Histolocalization and physico-chemical characterization of dihydrochalcones:
Insight into the role of apple major flavonoids
Matthieu Gaucher
a,b,c, Thomas Dugé de Bernonville
a,b,c,1, David Lohou
a,b,c, Sylvain Guyot
d, Thomas Guillemette
a,b,c, Marie-Noëlle Brisset
a,b,c,⇑, James F. Dat
a,b,caINRA, UMR1345 Institut de Recherche en Horticulture et Semences, F-49071 Angers, France
bUniversité d’Angers, UMR1345 Institut de Recherche en Horticulture et Semences, F-49071 Angers, France
cAgrocampus-Ouest, UMR1345 Institut de Recherche en Horticulture et Semences, F-49071 Angers, France
dUR117 URC-BFC INRA, F-35650 Rennes, France
a r t i c l e i n f o
Article history:
Received 4 September 2012
Received in revised form 15 February 2013 Available online 3 April 2013
Keywords:
Malus x domestica Rosaceae Histolocalization Antimicrobial activity Iron chelation Dihydrochalcones Phloridzin Sieboldin Phloretin
a b s t r a c t
Flavonoids , like other metabolites synthesized via the phenylp ropanoid pathway, possess a wide range of biological activities including functions in plant development and its interaction with the environment.
Dihydro chalcones (mainly phloridzin, sieboldin, trilobatin, phloretin) represe nt the major flavonoid sub- group in apple green tissues. Although this class of phenolic compounds is found in very large amounts in some tissues (200 mg/g of leaf DW), their physio logical significance remains unclear. In the present study, we highlight their tissue-specific localization in young growing shoots suggesting a specific role in important physiological proce sses, most notably in response to biotic stress. Indeed, dihydrochalcones could constitute a basal defense, in particular phloretin which exhibits a strong broad-range bactericidal and fungicidal activity. Our results also indicate that sieb oldin forms complexes with iron with strong affinity, reinforcing its antioxidant properties and conferring to this dihydrochalcone a potential for iron seclusion and/or storage. The importance of localization and biochemical properties of dihydroc halcones are discussed in view of the apple tree defense strategy against both biotic and abiotic stresses.
Ó2013 Elsevier Ltd. All rights reserved.
1. Introduction
Flavonoids are polyphenol ic compound s harboring diphenyl- propane skeleton (C6C3C6). This group of secondary metaboli tes is widely distributed in vascular plants and is known to be involved in a large panel of physiolog ical processes including cell wall rein- forcing, plant–microbes interactio ns, allelopathic plant–plant interactions , UV filtering or protectio n against oxidative stress (for review see Treutter, 2006; Hückelhoven, 2007; Yu and Jez, 2008). Due to their particular substitution patterns (e.g. variable number of hydroxyl groups attached to aromatic rings) flavonoids are very good hydrogen and electron donors conferrin g them sig- nificant antioxidant properties. These include radical scavenging (Re et al., 1999; Gao et al., 1999; Choi et al., 2002 ), singlet oxygen quenching (Foley et al., 1999 ) and metal chelation (Deng and Van Berkel, 1998; Moridani et al., 2003; Mira et al., 2002 ), allowing them to be strong candidat es for protection against oxidative dam-
age. Flavonoids have also been shown to exhibit broader bioactiv- ities such as protection of vascular integrity, antihepatot oxicity, anti-inflammatory activity, antitumor effect, antiallergic propertie s and antimicrobial effects (reviewed in Di Carlo et al., 1999 ).
Flavonoids constitute a chemically diverse family that can be di- vided into sub-families including flavonols, flavanols, flavanones, flavones, anthocyanidi ns, chalcones and dihydrochal cones. To date, approximat ively 200 dihydroc halcones are known to be formed in over 30 plant families, such as Pieris japonica [Ericaceae] (Williams, 1964), Lindera lucida [Lauracea e] (Leong et al., 1998 ), Fragaria x ananassa[Rosaceae] (Hilt et al., 2003 ),Piper elongatum [Piperacea e]
(Hermoso et al., 2003 ),Lithocarpus polystachyu s[Fagaceae] (Dong et al., 2007 ),Lactuca sativa [Asteraceae] (Altunkaya and Gökmen, 2009). Numerous studies have described the potential benefits of dihydroc halcones in human health particular ly due to their antiox- idant properties. Indeed these compounds may be effective in pre- venting various physiopathologi cal processes , notably diabetes (Ehrenkra nz et al., 2005 ), bone resorption (Puel et al., 2005 ) or free radical-invo lving diseases by inhibiting the formation of Advanced Glycation End-products (Dugé de Bernonville et al., 2010 ). However their role in planta still remains unresolved.Malusspecies possess high levels of dihydrochal cones in young leaves and immature fruits (Treutter, 2001; Pontais et al., 2008; Dugé de Bernonville 0031-9422/$ - see front matter Ó2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.02.009
⇑Corresponding author. Address: INRA IRHS, 42 rue Georges Morel, BP60057, F-49071 Beaucouzé, France. Tel.: +33 2 41 22 53 17.
E-mail address:brisset@angers.inra.fr(M.-N. Brisset).
1 Present address: Université François Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, F-37200 Tours, France.
Contents lists available at SciVerse ScienceDi rect
Phy tochemistry
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h y t o c h e m
et al., 2011 ). Among known dihydrochal cones, the biosynthesis of phloridzin (phloretin 20-O-glucoside), which seems to be the most quantitative ly important dihydroc halcone in commercial varieties, and its aglycon phloretin has recently been described in Malus(Jug- dé et al., 2008; Gosch et al., 2009 ). Addition al derivatives such as trilobatin (phloretin 40-O-glucoside), sieboldin (3-hydroxyphloretin 40-O-glucoside) and 3-hydroxyphlor etin have been identified in specific apple tree cultivars (Williams, 1961; Hunter, 1975 ).
