Journal of Chemical Ecology, Vol. 17, No. 3, 1991
R E S P O N S E OF T O T A L T A N N I N S A N D P H E N O L I C S IN L O B L O L L Y PINE F O L I A G E E X P O S E D TO O Z O N E A N D ACID RAIN l
D.N. JORDAN, 2 T.H. GREEN, B.G. LOCKABY, D.H.
A.H. CHAPPELKA,*
R.S. MELDAHL,
and
GJERSTAD
School of Forestry Auburn University Auburn, Alabama 36849-5418 (Received August 6, 1990; accepted October 29, 1990) Abstract--Tannin and total phenolic levels in the foliage of loblolly pine (Pinus taeda L.) were examined in order to evaluate the effect of atmospheric pollution on secondary plant metabolism. The trees were exposed to four ozone concentrations and three levels of simulated acid rain. Tannin concentration (quantity per gram) and content (quantity per fascicle) were increased in foliage exposed to high concentrations of ozone in both ozone-sensitive and ozone-tolerant families. No effect of acid rain on tannins was observed. Neither total phenolic concentration nor content was significantly affected by any treatment, indicating that the ozone-related increase in foliar tannins was due to changes in allocation within the phenolic group rather than to increases in total phenolics. The change in allocation of resources in the production of secondary metabolites may have implications in herbivore defense, as well as for the overall energy balance of the plant. Key Words--Plant defense, air pollution, acidic deposition, biological indicators, plant polyphenols, total phenolics, proanthocyanidins, condensed tannins, secondary metabolites, Pinus taeda.
INTRODUCTION T r e e s p o s s e s s b o t h c h e m i c a l a n d structural d e f e n s e s a g a i n s t p a t h o g e n s a n d herb i v o r y ( L o e h l e , 1988; M c L a u g h l i n a n d S h r i n e r , 1980; Stafford, 1988). W h i l e *To whom correspondence should be addressed. t AAES Journal no. 9-902690P. 2Current address: Botany Department, University of Wyoming, Laramie, Wyoming 82017. 505
0098-0331/9t/0300-0505506.50/09 1991PlenumPublishingCorporation
506
JORDANET AL.
not the only defense available to plants, tannins may contribute to plant defense by increasing tissue toughness (Stafford, 1988) or by reducing nutritive value of foliage (Mole and Waterman, 1988). Tannins consist of polymerized phenols and are divided into two groups, condensed and hydrolyzable tannins (Goodwin and Mercer, 1983). Haslam (1988) states that hydrolyzable tannins are confined to the dicotyledons, indicating that the tannins produced by loblolly pine are condensed tannins (CT). Foliar tannin content is affected by various environmental factors. Schultz and Baldwin (1982) and Schultz (1988) found increased tannin content in red, oak leaves (Quercus rubra) following insect defoliation. Walters and Stafford (1984) found increases in condensed tannin concentration of Douglas-fir (Pseudotsuga menziesii) needles related to insect defoliation and also to mechanical damage. Mole et al. (1988) showed changes in condensed tannins in the foliage of several rain-forest species positively correlated with light intensity. Coley et al. (1985) suggest that chemical defenses are inversely correlated with resource availability. Gershenzon (1984) reported that deficiencies in nitrogen, sulfur, and other nutrients lead to increased production of phenolics in many plant species. Plant susceptibility to disease or herbivory may be mediated by atmospheric pollution. Manning et al. (1969, 1970) found that ozone damage increased disease rates and infection severities of potato (Solanum tuberosum) and geranium (Pelargonium hortorum) plants inoculated with Botrytis cinerea. Hain (1987) discusses ozone effects on ponderosa pine (P. ponderosa) in conjunction with attacks by the westem pine beetle (Dendroctonus brevicomis) and mountain pine beetle (D. ponderosae). He suggests that increased susceptibility is offset by decreased suitability, preventing an increase in overall infestation rate. Ozone has been shown to affect tannin and total phenolic content of several plant species (Howell, 1974). Tingey (1989) discusses the importance of discovering an unambiguous indicator of pollution stress. Loehle (1988) states that leaf defenses should be a good variable for assessing pollution impact, because the allocation of carbon to defensive compounds in the foliage should be reduced as a result of decreased vigor in pollution-stressed trees. Jones and Coleman (1989) suggest that phenolics, in conjunction with some other biochemical indices, might be useful as an indicator of pollution effects, but differentiate between the effects of direct damage to plant structure and carbon stress due to interference with normal plant function by the pollutant. These authors predicted an increase in polymerized carbon-based secondary metabolites with pollution damage. The current study was conducted to determine whether atmospheric pollutants could alter foliar levels of defensive compounds (tannins and phenolics) in loblolly pine trees and to establish the basis for further inves-
OZONE, ACID RAIN EFFECTS
507
tigations into the utility of plant phenolics as indicators of pollution stress, as well as the impacts of these stresses on forest pests and/or pathogens.
