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Molecular dissection of pathway components unravel atisine biosynthesis in a non-toxic Aconitum species, A. heterophyllum Wall

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ABSTRACT

Aconitum heterophyllum is an important component for various herbal drug formulations due to the occurrence of non-toxic aconites including marker compound, atisine. Despite huge pharmacological potential, the reprogramming of aconites production is limited due to lack of understanding on their biosynthesis. To address this problem, we have proposed here the complete atisine biosynthetic pathway for the first time connecting glycolysis, MVA/MEP, serine biosynthesis and diterpene biosynthetic pathways. The transcript profiling revealed phosphorylated pathway as a major contributor towards serine production in addition to repertoire of genes in glycolysis (G6PI, PFK, ALD and ENO), serine biosynthesis (PGDH and PSAT) and diterpene biosynthesis (KO and KH) sharing a similar pattern of expression (2-4-folds) in roots compared to shoots vis-à-vis atisine content (0–0.37 %). Quantification of steviol and comparative analysis of shortlisted genes between roots of high (0.37 %) vs low (0.14 %) atisine content accessions further confirmed the route of atisine biosynthesis. The results showed 6-fold increase in steviol content and 3–62-fold up-regulation of all the selected genes in roots of high content accession ascertaining their association towards atisine production. Moreover, significant positive correlations were observed between selected genes suggesting their co-expression and crucial role in atisine biosynthesis. This study, thus, offers unprecedented opportunities to explore the selected candidate genes for enhanced production of atisine in cultivated plant cells.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-016-0417-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


Plausible biosynthetic pathway leading to atisine in A. heterophyllum. The metabolic network has been constructed by including glycolysis, phosphorylated, glycerate, glycolate, MVA/MEP and diterpene biosynthetic pathways. Question marks indicate missing enzymes with no available information. Green color represents the positions of enzymes identified as candidate genes in atisine biosynthesis. Abbreviations are elaborated in supplementary Table 4
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Fig1: Plausible biosynthetic pathway leading to atisine in A. heterophyllum. The metabolic network has been constructed by including glycolysis, phosphorylated, glycerate, glycolate, MVA/MEP and diterpene biosynthetic pathways. Question marks indicate missing enzymes with no available information. Green color represents the positions of enzymes identified as candidate genes in atisine biosynthesis. Abbreviations are elaborated in supplementary Table 4

Mentions: Atisine is a diterpenoid alkaloid. The biosynthesis of diterpenoid alkaloids (DAs) has been scarcely investigated and so far there is dearth of reports available on their biosynthesis in A. heterophyllum. However, the atisine-type DAs corresponding to the basic skeleton of atisane-type diterpenes have been isolated from the Spiraea japonica complex (Hao et al. 2003). Atisine is thought to be produced from mevalonate (MVA) and non-mevalonate (MEP) pathways leading to the formation of diterpene precursor geranylgeranyl diphosphate (GGPP) (Malhotra et al. 2014), but information is not yet available on the biosynthesis of atisine beyond GGPP. To fill this gap, we constructed a metabolic network for the first time which showed the coordination and connecting links between different pathways integrating into atisine production to provide a more robust view of atisine biosynthesis in A. heterophyllum (Fig. 1).Fig. 1


Molecular dissection of pathway components unravel atisine biosynthesis in a non-toxic Aconitum species, A. heterophyllum Wall
Plausible biosynthetic pathway leading to atisine in A. heterophyllum. The metabolic network has been constructed by including glycolysis, phosphorylated, glycerate, glycolate, MVA/MEP and diterpene biosynthetic pathways. Question marks indicate missing enzymes with no available information. Green color represents the positions of enzymes identified as candidate genes in atisine biosynthesis. Abbreviations are elaborated in supplementary Table 4
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835424&req=5

Fig1: Plausible biosynthetic pathway leading to atisine in A. heterophyllum. The metabolic network has been constructed by including glycolysis, phosphorylated, glycerate, glycolate, MVA/MEP and diterpene biosynthetic pathways. Question marks indicate missing enzymes with no available information. Green color represents the positions of enzymes identified as candidate genes in atisine biosynthesis. Abbreviations are elaborated in supplementary Table 4
Mentions: Atisine is a diterpenoid alkaloid. The biosynthesis of diterpenoid alkaloids (DAs) has been scarcely investigated and so far there is dearth of reports available on their biosynthesis in A. heterophyllum. However, the atisine-type DAs corresponding to the basic skeleton of atisane-type diterpenes have been isolated from the Spiraea japonica complex (Hao et al. 2003). Atisine is thought to be produced from mevalonate (MVA) and non-mevalonate (MEP) pathways leading to the formation of diterpene precursor geranylgeranyl diphosphate (GGPP) (Malhotra et al. 2014), but information is not yet available on the biosynthesis of atisine beyond GGPP. To fill this gap, we constructed a metabolic network for the first time which showed the coordination and connecting links between different pathways integrating into atisine production to provide a more robust view of atisine biosynthesis in A. heterophyllum (Fig. 1).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Aconitum heterophyllum is an important component for various herbal drug formulations due to the occurrence of non-toxic aconites including marker compound, atisine. Despite huge pharmacological potential, the reprogramming of aconites production is limited due to lack of understanding on their biosynthesis. To address this problem, we have proposed here the complete atisine biosynthetic pathway for the first time connecting glycolysis, MVA/MEP, serine biosynthesis and diterpene biosynthetic pathways. The transcript profiling revealed phosphorylated pathway as a major contributor towards serine production in addition to repertoire of genes in glycolysis (G6PI, PFK, ALD and ENO), serine biosynthesis (PGDH and PSAT) and diterpene biosynthesis (KO and KH) sharing a similar pattern of expression (2-4-folds) in roots compared to shoots vis-à-vis atisine content (0–0.37 %). Quantification of steviol and comparative analysis of shortlisted genes between roots of high (0.37 %) vs low (0.14 %) atisine content accessions further confirmed the route of atisine biosynthesis. The results showed 6-fold increase in steviol content and 3–62-fold up-regulation of all the selected genes in roots of high content accession ascertaining their association towards atisine production. Moreover, significant positive correlations were observed between selected genes suggesting their co-expression and crucial role in atisine biosynthesis. This study, thus, offers unprecedented opportunities to explore the selected candidate genes for enhanced production of atisine in cultivated plant cells.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-016-0417-7) contains supplementary material, which is available to authorized users.

No MeSH data available.