Physiologia Plantarum 128: 604–617. 2006 Copyright ª Physiologia Plantarum 2006, ISSN 0031-9317

Induction of polyphenol gene expression in apple(Malus x domestica) after the application of adioxygenase inhibitorThilo C. Fischer,a,† Heidrun Halbwirth,b,† Susanne Roemmelt,c,† Emidio Sabatini,d,†

Karin Schlangen,b,† Carlo Andreotti,d Francesco Spinelli,d Guglielmo Costa,d Gert Forkmann,a

Dieter Treutterc and Karl Stichb,*

aLehrstuhl fur Zierpflanzenbau, Department fur Pflanzenwissenschaften, Technische Universitat Munchen Weihenstephan, Am Hochanger 4,

D-85350 Freising, GermanybInstitut fur Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, Technische Universitat Wien, Getreidemarkt 9/1665,

A-1060 Vienna, AustriacFachgebiet Obstbau, Department fur Pflanzenwissenschaften, Technische Universitat Munchen Weihenstephan, Alte Akademie 16,

D-85350 Freising, GermanydDipartimento Colture Arboree, Universita di Bologna, via Fanin 46, I-40127 Bologna, Italy

Correspondence

*Corresponding author,

e-mail: kstich@mail.zserv.tuwien.ac.at

Received 24 March 2006; revised 12

June 2006

doi: 10.1111/j.1399-3054.2006.00787.x

A comprehensive study of the complex polyphenol biosynthesis in developing

leaves of apple (Malus domestica) was performed comprising gene expression,

enzyme activities and polyphenol composition. During leaf development, an

early increase in gene expression was observed for phenylalanine ammonialyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), flavanone 3-

hydroxylase (FHT, EC 1.14.11.9) and dihydroflavonol 4-reductase/flavanone

4-reductase (DFR/FNR, EC 1.1.1.219). Their enzyme activities showed

a corresponding trend during the time course. A parallel set of experiments

was carried out with leaves treated with prohexadione-Ca (ProCa), which is an

enzyme inhibitor of 2-oxoglutarate dependent dioxygenases (2-ODDs). ProCa is

known to induce changes in polyphenol biosynthesis, which are accompanied

bya reduced incidenceof fire blight and scab, the twomajor pome fruit diseases.The application of ProCa led to an increase in activities of PAL, CHS, FHT and

DFR/FNR, which was based on an enhanced gene expression. In contrast, an

inhibition of gene expression was detected for anthocyanidin synthase (EC

1.14.11.19). These effects are interpreted as a feedback regulation by changed

polyphenol levels. Because of the inhibition of the 2-ODDs FHT and flavonol

synthase (EC 1.14.11.23), some pronounced changes in polyphenol composi-

tion were observed. Eriodictyol, the substrate of FHT, accumulated as

eriodictyol-7-O-glucoside and 6$-O-trans-p-coumaroyleriodictyol 3#-O-glu-coside. In addition, the 3-deoxycatechin luteoliflavan was formed which is not

present in untreated apple leaves. Hence, beyond the redirection of polyphenol

biosynthesis by the enzyme inhibitor, changed polyphenol levels obviously

cause a distinct induction of gene expression by feedback regulation.

Abbreviations – 2-ODD, 2-oxoglutarate dependent dioxygenase; ANS, anthocyanidin synthase; cDNA, complementary DNA;

CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; DW, dryweight; EDTA, ethylenediaminetetraacetic acid; FHT, flavanone

3-hydroxylase (¼F3H); FLS, flavonol synthase; FNR, flavanone 4-reductase; F3#H, flavonoid 3#-hydroxylase; GA, gibberellic acid;HPLC, high-performance liquid chromatography; mRNA, messenger RNA; NAR, naringenin; PAL, phenylalanine ammonium lyase;

ProCa, prohexadione-Ca; rDNA, ribosomal DNA; SDS, sodium dodecyl sulphate.

†Contributed equally to the results.

604 Physiol. Plant. 128, 2006

Introduction

The constitutive phenolic compounds of apple leaves

(Malus domestica) have been investigated in detail

(Gutmann et al. 1990, Mayr et al. 1995, Schieber et al.

2001, Treutter 2001). Although some of them, in

particular proanthocyanidins, were shown to play animportant part in resistance against pathogens (Feucht and

Treutter 1999, Feucht et al. 1996, 1998, Mayr et al. 1997,

Michalek et al. 1998, Picinelli et al. 1995), knowledge

about their biosynthesis in apple leaves is still limited

(Fischer et al. 2003,Halbwirth et al. 2003). Little is known

about resistance induction under field conditions, and the

accumulation of resistance-related polyphenols during

leaf development is poorly investigated.Recently, interest has focused on possibilities tomodify

the polyphenol composition to induce resistance to

pathogens (Halbwirth et al. 2006, Norelli and Miller

2004, Rademacher et al. 2000a, 2000b). One effective

tool for themodification of the polyphenol composition is

the application of enzyme inhibitors such as prohex-

adione-Ca (ProCa), which was originally developed as

a growth regulator. As a 2-oxoglutarate analogue, itinhibits 3b-hydroxylation of gibberellic acidGA20,which

is catalysed by a 2-oxoglutarate dependent dioxygenase

(2-ODD), and thus impedes the formation of the active

gibberellic acid GA1 (Rademacher et al. 1992, 2000). In

addition, 2-ODDs involved in the polyphenol biosynthe-

sis such as flavanone 3-hydroxylase (FHT), flavonol

synthase (FLS) and anthocyanidin synthase (ANS) are

affected (Halbwirth et al. 2006). Treatments with ProCacause an effective inhibition of FHT in apple leaves,

which leads to changes in the polyphenol composition

and to the occurrence of 3-deoxyflavonoids, which are

not formed in untreated apple leaves (Fischer et al. 2003,

Halbwirth et al. 2003, Roemmelt et al. 2003). 3-

Deoxyflavonoids are known to contribute to resistance in

maize and sorghum species (Lo et al. 1999, Mueller-

Harvey and Reed 1992, Lopes 1993, Snyder andNicholson1990, Styles and Ceska 1972, Tenkouano et al. 1993), and

a strong anti-microbiological activity of the 3-deoxyflavo-

noid luteoforol (3-deoxyleucocyanidin) could be demon-

strated (Spinelli et al. 2005). ProCa-treated apple trees

showed reduced susceptibility to fire blight and scab (Bazzi

et al. 2003). Thus, the induction of 3-deoxyflavonoid

formation, in particular of luteoforol, may provide a very

strong defence mechanism against plant pathogens.Apart from FHT inhibition by ProCa (Halbwirth et al.