METHODS AND MATERIALS
Exposure Facilities. The effects of ozone and acid rain on loblolly pine in central Alabama were investigated using cylindrical open-top chambers (4.5 m diam. x 4.8 m high). The site is located on the upper Coastal Plain of eastern Alabama. The soil series present is a Cowarts, a Typic Kanhapludult. Sixmonth-old seedlings from two families of loblolly pine were planted in 24 chambers in January 1988. The families used were GAKR 15-23, considered to be ozone-tolerant (Reinert et al., 1988) and GAKR 15-91, regarded as ozonesensitive (McLaughlin et al., 1988). The chambers were constructed with rain exclusion covers and were exposed to three target pH levels of artificial rain (pH 3.3, 4.3, and 5.3). Rain solutions were prepared by adjusting the pH of deionized water containing selected background ions (Cogbill and Likens, 1974) with a 1 N mixture of HeSO 4 and HNO3 in a 3 : 1 ratio. Rain was applied twice weekly through stainless-steel cone nozzles mounted at the top of each chamber. Rain volumes were based on monthly averages for the preceding 30-year period (Chappelka et al., 1990a). In addition to the acid rain treatments, four ozone treatments were applied as multiples of the ambient concentration. Treatments were attained by filtering ambient air through charcoal filters (CF), applying nonfiltered ambient air (NF), and supplementing ozone concentrations to 1.7 and 2.5 times ambient (1.7 x NF and 2.5 x NF) (Chappelka et al., 1990a). The experiment was established as a 4 x 3 factorial with two replications. Actual ozone concentrations and rain pH, as well as a more detailed description of the application system, are reported by Chappelka et al. (1990b). Additional trees from both families, which had been grown on the site outside of chambers, were also sampled to evaluate chamber effect. These trees were planted in a similar configuration to those in chambers but were exposed to natural air and rainfall. Sampling Technique. In November of the second growing season, four trees in each chamber were sampled. Six fascicles per tree were removed from the second flush of current-year (1989) growth on a south-facing primary branch in order to limit potential differences in tannins and phenolics due to light exposure or leaf age (Mole et al., 1988). Fascicles from two trees from each family from each chamber were composited, resulting in one sample per family per chamber. Laboratory Analyses. The fascicles were weighed, stored on ice, and trans-
508
JORDAN ET AL.
ported to the laboratory, where they were immediately cut into 0.5-2.0-mm sections. One-gram samples were ground in 5 ml of 70 % acetone with a mortar and pestle and then centrifuged at 5000 rpm for 15 min. The resulting supernatants were stored in the dark at 4~ until analysis. Tannin samples were analyzed in duplicate by a modification of the radial diffusion assay of Hagerman (1987). A gel containing 1% (w/v) agarose and 0.05% bovine serum albumin (BSA, fraction V) in 50 mM acetic acid and 60 /zM ascorbic acid at pH 5.0 was formed by pouring 9.5-ml aliquots of warm solution into standard plastic Petri plates and allowing each to cool overnight at 4~ Four 3-mm wells per plate then were punched in the gel. Four 8-/zl aliquots of sample were applied to each well, with sufficient time allowed for absorption, but not for complete drying between applications. After incubation for 90-120 hr at 30~ two perpendicular radii of each sample were measured with a binocular microscope and platform micrometer to the nearest 0.01 ram. The radii were averaged, and the area of binding was calculated. The areas of the duplicate samples were averaged for the statistical analysis. Results are presented in mm 2 per gram fresh weight for concentration data and mm 2 per fascicle for content data. Phenolics in the extract were determined with a colorimetric assay using 2.0 N Folin and Ciocalteu's phenol reagent (Sigma Chemical Co., St. Louis, Missouri). This method yields molar absorptivity specific for each phenolic compound (Singleton and Rossi, 1965) and therefore gives results impossible to present in absolute concentration units. Phenolic results are presented in A760 per gram and A76o per fascicle for concentration and content respectively. Data were analyzed using SAS (SAS Institute, Cary, North Carolina), analysis of variance (ANOVA), and linear regression procedures. All significance was reported at the P = 0.05 level.
RESULTS
Ozone exposure caused an increase in concentration and content of foliar CT in loblolly pine trees (Table 1). Linear regression analysis showed a significant increase in CT concentration (CT/g = 33.4 + 5.3 X ozone, r 2 = 0.37, P _ 0.0001) and content (CT/fascicle = 12.8 + 1.6 x ozone, r 2 = 0.16, P = 0.005) with increasing ozone concentration. There was no significant difference between families in foliar tannin levels. However, the ozone-sensitive family exhibited the highest tannin levels of the experiment in the high ozone treatment. No significant differences in CT were observed among acid rain treatments, nor were significant acid rain x ozone interactions observed in this study. No significant differences were observed in the total phenolic (TP) con-
OZONE, ACID RAIN EFFECTS
509
centration or content among either ozone or acid rain treatments or between families (Table 1). The overall ratio of CT to TP was significantly increased (CT/TP = 37.4 + 6.7 x ozone, r 2 = 0.27, P _ 0.0001) with ozone exposure, indicating a redistribution within the phenolic group (Figure 1). Chambers themselves had no effect on either foliar tannins or total phenolics. The trees growing outside the chambers had levels of tannins and phenolics comparable to the ambient (NF, pH 5.3) chambers. Phenolic concentrations of these trees were 1.022 A 7 6 o / g and 1.027 A 7 6 o / g , and tannin concentrations were 40.02 mmZ/g and 41.23 mm2/g for the tolerant and sensitive families, respectively. These data were not used in statistical analyses for ozone or acid rain effects.
