Sesquiterpene lactones are a class of natural compounds well-known for their bioactivity and are characteristic for the Asteraceae family. Most sesquiterpene lactones are considered derivatives of germacrene A acid (GAA). GAA can be stereospecifically hydroxylated by the cytochrome P450 enzymes (CYP) Lactuca sativa costunolide synthase CYP71BL2 (LsCOS) and Helianthus annuus GAA 8β-hydroxylase CYP71BL1 (HaG8H) at C6 (in α-orientation) or C8 (in β-orientation), respectively. Spontaneous subsequent lactonization of the resulting 6α-hydroxy-GAA leads to costunolide, whereas 8β-hydroxy-GAA has not yet been reported to cyclize to a sesquiterpene lactone. Sunflower and related species of the Heliantheae tribe contain sesquiterpene lactones mainly derived from inunolide (7,8-cis lactone) and eupatolide (8β-hydroxy-costunolide) precursors. However, the mechanism of 7,8-cis lactonization in general, and the 6,7-trans lactone formation in the sunflower tribe, remain elusive. Here, we show that, in plant cells, heterologous expression of CYP71BL1 leads to the formation of inunolide. Using a phylogenetic analysis of enzymes from the CYP71 family involved in sesquiterpenoid metabolism, we identified the CYP71DD6 gene, which was able to catalyze the 6,7-trans lactonization in sunflowers, using as a substrate 8β-hydroxy-GAA. Consequently, CYP71DD6 resulted in the synthesis of eupatolide, thus called HaES ( Helianthus annuus eupatolide synthase). Thus, our study shows the entry point for the biosynthesis of two distinct types of sesquiterpene lactones in sunflowers: the 6,7-trans lactones derived from eupatolide and the 7,8-cis lactones derived from inunolide. The implications for tissue-specific localization, based on expression studies, are discussed.

Acting as chemical defense or signaling compounds, secondary metabolites (SMs) play an essential role in the evolutionary success of many angiosperm plant families. However, the adaptive advantages that SMs confer, and the influence of environmental and developmental factors on SMs expression, remains poorly understood. A study of taxa endemic to the variable Andean climate, using a metabolomics approach, may provide further insight. By analyzing gene expression patterns and metabolic fingerprints, we report herein the developmental and environmental regulation of the secondary metabolism of Smallanthus sonchifolius (yacón), a medicinal Andean plant. Our results demonstrate a clear developmental stage dependent regulation of the secondary metabolism of yacón leaves wherein the metabolic diversity increases with plant age. However, environmental factors seem to regulate biosynthetic pathways, creating differences in the expression of chemical classes, pointing to an association between transcription levels of relevant genes and the relative amounts of more than 40 different metabolites. This study suggests that the secondary metabolism of yacón is regulated by a complex interplay between environmental factors and developmental stage and provides insight into the regulatory factors and adaptive roles of SMs in Andean taxa.

Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

Smallanthus sonchifolius, also known as yacón, is an Andean crop species commercialized for its nutraceutical and medicinal properties. The tuberous roots of yacón accumulate a diverse array of probiotic and bioactive metabolites including fructooligosaccharides and caffeic acid esters. However, the metabolic diversity of yacón remains unexplored, including the site of biosynthesis and accumulation of key metabolite classes. We report herein a multidisciplinary approach involving metabolomics, gene expression and scanning electron microscopy, to provide a comprehensive analysis of the diversity, distribution and spatial regulation of the specialized metabolism in yacón. Our results demonstrate that different metabolic fingerprints and gene expression patterns characterize specific tissues, organs and cultivars of yacón. Manual inspection of mass spectrometry data and molecular networking allowed the tentative identification of 71 metabolites, including undescribed structural analogues of known bioactive compounds. Imaging by scanning electron microscopy revealed the presence of a new type of glandular trichome in yacón bracts, with a distinctive metabolite profile. Furthermore, the high concentration of sesquiterpene lactones in capitate glandular trichomes and the restricted presence of certain flavonoids and caffeic acid esters in underground organs and internal tissues suggests that these metabolites could be involved in protective and ecological functions. This study demonstrates that individual organs and tissues make specific contributions to the highly diverse and specialized metabolome of yacón, which is proving to be a reservoir of previously undescribed molecules of potential significance in human health.

Main conclusion Tissue-specific occurrence and formation of endogenous sesquiterpene lactones has been assessed and suggests physiological function as antagonists of auxin-induced plant growth in sunflower. Abstract Sunflower, Helianthus annuus, accumulate high concentrations of bioactive sesquiterpene lactones (STL) in glandular trichomes, but in addition, structurally different STL occur in only trace amounts in the inner tissues. The spatial and temporal production of these endogenous STL during early phases of plant development is widely unknown and their physiological function as putative natural growth regulators is yet speculative. By means of HPLC and MS analysis it was shown that costunolide, dehydrocostuslactone, 8-epixanthatin and tomentosin are already present in dry seeds and can be extracted in low amounts from cotyledons, hypocotyls and roots of seedlings during the first days after germination. Semi-quantitative and RT-qPCR experiments with genes of the key enzymes of two independent routes of the endogenous STL biosynthesis confirmed the early and individual expression in these organs and revealed a gradual down regulation during the first 72–96 h after germination. Light irradiation of the plants led to a fast, but transient increase of STL in parts of the hypocotyl which correlated with growth retardation of the stem. One-sided external application of costunolide on hypocotyls conferred reduced growth of the treated side, thus resulting in the curving of the stem towards the side of the application. This indicates the inhibiting effects of STL on plant growth. The putative function of endogenous STL in sunflower as antagonists of auxin in growth processes is discussed.