2006) and the fact that apple dihydroflavonol 4-reductase

(DFR) shows also a flavanone 4-reductase (FNR) activity

(Fischer et al. 2003), little information was available on

the underlying molecular mechanisms for the observed

resistance induction. We here provide the first study,

which investigates the effect of ProCa on the polyphenol

biosynthesis at the levels of gene expression, enzyme

activity and polyphenol accumulation. This knowledge

will form the basis of strategies for an effective defence

management against fire blight and scab.

Materials and methods

Plant material

The experiments were carried out on M. domestica cv.‘Golden Delicious’ in May 2000 during periods of

uniform and sunny weather conditions. One hundred

2-year-old scions grafted on ‘M9’ rootstocks kept in the

orchard of the Department of Arboriculture, University of

Bologna, were used. Half of the plants were sprayed once

in the evening with 250 ppm ProCa until the leaf surfaces

were completely wetted, whilst the control was treated

with water. The first three leaves were labelled. Youngest(leaf 1), second (leaf 2) and third leaves (leaf 3) were

collected separately. Representative samples of 15 leaves

were collected in total for each developmental stage and

time from five different trees and distributed for analyses

of polyphenol composition, enzyme activities and gene

expression, respectively. Samples were drawn before (0 h)

and five times after treatment (4 h, 1 day, 2 days, 5 days,

10 days). The plant material was frozen in liquid nitrogenand kept at 280�C. For high-performance liquid chroma-

tography (HPLC) analysis, the material was freeze dried.

Chemicals

For spray application of ProCa, the formulation BAS 125

10 W (Regalis�, BASF AG, Ludwigshafen, Germany),

a wettable granular containing 10% by weight of ProCa

was used. [2-14C]-Malonyl-coenzyme A (55 mCimmol21) was purchased from Amersham International

(Freiburg, Germany). [14C]-Naringenin (NAR) was pre-

pared as described (Britsch and Grisebach 1985), using

recombinant chalcone synthase (CHS) (Schroeder, Uni-

versity of Freiburg, Germany). [14C]-Eriodictyol was

synthesized from [14C]-NAR (Forkmann and Stotz

1981), using F3#H-activity of microsomal preparations

from Tagetes erecta. [14C]-Dihydrokaempferol was pre-pared from [14C]-NARwith recombinant FHT (Halbwirth

et al. 2006) and used for preparation of [14C]-dihydro-

quercetin (Forkmann and Stotz 1981), with microsomal

preparations from T. erecta.

Analyses of phenolic compounds

Determination and identification of phenolic compounds

were performed according to Roemmelt et al. (2003). For

Physiol. Plant. 128, 2006 605

extraction of phenolic compounds, lyophilised leaves

were ground in a ball mill. One milligram of the fine

powder was extracted with 500 ml of 100% methanol

containing 6-methoxyflavone (0.1 mg ml21) as an inter-

nal standard for 30 min in a cooled water bath during

sonication. After centrifugation the supernatant wasevaporated, the residue redissolved in small quantities

ofmethanol and injected forHPLC analysis. The phenolic

compounds were separated on a column (250 � 4 mm

inner diameter, I.D.) prepackedwithHypersil ODS, 3 mm

particle size, following a stepwise gradient, using

mixtures of solvent A (formic acid, 5% in water) and

solvent B (methanol), from 95:5 (v/v) to 10:90 (v/v), with

a flow rate of 0.5 ml min21 (Treutter et al. 1994). Thegradient profile usedwas 0–5 min, isocratically, 5%B; 5–

15 min, 5–10% B; 15–30 min, isocratically, 10% B; 30–

50 min, 10–15% B; 50–70 min, isocratically, 15% B;

70–85 min, 15–20% B; 85–95 min, isocratically, 20% B;

95–110 min, 20–25% B; 110–140 min, 25–30% B; 140–

160 min, 30–40% B; 160–175 min, 40–50% B; 175–

190 min, 50–90% B. For the selective estimation of

flavans, a postcolumn derivatisation method was em-ployed (Treutter et al. 1994). Flavanones, catechins and

procyanidins aswell as the novel compound luteoliflavan

were identified according to their chromatographic

behaviour on HPLC and thin layer chromatography and

in comparison with previously isolated standards (Mayr

et al. 1995, Roemmelt et al. 2003). Flavonols were

selectively detected at 360 nm and identified by their

ultraviolet absorbance spectra with a diode array de-tector. Quantificationwas performed as follows: catechin

and epicatechin were available as standards; procyani-

dins were calculated as procyanidin B2, luteoliflavan as

catechin, flavonols as rutin and flavanones as eriodictyol-

7-O-glucoside. For the HPLC determination of the

dihydrochalcone glycoside phloridzin the extract was

diluted 1:200 with methanol and the phloridzin was

analysed using a short column (12.5 � 4 mm I.D.)

prepacked with LiChrospher 100 RP18, 5 mm particle

size and a gradient ranging from 40 to 90% aqueous

methanol (Leser and Treutter 2005).