DISCUSSION
The increase in foliar CT with ozone exposure corresponds to an observed increase in visible injury and reduction in foliar biomass (Chappelka et al., 1990b). This is contrary to what is proposed by Loehle (1988), who predicted decreases in chemical defenses, including tannins, associated with reduced vigor
TABLE ]L. TANNIN AND PHENOLIC CONCENTRATION (mm2/g AND A760/g, RESPECTIVELY) AND CONTENT (mm2/FAsCICLE AND A760/FAsCICLE , RESPECTIVELY) BY FAMILY, OZONE EXPOSURE, AND p H TREATMENT
Treatments a
Ozone Variable
CF
NF
1.7 x NF
Rain pH 2.5 x NF
3,3
4.3
5.3
Tolerant family TC b 32.8 (1.3) 38.7 (1.4) 38,5 (1.1) 45.5 (4.2) 38.7 (2.4) 38.4 (1.7) 39,6 (3.4) TCO 11.6 (1.2) 13,8 (1.3) 14.7 (0.3) 15.8 (1.3) 14.0 (0.9) 13.8 (1,2) 14.1 (1.2) PC 0.86 (0.06) 1.02 (0.03) 0,89 (0.06) 0.84 (0.09) 0.88 (0.06) 0.90 (0.05) 0.94 (0,06) PCO 0.31 (0.03) 0.36 (0.03) 0.34 (0.02) 0.29 (0.03) 0.32 (0,03) 0.33 (0.03) 0.33 (0.33) Sensitive family TC 37.3 (2.2) 38.3 (1.9) 36.4 (1.7) 54.6 (1.8) 42.5 TCO 14.8 (1.8) 15.1 (1.4) 13.2 (1.2) 19.6 (1.3) 15.2 PC 0.86 (0.06) 0.97 (0.05) 0.79 (0.07) 0.93 (0.07) 0.89 PCO 0.34 (0.04) 0.38 (0.02) 0.29 (0.04) 0.33 (0.03) 0.32
(3.8) 41.1 (1.6) 15.7 (0.04) 0.81 (0.03) 0.30
(3.2) 41.3 (2.8) (1.6) 16.2 (1.2) (0.08) 0.97 (0.03) (0.03) 0.38 (0.02)
aCF = charcoal-filtered air; NF = nonfiltered air; 1.7 x NF = nonfiltered air x 1.7 ambient ozone; 2.5 x NF = nonfiltered air x 2.5 ambient ozone. bTC = tannin concentration; TCO = tannin content; PC = phenolic concentration; PCO = phenolic content; means are followed in parenthesis by + SE.
510
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Tolerant Family 50
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25
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0
CF
NF
1.7 x NF 2.5x NF
FIG. 1. Tannin-phenolic ratio by ozone exposure and family. Vertical bars represent standard error of the mean. CF -= charcoal filtered air; NF = nonfiltered air; 1.7 x NF = 1.7 x ambient ozone; 2.5 x NF = 2.5 x ambient ozone.
resulting from pollution stress. Our data indicate that the increase in tannin production with increased ozone exposure is not a vigor-mediated response but is apparently a direct response to ozone-induced membrane damage. This interpretation agrees with Jones and Coleman (1989), who suggested that structural damage resulting from air pollution would cause an increase in polymerized carbon-based secondary metabolites. The increases in secondary metabolites might have a substantial impact on the overall energy budget of the plant (McLaughlin and Shriner, 1980). Resources dedicated to production of defensive compounds cannot be utilized in growth and/or repair processes. From an energy or carbon allocation per-
OZONE, ACID RAIN EFFECTS
51 1
spective, the ozone-treated trees have diverted a greater percentage of resources toward secondary metabolism. The qualitative correlation of visible injury, increased tannins, and high ozone exposure supports Howell's (1974) assertion that membrane damage and reallocation to secondary metabolites may contribute to ozone-related growth reductions. Gershenzon (1984) reported increased phenolic production related to nutrient limitation in a number of species. In the current study, increased resource availability (nitrogen and sulfur supplied in pH treatments) did not alter the allocation of resources to defense, as represented by tannin content, although growth of the low pH-treated trees was significantly increased (Chappelka et al., 1990b). This is contrary to the carbon-nutrient balance hypothesis of Bryant et al. (1983), which suggests that at higher nutrient availability, less carbon is available for production of carbon-based defensive compounds. Based on this hypothesis, the trees exposed to low pH (higher nutrients) would have been expected to have lower foliar CT levels. The ease of the radial diffusion assay of Hagerman (1987), the increase in tannins with ozone, and the lack of an acid rain effect indicate that foliar tannin levels would fit the criteria of Tingey (1989), who states that pollution indicators should: provide a readily detectable response to the pollutant, be easy to use and readily related to the response of interest, and have a distinctive syndrome not easily confused with other causes. Further studies are needed to establish whether foliar tannin levels are useful unambiguous chemical indicators of ozone stress, either alone or in combination with other measures. If foliar tannin concentration is an important factor in deterring insect or pathogen attack, ozone and acid rain exposure at the applied rates does not appear likely to result in increased pest damage. Increased insect feeding has been associated with ozone by some researchers (Chappelka et al., t988; Hain, 1987; Trumble et al., 1987). However, as Hain (1987) noted, increased host susceptibility does not imply increased infection or predation, provided that host suitability is also decreased. Decreased suitability may result from reduced foliar nutrient content or reduced digestibility of foliage (Coley, 1986). Increased attractiveness and decreased nutritive value would result in the type of susceptibility-suitability relationships described by Hain (1987). Further experiments are needed to determine whether tannin increases of this magnitude are effective against herbivory and if concurrent changes in foliar nutrient content or other defense mechanisms would further discourage attack or render the foliage more desirable. Acknowledgments--The authors would like to thank Daureen Nesdill for her demonstration of tannin-binding evaluation; John Kush, Mark Bass, Karen Westin, and Efrem Robbins for their data collection; and Alan Tiarks, Bob Jones, Greg Somers, and Nancy Stumpff for their reviews of the manuscript. The study was partially supported by funds provided by the Southeastern Forest Experiment Station, Southern Commercial Forest Research Cooperative of the Forest Response
512
JORDAN ET AL.
