Sesquiterpene Lactones – Insights into Biosynthesis, Regulation and Signalling Roles

Figures & data

Figure 1. Sesquiterpene Lactones (A) Stereochemistry of α-methylene-γ-lactone sesquiterpene lactones, (1a) 6,7 cis lactone, (1b) 6,7 trans lactone, (2a) 7,8 cis lactone (2b) 7,8 trans lactone. (B) Examples for sesquiterpene lactone backbones of increasing complexity levels as defined by Seaman, (1982). (3) germacranolide (here: costunolide), (4) guaianolide, (5) xanthanolide, (6) 3,4-secoambrosanolide. (C) Examples for chemical STL diversity found in nature: (7) argophyllin B, (8) dehydrocostus lactone, (9) lactucopicrin, (10) thapsigargin, (11) 8-epi-xanthatin, (12) tomentosin, (13) 2α-acetoxy-inuviscolide. Ac: acetate, Ang: angelate.

Figure 2. Phylogeny of cytochrome P450 enzymes (CYPs) that oxidize sesquiterpenes. Neighbour-joining tree with 500 bootstraps, outgroup: AaC4H, subfamilies with CYPs oxidizing sesquiterpenes but not involved in STL biosynthesis are colored grey. CYP subfamilies involved in STL biosynthesis are shown in green (CYP71AV), yellow (CYP71BZ), orange (CYP71BL), red (CYP71CB and AX), purple (CYP71CA and DD) and blue (CYP76AE). The positions of the oxidized carbons of the substrate are indicated by a red asterisk. (38): R₁=R₂=H, (40): R₁=O, R₂=OH.

Figure 3. Sesquiterpene synthase reactions and artemisinin biosynthesis. (A) Sesquiterpene synthase reactions involved in STL biosynthesis pathways leading from farnesyl pyrophosphate (FPP, (14)), to amorphadiene (15), germacrene A (16) and kunzeaol (17). (B) Artemisinin biosynthesis from amorphadiene (15) via artemisinic aldehyde (19) to artemisinin (23) with the side product artemisinic acid (20).

Table 1. CYP enzymes involved in STL biosynthesis.

CYP subfamily	Enzyme(s)	#^(1)	Substrates	Reactions
CYP71AV	GAO AaAMO CiVO	8 1 1	Germacrene A Amorphadiene Valencene	Multi-step oxidations of olefinic backbones
CYP71AX	HaC14H	1	Costunolide & Parthenolide	STL hydroxylation
CYP71BL	COS Ih8H HaG8H	8 1 1	Germacrene A acid	Lactonization (6-7 trans) Hydroxylation/ lactonization (7-8 trans) Hydroxylation/ lactonization (7-8 cis)
CYP71BZ	KLS	4	Kauniolide	STL backbone rearrangement
CYP71CA	CpPTS	1	Costunolide	STL epoxidation
CYP71CB	Tp8878	1	Costunolide & Parthenolide	STL hydroxylation
CYP71DD	HaES KO CiLCS	1 1 1	8β-OH-GAA Kauniolide 8-deoxylactucin	Lactonization (6-7 trans) STL hydroxylation STL hydroxylation
CYP76AE	CYP76AE2	1	Kunzeaol	Lactonization (6-7 cis)

¹Number of characterized homologous enzymes from different species catalyzing the same reactions.

Table 2. STL in major subfamilies of the Asteraceae family and two outgroup families.

			Secretory structures^(3)		STL enyzmes Characterized/Predicted^(4)
Subfamily^(1)	# species	STL^(2)	T	L	GAS	GAO	COS/G8H	KS	Genome^(5)	Genome Assembly Reference^(6)
Asteroideae	16,200	+	+	–	+	+	+	+	+	Badouin et al. 2017
Cichorioideae	3,200	+	+	+	+	+	+	+	+	Shen et al. 2023b
Pertyoideae	70	+	+	–	n.t.	n.t.	n.t.	n.t.	–	–
Carduoideae	2,500	+	+	–	+	+	+	–	+	Acquadro et al. 2017
Gochnatioideae	100	+	+	–	n.t.	n.t.	n.t.	n.t.	–	–
Wunderlichioideae	30	+	+	–	n.t.	n.t.	n.t.	n.t.	–	–
Mutisioideae	750	+	+	–	n.t.	n.t.	n.t.	n.t.	–	–
Stifftioideae	50	–	–	–	n.t.	n.t.	n.t.	n.t.	–	–
Barnadesioideae	91	–	–	–	+	+	–	–	–	–
Goodeniaceae	404	–	–	–	n.t.	n.t.	n.t.	n.t.	+	Shen et al. 2023b
Apiaceae	3,800	+	–	–	n.t.	n.t.	n.t.	n.t.	+	Iorizzo et al. 2016

¹Major subfamilies of the Asteraceae are defined as subfamilies with >25 species. A lower position in the table indicates a more ancestral lineage.

²Distribution of STL across the different subfamilies and outgroup families as previously reported (Seaman, 1982; Catalán et al., 1996).

³Distribution of secretory structures across the different subfamilies and outgroup families as previously reported (Martínez-Quezada et al., 2023). T: trichomes, L: laticifers.

⁴Enzymes from the following species were characterized: Cichorium intybus and Lactuca sativa, Cichorioideae; Saussurea lappa and Cynara cardunculus, Carduoideae; Barnadesia spinosa, Barnadesoideae. n.t (not tested): the presence of an enzyme homolog in the tribe is likely, but it has not been investigated so far.

⁵In the respective subfamilies or outgroup family, there is one (or more) representative species with a sequenced genome available.

⁶Representative genome sequences for subfamilies: Lactuca sativa, Cichorioideae (Shen et al., 2023b); Cynara cardunculus, Carduoideae (Acquadro et al., 2017).

