Trade-Offs in Phenolic Metabolism of Silver Birch: Effects of Fertilization, Defoliation, and...

1970

Ecology, 80(6), 1999, pp. 1970–1986q 1999 by the Ecological Society of America

TRADE-OFFS IN PHENOLIC METABOLISM OF SILVER BIRCH: EFFECTSOF FERTILIZATION, DEFOLIATION, AND GENOTYPE

MARKKU KEINANEN,1,5 RIITTA JULKUNEN-TIITTO,1 PIA MUTIKAINEN,2,3 MARI WALLS,2 JARI OVASKA,2,4 AND

ELINA VAPAAVUORI4

1Department of Biology, University of Joensuu, P.O. Box 111, FIN-80110 Joensuu, Finland2Department of Biology, University of Turku, FIN-20014 Turku, Finland

3ETH-Zurich, Experimental Ecology, ETH-Zentrum NW, CH-8092 Zurich, Switzerland4The Finnish Forest Research Institute, Suonenjoki Research Station, FIN-77600 Suonenjoki, Finland

Abstract. We examined the chemical responses of 10 silver birch (Betula pendula)clones to fertilization and defoliation in a field experiment. In defoliation, every secondleaf was removed from the saplings. Three days later, two undamaged short-shoot leaveswere collected, air-dried, and analyzed for condensed tannins and 34 nontannin phenoliccompounds by high-performance liquid chromatography. The clones showed substantialvariation in phenolic composition of the leaves and in chemical responses to fertilizationand defoliation. A cluster analysis by UPGMA indicated that the phenolic profiles of birchleaves were affected more by genotype than fertilization or defoliation, and the clones couldthus be distinguished from each other. In addition, on the basis of their overall phenoliccomposition, the clones were clustered loosely in three clone groups.

The leaves of fertilized saplings contained lower levels of condensed tannins thancontrols, as predicted by carbon/nutrient balance (CNB) hypothesis. However, fertilizationhad no effect on the total amount of nontannin phenolics. The concentrations of (1)-catechin, 3,49-dihydroxypropiophenone 3-glucoside (DHPPG), 3-cinnamoylquinic acids,and flavone aglycones were lower in fertilized saplings, whereas the opposite was true for5-cinnamoylquinic acids and the total amount of flavonol glycosides. Although our resultsprovide support for the CNB hypothesis, they also show that the accumulation of phenoliccompounds in birch leaves is strongly coordinated. Different branches of the biosyntheticpathway of phenolic compounds may compete for substrates, and such internal metabolictrade-offs may explain the differential accumulation of the compounds. In fertilized sap-lings, the concentration of condensed tannins was also negatively correlated with the amountof triterpenoid resin droplets measured from the same saplings. We suggest that a linkagevia malonyl-CoA between the biosynthetic routes to terpenoids and flavonoid derivatives,such as condensed tannins, may explain the different responses to fertilization reported forterpenoids and phenolics.

Undamaged leaves of partially defoliated saplings contained more DHPPG and flavoneaglycones and less cinnamic acid derivatives and (1)-catechin than did leaves of controlsaplings. The induction of DHPPG and flavonoid aglycones was significantly and negativelycorrelated with the concentration of myricetin glycosides in fertilized saplings, which mayindicate a trade-off between induced and constitutive defense. Moreover, in fertilized sap-lings, the three clone groups formed by UPGMA clustering differed significantly in themagnitude of induction of DHPPG and flavone aglycones. Different birch genotypes maythus have different modes of chemical defense, and the magnitude of chemical responseof a genotype may partly depend on resource availability. In general, our results show thatnew insights in the theory of chemical defense can be gained by accomplishing studies onplant–herbivore interaction with high chemical resolution.

Key words: Betula pendula; carbon/nutrient balance; clonal variation; defoliation; fertilization;flavonoids; induced defense; phenolic compounds; plant–herbivore interaction; secondary metabolism;tannins.

INTRODUCTION

Current hypotheses of variation in plant chemicaldefenses are based on resource limitation in the envi-

Manuscript received 17 February 1998; revised 7 August1998; accepted 10 August 1998.

5 Present address: Max-Planck-Institut fur Chemische Oko-logie, Tatzenpromenade 1a, 07745, Jena, Germany. E-mail:[email protected]

ronment (Coley et al. 1985). The carbon/nutrient bal-ance (CNB) hypothesis predicts that fertilization withgrowth-limiting nutrients will lead to decreased con-centrations of carbon-based secondary metabolites(Bryant et al. 1983, Tuomi et al. 1988). Although sub-stantial support exists for the CNB hypothesis (e.g.,Gershenzon 1984, Tuomi et al. 1984, Waterman et al.1984, Bryant 1987, Waterman and Mole 1989, Bryant

September 1999 1971PHENOLIC VARIATION IN BIRCH

et al. 1993), several studies have given mixed results(see Herms and Mattson 1992, Baldwin 1994, Ger-shenzon 1994, Karban and Baldwin 1997, and refer-ences therein). Some of the apparently inconsistent datamay be explained by internal metabolic trade-off (Bald-win et al. 1987, Berenbaum and Zangerl 1988), becausethe CNB hypothesis merely predicts that carbohydratesaccumulated in excess of growth requirements will beallocated to carbon-based secondary metabolism, butdoes not explain how this carbon is distributed amongdifferent pathways and compounds (Tuomi et al. 1988,Herms and Mattson 1992). Tuomi et al. (1988) sug-gested that the total level of carbon-based secondarycompounds varies as a function of plant carbon/nutrientbalance, whereas the proportional distribution of thesesubstances may depend on the selective importance ofspecific herbivores over the evolutionary history of theplant population.

Environmental factors, such as mineral nutrition,may also influence the production of secondary com-pounds at the level of metabolic regulation, e.g., byregulation of enzymatic activity (Waterman and Mole1989). In addition, because of the costs of the biosyn-thetic machinery, storage, transport, or maintenance ofsecondary metabolites, changes in carbon/nutrient ra-tios may not result in changes in secondary metaboliteconcentrations (Gershenzon 1994). Moreover, the con-centration of one compound may increase at the ex-pense of another; thus, changes in the amounts of in-dividual compounds are not necessarily correlated withoverall changes in total secondary metabolism (Hermsand Mattson 1992). Reichardt et al. (1991) suggestedthat increased concentrations of secondary chemicalsmay, in some cases, be masked by rapid metabolicturnover. The CNB hypothesis would thus predict mostaccurately the effects on stable ‘‘end products’’ withlittle or no metabolic turnover, such as condensed tan-nins and insoluble, nonvolatile resins secreted overplant surfaces.

Attempts to understand chemical responses of plantsto herbivory or resource availability have been hin-dered by a poor knowledge of the relative contributionsof specific secondary metabolites to these responses(Karban and Myers 1989, Haukioja 1990, Bryant et al.1993). Most studies on induced resistance have ana-lyzed the plant’s responses with low chemical resolu-tion (Baldwin 1994). Measurements of total amountsof secondary compounds (e.g., total phenolics) mayhide ecologically meaningful variation in individualcompounds. The compounds in different mixtures mayact additively or synergistically (McKey 1979, Ber-enbaum 1985). Thus, variation in the relative propor-tions of secondary compounds may have considerableecological consequences (Langenheim 1994).

