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Summary Polyphenolicparenchyma cells (PPcells) in Nor-way spruce (Picea abies(L.) Karst.) stem phloem play impor-tant roles in constitutive and inducible defenses. To determinewhether anatomical andmolecular changes in PP cells arecor-related with tree resistance, we infected two Norway spruceclones with the pathogenic fungusCeratocystis polonica(Siem.) C. Moreau. The fungus induced significantly differentlesion lengths in the two clones, indicating that one clone wasmore resistant to thefungus(short lesions) than theother (longlesions). After infection, the cross-sectional area of PP cellsand their vacuolar polyphenol bodies increased in the threemost recent annual rings of PP cells in both clones. The moreresistantclone hadlargerPP cells with denserpolyphenol bod-ies than theless resistantclone,whereas theless resistantcloneaccumulated relatively more polyphenols after infection.Comparedwith thelessresistantclone, themoreresistantclonecontained higher starch concentrations before infection thatwere reduced more quickly after infection before returning tooriginal values. Low transcript levels of chalcone synthasewere detected in uninfected tissues of both clones, but the lev-els increased dramatically after infection. Transcript levelswere higher and peaked 6 days earlier in the more resistantclone than in theless resistantclone.The activityof at leastonehighlybasic peroxidase isoform wasgreatly enhanced after in-fection, and this increase occurred earlier in the more resistantclone.

Keywords: Ceratocystis polonica, defense responses, phenolsynthesizing enzymes, phenolic content, phloem parenchymacells, Picea abies, starch concentration.

Introduction

Plants produce various structural hindrances and toxic or re-

pellent metabolites to defendagainst challengesby insects amicroorganisms (Karban and Baldwin 1997). Secondary plametabolites in particular areimportantcomponents of both tconstitutive and inducible defense system of most plan(Harborne 1993, Grayer and Kokubun 2001). An increasesecondary metabolites such as phenolic compounds (flavooids, tannins and the structural polymer lignin) seems to beimportant inducible plant response to insect and pathogen tack (Halbrock and Scheel 1989, Dixon and Paiva 199

Karban and Baldwin 1997). Phenolics are involved in boconstitutive defenses and in inducible stress responses by dterring herbivores and pathogens, protecting against ultravlet radiation, strengthening cell walls (lignin), tanninproteins, scavenging oxidative species and serving as signing molecules (Dixon and Paiva 1995, Grace and Log2000).

Many conifers, particularly in the family Pinaceae, are tacked and killed by scolytid bark beetles and their associaphytopathogenicfungi (Paineet al.1997).One relatively westudied conifer defense system is that of Norway spru(Picea abies(L.) Karst.). This species is attacked by the barbeetle Ips typographusL. and its fungalassociateCeratocystis

polonica (Siem.) C. Moreau, which is capable of killinhealthy trees (Horntvedt et al. 1983, Christiansen 1985). Geerally, the bark of Norway spruce and other conifers contalayers of suberized, lignified cells and phenol-filled parechyma cells (PP cells) that create a thick, constitutive defenbarrier to potential invaders (Franceschi et al. 1998). The Pcells are also a major site for energy storage, in the form starch and sugars. A new layer of PP cells is produced eveyear (Krekling et al. 2000).

Attacks by insects or microorganisms induce a wound rsponse in conifers that is characterized by morphologicchanges and accumulation of anti-fungal compounds that

Tree Physiology 24, 505–515© 2004 Heron Publishing—Victoria, Canada

Induced responses to pathogen infection in Norway spruce phloem:changes in polyphenolic parenchyma cells, chalcone synthasetranscript levels and peroxidase activity

NINA ELISABETH NAGY,1,2CARL GUNNAR FOSSDAL,1 PAAL KROKENE,1 TRYGVEKREKLING,3 ANDERS LÖNNEBORG1,4and HALVOR SOLHEIM11 Norwegian Forest Research Institute, Høgskoleveien 12, N-1432 Ås, Norway2 Corresponding author ([email protected])3 Institute of Chemistry and Biotechnology, Agricultural University of Norway,Ås, Norway4 Present address: DiaGenic A.S., Oslo, Norway

Received October 2, 2002; accepted September 21, 2003; published online March 1, 2004



hibit infection and promote wound-healing (Berryman 1972,Woodward 1992). Recent evidence indicates that the two pri-mary sites of inducible defenses in Norway spruce stems arethe PP cells and traumatic resin ducts that form in the xylemafter an attack. The size of the PP cells increasesand their phe-nolic contents change in response to wounding, fungal inocu-lation and bark beetle attack (Franceschi et al. 2000, Krokeneet al. 2003). Similar phenol-containing parenchyma cells de-velop in the xylem around the traumatic resin ducts (Nagy etal.2000). In addition, local acquired resistance seems to be as-sociated with PP cell activation and traumatic resin duct for-mation (Christiansen et al. 1999, Krokene et al. 1999, 2003,Franceschi et al. 2002). These studies suggest that phenolicsplay an important role in the defense strategy of Norwayspruce.

