View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Universidade do Minho: RepositoriUM Phytochemistry 70 (2009) 65–73 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress Gregory Franklin a, Luis F.R. Conceição a, Erich Kombrink b, Alberto C.P. Dias a,* a Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal b Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Köln, Germany article info abstract Article history: Xanthone production in Hypericum perforatum (HP) suspension cultures in response to elicitation by Received 13 May 2008 Agrobacterium tumefaciens co-cultivation has been studied. RNA blot analyses of HP cells co-cultivated Received in revised form 18 October 2008 with A. tumefaciens have shown a rapid up-regulation of genes encoding important enzymes of the gen- Available online 4 December 2008 eral phenylpropanoid pathway (PAL, phenylalanine ammonia lyase and 4CL, 4-coumarate:CoA ligase) and xanthone biosynthesis (BPS, benzophenone synthase). Analyses of HPLC chromatograms of methanolic Keywords: extracts of control and elicited cells (HP cells that were co-cultivated for 24 h with A. tumefaciens) have Agrobacterium revealed a 12-fold increase in total xanthone concentration and also the emergence of many xanthones Hypericum perforatum after elicitation. Methanolic extract of elicited cells exhibited significantly higher antioxidant and antimi- Biotic stress Xanthones crobial competence than the equivalent extract of control HP cells indicating that these properties have Antioxidant defense response been significantly increased in HP cells after elicitation. Four major de novo synthesized xanthones have Elicitation been identified as 1,3,6,7-tetrahydroxy-8-prenyl xanthone, 1,3,6,7-tetrahydroxy-2-prenyl xanthone, Phytoalexins 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and paxanthone. Antioxidant and antimicrobial charac- terization of these de novo xanthones have revealed that xanthones play dual function in plant cells dur- ing biotic stress: (1) as antioxidants to protect the cells from oxidative damage and (2) as phytoalexins to impair the pathogen growth. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction 2005), cardiovascular protective (Jiang et al., 2004), selective inhi- bition of cyclooxygenase-2 (Zou et al., 2005), inhibition of platelet- Hypericum, a genus of the family Clusiaceae, is widely used in activating factor (PAF)-induced hypotension (Ishiguro et al., 2002; traditional medicine throughout the world since ancient times. Oku et al., 2005) and cytotoxic activities (Yimdjo et al., 2004; The genus is known to produce several xanthones (Dias et al., Boonsri et al., 2006; Suksamrarn et al., 2006). 2000, 2001; Dias, 2003; Ferrari et al., 2005; Tanaka and Takaishi, Plant cells respond to pathogens predominantly by mobilizing 2006). their secondary metabolites, which tend to protect the plant Xanthones are a class of polyphenolics that exhibit well-docu- cells from pathogen attack. In this context, Beerhues and Berger mented pharmacological properties, mainly due to their oxygen- (1995) observed an increase in xanthone accumulation in sus- ated heterocyclic nature and diversity of functional groups. They pended cells of Centaurium species upon elicitation with yeast have been described as strong scavengers of free radicals (Jiang extract and methyl jasmonate (MeJ). Conceição et al. (2006) ob- et al., 2004). In addition, many of the xanthones have been re- served a similar response (xanthone accumulation) in Hypericum ported to be active against bacteria including methicillin/multi- perforatum suspended cells elicited with Colletotrichum gloeospo- drug resistant Staphylococcus aureus (Rukachaisirikul et al., 2003, rioides cell wall extracts. The induced xanthone accumulation 2005; Sukpondma et al., 2005; Xiao et al., 2008), vancomycin resis- was further increased (by at least 12-fold) when the HP cells tant Enterococci (Sakagami et al., 2005), Mycobacterium tuberculosis were primed with either MeJ or salicylic acid (well known de- (Suksamrarn et al., 2003), etc. Some xanthones even surpass the fense signaling compounds in plants), an important observation, antimicrobial activity of traditional antibiotics (Iinuma et al., which explains possible role of xanthones in plant cells under 1996; Xiao et al., 2008). Other pharmacological properties of xant- biotic stress. hones include anti-inflammatory (Banerjee et al., 2000), cancer- Here, we show that the antioxidant and antimicrobial potentials chemopreventive (Ito et al., 2003), hepatoprotective (Tian et al., have been significantly increased in HP cells after elicitation with Agrobacterium tumefaciens due to the rapid up-regulation of xan- thone metabolism. Putative roles of xanthone metabolism in HP * Corresponding author. Tel.: +351 253604317/18; fax: +351 53 678980. E-mail address: [email protected] (A.C.P. Dias). cells under conditions of biotic stress are discussed. 