Journal of Chemical Ecology. Vol. 22, No. II, 1996

VARIATION OF TOTAL PHENOLIC CONTENT AND INDIVIDUAL LOW-MOLECULAR-WEIGHT PHENOLICS IN FOLIAGE OF MOUNTAIN BIRCH TREES (Betula pubescens ssp. tortuosa)

K 1 M M O N U R M I , I'* V L A D I M I R and

O S S I P O V , ~'-~ ERKKI H A U K 1 O J A , 2

KALEVI

PIHLAJA

I

tLaborator3, of Physical Chemistry "-Laboratot3" of Ecological Zoology University of Turku FIN-20014. Turku, Finhmd (Received February 12, 1996; accepted June 21, 1996)

A b s t r a c t - - W e studied seasonal and between-tree variation in the composition and content of total and individual low-molecular-weight phenolics (LMWP) in leaves of mountain birch trees (Betula pubescetls ssp. Iorttto.~a). The m~tior phenolic compounds were chlorogenic acid, quereetin-3-O-t3-D-glucuronopyranoside, myricetin-3-O-(5-acetyl)-L-rhamnopyranoside, and l-O-galloyl/J-D-(2-O-acetyl)-glucopyvanose. The content of total phenolics, as well as the sum of individual LMWP, varied only slightly among trees while variation in contents of individual LMWP was large. Concentv,Jlions of almost all phenolics decreased during the growing season but pairwise correlations between individual phenolics remained similar over the whole season indicating tree-specific LMWP profiles over the season. Among flavonoids, the between-tree component of variation was 2.6 times as large as the seasonal component, while lot variation of nonflavonoids the between-tree component was larger than the seasonal one. To explain the significant correlations within both flavonoid and nonflavonoid compounds, we discuss the biogenesis of LMWP in birch leaves, as well us their ecological role.

Key Words--Betula pubescens ssp. tortuosa: birch; phenolics, flavonoid glycosides: among-tree variation; seasonal variation.

*To whom correspondence should be addressed.

2023 tX)98 0331/96/I I(X) 2(1235(F),5(ffO ~ 1~96 Plenum Publi,~hing Coq~r-allon

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NURMI, OSSIPOV,HAUKIOJA,AND PIHLAJA INTRODUCTION

The value of host plant tissue for herbivores is a function of its biochemical composition and physiological state of the plant. General theories of plant defenses share the basic assumption that secondary compounds are responsible for plant resistance, including induced defenses, and that these, rather than nutritive factors, represent the critical chemical factors explaining variance in herbivore performance (but see, e.g., Haukioja et al., 1991; Berenbaum, 1995). Plant phenolics are known for their negative effects on insects and they have played an important role in theories of plant-herbivore interactions (Feeny, 1976; Rhoades and Cates, 1976; Coley et al., 1985), and they are the defensive compounds generally measured from plant tissue. However, in addition to their possible defensive role against herbivores, phenolics in plants have a suite of other possible functions, including antidesiccation, UV protection, growth regulation, etc. (Kuiters, 1989; Li et al., 1993; Lois, 1994; Northup et al., 1995). Furthermore, they seem to accumulate in slowly growing plants and plant tissues. The original idea of all phenolics having a basically similar and nonspecific effect on herbivores via protein precipitation (Feeny, 1976; Schultz, 1989) is far too simple. Different phenolics can vary in their biological activity, and in the mechanism of action on herbivores. Some phenolics may have a direct toxic effect on herbivores or require oxidation for realization of their ecological activity (Felton et al., 1989; Appel, 1993). Total phenolics in birch leaves, and in many other plants, correlate negatively with insect performance (Haukioja et al., 1985). Furthermore, it is known that total phenolics may experience strong seasonal patterns, but what happens among individual phenolics is not well understood. We have recently identified 33 low-molecular-weight phenolics (LMWP) in the leaves of white (Betula pubescens) and silver (B. pendula) birches (Ossipov et al., 1995, 1996) by preparative HPLC and determined their structures on the basis of chromatographic (analytical HPLC), chemical (hydrolysis), and spectroscopic (UV, MS, ~H NMR and J3C NMR) techniques. In this paper we examine the composition and content of low-molecular-weight phenolics in leaves of individual mountain birch trees over a season to find out how well total phenolic deteminations describe seasonal and between-tree variation in the contents of individual lowmolecular-weight phenolics of the leaves of mountain birch.

