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

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

De Novo RNA Sequencing and Expression Analysis of Aconitum carmichaelii to Analyze Key Genes Involved in the Biosynthesis of Diterpene Alkaloids

“…Since the root is the main tissue where we see the accumulation of aconitine-type diterpene alkaloids, our qualitative results suggesting high expression of unigenes annotated as the key enzymes of the aconitine-type diterpene alkaloids biosynthetic pathway seem to be quite significant. While unigenes corresponding to HMGR were also reported to be highly upregulated in roots of A. heterophyllum [ 18 ], the expression of GGPPS and CDPS were downregulated in the root compared to the shoot, and the expression of KS showed non-significant modulation in the two tissues [ 51 ]. These results indicate the probability of CDPS being the rate-limiting step towards the biosynthesis of aconitine-type diterpene alkaloids.…”

“…The incorporation of β-aminoethanol moiety in the atisane and steviol facilitates the formation of an atisine skeleton. While the accumulation of steviol has been reported in A. heterophyllum , it has not been detected in A. carmichaelii [ 51 ]. Consistent with this, we were also not able to identify the unigene annotated as kaurenoic acid hydroxylase in our transcriptome dataset, indicating that atisine biosynthesis in A. carmichaelii most likely takes the atisenol route.…”

Functional identification of the terpene synthase family involved in diterpenoid alkaloids biosynthesis in Aconitum carmichaelii

“…Recently, several RNAsequencing (RNA-seq) approaches using the Illumina platform have analyzed the transcriptome of A. carmichaelii 12 , 13 , 14 , 15 , 16 , 17 and Aconitum heterophyllum 18 . Many candidate genes, such as terpenoid-related enzymes, monooxygenases, methyltransferases, and BAHD acyltransferases (named according to the first four identified enzymes of the family: BEAT, benzylalcohol O -acetyltransferase; AHCT, anthocyanin O -hydroxycinnamoyltransferase; HCBT, anthranilate N -hydroxycinnamoyl/benzoyltransferase; DAT, deacetylvindoline 4- O -acetyltransferase) related to DAs biosynthesis have been identified 13 , 14 , 15 , 16 , 17 , 19 , 20 . However, the limitations of short-read sequencing results in the vast majority of isotigs not representing full-length cDNA sequences, and none of these candidates have yet been functionally identified through in vivo or in vitro analysis.…”

Quality and Potency Analysis of IVY Leaf Extract

“…An Ivy extract was found concord of the permissible microbial confines. Table 3 Plant based drugs are typically accessible as a blend of more than one plant component and its medicinal action relies on its phytochemical components [13].…”