Program. The Forest Response Program, part of the National Acid Precipitation Assessment Program, is jointly sponsored by the USDA Forest Service, US Environmental Protection Agency, and the National Council of the Paper Industry for Air and Stream Improvement. This paper has not been subject to EPA or Forest Service policy/review and should not be construed to represent the policies of either agency or NCASI.
REFERENCES
BRYANT, J.P., CHAPIN, F.S., III, and KLEIN, D.R. 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40:357-368. CHAPPELKA, A.H., KRAEMER, M.E., MEBRAI-ITU,T., RANGAPPA,M., and BENEPAL, P.S. 1988. Effects of ozone on soybean resistance to the Mexican bean beetle (Epilachna varivestis Mulsant). Environ. Exp. Bot. 28:53-60. CHAPPELKA,A.H., KUSH, J.S., MELDAHL,R.S., and LOCKABY,B.G. 1990a. An ozone-low temperature interaction in loblolly pine (Pinus taeda L.). N. PhytoL 114:721-726. CHAPPELKA, A.H., LOCKABY, B.G., MITCHELL, R.J., MELDAHL, R.S., KUSH, J.S., and JORDAN, D.N. 1990b. Growth and physiological responses of loblolly pine exposed to ozone and simulated acid rain in the field, in Proceedings of Air and Waste Management Association. June 24-29, 1990, Pittsburgh, Pennsylvania. 90-187.5. COGBILL,C.V., and LIKENS, G.E, 1974. Acid precipitation in the northeastern United States. Water Resour. Res. 10:1133-1137. COLEY, P.D. 1986. Costs and benefits of defense by tannins in a neotropical tree. Oecologia. 70:238-241. COLEY, P.D., BYRANT,J.P., and CHAPIN, F.S., III. 1985. Resource availability and plant antiherbivore defense. Science. 230:895-899. GERSHENZON,J. 1984. Changes in the levels of plant secondary metabolites under water and nutrient stress. Recent Adv. Phytochem. 18:273-320. GOODWlN, T.W., and MERCER, E.I. 1983. Plant phenolics, pp. 567-626, in Introduction to Plant Biochemistry, 2rid ed. Pergamon Press, Oxford. HAGERMAN, A.E. 1987. Radial diffusion method for determining tannin in plant extracts. J. Chem. Ecol. 13:437-446. HAIN, F.P. 1987. Interactions of insects, trees and air pollutants. Tree Physiol. 3:93-102. HASLAM, E. 1988. Plant polyphenols (syn. vegetable tannins) and chemical defense--a reappraisal. J. Chem. Ecol. 14:1789-1805. HOWELL, R.K. 1974. Phenols, ozone, and their involvement in pigmentation and physiology of plant injury, pp. 94-105, in M. Dugger (ed.). Air Pollution Effects on Plant Growth. American Chemical Society, Washington, D.C. JONES, C.G., and COLEMAN, J.S. 1989. Biochemical indicators of air pollution effects in trees: unambiguous signals based on secondary metabolites and nitrogen in fast-growing species? pp. 261-274, in Biologic Markers of Air-Pollution Stress and Damage in Forests. National Academy Press, Washington, D.C. LOEHLE, C. 1988. Forest decline: Endogenous dynamics, tree defenses, and the elimination of spurious correlation. Vegetatio 77:65-78. MANNING, W.J., FEbER, W.A., PERKINS, I., and GLUer;MAN, M. 1969. Ozone injury and infection of potato leaves by Botrytis cinerea. Plant Dis. Rep. 53:691-693. MANNING, W.J., FEDER, W.A., and PERKINS, I., 1970. Ozone injury increases infection of geranium leaves by Botrytis einerea. Phytopathology 60:669-670. MCLAUGHLIN, S.B., and SHRINER, D.S. 1980. Allocation of resources to defense and repair, p.
