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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

CDF97 performance in quality reconstruction and transcript expression level. a Current resources (A = ccOrcae, B = mpgrCra, C = NIPGR, D = PMS454, E = PMSIllu), CD97 and CDF97 were used as databases to identify homologs of MIA genes, and the resulting bitscore obtained by BLAST was compared to that of an ideal sequence (bitscore of the reference sequence against itself, i.e. bitscore ratio = 1). Values inside each cell indicate the number of hits having a ratio >0.8. b Comparison of log2 expression levels measured by qPCR and RNA-seq for SLS and T16H isoforms in current assemblies, CD97 and CDF97. RNA-seq samples used for this comparison were: flowers (Fl, SRR122239 and SRR1271859), mature leaves (ML, SRR122251 and SRR1271857), roots (R, SRR122254 and SRR1271858) and young leaves (YL, SRR122252). Expression levels were normalized by RPS9, in both qPCR and RNA-seq measurements. The linear regression line is shown in blue with shading corresponding to 95 % confidence intervals. The r² values indicate correlation coefficients of the linear models
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Fig11: CDF97 performance in quality reconstruction and transcript expression level. a Current resources (A = ccOrcae, B = mpgrCra, C = NIPGR, D = PMS454, E = PMSIllu), CD97 and CDF97 were used as databases to identify homologs of MIA genes, and the resulting bitscore obtained by BLAST was compared to that of an ideal sequence (bitscore of the reference sequence against itself, i.e. bitscore ratio = 1). Values inside each cell indicate the number of hits having a ratio >0.8. b Comparison of log2 expression levels measured by qPCR and RNA-seq for SLS and T16H isoforms in current assemblies, CD97 and CDF97. RNA-seq samples used for this comparison were: flowers (Fl, SRR122239 and SRR1271859), mature leaves (ML, SRR122251 and SRR1271857), roots (R, SRR122254 and SRR1271858) and young leaves (YL, SRR122252). Expression levels were normalized by RPS9, in both qPCR and RNA-seq measurements. The linear regression line is shown in blue with shading corresponding to 95 % confidence intervals. The r² values indicate correlation coefficients of the linear models

Mentions: The gene expression levels of SLS and T16H isoforms were determined by qPCR and compared to FPKM values calculated according to the RSEM procedure for each transcript within each assembly. As each isoform has apparent specific expression patterns (Fig. 11b; [22]), the correct alignment of reads to high quality sequences should be able to yield similar results.Fig. 11


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)

CDF97 performance in quality reconstruction and transcript expression level. a Current resources (A = ccOrcae, B = mpgrCra, C = NIPGR, D = PMS454, E = PMSIllu), CD97 and CDF97 were used as databases to identify homologs of MIA genes, and the resulting bitscore obtained by BLAST was compared to that of an ideal sequence (bitscore of the reference sequence against itself, i.e. bitscore ratio = 1). Values inside each cell indicate the number of hits having a ratio >0.8. b Comparison of log2 expression levels measured by qPCR and RNA-seq for SLS and T16H isoforms in current assemblies, CD97 and CDF97. RNA-seq samples used for this comparison were: flowers (Fl, SRR122239 and SRR1271859), mature leaves (ML, SRR122251 and SRR1271857), roots (R, SRR122254 and SRR1271858) and young leaves (YL, SRR122252). Expression levels were normalized by RPS9, in both qPCR and RNA-seq measurements. The linear regression line is shown in blue with shading corresponding to 95 % confidence intervals. The r² values indicate correlation coefficients of the linear models
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig11: CDF97 performance in quality reconstruction and transcript expression level. a Current resources (A = ccOrcae, B = mpgrCra, C = NIPGR, D = PMS454, E = PMSIllu), CD97 and CDF97 were used as databases to identify homologs of MIA genes, and the resulting bitscore obtained by BLAST was compared to that of an ideal sequence (bitscore of the reference sequence against itself, i.e. bitscore ratio = 1). Values inside each cell indicate the number of hits having a ratio >0.8. b Comparison of log2 expression levels measured by qPCR and RNA-seq for SLS and T16H isoforms in current assemblies, CD97 and CDF97. RNA-seq samples used for this comparison were: flowers (Fl, SRR122239 and SRR1271859), mature leaves (ML, SRR122251 and SRR1271857), roots (R, SRR122254 and SRR1271858) and young leaves (YL, SRR122252). Expression levels were normalized by RPS9, in both qPCR and RNA-seq measurements. The linear regression line is shown in blue with shading corresponding to 95 % confidence intervals. The r² values indicate correlation coefficients of the linear models
Mentions: The gene expression levels of SLS and T16H isoforms were determined by qPCR and compared to FPKM values calculated according to the RSEM procedure for each transcript within each assembly. As each isoform has apparent specific expression patterns (Fig. 11b; [22]), the correct alignment of reads to high quality sequences should be able to yield similar results.Fig. 11

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