Determination of enzyme activities

Enzyme preparations and assays were performed as

previously described (Halbwirth et al. 2002). Crude

extracts were passed through a gel chromatography

column (Sephadex G25) to remove low molecular

compounds. This step was particularly important for the

determination of FHT activity from ProCa-treated leaves

because remnants of the inhibitor were shown to disturb

the analyses. Specific activities were calculated based onthe protein content, whichwas determined by amodified

Lowry procedure (Sandermann and Strominger 1972)

using bovine serum albumin as a standard. For compar-

ison of the datameasured on treated anduntreated leaves,

the activities at time 0 were taken as 100% and were

calculated relative to the activity at time 0.

Molecular cloning of complementary DNAs

The partial complementary DNA (cDNA) of phenylala-

nine ammonia lyase (PAL) and the full size clones of FHT,

DFR, FLS, ANS and an 18S rDNA fragment were isolated

from M. domestica cv. ‘M9’, a widely used rootstock

cultivar. The partial cDNA of CHS was obtained from

M.domestica cv. ‘Weirouge’. Isolation ofmessenger RNA

(mRNA) was performed using the mMACS mRNA Iso-lation Kit� (Miltenyi Biotec, 752-01; Bergisch Gladbach,

Germany). Reverse transcription was done with the

SuperScript II� Reverse Transcriptase (Gibco BRL 1864)

and an oligo-dT-anchor-primer (Table 1). The following

reverse transcription-PCR (RT-PCR) conditions were

used: 1.5 min 94�C, 30� (30 s, 94�C; 1 min, 45–60�C;2 min, 72�C), 7 min 72�C. RT-PCR products obtained

with the optimal annealing conditions (45–60�C) were

Table 1. PCR primers used for the cloning of complementary DNAs. ANS, anthocyanidin synthase; PAL, phenylalanine ammonium lyase; CHS, chalcone

synthase; DFR, dihydroflavonol 4-reductase.

Primer Sequence Reference for sequence

oligo-dT-anchor-primer GAC CAC GCG TAT CGA TGT CGA C(T)16V –

5# PAL GTC GAC GAG CAG CAC AAT CAG G Davies and Bradley, Genbank X68126

3# PAL ATG CAG CAT GTA AAC CGT GAC G Davies and Bradley, Genbank X68126

5# CHS ATG ATG TAC CAG CAG GGG TGC Podivinsky et al. 1993, X68977

3# CHS CTT CAA GCA GCC ACG CTG TG Podivinsky et al. 1993, X68977

5# ANS CGA GTA ATA TAC TAG CTGAG Lee et al. 1998, Genbank AF117269

3# ANS ATT AGG ACG ATA GTT CAC AAC Lee et al. 1998, Genbank AF117269

5# 18S rDNA GAC TGT GAA ACT GCG AAT GG Kim et al., Genbank AF179400

3# 18S rDNA GTA AGT TTC AGC CTT GCG ACC Kim et al., Genbank AF179400

5# degen DFR GGX TTYATH GGB TCW TGG CTY RTC ATG A Amino acid motif GF(I/V)GSWL(V/I)M

3# degen DFR TCD AYX GCH CCH XYR WAC ATR TCC TC Amino acid motif EDM XXG AXX

606 Physiol. Plant. 128, 2006

chosen for cloning,whichwas performedwith the ‘TOPO

TA Cloning� Kit’ (Invitrogen, 45-0071; Karlsruhe, Ger-

many). By this PCR-approach the fragments of PAL and

CHS cDNAs as well as the full size cDNA of ANS were

cloned. Sequences were determined by a commercial

supplier to verify the identity of the cloned cDNAs.Primers for RT-PCR amplification of PAL, CHS and ANS

and the sequence references are given in Table 1. TheM.

domestica M9 ANS is available under the accession

number DQ156905. Full size cDNAs of M. domestica

M9 DFR, FHT and FLS were available from previous

studies (Fischer et al. 2003, Halbwirth et al. 2006,

accession no AY227728, AY965340, AY965343). An

18S rDNA-fragment was amplified fromM. domestica cv.‘M9’ genomic DNA using primers that were derived from

the 18S rDNA sequence known from Pyrus pyrifolia

(Table 1). This fragmentwas used as a hybridization probe

to quantify and normalize the amount of gel-loaded total

RNA. DFR fragments were PCR amplified from cDNA

derived from ProCa-treated leaves and untreated control

leaves. The degenerated DFR primers were derived from

conserved amino acid sequences (Table 1).

Northern analysis

Gene expression studies were performed by Northern

analysis. Total RNAwas prepared from first leaves of apple

plants using the RNeasy Kit� (Qiagen; Hilden, Germany)

and stored for later use at –80�C. Denaturing 1% agarose

gels were run with 20 mM 3-morpholinopropane sulfonicacid,MOPS (pH7.0), 8 mM sodiumacetate (NaAc), 1 mM

ethylenediaminetetraacetic acid (EDTA), 6.6% formalde-

hydeand0.5 mg ml21 ethidiumbromide. Fivemicrograms

of RNAwas loaded after addition of 0.33 volumes of 37%

formaldehyde and 0.33 volumes of 100 mM MOPS (pH

7.0), 40 mM NaAc, 5 mM EDTA, heating to 65�C,subsequent cooling on ice, addition of 0.33 volumes of

TE/bromophenol blue and parallel prerun of gel electro-phoresis. All samples of one time course were loaded on

the same gel. Gel electrophoresis was performed at 40 V.

The gel was washed several times with H2O and photo-

graphed to check equal RNA loading. Further washings

were carried out for 20 min in 50 mM NaOH, 20 min in

H2O and 45 min in 20 � SSC. Membrane transfer was

performed with 20 � SSC on Immobilon Nylon N1�

membrane (Millipore, Schwalbach, Germany) over night.After blotting the membrane was briefly washed with

6 � SSC, air-dried and baked for 2 h at 80�C. DNA-

labelling was performed using the RediprimeTM II kit (RPN

1633, Amersham). Hybridizations were performed with32P-labelledDNA in 2 � SSC at 63�C for at least 15 h. The

blot was washed three times with 0.2 � SSC/1% sodium

dodecyl sulphate (SDS) at 63�C. Exposition was performed

with a phosphor-imager (Fuji BAS 1000 Bio-Imaging

Analyser, screens: BAS-MS 2040, Fuji). For the analysis,

the program TINA 2.09 (Raytest, Straubenhardt, Germany)

was used. For quantification equal areas were defined for

the bands, and resulting values were corrected for the

background. After hybridization with the gene-specificprobe, the blots were additionally hybridized with 32P-

labelled 18S rDNA. The resulting values were used for

normalization of the gene-specific values. The values for

the time0were takenas100% for the treated anduntreated

time course, respectively.

