# Roots of Indian heliotrope (Heliotropium indicum) produce simple pyrrolizidine alkaloids using the same homospermidine oxidase involved in biosynthesis of complex pyrrolizidine alkaloids in aerial parts

## TL;DR

Indian heliotrope uses the same enzyme to make different types of toxic plant compounds in its roots and leaves.

## Contribution

Discovery that a single homospermidine oxidase is responsible for pyrrolizidine alkaloid biosynthesis in both roots and aerial parts of Heliotropium indicum.

## Key findings

- H. indicum synthesizes complex PAs in aerial parts and simple PAs in roots.
- Only one of three CuAOs in H. indicum is involved in root PA biosynthesis.
- Root PA biosynthesis is less efficient than in aerial parts, allowing intermediate analysis.

## Abstract

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.

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.

Indian heliotrope uses the same oxidase to produce complex pyrrolizidine alkaloid esters in aerial parts and simple pyrrolizidine alkaloids in roots.