Journal of Chemical Ecology, Vol. 15, No. 1, 1989
PHENOLIC BIOSYNTHESIS, LEAF DAMAGE, AND INSECT HERBIVORY IN BIRCH (Betula pendula)
S.E. H A R T L E Y and R.D. F I R N Department of Biology University of York Heslington. York. Y01 5DD. England (Received August 5, 1987; accepted November 16, 1987) Abstract--The effect of both caterpillar herbivory and artificial damage on phenylalanine ammonia lysase (PAL) activity of birch foliage was measured, using an intact cell assay. After artificial damage there was a small increase in PAL activity in damaged leaves but no change in adjacent undamaged ones. Insect grazing produced a larger increase in PAL activity, and the enzyme activity was also increased in adjacent undamaged leaves. Artificial damage increased the phenolic levels of the damaged leaves. Insect grazing caused a larger, longer-lasting increase in phenolic levels and also elevated phenolic levels in undamaged leaves. The possible role of these woundinduced biochemical changes in birch is discussed. Key Words--Phenolics, PAL activation, insect herbivory, plant resistance, Betula pendula, Apocheima pilosaria, Lepidoptera, geometridae. INTRODUCTION Wound-induced reductions in leaf palatability to insect herbivores have been reported in Betula pubescens Ehrh. (Haukioja and Niemela, 1977) and Betula pendula Roth (Wratten et al., 1984). Such changes can occur in damaged and adjacent undamaged leaves, and the responses may be part o f a wound-induced antiherbivore defense mechanism (Haukioja and Hahnimaki, 1985; Bergelson et al., 1986; Lawton, 1987). It has been suggested that the decrease in palatability is a result o f an increase in the concentration o f phenolic compounds known to occur in d a m a g e d leaves (Niemela et al., 1979; Wratten et al., 1984; Bergelson et al., 1986). However, palatability to insect herbivores does not always correspond well with phenolic levels (Hartley and Lawton, 1987).
275 0098-033118910100-027500.0010 © 1989 Plenum Publishing Corporation
276
HARTLEYAND FInN
Changes in the phenolic metabolism of damaged birch leaves were investigated at a more fundamental level than has hitherto been possible. Simple phenolic compounds are derived from the shikimate pathway (Conn, 1981). Consequently, by studying the activity of enzymes in this pathway in damaged and undamaged leaves, it may be possible to define, in more precise biochemical terms, the way in which plants respond to insect attack. Differences in the chemical changes induced by artificial damage as opposed to insect-grazing can thus be characterized in terms of enzyme activity, which could be important in assessing whether the changes are likely to be involved in defense against herbivores or pathogens, or simply in repair of wounded tissues. For example, an increase in phenolic compounds in undamaged leaves due to increased enzyme activity in these undamaged tissues, rather than due to transport of phenolics from the site of damage, may indicate a mechanism which is not purely tissue repair. In the present study, the activity of phenylalanine ammonia lysase (PAL) was investigated, together with the concentration of phenolic compounds, in damaged and undamaged leaves from both insect-grazed and artificially damaged trees. Evidence is presented that PAL activity is stimulated by insect attack on birch, even in undamaged leaves, and the response differs from that caused by artificial damage to the leaves.
METHODS AND MATERIALS
PAL Measurements. On July 8, 1986, 12 birch trees, each about 2 m high, were chosen in the University of York tree nursery, where they had been transplanted several years earlier. Twenty-five Apocheima pilosaria larvae (Lepidoptera, Geometridae) Were caged on three branches on each of four of the trees and allowed to graze overnight, by which time 10-15% of the leaf area of the caged branches had been removed. A further four trees had the same amount and distribution of damage inflicted on three of their branches with a pair of scissors (sterilized in alcohol to reduce the risk of fungal infection). The four remaining trees were left as controls. Fifteen leaves were collected from each tree at the start of the experiment, and from the control trees one, three, five, and seven days later, while 15 damaged, and 15 undamaged leaves immediately adjacent to damaged ones, were removed from the treatment trees on the same days. The undamaged leaves included some at the same node, some above, and some below the damaged leaves, as available. Enzyme activity was measured immediately after the leaves were collected using a modified version of the intact cell assay of Amrhein et al. (1976). This "in vivo" method for assaying PAL was chosen in preference to an in vitro enzyme assay in order to obviate possible differences in enzyme recovery from the damaged and adjacent leaves.
