The cytochrome P450 CYP72A552 is key to production of hederagenin‐based saponins that mediate plant defense against herbivores

Abstract

Summary Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin‐based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C‐23 to hederagenin. In vitro‐produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin‐based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol

“…First, oxidation of the methyl group to an alcohol, then to the corresponding aldehyde, and finally oxidation to a carboxylic acid. This pattern has previously been described for the oxidation of carbons 23, 24, 28, and 30 of an oleanane backbone [45,[62][63][64][65]. Although all three CYP712Ks identified here oxidize the C-29 position on friedelin, we noted that each of them produced a different ratio of 29-hydroxy-friedelin to polpunonic acid (Fig.…”

Evolution of Structural Diversity of Triterpenoids

“…β-amyrin is a precursor of glycyrrhizin, a triterpenoid saponin found in the roots and stolons of liquorice (Glycyrrhiza glabra) well known for its pharmaceutical properties and as a natural sweetener (Hayashi et al, 2002). β-amyrin is also precursor for the antifungal saponin avenacin A-1 in oat and for the potent insect-feeding deterrent hederagenin glycosides in the Brassicaceae Barbarea vulgaris (Kuzina et al, 2009;Nielsen et al, 2010;Khakimov et al, 2016;Liu et al, 2019);. Additionally, β-amyrin seems to play a role in root development in oat (Kemen et al, 2014) and in Lotus japonicus (Krokida et al, 2013), suggesting that triterpenoids like lupeol and β-amyrin are not exclusively involved in plant defense.…”

“…in some species they are arranged in clusters of non-homologous genes (e.g., avenacins in oat, Qi et al, 2004; cucurbitacins in cucumber, Shang et al, 2014); the biosynthesis of other triterpenoids is mediated by genes scattered along the plant genome organized typically in tandem repeats (e.g., saponins in Barbarea vulgaris, Khakimov et al, 2015;Erthmann et al, 2018;Liu et al, 2019; and mogrosides in Siraitia grosvenorii, Itkin et al, 2016).…”

“…In recent years, the Barbarea genus has appeared as a unique plant model as it is the only genus in the agronomically important cabbage family (Brassicaceae) known to accumulate saponins (Khakimov et al, 2016). Some of these saponins (e.g., hederagenin cellobioside) are highly deterrent to Brassica specialist herbivores such as flea beetles (Phyllotreta nemorum) and the diamondback moth (Plutella xylostella) (Kuzina et al, 2009;Kuzina et al, 2011;Liu et al, 2019).…”

“…Conversely, LUP2 was preferentially expressed in the P-type plants (insect-susceptible), and produces mainly lupeol, the precursor for lupeol-derived saponins that appear not to be deterrent. Of the eight CYP72As, only CYP72A552 oxidizes oleanolic acid at the C23 position leading to the formation of insect deterrent hederagenin glycosides (Liu et al, 2019). CYP716A80 and CYP716A81 catalyze C28 carboxylation and two additional hydroxylation (Khakimov et al, 2015) while FIGURE 2 | Structural changes may affect the biological activity of triterpenoids.…”