0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2008.10.016 66 G. Franklin et al. / Phytochemistry 70 (2009) 65–73 2. Results and discussion HP cells that were elicited with fungal extracts or primed with plant defense signaling compounds such as MeJ and salicylic acid 2.1. Induction of xanthone biosynthesis is a defense response (Conceição et al., 2006). Likewise, xanthones were seen accumu- lated when cell cultures were treated with MeJ and yeast-extract 2.1.1. Xanthone profile of HP cells was altered after co-cultivation with in Centaurium species (Beerhues and Berger, 1995), H. androsae- A. tumefaciens mum and Centaurium erythraea (Abd El-Mawla et al., 2001); the lat- Major classes of phenolic compounds produced by cultured HP ter cell cultures exhibited new xanthones after MeJ treatment. All cells under normal conditions are flavonoids and xanthones these observations support the participation of xanthones in plant (Fig. 1a, F, flavonoid and X, xanthone). Though the flavonoid profile defense response. remained unaltered after elicitation, xanthone profile has signifi- Although mangiferin (peak denoted as ‘M’) found along with cantly changed within 24 h (Fig. 1b). With new xanthones, the total few other unidentified xanthones in both control and treated cells, xanthone content increased 12-fold (Fig. 2) indicating that xan- new xanthones appeared in the HP cells treated with A. tumefaciens thone biosynthesis has potential role in the biotic interaction. Sim- (Figs. 1a and b). Amongst the up-regulated xanthones, four were ilar patterns of xanthone accumulation has also been observed in identified (Fig. 1b, peaks 1–4, see Fig. 1c for structural formulae) a F 3000 2500 F M 2000 A U 1500 1000 500 M XX 0 0255075 minutes — 254 nm Band = 4 nm b F 3000 X 2500 F 1 M A 2000 U 1500 2 1000 3 M 500 XX 4 0 0255075 minutes — 254 nm Band = 4 nm c 1,3,6,7-tetrahydroxy-8-prenyl xanthone 1,3,6,7-tetrahydroxy-2-prenyl xanthone 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone paxanthone Fig. 1. Phenolic profile of H. perforatum cells before and after co-cultivation with A. tumefaciens. HPLC chromatogram showing the major phenolic compounds (F, flavonoids; X, xanthones and M, mangiferin) in control (a) and H. perforatum cells co-cultivated with A. tumefaciens for 24 h (b). Note the appearance of many new compounds after elicitation. Peaks 1, 2, 3 and 4 were identified as 1,3,6,7-tetrahydroxy-8-prenyl xanthone, 1,3,6,7-tetrahydroxy-2-prenyl xanthone, 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and paxanthone, respectively. Structural formulae of these xanthones are shown in (c). G. Franklin et al. / Phytochemistry 70 (2009) 65–73 67 5 control b a elicited 28S rRNA 4 18S rRNA 3 b 2 PAL mRNA aa 1 mg/g biomass (DW) a c 0 Xanthone Flavonoids 4CL mRNA Fig. 2. Graph representing total xanthones and flavonoids content of the control and treated H. perforatum cells showing a dramatic increase of xanthones in HP cells d co-cultivated for 24 h with A. tumefaciens. Values are represented in mean ± SE; bars indicated with different letters in a pair are significantly different (p < 0.001), note: BPS mRNA between control and elicited cells, flavonoid concentration is not significantly different (p > 0.05), whereas xanthone concentration is significantly different (p < 0.001) Bonferroni test. 0 h 4 h 12 h 24 h as 1,3,6,7-tetrahydroxy-8-prenyl xanthone (peak 1), 1,3,6,7-tetra- Fig. 3. Northern blot analysis of total RNA isolated from H. perforatum cells after co- cultivation with A. tumefaciens. Ethidium bromide stained rRNA on the Northern hydroxy-2-prenyl xanthone (peak 2), 1,3,7-trihydroxy-6-meth- blot to show loading (a); blots showing the up-regulation of phenylalanine oxy-8-prenyl xanthone (peak 3) and paxanthone (peak 4). ammonia lyase (b); 4-coumarate:CoA ligase (c) and benzophenone synthase and Concentrations of those xanthones before and after elicitation are (d) genes expression within 4 h reaching the maximum in 12 h and declining shown in Table 1. The major xanthone in the treated HP cells thereafter. Control H. perforatum cells (0 h) does not have any hybridization signal for BPS indicating that the xanthone metabolism is activated only after A. was 1,3,6,7-tetrahydroxy-8-prenylxanthone, which accounted for tumefaciens co-cultivation. 23% of the total xanthones (Table 1). This was found to be the same compound that was elicited by MeJ in H. androsaemum cell cultures (Abd El-Mawla et al., 2001), and, might be a common compound induced in both H. perforatum and H. androsaemum in response namic acid, which is used in the synthesis of benzoic acids, the pre- to the biotic stress. cursors of xanthone biosynthesis (Abd El-Mawla et al., 2001) thus, plays a key role in diverting photosynthate from primary metabo- 2.1.2.