PHENOLIC BIOSYNTHESIS IN BIRCH
277
One half gram (wet weight) of 6-mm leaf disks was incubated with 5/zCi L-phenyl(2,3-3H)alanine (Amersham; specific activity 43 Ci/mmoI) in 2 ml of 0.15 M potassium phosphate buffer (pH 5.5) for 3 hr. The labeled phenylalanine was absorbed by the cells, equilibrated with the endogenous substrate, and the action of PAL liberated labeled N3HH2 . This lost 3H to tissue water to produce 3HOH, a virtually irreversible process due to the considerable dilution of the tracer, so recycling is very unlikely. The 3HOH was recovered from the medium by sublimation by a procedure similar to that of Mitra et al. (1975). Twenty microliters of incubation medium, together with a drop of COC12"6H20, was placed on a filter paper disk that was positioned on a pin stuck into the lid of a scintillation vial. Vials were placed in a piece of polystyrene such that the bottom of the vial was in liquid nitrogen while the top was heated with an infrared lamp until the COC12 solution turned blue. The pin and filter paper were then removed, and the 3H activity of the ice formed was determined using a scintillation counter (LKB), following the addition of 2 ml of Optiphase MP (Fisons) to the vial. The PAL activity was measured as counts per minute (cpm) per 0.5 g fresh weight per 3 hr, after correction for background and control counts. (Control counts are the values recorded from incubations using boiled leaves with no PAL activity.) The results were expressed as mean percentage changes over the activity at the start of the experiment (to reduce the effect of the between-tree variation in initial PAL activities) and were analyzed in a one-way ANOVA, tested separately each day. The specific treatments responsible for producing significant effects in the ANOVA were identified using nominated comparison t tests. The leaf material remaining after the enzyme assay had been carried out was freeze-dried and assayed for total phenolics as described below. Field Experiment. On May 15, 1985, at Skipwith Common, 10 miles south of York (grid ref. SE 664379), 15 small Betula pendula trees (each about 1.5 m high) were selected, sprayed with insecticide (Sprayday, Pan Britannica Industries Ltd.; contains resmethrin and pyrethrum), and all natural damage marked with a small dot of paint on leaf petioles. Three weeks later, the leaves of five of the trees were left undamaged, five were cut with scissors, and five were grazed by Apocheima pilosaria, as in the experiment described above. Twenty leaves were collected from each of the control trees at the start of the experiment, and one, three, five, and eight days subsequently. In the case of the other treatments, 20 leaves were collected before damage, but on days one, three, five, and eight, 20 undamaged leaves from a branch adjacent to a treatment branch were collected, in addition to the 20 damaged leaves. Leaves were placed on ice immediately after collection, returned to the laboratory within 1 hr, and stored at - 2 0 ° C . Chemical Analyses. All chemical analysis (except for PAL activity) was carded out on freeze-dried material. Total phenolics were measured using the Folin-Denis method as described in Bergelson et al. (1986), except that 20 mg
278
HARTLEY AND FIRN
of leaf powder was extracted and after centrifugation the volume was adjusted to 10 ml, and then aliquots equivalent to 0.1 and 0.2 mg of leaf-powder were used in the analysis. Protein precipitation was measured using the Bradford method (Bradford, 1976). The procedure was similar to that of Martin and Martin (1982), but slightly modified as described in Hartley and Lawton (1987). The protein-precipitating properties were expressed as the coefficients (slopes) of regression lines fitted to the graphs of milligrams (dry weight) of leaf extracted vs. grams of BSA precipitated (see Martin and Martin 1982 for further details). RESULTS PAL activity was measured in control leaves and in damaged and adjacent undamaged leaves from insect and artificially damaged trees (Figure 1). The activity was found to vary somewhat on a daily basis (Figure 1), with large between-tree differences; hence, although there was a significant effect of treatment on activity on both days 1 ( F = 10.64, df = 4,15, P = 0.0003) and 5 ( F = 5.46, df = 4,14, P = 0.0084), there were no significant effects on day 3, or on day 7, by which time activities in all treatments had decreased to near control levels. In the artificially damaged trees, cut leaves had significantly higher PAL activities than leaves from the control trees one day after damage
o
F-
.E;
100
0 1357
1357
Contr~
Cut leaves
1357 Uncut leaves
k
1357 Grazed leaves
1357
Days elapsed
Ungrazed leaves
FIG. 1. The percentage change in PAL activity in damaged and adjacent undamaged birch leaves over the seven days subsequent to damage, and in control trees. Each value is the mean of measurements of four trees (the same four trees for each sample).
279
PHENOLIC BIOSYNTHESIS IN BIRCH
(t = 4.03, df = 6, P < 0.01), but by day 5 the activity had declined, and there was no longer a significant difference. At no time did the undamaged leaves from these trees show a significant increase in their PAL activity. On the insectgrazed trees, P A L activities were greatly increased, with the damaged leaves having higher activities than both control and cut leaves (t = 6.64, df = 6, P < 0.001, and t = 5.09, df = 6, P < 0.02, respectively). On these trees, the adjacent undamaged leaves had the largest increase in activity of all on day 1 (t = 3.39, df = 3.2; P < 0.05), but this was short-lived; by day 3 there was no significant difference from the controls. However, the elevation of PAL activity in the insect-damaged leaves was more persistent and, unlike the artificially damaged leaves, the level of activity five days after damage was still significantly higher than in control trees (t = 2.58, df = 6, P < 0.05). Samples of the leaves used for enzyme assays were analyzed for total phenolics, and the phenolic levels were found to be elevated in the leaves with increased P A L activity (Figure 2). However, since the amount of phenolics accumulating depends on factors such as the rate of phenolic degradation (which was not measured), and because of the imprecise method used to estimate phenolics, PAL activity and phenolic levels do not correspond exactly. The control leaves and the undamaged leaves on cut trees showed no significant changes in phenolic content, but the cut leaves, and both the damaged and undamaged leaves on the insect-grazed trees all had increased amounts of phenolic compounds (paired t tests, t = 6.64, df = 3, P < 0.01; t = 13.6, df = 3, P < 0.001; and t = 5.79; df = 3; P < 0.02, respectively). Insect attack produced a larger increase in phenolics than artificial damage (two sample t test, t = 3.52, df = 6, P < 0.02).