METHODS AND MATERIALS

Plant Material. The experimental mountain birches [Betula pubescens Ehrh ssp, fartuosa (Lebed.) Nyman] were mature, wild trees growing near the Kevo

PHENOLICS IN MOUNTAIN BIRCH

2025

Subarctic Research Station (69°45'N, 27°01 'E) in northem Finland. The study area lies at about 150 m above sea level and is covered with almost pure mountain birch forest. We sampled foliage of the same 20 birch trees that were originally randomly chosen from a homogeneous forest plot and marked individually by Hanhim/iki et al. (1996). Short shoot leaves from individual trees were sampled on July 1 and 20, and August 24, 1994, always between 9 and 10 AM. Leaves even at the earliest sampling date were almost full grown. Sampling dates were designed to coincide with the peak feeding of the two most important insect herbivores of the mountain birch, the autumnal moth, Epirrita autumnata (early July) and the sawfly, Dineura virididorsata (August), as well as to include an intermediate date when many mid-season sawflies consume foliage (Hanhim~iki et at., 1995). Since the short shoot leaves of the mountain birch burst simultaneously in spring, all the leaves within a tree were of the same age on a given sampling date and matured similarly between the sampling dates. Excised leaves were placed in plastic vials and returned to the laboratory on ice. For determination of phenolic compounds, the birch leaves were vacuum dried and stored in plastic vials at - 2 0 ° C . Extraction of Phenolics. The dried leaves (0.5 g) were ground for 2 min with Ultra-turrax T 25 in 25 ml of 70% aqueous acetone. The homogenate was centrifugated at 2500g for 10 min and the pellet was reextracted with 25 ml of 70% aqueous acetone for 60 min under continuous stirring. After centrifugation the combined extracts were evaporated to dryness under low pressure at 40°C. The residue was dissolved in 10 ml of water and centrifuged. Then the solution was filled to 25 ml of water and stored in a freezer ( - 2 0 ° C ) . Extraction efficiency of phenolic compounds was confirmed by reextracting the pellet three times with 25 ml of 70% aqueous acetone and checking these solutions by HPLC. Total Content ofPhenolics. A modification of the Folin-Ciocalteau method (Torres et al., 1987) was used for measurement of total content of phenolic compounds. A 0.1-ml extract was delivered to a 15-ml tube and mixed with 5.9 ml water. One milliliter of this diluted extract was mixed with 1.0 ml t N Folin reagent (Folin-Ciocalteau's phenol reagent, Fluka, BioChemica, Buchs, Switzerland) in the centrifuge tube (10 ml volume) and the mixture was allowed to stand for 2-5 min. Then 2 ml of 20% NazCO3 was added. After a 10-min incubation at room temperature, the mixture was centrifuged at 1500g for 8 min and the absorbance was measured at 730 nm on a Perkin-Elmer Spectrophotometer 550. Three replicates of each sample were analyzed. The standard curve was prepared with known concentrations of gallic acid. Quantification of LMWP. Before the HPLC analysis of phenolics, the extract was filtered through Millex-HVt3 0.45-/zm filter (Millipore, Bedford, Massachusetts). The HPLC system consisted of Merck-Hitachi Pump L-6200 A,

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NURMI, OSSIPOV, HAUKIOJA, AND PIHLAJA