Intraspecific variation in plant chemistry may pro-vide additional protection against herbivores, since theunpredictability of chemical composition may decreasethe likelihood that resistance to the defense evolves in

herbivores (Schultz 1983, Whitham 1983). Chemicalresponses to resource availability or herbivory mayvary among plant genotypes (Chapin et al. 1987). Dif-ferent genotypes may also have different thresholds ofdamage before exhibiting induced responses, may showdifferent responses once induced, or may be inducibleonly under specific environmental conditions (Colemanand Jones 1991). Moreover, different herbivore speciesmay attack host trees of specific phenotypes (Linhart1991). Specialist herbivores are not necessarily de-terred by high concentrations of defense compounds,but may overlook plants with low enough concentra-tions of specific compounds needed as recognition cues(van der Meijden 1996). Misleading the recognitionsystem of the herbivore may thus be the best and mostsuccessful defense for a plant (Haukioja et al. 1994).Insects can perceive rather subtle variations in chemicalcomposition of their food plants (Schoonhoven 1982,Harborne and Grayer 1994) and use this capacity intheir oviposition or feeding behavior (Schultz 1983).For example, the feeding of insect larvae may be eitherstimulated or inhibited by different glycosides of thesame flavonoid aglycone, and the same glycoside ofdifferent aglycones may also have different effects(Harborne and Grayer 1994).

One of the most studied systems for induced defensehas been the interaction of birches (Betula pendulaRoth and B. pubescens Ehrh., including ssp. tortuosaLedeb.) and birch-feeding insects (Edwards et al. 1991,Hartley and Lawton 1991, Neuvonen and Haukioja1991; see also Karban and Baldwin 1997). Rapid,wound-induced changes in the foliage of B. pendulaand B. pubescens influence food preferences and larvalperformance of several birch-feeding insects (Edwardset al. 1991, Hartley and Lawton 1991, Neuvonen andHaukioja 1991, Mutikainen et al. 1996). However, theeffects are often small and variable both within andbetween trees, and could also be different in differentpopulations of birches (Hartley and Lawton 1991), orcould be shown only under specific conditions (Hau-kioja 1990). Although induction in birch leaves hasbeen associated with total phenolics or the activity ofphenylalanine ammonia-lyase (PAL) (Hartley and Firn1989), the chemical responses have not been studiedin detail. Consequently, Hartley and Lawton (1991)suggested that more attention needs to be paid to thephenolic profiles of individual trees and to the exactnature of the compounds being induced.

The purpose of this study was to examine the re-sponses of phenolic compounds of silver birch (Betulapendula) leaves to defoliation and fertilization. In orderto investigate the relations between different phenoliccompounds and alternative branches of the biosyntheticpathway of phenolic metabolites, we analyzed the con-centrations of condensed tannins and a wide range ofnontannin phenolics, including cinnamic acid deriva-tives, flavonol glycosides, and flavone aglycones. Theprimary objective of the study was to examine whether

1972 Ecology, Vol. 80, No. 6MARKKU KEINANEN ET AL.

chemical responses of birch leaves to fertilization varyamong different phenolic compounds or groups of com-pounds according to their biosynthetic origin. Second-ly, we examined whether the phenolic composition ofundamaged birch leaves changes due to defoliation ofneighboring leaves, and whether the possible chemicalresponses depend on nutrient availability. Thirdly, inorder to examine genetic differences in constitutive lev-els of phenolic compounds and in responses inducedby defoliation, we used micropropagated saplings from10 B. pendula clones.

MATERIALS AND METHODS

Plant material

We studied the chemical responses of 10 Betula pen-dula clones (Hortus-Puutarha, Kaarina, Finland) to par-tial defoliation and nutrient availability in a field ex-periment. The experiment, established in May 1993with 1-yr-old saplings at the Suonenjoki Research Sta-tion of the Finnish Forest Research Institute, was or-ganized in a randomized complete-block design. The10 clones analyzed for leaf phenolic composition wererandomly chosen from a larger experiment consistingof 15 clones and 10 blocks (Mutikainen et al., in press).Saplings from eight blocks (9 3 24 m2) were used. Thetreatments were: (1) no fertilization 1 no defoliation;(2) no fertilization 1 defoliation; (3) fertilization 1 nodefoliation; and (4) fertilization 1 defoliation.

For the saplings assigned to the fertilization treat-ment, we applied NPK fertilizer (18:5:10, TyppirikasY-lannos, Kemira, Finland) twice during the growingseason in 1993 and once in 1994 before the samplingof the leaves. At the time of the sampling (end of June1994), each sapling had received 9 g of the fertilizer.The amount of fertilizer applied corresponds to 12 kgnitrogen·ha21·yr21, 3.3 kg phosphorus·ha21·yr21, and 6.7kg potassium·ha21·yr21. Defoliation of saplings wasperformed at the end of June 1994 in two phases, firstcutting half of the lamina from every second leaf, andthe next day removing the rest of the leaf, includingthe petiole. Two short-shoot leaves from differentbranches in the upper part of the saplings were col-lected 3 d after the start of defoliation and air-dried.Mature short-shoot leaves were used because the vari-ation in phenolic concentrations among birch short-shoot leaves is considerably smaller than among long-shoot leaves (Keinanen et al. 1998).

Chemical analysis

The two leaves from each sapling were extractedwith methanol and 80% methanol by using an Ultra-Turrax homogenizer (Janke and Kunkel Gmbh, Stau-fen, Germany), as in Keinanen and Julkunen-Tiitto(1996). Phenolic composition of the leaves was ana-lyzed by high-performance liquid chromatography(HPLC). The HPLC system was a Hewlett-Packard HP1050 instrument (Avondale, Pennsylvania, USA) with

a quaternary pump, a vacuum degasser, an autosampler,a thermostatic column oven, and a photodiode arraydetector (HP 1040A) combined with HP Chemstation.A 3-mm HP Hypersil ODS column (60 3 4.6 mm) wasused. The solvents were A (water 1 2% methanol, 1.3%tetrahydrofuran, and 0.25% orthophosphoric acid) andB (acetonitrile). The solvent gradient was as follows:0–5 min, 0–5% of B in A; 5–20 min, 5–14% of B inA; and 20–35 min, 14–40% of B in A. The flow ratewas 2 mL/min and the injection volume was 15 mL.The column oven was set at 308C. The analysis wasmonitored at 220, 280, 320, and 360 nm.