26.4%
10.4 10.0
c o
9.0
--
10.0%
17.8%
.~
(3
o n
8.0
~% 037 0 1 3 5 Control Cut leaves
01357 Uncut leaves
0 1 3 5 7 0 1 3 7 Days Grazed Ungrazed leaves
elapsed
leaves
FIG. 2. The effect of leaf damage on phenolic levels in birch leaves. The values are the means of measurements made on four trees, and the same trees were used on each sampling day. The percentage change in phenolic content after seven days is also shown.
280
HARTLEY AND FIRN
The results of the 1985 field experiment also show chemical differences between insect and artificially damaged leaves. The level of phenolic compounds in control trees (Figure 3) did not differ significantly over an eight-day experimental period (paired t test, t = 1.36, d f = 4; P > 0.1). In trees with leaves that were cut with scissors (Figure 3), the phenolic levels increased in the damaged leaves (paired t test, t = 5.91, d f = 4, P < 0.01), but not in undamaged ones from an adjacent branch (paired t test, t = 1.33, d f = 4, P > 0. I). Again, in insect-grazed leaves, the phenolics rose to higher levels than in cut leaves (two sample t test on eight-day values: t = 3.14, d f = 8, P < 0.02), and undamaged leaves on the neighboring branch to the grazing also had slightly increased phenolic levels after eight days (paired t test, t = 2.99; d f = 4, P < 0.05). However, the change in the undamaged foliage was less than in the 1986 experiment, perhaps because the undamaged leaves in that experiment were immediately adjacent to the insect-damaged ones. The protein-precipitating abilities of extracts of artificially damaged, insectgrazed, and control trees were also made. However, these measurements failed to reveal any significant increases in protein-precipitating ability resulting from insect grazing, although a slight, but statistically significant, increase was recorded in artificially damaged leaves (data not shown). These results suggested that assays of protein-precipitation ability and Folin-Denis measurements were not equivalent as indicators of insect-induced chemical changes, possibly because they were measures of different kinds of chemicals, and no further protein precipitation measurements were made.
f
¢5 •,~
26.5%
1 1.796
11.0
10.0
1.0%
0 i
o
9.0
-'0.7% ~
~= 8.4 0 1 3 6
Control
0
38 Cut leaves
0 1 3 8 Uncut leaves
0 1 3 Grazed leaves
0 1 3 8 Days elapsed Ungrazed leaves
FIG. 3. The effect of leaf damage on the phenolic content of birch leaves. Each value is the mean of measurements of five trees, each tree sampled on the days shown. The percentage change in phenolic content after eight days is also shown.
281
PHENOLIC BIOSYNTHESIS IN BIRCH
DISCUSSION
It is clear from previous work (e.g., Wratten et al., 1984), and from the results presented here, that phenolic concentrations are elevated in insect-grazed birch leaves and are also higher in undamaged leaves adjacent to those which were damaged. The finding that PAL activity is also increased in such leaves suggests that these elevated levels of phenolic compounds are the consequence of increased in situ biosynthetic activity, rather than mobilization of phenolics from the site of damage. The response to artificial damage was different, however, with smaller changes both in terms of PAL activity and phenolic concentration, and no changes occurring in adjacent undamaged leaves. It has been suggested that such a response, in which insect damage is a more effective inducer of chemical changes than artificial damage and in which synthesis of phenolics occurs at sites remote from damage, represents an active and specific defense, rather than a wound-repair mechanism (e.g., Haukioja and Neuvonen, 1985). However, a number of questions require further investigation before the role of these wound-induced changes is clear. First, is the increased PAL activity the result of enzyme activation or enzyme synthesis? A number of stimuli, such as light, chemicals, and fungal infection, can increase PAL activity in plants, and it has been shown in some cases that increased synthesis of mRNA coding for PAL is associated with this increase in PAL activity (e.g., Dixon, 1986). The second question is, what is the nature of the transmitted factor that passes from insect-grazed leaves to adjacent leaves, resulting in elevated PAL activity? Transmittable factors have been reported in plants following damage (Ryan, 1974), and, interestingly, "elicitors" capable of inducing PAL in plant ceils are found in some fungal-plant interactions (Hahlbrock et al., 1981). The fact that increased PAL activity is a common response of plants subject to fungal attack raises a third question: what is the relevance of the increased PAL activity found in the insect-grazed leaves? Although insect grazing had a more marked effect on PAL activity than an equivalent amount of damage inflicted with scissors, this could simply reflect the contamination of caterpillar mouthparts with fungal spores (Shain and Hillis, 1972). A second possibility is that grazing produces a more severe stress on the leaf by a sustained period of physical damage. The function of the damage-induced increase in phenolic levels is also hard to assess since the responses of leaves to damage might include the induction of many chemicals, some of which may be primarily antifungal and some of which might play a role in defense against insects. Phenolics have certainly been associated with resistance to fungal attack (Werder and Kern, 1985), as well as antiherbivore effects (Bennett, 1965; Todd et al., 1971; Roehrig and
282
HARTLEY AND FIRN
Capinera, 1983), as has leaf d a m a g e (Karban et al., 1987). H o w e v e r , the role o f phenolics is not entirely clear, as both insects (Bernays, 1981) and fungi (Shaw, 1985) can remain unaffected, and birch-feeding insects often seem indifferent to the chemical changes induced in d a m a g e d leaves (Lawton, 1987; Hartley and L a w t o n , 1987; but see Bergelson et al., 1986; Wratten et al., 1984). Although the ecological significance o f the reported changes in P A L activity remains to be established, this study has clearly s h o w n a differential response o f birch trees to insect and artificial d a m a g e and that insect attack produces marked changes in phenolic biosynthesis in both grazed and adjacent u n d a m aged leaves. Furthermore, these results suggest that the in vivo P A L assay may prove to be a sensitive measure of insect and mechanical d a m a g e - i n d u c e d c h e m ical changes. Further studies are needed on other plant species and on the mecha n i s m of increasing P A L activity to progress towards a better u n d e r s t a n d i n g o f the way in which plants detect and respond to insect attack. Acknowledgments--Permission to work at Skipwith Common is granted by the Yorkshire Wildlife Trust. S.E.H. was supported by a SERC studentship. We are grateful to John Lawton and Clive Jones for helpful comments on the manuscript, and to Philip Warren for technical assistance.
