seasonal variation in the content of the hydrolizable tannin

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Journal of Chemical Ecology, Vol. 30, No. 9, September 2004 (C 2004)

SEASONAL VARIATION IN THE CONTENT OF

HYDROLYZABLE TANNINS, FLAVONOID GLYCOSIDES,

AND PROANTHOCYANIDINS IN OAK LEAVES

JUHA-PEKKA SALMINEN,1 TOMAS ROSLIN,2, MAARIT KARONEN,1

JARI SINKKONEN,3 KALEVI PIHLAJA,1,3 and PERTTI PULKKINEN4

1Laboratory of Environmental ChemistryDepartment of Chemistry, University of Turku

FI-20014 Turku, Finland2Metapopulation Research Group

Department of Biological and Environmental SciencesP.O. Box 65 (Viikinkaari 1), University of Helsinki

FI-00014 Helsinki, Finland3Structural Chemistry Group, Department of Chemistry

University of Turku, FI-20014 Turku, Finland4

Finnish Forest Research InstituteHaapastensyrja Breeding StationKarkkilantie 247, FI-12600 Layliainen, Finland

(Received August 26, 2003; accepted May 23, 2004)

AbstractOaks have been one of the classic model systems in elucidating

the role of polyphenols in plantherbivore interactions. This study provides a

comprehensive description of seasonal variation in the phenolic content of the

English oak (Quercus robur). Seven different trees were followed over the full

course of the growing season, and their foliage repeatedly sampled for gallic

acid, 9 individual hydrolyzable tannins, and 14 flavonoid glycosides, as well as

for total phenolics, total proanthocyanidins, carbon, and nitrogen. A rare dimeric

ellagitannin, cocciferin D2, was detected for the first time in leaves ofQ. robur,

and relationships between the chemical structures of individual tannins were

used to propose a biosynthetic pathway for its formation. Overall, hydrolyzable

tannins were the dominant phenolic group in leaves of all ages. Nevertheless,

young oak leaves were much richer in hydrolyzable tannins and flavonoid gly-

cosides than old leaves, whereas the opposite pattern was observed for proan-

thocyanidins. However, when quantified as individual compounds, hydrolyzable

tannins and flavonoid glycosides showed highly variable seasonal patterns. This

large variation in temporal trends among compounds, and a generally weak

correlation between the concentration of any individual compound and the total

To whom correspondence should be addressed. E-mail: [email protected]

1693

0098-0331/04/0900-1693/0 C 2004 Springer Science+Business Media, Inc.


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1694 SALMINEN ET AL.

concentration of phenolics, as quantified by the FolinCiocalteau method, leads

us to caution against the uncritical use of summary quantifications of composite

phenolic fractions in ecological studies.

KeyWordsQuercus robur, hydrolyzable tannins, ellagitannins, flavonoid gly-cosides, proanthocyanidins, HPLC, quantification of phenolics, biosyntheticpathways, compound-specific patterns.

INTRODUCTION

Oaks in the genus Quercus have long been a popular target for ecologists studying

the chemical interplay between plants and herbivorous insects (e.g., Feeny, 1970;

Faeth, 1986; Rossiter et al., 1988; Tikkanen and Julkunen-Tiitto, 2003). One ofthe earliest and still most influential ecological explorations of oak leaf chemistry

was conducted by Feeny (1970). In his seminal paper, he related the phenology of

an oak-feeding moth (Operopthera brumata) to seasonal variation in the chemical

contents of oak leaves. By quantifying relatively crude fractions of phenolic com-

pounds, he inferred that the nutritional value of oak leaves declines throughout

the summer, and that this may be the ultimate factor causing spring-feeding in

O. brumata.

Phenolics may well play a central part in the oaks defense against its her-

bivores (e.g., Schultz and Baldwin, 1982; Rossiter et al., 1988). However, pastphytochemical and experimental studies leave no doubt that individual phenolic

compounds vary substantially with respect to biological activity (Zucker, 1983;

Ozawa et al., 1987; Clausen et al., 1990; Ayres et al., 1997; Feldman et al., 1999;

Kilkowski and Gross, 1999; Kraus et al., 2003) and that even chemically closely

related compounds may encounter a different fate in the digestive tract of an insect

(e.g., Salminen and Lempa, 2002). Hence, to understand the interplay between

the oak and its herbivores, it seems preferable to measure the concentrations of

individual compounds. The quantification of composite phenolic fractions will

only make sense if (a) concentrations of individual phenolics vary in concert and(b) the quantified compounds have matching biological activitiespresumably a

rare combination in real organisms.