Merck-Hitachi L-4250 UV-VIS Detector (Hitachi, Tokyo, Japan), Perkin-Elmer LC-235 Diode Array Detector (Shimadzu, Kyoto, Japan), and Shimadzu C-R6A Chromatopac Integrator (Shimadzu, Kyoto, Japan). The sample was injected on Spherisorb ODS-2 column (250 x 4.6 mm ID, 5 ~tm; Phase Sep. Ltd.) and the flow rate was 1.0 ml/min. Detection was performed at 280 nm, 0--40 min; 360 nm, 40-60 min; and 280 nm, 60-110 min. Two solvents were used; solvent A was 5% formic acid and solvent B was acetonitrile. The gradient was as follows: 0-5 min, 100% A (Isocratic); 5-60 min, 0-30% B in A (linear gradient); 6070 min, 30-60% B in A (linear gradient); 70-80 min, 60% B in A (Isocratic). Quantitative determinations were carried out by the external standard method using gallic, p-coumaric, and chlorogenic acids, quercetin, myricetin, and kaempferol as standards, assuming a similar molecular extinction coefficient for aglycones and glycosides (Winter and Herrmann, 1986). The relative standard error in the quantitation of LMWP, ranging from 2.2% to 11.2% (small peaks) based on HPLC analysis of five leaf samples of the same tree, provides an estimation of the error involved. Statistical Analysis. The distributions of a few phenolic compounds deviated from the normal. The homogeneity of variances was tested with Cochran's test; for a few phenolic compounds, the variances differed significantly. There were, however, no deviations from normality or homoscedasticity with these compounds. Significance of variations in the content of phenolic compounds were tested by a two-way ANOVA. The correlations between individual phenolic compounds during the growing season were tested with Pearson's correlation analysis (Proc Corr, SAS Institute, Cary, North Carolina).

RESULTS

Identification. Up to 30 individual low-molecular-weight phenolic compounds in the leaves of the mountain birch were detected by HPLC (Figure 1). Composition of LMWP in the leaves of mountain birch were closely consistent with our earlier analyses of white and silver birch leaf chemistry (Ossipov et al., 1995, 1996). The identified phenolics included both flavonoid (peaks 4, 5, 11, 13, 20-36) and nonfiavonoid (peaks 1, 2, 6, 8, 9, 12, 14, 18, 19) compounds. We detected 21 flavonoids and identified 11 of them on the basis of UV spectroscopy data as flavanols (peaks 4, 5, 13, 20-22, 25), quercetin glycosides (30, 32), or kaempferol glycosides (35, 36). The identified flavonoid compounds were the glycosides of myricetin, M-3-O-i3-D-gtucuronopyranoside (23), M-3O-~-D-galactopyranoside (24), and M-3-O-L-(acetyl)-rhamnopyranoside (26); glycosides of quercetin, Q-3-O-/3-o-glucuronopyranoside (27), Q-3-O-t3-D-galactopyranoside (28), Q-3-O-~-L-arabinofuranoside (29), Q-3-O-c~-L-(4"-O-ace-

PHENOLICS I N MOUNTAIN BIRCH

2027

14

27

26 18

6

f

9

10 13 11

[

29

34

i

i

I

i

i

i

t0

20

30

40

50

60

Time (rnln)

FIG. I. Typical trace of HPLC analysis of soluble low-molecular-weight phenolics (1-40) in the leaves of mountain birch trees. Spherisorb ODS-2 column (250 × 4.6 mm ID, 5 ~m) at a flow rate of 1.0 ml/min with gradient of acetonitrile in 5% aqueous formic acid; detection wavelength at 280 nm. Peak 1, l-O-galloyl-/~-D-(2-acetyl)-glucopyranose; 2, gallic acid; 4 and 5, flavanois; 6. neochlorogenic acid; 8, cis-5-p-coumaroylquinic acid; 9, trans-5-p-coumaroylquinic acid; 11, (+)-catechin; 12, l-(4"hydroxyphenyl)-3'-oxopropyl-B-D-glucopyranose:13, flavanol; 14, chlorogenic acid; 18, trans-3-p-coumaroylquinic acid; 19, cis-3-p-coumaroylquinic acid; 20-22, flavanols; 23, myricetin-3-O-~-o-glucuronopyranoside; 24, myricetin-3-O-~-D-galactopyranoside; 25, flavanol; 26, myricetin-3-O-u-L-(acetyl)-rhamnopyranoside; 27, quercetin-3-O-~-Dglucuronopyranoside; 28, quercetin-3-O-~-D-galactopyranoside; 29, quercetin-3-O-cc-Larabinofuranoside; 30, quercetin glycoside; 31, kaempfeml-3-O-~-D-glucopyranoside; 32, quercetin glycoside; 33, kaempferol-3-O-u-L-rhamnopyranoside; 34, quercetin-3-OeZ-L-(4"-O-acetyl)-rhamnopyranoside; 35 and 36, kaempferol glycosides. Peaks 3, 7, t0, 15, 16, 17, 37, and 38 were not identified. tyl)-rhamnopyranoside (34); and glycosides of kaempferol, K-3-O-/~-Dglucopyranoside (31) and K-3-O-a-L-rhamnopyranoside (33). Compound 11 was identified as the flavanol, (+)-catechin. The identified nonflavonoid compounds were gallic acid (peak 2) and its derivative, 1-O-galloyt-/~-D-(2-O-acetyl)-glucopyranose (1); derivatives of caf-