OZONE, ACID RAIN EFFECTS
5 13
407-431, in J.G. Horsfall and E.B. Cowling (eds.). Plant Disease: An Advanced Treatise, Vol. V. How Plants Defend Themselves. Academic Press, New York. McLAUGHLIN, S.B., ADAMS, M.B., EDWARDS, N.T., HANSON, P.J., LAYTON, P.A., O'NEILL, E.G., and RoY, W.K. 1988. Comparative sensitivity, mechanisms, and whole plant physiological implications of responses of loblolly pine genotypes to ozone and acid deposition. Environmental Sciences Division, Oak Ridge National Laboratory, Publication No. 3105. 301 pp. MOLE, S., and WATERMAN, P.G. 1988. Light-induced variation in phenolic levels in foliage of rain-forest plants. II. Potential significance to herbivores. J. Chem. Ecol. 14:23-34. MOLE, S., ROSS, J.A.M., and WATERMAN, P.G. 1988. Light-induced variation in phenolic levels in foliage of rain-forest plants. I. Chemical changes. J. Chem. Ecol. 14:1-21. REINERT, R.A., SCHOENEBERGER,M.M., SHAFER, S.R., EASON, G., HORTON, S.J., and WELLS, C. 1988. Responses of Ioblolly pine half-sib families to ozone, in Proceedings of the 81st Annual Meeting of the Air Pollution Control Association, Dallas, Texas. 88-t25.2. SCHULTZ, J.C. 1988. Plant responses induced by herbivores. Trends EcoL Evol. 3:45-49. SCHULTZ,J.C., and BALDWIN, I.T. 1982. Oak leaf quality declines in response to defoliation by gypsy moth larvae. Science 217:149-151. SINGLETON, V.L., and ROSSl, J.A., JR. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16:144-158. STAFFORD,H.A. 1988. Proanthocyanidins and the lignin connection. Phytochemistry 27:1-6. TINGLY, D.T. 1989. Bioindicators in air pollution research--applications and constraints, pp. 7380, in Biologic Markers of Air-Pollution Stress and Damage in Forests. National Academy Press, Washington, D.C. TRUMBLE,J.T., HARE,J.D., MUSSELMAN,R.C., and McCooL, P.M. 1987. Ozone-induced changes in host-plant suitability: Interactions of Keiferia lycopersicella and Lycopersicon esculentum. J. Chem. Ecol. 13:203-218. WALTERS, T., and STAFFORD, H.A. 1984. Variability in accumulation of proanthocyanidins (condensed tannins) in needles of Douglas fir (Pseudotsuga menziesii) following long-term budworm defoliation. J. Chem. Ecol. 10:1469-1476.
R E S P O N S E OF T O T A L T A N N I N S A N D P H E N O L I C S IN L O B L O L L Y PINE F O L I A G E E X P O S E D TO O Z O N E A N D ACID RAIN l
D.N. JORDAN, 2 T.H. GREEN, B.G. LOCKABY, D.H.
A.H. CHAPPELKA,*
R.S. MELDAHL,
and
GJERSTAD
School of Forestry Auburn University Auburn, Alabama 36849-5418 (Received August 6, 1990; accepted October 29, 1990) Abstract--Tannin and total phenolic levels in the foliage of loblolly pine (Pinus taeda L.) were examined in order to evaluate the effect of atmospheric pollution on secondary plant metabolism. The trees were exposed to four ozone concentrations and three levels of simulated acid rain. Tannin concentration (quantity per gram) and content (quantity per fascicle) were increased in foliage exposed to high concentrations of ozone in both ozone-sensitive and ozone-tolerant families. No effect of acid rain on tannins was observed. Neither total phenolic concentration nor content was significantly affected by any treatment, indicating that the ozone-related increase in foliar tannins was due to changes in allocation within the phenolic group rather than to increases in total phenolics. The change in allocation of resources in the production of secondary metabolites may have implications in herbivore defense, as well as for the overall energy balance of the plant. Key Words--Plant defense, air pollution, acidic deposition, biological indicators, plant polyphenols, total phenolics, proanthocyanidins, condensed tannins, secondary metabolites, Pinus taeda.
INTRODUCTION T r e e s p o s s e s s b o t h c h e m i c a l a n d structural d e f e n s e s a g a i n s t p a t h o g e n s a n d herb i v o r y ( L o e h l e , 1988; M c L a u g h l i n a n d S h r i n e r , 1980; Stafford, 1988). W h i l e *To whom correspondence should be addressed. t AAES Journal no. 9-902690P. 2Current address: Botany Department, University of Wyoming, Laramie, Wyoming 82017. 505
0098-0331/9t/0300-0505506.50/09 1991PlenumPublishingCorporation
506
JORDANET AL.
not the only defense available to plants, tannins may contribute to plant defense by increasing tissue toughness (Stafford, 1988) or by reducing nutritive value of foliage (Mole and Waterman, 1988). Tannins consist of polymerized phenols and are divided into two groups, condensed and hydrolyzable tannins (Goodwin and Mercer, 1983). Haslam (1988) states that hydrolyzable tannins are confined to the dicotyledons, indicating that the tannins produced by loblolly pine are condensed tannins (CT). Foliar tannin content is affected by various environmental factors. Schultz and Baldwin (1982) and Schultz (1988) found increased tannin content in red, oak leaves (Quercus rubra) following insect defoliation. Walters and Stafford (1984) found increases in condensed tannin concentration of Douglas-fir (Pseudotsuga menziesii) needles related to insect defoliation and also to mechanical damage. Mole et al. (1988) showed changes in condensed tannins in the foliage of several rain-forest species positively correlated with light intensity. Coley et al. (1985) suggest that chemical defenses are inversely correlated with resource availability. Gershenzon (1984) reported that deficiencies in nitrogen, sulfur, and other nutrients lead to increased production of phenolics in many plant species. Plant susceptibility to disease or herbivory may be mediated by atmospheric pollution. Manning et al. (1969, 1970) found that ozone damage increased disease rates and infection severities of potato (Solanum tuberosum) and geranium (Pelargonium hortorum) plants inoculated with Botrytis cinerea. Hain (1987) discusses ozone effects on ponderosa pine (P. ponderosa) in conjunction with attacks by the westem pine beetle (Dendroctonus brevicomis) and mountain pine beetle (D. ponderosae). He suggests that increased susceptibility is offset by decreased suitability, preventing an increase in overall infestation rate. Ozone has been shown to affect tannin and total phenolic content of several plant species (Howell, 1974). Tingey (1989) discusses the importance of discovering an unambiguous indicator of pollution stress. Loehle (1988) states that leaf defenses should be a good variable for assessing pollution impact, because the allocation of carbon to defensive compounds in the foliage should be reduced as a result of decreased vigor in pollution-stressed trees. Jones and Coleman (1989) suggest that phenolics, in conjunction with some other biochemical indices, might be useful as an indicator of pollution effects, but differentiate between the effects of direct damage to plant structure and carbon stress due to interference with normal plant function by the pollutant. These authors predicted an increase in polymerized carbon-based secondary metabolites with pollution damage. The current study was conducted to determine whether atmospheric pollutants could alter foliar levels of defensive compounds (tannins and phenolics) in loblolly pine trees and to establish the basis for further inves-
OZONE, ACID RAIN EFFECTS
507
tigations into the utility of plant phenolics as indicators of pollution stress, as well as the impacts of these stresses on forest pests and/or pathogens.
