Dynamic evolution of terpenoid biosynthesis in the Lamiaceae

With the increasing accessibility of whole-genome sequencing, it has become standard practice to apply such an approach to investigation of plants with important and/or interesting natural products. In part, this reflects recognition that plants have occasionally rearranged or shuffled their genes to generate biosynthetic gene clusters (BGCs) for certain natural products (Smit and Lichman, 2022; Zhan et al., 2022). Use of such a genome-sequence based ‘reverse-genetics’ approach to BGC discovery was first demonstrated in rice (Oryza sativa), particularly for diterpenoid production, enabling relatively rapid progress towards elucidation of the underlying metabolic network (Wang et al., 2023). Accordingly, discovery of an associated BGC is expected to enable more facile investigation of the relevant biosynthetic pathway for the targeted natural product (Zhan et al., 2022). However, it must be noted that plant metabolic evolution is not constrained by BGCs, as the associated pathways often rely on enzymes whose genes are not co-localized (Smit and Lichman, 2022). Indeed, it has been reported that co-expression is a more reliable signal of common biosynthetic function (Wisecaver et al., 2017).

Three papers recently published in Molecular Plant use genome sequencing to provide insight into the evolution of terpenoid biosynthesis in the Lamiaceae, representing the sixth largest family of flowering plants, with great ecological, economic, and cultural importance worldwide. Plants from this mint family are extensively cultivated for culinary, ornamental, fragrance and medicinal usages, and possess secondary metabolite diversity especially rich in terpenoids (Boachon et al., 2018). Pioneering work on tanshinone biosynthesis in the Chinese medicinal herb Danshen (Salvia miltiorrhiza) revealed the presence of an associated BGC, which is now recognized to encode such phenolic abietane-type diterpenoid metabolism more broadly in the Salvia genus, along with a cytochrome P450 (CYP) tandem gene array more specific to tanshinone production (Wang and Peters, 2022). Recent results further highlight conservation of this BGC and its association with production of phenolic abietanes in the Lamiaceae (Bryson et al., 2023; Li et al., 2022). Thus, it was noteworthy that duplicated copies of diterpene synthases found within this BGC in the Scutellaria barbata genome exhibited divergent activity, acting in clerodane rather than abietane diterpenoid biosynthesis, as just reported in Molecular Plant (Li et al., 2023). However, despite similar duplication, the CYPs from this BGC do not seem to act upon the resulting clerodanes, but rather retain their ancestral specificity for abietanes. In addition, the necessary upstream class II diterpene cyclases, which also were identified here, are not part of the BGC. While not identified here, the necessary downstream tailoring enzymes (e.g., CYPs), similarly must be located elsewhere in the genome. This emphasizes that gene duplication and neofunctionalization occurs within BGCs but does not impose constraints on co-evolution with the accompanying genes, so such co-localization cannot be taken as necessarily implying dedication to a common biosynthetic process.

Beyond diterpenoids, perhaps reflecting the prevalence of such metabolism in plants, the majority of BGCs are involved in terpenoid production more broadly (Zhan et al., 2022). This is seen here in discovery of a BGC associated with production of the monoterpenoid p-menthane found upon sequencing of the Schizonepeta tenuifolia genome, which further provides a novel example of a bipartite structure, with two separated and mirrored biosynthetic regions, as also just reported in Molecular Plant (Liu et al., 2023). The report also identifies a novel isopiperitenone reductase, whose distinct nature relative to the enzyme catalyzing the equivalent reaction in Mentha longifolia demonstrates convergent evolution of this biosynthetic step. Similarly, it was further reported that this S. tenuifolia BGC appears to have been independently assembled relative to the BGC previously identified in M. longifolia also associated with p-methane production (Vining et al., 2022), albeit of the opposite stereochemical configuration (Liu et al., 2023). This highlights the relatively frequent assembly of BGCs, as well as the intriguing observation of convergent such BGC assembly for certain biosynthetic pathways, as previously observed for momilactones and casbene derived diterpenoids, with the latter exhibiting similar entantiomeric distinction as observed here for p-menthol (Wang et al., 2023).