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NURM[, OSSIPOV, HAUKIOJA, AND PIHLAJA

feic acid, neochlorogenic acid (6, trans-5-p-caffeoylquinic acid) and chlorogenic acid (14, trans-3-caffeoylquinic acid); and derivatives of p-coumaric acid, cis5-p-coumaroylquinic (8), trans-5-p-coumaroylquinic (9), trans-3-p-coumaroylquinic (18), and cis-3-p-coumaroylquinic (19) acids. Peak 12 was identified as 1-(4"-hydroxyphenyl)-3'-oxopropyl-/3-D-glucopyranose. Seasonal Variation. The mean total phenolics content in the leaves of mountain birch in early, mid-, and late summer was 103. 93, and 89 mg/g of dry wt, respectively: the average decline from early July to late August was 14%. The mean content of the main individual low-molecular-weight phenolics and total content of phenolics in the leaves of the 20 trees used in our study are shown in Table 1. For quantitative calculations, the main 18 phenolic compounds were considered; the remaining 12 compounds (peaks 4, 5, 8, 9, 1113, 20-22. 25, 35, 37 and 38) were disregarded due to lower levels of accumulation and poor separability of the respective peaks. Most phenolic parameters showed significant differences among sampling dates (Table 2). The mean total LMWP content of tbliage in early, mid-, and late summer was approximately 48, 41, and 38 mg/g of dry wt, respectively. The average decline was 21%, i.e., steeper than in the case of total phenolics. The average seasonal decline of nonflavonoid compounds ranged from 52 % (6) to 70% (19), except for peak 14, the average content of which increased 12% in July, followed by a 15% drop in late August. The content of individual flavonoid compounds declined at the same time from 7% (28) to 37% (23). The amount of most phenolic compounds declined evenly during the three sampling dates, but compound 1 declined rapidly in July, over 80% of total decrease. The average content of flavonoid compounds 26, 28, and 30, however, increased over 10% in late summer compared to mid-summer. Between-Tree Variation. Differences in the composition of LMWP between the 20 trees were small, and most compounds could be detected from each tree. However, compounds 32, 34, and 36 were found in less than one third of the trees, and compounds 1, 2, 6, and 30 occurred in small or trace amounts in some of the trees. The quantitative variation in phenolic compounds was large throughout the growing season (Table 1). Between-tree variation was significant at P < 0.001 in all phenolics through the growing season, except for gallic acid (peak 2). In early summer the coefficients of between-tree variation (CV) of nonflavonoid compounds ranged from 42% (19) to 93% (6), and of flavonoid compounds from 36% (27) to 264% (36), being less than 70% for two thirds of the compounds. The tendency was similar throughout the growing season. Still, the coefficient of between-tree variation of total content of LMWP (the sum of individual compounds) and total phenolics varied during the season only between 23 and 27% and 14 and 17%, respectively.

Kaempferol glycosidea Total LMWP Total phenolics

3.70 0.26 1.03 13.74 1.36 0.35 2.72 0.71 5.17 9.58 1.88 2. II 0.73 2.02 0.34 1.19 0.65 0.11 47.64 103.12

+ 3,16 5:0.17 5:0.96 5:8.26 + 0.80 + 0,15 5:1.32 5:0,43 ± 2,75 _ 3,47 ± t.09 5:1.10 5:0.78 5:0.99 ± 0.53 5:0.63 ± 1.37 5:0.30 _+ 10.87 5:16.68

Early

"Values are mg/g dry weight (mean + 1 SD). Q, quercetin; K, kaempferol; M, myricetm ~'Refers to peak numbers in Figure 1~ ' Expressed as quercetin-3-O-D-rhamnopyranoside (M = 448.16 g/tool). aExpressed as kaempferol-3-O-o-glucopyranoside (M = 448.36 g/mob.

