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Transcriptome analysis of Catharanthus roseus for gene discovery and expression profiling.

Verma M, Ghangal R, Sharma R, Sinha AK, Jain M - PLoS ONE (2014)

Bottom Line: Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp.Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues.Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals.

View Article: PubMed Central - PubMed

Affiliation: Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT
The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals.

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Differential expression analysis of C. roseus transcriptome.(A) Number of differentially expressed genes in different tissues in pairwise comparisons. Number of up-regulated genes are in bold, while down-regulated are in normal font. (B) Venn diagram showing number of up- and down-regulated (in parentheses) genes in leaf and root tissues as compared to flower. Asterisk represents genes up-regulated in one tissue and down-regulated in another tissue. (C) Heat-map showing expression patterns of differentially up-regulated TF encoding genes in different tissues. The scale at the bottom represents log2 fold change. (D) Graphical view showing GO terms associated with biological process enriched in up-regulated genes of leaf. The GO enrichment was performed using BiNGO. Node size is proportional to the number of genes in each category and shades represent the scale denoting significance level (white- no significant difference).
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pone-0103583-g003: Differential expression analysis of C. roseus transcriptome.(A) Number of differentially expressed genes in different tissues in pairwise comparisons. Number of up-regulated genes are in bold, while down-regulated are in normal font. (B) Venn diagram showing number of up- and down-regulated (in parentheses) genes in leaf and root tissues as compared to flower. Asterisk represents genes up-regulated in one tissue and down-regulated in another tissue. (C) Heat-map showing expression patterns of differentially up-regulated TF encoding genes in different tissues. The scale at the bottom represents log2 fold change. (D) Graphical view showing GO terms associated with biological process enriched in up-regulated genes of leaf. The GO enrichment was performed using BiNGO. Node size is proportional to the number of genes in each category and shades represent the scale denoting significance level (white- no significant difference).

Mentions: RNA-Seq has been considered to be the method of choice for differential gene expression studies at whole genome level [22], [23]. In total, approximately 87–90% short reads mapped onto C. roseus transcriptome and nearly 84–87% mapped uniquely (Table 2). DESeq package, was used to identify the genes differential expressed in different tissue samples [24]. We identified differentially expressed genes among different tissues of C. roseus via pairwise comparisons (Fig. 3A). Leaf and root being the primary site for alkaloid production, differential gene expression in these tissues as compared to flower was analyzed in more detail. A total of 2443 and 2153 genes were differentially expressed in leaf and root, respectively, as compared to flower tissue (Fig. 3A, B). In leaves, higher number of genes (1635) were down-regulated than 808 up-regulated genes, whereas in roots, there were nearly equal number of up- (1125) and down-regulated (1028) genes (Fig. 3A). Out of the total 4596 differentially expressed genes in leaf and roots, 679 were common. Among 679 genes commonly differentially expressed in roots and leaves, 72 and 552 genes were up- and down-regulated, respectively (Fig. 3B). Fifty five genes were found to be up-regulated in one tissue and down-regulated in another tissue. The genes related to photosynthesis were up-regulated in leaf, whereas genes annotated as DNA binding proteins (TFs), disease-resistant proteins and wound response proteins were up-regulated in roots. A heat-map of 1861 up-regulated genes in at least one tissue is represented in Fig. S6. Among 1861 up-regulated genes, at least 148 were found to encode for TFs, which are key regulatory proteins. TFs are known to play an important role in accumulation of secondary metabolites in plants [25]–[27]. Among 148 TFs exhibiting significant differential expression in leaf and root tissues, majority of the transcription factors belonging to AP2-EREBP, HB, MYB, NAC, Tify and WRKY families were up-regulated in root tissues (Fig. 3C).


Transcriptome analysis of Catharanthus roseus for gene discovery and expression profiling.

Verma M, Ghangal R, Sharma R, Sinha AK, Jain M - PLoS ONE (2014)

Differential expression analysis of C. roseus transcriptome.(A) Number of differentially expressed genes in different tissues in pairwise comparisons. Number of up-regulated genes are in bold, while down-regulated are in normal font. (B) Venn diagram showing number of up- and down-regulated (in parentheses) genes in leaf and root tissues as compared to flower. Asterisk represents genes up-regulated in one tissue and down-regulated in another tissue. (C) Heat-map showing expression patterns of differentially up-regulated TF encoding genes in different tissues. The scale at the bottom represents log2 fold change. (D) Graphical view showing GO terms associated with biological process enriched in up-regulated genes of leaf. The GO enrichment was performed using BiNGO. Node size is proportional to the number of genes in each category and shades represent the scale denoting significance level (white- no significant difference).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4114786&req=5

pone-0103583-g003: Differential expression analysis of C. roseus transcriptome.(A) Number of differentially expressed genes in different tissues in pairwise comparisons. Number of up-regulated genes are in bold, while down-regulated are in normal font. (B) Venn diagram showing number of up- and down-regulated (in parentheses) genes in leaf and root tissues as compared to flower. Asterisk represents genes up-regulated in one tissue and down-regulated in another tissue. (C) Heat-map showing expression patterns of differentially up-regulated TF encoding genes in different tissues. The scale at the bottom represents log2 fold change. (D) Graphical view showing GO terms associated with biological process enriched in up-regulated genes of leaf. The GO enrichment was performed using BiNGO. Node size is proportional to the number of genes in each category and shades represent the scale denoting significance level (white- no significant difference).
Mentions: RNA-Seq has been considered to be the method of choice for differential gene expression studies at whole genome level [22], [23]. In total, approximately 87–90% short reads mapped onto C. roseus transcriptome and nearly 84–87% mapped uniquely (Table 2). DESeq package, was used to identify the genes differential expressed in different tissue samples [24]. We identified differentially expressed genes among different tissues of C. roseus via pairwise comparisons (Fig. 3A). Leaf and root being the primary site for alkaloid production, differential gene expression in these tissues as compared to flower was analyzed in more detail. A total of 2443 and 2153 genes were differentially expressed in leaf and root, respectively, as compared to flower tissue (Fig. 3A, B). In leaves, higher number of genes (1635) were down-regulated than 808 up-regulated genes, whereas in roots, there were nearly equal number of up- (1125) and down-regulated (1028) genes (Fig. 3A). Out of the total 4596 differentially expressed genes in leaf and roots, 679 were common. Among 679 genes commonly differentially expressed in roots and leaves, 72 and 552 genes were up- and down-regulated, respectively (Fig. 3B). Fifty five genes were found to be up-regulated in one tissue and down-regulated in another tissue. The genes related to photosynthesis were up-regulated in leaf, whereas genes annotated as DNA binding proteins (TFs), disease-resistant proteins and wound response proteins were up-regulated in roots. A heat-map of 1861 up-regulated genes in at least one tissue is represented in Fig. S6. Among 1861 up-regulated genes, at least 148 were found to encode for TFs, which are key regulatory proteins. TFs are known to play an important role in accumulation of secondary metabolites in plants [25]–[27]. Among 148 TFs exhibiting significant differential expression in leaf and root tissues, majority of the transcription factors belonging to AP2-EREBP, HB, MYB, NAC, Tify and WRKY families were up-regulated in root tissues (Fig. 3C).

Bottom Line: Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp.Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues.Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals.

View Article: PubMed Central - PubMed

Affiliation: Functional and Applied Genomics Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT
The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals.

Show MeSH