Southern analysis

Genomic DNA was prepared from young apple leaves

using the DNeasy� Plant DNA Kit (Qiagen). Five micro-

grams of DNA each was digested with 50 U restriction

enzyme (BamHI, EcoRI, HindIII, SalI, SacI and XbaI) in

the respective buffer for 5 h at 37�C. The restricted DNA

was ethanol precipitated, redissolved in 20 ml TE at 65�Cand used for agarose gel electrophoresis (1% agarose,

1 mg ethidium bromide l21, TAE buffer, 30 V). The gelwas soaked in 0.25 M HCl for 15 min afterward, rinsed

withwater,washed twice in 0.5 MNaOH/1.5 MNaCl for

20 min, rinsed with water and washed two times in 1 M

Tris pH 7.4/1.5 M NaCl for 20 min. Blotting transfer was

done with 0.4 M NaOH on Immobilon Nylon N1�

membrane (Millipore) overnight. After blotting the

membrane was briefly washed with 5 � SSPE, it was

air-dried and baked for 30 min at 80�C. Hybridizationswere performed with 32P-labelled DNA (see Northern

analysis) in hybridization buffer (3 � SSPE/0.02% Ficoll/

0.02% polyvinyl pyrrolidone (40 000 D)/0.1% SDS/

50 mg l21 preboiled calf thymus DNA) at 63�C for at

least 15 h. The blot waswashed twice with 2 � SSPE/1%

SDS and oncewith 2 � SSPE/0.1%SDS at 63�C for 5 min

each. Exposition and analysis were performed as

described for Northern analysis.

Results

The experiments were performed under orchard con-

ditions on the economically important apple cultivar

‘Golden Delicious’ to provide data representing natural

conditions. Polyphenol gene expression, activity of the

corresponding enzymes and polyphenol accumulationwere studied from first leaves of apple shoots over a time

course of 10 days. Because of the accumulation of

disturbing substances in RNA preparations from the

second and third leaves, quantification of gene expres-

sion was only possible in the first leaves. Enzyme

activities and corresponding polyphenol composition

were determined also from second and third leaves.

Physiol. Plant. 128, 2006 607

Cloning of cDNAs and gene number determination

Full sized cDNAs fromM. x domesticaM9were availablefor DFR, FHT and FLS from previous work (Fischer et al.

2003, Halbwirth et al. 2006). The function of these genes

had been confirmed by expression of the recombinant

enzymes. PAL and CHS cDNA fragments and a full sized

ANS cDNAwere RT-PCR-amplified from M9 leaf cDNA

using available sequence information, cloned and

confirmed by sequencing. To test whether further DFR/

FNR genes are induced after ProCa treatment, whichcould be responsible for the 3-deoxyflavonoid formation,

DFR/FNR fragments of approximately 900-bp length

were PCR amplified with degenerated DFR primers from

ProCa-treated leaves and untreated control leaves of the

cultivar ‘Golden Delicious’, cloned and sequenced.

There was only little sequence variation in comparison

with the known DFR/FNR sequence of M9, and variation

was even less for the sequences recovered from thetreated leaves (data not shown). No hint was found by this

approach for induction of further DFR/FNR genes after

ProCa treatment. The cloned cDNAs of the various

polyphenol genes were used as hybridization probes for

Southern analyses and for the gene expression studies

performed by Northern analyses.

Southern analyses of ‘M9’ apple genomicDNAcleaved

with six different restriction enzymes revealed theexistence of small multigene families for all the genes

studied except ANS (Table 2). For some genes that had

been included in previous studies on apple fruits,

comparable results of small multigene families were also

obtained for ‘Jonathan’ apple (Honda et al. 2002) and

‘Fuji’ apple (Kim et al. 2003). Most apple cultivars are

highly heterozygous. Therefore, some bands in Southern

analysis could be because of restriction fragment lengthpolymorphisms between parent lines, which would

reduce the definite gene number of the multigene family.

Gene expressions in first leaves

The expression studies included cDNAsof six genes of the

polyphenol pathway. 18S rDNA fragment hybridizationwas performed for quantification and normalization of

the total amount of RNA loaded on the gels (Fig. 1). For

comparison of the data for treated and untreated leaves,

the values for time ‘0 h’ were taken as 100%, all other

data refer to this, respectively (Fig. 2).

During the first 2 days, untreated unrolling first leaves

showed an increase in gene expression for PAL, CHS, FHT

and DFR/FNR, whereas FLS und ANS genes showed onlyminor changes (Fig. 2, dashed lines). Surprisingly, the

expression of all six genes was markedly decreased on

day 5, but thereafter expression increased again. In

ProCa-treated leaves, inductive effects on gene expres-

sions were observed (Fig. 2, full lines). CHS, FHT and

DFR/FNR showed strong induction of gene expression

during the first days,whereas for PAL only a late induction

effect was found. FLS showed only a weak and lateinduction, and ANS gene expression even decreased

slightly. As observed for untreated leaves, a—partly

considerable—decrease in expression of all six genes

was measured on day 5, followed by a late increase.

Despite filing of meteorological data during the orchard

experiment, no environmental influence could be iden-

tified that could explain the decrease at day 5.