We analyzed a total of 34 compounds by HPLC: 3,49-dihydroxypropiophenone-3-glucoside (DHPPG), (1)-catechin, nine cinnamic acid derivatives, 15 flavonolglycosides, and eight flavone aglycones. The flavoneaglycones (methoxylated apigenin and luteolin deriv-atives) are present on leaf surfaces (Keinanen and Julk-unen-Tiitto 1998). The flavonol glycosides are conju-gates of myricetin, quercetin, and kaempferol. At leastfour of the cinnamic acid derivatives are quinic acidesters: 5-caffeoylquinic acid (neochlorogenic acid),3-caffeoylquinic acid (chlorogenic acid), 5-coumaro-ylquinic acid, and 3-coumaroylquinic acid. Five cin-namic acid derivatives were identified tentatively ac-cording to their UV-Visible spectra. The leaf extractsof clone 26 contained two additional flavonol glyco-sides that coeluted with unknown compounds. Becausethese flavonols were tentatively identified as quercetinglycosides according to their UV-Visible spectra, theirconcentrations were included in the total amount ofquercetin glycosides, total flavonol glycosides, and to-tal nontannin phenolic compounds of clone 26. Thecompounds analyzed represent .90% of the absorptionof all the peaks present in the HPLC chromatogram at280 nm. Identification of the compounds is describedin Keinanen and Julkunen-Tiitto (1998). Flavonol gly-cosides were quantified as equivalents of quercetin3-galactoside, flavone aglycones as apigenin or luteo-lin, cinnamic acid derivatives as chlorogenic acid, andDHPPG as picein. In addition, condensed tannins weredetermined by the vanillin-HCl assay (Julkunen-Tiitto1985).

Statistical analyses

Differences in chemical concentrations betweentreatments were analyzed as a mixed-model random-ized-block ANOVA with Type III sums of squares,including fertilization and defoliation as fixed effectsand clones as a random effect (SPSS-Win 7.5, MAN-OVA procedure, SPSS 1997). The error terms weredetermined according to Zar (1984). The frequency dis-tributions of the concentrations of analyzed compoundswere of the lognormal type, as is generally found forconcentrations of secondary compounds (van der Me-ijden 1996). Due to significant non-normality or het-eroscedasticity, log(x)-transformed data were used inall of the ANOVAs. The Kruskal-Wallis test was used


FIG. 1. UPGMA dendrogram of the Betula pendula clonesby treatment combinations. Treatment abbreviations: F, fer-tilization; f, no fertilization; D, defoliation; d, no defoliation.

to test differences among the clone groups formed byUPGMA in the magnitude of induction of DHPPG andflavonoid aglycones (the concentration difference be-tween defoliated and control sapling within eachblock). Correlations of the magnitude of induction ofDHPPG and flavonoid aglycones with constitutive lev-els of phenolics were tested by using Kendall’s coef-ficient of rank correlation (t).

The Pearson correlation coefficient was used as ameasure of similarity in UPGMA clustering. Cloneshaving qualitative differences were not necessarilyclustered in different groups, because in order to com-pare the similarity of the overall phenolic profiles ofthe clones, the data were standardized to z-scores (zeromean and unit standard deviation) by variables (con-centrations of 34 compounds analyzed by HPLC). Thisremoves the effect of concentration differences be-tween compounds and takes into account the possibilitythat the compounds apparently absent from the clonescould be present in concentrations below the detectionlimit of our analytical method.

Cluster analysis produces clusters whether or not nat-ural groupings exist, and the results depend on boththe similarity measure chosen and the algorithm usedfor clustering (James and McCullogh 1990). The useof Pearson’s correlation coefficient as a similarity mea-sure has been criticized, because two individuals mayhave a correlation of unity even when their profiles ofmeasurements are not parallel (Everitt 1993). All thatis required for perfect correlation is that one set of thescores is linearly related to the second set. The ro-bustness of the result was therefore checked by ana-lyzing the data with or without the compounds havingqualitative differences among clones (flavonol arabi-nofuranosides and glucuronides, 5-caffeoylquinicacid), and without standardization to z scores, and byusing squared Euclidean distance as a similarity mea-sure instead of Pearson’s correlation coefficient. All ofthese analyses resulted in essentially the same resultas in Fig. 1.

RESULTS

Clonal variation

There were significant differences among the clonesin the concentrations of each of the compounds ana-lyzed (Figs. 2–7, Tables 1–4). The concentrations ofcondensed tannins showed twofold differences amongclones (Fig. 2). Although several individual phenoliccompounds showed even larger differences (Figs. 2–7), the amount of total nontannin phenolics varied,20% among clones (Fig. 2). Flavonol arabinofura-nosides, flavonol glucuronides, and 5-caffeoylquinicacid were not detected in all clones. The clones couldthus be classified to groups or chemotypes accordingto the presence or absence of these compounds.

UPGMA clustering of the clone–treatment combi-nations separated the clones to groups of the four treat-

ment combinations (Fig. 1). Except for clones 26 and124, fertilized saplings in each clone were groupedtogether, indicating that fertilization had a larger effecton phenolic profiles of leaves than did defoliation oftheir neighboring leaves. The clones were clusteredloosely in three groups: group 1 (clones 26, 86, 60,124), group 2 (clones 84, 80, 40), and group 3 (clones93, 28, 7). Because the clustering is based on similar-ities of the overall phenolic profiles of nontannin phe-nolics, the separation of the groups cannot be attributedto differences in specific compounds. The only cleardifferences are low concentration of 5-caffeoylquinicacid (Fig. 3) and high concentrations of myricetin gly-cosides (Fig. 4) in group 3, and the absence of flavonolarabinofuranosides (Figs. 4–6) in group 2.

Fertilization

The concentration of condensed tannins was signif-icantly lower in leaves of fertilized saplings (17.0 6


FIG. 2. Mean (11 SE) concentrations of foliar phenoliccompounds in different clones of B. pendula grouped by bio-synthetic origin. Shaded bars represent fertilized saplings;hatched bars represent defoliated saplings; shaded, hatchedbars represent fertilized defoliated saplings; open bars rep-resent controls.

FIG. 3. Mean (11 SE) concentrations of cinnamoylquinicacids in different clones of B. pendula (5-caffeoylquinic acid5 neochlorogenic acid; 3-caffeoylquinic acid 5 chlorogenicacid). Shaded bars represent fertilized saplings; hatched barsrepresent defoliated saplings; shaded, hatched bars representfertilized defoliated saplings; open bars represent controls.

0.72 mg/g dry mass; mean 6 1 SE) than in leaves ofcontrol saplings (32.2 6 1.08 mg/g dry mass). How-ever, fertilization had no significant effect on total non-tannin phenolics analyzed by HPLC (Table 1, Fig. 2).

Individual compounds or groups of compounds com-posing the total nontannin phenolics showed varied re-sponses to fertilization (Table 1, Fig. 2). Concentrationsof flavone aglycones, DHPPG, and (1)-catechin weresignificantly lower in fertilized saplings (Table 1, Fig.2). However, the total concentration of flavonol gly-cosides, the largest group of nontannin phenolics byconcentration, was significantly higher in fertilizedsaplings (Table 1, Fig. 2). By contrast, the amounts offlavonol 3-rhamnosides were significantly lower in fer-tilized saplings (Table 3, Figs. 4–6). Among cinnamicacid derivatives (Table 2, Fig. 3), concentrations of5-caffeoylquinic and 5-coumaroylquinic acids weresignificantly higher in fertilized saplings, whereas theopposite was true for corresponding quinates substi-tuted at 3-position (3-caffeoylquinic and 3-coumaro-ylquinic acids).

