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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.


Positively co-expressed network of shortlisted genes during atisine biosynthesis in roots of A. heterophyllum. In each metabolic pathway, shortlisted genes are boxed with different color and significant positive correlations between the gene expression levels are shown by connected lines. Abbreviations are elaborated in supplementary Table 4
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Fig5: Positively co-expressed network of shortlisted genes during atisine biosynthesis in roots of A. heterophyllum. In each metabolic pathway, shortlisted genes are boxed with different color and significant positive correlations between the gene expression levels are shown by connected lines. Abbreviations are elaborated in supplementary Table 4

Mentions: This study has revealed the complete biosynthetic pathway of atisine along with candidate genes in A. heterophyllum for the first time. The expression analysis results of all the 25 selected genes showed modulated expression vis-à-vis atisine content in root and shoot tissues of A. heterophyllum. The atisine content showed 0–0.37 % increase in root compared to shoot tissue. The higher expression level of genes encoding G6PI (1.67-fold), PFK (1.75-fold), ALD (3.60-fold) and ENO (2.73-fold) enzymes of glycolysis in roots might indicate that these genes contribute to atisine biosynthesis by producing higher levels of precursors for connecting pathways, viz. mevalonate, non-mevalonate (MEP) and serine biosynthetic pathways leading to their activation (Kumar et al. 2015c; Munoz-Bertomeu et al. 2013). This was also supported by correlation analyses which showed significant positive correlations (p < 0.05; p < 0.01; p < 0.001) between shortlisted transcripts of glycolysis, serine biosynthesis and diterpene biosynthesis (Fig. 5). The combined effect of increased expression levels of G6PI, PFK and ALD would produce higher levels of glyceraldehyde-3-phosphate, the substrate for DXPS and GAPDH enzymes (Mutuku and Nose 2012). DXPS, which catalyzes the rate limiting step in MEP pathway, could produce higher levels of pathway intermediates resulting in the activation of MEP pathway (Broun and Somerville 2001). Malhotra et al. (2014) also showed the activation of MEP pathway in root tissue of A. heterophyllum known to produce higher levels of atisine. Stimulation of MEP pathway might be linked with the increased supply of GGPP, which is a precursor for the diterpene biosynthesis (Malhotra et al. 2014; Zhao et al. 2009). GAPDH, on the other hand, serves as the housekeeping gene and did not show significant alteration in expression in root and shoot tissues of A. heterophyllum. This might be indicative of glycolysis homeostasis irrespective of its flux towards non-mevalonate pathway. The expression of ENO gene showed noticeable increase (2.73-fold) in root over shoot tissue which might produce higher levels of PEP, the substrate for the enzyme DAHPS and PK. The supply of PEP is limiting for the shikimate pathway. Voll et al. (2009) observed that antisense inhibition of ENO enzyme hampered the plastidic shikimate pathway. It has been reported that shikimate/phenylpropanoid pathway is supposed to be the entry point into alkaloid biosynthesis via the formation of tyrosine (Tzin and Galili 2010). Our observations are also in agreement with another study which showed that total alkaloid content is higher in roots of high content accessions compared to low content accessions of A. heterophyllum (Malhotra et al. 2014). The expression of PK gene showed no significant modulation in root and shoot tissue which might indicate that higher concentrations of PEP is likely to be associated with enhanced allocation into shikimate/phenylpropanoid pathway but might also not affect the pyruvate production. This was further supported by correlation analysis results which represented that no significant positive correlation of ENO gene was found with other studied genes (Fig. 5). This observation implied that enhanced expression of ENO gene might be correlated with elevated total alkaloid biosynthesis in high content accessions but not affecting the biosynthesis of atisine through pyruvate homeostasis.Fig. 5


Molecular dissection of pathway components unravel atisine biosynthesis in a non-toxic Aconitum species, A. heterophyllum Wall
Positively co-expressed network of shortlisted genes during atisine biosynthesis in roots of A. heterophyllum. In each metabolic pathway, shortlisted genes are boxed with different color and significant positive correlations between the gene expression levels are shown by connected lines. Abbreviations are elaborated in supplementary Table 4
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Related In: Results  -  Collection

