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

Abstract

Aconitum carmichaelii is a high-value medicinal herb widely used across China, Japan, and other Asian countries. Aconitine-type diterpene alkaloids (DAs) are the characteristic compounds in Aconitum . Although six transcriptomes, based on short-read next generation sequencing technology, have been reported from the Aconitum species, the terpene synthase (TPS) corresponding to DAs biosynthesis remains unidentified. We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A. carmichaelii transcriptome. Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b, TPS-c, and TPS-e/f subfamilies were identified . In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve diTPSs. Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent -copalyl diphosphate. Five AcKSLs separately reacted with ent -CPP to produce ent -kaurene, ent -atiserene, and ent -13- epi -sandaracopimaradie: a new diterpene found in Aconitum . AcTPSs gene expression in conjunction DAs content analysis in different tissues validated that ent -CPP is the sole precursor to all DAs biosynthesis, with AcKSL1, AcKSL2s and AcKSL3-1 responsible for C 20 atisine and napelline type DAs biosynthesis, respectively. These data clarified the molecular basis for the C 20 -DAs biosynthetic pathway in A. carmichaelii and pave the way for further exploration of C 19 -DAs biosynthesis in the Aconitum species.

Integrating Metabolomics and Transcriptomics to Unveil Atisine Biosynthesis in Aconitum gymnandrum Maxim

“…Further investigation found more C 20 -DAs (80%) induced by MJ than C 19 -DAs (54.9%). This is consistent with the inference that C 19 -DAs originated from the further rearrangement of C 20 -DAs [ 13 , 14 , 15 ]. To obtain more C 19 -DAs with noticeable induction effects, it may be necessary to use different induction concentrations or different types of elicitors for further study.…”

“…The highest expression level of AgCPS1 and AgKSL1 provides them more opportunities for the biosynthesis of DAs in the root. Compared with three different functional KSL enzymes in A. carmichaelii [ 15 ], we only identified AgKSL1 in A. gymnandrum . The fact that AgKSL1 could combine with all four identified CPS enzymes to produce the single diterpene ent -atiserene highlighted the possibility that ent -atiserene could be the main precursor in atisine biosynthesis in A. gymnandrum .…”

“…The fact that AgKSL1 could combine with all four identified CPS enzymes to produce the single diterpene ent -atiserene highlighted the possibility that ent -atiserene could be the main precursor in atisine biosynthesis in A. gymnandrum . The absence of napline-type C20 DAs in the seedling coincides with with the lack of an identified ent -kaurene synthase, which was regarded as the precursor of napline-type C20 DAs [ 14 , 15 ]. However, the ent -kaurene-mediated gibberellin phytohormones biosynthesis is universal in the plant kingdom [ 43 ], thus more KSL genes need to be further identified.…”

“…Like other diterpenoids, DAs were thought to be produced from the diterpene precursor geranylgeranyl pyrophosphate (GGPP), which is synthesized through IPP and DMAPP produced from the mevalonate (MVA) pathway in the cytoplasm and the methylerythritol 4-phosphate (MEP) pathway in the plastid. Copalyl diphosphate synthase (CPS) and kaurene synthase-like (KSL) enzymes then serially catalyze GGPP to form ent -copalyl diphosphate ( ent -CPP) and ent -atiserene or ent -kaurene, which establishes the skeleton structure of atisine and napelline type C 20 -DAs in A. carmichaelii [ 15 ]. Atisine-type alkaloids are considered to be the simplest group of C 20 -DAs [ 10 ].…”

Alternative Splicing and Its Roles in Plant Metabolism

“…Various transcriptomic studies have revealed that terpenoid biosynthetic genes are regulated by alternative splicing in different tissues and stress conditions. For instance, terpenoid biosynthetic genes are regulated by alternative splicing in different tissues of Ginkgo biloba [113], Sichuan pepper (Zanthoxylum armatum) [114], Artemisia argyi [115] and Lindera glauca [116]. They are also alternatively spliced under drought and heat stress in tea plants [117].…”