Consistent with the importance of co-expression as a predictor of genes encoding enzymes operating in common metabolic pathways in plant natural products biosynthesis (Wisecaver et al., 2017), such analysis supports the relevance of the identified genes, both from the BGC found in S. tenuifolia (Liu et al., 2023) and those not in a BGC from S. barbata (Li et al., 2023). Notably, such data was particularly important for investigation of oridonin diterpenoid biosynthesis in Chinese medicinal herb ‘dong ling cao’ (Isodon rubsecens) also recently reported in Molecular Plant (Sun et al., 2022). In particular, as genome sequencing did not yield a BGC, an alternative approach to biosynthetic gene discovery was necessary. Building on localization of oridonin accumulation, co-expressed CYPs with high transcript levels were investigated, revealing that two CYP706V subfamily members arising from a large tandem CYP array uniquely expanded upon in this genus act in such diterpenoid biosynthesis (Sun et al., 2022). The correlation between this CYP706V subfamily expansion and oridonin production is reminiscent of a previous report in S. miltiorrhiza, where a similarly expanded CYP tandem array characterizes tanshinone biosynthesis, catalyzing the distinctive heterocyclization reaction (Ma et al., 2021). Together these serve to further highlight the role that ‘simpler’ tandem gene duplication and neofunctionalization can play in the evolution of plant natural products biosynthesis.

These three reports help highlight how advances in sequencing technologies have enabled easier access to high-quality genome sequences, as well as transcriptomic data, providing significant insights into the evolution of plant natural products, which vary at a microevolutionary scale within families. However, in each of these cases full elucidation of the targeted biosynthetic pathway(s) has not yet been accomplished. Indeed, the results highlight the dynamic evolution of terpenoid biosynthesis in the Lamiaceae, with relatively frequent assembly of BGCs but also gene duplication and neofunctionalization both within and external to such loci (Figure 1), and emphasize the accompanying need for functional characterization to provide true insight. Nevertheless, these reports lay a solid foundation for further investigations of not only the targeted biosynthetic pathways, but also metabolic evolution in this important plant family more generally.

Figure 1. Summary of the reported investigations of terpenoid biosynthesis in the Lamiaceae.

(A) Relevant phylogeny of Lamiaceae (subfamilies Scutellarioideae, Nepetoideae and their species are highlighted in blue and purple, respectively), and images of Schizonepeta tenuifolia (from Nepetoideae), Scutellaria barbata (from Scutellarioideae), and Isodon rubescens (from Nepetoideae).

(B) Relevant gene clusters for p-menthol biosynthesis on S. tenuifolia chromosome 6, abietane biosynthesis on S. barbata chromosome 2, which has undergone gene duplication and neofunctionalization of diterpene synthases (diTPSs) to clerodane biosynthesis, and the tandem-array of CYP706V on I. rubescens chromosome 2, from which several have evolved functions in oridonin diterpenoid biosynthesis (red indicates terpene synthases, including diTPSs and copalyl diphosphate synthases (CPSs), as well as limonene synthase (StLS) and other monoterpene synthases (StmTPS); blue indicates all cytochromes P450 (CYPs) other than limonene 3-hydroxylase (StL3OH), which is green; old yellow enzyme (StOYE) is yellow; isopiperitenone dehydrogenase (StISPD) is purple).

(C) Expression levels of the biosynthetic gene cluster on S. tenuifolia chromosome 6 (D10A, 10-day-old aerial tissues; D20L, 20-day-old leaves; D35L, 35-day-old leaves; D35R, 35-day-old roots; HM, high methyl jasmonate-treated leaves; CK, untreated leaves; LM, low methyl jasmonate-treated leaves), relevant diTPSs from S. barbata (FL, flower; T, trichome), and relevant CYP706V subfamily members from the tandem-array on I. rubescens chromosome 2.

(D) p-Menthane biosynthesis in S. tenuifolia, and diterpenoid biosynthesis in S. barbata and I. rubescens (GPP, geranyl pyrophosphate; GGPP, geranyl geranyl pyrophosphate). Known biosynthetic enzymes are shown beside solid arrows (IPR, isopiperitenone reductase; IPI, isopulegone isomerase; PR, pulegone reductase; MR, menthone reductase; KPS, kolavenyl diphosphate synthase; IKPS, isokolavenyl diphosphate synthase; KLS, kolavenol synthase; IKLS, isokolavenol synthase; (ent-)CPS, (ent-)copalyl diphosphate synthase; KSL, kaurene synthase-like), while dotted arrows indicate unknown enzymes.