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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.

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Quantitative expression analysis of selected genes in different tissues of high content accession of A. heterophyllum; a glycolysis, b serine biosynthesis and, c diterpene biosynthesis. The vertical axis represents the normalized expression and horizontal axis represents the different genes. Expression level was normalized to housekeeping genes, i.e., 26S and GAPDH. Bar graphs show mean ± SD (n = 4). Significance was evaluated for each gene between different tissues (*p < 0.05, **p < 0.01, ****p < 0.0001). Abbreviations are elaborated in supplementary Table 4
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Fig3: Quantitative expression analysis of selected genes in different tissues of high content accession of A. heterophyllum; a glycolysis, b serine biosynthesis and, c diterpene biosynthesis. The vertical axis represents the normalized expression and horizontal axis represents the different genes. Expression level was normalized to housekeeping genes, i.e., 26S and GAPDH. Bar graphs show mean ± SD (n = 4). Significance was evaluated for each gene between different tissues (*p < 0.05, **p < 0.01, ****p < 0.0001). Abbreviations are elaborated in supplementary Table 4

Mentions: The expression of different genes belonging to integrated pathways showed significant modulations in congruence with atisine content between root and shoot tissues of A. heterophyllum. Among the glycolytic genes, 4 genes exhibited up-regulation, 2 genes were down-regulated while 4 genes showed non-significant modulation in roots compared to shoots (Fig. 3a). The G6PI, PFK, ALD and ENO genes were up-regulated by 1.67- (p < 0.01), 1.75- (p < 0.01), 3.60- (p < 0.0001) and 2.73-fold (p < 0.0001), respectively, whereas the expression of TPI and PGK was down-regulated by 0.22- (p < 0.0001) and 0.01-fold (p < 0.0001), respectively. Further, 2 genes corresponding to phosphorylated pathway of serine biosynthesis, i.e., PGDH and PSAT exhibited 1.50- (p < 0.05) and 3.33-fold (p < 0.0001) up-regulation, respectively, while transcript encoding PSP enzyme showed non-significant modulation in roots compared to shoots. Conversely, 3 genes of glycolate pathway, i.e., GO, GD and SHMT showed significant down-regulation with 0.04- (p < 0.0001), 0.03- (p < 0.0001) and 0.07-fold (p < 0.0001), respectively, while one gene, i.e., PGP showed non-significant modulation in roots compared to shoots (Fig. 3b). On the other hand, GAD of glycerate pathway showed significant expression only in shoots (3.53, p < 0.0001) of A. heterophyllum. The transcript encoding SDC showed non-significant change in expression level between root and shoot tissues.Fig. 3


Molecular dissection of pathway components unravel atisine biosynthesis in a non-toxic Aconitum species, A. heterophyllum Wall
Quantitative expression analysis of selected genes in different tissues of high content accession of A. heterophyllum; a glycolysis, b serine biosynthesis and, c diterpene biosynthesis. The vertical axis represents the normalized expression and horizontal axis represents the different genes. Expression level was normalized to housekeeping genes, i.e., 26S and GAPDH. Bar graphs show mean ± SD (n = 4). Significance was evaluated for each gene between different tissues (*p < 0.05, **p < 0.01, ****p < 0.0001). Abbreviations are elaborated in supplementary Table 4
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835424&req=5

Fig3: Quantitative expression analysis of selected genes in different tissues of high content accession of A. heterophyllum; a glycolysis, b serine biosynthesis and, c diterpene biosynthesis. The vertical axis represents the normalized expression and horizontal axis represents the different genes. Expression level was normalized to housekeeping genes, i.e., 26S and GAPDH. Bar graphs show mean ± SD (n = 4). Significance was evaluated for each gene between different tissues (*p < 0.05, **p < 0.01, ****p < 0.0001). Abbreviations are elaborated in supplementary Table 4
Mentions: The expression of different genes belonging to integrated pathways showed significant modulations in congruence with atisine content between root and shoot tissues of A. heterophyllum. Among the glycolytic genes, 4 genes exhibited up-regulation, 2 genes were down-regulated while 4 genes showed non-significant modulation in roots compared to shoots (Fig. 3a). The G6PI, PFK, ALD and ENO genes were up-regulated by 1.67- (p < 0.01), 1.75- (p < 0.01), 3.60- (p < 0.0001) and 2.73-fold (p < 0.0001), respectively, whereas the expression of TPI and PGK was down-regulated by 0.22- (p < 0.0001) and 0.01-fold (p < 0.0001), respectively. Further, 2 genes corresponding to phosphorylated pathway of serine biosynthesis, i.e., PGDH and PSAT exhibited 1.50- (p < 0.05) and 3.33-fold (p < 0.0001) up-regulation, respectively, while transcript encoding PSP enzyme showed non-significant modulation in roots compared to shoots. Conversely, 3 genes of glycolate pathway, i.e., GO, GD and SHMT showed significant down-regulation with 0.04- (p < 0.0001), 0.03- (p < 0.0001) and 0.07-fold (p < 0.0001), respectively, while one gene, i.e., PGP showed non-significant modulation in roots compared to shoots (Fig. 3b). On the other hand, GAD of glycerate pathway showed significant expression only in shoots (3.53, p < 0.0001) of A. heterophyllum. The transcript encoding SDC showed non-significant change in expression level between root and shoot tissues.Fig. 3

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.


Related in: MedlinePlus