So far, a relatively large number of compound-specific studies have inves-

tigated the hydrolyzable tannin composition of oak wood (e.g., Masson et al.,

1994; Viriot et al., 1994; Conde et al., 1998; Mosedale et al., 1998; Fern andez de

Simon et al., 1999; Mammela et al., 2000; Cadaha et al., 2001). This interest has

primarily been spurred by the needs of the wine industry, as most wine barrels are

made of English oak (Quercus robur) or sessile oak (Q. petraea). Of the named

hydrolyzable tannins, the heartwood ofQ. robur has been found to contain casta-lagin, vescalagin, grandinin, and roburins AE (Herve du Penhoat et al., 1991a,b;

Masson et al., 1994; Vivas et al., 1995).


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SEASONAL VARIATION IN OAK LEAF CONTENTS 1695

Substantially less attention has been aimed at the phenolic composition of

oak leaves. A handful of earlier studies have shown the foliage of Q. robur to

contain pedunculagin, castalagin, vescalagin, casuarictin, and flavonol glycosides

(Scalbert and Haslam, 1987; Scalbert et al., 1988; Grundhofer et al., 2001). How-ever, this information has only rarely been utilized in ecological studies that try to

relate the polyphenolic composition of oak leaves to the performance of folivorous

insects. Most often, only total phenolics or the total concentrations of summary

tannin groups have been quantified (e.g., Feeny, 1970; Faeth, 1986; Rossiter et al.,

1988; Lill and Marquis, 2001; Abrahamson et al., 2003; Forkner et al., 2004; but

see Tikkanen and Julkunen-Tiitto, 2003), and how well such composite measures

will reflect variation at the level of individual compounds has remained an open

question.

In this paper, we provide the first comprehensive description of seasonalvariation in the phenolic contents of oak leaves as quantified at the level of

both individual compounds and two summary groupstotal phenolics and total

proanthocyanidins. For the first time, we also report the presence of a rare dimeric

ellagitannin, cocciferin D2, in the foliage of Q. robur and propose a biosynthetic

pathway for its formation. On the basis of seasonal patterns observed in individual

compounds, we ask: (1) To what extent can seasonal variation in the concentrations

of individual compounds be described by a single summary measure such as the

total concentration of phenolic compounds? (2) How well is variation among

compounds explained by biochemical connectionswill the knowledge of centralmetabolic pathways help us understand temporal changes in concentrations of

individual compounds? (3) What does variation in phenolic content and variation

in the total content of carbon and nitrogen reveal about changes in the nutritional

value of oak leaves over the season?

METHODS AND MATERIALS

Study Object. The deciduous English oak is native to Europe and Western

Asia (Jalas and Suominen, 1976). In Finland, its natural distribution is limitedto the southernmost part of the country, where new oak leaves are produced

in late May and early June (Hoffman and Lyr, 1973; Niemela and Haukioja,

1982). Although the period of active leaf production is relatively short and well-

synchronized in oak as compared to other tree species (Niemela and Haukioja,

1982), its onset may vary by several weeks between both years and trees (cf.

Crawley and Akhteruzzaman, 1988). Within trees, leaf production is apparently

more synchronized on short shoots than on actively growing long shoots (Erkki

Haukioja, personal communication, 2003). As a result, leaves on short shoots will

be of similar age throughout the summer; only in some oak individuals is therea second growth period in August, resulting in so-called lammas shoots with

new leaves. All leaves typically senesce in October, but variation among trees and


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years is again substantial. While most leaves are shed in the autumn, some trees

retain a high proportion of dead leaves until the following spring.

Study Site. In the spring of 2001, seven oaks were selected within a small

(about 0.1 ha) stand of 21 oaks growing at the Haapastensyrja Tree BreedingCenter (60 36 N, 24 26 E), run by the Finnish Forest Research Institute. These

oaks had been planted in 1978 and were now approximately 78 m in height. Each

tree was originally created by grafting a small oak twig to a sapling reared from a

randomly collected acorn. Any twigs produced by the original sapling were later

pruned, and hence the current canopy of the oak consists of the grafted genotype

only. The grafted twigs had been collected in oak stands across the full Finnish

range of the English oak.

Leaf Sampling. Leaf samples were collected from each of the seven oaks

on 11 dates throughout the summer of 2001. The sampling dates ranged fromMay 29, which was the 1st d when each tree had leaves larger than 1 cm, to

September 26, when the leaves were already senescing. Individual sampling dates

were May 29, June 7, June 15, June 24, July 7, July 16, July 27, August 16,

August 31, September 13, and 26. All samples were collected between 8 and

12 A.M. A similar number of leaves (730, depending on the date and leaf size)

were randomly collected from the lower branches of each tree. To reduce variation

among leaves within a sample, we specifically avoided the hard and waxy sun

leaves of the outer canopy (cf. Feeny, 1970) as well as leaves on actively growing

long shoots. Leaves were sealed in polyethylene bags in the field and placed intoa cooler with ice. Upon return to the laboratory, they were air-dried for 3 d at

room temperature in a ventilated fume cupboard, and samples were weighed and

subsequently stored at 18C until ground into a fine powder before extraction.