METHODS AND MATERIALS
Exposure Facilities. The effects of ozone and acid rain on loblolly pine in central Alabama were investigated using cylindrical open-top chambers (4.5 m diam. x 4.8 m high). The site is located on the upper Coastal Plain of eastern Alabama. The soil series present is a Cowarts, a Typic Kanhapludult. Sixmonth-old seedlings from two families of loblolly pine were planted in 24 chambers in January 1988. The families used were GAKR 15-23, considered to be ozone-tolerant (Reinert et al., 1988) and GAKR 15-91, regarded as ozonesensitive (McLaughlin et al., 1988). The chambers were constructed with rain exclusion covers and were exposed to three target pH levels of artificial rain (pH 3.3, 4.3, and 5.3). Rain solutions were prepared by adjusting the pH of deionized water containing selected background ions (Cogbill and Likens, 1974) with a 1 N mixture of HeSO 4 and HNO3 in a 3 : 1 ratio. Rain was applied twice weekly through stainless-steel cone nozzles mounted at the top of each chamber. Rain volumes were based on monthly averages for the preceding 30-year period (Chappelka et al., 1990a). In addition to the acid rain treatments, four ozone treatments were applied as multiples of the ambient concentration. Treatments were attained by filtering ambient air through charcoal filters (CF), applying nonfiltered ambient air (NF), and supplementing ozone concentrations to 1.7 and 2.5 times ambient (1.7 x NF and 2.5 x NF) (Chappelka et al., 1990a). The experiment was established as a 4 x 3 factorial with two replications. Actual ozone concentrations and rain pH, as well as a more detailed description of the application system, are reported by Chappelka et al. (1990b). Additional trees from both families, which had been grown on the site outside of chambers, were also sampled to evaluate chamber effect. These trees were planted in a similar configuration to those in chambers but were exposed to natural air and rainfall. Sampling Technique. In November of the second growing season, four trees in each chamber were sampled. Six fascicles per tree were removed from the second flush of current-year (1989) growth on a south-facing primary branch in order to limit potential differences in tannins and phenolics due to light exposure or leaf age (Mole et al., 1988). Fascicles from two trees from each family from each chamber were composited, resulting in one sample per family per chamber. Laboratory Analyses. The fascicles were weighed, stored on ice, and trans-
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JORDAN ET AL.
ported to the laboratory, where they were immediately cut into 0.5-2.0-mm sections. One-gram samples were ground in 5 ml of 70 % acetone with a mortar and pestle and then centrifuged at 5000 rpm for 15 min. The resulting supernatants were stored in the dark at 4~ until analysis. Tannin samples were analyzed in duplicate by a modification of the radial diffusion assay of Hagerman (1987). A gel containing 1% (w/v) agarose and 0.05% bovine serum albumin (BSA, fraction V) in 50 mM acetic acid and 60 /zM ascorbic acid at pH 5.0 was formed by pouring 9.5-ml aliquots of warm solution into standard plastic Petri plates and allowing each to cool overnight at 4~ Four 3-mm wells per plate then were punched in the gel. Four 8-/zl aliquots of sample were applied to each well, with sufficient time allowed for absorption, but not for complete drying between applications. After incubation for 90-120 hr at 30~ two perpendicular radii of each sample were measured with a binocular microscope and platform micrometer to the nearest 0.01 ram. The radii were averaged, and the area of binding was calculated. The areas of the duplicate samples were averaged for the statistical analysis. Results are presented in mm 2 per gram fresh weight for concentration data and mm 2 per fascicle for content data. Phenolics in the extract were determined with a colorimetric assay using 2.0 N Folin and Ciocalteu's phenol reagent (Sigma Chemical Co., St. Louis, Missouri). This method yields molar absorptivity specific for each phenolic compound (Singleton and Rossi, 1965) and therefore gives results impossible to present in absolute concentration units. Phenolic results are presented in A760 per gram and A76o per fascicle for concentration and content respectively. Data were analyzed using SAS (SAS Institute, Cary, North Carolina), analysis of variance (ANOVA), and linear regression procedures. All significance was reported at the P = 0.05 level.