According to various authors, the concentratio ns of dihydrochal- cones in apple leaf homogenates could reach several hundreds of micromolar for phloretin and several millimola r for the other gly- cosylated dihydrochal cones i.e. phloridzin, sieboldin, trilobatin (Le- ser and Treutter, 2005; Pontais et al., 2008; Petkovsek et al., 2009;
Dugé de Bernonville et al., 2011 ). The physiologica l significance of such high constitutive concentratio ns still remains unresolved. Pre- vious studies have suggested the implication of dihydrochalcones in the resistance of apple against pathogens, more particularly the apple specific pathogen s responsib le for scab (Venturia inaequalis ) and fire blight (Erwinia amylovora ). Accumul ation of phloridzin and phloretin has shown to be related with quantitative resistance toV. inaequalis suggesting the creation of a harmful environment (Leser and Treutter, 2005; Petkovsek et al., 2009 ) and sieboldin , phloridzin and phloretin may behave as potential antibacterial compounds towards E. amylovora (Pontais et al., 2008; Dugé de Ber- nonville et al., 2011 ). However no clear evidence on the direct role of this class of flavonoids in the general resistance of apple against diseases has been described to date. Besides their role in planta , we have recently described the strong antioxidant activity of sieboldin and its in vitro capacity in preventing physiopathol ogical processes (Dugé de Bernonville et al., 2010 ). Addition ally, phloretin, one of the better characterized dihydrochalcones , exhibits anti-tumor activ- ity, inhibition of human leukemia cell growth, electrical and struc- tural pertubati ons on lipid membran es affecting thus their permeabilit y, and induction of apoptosis (Devi and Das, 1993; Cseh et al., 2000; Kern et al., 2007 ). Finally, the glycoside phloridzin has been reported to produce renal glycosuria and block intestinal glu- cose absorption (Ehrenkranz et al., 2005 ). Thus characteri zing the dihydrochal cones is of interest to both understand their role in plantaand analyze their potential bioactivi ties.
In an attempt to better understa nd dihydrochal cone physiolog- ical roles, we first assessed their tissue localization using two stain- ing methods (vanillin–sulfuric acid and Neu’s reagents). In addition, we investigated their ability to behave either as specific or as broad-range basal defense mechanis ms. To this aim,in vitro antimicrobial activity of diverse dihydrochalcones was evaluated against the two major apple pathogens E. amylovora andV. inaeq- ualisas well as against other bacterial and fungal species non-path- ogenic to apple. Finally, flavonoids exhibiting strong iron chelating properties and iron being an essential element for both plants and microbes we also investigated the iron chelating potential of dihydrochal cones using both UV–vis spectrophot ometry and mass spectrometry (ESI-MS) methods.
2. Results
2.1. Histolocaliza tion of dihydroch alcones
In an attempt to better understand the physiologica l properties of dihydrochal cones in planta , we first decided to analyze their localization in green tissues. Localization was studied in growing shoots of Evereste (Fig. 1), MM106 and Golden Delicious (not shown) genotypes by staining transverse-secti ons of limbs, midribs and stems with vanillin–sulfuric acid or Neu’s reagents.
The two reagents were used to enhance the analysis and compare the two staining methods. The vanillin–sulfuric acid reagent allows
an ‘‘aldehyde acid’’ reaction that depends on the protonation of the aromatic aldehyde (vanillin) due to the presence of electron with- drawing or acceptor groups. Condensation with certain organic molecule s such as alkaloids, flavonoids, steroids, essential oil com- ponents and phenols can then readily occur to form triphenyl - methane type dyes often leading to a variety of colors. Neu’s reagent (Neu, 1957 ) is a standard histochemical reagent for pheno- lic compound s. This borate salt forms complexes with phenolic s emitting a specific fluorescence with some phenolic groups. During immersion of transverse sections, this methano l-containing re- agent also dissolves pigments such as chloroph yll, thus allowing easyin situ localization of flavonoids by the specific greenish-white fluorescence emitted under UV light.
Used as revelation agents for TLC, the two reagents allow effi- cient detection of dihydrochal cones in methanolic tissue extracts (Fig. 2). For vanillin-sulfur ic acid detection flavonoids appeared in orange-red (Figs. 1A and 2A) under white light, whereas for Neu’s reagent these latter emitted a greenish-wh ite fluorescence when excited by UV light (Figs. 1B and 2B). According to the TLC results, the main flavonoids being revealed were sieboldin , phlo- ridzin and trilobatin in Evereste tissues or phloridzin alone in MM106 and Golden Delicious (data not shown). Phloretin was also detected but in our TLC conditions (15 mg of fresh tissue/ml of methano l), extracts were too diluted for an efficient detection of this compound that is known to be the less abundant constitu- tively amongst the four dihydrochalcones . Thus, histolocalization with the two staining methods is likely to reveal preferred sites of accumulati on of the major dihydroc halcones.
Histoloca lization of dihydrochalcones is only presente d for Evereste (Fig. 1) because of its more generous polyphenol pattern, however similar results were obtained for MM106 and Golden Delicious (data not shown). Dihydrochalcones were present in par- ticularly large amounts in the palisade mesophyll cells in the limb section, although staining with Neu’s reagent could also suggest a potential location of these flavonoids in epidermal cells. We also observed a red coloration and a greenish -white fluorescence (Fig. 1A and B, respectively ) in cortical parenchymal cells sur- rounding the vascular system in transverse sections of midrib, pet- iole and stem thus suggesting a strong accumulation of dihydroc halcones near transport tissues. Once more, a location of these compounds in epidermis cells may be suspected, particularly in petiole and stem whatever the staining methods used. Our work showed that combining TLC with histological observations pro- vides a fast and simple method to monitor the presence and loca- tion of flavonoids in fresh tissues. Our histological results raise some interesting questions regarding the relationship between the localization versus the role of dihydrochal cones which prompted us to further investigate the antimicrobial and iron scav- enging activities of the dihydrochal cones. Indeed, dihydrochal - cones localized in the vicinity of the vascular system and the upper layers of leaves may suggest a role in pathogen resistance by antimicrobial activity and/or iron scavenging. In addition, dihydroc halcones localized in palisade parenchyma may be in- volved in the protection of photosyntheti c tissues against iron- mediated generation of reactive oxygen species.
2.2. Antimicrobi al properties of dihydrochalcon es
Because dihydrochal cones are mainly found in tissues that may be subjected to pathogen invasion, their antimicrobi al propertie s towards different bacterial and fungal strains were examinated, accordin g to their potential in planta concentratio n (Fig. 3). The ability of dihydrochal cones to behave as a defense system in Malus was examinated by monitoring their antimicrobial activity against the two main apple pathogens i.e.E. amylovora andV. inaequalis and against other microbes non-pathog enic to apple trees.