Although air-drying may not be the optimal drying method, it is known to alter the

levels of hydrolyzable tannins and flavonoid glycosides only minimally in birch

leaves (Salminen, 2003, unpublished data) or oak leaves (Salminen, unpublished

pilot study) as compared to levels observed in freeze-dried samples. For each

sample, the average biomass per leaf was calculated by dividing the total weight

of the sample by the number of leaves it contained.Extraction. Dried and ground oak leaves (200 mg per sample) were extracted

four times (4 1 hr) with 70% aqueous acetone (4 8 ml) on a planary shaker.

The freeze-dried aqueous phase of the extract was dissolved in water (3 2 ml);

the supernatant of the centrifuged (10 min at 2000 g) sample was filtered through

a 0.45-m PTFE filter and kept frozen at 20C until analyzed with HPLC-DAD

or HPLCESI-MS.

Analysis of Phenolics with HPLC-DAD and HPLCESI-MS. HPLC-DAD

analysis of oak leaf extracts was performed at 280 and 349 nm with Merck-

Hitachis LaChrom HPLC system (Merck-Hitachi, Tokyo, Japan). Column andchromatographic conditions were as described earlier (Salminen et al., 1999),


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except that 0.1 M H3PO4 was replaced with 0.05 M H3PO4. Phenolic compounds

were quantified using pedunculagin, 1-O-galloylglucose, gallic acid, quercetin,

and kaempferol as external standards.

HPLCESI-MS analysis was performed using a Perkin-Elmer Sciex API365 triple quadrupole mass spectrometer (Sciex, Toronto, Canada) equipped with

an ion-spray interface. The HPLC system consisted of two Perkin-Elmer Series

200 micro pumps (Perkin-Elmer, Norwalk, CT, USA) connected to a Series 200

autosampler. The column used and chromatographic and ESI-MS conditions were

as described previously (Salminen et al., 1999).

Isolation and Identification of Cocciferin D2. Cocciferin D2 (120 mg) was

isolated from oak leaves with a combination of Sephadex LH-20 (40 2.5 cm i.d.)

and Merck LiChroprep RP-18 (44 3.7 cm i.d., 4063 m) columns following

the methods outlined by Salminen et al. (1999, 2001). Part of the pure isolate waspartially hydrolyzed in mild conditions (40C water, 1 hr) and the reaction products

analyzed by HPLC-DAD and HPLCESI-MS. The NMR spectra of Cocciferin D2were acquired using a JEOL JNM-A-500 spectrometer operating at 500.16 MHz

for1H and 125.78 MHz for13C. Spectra were recorded at 25C using acetone-d6 as

a solvent. In addition to standard proton and carbon spectra, DEPT, DQF-COSY,

HMQC, and HMBC spectra were measured.

Analyses of Total Phenolics, Proanthocyanidins, Carbon, and Nitrogen. The

total phenolic content of the extracts was determined by a modification of the

FolinCiocalteau method (Nurmi et al., 1996), using a Perkin-Elmer Lambda 12UVVIS spectrometer (Norwalk, CT, USA). Three replicates of each sample were

analyzed and their average was used as the final reading. A standard curve was

prepared on the basis of known concentrations of gallic acid. The total content of

proanthocyanidins was measured with the butanol-HCl assay as in Ossipova et al.

(2001). Again, measurements were based on the average reading of three replicate

samples, and a standard curve prepared on the basis of known concentrations

of purified birch leaf proanthocyanidins. The total concentration of carbon and

nitrogen in the leaves was performed with a Perkin-Elmer Series II CHNS/O

Analyzer 2400 (Norwalk, CT, USA). A subset of six samples was included inthis analysis, corresponding to sampling dates May 29, June 7, June 15, July 7,

August 16, and September 26.

Data Analysis. Seasonal changes in leaf chemistry were described by vi-

sual plots of compound-specific patterns. To further evaluate the extent to which

the concentrations of different compounds vary in unison among sampling dates,

we calculated simple Spearman rank correlation coefficients (rS). All trees were

first ranked within compounds and sampling dates, from the tree richest in this

particular compound to the tree with the lowest concentration. Then, two types

of comparisons were made on the basis of samples taken on the date with thehighest phenolic readings (May 29). First, to illustrate the concordance between


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the concentration of an individual compound and the pooled concentration of

larger phenolic groups, correlation coefficients were calculated for the rank of the

compound in question and the rank of the pooled concentrations of all individual

compounds as quantified by HPLC. Second, to depict the consistency between theconcentration of an individual compound and the total concentration of phenolics

as quantified by a summary method, correlation coefficients were calculated for a

trees rank for the compound in question and its rank for total phenolics as quanti-

fied by the FolinCiocalteau method. From an applied perspective, what we ask is

specifically: if we know the precise concentrations of individual compounds, will

patterns at the level of total phenolics summarize patterns at the level of individual

compounds?