Among different biosynthetic groups of compounds,flavonoid aglycones and DHPPG showed significantinteraction between clone and fertilization, and a sim-ilar tendency was shown in total concentration of cin-namic acid derivatives (Table 1). The results of flavonol


FIG. 4. Mean (11 SE) concentrations of myricetin gly-cosides in different clones of B. pendula. Shaded bars rep-resent fertilized saplings; hatched bars represent defoliatedsaplings; open bars represent controls.

glycosides having different aglycones differed in thisrespect. The response of quercetin glycosides to fer-tilization did not differ among the clones. By contrast,the clone 3 fertilization interaction was significant forall myricetin and kaempferol glycosides except myric-etin 3-glucoside (Table 3).

Defoliation

Defoliation of saplings had no significant effect oncondensed tannins, total nontannin phenolics, or totalflavonol glycosides (Table 1). However, defoliated sap-lings had significantly higher concentrations of totalflavone aglycones and DHPPG and significantly lowerconcentrations of cinnamic acid derivatives and(1)-catechin than did control saplings (Table 1, Fig.2). The concentrations of DHPPG and all individualflavone aglycones showed significant interaction be-tween clone and defoliation (Tables 1 and 4). Apigenin49-methyl ether and apigenin derivative 2 showed alsosignificant fertilization 3 defoliation interaction (Table4). In addition, there was a slight tendency for three-way interaction among clone, fertilization, and defo-liation for apigenin and apigenin 49-methyl ether (Table4).

Among the quinates analyzed, concentrations of5-coumaroylquinic acid and 5- and 3-caffeoylquinicacids were significantly lower in defoliated saplings,whereas the concentration of 3-coumaroylquinic acidshowed no significant difference between defoliatedand control saplings (Table 2, Fig. 3). Individual quer-cetin and kaempferol glycosides did not show any sig-nificant responses to defoliation (Table 3, Figs. 5–6).The concentration of myricetin 3-glucoside was sig-nificantly higher in defoliated saplings, whereas my-ricetin 3-rhamnoside showed an opposite tendency. Inaddition, the concentrations of myricetin glycosidesshowed significant interaction between clone and de-foliation (Table 3, Fig. 4).

Differences in the magnitude of chemical inductionof flavone aglycones and DHPPG (i.e., the concentra-tion difference between defoliated and control sap-lings) were tested separately for fertilized and unfer-tilized saplings among the three clone groups formedby the UPGMA clustering (Fig. 1). In unfertilized sap-lings, the concentrations were consistently higher indefoliated saplings (Fig. 2), and the three clone groupsdid not differ in the intensity of induction (DHPPG, P5 0.664; flavone aglycones, P 5 0.453; Kruskal-Wallistest). In fertilized saplings, the response to defoliationwas dependent on the clone group. The groups differedsignificantly in the intensity of induction of DHPPGand flavone aglycones (P 5 0.019 and P 5 0.004,respectively; Kruskal-Wallis test). In addition, we cor-related the intensity of induction of DHPPG and fla-vone aglycones with constitutive levels of phenoliccompounds to test for trade-offs between induced andconstitutive resistance (Karban and Myers 1989). Theinduction of DHPPG and flavonoid aglycones was sig-nificantly and negatively correlated only with the con-centration of myricetin glycosides in fertilized saplings(t 5 20.258, P , 0.001 and t 5 20.256, P , 0.001,for the induction of DHPPG and flavonoid aglycones,respectively).


TABLE 1. ANOVA results for phenolic compounds in Betula pendula leaves grouped by biosynthetic origin.

Factor dfCondensed

tanninsNontanninphenolics HCA

Flavonolglycosides

Flavoneaglycones DHPPG (1)-catechin

Block (B)Clone (C)Fertilization (F)Defoliation (D)C 3 FC 3 DF 3 DC 3 F 3 DError

79119919

268

8.54***12.15***

270.12***1.770.790.681.400.45

7.71***7.28***0.582.050.951.291.080.56

2.69**58.36***

3.1618.73**

1.91†1.040.421.17

11.42***12.40***

9.64*1.440.811.284.65†0.38

3.76**19.65***

9.90*6.37*2.89**2.66**2.251.14

5.55***12.16***20.79**

7.84*2.04*2.10*1.070.76

2.53*28.31***29.01***58.61***

1.410.580.130.78

Notes: The F values are presented for main effects and their interactions, with levels of significance indicated as follows:†P , 0.1, * P , 0.05, ** P , 0.01, *** P , 0.001. Clone 3 Fertilization, Clone 3 Defoliation, and Clone 3 Fertilization3 Defoliation were used as error terms for Fertilization, Defoliation, and Fertilization 3 Defoliation, respectively. HCAcomprises cinnamic acid derivatives, such as cinnamoylquinic acids.

TABLE 2. ANOVA results for cinnamoylquinic acids.

Factor df5-Caffeoyl-quinic acid

3-Caffeoyl-quinic acid

5-Coumaroyl-quinic acid

3-Coumaroyl-quinic acid


79 (8)119 (8)9 (8)19 (8)

268 (241)

4.00***73.67***47.25***13.30**1.152.54*0.101.06

2.09*48.79***

171.19***32.70***

0.381.290.170.76

2.98**468.80***287.78***

6.02*0.320.820.370.94

1.85†39.11***

156.57***0.090.730.410.081.00

Notes: The F values are presented for main effects and their interactions, with levels of significance indicated as follows:†P , 0.1, * P , 0.05, ** P , 0.01, *** P , 0.001. Clone 3 Fertilization, Clone 3 Defoliation, and Clone 3 Fertilization3 Defoliation were used as error terms for Fertilization, Defoliation, and Fertilization 3 Defoliation, respectively. Degreesof freedom for 5-Caffeoylquinic acid are given in parentheses. (Note that 5-caffeoylquinic acid 5 neochlorogenic acid; 3-caffeoylquinic acid 5 chlorogenic acid.)

DISCUSSION

Fertilization

According to the CNB hypothesis, the accumulationof phenolic compounds is determined by the amountof carbon substrate over growth requirements (Bryantet al. 1983, Tuomi et al. 1988). This is consistent withthe view that the supply of phenylalanine may controlphenolic accumulation (Margna 1977, da Cunha 1987,Yao et al. 1995). During periods of rapid growth, phe-nylalanine is preferentially incorporated into proteinsynthesis, but when growth is suppressed, more phe-nylalanine becomes available for phenolic synthesis.Phenolic accumulation may thus be seen as a by-prod-uct of suppressed primary metabolism (Tuomi et al.1988) or the result of ‘‘overflow’’ metabolism (Haslam1993), in which the process of secondary metabolismis emphasized, rather than the secondary products.However, environmental conditions conducive to ac-cumulation of phenolic compounds may also affect theactivity of biochemical machinery used to synthesizethese compounds, e.g., via hormonal control (Water-man and Mole 1989, Fajer et al. 1992). There is strongevidence from molecular biological studies that thepathway of phenolic biosynthesis is highly regulated

and coordinated temporally and spatially to give spe-cific end products in response to different develop-mental or environmental cues, such as UV light,wounding, or infection (Hahlbrock and Scheel 1989,Koes et al. 1994, Dixon and Paiva 1995). Consequently,the significance of variation in phenolic compositionor responses to abiotic or biotic factors cannot be de-termined without better understanding of the regulationof phenolic metabolism. Therefore, we find it necessaryto view our results in light of current knowledge onthe biosynthesis of phenolic compounds (Fig. 8).