Firstly, antimicrobial activity of sieboldin, phloridzin and phloretin was evaluated against E. amylovora and V. inaequalis (Fig. 3A). We used several E. amylovora strains displaying differen- tial aggressiveness on the Evereste genotype including the Ea1430 strain, non virulent on this resistant genotype (Venisse et al., 2001) and the Ea1197/Ea3 049/Ea3792 strains which are some- times able to overcome this resistance (Suppl. Fig. 1). Phloretin was found to be the strongest antibacterial compound on the tested strains as demonstrated by the 50% bacterial growth inhibi- tion (BGI50) for concentrations approximat ely ranging from 250 to 500
l
M. Sieboldin and phloridzin exhibited lower effects, siebol- din being more efficient with a BGI 50 for concentr ations ranging from 5 to 10 mM versus>10 mM for phloridzin. Thus, we observed at least a 10-fold differenc e in antibacte rial activity between gly- cosylated forms and the aglycon. Although the E. amylovora tested strains display a differential aggressiven ess on Evereste, this dif- ference in behavior was not reflected in the antibacterial activity assays. As far as V. inaequalis is concerned, phloretin also appeared to display the highest antimicrobi al activity. Indeed, we observed a 50% inhibition of conidial germinat ion inhibition (CGI50) at ca.1 mM with phloretin, 5 mM for phloridzin and >5 mM for sieboldin.
Following these interesting results we decided to test the vari- ous dihydrochalcones on a wider range of microbes. These latter were chosen among phytopathogen s and opportunisti c micro- organisms , non-pathog enic on apple. For the bacterial strains we observed different tendencies . The Gram-pos itive bacteria (Rf2401; Ai1380; Cfb3401) seemed more resistant to phloridzin and phloretin (BGI50> 1 mM for phloretin; > 10 mM for phloridzin) than the Gram-negative ones (Pst2212; Xc2537; Rs2047) (250 < BGI 50< 500
l
M for phloretin; BGI 50> 5 mM for phlorizin).Sieboldin exhibited the strongest bactericidal activity against Xc2537 (BGI50< 500
l
M), an intermedi ate activity against Pst2212 and Rf 2401 (BGI50> 1 mM), the slightest activity against Rs2047 (BGI50> 5 mM) and no clear effect against Ai1380 and Cfb3401. Phloretin again appeared to be more active than the two glycosylated dihydrochalcones , as observed for E. amylovora . Concerning the other fungi, a pattern similar to V. inaequalis was observed . Once again, phloretin exhibited the highest antifungal activity (50% of mycelium growth inhibition (MGI50), for a concen- tration > 500l
M). In fact, both sieboldin and phloridzin were over- all unable to inhibit fungal growth at the tested concentr ations.pp sp
ph x
pp sp
ph x
ph x pp
sp
pp sp
ph x
ph
ph x
x
A B
Limb
Midrib
Petiole
Stem
Fig. 1.Histolocalization of dihydrochalcones in Evereste growing shoots. Slice vibratom-sections of limb, midrib, petiole and stem colored with (A) vanillin–sulfuric acid reagent and observed by optic microscopy under white light and (B) Neu-reagent and observed by epifluorescence microscopy under UV at 366 nm. Bars = 100 lm. pp, palisade parenchyma; sp, spongy parenchyma; ph, phloem; x, xylem..Potential location of dihydrochalcones. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)
2.3. Iron chelating properties of dihydroch alcones
Among known flavonoid properties, the interaction with transi- tion metal ions to produce Fe(III)–flavonoids complexes is consid- ered a detoxifying mechanism in biological systems. Indeed, the formation of such complexes prevents the participa tion of metal ions in potentially toxic free radical generating reactions (Afanas’ev et al., 1989; Van Acker et al., 1996; Sugihara et al., 1999 ). These data prompted us to evaluate iron chelating properties of dihydr- ochalcones by UV/visible spectrophot ometry and electrospray mass spectrometry. The iron chelating properties of this class of flavonoids were compared with those of other known iron chelat- ing compounds : epicatechin and desferrioxamin, a flavonoid and a well-known sideroph ore, respectively (Basu-Modak et al., 2006;
Zhao et al., 1998; Mlade ˇnka et al., 2011 ).
2.3.1. Spectroph otometric analysis
The UV–vis spectra of 400
l
M solutions of the various com- pounds in the presence of increasing Fe(III) concentrations were assessed at different pH to mimic the pH state/variation in apo- plastic and cytoplasmic cell compartments , especially in cells undergoing biotic or abiotic stress (Fig. 4). The metal-chelatin g properties of polyphenols is generally evaluated by analyzing the shifts of UV bands I (B ring absorption) and II (A ring absorption), which characterize the polyphenoli c spectra (Rice-Evans et al., 1996; Mira et al., 2002; Moridani et al., 2003 ). The powerful chela- tor desferrioxami n exhibited a single absorbance maximum in the region 220–260 nm. With the addition of Fe(III), an additional band in the region 365–580 nm with a maximum at 410–450 nm wasdetected. The increase of absorbance in this region was associated with ascending iron concentrations , indicating that desferrio xamin produced complexes with iron that gave a visible color. The absor- bance increased up to 1.2 mM Fe(III) at pH 3.5 and pH 5.5 and to 1.6 mM Fe(III) at pH 7.5, suggestin g a better chelation ability of desferrio xamin at pH 7.5.
The UV–vis spectra of epicatechin and dihydrochal cones showed two absorbance maxima in the region 220–350 nm. Upon addition of iron and for all tested pHs, a rise in absorbance in the sieboldin absorption spectra (365–580 nm) was observed particu- larly at pH 5.5. Slight increases in absorbance in this area were also observed in the epicatechi n absorption spectra in the presence of iron but only at pH 5.5 and pH 7.5. No rise in the 365–580 nm range was observed when either the phloridzin or phloretin solu- tions were incubated with iron. Previous studies have shown that the interaction of iron with some polyphenol ic compounds pro- duced bathochrom ic shifts of the two characteri stic bands (region 220–350 nm) for which the increase in absorbance was related to an increase in iron concentratio n (Andjelko vic et al., 2006; Lu et al., 2009 ). Furthermore an additional band in the visible spectra was also observed. Here, an increase in absorbance of the two char- acteristic bands was observed for all compound s tested in the pres- ence of Fe(III) but bathochrom ic shifts were not clearly distingui shable. Nevertheles s, a new band in the visible spectra was only detected for epicatechi n and sieboldin. Spectral changes in the absorption spectra vary from one study to another as some authors have reported significant decreases in the absorbance of the two characterist ic flavonoids spectral bands in the presence of metal ions (Mira et al., 2002 ).
The spectrophot ometric data obtained here suggest that siebol- din is more efficient in complexing iron than the other dihydroch- alcones. To refine these results we decided to use the ESI-MS method to identify the formatio n of compound–iron complexes.
2.3.2. Electrospray ionization mass spectrometry (ESI-MS) analysis As may be seen in Table 1, all compound s studied form com- plexes with iron with stoechiometr ies 1:0.1, 1:0.5, 1:1 and 1:2 (compound/iron), under our ESI-MS conditions. However the com- plexes formed were different depending on the tested compound.
