Subcellular localization studies showed that SLS2 is located to the endoplasmic reticulum as previously observed for SLS1 [36] (Fig. 4). Since both SLS1 and SLS2 are anchored to this subcellular compartment and release their product in the cytosol, active transport of secologanin to the vacuolar compartment with high efficiency might allow its import before additional oxidation by SLS1 or SLS2. This would be a direct and interesting consequence of the complex subcellular organization of the MIA biosynthetic pathway regarding regulation of the metabolic flux.Fig. 4

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.


Related in: MedlinePlus