Several studies have shown that instead of mere sub-strate control, the key factor in regulation of the phe-nolic pathway is the activity of PAL (Hahlbrock andScheel 1989, Bate et al. 1994). PAL is the first com-mitted enzyme in the pathway leading to biosynthesisof phenolic compounds in higher plants, and, as abranch point enzyme between primary and secondarymetabolism, is a potential site for pathway regulation(Jones 1984, Hahlbrock and Scheel 1989). However,in our study, the group of compounds most stronglyaffected by nutrient availability, condensed tannins, issynthesized 7–8 steps downstream of PAL along thebiosynthetic pathway (Fig. 8). The accumulation of fla-vonoid derivatives, such as condensed tannins (see Fig.


TABLE 3. ANOVA results for flavonol glycosides.

Factors df TotalGalacto-

side Glucoside GlucuronideArabino-side(p)

Arabino-side(f) Rhamnoside

Myricetin-3-O-glycosidesBlock (B)Clone (C)Fertilization (F)Defoliation (D)C 3 FC 3 DF 3 DC 3 F 3 DError

79 (8, 4)119 (8, 4)919 (8, 4)

268 (241, 129)

6.53***126.44***

0.071.322.48*2.05*1.320.40

5.93***101.03***

0.042.492.01*1.97*1.840.33

1.4331.27***

0.2912.99**

1.051.72†0.170.33

9.51***542.01***

1.540.603.33**1.88†1.290.47

4.82***98.87***

3.37†1.553.01**1.260.010.62

4.49**288.92***

0.070.002.94*3.21*0.401.39

4.85***141.48***

16.81**4.55†4.58***0.760.341.23

Quercetin-3-O-glycosidesBlock (B)Clone (C)Fertilization (F)Defoliation (D)C 3 FC 3 DF 3 DC 3 F 3 DError

79 (8, 4)119 (8, 4)9 (8, 4)19 (8, 4)

268 (241, 129)

11.53***41.15***8.66*0.871.391.035.93*0.52

10.43***57.48***14.39**

1.281.221.135.28*0.44

8.48***173.05***

16.12**1.381.351.470.690.40

9.12***1394.86***

5.58*1.451.691.032.930.94

13.32***84.99***49.46***

0.481.281.503.56†0.47

4.47***294.76***

4.98†0.330.781.062.220.80

3.58**64.16***76.39***

0.711.221.610.210.78

Kaempferol-3-O-glycosidesBlock (B)Clone (C)Fertilization (F)Defoliation (D)C 3 FC 3 DF 3 DC 3 F 3 DError

79 (7, 4)119 (7, 4)9 (7, 4)19 (7, 4)

268 (213, 129)

6.33***129.13***

2.010.033.59***0.493.290.19

5.51***74.02***

5.32†0.423.98***0.734.54†0.50

3.45**8.89***0.360.002.90*0.675.20†0.12

3.76**25.59***43.01***

0.082.45*0.640.010.43

Notes: The F values are presented for main effects and their interactions, with levels of significance indicated as follows:†P , 0.1, * P , 0.05, ** P , 0.01, *** P , 0.001. Clone 3 Fertilization, Clone 3 Defoliation, and Clone 3 Fertilization3 Defoliation were used as error terms for Fertilization, Defoliation, and Fertilization 3 Defoliation, respectively. Degreesof freedom for flavonol glucuronidase and arabinofuranosides are given in parentheses, respectively.

TABLE 4. ANOVA results for flavone aglycones.

Factor df Apigenin

Apigenin derivatives

49-OMe Derivative 1 Derivative 2

Luteolin derivatives

Derivative 1 Derivative 2 Derivative 3


79119919

268

6.01***81.92***9.85*5.14*1.201.99*0.721.80†

7.59***67.53***4.10†0.812.33*2.93**5.32*1.68†

3.74**21.17***

1.715.02†3.10**2.70**3.001.16

4.11***78.99***11.50**

2.913.41**2.37*5.61*1.42

2.39*62.88***64.54***

3.68†2.51**3.03**0.421.52

5.20***32.32***35.40***

4.15†2.023.17**0.990.85

1.0227.40***56.16***10.87**

1.74†1.95*0.280.44

Notes: The F values are presented for main effects and their interactions, with levels of significance indicated as follows:†P , 0.1, * P , 0.05, ** P , 0.01, *** P , 0.001. Clone 3 Fertilization, Clone 3 Defoliation, and Clone 3 Fertilization3 Defoliation were used as error terms for Fertilization, Defoliation, and Fertilization 3 Defoliation, respectively.

8), may not be directly controlled by PAL activity, butinstead by the activity of chalcone synthase (CHS), thefirst enzyme of flavonoid metabolism. However, be-cause only 60% of the carbon of chalcone originatesfrom phenylalanine and 40% from malonyl-CoA viathe acetate pathway, changes in synthesis of such mixedmetabolites require a coordinated supply of substratefrom different routes (Waterman and Mole 1989). Thisindicates that, in addition to possible substrate avail-

ability at the level of PAL (Margna 1977), flavonoidaccumulation may also be affected by substrate com-petition with synthesis of other carbon-based metab-olites, such as lipids and terpenoids, via malonyl-CoA(Fig. 8). Such trade-off could partly explain the dif-fering responses to fertilization observed for terpenoidsand phenolic compounds (e.g., Muzika 1993, Lavolaand Julkunen-Tiitto 1994, Kainulainen et al. 1996). Ourresults show that in fertilized saplings, the concentra-


FIG. 5. Mean (11 SE) concentrations of quercetin gly-cosides in different clones of B. pendula. Shaded bars rep-resent fertilized saplings; hatched bars represent defoliatedsaplings; open bars are controls.

FIG. 6. Mean (11 SE) concentrations of kaempferol gly-cosides in different clones of B. pendula. Shaded bars rep-resent fertilized saplings; hatched bars represent defoliatedsaplings; open bars are controls.

tion of condensed tannins was negatively correlated (r5 20.543, P 5 0.050) with the amount of triterpenoidresin droplets measured from the same saplings (Mu-tikainen et al., in press).

At low nutrient availability, the accumulation of con-densed tannins might be preferred over flavonoid gly-

cosides and other phenolic compounds. The results ofseveral studies indicate a tendency for total phenolicsto give weaker responses to fertilization than con-densed tannins (Bryant 1987, Bryant et al. 1987a, b,Glyphis and Puttick 1989, Iason and Hester 1993, Fol-garait and Davidson 1995). Because these results wereobtained by simple color tests, such as Folin-Denisassay, that do not discriminate between tannin and non-tannin phenolics (Mole and Waterman 1987), nontan-nin phenolics may have shown little or no response tofertilization. In this study, .50% of the nontannin phe-nolics of birch leaves consisted of flavonol glycosides,the concentration of which increased by fertilization(Figs. 2, 8).