RESULTS

Characterization of Phenolic Compounds. Twenty-four phenolic compounds

were detected in the HPLC-DAD analyses. From their UV spectra, two were pre-

liminarily identified as gallic acid or its derivatives (compounds 12; Salminen

et al., 1999), and eight as ellagitannins (310; Salminen et al., 1999). The rest of

the compounds were classified on the basis of both UV and mass spectral charac-

teristics as quercetin (1119; Ossipov et al., 1995, 1996) or kaempferol glycosides

(2024; Ossipov et al., 1995, 1996).Since tannins are generally supposed to play a more important role in plant

herbivore interactions than flavonoid glycosides, we focused more on the identi-

fication of compounds 110 than on 1124. On the basis of their retention times

(Rts; compared to those given in the literature) and molecular masses recorded

from a negative ion HPLCESI-MS run, the structures of15 were identified as

gallic acid (1, 6.7 min, 170 g/mol), 1-O-galloylglucose (2, 6.4 min, 332 g/mol),

tellimagrandin II (3, 19.2 min, 938 g/mol), casuarictin (4, 18.0 min, 936 g/mol),

and pedunculagin (5, 9.4 and 11.7 min, anomeric mixture, 784 g/mol; Salminen

et al., 2001). Ellagitannin 6 (14.3 min, 936 g/mol) could be either stachyurinor casuarinin; only one of these isomers was detected in the Q. robur extract,

thus making it impossible to utilize the Rt data of Okuda et al. (1982) for dif-

ferentiating between the two. In contrast, the other two isomeric ellagitannins,

i.e., 7 (8.4 min, 934 g/mol) and 8 (10.2 min, 934 g/mol), were clearly detected

and identified as vescalagin and castalagin, respectively (Fernandez de Simon

et al., 1999; Zhentian et al., 1999). At this point, the structures of two ellagi-

tannins, i.e., 9 (7.6 min, 1102 g/mol) and 10 (15.2 min, 1868 g/mol), remained

unresolved.

The mild hydrolysis of10 yielded 5 and an unidentified ellagitannin with amolecular mass of 1084 g/mol; thus, 10 was shown to be a dimeric ellagitannin


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with 5 as one of the monomeric constituents. The whole chemical structure of

10 was unravelled by a combination of NMR experiments. All proton and carbon

chemical shifts, as well as the protonproton coupling constants, matched perfectly

with those reported for cocciferin D2, an ellagitannin recently found in the leavesof Q. coccifera and Q. suber (Ito et al., 2002). Therefore, 10 was identified as

cocciferin D2, now detected for the first time in leaves of Q. robur. This dimeric

ellagitannin consists of two monomeric units, i.e., 5 and castavaloninic acid. The

latter of these compounds has a molecular mass of 1102 g/mol, corresponding to

that of monomeric ellagitannin 9. Interestingly, according to Yoshida et al. (1992),

the valoneoyl group of castavaloninic acid may undergo lactonization (loss of

water) upon hydrolysis resulting in a depsidone molecule having a molecular

mass of 1084 g/mol (see also hydrolysis of 10 above). This is exactly the same

as that of a significant fragment of9 produced in negative ion HPLCESI-MS,which is known to fragment hydrolyzable tannins in a manner similar to chemical

hydrolysis (cf. Salminen et al., 1999, 2001; Salminen, 2002). For these reasons, 9

was identified as castavaloninic acid.

A Biosynthetic Pathway for the Hydrolyzable Tannins of Oak Leaves. By

examining the relationships among the chemical structures of individual tannins

identified in this study, we propose a biosynthetic pathway for their formation. It is

well accepted (as reviewed by Gross, 1999) that the first compound in the general

hydrolyzable tannin pathway, 2, is formed from 1 and UDP-glucose, and that the

galloylations then continue consecutively and position-specifically to finally yield1,2,3,4,6-penta-O-galloylglucose. On the other hand, the formation of the first

ellagitannin of the pathway, 3, directly from pentagalloylglucose was proven only

recently (Niemetz et al., 2001). The subsequent biosynthetic steps from 3 onwards

have not been experimentally proven, but it is generally assumed that e.g., 4 is

a product of further oxidative coupling of two spatially adjacent galloyl groups

of 3 (see, e.g., Helm et al., 1999). Furthermore, it has been suggested that the

C-glycosidic ellagitannins 7 and 8 are formed from 5, and that this step contains at

least stachyurin and casuarinin (6) as intermediates (see also Okuda et al., 1982;

Hatano et al., 1986; Haslam, 1992; Vivas et al., 1995; Helm et al., 1999). We arenot aware of any biosynthetic speculations about the origin of9 and 10, but on the

basis of their structures it seems safe to assume that 9 is formed by galloylation

at the hexahydroxydiphenoyl (HHDP) group of 8, and that 10 is a product of

dimerization of 5 and 9. On the basis of these considerations, we arrive at the

biosynthetic pathway depicted in Figure 1.