Stilbenes and flavonoids, together with the enzymes in-volved in their synthesis (e.g., PAL = phenylalanine ammonialyase, CHS = chalcone synthase and STS = stilbene synthase;Lindberg et al. 1992, Brignolas et al. 1995a, 1995b,Franceschi et al. 1998, Chiron et al. 2000, Nagy et al. 2000,Viiri et al. 2001a), are among the most intensively studiedanti-fungal phenolic compounds in Norway spruce bark.Stilbenes and flavonoids, which are constitutively present asglycosides, represent a primary chemical barrier to invasionand are directly involved in defense against injury and fungalinfection (Nicholson and Hammerschmidt 1992, Brignolas etal. 1995a, 1995b, Evensen et al. 2000). Chemical analyseshave shown that there are differences in phenolic compositionandenzyme activities between resistantandsusceptible clonesof Norway spruce, both before and after fungal inoculation(Brignolas et al. 1995a, 1995b, Evensen et al. 2000). Phenyl-

alanine ammonia lyase, a key enzyme in phenolic synthesis, isfound in PP cells and ray cells of Norway spruce bark(Franceschi et al. 1998), in xylem and in epithelial cells of traumatic resin ducts (Nagy et al. 2000). The peroxidases con-stitute another important group of enzymes associated withphenolic chemistry that are involved in defense-related pro-cessessuch as lignification, cross-linking of cell wallproteins,auxin catabolism, production of oxygen radicals, as well as di-rect defense against pathogens (Campa 1991, Mohan et al.1993, Otter and Polle 1997). Increased accumulation of peroxidases has been reported in Norway spruce seedlings in-fected with a root pathogenic fungus (Asiegbu et al. 1999,Fossdal et al. 2001).

The recently discovered role of PP cells in the defense of Norway spruce (Franceschi et al. 1998, 2000) prompted us toinvestigate the anatomical features of these cells in relation tophenolic synthesizing agents. By studying Norway spruceclones differing in resistance to fungal infection, we hoped tobetter understand the underlying defense mechanisms. Weshow that PP cell activation, which involves enlargement andchanges in polyphenolic content, is preceded by up-regulationof chalcone synthase and highly basic peroxidases. These re-actions occurred more rapidly in a relatively resistant clonethan in a less resistant clone.

Materials and methods

Plant material and fungal inoculationField experiments were started on June 1, 1999 with30-year-old Norway spruce trees from a clonal stand at theHogsmark plantation of the Norwegian Forest Research Insti-tute, Ås, Norway (see Franceschi et al. 1998 for a descriptionof the stand). The studied trees were of Clone 579, with highresistance toC. polonica, and Clone 267, with limited resis-tance toC. polonica. Four trees of each clone were inoculatedwith pathogenic fungus growing on malt agar, which was ap-plied with a 5-mm cork borer as described by Krokene andSolheim (1998). Each tree received 15 inoculations evenlyspaced around the trunk in four rings, situated 1 m apart be-tween 3 and 6 m above ground (six inoculations in the upper-most ring and three in each of the lower rings). Of the 15inoculation sites per tree, five were inoculated withC. polonica(Isolate No. 93-208/115 from the Culture Collec-tion of the Norwegian Forest Research Institute), five with Heterobasidion annosum(Fr.) Bref. (heterocaryotic strain87-257/1, S intersterility group) and five with sterile agar aswounded controls. Only samples fromC. polonicaand sterileagar inoculated sites were included in the present study.

Tissue samples were collected from each tree 3, 6, 10, 16and 37 days after inoculation (starting with the uppermostring) by removing rectangular strips of bark and sapwood(1.6 cm wide× 10 cm long; Franceschi et al. 1998) immedi-ately below and above the inoculation site. Upper sampleswere used for microscopy, and lower samples were used formolecular analysis. In addition, control samples of uninocu-lated tissues from the same trees were collected on the day of inoculation and each subsequent sampling day. Control sam-ples were always taken at least 10 cm laterally from woundedsites. For microscopy, bark strips were stored in fixative untilprocessed. For molecular analysis, whole tissue strips werefrozen in liquid nitrogen immediately after sampling andstored at –80 °C.

Relative resistance of the two clones was determined bymeasuring the lengths of necrotic lesions in the inner bark of the samples for microscopy. Necrosis lengths were measuredupward from the inoculation sites. The two clones were se-lectedfrom a group of 15 clones that were testedforresistancein 1997 by measuring necrosis lengths 60 days after inocula-tion withC. polonica(eight inoculations per tree at 2 m heightin 2–3 trees per clone; H. Solheim, unpublished data).

Light microscopy and image analysisSubsamples, containing phloem, cambium, and at least oneannual ring of xylem, were dissected 50 mm above the inocu-lation site on the upper samples. These samples were fixed inparaformaldehyde (2%) and glutaraldehyde (1.25%) inL-piperazine- N , N ′-bis (2-ethanesulfonic)acid buffer(50mM,pH 7.2) for 12 h at room temperature, washed in the samebuffer (3 × 15 min), dehydrated in an ethanol series, infiltratedin a resin:ethanol series and embedded in LR White acrylicresin (polymerization at 60 °C for 24 h; TAAB Laboratories,

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Aldermarston, U.K.). Semi-thin cross sections (1.5 µm) of thephloem–cambium region were cutwith a diamond knife,driedonto gelatin-coated glass slides, stained with periodic acidSchiff’s(PAS)carbohydratestain (Hotchkiss 1948, Nagy et al.2000), and mounted with immersion oil. All reagents werefrom Sigma-Aldrich (St. Louis, MO).

