Metabolomic and Gene Expression Studies Reveal the Diversity, Distribution and Spatial Regulation of the Specialized Metabolism of Yacón (Smallanthus sonchifolius, Asteraceae).

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.

Trichome differentiation on leaf primordia of Helianthus annuus (Asteraceae): morphology, gene expression and metabolite profile.

MAIN CONCLUSION: Sunflower trichomes fully develop on embryonic plumula within 3 days after start of germination. Toxic sesquiterpene lactones are produced immediately thereafter thus protecting the apical bud of the seedling against herbivory. Helianthus annuus harbors non-glandular and two different types of multicellular glandular trichomes, namely the biseriate capitate glandular trichomes and the uniseriate linear glandular trichomes. The development of capitate glandular trichomes is well known from anther tips on sunflower disk florets, but not from leaves and no information is yet available on the development of the linear glandular trichomes. Scanning electron microscopy of sunflower seedlings unravelled that within the first 40 h of seed germination all three types of trichomes started to emerge on primordia of the first true leaves. Within the following 20-30 h trichomes developed from trichoblasts to fully differentiated hairs. Gene expression studies showed that genes involved in the trichome-based sesquiterpene lactone formation were up-regulated between 72 and 96 h after start of germination. Metabolite profiling with HPLC confirmed the synthesis of sesquiterpene lactones which may contribute to protect the germinating seedlings from herbivory. The study has shown that sunflower leaf primordia can serve as a fast and easy to handle model system for the investigation of trichome development in Asteraceae.

Lipid-dissolved γ-carotene, ß-carotene, and lycopene in globular chromoplasts of peach palm (Bactris gasipaes Kunth) fruits.

MAIN CONCLUSION: High levels of ß-carotene, lycopene, and the rare γ-carotene occur predominantly lipid-dissolved in the chromoplasts of peach palm fruits. First proof of their absorption from these fruits is reported. The structural diversity, the physical deposition state in planta, and the human bioavailability of carotenoids from the edible fruits of diverse orange and yellow-colored peach palm (Bactris gasipaes Kunth) varieties were investigated. HPLC-PDA-MS(n) revealed a broad range of carotenes, reaching total carotenoid levels from 0.7 to 13.9 mg/100 g FW. Besides the predominant (all-E)-ß-carotene (0.4-5.4 mg/100 g FW), two (Z)-isomers of γ-carotene (0.1-3.9 mg/100 g FW), and one (Z)-lycopene isomer (0.04-0.83 mg/100 g FW) prevailed. Approximately 89-94 % of total carotenoid content pertained to provitamin A carotenoids with retinol activity equivalents ranging from 37 to 609 µg/100 g FW. The physical deposition state of these carotenoids in planta was investigated using light, transmission electron, and scanning electron microscopy. The plastids found in both orange and yellow-colored fruit mesocarps were amylo-chromoplasts of the globular type, containing carotenoids predominantly in a lipid-dissolved form. The hypothesis of lipid-dissolved carotenoids was supported by simple solubility estimations based on carotenoid and lipid contents of the fruit mesocarp. In our study, we report first results on the human bioavailability of γ-carotene, ß-carotene, and lycopene from peach palm fruit, particularly proving the post-prandial absorption of the rarely occurring γ-carotene. Since the physical state of carotenoid deposition has been shown to be decisive for carotenoid bioavailability, lipid-dissolved carotenoids in peach palm fruits are expected to be highly bioavailable, however, further studies are required.

Ultrastructural Alterations in Cells of Sunflower Linear Glandular Trichomes during Maturation.

Sunflower and related taxa are known to possess a characteristic type of multicellular uniseriate trichome which produces sesquiterpenes and flavonoids of yet unknown function for this plant. Contrary to the metabolic profile, the cytological development and ultrastructural rearrangements during the biosynthetic activity of the trichome have not been studied in detail so far. Light, fluorescence and transmission electron microscopy were employed to investigate the functional structure of different trichome cells and their subcellular compartmentation in the pre-secretory, secretory and post-secretory phase. It was shown that the trichome was composed of four cell types, forming the trichome basis with a basal and a stalk cell, a variable number (mostly from five to eight) of barrel-shaped glandular cells and the tip consisting of a dome-shaped apical cell. Metabolic activity started at the trichome tip sometimes accompanied by the formation of small subcuticular cavities at the apical cell. Subsequently, metabolic activity progressed downwards in the upper glandular cells. Cells involved in the secretory process showed disintegration of the subcellular compartments and lost vitality in parallel to deposition of fluorescent and brownish metabolites. The subcuticular cavities usually collapsed in the early secretory stage, whereas the colored depositions remained in cells of senescent hairs.

Localization of sesquiterpene lactone biosynthesis in cells of capitate glandular trichomes of Helianthus annuus (Asteraceae).

Capitate glandular trichomes (CGT) of sunflower, Helianthus annuus, synthesize bioactive sesquiterpene lactones (STLs) within a short period of only a few days during trichome development. In the current project, the subcellular localization of H. annuus germacrene A monooxygenase (HaGAO), a key enzyme of the STL biosynthesis in sunflower CGT, was investigated. A polyclonal antibody raised against this enzyme was used for immunolabelling. HaGAO was found in secretory and stalk cells of CGT. This correlated with the appearance of smooth endoplasmic reticulum in both cell types. Stalk cells and secretory cells differed in form, size and types of plastids, but both had structures necessary for secretion. No HaGAO-specific immunoreaction was found in sunflower leaf tissue outside of CGT or in developing CGT before the secretory phase had started. Our results indicated that not only secretory cells but also nearly all cells of the CGT were involved in the biosynthesis of STL and that this process was not linked to the presence or absence of a specific type of plastid.

Capitate glandular trichomes of Helianthus annuus (Asteraceae): ultrastructure and cytological development.

Previous studies have shown that capitate glandular trichomes (CGT) of the common sunflower, Helianthus annuus, produce sesquiterpene lactones (STL) and flavonoids, which are sequestered and accumulated between the apical cuticle and the wall of the tip cells. To explore the cellular structures required and putatively involved in the STL biosynthesis and secretion, the present study was focused on the development of CGT and the comparison of the ultrastructure of its different cell types. Gradual maturation of flowers in the capitulum of the sunflower provided the possibility to study the simultaneous differentiation from the primordial to the secretory stage of CGT located by light microscopy (bright field, differential interference contrast and fluorescence) as well as transmission electron microscopy. It was shown that the CGT of sunflower anthers had a biseriate structure with up to 14 cell pairs. In mature trichomes, the apical cells called secretory cells were covered entirely by a large cuticle globe, which enclosed the resinous terpenoids and was specialised in thickness and structure. The secretory cells lacked chloroplasts and contained mainly smooth endoplasmic reticulum (sER). Conspicuous cell wall protuberances and an accumulation of mitochondria nearby occurred in the horizontally oriented cell walls. The cytological differences between stalk cells and secretory cells indicate a different function. The dominance of sER suggests its involvement in STL biosynthesis and cell wall protuberances enlarge the surface of the plasmamembrane of secretory cells and may be involved in the secretion processes of STL into the subcuticular space.