Seasonal Changes in Phenolic Contents. Throughout the summer, the phe-

nolic contents of oak leaves were dominated by hydrolyzable tannins (Figure 2A

and B). Although the pooled concentration of all hydrolyzable tannins declined by

54% between late May and September, their total concentration still overshadowedthat of proanthocyanidins by a factor of 7.7 on September 26. The concentrations


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FIG. 1. The proposed biosynthetic pathway for the formation of ellagitannins in leaves

of Quercus robur L. The biosynthetic steps involve (a) galloylation at C-1 of glucose;

(b) four consecutive galloylation steps; (c) oxidative coupling between galloyl groups atC-4 and C-6; (d) oxidative coupling between galloyl groups at C-2 and C-3; (e) cleavage

of galloyl group at C-1; (f) glucopyranose ring opening at C-1, followed by galloylation at

C-5; (g) oxidative coupling between benzene rings at C-4 and C-5; (h) further galloylation

thus forming a valoneoyl group at C-4/C-5; and (i) dimerization of pedunculagin and

castavaloninic acid to form cocciferin D2.

of proanthocyanidins showed an opposite seasonal pattern, steadily increasing

over the summer from undetectable levels in the young leaves to an average of

10.8 mg/g in old leaves.The most rapid changes in leaf chemistry occurred during the early part

of the summer, as the oak leaves grew and matured (Figure 2). In the majority


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FIG.2.

Seasonalvariationinleaftra

its.Eachdatapointrepresen

tsthemeanofseventrees,a

ndverticalbarsshow

standa

rderrors.

(A)Concentrationsoftotalhydrolyzab

letannins(sum

ofcompound

s2

10)andproanthocyanidins(quantifiedbythebutanol-H

Classay).

(B)Concentrationsoftotalphenolics(

quantifiedbytheFolinCiocalteauassay)andflavonoidglycosides(sum

ofcompounds11

24).(C)

Totalconcentrationsofcarbonandnitrogen.(D)Biomassperdried

oakleaf.(EandF)Concentra

tionsofindividualhydrolysab

letannins

andgallicacid.(G)Concentrationso

findividualkaempferolglycosides.(H

andI)Concentrationsofindividualquerceting

lycosides.

Compou

ndsnumberedasinFigure1.


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of individual compounds, there was a distinct peak in concentration around the

time of maximum leaf expansion (Figure 2), followed by an extended period of

relatively stable values. After July 7, when the leaves had reached their final size,

their phenolic composition remained more or less unchanged until leaf senescence(Figure 2). However, when inspected in more detail, different seasonal patterns

emerge among individual compounds, with a few compounds peaking later in the

season than the rest (notably 2, 9, and 10 among the tannins; Figure 2EI).

Interestingly, the maximal concentrations of many phenolic compounds in

late May and early June coincide with a minimum in the total amount of car-

bon, followed by a continuous buildup of carbon over the course of the summer

(Figure 2C). For nitrogen, the pattern was exactly the opposite, with the level

steadily decreasing over the summer (Figure 2C).

Consistency Among Individual Compounds and Larger Phenolic Groups.Variation at the level of total phenolic content did not capture variation at the

level of individual compounds: a sample rich in total phenolics, or in the pooled

concentration of all individual compounds, was not necessarily rich in any indi-

vidual compound (Figure 3). Some of the correlations between the concentration

of an individual compound and the total concentration of a larger phenolic group

are trivial because we are comparing an element to a sum of which it is a part.

In the absence of any correction for multiple tests, chance alone is also expected

to render one in every 20 results significant. Still, among correlations between

individual compounds and total phenolic content, only one out of 48 rS values wassignificant, and most values were relatively low or even negative (Figure 3). At

the level of the pooled concentrations of chemically similar compounds, only the

concentration of hydrolyzable tannins was significantly correlated with the pooled

concentration of all individual compounds (and perfectly so; rS = 1.0). Finally,

our two different measures of total phenolic contentstotal phenolics as quan-

tified by the FolinCiocalteau method and pooled phenolics as calculated by

pooling the HPLC readings of individual compoundswere not significantly cor-

related with each other (rS = 0.71, N= 7, P = 0.07). Hence, we conclude that

the rough quantification of a composite phenolic fraction tells us virtually noth-ing about how different trees rank compared to each other in terms of individual

compounds.

