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Planta Med 2019; 85(14/15): 1177-1186
DOI: 10.1055/a-0998-5125
Natural Product Chemistry and Analytical Studies
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Reduction of Pyrrolizidine Alkaloid Levels in Comfrey (Symphytum officinale) Hairy Roots by RNAi Silencing of Homospermidine Synthase

Lars H. Kruse
1   Botanisches Institut, Kiel University, Kiel, Germany
3   Plant Biology Section, School of Integrative Plant Science, Cornell University, USA
,
Thomas Stegemann
1   Botanisches Institut, Kiel University, Kiel, Germany
,
Julia Jensen-Kroll
1   Botanisches Institut, Kiel University, Kiel, Germany
,
Annika Engelhardt
1   Botanisches Institut, Kiel University, Kiel, Germany
,
Anne-Maria Wesseling
1   Botanisches Institut, Kiel University, Kiel, Germany
,
Annemarie Lippert
2   Institut für Botanik, Technische Universität Dresden, Dresden, Germany
,
Jutta Ludwig-Müller
2   Institut für Botanik, Technische Universität Dresden, Dresden, Germany
,
Dietrich Ober
1   Botanisches Institut, Kiel University, Kiel, Germany
› Author Affiliations
Further Information

Publication History

received 06 May 2019
revised 05 August 2019

accepted 15 August 2019

Publication Date:
26 August 2019 (online)

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Abstract

Comfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 – 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.

Key words

Symphytum officinale - alkaloid extraction - transgenic tissue culture - transcript quantification - Boraginaceae

Supporting Information

    The design of amiRNAs, their respective HSS transcript level, and PA content can be found in Fig. 1S. Methodological details about transcript quantification, PA quantification, and experimental growth conditions for amiRNA mutant lines are described in Supplementary Methods 1. The abundance of the predominant PAs in HRs of S. officinale is illustrated in Fig. 2S. A GC-MS base peak chromatogram (50 – 300 m/z) of a strong cation exchange-solid phase extraction from a HR control line is given in Fig. 3S. Primer sequences used in this manuscript are given in Table 1S.

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