RESULTS
Ozone exposure caused an increase in concentration and content of foliar CT in loblolly pine trees (Table 1). Linear regression analysis showed a significant increase in CT concentration (CT/g = 33.4 + 5.3 X ozone, r 2 = 0.37, P _ 0.0001) and content (CT/fascicle = 12.8 + 1.6 x ozone, r 2 = 0.16, P = 0.005) with increasing ozone concentration. There was no significant difference between families in foliar tannin levels. However, the ozone-sensitive family exhibited the highest tannin levels of the experiment in the high ozone treatment. No significant differences in CT were observed among acid rain treatments, nor were significant acid rain x ozone interactions observed in this study. No significant differences were observed in the total phenolic (TP) con-
OZONE, ACID RAIN EFFECTS
509
centration or content among either ozone or acid rain treatments or between families (Table 1). The overall ratio of CT to TP was significantly increased (CT/TP = 37.4 + 6.7 x ozone, r 2 = 0.27, P _ 0.0001) with ozone exposure, indicating a redistribution within the phenolic group (Figure 1). Chambers themselves had no effect on either foliar tannins or total phenolics. The trees growing outside the chambers had levels of tannins and phenolics comparable to the ambient (NF, pH 5.3) chambers. Phenolic concentrations of these trees were 1.022 A 7 6 o / g and 1.027 A 7 6 o / g , and tannin concentrations were 40.02 mmZ/g and 41.23 mm2/g for the tolerant and sensitive families, respectively. These data were not used in statistical analyses for ozone or acid rain effects.
DISCUSSION
The increase in foliar CT with ozone exposure corresponds to an observed increase in visible injury and reduction in foliar biomass (Chappelka et al., 1990b). This is contrary to what is proposed by Loehle (1988), who predicted decreases in chemical defenses, including tannins, associated with reduced vigor
TABLE ]L. TANNIN AND PHENOLIC CONCENTRATION (mm2/g AND A760/g, RESPECTIVELY) AND CONTENT (mm2/FAsCICLE AND A760/FAsCICLE , RESPECTIVELY) BY FAMILY, OZONE EXPOSURE, AND p H TREATMENT
Treatments a
Ozone Variable
CF
NF
1.7 x NF
Rain pH 2.5 x NF
3,3
4.3
5.3
Tolerant family TC b 32.8 (1.3) 38.7 (1.4) 38,5 (1.1) 45.5 (4.2) 38.7 (2.4) 38.4 (1.7) 39,6 (3.4) TCO 11.6 (1.2) 13,8 (1.3) 14.7 (0.3) 15.8 (1.3) 14.0 (0.9) 13.8 (1,2) 14.1 (1.2) PC 0.86 (0.06) 1.02 (0.03) 0,89 (0.06) 0.84 (0.09) 0.88 (0.06) 0.90 (0.05) 0.94 (0,06) PCO 0.31 (0.03) 0.36 (0.03) 0.34 (0.02) 0.29 (0.03) 0.32 (0,03) 0.33 (0.03) 0.33 (0.33) Sensitive family TC 37.3 (2.2) 38.3 (1.9) 36.4 (1.7) 54.6 (1.8) 42.5 TCO 14.8 (1.8) 15.1 (1.4) 13.2 (1.2) 19.6 (1.3) 15.2 PC 0.86 (0.06) 0.97 (0.05) 0.79 (0.07) 0.93 (0.07) 0.89 PCO 0.34 (0.04) 0.38 (0.02) 0.29 (0.04) 0.33 (0.03) 0.32
(3.8) 41.1 (1.6) 15.7 (0.04) 0.81 (0.03) 0.30
(3.2) 41.3 (2.8) (1.6) 16.2 (1.2) (0.08) 0.97 (0.03) (0.03) 0.38 (0.02)
aCF = charcoal-filtered air; NF = nonfiltered air; 1.7 x NF = nonfiltered air x 1.7 ambient ozone; 2.5 x NF = nonfiltered air x 2.5 ambient ozone. bTC = tannin concentration; TCO = tannin content; PC = phenolic concentration; PCO = phenolic content; means are followed in parenthesis by + SE.
510
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CF
NF
1.7 x NF 2.5x NF
FIG. 1. Tannin-phenolic ratio by ozone exposure and family. Vertical bars represent standard error of the mean. CF -= charcoal filtered air; NF = nonfiltered air; 1.7 x NF = 1.7 x ambient ozone; 2.5 x NF = 2.5 x ambient ozone.