Sections were imaged at 16× with a Leica DC200 CCDcamera on a Leitz Aristoplan photomicroscope. The imagesrepresented 540× 435 µm of the phloem,which included the3–4 youngest rows of fully differentiated PP cellsin the tan-gential direction. The current annual PP cell layer (PP99) wasomitted from the analysis because the cells were undifferenti-ated at the time of the experiment. In the next three annual lay-ers of PP cells (PP96, PP97, PP98), the contour of individual PPcells, and polyphenol bodies and starch grains within thesecells, were outlined on transparencies. Cross-sectional areaswere determined from scanned images of the transparenciesusing image analysis software (ImagePro Plus, Version 3.0,Media Cybernetics, Leiden, The Netherlands). For each sam-

pling day, total cross-sectional areas of PP cells, polyphenolbodies and starch grains within the three annual layers of PPcells were expressed as proportions of the total image area.Changes over the course of the experiment are presented rela-tive to Day 0 for each clone.

RNA extraction and Northern analysisWe extracted RNA from inoculated and control samples fromtwo trees per clone and time, following the procedure of Lönneborg and Jensen (2000). Briefly, the outer bark was re-moved from the frozen samples, and 1–2 g of the inner barkwas separated from the wood, ground with liquid N2 for10 min, and extracted for 30 min in homogenizing buffer(0.5 M TRIS-buffered saline (pH 8.0), 300 mM NaCl, 5mMEDTA, 2% SDS, 0.5% polyvinylpyrrolidone (PVP), 0.5 mMaurintricarboxylic acid (ATA) and 14.3 mM 2-mercapto-ethanol) (15 ml of homogenizing buffer per g freshmass of in-ner bark). After multiple (6–7 times) chloroform:isoamylalcohol extractions, the extracts were treated with 100 mg g–1

polyvinylpolypyrrolidone (PVPP) for 20 min at 75°C,and theRNA pellets were washed once with 2 M LiCl and once with70% ethanol. The resuspended pellet was reprecipitated oncewith 2 M LiCl.

For RNA gel blot hybridization, a total of 15 µg total RNA

was used and standard protocols (Ausubel et al. 1987) werefollowed. A32P-labeled cDNA probe of Norway sprucechalcone synthase (CHS) was used for the hybridization. TheCHS probe used (SPI3; GenBank accession number AF417107) corresponds to the last 217 bp of the 546 partialcDNA. This probe, which contains the 3′-untranslated regionwith < 30% similarity to other sequences, was used to avoidcross hybridization. The only knownCHS-like sequence iso-lated from Norway spruce is SP13, and the 5′ partial 346 bpcoding region of this Norway spruceCHSshows 90% similar-ity to CHS sequences from other spruces andPinus syl-vestrisL. (data not shown).

Filters were washed under stringent conditions with 0.SSC with 0.1% SDS at 65°C in the last two washes. Relativeamounts of transcripts (measured as counts per minute, CPwere determined from the32P-signals with an Instant Imager(Packard Instrument, Meriden, CT) at an exposure time 15 min in each case. The CPM data were obtained from th

membranes hybridized in parallel and adjusted for the sigintensity from a32P-labeled 18S rRNA probe as described byFossdal et al. (2001). Film exposure was performed f16–24 h at –80°C after CPM data were obtained from the filters. Allreagents were from Sigma-Aldrich (St. Louis,MO)Fisher Scientific (Pittsburg, PA) unless otherwise stated.

Real time PCROligonucleotide primers and probes for real-time PCR wedesigned with Primer Express software Version 1.5a providwith Applied Biosystems(Foster City, CA)Real Time Quantative PCR systems. Primers were designed for the Norw

spruceCHScDNA sequence, Gene Bank accession numbeAF417107. The designedCHSforward and reverse primers(5′-CCGCCTCTCAAATAAATCGTATTAGT-3′ and 5-′ATTATCAATTATTTGGGTTTCAGTTCTG-3′, respectively) am-plify only the 3′ untranslated end of this partial cDNA (frombase 354 to base 446). After amplification, only one peak wseen in themelting point analysis,verifying that only one PCproduct originated from theCHSprimer pairs. For each sam-ple, a secondinternal control reaction wasperformedusing tNorway spruce alpha-tubulin (aT ) transcript (Gene Bank ac-cession number X57980) as a target under identical reacticonditions as forCHS.TheaT5′-GGCATACCGGCAGCTCTTC-3′ and 5′-AAGTTGTTGGCGGCGTCTT-3′ forward and

reverse primers, respectively, amplify the cDNA region frobase 187 to 252 of theaT cDNA. Only one peak appeared inthe melting point analysis, verifying that one PCR produoriginated from theaT primer pairs. The primers were orderedfrom ABI PRISM Primers & TaqMan Probes Synthesis Svice (Applied Biosystems).