In the presence of iron, desferrioxami n gave one complex detected through the peak at m/z 614 correspondi ng to the ion [Fe(III)+(M2H)]+(Fig. 5A). The spectrum of epicatechin (Mr 290) in the presence of iron presented two signals at m/z345 and m/z 634, correspondi ng to the ions [Fe(II)+(MH)]+ and [Fe(III)+2(MH)]+, respectively . The same complexes were de- tected with phloridzin (Mr 436) and iron through peaks at m/z 491 and m/z 926 although these complexes were found in low abundan ce. Sieboldin (Mr 452) and phloretin (Mr 274) gave two complexes with iron correspondi ng to ions [Fe(II)+(MH)]+ and [Fe(III)+M+(MH)]+. These complexes were detected at m/z 507 and m/z959 for sieboldin (Fig. 5B) and at m/z 329 and m/z603 for phloretin (not shown). The relative abundances listed in Table 1 show that sieboldin was the most efficient compound in complex- ing iron and that this ability increased with iron concentratio n.
Overall, in our experimental conditions, the compounds tested may be ranked as follows depending on their chelation activity:
sieboldin > epicatechin > desferrioxami n > phloretin > phloridzin.
These results are in agreement with the fact that sieboldin and epi- catechin contain a catechol group which is well-known to favor the formatio n of complexes with metal ions.
3. Discussion
Dihydroc halcones are quantitat ively major phenolic com- pounds found in apple leaves. According to the literature, these
A
B B
Fig. 2.Specific coloration of dihydrochalcones by (A) vanillin–sulfuric acid reagent or (B) Neu’s reagent verified by thin layer chromatography of methanolic extracts of MM106 and Evereste known to have differential phenolic profiles. SIE, sieboldin;
TRI, trilobatin; PLZ, phloridzin; PLT, phloretin; L, limb; M, midrib; P, petiole.
Standard concentrations: 1 mM.
Sieboldin Phloridzin Phloretin
Sieboldin Phloridzin Phloretin
A A
%)
500 µM 1 mM 2.5 mM 5 mM 10 mM 50 µM 100 µM 250 µM 500 µM
100
owth(% 90 Erwinia amylovora strains
90 80 70 rialgro 60
BGI50 60
50 f bacter 40
30 20 ition of 1010 0
Inhibi Ea1430 Ea1197 Ea3049 Ea3792 Ea1430 Ea1197 Ea3049 Ea3792 Ea1430 Ea1197 Ea3049 Ea3792
%) 100 µM 500 µM 1 mM 2 5 mM 5 mM
ation (% 100
Venturia inaequalis strain
100 µM 500 µM 1 mM 2.5 mM 5 mM
100 90
ermina 7080
60 nidialg 50
CGI50
40 30
of con 2020
10 0
hibition
NT
Inh ViEUB04ViEUB04ViEUB04
B B
(%) Bacterial strains / non pathogenic on apple
500 µM 1 mM 2.5 mM 5 mM 10 mM 50 µM 100 µM 250 µM 500 µM
100 90
rowth( 80 Bacterial strains / non-pathogenic on apple
BGI50 80
70 erialgr 60
BGI50 50
40
of bact 3030
20 bition o 10
0
Inhib Pst
2212 Xc 2537
Rs 2047
Rf 2401
Ai 1380
Cfb 3401
Pst 2212
Xc 2537
Rs 2047
Rf 2401
Ai 1380
Cfb 3401
Pst 2212
Xc 2537
Rs 2047
Rf 2401
Ai 1380
Cfb 2212 2537 2047 2401 1380 3401 2212 2537 2047 2401 1380 3401 2212 2537 2047 2401 1380 34013401
100 µM 500 µM 1 mM 2.5 mM 5 mM
Fungal strains / non-pathogenic on apple
%) Fungal strains / non-pathogenic on apple
100 90
owth(%
FGI50 80
70 ngalgro 60
FGI50 50
40 n of fun 3030 20
hibition 10 NT NT
Ab 43 Nc988 Inh 0
Ab 43 Nc988 Ab 43 Nc988
NT NT
Fig. 3.Antimicrobial effect of dihydrochalcones on a range of microbial isolates. Means of percentage of microbial growth inhibition compared to a control from three independent experiments. Bars represent standard deviations. Ea,Erwinia amylovora ; Pst,Pseudomonas syringae pv.tomato; Xc,Xanthomonas campestris pv. vesicatoria ; Rs, Ralstonia solanacearum ; Rf,Rhodococcus fascians ; Ai,Arthrobacter illicis ; Cfb,Curtobacterium flaccumfacienspv. betae ; Vi,Venturia inaequalis ; Ab,Alternaria brassicicola ; Nc, Neurospora crassa ; NT, not tested.
Epicatechin Sieboldin Phloridz in Phloretin Desferrioxamin
pH 3.5 pH 5.5 pH 7.5
220 320 420 520 620 720 320 420 520 620 720 320 420 520 620 720
Wavelength (nm)
0 0.2 0.4 0.6 0.8 1 1.2 0.2 0.4 0.6 0.8 1 1.2 0.2 0.4 0.6 0.8 1 1.2 0.2 0.4 0.6 0.8 1 1.2 0.2 0.4 0.6 0.8 1 1.2
A bsorbance
Fe(III) 2 mM alone Compound alone Compound + Fe 100 µM
Compound + Fe 200 µM Compound + Fe 300 µM Compound + Fe 400 µM
Compound + Fe 800 µM Compound + Fe 1.2 mM Compound + Fe 1.6 mM Compound + Fe 2 mM
Fig. 4.Absorption spectrum of dihydrochalcones, desferrioxamin and epicatechin at 400 lM in the presence of increasing concentrations of ferric iron at different pH (3.5/
5.5/7.5).
compounds could be involved in different physiological processes : defense against bioagressors, redox homeostasis, dissipation of ex- cess excitatio n energy, signaling, UV protection and pigmenta tion for pollinisator attraction (for review see Treutter, 2006; Hückelho- ven, 2007; Yu and Jez, 2008 ). In the present study, we decided to investigate their localization in apple green tissues and to charac- terize their antimicrobial properties against different micro-organ- isms as well as their iron chelating propertie s. Our aim was to gain some insight into the location versusrole relationship in order to better understand the significance of these compounds present in very high constitutive concentratio ns.