Although our results provide support for the CNBhypothesis, they also clearly indicate that the accu-mulation of phenolic compounds in birch leaves isstrongly coordinated, possibly by enzymic regulation(Fig. 8). The opposite responses of condensed tanninsand flavonol glycosides indicate that the branchpointat the dihydroflavonol stage (Fig. 8) must be strictlycontrolled. Different branches of the pathway maycompete for substrates, such as dihydroflavonol, andthe enzymes involved might be directly regulated byenvironmental cues, such as nutrient availability. Thus,


FIG. 7. Mean (11 SE) concentrations of flavone aglyconesin different clones of B. pendula. Shaded bars represent fer-tilized saplings; hatched bars represent defoliated saplings;open bars are controls.

FIG. 8. A simplified scheme of the biosynthetic pathwayleading to phenolic compounds in B. pendula leaves. Thearrows next to the analyzed end products indicate significantchanges in concentration following the treatments. Shadedarrows represent fertilization; open arrows represent defoli-ation. Abbreviations: PAL, phenylalanine ammonia-lyase;CHS, chalcone synthase; CHI, chalcone isomerase; FNS, fla-vone synthase; FHT, flavanone 3-hydroxylase; FLS, flavonolsynthase; DFR, dihydroflavonol 4-reductase; LAR, leucoan-thocyanidin 4-reductase.

the accumulation of different types of phenolic com-pounds may be determined by different mechanisms.Bate et al. (1994) found that in transgenic tobaccoplants with differing levels of PAL activity, lignin con-tent is not greatly affected until PAL activity is reduced

to 20–25% of wild-type levels. By contrast, the ac-cumulation of the major soluble phenolic compounds,chlorogenic acid and rutin, a flavonol glycoside, wasaffected even by small changes in PAL activity. More-over, transgenic tobacco plants overexpressing PALwere found to contain increased levels of chlorogenicacid, but not of rutin (Howles et al. 1996). Phenolicpolymers, such as lignin and condensed tannins, maythus be affected more by substrate availability, in ac-cordance with the CNB hypothesis, but the accumu-lation of various phenolics of low molecular mass, suchas flavonol glycosides (Fig. 8), may be regulated ad-ditionally by direct control of enzyme activities. Stableend products with low potential turnover may thus fol-low most closely the predictions of the CNB hypoth-esis, as suggested by Reichardt et al. (1991). The resultsof this study are in accordance with this suggestion,because the compounds with low potential rate of turn-


over, flavone aglycones, which are deposited on leafsurfaces as a nonvolatile resin (Wollenweber 1990,Keinanen and Julkunen-Tiitto 1998), and condensedtannins both decreased in response to fertilization.However, possibly due to internal metabolic trade-off,individual compounds within different biosyntheticgroups showed differences in response to fertilization,such as the opposite tendencies of 5- and 3-cinnamo-ylquinic acids (Fig. 3).

The idea of overflow metabolism (Haslam 1993) isrelated to the notion that the plant must do somethingwith redundant light energy, and phenolics may simplybe one of the few useful products that can be synthe-sized under growth-limiting nutrient supply (Watermanet al. 1984). Conversely, in conditions of better nutrientavailability, the amount of surplus carbon shunted intothe phenolic pathway decreases. However, if light con-ditions do not change, the plant still needs protectionfrom harmful UV radiation, which may damage DNAand impair several physiological processes (Koes et al.1994). Because UV protection is one of the primaryfunctions implicated for flavonoid glycosides in plants(Koes et al. 1994), it is possible that the plant couldnot afford to lower their concentration, even thoughthe total amount of phenolics would decrease at thesame time. This could explain why the amount of fla-vonol glycosides in birch leaves was not lower in fer-tilized saplings, as were the concentrations of almostall other phenolic compounds. Although the total con-centration of flavonol glycosides may be primarily de-termined by light environment (Lavola et al. 1997,1998) and not by nutrient availability, individual com-pounds did not respond consistently to fertilization(Figs. 4–6). The accumulation of flavonol rhamnosidesin birch leaves is apparently controlled differently thanfor other flavonol glycosides. This is in agreement withthe observation that in poinsettia (Euphorbia pulcher-rima) cultivars, the synthesis of quercetin-3-rhamno-side is linked with the synthesis of kaempferol-3-rham-noside, but not with synthesis of quercetin-3-galacto-side (Stewart et al. 1980).

Defoliation

The changes in concentration of both flavone agly-cones and DHPPG due to defoliation and fertilizationwere very similar in each clone (Fig. 2). This suggeststhat the accumulation of flavone aglycones and DHPPGis controlled by some common factor that is responsiveto environmental cues, such as nutrient availability ordefoliation, but it is difficult to rationalize a physio-logical mechanism for such a tight linkage. AlthoughDHPPG is a phenylpropanoid derivative and, as such,is probably synthesized from cinnamates, its synthesisis separated by several steps from flavone synthesis(Fig. 8). Moreover, the end products are accumulatedin different compartments. DHPPG is a vacuolar com-pound, but the flavone aglycones are present on leafsurfaces (Keinanen and Julkunen-Tiitto 1998). How-

ever, the induction of DHPPG and flavone aglyconescoincides with significant decrease in concentration ofcinnamic acid derivatives, especially that of caffeo-ylquinic acids. Synthesis of DHPPG or flavone agly-cones from cinnamic acid derivatives may be fasterthan to build them from phenylalanine (Fig. 8). Thisis consistent with the view that chlorogenic acid (3-caf-feoylquinic acid) may function as a reservoir for thesynthesis of other phenolics when phenylpropanoidmetabolism is activated (Friend 1981, Yao et al. 1995).Furthermore, Haslam (1993) has suggested that quinicacid may be formed as a result of overflow metabolismof the shikimate pathway. Storing surplus carbon ascinnamoylquinates would thus combine shunt metab-olites of two distinct segments of the plant aromaticpathway from carbohydrates to phenolics (Fig. 8).These reserves would be better defended against her-bivory than the ordinary storage carbohydrates (Ger-shenzon 1984), and may thus allow rapid responses toenvironmental stimuli.

Increases in the concentration of DHPPG and flavoneaglycones induced by defoliation were about as largeas differences in constitutive levels among clones(,50%). In bioassays conducted with leaves from thesame field experiment (Mutikainen et al., in press), theperformance of Epirrita autumnata larvae was not sig-nificantly negatively correlated with the concentrationsof DHPPG and flavone aglycones, but in fertilized sap-lings, the intensity of DHPPG induction was positivelycorrelated with the induced reduction in relative growthrate of the larvae. However, our experiment may havebeen conservative. We used mature short-shoot leaves,which usually do not show much plasticity to environ-mental conditions (Coleman and Jones 1991). The in-duced response in Betula pendula leaves has beenshown to be strongest early in the season (Edwards etal. 1991, Hartley and Lawton 1991, Neuvonen andHaukioja 1991). Because our defoliation treatment wasconducted at the end of June, the intensity of responsemay have already declined. Furthermore, herbivorefeeding may cause a stronger chemical response thanthe artificial defoliation we used, because there couldbe specific elicitors present in the regurgitant of a feed-ing caterpillar (Korth and Dixon 1997), or because theherbivore may contaminate the plants with pathogens(Hartley and Firn 1989).