DISCUSSION

Oaks have formed one of the classic model systems in elucidating the role

of polyphenols in plantherbivore interactions. This study provides, to our knowl-

edge, the most elaborate description of temporal variation in the phenolic contentsof oak leaves conducted so far. As such, it adds substantial detail to the already

classical image of oak leaf chemistry drawn by Feeny in 1970. However, several


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FIG. 3. Spearman rank correlations (rS) among different phenolic fractions in samples

from May 29. PPh (i.e., Pooled Phenolics) shows the consistency in rank (rS) between the

concentration of individual compounds and the pooled concentration of all individually

quantified phenolic compounds; FC (i.e., FolinCiocalteau) shows rS values for the com-

pound in question compared to FolinCiocalteau readings of total phenolics. For each data

point, the number shows the identity of the compound (numbers as in Figures 1 and 2). Data

points labelled with letters refer to pooled concentrations of different phenolic subgroups;

PFG=Pooled Flavonoid Glycosides (1124), PHT=Pooled Hydrolyzable Tannins (210),

PQG = Pooled Quercetin Glycosides (1119), and PKG = Pooled Kaempferol Glycosides

(2024). For all values of rS, N= 7 trees. The horizontal bars at the top and bottom of

each panel shows the critical rS

for P < 0.05 before (solid line) and after (stippled line)

Bonferroni correction for 29 individual tests.

discrepancies with Feeny show the need for some substantial specifications to

current descriptions of oak leaf chemistry.

In his seminal account of seasonal variation in oak leaf quality, Feeny (1970,

p. 574) reported a general increase in the tannin contents of oak leaves over

the course of the summer. On the basis of two-dimensional paper chromatogra-

phy, he attributed this pattern to a progressing dominance of proanthocyanidinsover hydrolyzable tannins, the levels of which appeared to remain approximately

constant over time. Our results strongly contrast with those findings. First, we


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found hydrolyzable tannins, not proanthocyanidins, to be the dominant group of

phenolic compounds over the full course of the growing season. The peak levels

of hydrolyzable tannins were strikingly highwhile heartwood of Q. robur can

contain up to 10% of ellagitannins by weight (Scalbert et al., 1988), young oakfoliage contained levels as high as ca 18% (see Figure 2A). Second, the concen-

tration of hydrolyzable tannins did not remain stable over time, but dropped to

less than half between late May and September. Early-summer maxima in the

levels of hydrolyzable tannins have been reported from Q. robur (e.g., Tikkanen

and Julkunen-Tiitto, 2003), from other oak species (e.g., Faeth, 1986; Rossiter

et al., 1988; Maufette and Oechel, 1989), and from other trees (e.g., Riipi et al.,

2002). In all these cases, the early peak in hydrolyzable tannins is associated with

a late-summer peak in condensed tannins, suggesting a common pattern across

species and upsetting Feenys (1970) initial notion. Yet, there is one importantdifference between our material and Feenys: where Feeny focused on the upper

sun leaves of the canopy, we collected our leaf samples on the lower branches of

the trees. As light levels have been found to affect foliar phenolics (e.g., Dudt and

Shure, 1994), part of the observed difference may be due to differences in leaf

exposure. This calls for further analyses of seasonal changes in different parts of

the canopy.

Feeny (1970) focused on seasonal patterns at the level of summary pheno-

lic groups. When we examined patterns at the level of individual compounds,

temporal changes at the level of pooled phenolic contents were found to maskvariation in the concentration of individual hydrolyzable tannins and flavonoid

glycosides over time (Figures 2 and 3). The ultimate, evolutionary reasons for the

observed variation in compound-specific patterns are largely unknown, as consen-

sus has yet to be reached even regarding the exact biological roles of hydrolyzable

tannins and flavonoid glycosides (cf. Appel, 1993; Close and McArthur, 2002).

Proximate reasons are better understood, as seasonal changes in the concentration

of individual hydrolyzable tannins can sometimes be mapped onto proposed or

established biogenetic pathways (cf. Hatano et al., 1986; Salminen et al., 2001).

In Q. robur, differences in the way individual compounds changed over timeappeared closely connected to their biosynthetic relationships. First, compound

2 was the only galloylglucose present in oak leaves in levels detectable with

HPLC-DAD. This was surprising, since the biosynthetic pathway of hydrolyz-

able tannins also contains di-, tri-, tetra-, and pentagalloylglucoses before the first

ellagitannin, i.e., 3 (Figure 1; cf. Gross, 1999; Niemetz et al., 2001; Salminen

et al., 2001). Evidently the synthesis of ellagitannins in oak is effective enough

not to let these galloylglucose intermediates accumulate in detectable amounts.