Different imaging techniqu es relying on physico- chemical prop- erties of flavonoids (i.e. optical or UV microscopy after histological staining, multispectral fluorescence and spectrometry imaging) can be used to decipher their localization in planta . Here, we chose histological staining as a fast and simple method to reveal dihydr- ochalcones in situ . Indeed our preliminary TLC results indicated that dihydrochal cones can be efficiently visualized using two stain- ing methods involving vanillin–sulfuric acid reagent and Neu’s re- agent, respectively. As dihydrochalcones were the only compounds visualized under our TLC conditions, the tissue-sectio n stainings are likely to reflect their location. Although we cannot exclude that the histological staining may also reveal the presence of other min- or compounds not detected by our TLC system, our results strongly suggest that dihydroc halcones accumulate mainly in the upper layers of apple leaves as well as in parenchymal non-conduc tive cells found in the vascular bundles (and to lesser extend in the epidermis of petiole and stem). This is in agreement with other re- ports showing that flavonoids are localized in upper layers of leaves and/or in tissues surrounding the vascular system e.g. in Quintinia serrata (Gould et al., 2000 ), Phillyrea latifolia (Tattini et al., 2005 ),Coffea canephor a(Mondolot et al., 2006 ). Taken to- gether all these data suggest that flavonoid accumulation sites in leaves are similar among different plant genera suggesting con- served physiological roles.
Flavonoids may be involved in the control of the redox status in photosyn thetic tissues in plants undergoing excess light stress or in protectio n against UV exposure. The location of dihydrochal - cones in upper layers of leaves is in agreement with a similar role for these compounds in protectin g against UV radiations, oxidative burst and high light levels particularly the glycosylated forms (phloridzin, sieboldin , trilobatin) representi ng the most abundant flavonoids in apple leaves. In apple fruit skin, flavonoid accumula- tion was reported to protect against UV-B-induced damage (Solovche nko and Schmitz- Eiberger, 2003 ) and interestingl y, phloridzin seemed to be more abundant in the peel of sun-expos ed apples (Malus domestica Borkh., cv. Aroma) than in that of shade- grown apples (Hagen et al., 2007 ). Thus, dihydrochal cones which are located in upper layers of leaves and able to efficiently absorb UV wavelengths may act as sunscreens. In addition, a localization of dihydrochalcones in the palisade mesophyl l is also in favor of a role for these compounds in the control of the redox status in photosyn thetic tissues. These results are supported by our previ- ous results indicating that sieboldin and phloridzin are very effi- cient radical scavengers (Dugé de Bernonville et al., 2010 ).
Complemen tary to these properties, we show here that dihydr- ochalcon es are efficient iron chelating agents. Mass spectrometr y data indicated that these phenolic s, in particular sieboldin, are able to form complexes with iron in vitro . In biologica l systems, transi- tion metals are involved in the production of free radicals (Fenton reaction) by decomposition of lipid hydroperox ide (LOOH) or hydrogen peroxide (H2O2) to give the most reactive and deleteri- ous reactive oxygen species, alkoxyl radicals or hydroxyl radicals, respectivel y (Halliwell and Gutteridge, 1984; Minotti and Aust, 1989). Thus, in addition to its radical scavenging ability and asso- ciated bioactivities (seeDugé de Bernonville et al., 2010 ), sieboldin could constitute a good candidat e to protect apple leaves against oxidative stress by limiting the Fenton reaction. In our condition s, phloridzin had a weaker iron chelating activity than sieboldin. As a conseque nce apple genotypes containing sieboldin are likely to be Table 1
Relative abundance (% with respect to the (M+H)+peak) of complex ions, compound/iron.
Compound (Mr, Da) 1:0.1 a 1:0.5 1:1 1:2
1b 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Desferrioxamin (560) – 3.3 – – – 8.2 – – – 22.7 – – – 34.3 – –
Epicatechin (290) 11.3 – 13.8 – 14.5 – 13.1 – 18.6 – 12.4 – 183.2 – 164.7 –
Sieboldin (452) 46.1 – – 142.4 49.3 – – 225.9 135.0 – – 453.6 210.6 – – 485.8
Phloridzin (436) 0.2 – 1.4 – 0.6 – 3.2 – 1.0 – 4.4 – 2.8 – 9.1 –
Phloretin (274) 47.4 – – 19.8 26.8 – – 17.1 21.9 – – 18.7 14.3 – – 11.7
aStoechiometry compound/iron.
b 1 = [Fe(II)+(MH)]+; 2 = [Fe(III)+(M2H)]+; 3 = [Fe(III)+2(MH)]+; 4 = [Fe(III)+M+(MH)]+.
100
0
Relative abundance (%)
200 300 400 500 600 700 800 900 1000
m/z
561
614 583
[M+H]+
[Na+M]+
[Fe(III)+(M-2H)]+
A
100
0 200 300 400 500 600 700 800 900 1000
m/z
291
453
[M+H]+475
[Na+M]+ [Fe(III)+M+(M-H)]+
959
507
[Fe(II)+(M-H)]+ [(M+H)-glc]+
B
Relative abundance (%)
Fig. 5.Electrospray mass spectrum of solutions of (A) FeCl 3–desferrioxamin and (B) FeCl 3–sieboldin (stoechiometry 1:2).
more tolerant to light induced stress. This is supported by the re- sults of Dugé de Bernonvil le et al. (2010)which showed that sie- boldin-cont aining leaf disks were more resistant to paraquat- induced oxidative stress than leaf disks containing phloridzin only.
In addition to their probable role in controlling the redox homeostasi s, flavonoids are also known to exhibit antimicrobial properties suggestin g a role in plant defense against bioagressors.