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Fig5: Positively co-expressed network of shortlisted genes during atisine biosynthesis in roots of A. heterophyllum. In each metabolic pathway, shortlisted genes are boxed with different color and significant positive correlations between the gene expression levels are shown by connected lines. Abbreviations are elaborated in supplementary Table 4
Mentions: This study has revealed the complete biosynthetic pathway of atisine along with candidate genes in A. heterophyllum for the first time. The expression analysis results of all the 25 selected genes showed modulated expression vis-à-vis atisine content in root and shoot tissues of A. heterophyllum. The atisine content showed 0–0.37 % increase in root compared to shoot tissue. The higher expression level of genes encoding G6PI (1.67-fold), PFK (1.75-fold), ALD (3.60-fold) and ENO (2.73-fold) enzymes of glycolysis in roots might indicate that these genes contribute to atisine biosynthesis by producing higher levels of precursors for connecting pathways, viz. mevalonate, non-mevalonate (MEP) and serine biosynthetic pathways leading to their activation (Kumar et al. 2015c; Munoz-Bertomeu et al. 2013). This was also supported by correlation analyses which showed significant positive correlations (p < 0.05; p < 0.01; p < 0.001) between shortlisted transcripts of glycolysis, serine biosynthesis and diterpene biosynthesis (Fig. 5). The combined effect of increased expression levels of G6PI, PFK and ALD would produce higher levels of glyceraldehyde-3-phosphate, the substrate for DXPS and GAPDH enzymes (Mutuku and Nose 2012). DXPS, which catalyzes the rate limiting step in MEP pathway, could produce higher levels of pathway intermediates resulting in the activation of MEP pathway (Broun and Somerville 2001). Malhotra et al. (2014) also showed the activation of MEP pathway in root tissue of A. heterophyllum known to produce higher levels of atisine. Stimulation of MEP pathway might be linked with the increased supply of GGPP, which is a precursor for the diterpene biosynthesis (Malhotra et al. 2014; Zhao et al. 2009). GAPDH, on the other hand, serves as the housekeeping gene and did not show significant alteration in expression in root and shoot tissues of A. heterophyllum. This might be indicative of glycolysis homeostasis irrespective of its flux towards non-mevalonate pathway. The expression of ENO gene showed noticeable increase (2.73-fold) in root over shoot tissue which might produce higher levels of PEP, the substrate for the enzyme DAHPS and PK. The supply of PEP is limiting for the shikimate pathway. Voll et al. (2009) observed that antisense inhibition of ENO enzyme hampered the plastidic shikimate pathway. It has been reported that shikimate/phenylpropanoid pathway is supposed to be the entry point into alkaloid biosynthesis via the formation of tyrosine (Tzin and Galili 2010). Our observations are also in agreement with another study which showed that total alkaloid content is higher in roots of high content accessions compared to low content accessions of A. heterophyllum (Malhotra et al. 2014). The expression of PK gene showed no significant modulation in root and shoot tissue which might indicate that higher concentrations of PEP is likely to be associated with enhanced allocation into shikimate/phenylpropanoid pathway but might also not affect the pyruvate production. This was further supported by correlation analysis results which represented that no significant positive correlation of ENO gene was found with other studied genes (Fig. 5). This observation implied that enhanced expression of ENO gene might be correlated with elevated total alkaloid biosynthesis in high content accessions but not affecting the biosynthesis of atisine through pyruvate homeostasis.Fig. 5

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-&agrave;-vis atisine content (0&ndash;0.37&nbsp;%). Quantification of steviol and comparative analysis of shortlisted genes between roots of high (0.37&nbsp;%) vs low (0.14&nbsp;%) atisine content accessions further confirmed the route of atisine biosynthesis. The results showed 6-fold increase in steviol content and 3&ndash;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.