Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

SUMMARY: Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey ( Symphytum officinale , Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper‐containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, So CuAO1 is expressed in young leaves, while So CuAO5 is expressed in roots. CRISPR/Cas9‐mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA‐free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of So CuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species‐specific, tight spatio‐temporal regulation, and independent evolutionary origins in multiple plant lineages.

Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites found in several plant lineages with independent evolutionary origins. In comfrey ( Symphytum officinale ), two independent homospermidine oxidase (HSO) paralogs are responsible for oxidation of homospermidine (Hspd) to the bicyclic pyrrolizidine in roots and young leaves. As PA biosynthesis in S. officinale and Heliotropium indicum (Indian heliotrope) share a common ancestor, we tested whether H. indicum is also able to synthesize PAs, not only in aerial parts but also in roots. H. indicum constitutively synthesizes not only complex PAs, in aerial parts but also simple PAs in roots. Of five copper‐containing amine oxidases (CuAOs) identified in H. indicum , three have the ability to convert Hspd to the bicyclic pyrrolizidine in vitro . CRISPR/Cas9 genome editing confirmed that, in planta , only one of these CuAOs is involved in PA biosynthesis in roots, which is identical to the HSO involved in PA biosynthesis in leaves. PA biosynthesis in roots is less efficient than in aerial parts, an observation that allowed the analysis of various pathway intermediates using tracer feeding experiments. The CuAO phylogeny, together with comparative gene structure analyses, suggest a common evolutionary origin of PA‐producing CuAOs. However, independent scenarios of PA metabolism were observed in Indian heliotrope and comfrey, adding a further aspect of diversity in the regulation of PA biosynthesis.