As our localization results suggest, dihydrochalcones could consti- tute a physico-chem ical barrier against pathogens colonizin g leaf upper layers as V. inaequalis and against pathogens associated with vascular tissues such as E. amylovora . The role of polyphenols in plant defense is well established but their mode of action has yet to be clarified. Bioactive compounds may act in different ways including a direct bactericidal activity against pathogens or a depri- vation of essential elements such as iron. Our result showed that the major glycosylated dihydroc halcones possess a relatively weak antimicrobial activity against the different micro-organi sms stud- ied. However as previously discussed, sieboldin exhibits interesting iron chelating properties. One of the most common strategie s of mi- crobes for iron sequestration is through the synthesis of chelators with high affinity for Fe(III), known as siderophore s. Therefore, the more effective chelation activity of sieboldin compared to that of desferrioxamin (a siderophore of some microbes such as E. amy- lovora(Feistner et al., 1993 )) observed in our study is clearly in fa- vor of a probable competition for iron between sieboldin and pathogen siderophores. Although the constitutive glycosylated dihydrochal cones show poor antimicrobial effectivenes s, the agly- con phloretin exerts the strongest antimicrobial activity towards apple pathogens according to our results and in agreement with previously published data (Pontais et al., 2008 ). Flavonoids are well known to undergo enzymati c reactions, the main transformat ions of phloridzin being deglucosylati on and oxidation (Raa and Overe- em, 1968; Sarapuu and Kheinaru, 1971; Sarapuu, 1971 ). For the fla- vonoid transformat ion processes to occur, enzymes have to be present in the same environment as the substrates and co-sub- strates. In healthy organs, enzymes and substrate s are potential ly distributed in different tissues or subcellular compartme nts. Many abiotic and biotic stresses can provoke cell or tissue disorganizati on and initiate the destruction of the physical barriers separating en- zymes and substrate s. In this manner, pathogen attacks could lead to the release of high amounts of the aglycon phloretin after deglu- cosylation of the dihydroc halcone-glucosi des via ab-glucosidase activity, this compound being constitutivel y found in trace amount in apple green tissues. In addition, enzymatic oxidations of dihydr- ochalcones could lead in a wide variety of oxidized forms including quinones generate d by polyphenolox idase which are highly reac- tive and might possess antimicrobial properties (Cowan, 1999;
Schweigert et al., 2001 ). Moreover, oxidized forms could polymer- ize with each other and with other host constituents to form ‘‘ligni- fied’’ structure and thus participate in cell wall reinforcing (Beckman et al., 1974; Hückelhoven, 2007 ). It would thus be inter- esting to study the transformation reactions of dihydrochal cones during the pathogen–apple tree interactio ns.
The involvement of dihydrochalcones in apple resistance to pathogens is also reinforced by other studies. Indeed, a strong accu- mulation of both phloridzin and phloretin was observed in apple scab-infected peels and leaves of Jonagold and Golden Delicious cultivars, in lesions and in the marginal tissue around lesions (Pet- kovsek et al., 2009; Leser and Treutter, 2005 ). During the initial stages of developmen t on the leaf of V. inaequalis , the conidia germi- nates, the appressoria penetrates the cuticle and then the fungus forms a flattened cell layer (primary stroma;Ortega et al., 1998 ) di- rectly on the epidermal cells in the vicinity of dihydrochalcones accumulation sites. The in vitro metabolism of phloridzin by V.
inaequaliswas studied by Hunter (1975)who reported that the fun- gus was able to convert phloridzin to phloretin viab-glucosidase.
Thus, in apple scab-infected apple leaves, large amounts of phlore- tin could be released by b-glucosidase activities (from either fungal or plant origin) resulting in a specific environment which could lim- it the growth of the fungus, in particular by inhibiting conidial ger- mination as indicated by our results. Although to a lesser extent, the remaining phloridzin may also inhibit the conidial germination . However in another apple–pathogen interaction (E. amylovora ), Pontais et al. (2008) observed an absence of accumulation of phloretin despite a decrease in the constituti ve stock of glycosyl- ated dihydrochalcones in two apple genotypes displayin g con- trasted susceptibi lity to fire blight, rather suggestin g oxidative transformat ions of this constituti ve stock. The authors proposed that a differential transformat ion leading to toxic end-products oc- cur in resistant genotypes only.
Phloretin also displayed a strong antimicrobi al activity against other micro-organisms . We show here that the effect of the aglycon on fungal developmen t was similar whicheve r the fungus studied. In addition, phloretin has approximat ively the same antibacte rial activity against E. amylovora and other Gram-ne gative bacteria and we found no different ial resistance between various E. amylovora strains exhibiting specific aggressiven ess. Therefore, all these data suggest that dihydroc halcones could be considered as participa ting in a broad-spectr um defense system. The better lenience of the Gram-pos itive bacteria to phloretin observed could be explained by the differenc e in the cell-wall compositi on with Gram-negative bacteria that possess a thin peptidoglyc an layer embedded by two li- pid membranes . Certain essential oil components, such as small ter- penoid and phenolic compounds have been reported to be active upon outer lipid membrane of Gram-negati ve bacteria (Helander et al., 1998 ) as it could be the case for phloretin according to its mode of action on the permeabilit y of lipid membranes (Andersen et al., 1976; Cseh et al., 2000 ). More extensive ly, a recent study suggests that a chloroform extract of M. domestica fruits and the phenolic compound phloretin could be used as biopesticide s for control of rice blast as well as tomato late blight caused by the fungus Magnaporthe griseaandPhytophtho ra infestans , respectivel y (Shim et al., 2010 ). In this latter work, phloretin is described to possess in vitro antifungal activity against different pathogen ic fungi including among others M. grisea ,Rhizoctonia solani ,Botrytis cinerea ,P. infestans andPhytoph- thora capsicis . These data reinforce the hypothesis of a possible role of dihydrochal cones as a non-specific basal defense system.
The accumulation of dihydrochalcones that we observed around vascular cells could suggest that apple trees possess a strategic de- fense system against vascular-associ ated pathogens. Xylem vessels are conducive to a rapid invasion of pathogens in the plant.Beck- man (2000)proposed a model in which phenolics can be stored in
‘‘phenoli c-storing cells’’ and be diffused in the vessel element.
Phenolics could thus participate in lignification processes or di- rectly act on pathogen development. The localization around vas- cular system could also suggest a long-ran ge transport of these phenolic s from leaves to stem, and maybe from stem to roots as suggested by Buer et al. (2007). Indeed the long-range transport from stem to roots of naringenin and dihydroflavonol (intermedi- ates of the phenolic biosynthetic pathway) in Arabidosps is , required the participatio n of some ABC family proteins to provide cell-to- cell transport. However, that apple dihydroc halcones undergo remobiliz ation from one tissue to another by long-ran ge transports remains to be determined .
4. Conclusion
Dihydroc halcones are found in very large amounts in apple leaves, however, the ecophysiologi cal significance of this remains an opened question. Our present work is, at our knowledge and apart in the fruit, the first to report extensive characteri zation of
apple dihydrochalcones and their localization in green tissues.
