Plant metabolism is usually differentiated into primary and secondary metabolism. Secondary metabolites (also known as natural products, phytochemicals or specialized metabolites) underpin important plant traits (e.g. resistance, abiotic stress tolerance, nutritional quality and flavour). The great diversity of secondary metabolites in plants is synthesized from a limited number of building blocks/skeletons that are ubiquitous in the majority of plants but differ in a species-specific manner in the enzymatic permutation and decoration of these basic structures. Thus, despite their common origin a multitude of diverse structures is formed that have divergent biological activities and have traditionally provided a matchless starting point for drug and agrochemical discovery.
Plant secondary metabolism, focusing on triterpene metabolism, has been a core activity for the last 15 years in the lab. Triterpenes are one of the largest classes of plant natural product and have important functions, both in plants (by providing protection against pests and diseases) and for human use (as drugs, adjuvants, anti-microbials, anticancer agents, surfactants, preservatives etc). Triterpenes are low-molecular weight metabolites that are synthesized from mevalonate via a 30 carbon intermediate, 2,3-oxidosqualene. The cyclization of 2,3-oxido-squalene to sterols and triterpene by enzymes known as oxidosqualenecyclases (OSCs) represents a critical branch-point between primary and secondary metabolism. Yet, the functions of these genes are largely unknown. Work in the lab focuses on triterpene biosynthesis and function in legumes.
We participate in a multi-national, academic-industrial research collaboration, funded by EU FP7 (ΤRIFORC project, www.triforc.eu) aiming in providing new biological tools (genes, enzymes, metabolites) as well as in developing sustainable sources of identified bioactive triterpenes, using combinatorial and synthetic biology approaches in plant and fungal expression systems. In partnership with other labs in our department, we hope to exploit triterpene diversity, in order to identify bioactive triterpenes as candidate anti-gluconeogenic and anti-diabetics in in vitro assays (collaboration with Prof. D. Leonidas), and as selective, glucocorticoid, receptor agonist (SEGRA) as anti-inflammatory and anti-proliferative agents that can be used as substitutes to glucocorticoids in medicine (collaboration with Prof. A-M. Psarra).
AMY2 gene cluster project
A novel feature of specialized metabolism in plant biology is that the genes for the synthesis of a number of major classes of plant-derived secondary metabolites, including triterpenes, are organised in clusters, reminiscent of the operons and metabolic gene clusters found in microbes.
This work focuses on how triterpene biosynthesis is regulated in the model legume, Lotus japonicus, at gene and transcript levels and how it is tailored in response to symbiotic, nitrogen-fixing Rhizobia. Previous work in the lab (Krokida et al., 2013) showed that a multi-functional β-amyrin synthase, AMY2, is clustered with two cytochrome P450 enzymes and an unidentified reductase in the Lotus japonicus genome. AMY2 was strongly co-expressed with both P450 enzymes in response to a range of plant hormone treatments, abiotic stresses, and Rhizobium infection. Current experiments aim to identify the triterpene products of this gene cluster and to elucidate the role of these triterpenes especially in the context of Rhizobium infection. To this end, we have also established a robust root culture contained system that allows to manipulate gene expression under controlled conditions.
In addition, overexpression of AMY2 in heterologous systems in E.coli and in appropriate yeast cells and a targeted mutagenesis of LjAMY2 has been initiated.
While many enzymes involved in triterpene biosynthesis have been identified to date, few molecular regulators of this facet of plant metabolism have been isolated in any species. Recent efforts in A. Goosens lab (VIB, Ghent) have identified two Triterpene Saponin Activating Regulators (TSARs) in the legume Medicago truncatula. These basic helix-loop-helix transcription factors selectively induce expression of enzymes involved in biosynthesis of triterpene saponins and of HMGR, the enzyme supplying the main precursor for triterpene biosynthesis, mevalonate, and thus lead to accumulation of triterpene saponins (Mertens et al., 2016). BLAST searches against the Lotus japonicus genome reveal three genes similar to MtTSARs, on which we are conducting phylogenetic and co-expression analyses. To further test whether TSAR-mediated signaling is conserved in legumes, we are currently evaluating the impact of heterologously expressed MtTSAR1 and MtTSAR2 in Lotus japonicus hairy roots on known triterpene biosynthetic gene expression and triterpene content.
Two legume crop plants are under study: Trigonella foenum- graecum, (producing a high content (0.6-1.7%) of steroidal saponins) and Vigna unguiculata (producing triterpene saponins in leaves and roots). As regards V. unguiculate, we work in close collaboration with Dr. M. Omirou and I. Ioannidis at ARI, Cyprus. A number of genotypes is in use to be tested under various conditions but also under field conditions and analysed for the saponin content. Both plants have been grown to evaluate triterpene production and select the appropriate tissues to analyse via RNAseq in order to identify the biosynthetic genes involved in the two plants. A hairy-root transformation system has been established for Trigonella foenum-graecum.
Georgatza D, Gorgogietas VA, Kylindri P, Charalambous MCh, Papadopoulou KK, Hayes JM, Psarra AMG. (2016) The triterpene echinocystic acid and its 3-O-glucoside derivative arerevealed as potent and selective glucocorticoid receptor agonists. The International Journal of Biochemistry &Cell Biology 79 (2016) 277–287
Mosses T, Papadopoulou KK, Osbourn AE (2014) Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives. Critical Reviews in Biochemistry and Molecular Biology DOI: 10.3109/10409238.2014.953628
Krokida A, Delis C, Geisler K, Garagkounis C, Tsikou D, Peña-Rodríguez LM, Field B, Osbourn AE, Papadopoulou KK* (2013) A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis New Phytologist 200: 675-690.
Osbourn AE*, Papadopoulou K, Qi X, Field B, Wegel E. (2012) Finding and analysing plant secondary metabolic gene clusters (Review) Methods in Enzymology 517: 113-138
Delis C, Krokida A, Georgiou S, Peña-Rodríguez LM, Kavroulakis N, Ioannou E, Roussis V, Osbourn AE, Papadopoulou KK (2011) Role of lupeol synthase in Lotus japonicus nodule formation, New Phytologist 189:335-346