Dataset Information

Silencing amorpha-4,11-diene synthase Genes in Artemisia annua Leads to FPP Accumulation.

ABSTRACT: Artemisia annua is established as an efficient crop for the production of the anti-malarial compound artemisinin, a sesquiterpene lactone synthesized and stored in Glandular Secretory Trichomes (GSTs) located on the leaves and inflorescences. Amorpha-4,11-diene synthase (AMS) catalyzes the conversion of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene and diphosphate, which is the first committed step in the synthesis of artemisinin. FPP is the precursor for sesquiterpene and sterol biosynthesis in the plant. This work aimed to investigate the effect of blocking the synthesis of artemisinin in the GSTs of a high artemisinin yielding line, Artemis, by down regulating AMS. We determined that there are up to 12 AMS gene copies in Artemis, all expressed in GSTs. We used sequence homology to design an RNAi construct under the control of a GST specific promoter that was predicted to be effective against all 12 of these genes. Stable transformation of Artemis with this construct resulted in over 95% reduction in the content of artemisinin and related products, and a significant increase in the FPP pool. The Artemis AMS silenced lines showed no morphological alterations, and metabolomic and gene expression analysis did not detect any changes in the levels of other major sesquiterpene compounds or sesquiterpene synthase genes in leaf material. FPP also acts as a precursor for squalene and sterol biosynthesis but levels of these compounds were also not altered in the AMS silenced lines. Four unknown oxygenated sesquiterpenes were produced in these lines, but at extremely low levels compared to Artemis non-transformed controls (NTC). This study finds that engineering A. annua GSTs in an Artemis background results in endogenous terpenes related to artemisinin being depleted with the precursor FPP actually accumulating rather than being utilized by other endogenous enzymes. The challenge now is to establish if this precursor pool can act as substrate for production of alternative sesquiterpenes in A. annua.

SUBMITTER: Catania TM

PROVIDER: S-EPMC5986941 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Silencing amorpha-4,11-diene synthase Genes in Artemisia annua Leads to FPP Accumulation.

Catania Theresa M TM Branigan Caroline A CA Stawniak Natalia N Hodson Jennifer J Harvey David D Larson Tony R TR Czechowski Tomasz T Graham Ian A IA

Frontiers in plant science 20180529

Artemisia annua is established as an efficient crop for the production of the anti-malarial compound artemisinin, a sesquiterpene lactone synthesized and stored in Glandular Secretory Trichomes (GSTs) located on the leaves and inflorescences. Amorpha-4,11-diene synthase (AMS) catalyzes the conversion of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene and diphosphate, which is the first committed step in the synthesis of artemisinin. FPP is the precursor for sesquiterpene and ste ...[more]

PMID: 29896204

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Project description:BackgroundAmorpha-4,11-diene synthase (ADS) is a key enzyme in the artemisinin biosynthetic pathway. ADS promotes the first step of artemisinin synthesis by cyclizing faresyl pyrophosphate to synthesize the sesquiterpene product amorpha-4,11-diene. Thanks to the continuous improvement of genomic information, its evolutionary trace can be analyzed in a genome view.MethodsPhylogenetic analysis was used to identify ADS-like genes in other Asteraceae. Gene structure and motif analysis was used to analyze the structural similarity of these identified genes. Heterologous expression and GC-MS analysis were performed to determine whether the functions of ADS and Cna4666 are consistent. Validation of ADS genes evolutionary trajectories was achieved by selective pressure and synteny analysis.ResultIn this study, we extracted 8 ADS genes from the Artemisia annua L. genome annotation and 121 ADS similar genes from the genomes of Artemisia annua L. and other plants in the Asteraceae, and further exploring their evolutionary relationship. Phylogenetic analysis showed that the genes most closely related to ADS genes were found in the genome of Chrysanthemum nankingense. Among them, the gene structure and motif composition of Cna4666 is very similar to ADS, we wondered whether it has the potential to synthesize amorpha-4,11-diene. Therefore, we extracted the products of recombinant p0_ADS.1 and Cna4666 proteins by HS-SPME combined with GC-MS analysis, the results indicate that Cna4666 is an α-bisabolol synthase, which cannot synthesize amorpha-4,11-diene. Through synteny analysis, we did not find collinear blocks of ADS genes in the Helianthus annuus and C. nankingense genomes. Furthermore, Ka/Ks ratios indicated that the evolution of ADS genes from their similar genes principally underwent purifying selection, and there was a strong positive selection between ADS genes.ConclusionsThis study proved that ADS is a multi-copy gene in Artemisia annua L., and they are not widely distributed in Asteraceae. The data will increase our understanding of the evolutionary selection pressure on ADS genes. The results suggest that ADS genes are subject to strong positive selection internally, and it is possible that they are a recently evolved gene in the Artemisia.

Dataset Information

Silencing amorpha-4,11-diene synthase Genes in Artemisia annua Leads to FPP Accumulation.

Publications

Silencing <i>amorpha-4,11-diene synthase</i> Genes in <i>Artemisia annua</i> Leads to FPP Accumulation.

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