REFERENCES AMRHEIN,N., GOEDEKE,K.H., and GERHARDT,J. 1976. The estimation of phenylalanine ammonia lysase (PAL) activity in intact ceils of higher plant tissue.Planta 131:33-40. BENNETT, S.E. 1965. Tannic acid as a repellant and toxin to alfalfa weevil. J. Econ. Entomol. 58:372-373. BERGELSON,J., FOWLER,S., and HARTLEY,S. 1986. The effects of foliage damage on casebearing moth larvae, Coleophora serratella, feeding on birch. Ecol. Emomol. 11:241-250. BERNAYS, E.A. 1981. Plant tannins and insect herbivores: An appraisal. Ecol. Entomol. 6:353360. BRADFORD,M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. CONN, E.E. 1981. The Biochemistry of Plants, Vol 7, Secondary Plant Products. Academic Press, London. DtxoN, R.A. 1986. The phytoalexin response: Elicitation, signalling, and control of gene expression. Biol. Rev. 61:239-291. HAHLBROCK,K., LAMB,C.J., PURWIN,C., EBEL, J., FAUTZ,E., and SCHAFFER,E. 1981. Rapid response of suspension-cultured parsley cells to the elicitor from Phytophthora megasperma var. sojae. Induction of the enzymes of the.general phenylpropanoid metabolism. Plant Physiol 67:768-773. HARTLEY,S.E., and LAWTON,J.L. 1987. Effects of different types of damage on the chemistry of birch foliage, and the responses of birch feeding insects. Oecologia (Berlin). 74:432-437. HAUKIOJA,E., and HAHNIMAKI,S. 1985. Rapid wound-induced resistance in white birch (Betula pubesence) foligge to the geometrid Epirrata autumnata: A comparison of trees and moths within and outside the outbreak range of the moth. Oecologia (Berlin). 65:223-228. HAUKIOJA, E., and NEUVONEN,S. 1985. Induced long-term resistance of birch foliage against defoliators: Defensive or incidental? Ecology 66:1303-1308.
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HAUKIOJA, E., and NIEMELA,P. 1977. Retarded growth of a geometrid larva after damage to leaves of its host tree. Ann. Zool. Fenn. 14:48-52. KARBAN, R., ADAMCHAK,R., and SCHNATHORST,W.C. 1987. Induced resistance and interspecific composition between spider mites and a vascular wilt fungus. Science 235:678-680. LAWTON, J.H. 1987. Food shortage in the midst of apparent plenty?: The case for birch feeding insects, pp. 219-228, in H.W. Velthuis (ed.). Proceedings of the Third European Congress of Entomology. Nededandse Entomologische Vereniging, Amsterdam. MARTIN, J.S., and MARTIN, M.M. 1982. Tannin assays in ecological studies: Lack of correlation between phenolics, proanthocyanidins and protein-precipitating constituents in mature foliage of six oak species. Oecologia (Berlin) 54:205-211. MITRA, R., GROSS, R.D., and VARNER,J.E. 1975. An intact tissue assay for enzymes that labilize C-H bonds. Anal. Biochem. 64:102-109. NIEMELA, P., ARO, E.M., and HAUKIOJA, E. 1979. Birch leaves as a resource for herbivores. Damage-induced increase in leaf phenols with trypsin-inhibiting effects. Rep. Kevo Subartic Res. Stat. 15:37-40. ROEHRIG, N.E., and Capinera, J.L. 1983. Behavioral and developmental responses of range caterpillar larvae, Hemileuca oliviae, to condensed tannin. J. Insect Physiol. 29:901-906. RYAN, C.A. 1974. Assay and biochemical properties of the proteinase inhibitor-inducing factor, a wound hormone. Plant Physiol. 54:328-332. SHAIN, L., and HILLIS, W.E. 1972. Ethylene production in Pinus radiata in response to Sirex, d n,,fl~ t~,,urn,,atta.qk , P,bj to#a Ihn/o~,y,6_2..tdD,7-Jd.D,q SHAW, C.G. 1985. In vitro responses of different Armillaria taxato gallic acid, tannic acid and ethanol. Plant Pathol. 34:594-602. TODD, G.W., GETArtUN, A., and CRESS, D.C. 1971 Resistance in barley to the greenbug Schizaphis granimum I. Toxicity of phenolic and flavanoid compounds. Ann. Entomol. Soc. Am. 64:718722. WERDER, J., and KERN, H. 1985. Resistance of maize to Helminthosporium carbonum: Changes in host phenolics and their antifungal activity. J. Plant Dis. Prot. 92:477-484. WRATTEN, S.D., EDWARDS, P.J., and DUNN, I. 1984. Wound-induced changes in the palatability of Betula pubescens and Betula pendula. Oecologia (Berlin) 61:372-375.
