Next-generation sequencing (NGS) transcriptomes reveal association of multiple genes and pathways contributing to secondary metabolites accumulation in tuberous roots of Aconitum heterophyllum Wall.

Abstract

The transcriptomes of Aconitum heterophyllum were assembled and characterized for the first time to decipher molecular components contributing to biosynthesis and accumulation of metabolites in tuberous roots. Aconitum heterophyllum Wall., popularly known as Atis, is a high-value medicinal herb of North-Western Himalayas. No information exists as of today on genetic factors contributing to the biosynthesis of secondary metabolites accumulating in tuberous roots, thereby, limiting genetic interventions towards genetic improvement of A. heterophyllum. Illumina paired-end sequencing followed by de novo assembly yielded 75,548 transcripts for root transcriptome and 39,100 transcripts for shoot transcriptome with minimum length of 200 bp. Biological role analysis of root versus shoot transcriptomes assigned 27,596 and 16,604 root transcripts; 12,340 and 9398 shoot transcripts into gene ontology and clusters of orthologous group, respectively. KEGG pathway mapping assigned 37 and 31 transcripts onto starch-sucrose metabolism while 329 and 341 KEGG orthologies associated with transcripts were found to be involved in biosynthesis of various secondary metabolites for root and shoot transcriptomes, respectively. In silico expression profiling of the mevalonate/2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway genes for aconites biosynthesis revealed 4 genes HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), MVDD (mevalonate diphosphate decarboxylase) and HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase) with higher expression in root transcriptome compared to shoot transcriptome suggesting their key role in biosynthesis of aconite alkaloids. Five genes, GMPase (geranyl diphosphate mannose pyrophosphorylase), SHAGGY, RBX1 (RING-box protein 1), SRF receptor kinases and β-amylase, implicated in tuberous root formation in other plant species showed higher levels of expression in tuberous roots compared to shoots. A total of 15,487 transcription factors belonging to bHLH, MYB, bZIP families and 399 ABC transporters which regulate biosynthesis and accumulation of bioactive compounds were identified in root and shoot transcriptomes. The expression of 5 ABC transporters involved in tuberous root development was validated by quantitative PCR analysis. Network connectivity diagrams were drawn for starch-sucrose metabolism and isoquinoline alkaloid biosynthesis associated with tuberous root growth and secondary metabolism, respectively, in root transcriptome of A. heterophyllum. The current endeavor will be of practical importance in planning a suitable genetic intervention strategy for the improvement of A. heterophyllum.

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

“…Such analysis also assists in the identification of orthologous genes and reduces the number of false positives for further differential expression analysis. While our study is the first report to date on the de novo transcriptome assembly and expression analysis of aconitine-producing aconites, a transcriptome assembly of the non-aconitine-producing plant from the Aconitum genus, A. heterophyllum , offers us a unique resource [ 18 ]. Comparing the de novo transcriptome assembly of A. carmichaelli obtained in this study with that of A. heterophyllum would allow us to identify transcripts that are shared between these two species or are unique to A. carmichaelii , which will be important in identifying strong candidate genes associated with aconitine biosynthesis.…”

“…A schematic diagram of the proposed pathway for the biosynthesis of aconitine and its derivatives is shown in Figure 8 a. Precursors for the biosynthesis of aconitine-type diterpene alkaloids are derived from IPP, which is the end product of both the mevalonate (MVA) and methylerythritol (MEP) biosynthetic pathway. While classical biochemists describe the MEP pathway as the only contributor of IPP for biosynthesis of diterpene alkaloids, numerous studies have now established that the MVA pathway may also contribute to the biosynthesis of diterpene alkaloids in plants through the exchange of intermediates with the MEP pathway [ 18 ]. Among the earliest biochemical reactions towards the biosynthesis of diterpene alkaloids, three molecules of IPP condense to form GGPP in the presence of enzyme GGPPS [ 10 , 11 ].…”

“…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.…”

“…Pal et al recently described de novo transcriptome assembly characteristics of A. heterophyllum , a non-toxic plant species from Aconitum genus using root and shoot tissues. A. heterophyllum does not produce toxic diester–diterpene alkaloids, with atisine being the major alkaloid [ 18 , 19 ]. While de novo transcriptome assembly and expression analysis of A. heterophyllum are important resources, they are of limited use when studying the biosynthesis of highly toxic aconitine-type diterpene alkaloids, which are the signature metabolites for the majority of aconites.…”

Modular Design of Picroside-II Biosynthesis Deciphered through NGS Transcriptomes and Metabolic Intermediates Analysis in Naturally Variant Chemotypes of a Medicinal Herb, Picrorhiza kurroa

“…The construction of libraries and their sequencing for transcriptome analysis was performed as per the protocol described in Pal et al (2015).…”