resulting from pollution stress. Our data indicate that the increase in tannin production with increased ozone exposure is not a vigor-mediated response but is apparently a direct response to ozone-induced membrane damage. This interpretation agrees with Jones and Coleman (1989), who suggested that structural damage resulting from air pollution would cause an increase in polymerized carbon-based secondary metabolites. The increases in secondary metabolites might have a substantial impact on the overall energy budget of the plant (McLaughlin and Shriner, 1980). Resources dedicated to production of defensive compounds cannot be utilized in growth and/or repair processes. From an energy or carbon allocation per-
OZONE, ACID RAIN EFFECTS
51 1
spective, the ozone-treated trees have diverted a greater percentage of resources toward secondary metabolism. The qualitative correlation of visible injury, increased tannins, and high ozone exposure supports Howell's (1974) assertion that membrane damage and reallocation to secondary metabolites may contribute to ozone-related growth reductions. Gershenzon (1984) reported increased phenolic production related to nutrient limitation in a number of species. In the current study, increased resource availability (nitrogen and sulfur supplied in pH treatments) did not alter the allocation of resources to defense, as represented by tannin content, although growth of the low pH-treated trees was significantly increased (Chappelka et al., 1990b). This is contrary to the carbon-nutrient balance hypothesis of Bryant et al. (1983), which suggests that at higher nutrient availability, less carbon is available for production of carbon-based defensive compounds. Based on this hypothesis, the trees exposed to low pH (higher nutrients) would have been expected to have lower foliar CT levels. The ease of the radial diffusion assay of Hagerman (1987), the increase in tannins with ozone, and the lack of an acid rain effect indicate that foliar tannin levels would fit the criteria of Tingey (1989), who states that pollution indicators should: provide a readily detectable response to the pollutant, be easy to use and readily related to the response of interest, and have a distinctive syndrome not easily confused with other causes. Further studies are needed to establish whether foliar tannin levels are useful unambiguous chemical indicators of ozone stress, either alone or in combination with other measures. If foliar tannin concentration is an important factor in deterring insect or pathogen attack, ozone and acid rain exposure at the applied rates does not appear likely to result in increased pest damage. Increased insect feeding has been associated with ozone by some researchers (Chappelka et al., t988; Hain, 1987; Trumble et al., 1987). However, as Hain (1987) noted, increased host susceptibility does not imply increased infection or predation, provided that host suitability is also decreased. Decreased suitability may result from reduced foliar nutrient content or reduced digestibility of foliage (Coley, 1986). Increased attractiveness and decreased nutritive value would result in the type of susceptibility-suitability relationships described by Hain (1987). Further experiments are needed to determine whether tannin increases of this magnitude are effective against herbivory and if concurrent changes in foliar nutrient content or other defense mechanisms would further discourage attack or render the foliage more desirable. Acknowledgments--The authors would like to thank Daureen Nesdill for her demonstration of tannin-binding evaluation; John Kush, Mark Bass, Karen Westin, and Efrem Robbins for their data collection; and Alan Tiarks, Bob Jones, Greg Somers, and Nancy Stumpff for their reviews of the manuscript. The study was partially supported by funds provided by the Southeastern Forest Experiment Station, Southern Commercial Forest Research Cooperative of the Forest Response
512
JORDAN ET AL.
Program. The Forest Response Program, part of the National Acid Precipitation Assessment Program, is jointly sponsored by the USDA Forest Service, US Environmental Protection Agency, and the National Council of the Paper Industry for Air and Stream Improvement. This paper has not been subject to EPA or Forest Service policy/review and should not be construed to represent the policies of either agency or NCASI.
REFERENCES
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407-431, in J.G. Horsfall and E.B. Cowling (eds.). Plant Disease: An Advanced Treatise, Vol. V. How Plants Defend Themselves. Academic Press, New York. McLAUGHLIN, S.B., ADAMS, M.B., EDWARDS, N.T., HANSON, P.J., LAYTON, P.A., O'NEILL, E.G., and RoY, W.K. 1988. Comparative sensitivity, mechanisms, and whole plant physiological implications of responses of loblolly pine genotypes to ozone and acid deposition. Environmental Sciences Division, Oak Ridge National Laboratory, Publication No. 3105. 301 pp. MOLE, S., and WATERMAN, P.G. 1988. Light-induced variation in phenolic levels in foliage of rain-forest plants. II. Potential significance to herbivores. J. Chem. Ecol. 14:23-34. MOLE, S., ROSS, J.A.M., and WATERMAN, P.G. 1988. Light-induced variation in phenolic levels in foliage of rain-forest plants. I. Chemical changes. J. Chem. Ecol. 14:1-21. REINERT, R.A., SCHOENEBERGER,M.M., SHAFER, S.R., EASON, G., HORTON, S.J., and WELLS, C. 1988. Responses of Ioblolly pine half-sib families to ozone, in Proceedings of the 81st Annual Meeting of the Air Pollution Control Association, Dallas, Texas. 88-t25.2. SCHULTZ, J.C. 1988. Plant responses induced by herbivores. Trends EcoL Evol. 3:45-49. SCHULTZ,J.C., and BALDWIN, I.T. 1982. Oak leaf quality declines in response to defoliation by gypsy moth larvae. Science 217:149-151. SINGLETON, V.L., and ROSSl, J.A., JR. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16:144-158. STAFFORD,H.A. 1988. Proanthocyanidins and the lignin connection. Phytochemistry 27:1-6. TINGLY, D.T. 1989. Bioindicators in air pollution research--applications and constraints, pp. 7380, in Biologic Markers of Air-Pollution Stress and Damage in Forests. National Academy Press, Washington, D.C. TRUMBLE,J.T., HARE,J.D., MUSSELMAN,R.C., and McCooL, P.M. 1987. Ozone-induced changes in host-plant suitability: Interactions of Keiferia lycopersicella and Lycopersicon esculentum. J. Chem. Ecol. 13:203-218. WALTERS, T., and STAFFORD, H.A. 1984. Variability in accumulation of proanthocyanidins (condensed tannins) in needles of Douglas fir (Pseudotsuga menziesii) following long-term budworm defoliation. J. Chem. Ecol. 10:1469-1476.