Enzyme activities in leaves

The enzymatic studies focused on the activities of PAL,

CHS, FHT and DFR/FNR. FLS and ANS activities could

not be detected with the enzyme preparations from apple

leaves. For all enzymes, specific enzyme activities were

calculated per total protein concentration. For compar-

ison of the datameasured on treated anduntreated leaves,the activities at time 0 were taken as 100% and were

calculated relative to the activity at time 0.

During the development of untreated unrolled first

leaves, an early increase in PAL, CHS and FHT enzyme

activities was observed (Fig. 3 left, dashed lines), which

corresponded to the respective gene expressions (Fig. 2,

dashed lines). Treatment with ProCa (Fig. 3 left, full lines)

led to an increase in the activities of PAL, FHT, DFR/FNRand CHS in comparison with the untreated first leaves.

Most pronounced was a late increase in PAL activity by

ProCa treatment. The time courses of enzyme activities in

first leaves closely followed the time courses of gene

expression (Fig. 2). However, for FHT the pronounced

increase in gene expression was reflected only by a

moderate increase in enzyme activity. In treated and

untreated leaves, a decrease in the activities of all fourenzymeswasmeasured at day 5 (Fig. 3), as observed also

for gene expression.

All enzyme activities were still present in the enzyme

preparations of second and third leaves. During leaf

development, a distinct decrease in protein concentration

was observed from 1.4 mg ml21 enzyme preparation in

first leaves to 0.7 mg ml21 enzyme preparation in third

Table 2. Estimation of gene copy numbers in Malus x domestica cv.

Golden Delicious, each relying on Southern analysis of six different

genomicdigests. PAL, phenylalanineammonium lyase;CHS, chalcone syn-

thase; FHT, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase;

FLS, flavonol synthase; ANS, anthocyanidin synthase.

Gene PAL CHS FHT DFR FLS ANS

Estimated number of homologues 3-4 4-5 2 3-5 2-3 1

608 Physiol. Plant. 128, 2006

leaves. Because protein concentration and reaction rates

simultaneously decreased over the investigation period of

10 days, specific activities (kat kg21 protein) of all enzymes

remained rather stable. Time courses for first, second and

third leaf enzyme activities are quite similar. Generally, the

inductive effect of ProCa could be seen in all three leaves.

For the enzymatic investigations, ProCa was removed

by gel chromatography from the enzyme preparations todetermine the FHTenzyme activity in the absence of the

enzyme inhibitor. However, the in vivo inhibition could

be demonstrated by omission of the gel chromatography

step in the enzyme preparation. When ProCa residues

were not removed from the enzyme preparations of

treated leaves, a distinct FHTenzyme inhibition lasting for

at least 5 days after treatment of the leaves with ProCa

was observed (Fig. 4). In these assays, the FHT inhibitionmasks the enhanced FHTactivity,which is the result of the

induced FHT gene expression.

Composition and concentration of phenoliccompounds in developing apple leaves

Seventeen main, constitutively formed phenolic com-

pounds of apple leaves were analysed. This included

hydroxycinnamic acids (p-coumaric acid, chlorogenic

acid, p-coumaroylglucose), dihydrochalcones (phloretin,

phloridzin, 4-O-cis-p-coumaroyl-phloridzin, 4-O-trans-

p-coumaroylphloridzin), the flavanone naringenin 7-O-

glucoside, derivatives of the flavonol quercetin (hyperin,

isoquercitrin, rutin, quercitrin), flavan 3-ols (catechin,

epicatechin) and derived polymeric procyanidins (B2, B5

and E-B5). In addition, three compounds occurring onlyafter ProCa treatment (Roemmelt et al. 2003) were

determined: the flavanones eriodictyol-7-O-glucoside

and 6$-O-trans-p-coumaroyleriodictyol 3#-O-glucoside

and the 3-deoxycatechin luteoliflavan (Fig. 5). The poly-

phenol contents given in Figs 6 and 7 are based on dry

weight (DW).

Dihydrochalcones are the main phenolic compounds

in apple leaves. Their concentrations remained quitestable during the whole investigation period, showing

values between 200 and 250 mg g21 DW in first leaves,

150–250 mg g21 DW in second leaves and 150–200 mg

g21 in third leaves. The flavonol content was lowest in

the youngest leaves and increased slowly during leaf

development (Fig. 6 first row, dashed lines). The catechin

content was rather low (below 0.15 mg g21 DW) and

remained quite constant during the developmental

Fig. 1. Northern blot hybridizations of polyphenol genes of Golden Delicious apple first leaves without (left) and after treatment (right) with 250 ppm

prohexadione-Ca. For each gene and treatment, the time course of the expression level is shown from left (0 h) to right (10 days) in six distinct

hybridization bands (lines 1–6). Equal loading is demonstrated by ethidium bromide (EtBr) gel staining (line 7). 18S rDNA hybridization (hybr.) for the

quantification and normalization of gene values on the base of the total amount of RNA loaded on the gels is shown in line 8.

Physiol. Plant. 128, 2006 609

process, while epicatechin and the derived procyanidinsincreased during the investigation period (Fig. 6, dashed

lines). Hydroxycinnamic acids also increased during the

investigation period from about 1 to 5 mg g21 DW.

Pronounced changes in the concentrations of the

constitutive phenolic compounds were observed after

ProCa treatment. Naringenin 7-O-glucoside concen-

tration was markedly increased (Fig. 7 first row). The

flavonols showed much lower contents in treated leavesduring the whole period of the experiment (Fig. 6 first

row). Catechin and epicatechin concentrations markedly

increased, particularly in first and second leaves (Fig. 6

second and third row, full lines). The formation of the

oligomeric flavan 3-ols (procyanidins) slightly increased

in first leaveswhile therewas no effect in second and third

leaves (Fig. 6 last row, full lines). In contrast, procyanidin

E-B5 decreased in all leaves treated with ProCa (data notshown). The content of hydroxycinnamic acids was only

weakly affected by ProCa, and the content of dihydro-

chalcones was not influenced at all (data not shown).