ForcDNA synthesis, 2.5µg of total extractedRNA persaple was used. The cDNA synthesis was performed with tTaqMan reverse transcription reagents kit using a poly dprimer for cDNA synthesis. Equal volumes (5 µl) of cDNwere used as the template and a 25-µl PCR reaction was pformed with a SYBR Green PCR Master Mix on 96-well PCplates (Applied Biosystems). The master mix contain

AmpliTaq Gold DNA polymerase, AmpErase uracil-N-glysylase (UNG), dNTPs with dUTP, passive reference 1 anbuffer components. The AmpErase UNG prevents the ramplification of any carryover PCR products containing ucil. The concentration of each primer was 50 mM. Thsamples were run as duplicates in each PCR run, and the Creaction and theaT control for the same sample were alwayrun on the sameplate.The PCR cycling parameterswere50for 3 min for UNG enzyme activity, 95 °C for 10 min to denture the UNG enzyme and to activate the polymerase, andcyclesat95°Cfor15sand60°Cfor1min.ThePCRreactiowere run on agarose gels, and a single cDNA band of the

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INDUCED DEFENSE RESPONSES IN NORWAY SPRUCE 5



pected size was obtained after each run. No band was detectedfrom the no template controls or the samples containing onlythe fungus (data not shown).

Real-time PCRdetectionwasperformed on theABIPRISM7700 (Applied Biosystems). Data acquisition and analysiswere performed with Sequence Detection System 1.7a Soft-

ware Package (Applied Biosystems). For each sample, thecritical threshold cycle value forCHS (dCt) was subtractedfrom the corresponding dCt value foraT , giving the relativeddCt value. This value gives the relative level of CHScom-pared withaT in thesample, and thehigher the ddCt value, themoreCHStranscript was present in the sample. For each timepoint, the ddCT values from the unwounded control sampleswere subtracted from the ddCT values fromC. polonicaandsterile agar inoculated samples, and the baseline of the graphwas set at zero.

Protein extraction and isoelectric focusing electrophoresis(IEF) of peroxidase isoforms

Native, crude protein was extracted from 0.3–0.4 g of phloemtissue(preparedandgroundwith liquidN2 as described above)from inoculated and wounded control samples from two treesper clone and time, in low pH buffer (0.001 M sodium citrate,pH 5.0) with 3% CHAPS (3-[(3-cholamidopropyl) dimethylamino]-1-propane-sulfonate). Insolublematerialwasremovedby centrifuging twice at 10,000g for 20 min. Total proteinconcentration was determined spectrophotometrically withCoomassie Plus Protein assay reagent (Pierce, Rockford, IL),and bovine serum albumin as the standard.

To detect peroxidase isoforms, equal amounts of total pro-tein (∼2.6 µg) were applied to, and separated on, non-denatur-ing IEF gels (10% polyacrylamide gels, 1 mm thick, pH3.5–9.5; Amersham Biosciences) prepared according to themanufacturer’s instructions. Isoelectric focusing was run at4 °C and 20 W for 2.5 h with an LKB 2117 Multiphor system(LKB, Bromma, Sweden). Peroxidase isoform activitywas as-sayed as described by Kerby and Sommerville (1989), with3-amino-9-ethylcarbazole (carbazol) and hydrogen perox-idase as substrates. All reagents were from Sigma-Aldrich un-less otherwise stated.

Statistical analysisLesion length data and image quantification data were evalu-ated by analysis of variance by the mixed procedure of the

SAS software package (SAS Institute, Cary, NC). Data fromDays 10 and 37 after inoculation were analyzed on a sin-gle-tree basis, by considering contrasts between treatmentswithin trees (pathogen treatment versus sterile agar or un-treated bark controls) as the response variable. The statisticalmodel wasyij = µ +ci + eij ,whereyij is the contrast of Clone i(i = Clone 267 or Clone 579) and ramet j (j = 1, 2, 3, or 4)withinClonei,µisthetotalmean,ci is thefixedeffect of Clonei, eij is the random experimental error (residual) for all traitsanalyzed. The values of clones were tested for difference from0 witht -tests (P < 0.05), and differences between clones weretested with anF -test (P < 0.05). In both cases, the residuals

were used as the experimental error term and valuessignificantly different from 0 imply a treatment effect. Wheredifferences between theanalyses at Days 10 and37 aresignifi-cant, an effect of time is implied.

Results

Necrotic zone formation in response to inoculationNecrotic zones, observed as areas of yellow-brownish discol-oration in the phloem, were produced in response to inocula-tion withC. polonica or with sterile agar, but were notobserved in uninoculated control samples. In both inoculationtreatments and clones, a narrow yellowish zone appearedaroundthe inoculation point 3 daysafter inoculation.Later on,this zone became darker brown, and gradually increased insize, primarily in the longitudinal direction. Both clones andtreatments had similar-sized necrotic zones during the first16 days after inoculation (Figure 1),but theless resistantcloneproduced darker and significantly longer zones in response tothe fungal inoculations compared with the sterile inoculationsat Day 16 (P = 0.003). At Day 37, fungal inoculations had in-duced significantly longer reaction zones than sterile inocula-tions in the less resistant clone (P < 0.0001), but onlymarginally longer zones in the more resistant clone (P =0.057). The difference between treatments was significantlygreater in the less resistant Clone 267 than in the more resis-tant Clone 579 (Figure 1;P = 0.0009).