Strong accumulation of this class of compounds was mainly ob- served in palisade parenchyma cells and in cells adjacent to the vascular system. This location prompted us to investiga te the po- tential role of dihydrochalcones in protecting apple tissues against mesophyll and potentially vascular pathogen s and in improving resistance to oxidative stress through iron chelation activity. Our results indicated that glycosyla ted dihydrochal cones are relatively poor antibacterial/anti fungal compound but are a pretty good source of phloretin which exhibited high antimicrobial activity to- ward both organisms. Besides, the antimicrobial activity of phlore- tin against a wide range of pathogens suggests that this phenolic compound could be use as biopestici des for control of many plant diseases. Glycosyla ted dihydroc halcones may instead behave as better antioxidants, in particular when possessing a catechol group like sieboldin , because this compound displayed a high iron chela- tion activity in vitro . Thus, dihydrochal cones could constitute a bipolar protective system of apple (Fig. 6), one permitting via the glycosylated forms resistance to oxidative stress by radical scav- enging activity and resistance to pathogen invasions by depriva- tion of iron, and the other limiting pathogen developmen t by direct antimicrobi al action of the aglycon phloretin. Because phloretin is constitutivel y found in trace amount in apple green tis- sue, this system should be activated via b-glucosidase activity prior to be effective as it is the case of numerous defense systems where this kind of enzyme is related to a detonato r.
5. Experimen tal
5.1. General exprimental procedure
Vanillin (4-hydroxy-3-methoxybenzaldeh yde), Neu’s reagent (2-amino-ethyldiphenyl borinate), epicatechin, desferrioxamin, PEG4000 (Poly(ethylene glycol)), ferric chloride (iron(III) chloride), HPLC grade ethyl acetate and methanol were purchased from Sig- ma–Aldrich (Steinheim, Germany). HPLC grade acetic acid was pur- chased from Carlo Erba (Val de Reuil, France). Sieboldin, phloridzin, phloretin and trilobatin were obtained from Extrasynthèse (Genay, France). Highly purified and deionised water was obtained from a Milli-Q water system (Millipore, Bedford, USA). For histolocaliza- tion, transverse sections of plant tissues (30
l
m) were performed with a vibratom HM650 V (Microm,http://ww w.microm-online .- com/) and observed after staining with a BH2-RFCA microscope (Olympus, Rungis, France) coupled to a MicroPublishe r 5.5 RTV camera (QIMAGING, Surrey, BC, Canada) and a USH-102 UV light source (Olympus, Rungis, France) (Neu staining) or with a Leica DM1000 microscope coupled to a MicroPublishe r 3.3 RTV camera (QIMAGING, Surrey, BC, Canada) (vanillin–sulfuric acid staining).For extraction and separation of phenolic compounds from plant tissue, tissue were grounded with a mixer mill (MM301, Restch, Haan, Germany) using 3 mm-tungste n carbide beads (Qiagen, Courtabo euf, France) and HPTLC was performed on pre-coate d sil- ica gel HPTLC 60 F254 (2010 cm) plates 0.2 mm in thickness
Fig. 6.Localization and hypothetical roles of dihydrochalcones in apple leaves. Dashed lines represent potential transformation reactions not studied in the present work.
using a Camag HPTLC (Camag, Muttenz, Switzerland ) system equipped with an automatic TLC sampler ATS4, a plate heater, TLC visualizer, and the integrated software win CATS version 1.4.2. Bacterial growth was assessed spectrophot ometrically with a Microbiolog y Analyzer Bioscreen C (Labsystems, Helsinki, Fin- land) and fungi growth (exceptV. inaequalis ) using a nephelom etric reader (NEPHELOstar Galaxy, BMG Labtech, Offenburg, Germany), equipped with a 635-nm laser as radiation source, as previously described by Joubert et al. (2010). Chelation of ferric ions was ana- lyzed using a Nanodrop ND-1000 spectrophotom eter (ThermoSci- entific, Rockford, IL, USA) and a LCQ DECA ion trap mass spectromete r (Thermofinnigan, San Jose, CA, USA) equipped with an ESI source and run by Xcalibur Ò (Thermofinnigan, San Jose, CA, USA) version 1.2 software. Before analysis, samples were fil- tered with filters PTFE Uptidisc (Interchim, Montluçon, France).
5.2. Biological material
ThreeMalus x domestica genotypes were chosen for their con- trasting phenolic profiles, the ornamental Evereste, the rootstock MM106 and the table apple Golden Delicious (Picinelli et al., 1995; Dugé de Bernonville et al., 2010 ). Experiments were per- formed on actively growing shoots of young scions grafted on MM106 and grown under greenhouse condition s (natural photope- riod, temperature s between 17 and 22°C). The 10 phytopathogen ic bacteria were provided by the CFBP (Collection Française de Bacté- ries Phytopatho gènes, Institut National de la Recherche Agronom i- que, Angers, France) and are listed with their main characterist ics in Table 2. Bacteria were cultivated at 26 °C overnight on solid King’s B medium for E. amylovora strains (King et al., 1954 ) or on the classical yeast extract peptone glucose agar (YPGA) medium for the others. The Alternaria brassicicola wild-type strain Abra43 was isolated from Raphanus sativus seeds (Joubert et al., 2010 ), theV. inaequalis wild-type strain EU-B-04 from Golden Delicious (Calenge et al., 2004 ) and the Neurospora crassa wild-type strain FGSC988 was provided by the FGSC (Fungal Genetics Stock Center, University of Missouri , Kansas City, USA). Conidia were obtained after 7–8 days of culture of mycelial fragments on potato dextrose (PD) agar medium at 24 °C for A. brassicicola andN. crassa and at 18°C for V. inaequalis.
5.3. Histochemic al and TLC analysis
Transverse sections from fresh plant tissues were immediately immersed at least 2 h either in 1% Neu’s reagent in absolute meth- anol or in 1% vanillin–sulfuric acid reagent in absolute methanol with 5% sulphuric acid before microscopic observation. For TLC
analysis, fresh leaf tissue (15 mg) in 2-ml centrifuge tubes was fro- zen in liquid nitrogen, ground to powder with the mixer mill (30 Hz for 30 s) and extracted with 1 ml of acidic methanol (0.1%
acetic acid) for 15 min in the mixer mill (10 Hz) at room tempera- ture. Homogenates were centrifuged 5 min at 13,000 gand super- natants (and standards) applied on TLC plates as 7 mm wide bands (8 mm between bands) with the automatic sampler under N2gas flow, 10 mm from the bottom, 10 mm from the side (appli- cation speed 150 nm/s). TLC plates were then let to dry in an air current and migration was performed using acetate–methanol–
water (10:1.35:1) as mobile phase. Bands were visualized by spraying the plate with a 1% Neu’s reagent (methanol)/5% PEG 4000 (ethanol) solution (1:0.8) or with a 1% vanillin 5% sulfuric acid reagent (methanol) followed by heating at 110 °C for 2 min.
Evaluation was at UV 366 nm or white light, respectively.
5.4. Inoculation procedure
Fire blight susceptibility evaluation of Evereste was performed by shoot injection as described in Brisset et al. (2000). Disease inci- dence was expressed as the percentage of the number of infected shoots/total number of inoculated shoots and disease severity as the mean of necrosis length of infected shoots. Ten shoots per strain were inoculated and the experiment was repeated indepen- dently three times.