Attack by gypsy moth (Lymantria dispar) larvae hasbeen shown to increase the amount of DHPPG in leavesof Betula platyphylla var. japonica (Mori et al. 1992).DHPPG was a weak antifeedant against fourth-instarlarvae of the gypsy moth at the concentration of 40 000mg/g (4% aqueous solution), but it repelled first-instarlarvae at 5000 mg/g (Mori et al. 1992). The flavoneaglycones on leaf surfaces of B. pendula are possiblyexcreted with terpenoids by glandular structures mainlyon the petiole and the midrib of young leaves (Wol-lenweber 1990, Lapinjoki et al. 1991). Morphology ofthe leaf glands is similar to that of young shoots (Lap-


injoki et al. 1991), which excrete triterpenoids such aspapyriferic acid, a highly deterrent defense chemicalfor mammalian herbivores (Reichardt et al. 1984, Tah-vanainen et al. 1991). If the flavone aglycones or ter-penoids excreted by these glands have harmful effectson insect herbivores (see Mutikainen et al., in press),deterioration of the glands during leaf development(Lapinjoki et al. 1991) might explain the declining in-tensity of induced response reported from insect bio-assays (Edwards et al. 1991, Hartley and Lawton 1991,Neuvonen and Haukioja 1991).

Interestingly, Gunthardt-Goerg et al. (1993) reportedthat ozone stress increases the density of hairs andpeltate scales (glands described by Lapinjoki et al.1991) on developing leaves of B. pendula. This re-sponse may thus be reflected in chemical constituentsof resin on leaf surfaces, analogously to induction bydefoliation shown in this study. Many plant secondarycompounds that have been implicated in defenseagainst herbivores or pathogens may well have a largevariety of other functions, such as protection againstharmful UV radiation, mediation of allelopathy, or re-duction of transpiration (Rhoades 1977, Wollenweber1990, Herms and Mattson 1992, Gershenzon 1994).Hartley and Lawton (1991) have proposed that the pri-mary role of damage-induced changes in birch leavesmay be antifungal or antimicrobial, rather than directedprimarily at insect herbivores. Herbivores might evenbenefit from this kind of induction, because it couldact against their own pathogenic organisms (Hunter andSchultz 1993). The flavone aglycones induced in thisstudy are relatively lipophilic, which is typical formany antifungal compounds in plants (Grayer and Har-borne 1994).

Because the feeding by herbivores results in wound-ing, and the wounds not only permit entry of pathogens,but also loss of water (Baldwin 1994), an induced re-sponse acting against any one of these stresses mayprovide selective advantage to the plant. Leaf resinscontaining flavonoid aglycones and terpenoids usuallyoccur in plants of arid or semiarid habitats (Wollen-weber 1990). Moreover, water-stressed creosote bushes(Larrea tridentata) produce more phenolic resins onleaf surfaces than do nonstressed plants (Waring andPrice 1990). This indicates that water stress may triggerthe induction of resin production. Nitrogen stress, inturn, causes a decline in hydraulic conductance of theroots and an associated decline in stomatal conduc-tance, and therefore transpirational water loss (Chapin1991). Because stomata also serve as a pathway forCO2 diffusion, decreasing water loss by closing stomatapotentially limits photosynthesis (Chapin et al. 1987).Thus, the tendency for stronger induction of flavoneaglycones under low nutrient availability (Fig. 2, Table4) may be connected to water shortage, because it mayact as a protective mechanism against water loss byepicuticular transpiration, and may alleviate the declineof CO2 diffusion.

Clonal variation

The clear separation of the clones in UPGMA clas-sification (Fig. 1) indicates that the accumulation pat-tern of nontannin phenolic compounds in birch leaveswas affected more by genotype than nutrient avail-ability or defoliation treatment. The accumulation pat-terns of the glycosides of the three flavonols (myricetin,quercetin, and kaempferol) closely resembled each oth-er. The only flavonol glycosides showing qualitativevariation among clones were flavonol arabinofurano-sides and glucuronides (Figs. 4–6). Moreover, theclones showed the presence or absence of glycosidesof each sugar moiety with all of the three flavonols.This suggests that the enzymes transferring the sugarsto the aglycones are relatively specific to the sugardonor (e.g., UDP-glucose), but may accept all threeflavonols as substrates, as has been suggested to occurin needles of Norway spruce (Heilemann and Strack1991). Thus, the enzyme transferring arabinose in fu-ranose form or glucuronic acid to the flavonols may bemissing or not functioning in all clones. Kaempferol-3-glucuronide was not detected from saplings of clone84, but it could be present in small concentration belowour detection limit, because the clone accumulatedsmall amounts of myricetin-3-glucuronide and quer-cetin-3-glucuronide. Apigenin and luteolin derivatives,and quinic acid esters of caffeic and p-coumaric acidsshowed also similarity in accumulation pattern amongclones. The absence of 5-caffeoylquinic acid fromclone 93 could thus be due to the detection limit of ouranalytical method, because 5-coumaroylquinic acidwas present in low concentration.

Genetic variation in compositional profiles of sec-ondary chemicals may reduce the incidence of attackof insect herbivores adapted to the average profile ofa plant population (Langenheim 1994). Changes incompositional profile that do not alter resource allo-cation between growth and secondary metabolism mayhave no cost to the plant in the form of reduced com-petitive ability (Herms and Mattson 1992), except forpossible differences in costs of biosynthetic machinery(Gershenzon 1994). Variation in relative proportions ofnontannin phenolics among birch clones may thus rep-resent an alternative to quantitative increases in chem-ical defense. Consequently, herbivorous insects orpathogens may select for or against particular phenoliccomposition in birch leaves. The performance of Epir-rita autumnata has been shown to vary among Betulapubescens individuals (Ayres et al. 1987) and amongclones of B. pendula (Mutikainen et al., in press). Fur-thermore, B. pendula clones have been shown to varyin resistance against birch rust, Melampsoridium be-tulinum (Poteri and Rousi 1996).

The significant differences among clone groups inthe magnitude of induced response in fertilized saplingsmay indicate the existence of birch chemotypes whosechemical responses partly depend on resource avail-


FIG. 9. Chemical structures of B. pendula leaf flavonolsin order of increasing B-ring hydroxylation. Kaempferol, R5 R9 5 H; quercetin, R 5 OH, R9 5 H; myricetin, R 5 R95 OH. B indicates the position of the corresponding ring inthe flavonoid C15-skeleton.

ability. Moreover, significant interactions betweenclone and fertilization for several compounds, includ-ing myricetin and kaempferol glycosides (Table 3),DHPPG (Table 1), and flavone aglycones (Tables 1 and4), might indicate differentiation of birch genotypes inparticular nutrient regimes, as has been suggested forwillow genotypes (Hakulinen et al. 1995).

Implications for chemical defense

If the chemical defense of plants is assumed to becostly, plants that are well defended by constitutivedefense against a particular herbivore should not al-locate resources to induced defenses against the sameherbivore (Karban and Myers 1989). In this study, theinduction of DHPPG and flavone aglycones by defo-liation was significantly negatively correlated only withmyricetin glycosides, and not with condensed tannins,which are usually implicated in plant chemical defense(e.g., Bryant et al. 1993). However, in addition to con-densed tannins, bioassays conducted with birches fromthe same field experiment also showed significant neg-ative correlations between the concentration of myric-etin glycosides and the pupal mass of Epirrita autum-nata in fertilized saplings (Mutikainen et al., in press).