PHENOLIC BIOSYNTHESIS, LEAF DAMAGE, AND INSECT HERBIVORY IN BIRCH (Betula pendula)
S.E. H A R T L E Y and R.D. F I R N Department of Biology University of York Heslington. York. Y01 5DD. England (Received August 5, 1987; accepted November 16, 1987) Abstract--The effect of both caterpillar herbivory and artificial damage on phenylalanine ammonia lysase (PAL) activity of birch foliage was measured, using an intact cell assay. After artificial damage there was a small increase in PAL activity in damaged leaves but no change in adjacent undamaged ones. Insect grazing produced a larger increase in PAL activity, and the enzyme activity was also increased in adjacent undamaged leaves. Artificial damage increased the phenolic levels of the damaged leaves. Insect grazing caused a larger, longer-lasting increase in phenolic levels and also elevated phenolic levels in undamaged leaves. The possible role of these woundinduced biochemical changes in birch is discussed. Key Words--Phenolics, PAL activation, insect herbivory, plant resistance, Betula pendula, Apocheima pilosaria, Lepidoptera, geometridae. INTRODUCTION Wound-induced reductions in leaf palatability to insect herbivores have been reported in Betula pubescens Ehrh. (Haukioja and Niemela, 1977) and Betula pendula Roth (Wratten et al., 1984). Such changes can occur in damaged and adjacent undamaged leaves, and the responses may be part o f a wound-induced antiherbivore defense mechanism (Haukioja and Hahnimaki, 1985; Bergelson et al., 1986; Lawton, 1987). It has been suggested that the decrease in palatability is a result o f an increase in the concentration o f phenolic compounds known to occur in d a m a g e d leaves (Niemela et al., 1979; Wratten et al., 1984; Bergelson et al., 1986). However, palatability to insect herbivores does not always correspond well with phenolic levels (Hartley and Lawton, 1987).
275 0098-033118910100-027500.0010 © 1989 Plenum Publishing Corporation
276
HARTLEYAND FInN
Changes in the phenolic metabolism of damaged birch leaves were investigated at a more fundamental level than has hitherto been possible. Simple phenolic compounds are derived from the shikimate pathway (Conn, 1981). Consequently, by studying the activity of enzymes in this pathway in damaged and undamaged leaves, it may be possible to define, in more precise biochemical terms, the way in which plants respond to insect attack. Differences in the chemical changes induced by artificial damage as opposed to insect-grazing can thus be characterized in terms of enzyme activity, which could be important in assessing whether the changes are likely to be involved in defense against herbivores or pathogens, or simply in repair of wounded tissues. For example, an increase in phenolic compounds in undamaged leaves due to increased enzyme activity in these undamaged tissues, rather than due to transport of phenolics from the site of damage, may indicate a mechanism which is not purely tissue repair. In the present study, the activity of phenylalanine ammonia lysase (PAL) was investigated, together with the concentration of phenolic compounds, in damaged and undamaged leaves from both insect-grazed and artificially damaged trees. Evidence is presented that PAL activity is stimulated by insect attack on birch, even in undamaged leaves, and the response differs from that caused by artificial damage to the leaves.
METHODS AND MATERIALS
PAL Measurements. On July 8, 1986, 12 birch trees, each about 2 m high, were chosen in the University of York tree nursery, where they had been transplanted several years earlier. Twenty-five Apocheima pilosaria larvae (Lepidoptera, Geometridae) Were caged on three branches on each of four of the trees and allowed to graze overnight, by which time 10-15% of the leaf area of the caged branches had been removed. A further four trees had the same amount and distribution of damage inflicted on three of their branches with a pair of scissors (sterilized in alcohol to reduce the risk of fungal infection). The four remaining trees were left as controls. Fifteen leaves were collected from each tree at the start of the experiment, and from the control trees one, three, five, and seven days later, while 15 damaged, and 15 undamaged leaves immediately adjacent to damaged ones, were removed from the treatment trees on the same days. The undamaged leaves included some at the same node, some above, and some below the damaged leaves, as available. Enzyme activity was measured immediately after the leaves were collected using a modified version of the intact cell assay of Amrhein et al. (1976). This "in vivo" method for assaying PAL was chosen in preference to an in vitro enzyme assay in order to obviate possible differences in enzyme recovery from the damaged and adjacent leaves.