Anatomical characterization of PP cellsLight microscopy studies of phloem cross sections dissected50 mm above the inoculation site revealed induced swelling of PPcellsinbothclonesatDays16and37afterinoculationwithC. polonica(Figure 2). Very little or no swelling was observedin the corresponding samples inoculated with sterile agar or inuninoculated control samples. At the start of the experiment

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Figure 1. Phloem necrosis lengths in two Norway spruce clones (=579, = 267) inoculated withCeratocystis polonica(filled symbols)or sterile agar (open symbols). Necrosis lengths were measured up-ward from the inoculation point on one sample per treatment per dayafter treatment, and are presented as means – SE (n = 4 trees perclone).



(Figure 2A), the phloem contained regular annual layers of PPcells, with rows of sieve cells and radial oriented ray cells inbetween. At Day 37 after inoculation withC. polonica(Figure 2B), the PP cells in all observed rows had swollen andcompressed the surrounding sieve cells into dense layers of cell walls. The rays appeared wavy because of this compres-sion and they also contained starch grains. In all observed PPcell layers, the cells were alive at all sampling times, as indi-cated by the presence of intact cytoplasm containing nuclei,lipid bodies and starch grains.

In both clones,the starchpool andpolyphenol concentrationof the PP cells were affected by infection withC. polonica(Figure 2). At Day 0, polyphenols were observed as large ho-mogeneous bodies within the vacuole of the more resistantclone (Figure 2C), but appeared as scattered droplets in theless resistant clone (data not shown). At Day 37 after infection

(Figure 2D), the polyphenol bodies in the swelling PP cellsboth clones appeared as porous aggregates that filled twhole vacuole,as distinct dense rings along thecell peripheor as droplets. Starch grains appeared clustered before inolation in both clones (Figures 2A and 2C), but were more sctered and less prominent after infection (Figure 2D). In tmore resistant clone, starch grains were less pronounc16 days after infection (Figure 2B and 2D), but seemed tomore prominent again 37 days after infection, whereas thcontinued to be less obvious in the less resistant clone. In cotrol tissue at Day 37, only small and scattered starch graiwere observed in both clones (data not shown).

Quantification of morphological changes in PP cellsTo quantify morphological changes in PP cells, we measurthe cross-sectional areas of PP cells, polyphenol bodies a



Figure 2. Light microscopy images of polyphenol-containing parenchyma (PP) cells in the 1996, 1997 and 1998 annual cell layers of

before and after inoculation with the pathogenic fungusCeratocystis polonica. All figures represent cross sections of tissue stained with perioacid Schiff’s stain. (A) At Day 0, the phloem consists of sieve cells and layers of small rounded PP cells with polyphenolic inclusiongrains.One cell thick parenchyma raycells runradially throughtherows of PP cells andsievecells (400×,bar=25µm).(B)AtDay37,thePPcellsare swollen and thesieve cell layers are compressed becauseof theenlargement of thePP cells(400×, bar = 25 µm). (C) High magnificationof thePP cell layerdeposited in 1997 at Day 0 of the experiment showing PP cells filledwith densely staining, evenly dispersed phenolicmavacuole and clusters of starch grains in the cytoplasm (800×, bar= 12.5 µm). (D) At Day 37, the PP cells areenlarged, phenols areof a poroustype or dispersed around the surface of the vacuole, and starch grains are scattered (800×, bar = 12.5 µm).



starch grains within the 1996–1998 annual layers (PP1996–PP1998). Because there was no increase in PP cell size (P =0.49–0.77) or polyphenol body size (P = 0.15–0.61) inuninoculated control samples at 10 or 37 days after the start of the experiment (Figures 3A and 3B), we assume there were nowhole-tree systemic effects in either clone after treatment.

UptoDay10,themeancross-sectionalareaofPPcellsinallthree annual layers was comparable between clones and treat-

ments (~600 µm2 per cell, or ~10% of the imaged areas) (Fig-ure 3A).AtDay 37 after fungal inoculation,all PPcells inbothclones had enlarged significantly compared with values atDay 0 (P = 0.0001 for both clones) (Figure 3A), and PP cellshad a mean cross-sectional area of about 1500 µm2 in the moreresistant clone and 1200 µm2 in the less resistant clone. Inocu-

lations withC. polonicainduced much more swelling thansterile inoculations at Day 37 (P = 0.01 and 0.057 for the moreand the less resistant clone, respectively), and the differencebetween the treatments did not differ significantly betweenclones (P = 0.39). Sterile inoculation induced little increase inPP cell size 50 mm from the inoculation site and did not differsignificantly (P = 0.43–0.79 for the two clones) from theuninoculated controls (Figure 3A).

The area of the polyphenolic bodies within PP cells in-creased following inoculation withC. polonica(Figure 3B).At Day 0, the mean cross-sectional area of phenolic bodies perPP cell was ~240 µm2 in all three annual layers. Thirty-sevendaysafterinoculationwithC. polonica, this area hadincreasedsignificantly to about 1000 µm2 inbothclones(P < 0.0005).Atthis time, the phenolic bodies were significantly larger in re-sponse to fungal inoculation than in response to sterile inocu-lation (P < 0.009 for both clones) (Figure 3B). The differencein phenolic body size in fungal and sterile inoculated samplesdidnotdiffer significantly between clones(P = 0.90).Phenolicbody size in sterile inoculated samples did not increase afterinoculation, and was not significantly larger than in un-inoculated controls 37 days after inoculation (P >0.99forbothclones).