Consistent with this view of active ellagitannin synthesis, the concentration of2

(i.e., the compound from which a galloyl group is utilized in the formation of othergalloylglucoses and ellagitannins therefrom) decreased dramatically after June 15

(see Figure 2F).


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Second, temporal variation in the concentrations of compounds 7, 8, 9, and

10 appears intimately interrelated (Figure 2). Although all the other ellagitannins

showed almost linear reductions in their concentrations already from May 29 on-

wards, ellagitannin9, i.e., the monomeric building block of10, showed an increaseuntil June 7, and its biosynthetic successor10 until June 15. Simultaneously, the

concentration of8, the building block of9, showed a more steep decrease than that

of7. This was presumably because 8 was utilized further in the biosynthesis of oak

leaf ellagitannins (for formation of9 and 10) unlike its isomer7 (see Figure 1).

For the same reason, the concentration of7 was approximately four times as high

as that of8 throughout the season (e.g., May 29; 57.6 mg/g vs. 14.0 mg/g). How-

ever, by summing the concentration of8 with that of its biosynthetic successors

9 and 10, almost equal values were obtained as with 7 only (e.g., May 29; 55.8

mg/g vs. 57.6 mg/g, respectively). This implies that oak leaves invest a closelysimilar amount of resources in the production of7 and 8, although this cannot be

seen in their foliar levels as such. Importantly, the potential for such invisible

investment in a given compound suggests that firm conclusions on the specific

pattern of resource allocation in oak trees cannot be reached until compounds are

quantified individually and biosynthetic pathways unravelled.

Considering the proposed biogenetic pathway forQ. robur leaves (Figure 1),

it is intriguing to note that it does not contain grandinin or roburins AE

compounds that occur as biosynthetic successors of7 and 8 in the heartwood of

this species (Herve du Penhoat et al., 1991a,b; Masson et al., 1994; Vivas et al.,1995). At the same time, ellagitannins 9 and 10 have not been encountered in

the heartwood, but have now been found for the first time in leaves of Q. robur.

Therefore, it is evident that enzymatic systems controlling the directions of the

ellagitannin pathways onwards from e.g., 7 and 8 differ even between plant parts,

not just among plant species as stated by Hatano et al. (1992). Interestingly,

Scalbert et al. (1988) noted that the proportions of7 and 8 vary between samples

from different parts of Q. robur, the former being predominant in the leaves and

the latter in the wood. However, they were unable to identify one major peak

from the HPLC chromatogram of the leaf extract, thus possibly neglecting thepresence of the dimeric ellagitannin 10. If the unidentified peak was indeed due

to 10, that might explain the lower level of 8 in the foliage. Nevertheless, the

observed differences in ellagitannin synthesis pathways caution against uncritical

generalizations among different plant tissues when interpreting, e.g., patterns of

seasonal change in phenolic composition.

Variation in oak leaf chemistry will affect a broad range of oak-associated

taxa, including herbivorous mammals and arthropods, pathogenic and endosym-

biotic fungi, and other microorganisms. Among Lepidoptera alone, more than

200 species feed on oak leavesa figure higher than for any other European treespecies (Feeny, 1970). The majority of these species attack young oak leaves in

the spring (Feeny, 1970; Niemela and Haukioja, 1982), and the ratio between oak


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specialists and generalists similarly peaks early in the season (Niemela, 1983).

Feeny (1970) attributed this pattern to a clearcut decline in the nutritional quality

of oak leaves over the course of the summer, largely because of an accumulation

of phenolics in general and proanthocyanidins in particular, is presumed to pre-cipitate proteins in the digestive system of the larva (Feeny, 1970; Niemela, 1983;

cf. Herms and Mattson, 1992). This view is partly upset by the current results, as

the total concentrations of phenolics clearly decreased over time, and the concen-

tration of hydrolyzable tannins was found to dominate over proanthocyanidins.

Hence, if there is a general decline in oak leaf quality over time, it can hardly

be linked to crude changes in phenolic contentsif it were, we would instead

expect an increase (cf. Figure 2A and B). Given different seasonal patterns both

among proanthocyanidins and hydrolyzable tannins as groups, and among indi-

vidual compounds within these groups, the total effect of phenolics on seasonalvariation in oak leaf quality will also depend on the relative biological activities

of each individual compound.