In addition, treatmentwithProCa led todistinct changesin the polyphenol composition and concentration. In

agreement with earlier studies (Roemmelt et al. 1999,

2003), three novel compounds were detected. In first and

second leaves, considerable amounts of luteoliflavanwere

formed, reaching a maximum of approximately 1 mg g21

DW5 days after treatment (Fig. 7 second row, full lines). In

third leaves, much lower amounts of luteoliflavan were

found (up to 0.2 mg g21 DW). Furthermore, the formationof eriodictyol glycosides was induced by ProCa. Highest

concentrations were observed in the youngest leaves

where they reached up to 1.4 mg g21 DW 10 days after

treatment (Fig. 7 third row, full lines). In third leaves,

eriodictyol glycosides accumulated only to a small extent.

Discussion

The effect of ProCa on the polyphenol composition

in apple leaves and the related beneficial effects

for plant health have been intensively investigated

PALfirst leaf

00 2 4 6 8 10

100

200

300

400

500

Time course (days)

0 2 4 6 8 10

Time course (days)

% r

elat

ive

% r

elat

ive

% r

elat

ive

0 2 4 6 8 10

Time course (days)

0 2 4 6 8 10

Time course (days)

0 2 4 6 8 10

Time course (days)0 2 4 6 8 10

Time course (days)

0

100

200

300

400

500

% r

elat

ive

0

100

200

300

400

500

0

100

200

300

400

500

% r

elat

ive

0

100

200

300

400

500

% r

elat

ive

0

100

200

300

400

500

CHSfirst leaf

FHTfirst leaf

DFRfirst leaf

FLSfirst leaf

ANSfirst leaf

Fig. 2. Time course of polyphenol gene expression in apple first leaveswithout (dashed lines) and after treatment (full lines)with 250 ppmprohexadione-

Ca. Relative gene expressions (%) were calculated with reference to the value measured for 0 h.

610 Physiol. Plant. 128, 2006

before (Halbwirth et al. 2003, Roemmelt et al. 2003).

However, there have been few studies on how the

enzymes and genes from the polyphenol pathway are

affected by ProCa (Fischer et al. 2003, Halbwirth et al.

2006). The parallel sets of data are valuable to under-

stand the changes in the levels of constitutive compound

and the formation of novel compounds after ProCa

treatment.

Polyphenol biosynthesis in young apple leaves

Generally, gene expressions and enzyme activities

showed parallel time courses, whereas changes in the

phenolic contentwere only observedwith a certain delay.

As the enzyme activities in leaves of different ages

showed quite similar time courses, it must be concluded

that the observed time courses are because of environ-

mental factors rather than leaf development. This is

PALfirst leaf

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

0

0 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

00 5 10

50

100

150

200

250

Time course (days)

% r

elat

ive

PALsecond leaf

PALthird leaf

CHSfirst leaf

CHSsecond leaf

CHSthird leaf

FHTfirst leaf

DFRfirst leaf

FHTsecond leaf

DFRsecond leaf

FHTthird leaf

DFRthird leaf

Fig. 3. Time course of polyphenol enzyme activities in apple first (left), second (middle) and third (right) leaves without (dashed lines) and after treatment

(full lines) with 250 ppm prohexadione-Ca. Relative enzyme activities (%) were calculated with reference to the value measured for OH.

Physiol. Plant. 128, 2006 611

supported by the high enzyme activities observed even in

youngest leaves on day 0,which indicate an early onset of

polyphenol biosynthesis in the still closed leaf. Further-

more, no distinct correlation could be observed between

the accumulation of phenolic compounds and their

positioning in the sequence of the polyphenol pathway.

For example, the formation of phloridzin and catechin,

which represent an early and a late biosynthetic step

(Fig. 5), respectively, could both already be determined in

the youngest leaves, and no significant further increases

gel chromatography

00 2 4 6 8 10

20406080

100120140160

Time course (days)0 2 4 6 8 10

Time course (days)

% r

elat

ive

no gel chromatography

020406080

100120140160

% r

elat

ive

Fig. 4. Time course of flavanone 3-hydroxylase (FHT) activity in apple first leaveswithout (dashed lines) and after treatmentwith 250 ppmprohexadione-

Ca (ProCa) (full lines). Left: Assays performed with enzyme preparations purified via gel chromatography (residual ProCa removed) showed strongly

induced FHT activities (corresponding to induced FHT gene expression) for ProCa applications. Right: Assays with enzyme preparations not purified by

a gel chromatography step clearly showed the FHTenzyme inhibition by remaining ProCa from the leaf treatments, reflecting the in vivo conditions.

Luteoliflavan 5-O-glucoside(3-Deoxycatechin)

Luteoliflavan(3-Deoxycatechin)

O

OH

OH

OH

HO

O

OR

FLS

ANS

PAL

HO

OH

O

p-Coumaric acid

Phenylalanine

O

O

OH

OH

OH

HO

COOHHO

Chlorogenic acid

HO

O

OGlu

p-Coumaroyl glucose

OH

OH

HO OH

O

Phloretin (Dihydrochalcone)

Phloridzin(Dihydrochalcone)

HO OH

OOGlu

O O

OH4-O-p-Coumaroyl-phloridzin

(Dihydrochalcone)

Epicatechin

Procyanidins

DFR

OGluO

O

OH

OH Naringenin(Flavanone)Naringenin 7-O-glucoside

(Flavanone)

Eriodictyol(Flavanone)

OGluO

O

OH

OH

OH

Eriodictyol 7-O-glucoside(Flavanone)

Dihydroquercetin(Dihydroflavonol)

Flavan 3, 4-diol

Catechin

CHS/CHI

FNR=DFR

Quercetin 3-O-Glycosides(Flavonol)

6’’-O-trans-p-Coumaroyl-eriodictyol 3’glucoside

(Flavanone)

Luteoforol (Flavan 4-ol)

O

OH

OH

OH

HO

FHT

O

OH

OH

OH

HO

OH

O

OH

OH

OH

HO

OH

Fig. 5. Overviewon the polyphenol biosynthesis in apple leaves. Compounds occurring exclusively after prohexadione-Ca application aremarked in grey.