PHENOLIC BIOSYNTHESIS IN BIRCH
277
One half gram (wet weight) of 6-mm leaf disks was incubated with 5/zCi L-phenyl(2,3-3H)alanine (Amersham; specific activity 43 Ci/mmoI) in 2 ml of 0.15 M potassium phosphate buffer (pH 5.5) for 3 hr. The labeled phenylalanine was absorbed by the cells, equilibrated with the endogenous substrate, and the action of PAL liberated labeled N3HH2 . This lost 3H to tissue water to produce 3HOH, a virtually irreversible process due to the considerable dilution of the tracer, so recycling is very unlikely. The 3HOH was recovered from the medium by sublimation by a procedure similar to that of Mitra et al. (1975). Twenty microliters of incubation medium, together with a drop of COC12"6H20, was placed on a filter paper disk that was positioned on a pin stuck into the lid of a scintillation vial. Vials were placed in a piece of polystyrene such that the bottom of the vial was in liquid nitrogen while the top was heated with an infrared lamp until the COC12 solution turned blue. The pin and filter paper were then removed, and the 3H activity of the ice formed was determined using a scintillation counter (LKB), following the addition of 2 ml of Optiphase MP (Fisons) to the vial. The PAL activity was measured as counts per minute (cpm) per 0.5 g fresh weight per 3 hr, after correction for background and control counts. (Control counts are the values recorded from incubations using boiled leaves with no PAL activity.) The results were expressed as mean percentage changes over the activity at the start of the experiment (to reduce the effect of the between-tree variation in initial PAL activities) and were analyzed in a one-way ANOVA, tested separately each day. The specific treatments responsible for producing significant effects in the ANOVA were identified using nominated comparison t tests. The leaf material remaining after the enzyme assay had been carried out was freeze-dried and assayed for total phenolics as described below. Field Experiment. On May 15, 1985, at Skipwith Common, 10 miles south of York (grid ref. SE 664379), 15 small Betula pendula trees (each about 1.5 m high) were selected, sprayed with insecticide (Sprayday, Pan Britannica Industries Ltd.; contains resmethrin and pyrethrum), and all natural damage marked with a small dot of paint on leaf petioles. Three weeks later, the leaves of five of the trees were left undamaged, five were cut with scissors, and five were grazed by Apocheima pilosaria, as in the experiment described above. Twenty leaves were collected from each of the control trees at the start of the experiment, and one, three, five, and eight days subsequently. In the case of the other treatments, 20 leaves were collected before damage, but on days one, three, five, and eight, 20 undamaged leaves from a branch adjacent to a treatment branch were collected, in addition to the 20 damaged leaves. Leaves were placed on ice immediately after collection, returned to the laboratory within 1 hr, and stored at - 2 0 ° C . Chemical Analyses. All chemical analysis (except for PAL activity) was carded out on freeze-dried material. Total phenolics were measured using the Folin-Denis method as described in Bergelson et al. (1986), except that 20 mg
278
HARTLEY AND FIRN
of leaf powder was extracted and after centrifugation the volume was adjusted to 10 ml, and then aliquots equivalent to 0.1 and 0.2 mg of leaf-powder were used in the analysis. Protein precipitation was measured using the Bradford method (Bradford, 1976). The procedure was similar to that of Martin and Martin (1982), but slightly modified as described in Hartley and Lawton (1987). The protein-precipitating properties were expressed as the coefficients (slopes) of regression lines fitted to the graphs of milligrams (dry weight) of leaf extracted vs. grams of BSA precipitated (see Martin and Martin 1982 for further details). RESULTS PAL activity was measured in control leaves and in damaged and adjacent undamaged leaves from insect and artificially damaged trees (Figure 1). The activity was found to vary somewhat on a daily basis (Figure 1), with large between-tree differences; hence, although there was a significant effect of treatment on activity on both days 1 ( F = 10.64, df = 4,15, P = 0.0003) and 5 ( F = 5.46, df = 4,14, P = 0.0084), there were no significant effects on day 3, or on day 7, by which time activities in all treatments had decreased to near control levels. In the artificially damaged trees, cut leaves had significantly higher PAL activities than leaves from the control trees one day after damage
o
F-
.E;
100
0 1357
1357
Contr~
Cut leaves
1357 Uncut leaves
k
1357 Grazed leaves
1357
Days elapsed
Ungrazed leaves
FIG. 1. The percentage change in PAL activity in damaged and adjacent undamaged birch leaves over the seven days subsequent to damage, and in control trees. Each value is the mean of measurements of four trees (the same four trees for each sample).