Nevertheless, under no circumstances will herbivore performance be de-

termined by phenolic contents aloneas emphasized by Haukioja (2003), the

impacts of phenolics should be interpreted against the background of seasonal

changes in crucial nutrients. Our elemental analysis showed that the nitrogen con-

centration of leaves declined by more than 50% between late May and September,

suggesting a rapid decline in the availability of proteins and free amino acids. At

the same time, the carbon contents of the leaves increased, indicating a buildup oflignin causing increased toughness of the leaves. Changes in nitrogen content and

leaf toughness were also observed by Feeny (1970) and may interact with other

attributes of the leaves such as water content (Mattson and Scriber, 1987; Haukioja

et al., 2002; Henriksson et al., 2003) in determining their nutritional quality. In

the end, a wealth of physical and biochemical factors may influence the quality

of growing leaf tissue from an herbivores perspective (Kause et al., 1999), and

much work remains to be done before the chemical contents of an oak leaf can be

linked to its perceived nutritional value.

From a methodological perspective, the observed idiosyncrasies among in-dividual compounds cast some doubt on the common use of so-called total

methodse.g., FolinCiocalteau for total phenolics, the sodium nitrite method

for total ellagitannins (Wilson and Hagerman, 1990), and the rhodanine method for

total gallotannins (Inoue and Hagerman, 1988). Clearly, when concentrations of

individual compounds vary more or less independently of each other, any method

quantifying their pooled concentration will be a poor descriptor of patterns at

the level of single compounds. This fact was graphically demonstrated by a gen-

eral lack of correlation between the contents of individual compounds and total

phenolics as quantified from the same samples (Figure 3).Furthermore, total methods do not provide an unbiased measure even

of the sum of individual compounds, as shown by a discrepancy between the


15/19


pooled amount of individual hydrolyzable tannins, flavonoid glycosides, and

condensed tannins on the one hand, and total phenolics as quantified by the

FolinCiocalteau method on the other (Figure 3). The problems are compounded

by the type of reactions used in the quantification processes. To illustrate thispoint, let us consider the determination of total ellagitannins with the sodium

nitrite method. This method relies on the hydrolysis of ellagitannins and on the

reaction of ellagic acid (the most common hydrolysis product of ellagitannins)

with sodium nitrite to yield a nitrosylated chromophore that is subsequently quan-

tified by a spectrophotometer. The production of ellagic acid requires the presence

of hexahydroxydiphenoyl (HHDP) group(s) in the hydrolyzed ellagitannins. Un-

fortunately, HHDP groups are not found in the structures of all ellagitannins,

and, even when they are, they may be just a small part of a larger ellagitannin

molecule. This may lead us to underestimate the ellagitannin content of plantsamples, orin extreme caseseven to overlook the mere presence of ellagitan-

nins. In Q. robur, we found the leaves to contain substantial amounts of 5, 7, 8,

9, and 10the structures of which contain highly variable proportions of HHDP

groups (77.6, 32.5, 32.5, 0, and 16.3%, respectively). Compound 9 is the ex-

treme example as it contains only biosynthetically modified HHDP groups, i.e., a

nonahydroxytriphenoyl and a valoneoyl group. Hence, for samples of oak leaves,

the sodium nitrite method is likely to yield a highly biased estimate of total ellag-

itannin content. Earlier, the same pattern of underestimation was shown to be true

with quantification of total galloylglucoses by the rhodanine method (Salminen,2003) and with quantification of total phenolics by the Folin (FolinCiocalteau

or FolinDenis) assay (Appel et al., 2001). Moreover, the Folin assay also un-

derestimated the total phenolic content of our oak leaves (compare Figure 2A

and B).

Despite these shortcomings, total methods are still commonly used in eco-

logical studies (e.g., McKinnon et al., 1999; Fisher et al., 2000; Inbar et al., 2001;

Lill and Marquis, 2001; Abrahamson et al., 2003; Forkner et al., 2004). We realize

that such methods may be useful under certain circumstances (Appel et al., 2001;

Salminen, 2003), but stress their limitations in studies aiming to pinpoint the roleof, e.g., hydrolyzable tannins as determinants of herbivore performance. Simply

speaking, if we do not know the structures of the hydrolyzable tannins of our

target species, we know neither what we are quantifying nor our measurement

error. Hence, we recommend that chemically minded ecologists and ecologically

minded chemists should focus their future analyses on individual tannins and es-

tablish the specific biological activities of these polyphenolic compounds through

rigorous bioassays (cf. Salminen and Lempa, 2002).

AcknowledgmentsWe thank Sofia Gripenberg, Aulis Leppanen, and Markku Salo for help

with the field work, Paivi Franti for measuring total phenolics and proanthocyanidins, and Tiina Buss

for conducting the elemental analyses. Comments by Ann Hagerman, Erkki Haukioja, Kyosti Lempa,


16/19


Vladimir Ossipov, Janne Suomela, and an anonymous referee helped us improve earlier drafts of this

article; comments by Gavin Hinten helped us improve the language. This project was funded by the

Academy of Finland (project numbers 45748, 51789, and 204209).

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seasonal variation in the content of the hydrolizable tannin

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