Dashed arrows indicate unknown biosynthetical steps.

612 Physiol. Plant. 128, 2006

Flavonolsleaf 1

00 2 4 6 8 10

2

4

6

8

10

12

14

Time course (days)

0 2 4 6 8 10Time course (days)

0 2 4 6 8 10Time course (days)

0 2 4 6 8 10

Time course (days)

Mg

g–1D

W

00 2 4 6 8 10

2

4

6

8

10

12

14

Time course (days)M

gg–1

DW

00 2 4 6 8 10

2

4

6

8

10

12

14

Time course (days)

Mg

g–1D

W

Catechinleaf 1

0

0.2

0.4

0.6

0.8

0.2

0.4

0.6

0.8

Mg

g–1D

W

0 2 4 6 8 10

Time course (days)

0

0.2

0.4

0.6

0.8

Mg

g–1D

W

0 2 4 6 8 10Time course (days)

0

0.2

0.4

0.6

0.8

Mg

g–1D

WEpicatechin

leaf 1

Flavonolsleaf 2

Catechinleaf 2

Epicatechinleaf 2

Flavonolsleaf 3

Catechinleaf 3

Epicatechinleaf 3

0

1

Mg

g–1D

W

0 2 4 6 8 10Time course (days)

0.2

0.4

0.6

0.8

0

1

Mg

g–1D

W

0 2 4 6 8 10Time course (days)

0.2

0.4

0.6

0.8

0

1

Mg

g–1D

W

Procyanidinsleaf 1

Procyanidinsleaf 2

Procyanidinsleaf 3

0

1

2

3

4

5

Mg

g–1D

W

0 2 4 6 8 10Time course (days)

0

1

2

3

4

5

Mg

g–1D

W

0 2 4 6 8 10

Time course (days)

0

1

2

3

4

5

Mg

g–1D

W

Fig. 6. Time course of flavonol and flavan 3-ol contents in apple first (left), second (middle) and third (right) leaves without (dashed lines) and after

treatment (full lines) with 250 ppm prohexadione-Ca.

Physiol. Plant. 128, 2006 613

were observed. In contrast, concentrations of flavonols,

epicatechin and derived procyanidins slightly increased

during leaf development. This coincides with previous

findings on very young shoot tips (Roemmelt et al. 2003).

Polyphenol biosynthesis in ProCa-treated leaves

After treatment with ProCa, two different effects on the

polyphenol pathway were observed. On the one hand,

partial inhibition of the 2-ODDs FHT, FLS and ANS led to

distinct changes of the polyphenol spectrum in the leaves.

On the other hand, therewas a general increase in enzymeactivities of thepolyphenol pathwaybasedonanenhanced

expression of their genes. This is an interesting effect of

ProCa treatment, which has not been described before.

Changes of the polyphenol spectrum

The inhibition of FHT primarily results in an accumula-

tion of flavanones, which are further modified viaglycosylation and acylation. This leads to the increased

formation of the constitutively present naringenin 7-O-

glucoside and to the occurrence of the two novel

compounds eriodictyol-7-O-glucoside and 6$-O-trans-

p-coumaroyleriodictyol 3#-O-glucoside (Roemmelt et al.

2003). Derivatives of hydroxycinnamic acid start to

accumulate 3 days later than flavanone derivatives as

they are involved in upstream steps and thus are affectedwith a certain delay by substrate tailback. The late

increase in hydroxycinnamic acids up to 10 days after

treatment reflects the late induction of PAL.

Naringenin 7-O-glucoside leaf 1

0.00 2 4 6 8 10

0 2 4 6 8 10

0 2 4 6 8 10

0.2

0.4

0.6

0.8

Time course (days)

mg

g–1D

W

0.00 2 4 6 8 10

0.2

0.4

0.6

0.8

Time course (days)

mg

g–1D

W

0.00 2 4 6 8 10

0.2

0.4

0.6

0.8

Time course (days)

mg

g–1D

W

Naringenin 7-O-glucosideleaf 2

Naringenin 7-O-glucosideleaf 3

Eriodictyol glycosidesleaf 1

0.0

0.4

0.8

1.2

1.6

Time course (days)

mg

g–1D

W

0 2 4 6 8 100.0

0.4

0.8

1.2

1.6

Time course (days)

mg

g–1D

W

0 2 4 6 8 100.0

0.4

0.8

1.2

1.6

Time course (days)

mg

g–1D

W

Eriodictyol glycosidesleaf 2

Eriodictyol glycosidesleaf 3

Luteoliflavanleaf 1

0.0

0.4

0.8

1.2

Time course (days)

mg

g–1D

W

0 2 4 6 8 100.0

0.4

0.8

1.2

Time course (days)

mg

g–1D

W

0 2 4 6 8 100.0

0.4

0.8

1.2

Time course (days)

mg

g–1D

W

Luteoliflavanleaf 2

Luteoliflavanleaf 3

Fig. 7. Time course of induced compounds after treatment with 250 ppm prohexadione-Ca in apple leaves (full lines) in comparison with untreated

controls (dashed lines).

614 Physiol. Plant. 128, 2006

Another consequence of the accumulation of flava-

nones is the formation of luteoliflavan (3-deoxycatechin)

from eriodictyol via the anti-microbial intermediate

luteoforol (3-deoxyleucocyanidin) occurring as early as

1 day after treatment and decreasing after 5 days.

Luteoliflavan and luteoforol belong to the rare class of3-deoxyflavonoids, which are commonly not formed in

Rosaceous species (Roemmelt et al. 2003). Previous

studies on Zea mays, Sinningia cardinalis (Halbwirth

et al. 2003,Winefieldet al. 2005) andSorghum sp. (Lo et al.

1999) suggested that natural occurrence of 3-deoxyfla-

vonoids is correlated with a low or even absent FHT

activity. Apple leaves generally show strong FHTactivities.