279
PHENOLIC BIOSYNTHESIS IN BIRCH
(t = 4.03, df = 6, P < 0.01), but by day 5 the activity had declined, and there was no longer a significant difference. At no time did the undamaged leaves from these trees show a significant increase in their PAL activity. On the insectgrazed trees, P A L activities were greatly increased, with the damaged leaves having higher activities than both control and cut leaves (t = 6.64, df = 6, P < 0.001, and t = 5.09, df = 6, P < 0.02, respectively). On these trees, the adjacent undamaged leaves had the largest increase in activity of all on day 1 (t = 3.39, df = 3.2; P < 0.05), but this was short-lived; by day 3 there was no significant difference from the controls. However, the elevation of PAL activity in the insect-damaged leaves was more persistent and, unlike the artificially damaged leaves, the level of activity five days after damage was still significantly higher than in control trees (t = 2.58, df = 6, P < 0.05). Samples of the leaves used for enzyme assays were analyzed for total phenolics, and the phenolic levels were found to be elevated in the leaves with increased P A L activity (Figure 2). However, since the amount of phenolics accumulating depends on factors such as the rate of phenolic degradation (which was not measured), and because of the imprecise method used to estimate phenolics, PAL activity and phenolic levels do not correspond exactly. The control leaves and the undamaged leaves on cut trees showed no significant changes in phenolic content, but the cut leaves, and both the damaged and undamaged leaves on the insect-grazed trees all had increased amounts of phenolic compounds (paired t tests, t = 6.64, df = 3, P < 0.01; t = 13.6, df = 3, P < 0.001; and t = 5.79; df = 3; P < 0.02, respectively). Insect attack produced a larger increase in phenolics than artificial damage (two sample t test, t = 3.52, df = 6, P < 0.02).
26.4%
10.4 10.0
c o
9.0
--
10.0%
17.8%
.~
(3
o n
8.0
~% 037 0 1 3 5 Control Cut leaves
01357 Uncut leaves
0 1 3 5 7 0 1 3 7 Days Grazed Ungrazed leaves
elapsed
leaves
FIG. 2. The effect of leaf damage on phenolic levels in birch leaves. The values are the means of measurements made on four trees, and the same trees were used on each sampling day. The percentage change in phenolic content after seven days is also shown.
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HARTLEY AND FIRN
The results of the 1985 field experiment also show chemical differences between insect and artificially damaged leaves. The level of phenolic compounds in control trees (Figure 3) did not differ significantly over an eight-day experimental period (paired t test, t = 1.36, d f = 4; P > 0.1). In trees with leaves that were cut with scissors (Figure 3), the phenolic levels increased in the damaged leaves (paired t test, t = 5.91, d f = 4, P < 0.01), but not in undamaged ones from an adjacent branch (paired t test, t = 1.33, d f = 4, P > 0. I). Again, in insect-grazed leaves, the phenolics rose to higher levels than in cut leaves (two sample t test on eight-day values: t = 3.14, d f = 8, P < 0.02), and undamaged leaves on the neighboring branch to the grazing also had slightly increased phenolic levels after eight days (paired t test, t = 2.99; d f = 4, P < 0.05). However, the change in the undamaged foliage was less than in the 1986 experiment, perhaps because the undamaged leaves in that experiment were immediately adjacent to the insect-damaged ones. The protein-precipitating abilities of extracts of artificially damaged, insectgrazed, and control trees were also made. However, these measurements failed to reveal any significant increases in protein-precipitating ability resulting from insect grazing, although a slight, but statistically significant, increase was recorded in artificially damaged leaves (data not shown). These results suggested that assays of protein-precipitation ability and Folin-Denis measurements were not equivalent as indicators of insect-induced chemical changes, possibly because they were measures of different kinds of chemicals, and no further protein precipitation measurements were made.
f
¢5 •,~
26.5%
1 1.796
11.0
10.0
1.0%
0 i
o
9.0
-'0.7% ~
~= 8.4 0 1 3 6
Control
0
38 Cut leaves
0 1 3 8 Uncut leaves
0 1 3 Grazed leaves
0 1 3 8 Days elapsed Ungrazed leaves
FIG. 3. The effect of leaf damage on the phenolic content of birch leaves. Each value is the mean of measurements of five trees, each tree sampled on the days shown. The percentage change in phenolic content after eight days is also shown.