Representative genome sequences for outgroup families: Scaevola taccada, Goodeniaceae (Shen et al., 2023b); Daucus carota, Apiaceae (Iorizzo et al., 2016). Detailed references to genome sequences are shown in Supporting Data S1.

Table 3. STL in major tribes of the Asteroideae subfamily.

			Secretory structures^(3)		STL enzymes Characterized/Predicted^(4)
Tribe^(1)	# species	STL^(2)	T	L	GAS	GAO	COS/G8H	KS	Genome^(5)	Reference Genome Assembly^(6)
Heliantheae	1,400	+	+	–	+	+	+	–	+	Badouin et al. 2017
Coreopsideae	550	+	+	–	n.t.	n.t.	n.t.	n.t.	+	Bellinger et al. 2022
Eupatorieae	220	+	+	–	n.t.	n.t.	n.t.	n.t.	+	Liu et al. 2020
Tagetae	267	–	–	–	n.t.	n.t.	n.t.	n.t.	+	Xin et al. 2023
Millerieae	380	+	+	–	n.t.	n.t.	n.t.	n.t.	+	Fan et al. 2022
Inuleae	687	+	+	–	+	+	+	–	+	McEvoy et al. 2023
Gnaphileae	1,240	+	+	–	n.t.	n.t.	n.t.	n.t.	+	Berman et al. 2023
Astereae	3,080	+	+	–	n.t.	n.t.	n.t.	n.t.	+	Peng et al. 2014
Anthemideae	1,800	+	+	–	+	+	+	+	+	Liao et al. 2022
Senecioneae	3,500	+	+	–	n.t.	n.t.	n.t.	n.t.	+	unpublished

¹Major tribes of the Asteroideae are defined as tribes with >200 species. A lower position in the table indicates a more ancestral lineage.

²Distribution of STL across the different tribes as previously reported (Seaman, 1982; Catalán et al., 1996).

³Distribution of secretory structures across the different tribes as previously reported (Martínez-Quezada et al., 2023). T: trichomes, L: laticifers.

⁴Enzymes from the following species were characterized: Heliantheae: Helianthus annuus and Xanthium strumarium, Inuleae: Inula hupehensis, Anthemideae: Artemisia annua, Tanacetum parthenium and T. cinerariifolium, n.t (not tested): the presence of an enzyme homolog in the tribe is likely, but it has not been investigated so far.

⁵In the respective tribe, there is one (or more) representative species with a sequenced genome available.

⁶Representative genome sequences for each tribe: Helianthus annuus, Heliantheae (Badouin et al., 2017); Bidens hawaiensis, Coreopsideae (Bellinger et al., 2022); Mikania micrantha, Eupatorieae (Liu et al., 2020); Tagetes erecta, Tageteae (Xin et al., 2023); Millerieae: Smallanthus sonchifolius (Fan et al., 2022); Dittrichia graveolens, Inuleae (McEvoy et al., 2023); Helichrysum umbraculigerum, Gnaphalieae (Berman et al., 2023); Erigeron canadensis, Astereae (Peng et al., 2014); Artemisia annua, Anthemideae (Liao et al., 2022); Senecio squalidus, Senecioneae. Detailed references to genome sequences are shown in Supporting Data S1.

Figure 4. Lactonization reactions in Asteraceae STL. (A) Conversion of germacrene A (16) to germacrene A acid (26). (B) Lactonization of germacrene A acid (26) to the STL inunolide (29), eupatolide (30), epi-inunolide (32) and costunolide (3).

Figure 5. Biosynthesis pathways downstream costunolide in Asteraceae and STL biosynthesis in Apiaceae. (A) Conversion of costunolide (3) to 14-hydroxycostunolide (33) and 3β-hydroxycostunolide (35) (via hydroxylation), to parthenolide (34) (via epoxidation) and kauniolide (37) (via backbone rearrangement). (B) Oxidation of kauniolide (37) to lactucin (40). C Conversion of kunzeaol (17) to epi-dihydrocostunolide (41).

Figure 6. Helianthus annuus (sunflower) as a model for the developmental regulation of sesquiterpene lactone (STL) biosynthesis in (biseriate) capitate glandular trichomes (CGT) of Asteraceae. Scheme of CGT in (A) pre-secretory and (B) in secretory stage, respectively. Fully developed trichomes consist of 5-6 biseriate layers of stalk cells and 2 layers of secretory cells. STL are sequestered between cell wall and cuticle of secretory cells forming an extracellular glandular globe. (C) Scheme of (I) disk floret development in the capitulum. (II) Development of CGT on anthers, (III) biosynthetic STL gene expression (yellow/green gradient) in secretory cells and (IV) STL accumulation (red/blue gradient) in the glandular globe are tightly co-regulated. When the outermost disk floret opens, CGTs of the outer half of disk floret rows are already in the post-secretory stage while the innermost disk florets are still in the pre-secretory stage. (Göpfert et al. 2005, 2009). Note: The scheme of a glandular trichome in Figure 4B is modified from Spring et al. (2020), The scheme of the cross section of a capitulum (flower head) in Figure 4C is modified from Göpfert et al. (2005). Both original figures were published under the Creative Commons Attribution License.

Figure 7. Schematic representation of the spatial distribution of gene expression (yellow/green gradient map) and STL accumulation (red/blue gradient map) over a schematic root structure from chicory based on data of previous publications (Bogdanović et al., 2019; Cankar et al., 2022; 2023; Vahabi et al., 2024). Genes involved in early steps of STL biosynthesis are expressed outside of the laticifers. Downstream of costunolide the expression pattern of STL biosynthesis genes is higher in the latex relative to other tissues (yellow/green gradient map). The accumulation of STLs is higher in the laticifers (red/blue gradient map) than in the other root tissues.

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