The chemical properties of myricetin provide supportfor a defensive role for myricetin derivatives in plants.In general, the degree of reactivity of a phenolic com-pound increases with the addition of hydroxyl groupsto the benzene nucleus. Among flavonoids (Fig. 9),chemical activity is often proportional to the numberof adjacent hydroxyl substituents in the B-ring (Elligeret al. 1980, Laks 1989). Thus, flavonoids that have B-rings derived from catechol or pyrogallol (two andthree adjacent hydroxyls, respectively) are potentiallymost active. Myricetin, with a pyrogallol B-ring (Fig.9), was the most active of the 33 flavonoid aglyconestested for antigrowth activity against Heliothis zea lar-vae (Elliger et al. 1980). Myricetin is also prone toautooxidation, which results in the generation of cy-totoxic oxygen radicals (Hodnick et al. 1986). Myric-etin autooxidizes substantially faster and at lower pH(7.5) than quercetin; under the same conditions, kaemp-ferol does not autooxidize at all (Canada et al. 1990).

Because the rate of autooxidation is markedly elevatedat higher pH (Hodnick et al. 1986), myricetin deriva-tives in birch leaves might function most effectivelyas a defense at alkaline conditions. High alkalinity ofthe midguts of many lepidopteran larvae has been sug-gested to be a counteradaptation to plant tannins, be-cause many tannin–protein complexes may dissolveunder alkaline conditions (Berenbaum 1980). Becausethe same conditions facilitate autooxidation of phenoliccompounds (e.g., Appel 1993), tannins and nontanninphenolics, such as myricetin derivatives, may be seenas alternative or complementary defenses against her-bivorous insects, and the mode and efficiency of de-fense could thus depend on pH and redox potential oflarval midguts, and on the ability of the phenolic com-pound to produce reactive oxygen species. Rapid gen-eration of reactive oxygen species is one of the firstresponses to invasion of pathogens in plants (Mehdy1994), and it has been suggested that oxidative re-sponse also occurs following attack by herbivores (Ah-mad 1992, Appel 1993, Bi and Felton 1995).

According to the CNB hypothesis, plants in low-resource environments are selected for resistanceagainst herbivory rather than regrowth, and increasedlevels of carbon-based secondary metabolites, such ascondensed tannins, are considered primarily as chem-ical defenses (Bryant et al. 1983, Coley et al. 1985).The results of this study show that, among phenoliccompounds in birch leaves, condensed tannins respondmost strongly to fertilization. There is evidence thatcondensed tannins are the most important chemicalcause of delayed inducible resistance (DIR) in Alaskapaper birch (Bryant et al. 1993). However, Ruohomakiet al. (1996) showed that defoliation of mountain birchcaused significant DIR, irrespective of nitrogen fertil-ization, and concomitant decrease in concentrations ofcondensed tannins and total phenolics. They concludedthat several different mechanisms may simultaneouslycontribute to the constitutive defense and DIR of moun-tain birch. The results of the present study indicate thatvariation in response to environmental cues among sev-eral phenolic compounds may partly explain this dis-crepancy. Although condensed tannins and several non-tannin phenolic compounds followed the predictions ofthe CNB hypothesis, potential defensive compoundspresent in high concentrations, such as flavonol gly-cosides, showed opposite responses to fertilization.

Although the accumulation of condensed tannins atlow nutrient availability may also be seen as a resultof undirected overflow metabolism (Haslam 1993),Northup et al. (1995) have suggested an alternative,adaptive explanation. Concentration of polyphenols,such as condensed tannins, of decomposing Pinus mur-icata needle litter was shown to control the proportionof nitrogen released in dissolved organic form (DON)relative to mineral forms (NH4, NO3). This feedbackmechanism may facilitate nitrogen recovery throughthe pine–mycorrhizal association minimizing nitrogen


availability to competing organisms, and may reducenitrogen losses from leaching and denitrification. Fur-thermore, Ayres et al. (1997) have argued that selectivepressures from folivorous insects may not be the mainexplanation for the diversion of so much carbon intothe synthesis of condensed tannins, in so many plantspecies. Thus, although condensed tannins may func-tion as defense against insect herbivores in birch leaves(Bryant et al. 1993; Mutikainen et al., in press), theirincreased accumulation under low nutrient availability(e.g., Bryant et al. 1993, Ruohomaki et al. 1996, thisstudy) may also reflect an adaptation to nitrogen lim-itation through increased nitrogen recovery from leaflitter (Northup et al. 1995).

In conclusion, our results show that although fertil-ization decreases the total level of phenolic compoundsin birch leaves, as predicted by the CNB hypothesis,different groups of compounds may show opposite re-sponses. These responses may be due to substrate lim-itation because of trade-off between different branchesof the biosynthetic pathway (Berenbaum and Zangerl1988). The results thus support the view that any the-oretical framework built to predict levels of secondarycompounds should incorporate the balance between theintrinsic demands of the plant and the actual acquisitionof resources (Iason et al. 1993, Lerdau et al. 1994).Demand-side processes complement source-drivenmodels, such as the CNB hypothesis, by exerting anadditional control over the flow of carbon into differ-entiated products (Lerdau et al. 1994). This is consis-tent with the suggestion that the carbon/nutrient bal-ance mainly affects the amount of substrate availableto the synthesis of secondary metabolites, but the dis-tribution of carbon to specific substances may dependon the selective importance to the plant (Tuomi et al.1988), and that specific defensive responses are pos-sibly enzymatically regulated (Tuomi et al. 1991). Al-though these workers discuss the responses in relationto herbivory, any demand-side process, such as otherbiotic stresses, or protection against UV radiation, mayselect for differential accumulation of secondary me-tabolites (Lavola et al. 1997). In the present study,defoliation induced an increase in concentration ofDHPPG and flavone aglycones and a concomitant de-crease in concentration of cinnamic acid derivativesand (1)-catechin. This underscores the importance ofaccomplishing studies on plant–herbivore interactionwith high enough chemical resolution, because themagnitude of herbivore response to changes in plantchemistry will depend on how compounds with specificimportance to different herbivores are affected (Rousiet al. 1993, 1997, Kinney et al. 1997). Furthermore,our results emphasize the significance of genetic dif-ferences in the phenolic chemistry of birch leaves. Thebirch clones showed substantial variation in phenoliccomposition of the leaves and in chemical responsesto fertilization and defoliation.

ACKNOWLEDGMENTS

John Bryant, Erkki Haukioja, Anu Lavola, Jorma Tahvan-ainen, and two anonymous referees provided valuable com-ments that helped to improve the manuscript. We also thankthe technical staff of the Suonenjoki Research Station, es-pecially Maija Piitulainen, for running the field experiment,and Sinikka Sorsa for skillful assistance in the laboratory.The study was financed by the Ministry of Agriculture andForestry, Academy of Finland, and Finnish Cultural Foun-dation.

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