Accordingly, the spectrum of polyphenols is shifted fromthe common series toward the rare 3-deoxy-series only

when FHT enzyme activity is artificially reduced by

treatment with ProCa.

The accumulation of luteoliflavan can be regarded as

a marker for the sensitivity of the leaves with respect to

their inducible polyphenol biosynthesis. In previous

studies (Roemmelt et al. 2003), where very young shoot

tips were treated with ProCa, luteoliflavan concentrationreached up to 8 mg g21 DW. In our study, the maximum

values for first and second leaveswere 1 mg g21DW, and

only 0.2 mg g21 DW for third leaves on day 5 after

treatment. The restricted accumulation in third leaves

occurs despite an enhanced activity of polyphenol

enzymes (Fig. 3). This may be because of the reduced

substrate supply for PAL in older leaves compared with

younger ones where a higher sink potential can beassumed. The reduced synthesis of luteoliflavan from

eriodictyol via luteoforol may therefore be explained

by a regulation at the level of substrate supply as proposed

by Jones and Hartley (1999) and Margna (1977). This is

an important feature with respect to the defensive role

of these compounds and to resistance induction by

ProCa.

Interestingly, the FHT inhibition did not result ina simultaneous decrease for all 3-hydroxyflavonoids.

Most pronounced was the decrease in flavonol contents

after treatment. This confirms the simultaneous inhibition

of FHT and FLS (two bottlenecks in the pathway), which

results in a rapid turnover of the soluble flavonols. After

overcoming theminimum, the flavonol curves of first and

second leaves parallel those of the untreated controls.

Only in the third leaf, where the effect of ProCa wasgenerallyweakest, the flavonol levels of the treated leaves

reached those of the untreated ones.

The flavan 3-ols catechin and epicatechin accumulate

on a very low level. Surprisingly, their concentrations rise

after ProCa treatment in the first and second leaves but

their amounts remain very low. In case of catechin, this

can easily be explained by the inhibition of FLS and ANS

(concurring reactions) leading to a rise in catechin,

despite the diminished precursor synthesis because of

reduced FHTactivity (one bottleneck in the pathway). The

flavan 3-ol epicatechin, however, is producedvia the FHT

and ANS reactions (two bottlenecks in the pathway).

Thus, the observed rise in epicatechin concentration isnot yet understood.

Influence of ProCa treatment on polyphenol gene

expression and corresponding enzyme activities

Inhibition of theMalus FHTand FLS by ProCawas already

shown in vitro with leaf enzyme extracts as well as with

the recombinant enzymes (Halbwirth et al. 2002, 2006).In our study, the inhibition of FHT by residual ProCa

(Fig. 4 right) in the enzyme assays was avoided (Fig. 3,

Fig. 4 left) by generally purifying the enzyme preparations

from ProCa by gel chromatography. However, the in vivo

inhibition could be visualized in vitro by using enzyme

preparations obtained without removing of residual

ProCa by gel chromatography (Fig. 4, right). The enzyme

inhibition observed in these assays indicates that thereis no complete blockage but a strong reduction of

FHT activity during the first 5 days. Later on, the ProCa

inhibition decreases, which seems to reflect degradation

of ProCa. This interpretation is supported by the coinci-

dence with the peak of the ProCa-induced luteoliflavan

also occurring at day 5.

Apart from the quantitative and qualitative changes in

the polyphenol spectrum, the second striking observationafter ProCa treatment was the unexpected, pronounced

increase in gene expression and enzyme activities. On

the one hand, the severe changes in the phytohormone

balance caused by ProCa treatment (Rademacher 2000)

may result in changes in thewhole secondarymetabolism

and thus may be responsible for the observed influence

on polyphenol gene expression and enzyme activity.

On the other hand, the changes in polyphenol levelsafter ProCa application might be responsible for the

changes in the gene expression by feedbackmechanisms.

This seems especially evident for FHT. Such influences

of intermediates on the expression of genes and corre-

sponding enzymes in the polyphenol pathway have been

discussed before (Dedaldechamp andUhel 1999, Kubasek

et al. 1992, Pelletier et al. 1999).

With respect to the induction of gene expression, it is aninteresting question, if genes usually not expressed in

apple leaves are induced after ProCa treatment. Especially

theDFR/FNR is the keyenzyme for the alternative pathway

leading to 3-deoxyflavonoids. However, using degener-

ated primers against conserved motifs of DFR/FNR

sequences for RT-PCR no hint was found for induction of

additional DFR/FNR genes by ProCa treatment.

Physiol. Plant. 128, 2006 615

Conclusions

Our study provided an in-depth insight into the formation

of resistance-related compounds in apple leaves. On the

one hand, the constitutive polyphenols catechin, epica-techin and derived procyanidins are built very early in leaf

development, prior to leaf unrolling. All polyphenol

enzymes investigated were already present in young

unrolled leaves.On the other hand, it could be shown that

inhibition of the 2-ODDs FHT, FLS and ANS by ProCa

induces the formation of 3-deoxyflavonoids. The 3-

deoxycatechin luteoliflavan serves as an indicator for the

rather unstable precursor 3-deoxyflavonoid luteoforol,which is anti-microbially active. Apart from the 2-ODD

inhibition, a strong induction of polyphenol gene expres-

sion occurs. Most likely, this is because of a feedback

mechanism relying on polyphenol concentrations. The

stimulating effect on polyphenol gene expression may be

of practical relevance for future applications.

Acknowledgements – These investigations were supported

by the European Union Commission (QLK5-CT-1999-01583).

The authors are grateful to Laszlo Janvari for design and supply

of degenerated DFR primers. The authors thank Christine

Statnik and Jurgen Greiner for their excellent technical

assistance. Special thanks go to Thorsten Strissel for his

support during the performance of the enzymatic investiga-

tions. Finally, the authors would like to thank Eva Meggeneder

for critically reading the manuscript.

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