The area within PP cells that was covered by starch grainsshowed an initial increase during the first 10 days after inocu-lation, followed by a large decrease in most cases (Figure 3C).Starch concentrations decreased more and sooner after infec-tion in the more resistant clone (Figure 3C), but then showed asignificant increase in fungal-inoculated tissues between Days16and37(P < 0.04).In the less resistant clone,there was a sig-nificant reduction in starchconcentrations over thesame inter-val (P < 0.03). In both clones, however, fungal inoculationinduced significantly less starch depletion than sterile inocula-tions 37 days after inoculation (P < 0.02). Starch concentrationdecreased significantly in uninoculated control tissues of bothclones from Day 0 to Day 37 (P < 0.03).

Temporal levels of chalcone synthase transcripts after infectionNorthern blotting with theCHSprobe detected low transcriptlevels in phloemextracts of uninoculatedcontrol tissues. Afterinfection, CHS transcripts increased dramatically in bothclones (Figures 4A–C). Transcript levels started to increasefrom Day 3, peaked at Days 10–16, and then declined towardDay37 (Figure 4B). Transcripts increasedto a higher levelandpeaked earlier in the more resistant clone than in the less resis-tant clone (Figure 4B). In the more resistant clone, maximumtranscript intensity was detected at Day 10, whereas in the lessresistant clone, maximum intensity was detected at Day 16.

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Figure 3. Changes in cross-sectional areas of polyphenolic paren-chyma (PP) cells and cell components in three annual cell layers(PP1996–PP1998) in the phloem of Norway spruce. Data are presentedas percent change in total image area that is covered by (A) PP cells,(B)polyphenol bodies and (C)starchgrains,relative to the areas mea-sured at the start of the experiment. Cross sections were taken fromtwo clones ( = 579, = 267) inoculated withCeratocystis polonica(filled symbols) or sterile agar (open symbols).Ceratocystis polonicainoculated tissue sites were analyzed 3, 6, 10, 16 and 37 days aftertreatment. Sterile agar and uninfected control tissue were analyzed atDays 10 and 37 (open symbols without continuous line). On eachcross section, PP cells extending 0.5 mm in the tangential direction of the tissue were included in the analysis. The data are presented asmeans + or – SE,n = 4 individual trees per clone.



To increase the temporal resolution and to comparewounded andinoculated samples we dida real-time PCRanal-ysis of CHStranscripts relative to a tubulin transcript (ddCt).The relative ddCT-levels showed the same trend as the North-ern analyses (Figure 4);CHStranscript levels increased fasterand to a greater extent after infection in the more resistantclone than in the less resistant clone. There was a clear effectof wounding onCHStranscript levels, but the response wasweaker than with fungal infection.

Temporal change in peroxidase isoforms after infectionNon-denaturing IEF gelsof peroxidasesrevealedthe existenceof multiple isoforms in the bark of both clones (Figure 5). Tento 11 peroxidase activity bands were observed in control tis-sues of both clones. An extra basic band (pI ~8.6) was ob-served only in the less resistant clone, and an acidic band (pI~3.4) was seen only in the more resistant clone. After infec-tion, the intensity of two highly basic isoforms (pI ~9.5) in-creased relative to the control, whereas none of the acidicisoforms were affected by infection (Figure 5). The increase

was most pronounced 10 and 16 days after infection in tmore resistant clone, whereas in the less resistant clone tsame bands were only weakly detected at Day 16 (Figure One weak band(pI ~ 9.0)becameless intenseafter infectionboth clones.

Discussion

Cellular and tissue reactionsDifferent types of stress (e.g., wounding, pathogen infectiodrought) may induce similar responses in plants. This raisthe question of whether it was wounding, fungal infectionboth, that induced the responses we observed. Responsesmechanical wounding are well characterized in conife(Wainhouse et al.1998) andaregenerallyweakerand morestricted than those observed when pathogenic fungi are intduced into the wound (Solheim 1988). Similarly, we observthat control samples from sterile agar inoculations showlimited responses compared with responses of samples ifected withC. polonica, indicating that the changes we ob-



Figure 4. (A) Northern-blot analysis showing chalconesynthase (CHS) transcripts in uninoculated bark (Con-trol) and in bark inoculated withCeratocystis polonica(Infected) from two Norway spruce clones (579 and267) at 0, 3, 6, 10, 16 and 37 days after inoculation. ThRNA transcripts were detected with32P-labeled cDNAprobes forCHS. Hybridization with32P-labeled 18SrRNA was carried out as a control for loading. (B) Relative32P-activity (counts per minute, CPM) of the tran-scripts was detected by Instant Image quantification(relative to 18S rRNA). Values represent means of n = 3blots (three replicates from the same extraction) perclone ( = 579, = 267). Open and filled symbols in-dicate uninoculated control samples and samples inoculated withC. polonica, respectively. (C) Real-time PCRanalysis of CHStranscripts in clone 579 () and 267( ). Open and filled symbols indicate samples inocu-lated with sterile agar andC. polonica, respectively. TheCHStranscript was detected by real-time PCR and sub-tracted from alpha-tubulin (aT ), to express relative lev-els of CHS(ddCT) at 3, 6, 10, 16 and 37 days aftertreatment. The ddCT values obtained from the un-wounded control samples were subtracted from theddCT values fromC. polonicaand sterile agar inocu-lated samples at each time, and the baseline of the grapwas set at 0. Data are presented as means ± SE,n = 2trees per clone.