5.5. Antibacteri al assays
Dihydroc halcones were prepared in 450
l
l acetate buffer (50 mM, pH 5.5) at final concentr ations ranging from 50l
M to 10 mM (or 1 mM max for phloretin using a 10 mM stock solution prepared in methanol), inoculated with 50l
l of 10 8cfu/ml bacte- rial suspensions prepared in acetate buffer and incubate d at room temperat ure under continuo us stirring. Controls consisted in ace- tate buffer instead of bacteria or glycosyla ted dihydrochal cones, or methanol instead of phloretin. After 2 h, reaction mixtures were diluted 10-fold into liquid LB (forE. amylovora strains) or liquid YPG (for others strains) media and bacterial growth measure d spectrophot ometrically in triplicates at 600 nm every 2 h. For each condition , area under growth curve representat ive of the lag phase and the maximal growth rate was calculated. A percentage of growth inhibition was calculated for each independen t experiment (100((AUCDHC/AUCc)100 where AUC DHC=area under growth curve after contact with a dihydrochalcone and AUC c=area under growth curve of a control culture). The BGI 50 value correspond s to the dihydrochal cone concentratio n for which 50% of Bacterial Growth Inhibition was observed.Table 2
Bacterial strains used in this study.
Strain Abbreviation Isolated from Gram Reference or source
Erwinia amylovora
CFBP1197 Ea1197 Crataegus Negative CFBP
CFBP1430 Ea1430 Crataegus Negative Paulin and Samson (1973)
CFBP3049 Ea3049 Malus domestiqua Negative Norelli et al. (1984)
CFBP3792 Ea3792 Prunus salicina Negative Mohan and Thomson (1996)
Pseudomonas syringae pv.tomato
CFBP2212 Pst2212 Solanum lycopersicum Negative CFBP
Ralstonia solanacearum
CFBP2047 Rs2047 Solanum lycopersicum Negative CFBP
Xanthomonas campestris pv.vesicatoria
CFBP2537 Xc2537 Solanum lycopersicum Negative CFBP
Rhodococcus fascians
CFBP2401 Rf2401 Lathyrus odoratus Positive CFBP
Curtobacterium flaccumfacienspv.betae
CFBP3401 Cfb3401 Beta vulgaris Positive CFBP
Arthrobacter illicis
CFBP1380 Ai1380 Lex opaca Positive CFBP
5.6. Antifungal assays
For V. inaequalis , dihydrochalcones were prepared in 4.5 ml sterile water at final concentratio ns ranging from 0.1 to 5 mM.
(or 2.5 mM max for phloretin using a 25 mM stock solution pre- pared in methanol), inoculated with 0.5 ml of a 3.10 4spores/ml prepared in sterile water and incubated at room temperature un- der continuo us stirring. Controls consisted in water instead of con- idia or glycosyla ted dihydrochalcones , or methanol instead of phloretin. After 2 h, one hundred microliters of the reaction mix- tures were spread over agar–malt medium supplemented with antibiotics (15 g/l agar, 10 g/l malt, 25 mg/l streptomycin, 5 mg/l chlortetracy cline, 12.5 mg/l penicillin). The germinated spore enu- meration was performed 1 week after incubation at 18 °C. The antifungal effect of dihydroc halcones was calculated as the per- centage inhibition of conidial germinat ion compared to a water control. The CGI 50value corresponds to the dihydrochalcone con- centration for which 50% of Conidial Germination Inhibitio n was observed.
For A. brassicicola and N. crassa , dihydrochal cones were pre- pared in methano l at 100 concentr ations ranging from 10 to 500 mM. Ten microliters of 100 solutions of each compound were added to 1 ml of a 10 5spores/ml prepared in PD broth. Fungal growth was measured directly in reaction mixtures in triplicates by laser nephelom etry every hour according to Joubert et al.
(2010). Percentages of growth inhibition were calculated with the same formula than for bacterial growth curves. The MGI 50va- lue correspond s to the dihydroc halcone concentration for which 50% of Mycelium Growth Inhibition was observed.
5.7. Chelation of ferric ions by dihydrochalco nes
Spectrophotom etric analysis was investigated as described by Paiva-Marti ns and Gordon (2005)with some modifications. Briefly experiments were performed in acetate buffer (50 mM) at pH 3.5 and 5.5 and in MOPS buffer (50 mM) at pH 7.5. Phenolic com- pounds (dihydrochalcones and epicatechin) and desferrio xamin were dissolved in methanol at a concentration of 4 mM, followed by a 10-fold dilution in the appropriate buffer (final concentratio n of 400
l
M for each compound ). A solution of ferric chloride (FeCl3) in acetate buffer pH 3.5 was also prepared at a concentratio n of 60 mM and a volume of this solution was added to the appropriate compound solutions such that ferric ions concentrations respected the following ratio compound/iron of 1:0.25, 1:0.5, 1:0.75, 1:1, 1:2, 1:3, 1:4 and 1:5. UV/vis spectra of the pure compounds , ferric chlo- ride, compound /FeCl 3 solutions were determined spectrophoto- metrically in the region 220–750 nm.For electrospr ay ionisation mass spectrometr y analysis , com- pound (65
l
M) and compound/FeCl 3(ratios 1:0.1, 1:0.5, 1:1, 1:2) solutions were prepared in MeOH–H2O (1:1), filtered and directly introduced into the ESI source by a built-in syringe pump at a flow rate of 5l
l/min. Infusion analyses were performed in positive mode with an ion spray voltage of approximat ely 4500 kV, a 60 V orifice voltage, a 225 °C capillary temperature , a 50 au (arbi- trary units) sheath nitrogen gas flow rate and a nominal mass range up to m/z1800.Acknowled gements
M.G. was a recipient of a Grant from the Ministère de l’Educa- tion Nationale, de l’Enseignement Supérieur et de la Recherche, France. This work was also supported by a research Grant of the Université d’Angers. The authors wish to thank Roland Chartier and Brice Marollea u for excellent technical assistance. The authors are also grateful to Vegepolys Innovation and to the I.M.A.C plat- form (SFR 4207 Quasav) for access to laborator y facilities and to
I.N.E.M and to the Horticult ural Experimental Unit of I.N.R.A An- gers (UE 449) for plant maintenanc e.
Appendi x A. Supplementar y data
Supplement ary data associated with this article can be found, in the online version, at http://dx.doi.o rg/10.1016/ j.phytochem.20 13.
02.009.
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