Automatic metabolite annotation in complex LC-MS(n ≥ 2) data using MAGMa

Poster presented at the Analytical Tools for Cutting-edge Metabolomics meeting in London, 30 April 201

Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves

Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors. In this study we introduced Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized transcription factors from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl-putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species

Towards elucidating carnosic acid biosynthesis in Lamiaceae: Functional characterization of the three first steps of the pathway in Salvia fruticosa and Rosmarinus officinalis

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPSand SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera

The influence of light microclimate on the lipid profile and associated transcripts of photosynthetically active grape berry seeds

Lipids and oils determine the quality and industrial value of grape seeds. Studies with legume seeds demonstrated the influence of light on lipid metabolism and its association with seed photosynthesis. Grape berry seeds are photosynthetically active till mature stage, but mostly during the green stage and veraison. The objective of this work was to compare the lipid profiles of seeds from white grape berries (cv. Alvarinho) growing at two contrasting light microclimates in the canopy (low and high light, LL and HL respectively), previously reported to have distinct photosynthetic competences. Berries were collected at three developmental stages (green, veraison and mature) and from both microclimates, and the seeds were analyzed for their lipid profiles in an untargeted manner using liquid chromatography coupled to high resolution mass spectrometry (LCMS). The seed lipid profiles differed greatly among berry developmental stages, and to a lesser extend between microclimates. The LL microclimate coincided with a higher relative levels of fatty acids specifically at mature stage, while the HL microclimate led to an up-regulation of ceramides at green stage and of triacylglycerols and glycerophospholipids at mature stage. The seed transcript levels of four key genes (VvACCase1, VvΔ9FAD, VvFAD6 and VvLOXO) involved in fatty acid metabolism were analyzed using real-time qPCR. The lipoxygenase gene (VvLOXO) was down- and up-regulated by HL, as compared to LL, in seeds at green and veraison stages, respectively. These results suggest that seed photosynthesis may play distinct roles during seed growth and development, possibly by fueling different lipid pathways: at green stage mainly towards the accumulation of membrane-bound lipid species that are essential for cell growth and maintenance of the photosynthetic machinery itself; and at veraison and mature stages mainly towards storage lipids that contribute to the final quality of the grape seeds

Natural diversity in health related phytochemicals in Turkish tomatoes

BACKGROUND: Tomatoes are important fruits in the Mediterranean diet and are considered to reduce the risk of different human diseases due to their antioxidative powers. OBJECTIVE: The purpose of the present study was to investigate the biodiversity within a set of 50 tomato fruit accessions collected across Turkey concerning their antioxidant capacities and the levels of potential health-beneficial compounds including phenolic compounds, carotenoids, ascorbic acid and tocopherols. METHODS: All accessions were simultaneously grown in an open experimental field in 2017 and ripe fruits were harvested for analysis. Antioxidant capacities of both hydrophilic and lipophilic extracts were determined using spectrophotometric assays after which individual antioxidants were identified by HPLC using an on-line antioxidant detection system. Phenolic acids, flavonoids, carotenoids and vitamins C and E were quantified using HPLC. RESULTS: The results indicated that there is a wide diversity within this small collection with respect to their hydrophilic and lipophilic antioxidants. CONCLUSION: Hydrophilic antioxidant capacity of the tomatoes was generally related to chlorogenic acid and Vitamin C levels, while lipophilic antioxidants were correlated to all-trans lycopene.Istanbul Technical University 4135

Variation in secondary metabolites in a unique set of tomato accessions collected in Turkey

In this study, 50 tomato landraces grown in Turkey were investigated in terms of their secondary metabolite profiles. Each accession was planted in 2016 and 2017 in 3 replicates in an open field. In this study, color, pH and brix of the fruit samples were measured and an unbiased LCMS-based metabolomics approach was applied. Based on Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) of the relative abundance levels of >250 metabolites, it could be concluded that fruit size was the most influential to the biochemical composition, rather than the geographical origin of accessions. Results indicated substantial biodiversity in various metabolites generally regarded as key to fruit quality aspects, including sugars; phenolic compounds like phenylpropanoids and flavonoids; alkaloids and glycosides of flavour-related volatile compounds. The phytochemical data provides insight into which Turkish accessions might be most promising as starting materials for the tomato processing and breeding industries.</p

Metabolite variation in the lettuce gene pool : towards healthier crop varieties and food

Introduction: Lettuce (Lactuca sativa L.) is generally not specifically acknowledged for its taste and nutritional value, while its cultivation suffers from limited resistance against several pests and diseases. Such key traits are known to be largely dependent on the ability of varieties to produce specific phytochemicals. Objectives: We aimed to identify promising genetic resources for the improvement of phytochemical composition of lettuce varieties. Methods: Phytochemical variation was investigated using 150 Lactuca genebank accessions, comprising a core set of the lettuce gene pool, and resulting data were related to available phenotypic information. Results: A hierarchical cluster analysis of the variation in relative abundance of 2026 phytochemicals, revealed by untargeted metabolic profiling, strongly resembled the known lettuce gene pool structure, indicating that the observed variation was to a large extent genetically determined. Many phytochemicals appeared species-specific, of which several are generally related to traits that are associated with plant health or nutritional value. For a large number of phytochemicals the relative abundance was either positively or negatively correlated with available phenotypic data on resistances against pests and diseases, indicating their potential role in plant resistance. Particularly the more primitive lettuces and the closely related wild relatives showed high levels of (poly)phenols and vitamin C, thus representing potential genetic resources for improving nutritional traits in modern crop types. Conclusion: Our large-scale analysis of phytochemical variation is unprecedented in lettuce and demonstrated the ample availability of suitable genetic resources for the development of improved lettuce varieties with higher nutritional quality and more sustainable production.</p