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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.


The phylogenetic relationship of kalmegh class II diTPSs with diTPSs of angiosperms, gymnosperms and moss. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA6. Ent-CPS, syn-CPS, (+)-CPS denote ent-, syn- and (+)/normal copalyl diphosphate synthase, respectively. CLS means copal-8-ol diphosphate synthase
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Fig7: The phylogenetic relationship of kalmegh class II diTPSs with diTPSs of angiosperms, gymnosperms and moss. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA6. Ent-CPS, syn-CPS, (+)-CPS denote ent-, syn- and (+)/normal copalyl diphosphate synthase, respectively. CLS means copal-8-ol diphosphate synthase

Mentions: Annotation of the kalmegh transcriptome revealed three diTPSs that showed close phylogenetic relationship with the dicotyledons monofunctional class II diTPSs of ent-CPP product specificity (Fig. 7). These are ApCPS1 (ApU55291), ApCPS2 (ApU48901) and ApCPS3 (ApU53774) (Table 3). Similar to class II diTPSs, the highly conserved DXDD motif that is essential for the protonation-initiated cyclization of GGPP was identified in ApCPS1, ApCPS2 and ApCPS3, following multiple sequence alignment (Additional file 1: Figure S5). Sequence analysis revealed that ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins with calculated molecular masses of 95.45, 93.43 and 90.81 kD, respectively. At the amino acid sequence level, ApCPS1 shared 55.2 and 57.21 % identities with ApCPS2 and ApCPS3, respectively (ClustalW score, http://www.ebi.ac.uk/). However, ApCPS2 shared 63.36 % amino acid identity with ApCPS3. Like other plant diTPSs, N-terminal transit peptides for the chloroplast localization were recognised in ApCPS1, ApCPS2 and ApCPS3, following iPSORT (http://ipsort.hgc.jp/) and Predotar v. 1.30. (http://urgi.versailles.inra.fr/predotar/predotar.html) analysis.Fig. 7


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)

The phylogenetic relationship of kalmegh class II diTPSs with diTPSs of angiosperms, gymnosperms and moss. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA6. Ent-CPS, syn-CPS, (+)-CPS denote ent-, syn- and (+)/normal copalyl diphosphate synthase, respectively. CLS means copal-8-ol diphosphate synthase
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: The phylogenetic relationship of kalmegh class II diTPSs with diTPSs of angiosperms, gymnosperms and moss. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA6. Ent-CPS, syn-CPS, (+)-CPS denote ent-, syn- and (+)/normal copalyl diphosphate synthase, respectively. CLS means copal-8-ol diphosphate synthase
Mentions: Annotation of the kalmegh transcriptome revealed three diTPSs that showed close phylogenetic relationship with the dicotyledons monofunctional class II diTPSs of ent-CPP product specificity (Fig. 7). These are ApCPS1 (ApU55291), ApCPS2 (ApU48901) and ApCPS3 (ApU53774) (Table 3). Similar to class II diTPSs, the highly conserved DXDD motif that is essential for the protonation-initiated cyclization of GGPP was identified in ApCPS1, ApCPS2 and ApCPS3, following multiple sequence alignment (Additional file 1: Figure S5). Sequence analysis revealed that ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins with calculated molecular masses of 95.45, 93.43 and 90.81 kD, respectively. At the amino acid sequence level, ApCPS1 shared 55.2 and 57.21 % identities with ApCPS2 and ApCPS3, respectively (ClustalW score, http://www.ebi.ac.uk/). However, ApCPS2 shared 63.36 % amino acid identity with ApCPS3. Like other plant diTPSs, N-terminal transit peptides for the chloroplast localization were recognised in ApCPS1, ApCPS2 and ApCPS3, following iPSORT (http://ipsort.hgc.jp/) and Predotar v. 1.30. (http://urgi.versailles.inra.fr/predotar/predotar.html) analysis.Fig. 7

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.