14 18 19 23 24 26 27 28 29 30 31 32 33 34 36

Q-3-O-c~-L-(4"-O-ac.)-rhamnopyranoside

I-O-galloyl-D-(2-ac.)-glucopyranoside gallic acid trans-5-caffeoylquinic acid chlorogenic acid trans-3-p-coumaroylaquinic acid cis-3-p-coumart~ylquinic acid M-3-O-D-glucuronopyranoside M-3-O-D-galactopyranoside M-3-O-(5-ac.)-L-rhamnopyranoside Q-3-O-D-glucuronopyranoside Q-3-O-galactopyranoside Q-3-O-cc-L-arabinofuranoside Quercetin-glycoside' K-3-O-D-glucopyranoside Quercetin glycoside' K-3-O-L-rhamnopyranoside

I 2

6

Compound

Peak t' 1.80 0,05 0,67 15.36 0.73 0.25 2.15 0.60 3.99 8.14 I54 1.68 0.54 1.52 0.32 0.93 0.56 0. I I 40.93 92.63

± 1,56 + 0.03 _+ 0,66 5:9.74 5:0,33 5:0.11 5:0.85 5:0.33 _+ 1.99 ± 3,07 5: 0,9I _++0.86 +_ 0,65 5:0.79 5:0,49 __++ ._0.49 + 1,14 + 0,23 _+ 11,16 ± 13.06

Mid

Sampling date

1.36 0.01 0.49 12.98 0.41 0.13 1.71 0.53 4.66 8.23 1.76 1.48 0.60 1.56 0.27 0.82 0.50 0.09 37.58 88.50

5:1.45 5:0.00 5:0,73 5:8.12 5:0.11 +_ 0,03 5:0.66 _ 0.35 +_ 1.98 5:3,03 ± t,03 5:0.83 _+ 0.62 5:0.85 _+ 0.41 5:0.44 5:1,04 ± 0.20 ± 9.69 5:14,89

Late

63 100 52 6 70 62 37 25 10 14 7 3O 17 23 I8 31 24 20 21 14

Dm'~p (

TABLE 1. CONTENT OF Low-MoLECULAR-WEIGHT PHENOLICS AND TOTAL PHENOLICS IN LEAVES OF 20 MOUNTAIN BIRCH TREES ON THE THREE SAMPLING DATES (EARLY, MID, AND LATE SEASON)"

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NURMI, OSSIPOV, HAUKIOJA, AND PIHLAJA

TABLE 2. RESULTSOF Two-WAY ANOVA WITHOUT REPLICATION FOR DIFFERENCES IN CONTENTS OF Low-MoLECULAR-WEIGHT PHENOLICSAND TOTAL PHENOLICSOF LEAVES OF 20 MOUNTAIN BIRCHES DURINGTHE SEASON. Among trees

Tempe,rat variation

Peak"

MS

F

MS

F

1 2 6 14 18 19 23 24 26 27 28 29 30 31 32 33 34 36 Total LMWP Total phenolics

11.99 0.01 1.64 219.66 0.48 0.02 2.56 0.38 14.33 30.06 3.01 1.38 2.49 2.25 0.67 0.79 4.20 0.16 316.61 610.88

9.49 ***h 1.22 13.29"** 46.87*** 3.36*** 4.11"** 15.08"** 25.68"** 25.5t*** 94.96*** 78.53*** 74.89"** 37.45*** 61.78"** 72.72*** 40.52*** 111.17"** 15.81"** 31.99*** 20.43***

3071 0,38 1,48 29.52 4.68 0.23 5.13 0.16 7.02 I2.99 0.59 0.18 2.05 1.53 0.02 0.74 0.12