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Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

Garg A, Agrawal L, Misra RC, Sharma S, Ghosh S - BMC Genomics (2015)

Bottom Line: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts.Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh.Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India. anchalfzd@gmail.com.

ABSTRACT

Background: Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs.

Results: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent-LRDs biosynthesis and ApCPS3 in root diterpene biosynthesis. Moreover, simple sequence repeats (SSRs) that might assist in genotyping and developing specific chemotypes were identified in transcripts of the specialized metabolic pathways, including ent-LRDs.

Conclusions: Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh. Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.

No MeSH data available.


Correlation of DGE- and qRT-PCR-based expression profiles of transcripts. Expression profiles of 35 transcripts that are related to specialized metabolism were selected for the analysis. Data are presented as log2FoldChange (leaf vs root). qRT - PCR data represents average of three biological replicates. Differential transcript expression determined by qRT - PCR in root and leaf tissues was statistically evaluated either at P<0.05 or P<0.01
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Fig4: Correlation of DGE- and qRT-PCR-based expression profiles of transcripts. Expression profiles of 35 transcripts that are related to specialized metabolism were selected for the analysis. Data are presented as log2FoldChange (leaf vs root). qRT - PCR data represents average of three biological replicates. Differential transcript expression determined by qRT - PCR in root and leaf tissues was statistically evaluated either at P<0.05 or P<0.01

Mentions: To generate a non-redundant reference transcriptome of kalmegh, leaf and root assembled transcripts were clustered using CD-HIT at 95 % sequence identity [34]. By following this approach, a total of 84,628 transcripts with an average length of 688.53 bp were generated (Table 2). For the identification of differential transcripts, these assembled unique transcripts (84,628) were considered as master control transcript and the read count profiles were first determined for the leaf and root Illumina reads using Bowtie tool [37] and further proceed for the differential gene expression analysis using DegSeq tool [38] (Additional file 1: Figure S2). This digital gene expression (DGE) analysis revealed 6277 and 5418 transcripts with expression only in leaf and root tissues, respectively. However, 19,564 and 16,678 transcripts were up-regulated and down-regulated, respectively, in leaf as compared to root. To authenticate DGE-based expression profiles of the transcripts, a correlation with quantitative RT-PCR (qRT-PCR)-based expression patterns was determined. For this, 35 transcripts that are related to the specialized metabolism were selected (Fig. 4). The overall correlation coefficient (r) of 0.9398 (r2 = 0.8832) indicated a very high level of correlation between DGE- and qRT-PCR-based expression profiles of the transcripts. Therefore, DGE-based expression profiles may be considered to identify transcripts involved in tissue-specific accumulation of specialized metabolites in kalmegh. To identify candidate transcripts of the ent-LRD biosynthetic pathway, transcripts were classified according to the DGE-based expression patterns in leaf and root tissues. DGE-based expression profiles of the transcripts with annotation to the diterpene biosynthetic pathway and to different CYP450, GT and transcription factor families are presented in Table 3; Additional file 1: Figure S4, Additional file 7: Table S6, Additional file 8: Table S7 and Additional file 9: Table S8.Table 2


Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

Garg A, Agrawal L, Misra RC, Sharma S, Ghosh S - BMC Genomics (2015)

Correlation of DGE- and qRT-PCR-based expression profiles of transcripts. Expression profiles of 35 transcripts that are related to specialized metabolism were selected for the analysis. Data are presented as log2FoldChange (leaf vs root). qRT - PCR data represents average of three biological replicates. Differential transcript expression determined by qRT - PCR in root and leaf tissues was statistically evaluated either at P<0.05 or P<0.01
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4557604&req=5

Fig4: Correlation of DGE- and qRT-PCR-based expression profiles of transcripts. Expression profiles of 35 transcripts that are related to specialized metabolism were selected for the analysis. Data are presented as log2FoldChange (leaf vs root). qRT - PCR data represents average of three biological replicates. Differential transcript expression determined by qRT - PCR in root and leaf tissues was statistically evaluated either at P<0.05 or P<0.01
Mentions: To generate a non-redundant reference transcriptome of kalmegh, leaf and root assembled transcripts were clustered using CD-HIT at 95 % sequence identity [34]. By following this approach, a total of 84,628 transcripts with an average length of 688.53 bp were generated (Table 2). For the identification of differential transcripts, these assembled unique transcripts (84,628) were considered as master control transcript and the read count profiles were first determined for the leaf and root Illumina reads using Bowtie tool [37] and further proceed for the differential gene expression analysis using DegSeq tool [38] (Additional file 1: Figure S2). This digital gene expression (DGE) analysis revealed 6277 and 5418 transcripts with expression only in leaf and root tissues, respectively. However, 19,564 and 16,678 transcripts were up-regulated and down-regulated, respectively, in leaf as compared to root. To authenticate DGE-based expression profiles of the transcripts, a correlation with quantitative RT-PCR (qRT-PCR)-based expression patterns was determined. For this, 35 transcripts that are related to the specialized metabolism were selected (Fig. 4). The overall correlation coefficient (r) of 0.9398 (r2 = 0.8832) indicated a very high level of correlation between DGE- and qRT-PCR-based expression profiles of the transcripts. Therefore, DGE-based expression profiles may be considered to identify transcripts involved in tissue-specific accumulation of specialized metabolites in kalmegh. To identify candidate transcripts of the ent-LRD biosynthetic pathway, transcripts were classified according to the DGE-based expression patterns in leaf and root tissues. DGE-based expression profiles of the transcripts with annotation to the diterpene biosynthetic pathway and to different CYP450, GT and transcription factor families are presented in Table 3; Additional file 1: Figure S4, Additional file 7: Table S6, Additional file 8: Table S7 and Additional file 9: Table S8.Table 2

Bottom Line: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts.Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh.Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India. anchalfzd@gmail.com.

ABSTRACT

Background: Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs.

Results: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent-LRDs biosynthesis and ApCPS3 in root diterpene biosynthesis. Moreover, simple sequence repeats (SSRs) that might assist in genotyping and developing specific chemotypes were identified in transcripts of the specialized metabolic pathways, including ent-LRDs.

Conclusions: Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh. Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.

No MeSH data available.