281
PHENOLIC BIOSYNTHESIS IN BIRCH
DISCUSSION
It is clear from previous work (e.g., Wratten et al., 1984), and from the results presented here, that phenolic concentrations are elevated in insect-grazed birch leaves and are also higher in undamaged leaves adjacent to those which were damaged. The finding that PAL activity is also increased in such leaves suggests that these elevated levels of phenolic compounds are the consequence of increased in situ biosynthetic activity, rather than mobilization of phenolics from the site of damage. The response to artificial damage was different, however, with smaller changes both in terms of PAL activity and phenolic concentration, and no changes occurring in adjacent undamaged leaves. It has been suggested that such a response, in which insect damage is a more effective inducer of chemical changes than artificial damage and in which synthesis of phenolics occurs at sites remote from damage, represents an active and specific defense, rather than a wound-repair mechanism (e.g., Haukioja and Neuvonen, 1985). However, a number of questions require further investigation before the role of these wound-induced changes is clear. First, is the increased PAL activity the result of enzyme activation or enzyme synthesis? A number of stimuli, such as light, chemicals, and fungal infection, can increase PAL activity in plants, and it has been shown in some cases that increased synthesis of mRNA coding for PAL is associated with this increase in PAL activity (e.g., Dixon, 1986). The second question is, what is the nature of the transmitted factor that passes from insect-grazed leaves to adjacent leaves, resulting in elevated PAL activity? Transmittable factors have been reported in plants following damage (Ryan, 1974), and, interestingly, "elicitors" capable of inducing PAL in plant ceils are found in some fungal-plant interactions (Hahlbrock et al., 1981). The fact that increased PAL activity is a common response of plants subject to fungal attack raises a third question: what is the relevance of the increased PAL activity found in the insect-grazed leaves? Although insect grazing had a more marked effect on PAL activity than an equivalent amount of damage inflicted with scissors, this could simply reflect the contamination of caterpillar mouthparts with fungal spores (Shain and Hillis, 1972). A second possibility is that grazing produces a more severe stress on the leaf by a sustained period of physical damage. The function of the damage-induced increase in phenolic levels is also hard to assess since the responses of leaves to damage might include the induction of many chemicals, some of which may be primarily antifungal and some of which might play a role in defense against insects. Phenolics have certainly been associated with resistance to fungal attack (Werder and Kern, 1985), as well as antiherbivore effects (Bennett, 1965; Todd et al., 1971; Roehrig and
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HARTLEY AND FIRN
Capinera, 1983), as has leaf d a m a g e (Karban et al., 1987). H o w e v e r , the role o f phenolics is not entirely clear, as both insects (Bernays, 1981) and fungi (Shaw, 1985) can remain unaffected, and birch-feeding insects often seem indifferent to the chemical changes induced in d a m a g e d leaves (Lawton, 1987; Hartley and L a w t o n , 1987; but see Bergelson et al., 1986; Wratten et al., 1984). Although the ecological significance o f the reported changes in P A L activity remains to be established, this study has clearly s h o w n a differential response o f birch trees to insect and artificial d a m a g e and that insect attack produces marked changes in phenolic biosynthesis in both grazed and adjacent u n d a m aged leaves. Furthermore, these results suggest that the in vivo P A L assay may prove to be a sensitive measure of insect and mechanical d a m a g e - i n d u c e d c h e m ical changes. Further studies are needed on other plant species and on the mecha n i s m of increasing P A L activity to progress towards a better u n d e r s t a n d i n g o f the way in which plants detect and respond to insect attack. Acknowledgments--Permission to work at Skipwith Common is granted by the Yorkshire Wildlife Trust. S.E.H. was supported by a SERC studentship. We are grateful to John Lawton and Clive Jones for helpful comments on the manuscript, and to Philip Warren for technical assistance.
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HAUKIOJA, E., and NIEMELA,P. 1977. Retarded growth of a geometrid larva after damage to leaves of its host tree. Ann. Zool. Fenn. 14:48-52. KARBAN, R., ADAMCHAK,R., and SCHNATHORST,W.C. 1987. Induced resistance and interspecific composition between spider mites and a vascular wilt fungus. Science 235:678-680. LAWTON, J.H. 1987. Food shortage in the midst of apparent plenty?: The case for birch feeding insects, pp. 219-228, in H.W. Velthuis (ed.). Proceedings of the Third European Congress of Entomology. Nededandse Entomologische Vereniging, Amsterdam. MARTIN, J.S., and MARTIN, M.M. 1982. Tannin assays in ecological studies: Lack of correlation between phenolics, proanthocyanidins and protein-precipitating constituents in mature foliage of six oak species. Oecologia (Berlin) 54:205-211. MITRA, R., GROSS, R.D., and VARNER,J.E. 1975. An intact tissue assay for enzymes that labilize C-H bonds. Anal. Biochem. 64:102-109. NIEMELA, P., ARO, E.M., and HAUKIOJA, E. 1979. Birch leaves as a resource for herbivores. Damage-induced increase in leaf phenols with trypsin-inhibiting effects. Rep. Kevo Subartic Res. Stat. 15:37-40. ROEHRIG, N.E., and Capinera, J.L. 1983. Behavioral and developmental responses of range caterpillar larvae, Hemileuca oliviae, to condensed tannin. J. Insect Physiol. 29:901-906. RYAN, C.A. 1974. Assay and biochemical properties of the proteinase inhibitor-inducing factor, a wound hormone. Plant Physiol. 54:328-332. SHAIN, L., and HILLIS, W.E. 1972. Ethylene production in Pinus radiata in response to Sirex, d n,,fl~ t~,,urn,,atta.qk , P,bj to#a Ihn/o~,y,6_2..tdD,7-Jd.D,q SHAW, C.G. 1985. In vitro responses of different Armillaria taxato gallic acid, tannic acid and ethanol. Plant Pathol. 34:594-602. TODD, G.W., GETArtUN, A., and CRESS, D.C. 1971 Resistance in barley to the greenbug Schizaphis granimum I. Toxicity of phenolic and flavanoid compounds. Ann. Entomol. Soc. Am. 64:718722. WERDER, J., and KERN, H. 1985. Resistance of maize to Helminthosporium carbonum: Changes in host phenolics and their antifungal activity. J. Plant Dis. Prot. 92:477-484. WRATTEN, S.D., EDWARDS, P.J., and DUNN, I. 1984. Wound-induced changes in the palatability of Betula pubescens and Betula pendula. Oecologia (Berlin) 61:372-375.