served were primarily an effect of pathogen infection. Whenindividual trees are sampled repeatedly over time, as in ourstudy, there is a possibility of eliciting whole-tree systemic re-sponses (Bonello et al. 2001). However, control samples fromunwounded fresh bark appeared normal, indicating that the re-sponses we observed were primarily local. Similar conclu-sions have been reached in other studies based on acomparable experimentaldesign(Franceschi et al.2000,Nagyet al. 2000, Krekling et al. 2003, Krokene et al. 2003).

Defense responses of coniferous trees to pathogen infectioninclude rapid cell death andaccumulation of secondarymetab-olites at the infection site (e.g., phenolic and resinous com-pounds), resulting in discrete necrotic lesions (Berryman1972, Nicholson and Hammerschmidt 1992, Krokene and

Solheim 1997). Tissue discoloration and necrosis are associ-ated with oxidation and polymerization of accumulating phe-nols. These processes make the attacked tissues resistant tomicrobialdecay, constrainfungal invasion,andmaylead to ac-quired resistance against future attacks (Klement and Good-man 1967, Nicholson and Hammerschmidt 1992, Krokene etal. 1999).

The PP cells form the major living tissue in the phloem, andare a target for invasive organisms as well as an important sitefor resistance to infection (Franceschi et al. 1998, 2000,Krekling et al. 2000, Krokene et al. 2003). When an intruderdamages PP cells, it may cause the release of stored phenolsand activate inducible defense responses (Franceschi et al.

1998, 2000). We observed that pathogen infection resulted inpronounced expansion of PP cells, as has been reported forother Norway spruce clones (Franceschi et al. 1998, Krokeneet al. 2003). Expansions of PP cells might be a secondary re-sponse induced by growth substances liberated from dyingcells (Biggs 1992, Woodward 1992), or result from changes inturgor pressure caused by an accumulation of osmotically ac-tive substances like soluble phenols in thevacuole (Franceschiet al. 1998). In our study, PP cell expansion was accompaniedby an increased area of polyphenols within cells and changesin the appearance of the phenols from large homogeneousbodies and scattered droplets to porous aggregates and ring-

like structures along the cell periphery.The quantitative and qualitative changes observed in the

phenolic bodies may be related to alterations in their chemicalstate,such as productionandaccumulation of insolublephenolpolymers or phenol monomers with higher solubility. In sup-port of this, other studies have demonstrated both quantitativeand qualitative changes in phenolic chemistry in the reactionzones of Norway spruce phloem. Trees inoculated withC. polonica show an early increase in concentrations of flavonoids (e.g., catechin and stilbene phenolic monomers),and it has been suggested that flavonoids are gradually con-verted to tannins and other insoluble compounds (Brignolas etal.1995a, Evensenet al. 2000).The increase in flavonoids andtannins is interpreted as the formation of a chemical barrier,because these compounds can inactivate fungal enzymes andhave fungistatic effects (Brignolas et al. 1995c, Evensen et al.2000). The ensuing polymerization may lead to stiffening of the cell matrix, which may inhibit the progress of invadingpathogens (Brignolas et al. 1995b).

Different clones of Norway spruce show highly variable re-sistance to colonization, and clonal differences in phenoliccontent have been reported.Brignolaset al. (1995a) found thatresistant clones had a higher mean concentration of flavon-oids, whereas Evensenet al.(2000)concludedthat theconcen-tration of stilbene was more closely related to resistance. Wefound a larger relative increase in polyphenolic concentrationin the less resistant clone, which initially had a lower concen-tration of polyphenols than themore resistantclone.This find-ing could be explained by the need to produce additionalamounts of phenols to reach a high enough concentration toprovide efficient defense in the less resistant clone (cf.Krokene et al. 2003). We note that, because of the low numberof inoculations per tree in our study, all trees were able to de-fend themselves and contain the pathogen within discretelesions.

Concomitantly with phenolaccumulation in PP cells, starchconcentrations decreased in both clones after infection. Thisobservation is in accordance with the findings of Viiri et al.(2001b) that the concentration of soluble carbohydrates in

512 NAGY ET AL.


Figure 5. Isoelectric focusing electrophoresis(IEF) analysis of peroxidase isoforms in unin-fected bark (Control) and bark infected withCeratocystis polonica(Infected) from twoNorway spruce clones (579 and 267) at 0, 3,6, 10, 16 and 37 days after inoculation. Na-tive proteins were extracted in low pH buffercontaining CHAPS. Crude protein extractsfrom 0.1 g of bark tissue were separated on a10% IEF gel (pH 3.5–10). Equal amounts of total protein (5 µg) were loaded in each lane.The IEF gel is a representative of the analysisof samples fromn = 2 trees per clone. Esti-mated pI values and two highly basicperoxidase isoforms, one with increased in-tensity ( +) and one with decreased inten-sity ( −) after infection, are indicated.



Norway spruce phloemdecreased considerably afterC. polon-ica inoculation. Starch reserves in the phloem constitute a car-bon source that may be mobilized rapidly during defensereactions for use in the synthesis of secondary metabolitessuch as resin (BordaschandBerryman 1977) andpolyphenols.Starch is hydrolyzed in cells bordering lesions induced by me-chanical wounding, and the resulting sugars are used for in-creased respiration during repair reactions, for production of protecting substances (e.g.,phenols, tannin) and foradditionalcell wall material (Wardell and Hart 1970, Johansson andStenlid 1985). After infection, the reduction in starch concen-trations was faster in the more resistant clone than in the lessresistant clone. However, after Day 16, starch concentrationsincreased again in the more resistant Clone 579, whereas theycontinued to decrease in the less resistant Clone 267. An ex-planation for the lower starch concentrations in the less resis-tant clone may be that more carbon was used for defense.Alternatively, a re-accumulation of starch reserves may haveoccurred in themore resistantclone after themost active phaseof the defense response was completed. These renewed starchreserves couldpotentiallycontribute to counteract laterattacksand thus form part of the acquired resistance response ob-served in Norway spruce (e.g., Krokene et al. 1999). In thecontrols of both clones there was a continuous decrease instarch concentrations, which is expected in healthy tissue as aresult of normal consumption during the growing season(Christiansen and Ericsson 1986).

Changes in CHS transcript levels and peroxidase activityAlthough transcript levels were analyzed from only two treesper clone per time after inoculation, our results highlight im-portant temporal aspects of defense responses in treeswith dif-

ferent degrees of resistance to pathogen infection. Transcriptsof CHS, which were found at low constitutive levels in un-wounded control tissues, were strongly up-regulated 3–16 days after fungal inoculation in both clones. This is consis-tent with other studies showing that infection results in ele-vated transcript levels, enzyme activity, and amounts of flavonoids and stilbenes (Lindberg et al. 1992, Schwekendieket al. 1992, Brignolas et al. 1995a, 1995b, Dixon and Paiva1995, Viiri et al. 2001a). The changes inCHStranscript levelsoccurred about a week before observable morphologicalchanges appeared in the phenolic bodies of the PP cells. Thisdelay may correspond to the time required for increased en-zymeactivity to produce observablemorphological changes in

phenol bodies.Transcript levels peaked sooner after infection in the moreresistant clone (Day 10) than in the less resistant clone (Day16). We also detected an earlier increase after infectionin a ba-sic peroxidase isoform (pI 9.5) in the more resistant clone.Brignolas et al. (1995a) found higher activities of chalconesynthase and stilbene synthase in response toC. polonicain-fection in a moreresistant clone than ina less resistantclone of Norway spruce. Based on this observation, they proposed thatresistantclonesarebetterable to activatethephenolic pathwaythan less resistant clones. Our results onCHStranscript levelsand peroxidaseactivities indicate thatdifferences in resistance

are related to the speed ofexpressionof these enzymes. Ingeeral, the more rapidly a defense response develops, the lowthe possibility for pathogen invasion (Biggs 1992, Woodwa1992, Woodward and Pocock 1996). A decline in transcrlevels and peroxidase activity was observed by Day 37 afinfection. This decline is in accordance with observations

angiosperm systems, suggesting that defense responses to fection are transient (Dixon and Harrison 1990) and ceawhen the further accumulation of metabolites becomes unnessary.

The basic peroxidase that increased in the more resistaclone after infection may be similar to the defense-relatspruce peroxidase (SPI2, pI 9.5) that accumulates in Norwspruce seedlings after infection with the pathogenPythiumdimorphumHendrix and Campbell (Fossdal et al. 2001)Spruce peroxidase SPI2 accumulated in the shoot as a sytemic response to infection in the roots, indicating that up-reulation of these peroxidases forms part of the host defensystem (Fossdal et al. 2001). This is consistent with the actrole peroxidases play in defense reactions andfundamentalsistance mechanisms in plants. Previous studies suggest thbasicperoxidasesparticipatein lignin formationduring needdevelopment in Norway spruce (Polle et al. 1994, Otter aPolle 1997), and our study demonstrates that they also accmulate in stems of mature Norway spruce after funginfection.

In conclusion, PP cells, vacuolar polyphenolic bodiestarch conversion, and activity of phenol-synthesizing metolites converge to enhance resistance to pathogen infectionNorway spruce. Taken together, these responses probabform an important part of the resistance capacity of individclones. Rapid mobilization of defense responses appears tocrucial for resistance, and an early response was typical of more resistant clone whereCHS transcript levels changedmore rapidly, peroxidase activity appeared earlier, anpolyphenol and starch pools of PP cells were more dynamthan in the less resistant clone.

Acknowledgments

The authorsgratefully acknowledge Drs. Erik Christiansenand ViR. Franceschi for critical review of the manuscript and helpfuldiscsions, and Marianne Jensen and Inger Heldal for technical assistanWe also thank Dr. Øystein Johnsen for valuable suggestions and hwith the statistical analysis. This research was funded by The Rsearch Council of Norway under Project No. 104023/110, 11375

111, 117925/140 and 133338/110.

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