Oxidation of Ingested Phenolics in the Tree-Feeding Caterpillar <Emphasis Type="Italic">Orgyia leucostigma</Emphasis> Depends on Foliar Chemical Composition

Tannins are believed to function as antiherbivore defenses, in part, by acting as prooxidants. However, at the high pH found in the midguts of caterpillars, the oxidative activities of different types of tannins vary tremendously: ellagitannins > galloyl glucoses > condensed tannins. Ingested ascorbate is utilized by caterpillars to minimize phenolic oxidation in the midgut. Thus, leaves that contain higher levels of reactive tannins and lower levels of ascorbate were hypothesized to produce higher levels of phenolic oxidation in caterpillars. We tested this hypothesis with eight species of deciduous trees by measuring their foliar phenolic and ascorbate compositions and measuring the semiquinone radical (oxidized phenolic) levels formed in caterpillars that ingested each species. When the generalist caterpillars of Orgyia leucostigma (Lymantriidae) fed on the leaves of tree species in which condensed tannins were predominant (i.e., Populus tremuloides, P. deltoides, and Ostrya virginiana), semiquinone radical levels were low or entirely absent from the midgut contents. In contrast, species that contained higher levels of ellagitannins (or galloyl rhamnoses; i.e., Quercus alba, Acer rubrum, and A. saccharum) produced the highest levels of semiquinone radicals in O. leucostigma. Low molecular weight phenolics contributed relatively little to the overall oxidative activities of tree leaves compared with reactive tannins. Ascorbate levels were lowest in the species that also contained the highest levels of oxidatively active tannins, potentially exacerbating phenolic oxidation in the gut lumen. We conclude that the tannin compositions of tree leaves largely determine the effectiveness of foliar phenolics as oxidative defenses against caterpillars such as O. leucostigma.     

Plant Community Chemical Composition Influences Trembling Aspen (<Emphasis Type="Italic">Populus tremuloides</Emphasis>) Intake by Sheep

Nutrients and plant secondary compounds in aspen (Populus tremuloides) may interact with nutrients in the surrounding vegetation to influence aspen use by herbivores. Thus, this study aimed to determine aspen intake and preference by sheep in response to supplementary nutrients or plant secondary compounds (PSC) present in aspen trees. Thirty-two lambs were randomly assigned to one of four molasses-based supplementary feeds to a basal diet of tall fescue hay (N = 8) during three experiments. The supplements were as follows: (1) high-protein (60% canola meal), (2) a PSC (6% quebracho tannins), (3) 25% aspen bark, and (4) control (100% molasses). Supplements were fed from 0700 to 0900, then lambs were fed fresh aspen leaves collected from stands containing high (Experiment 1, 2) or low (Experiment 3) concentrations of phenolic glycosides (PG). In Experiment 2, lambs were simultaneously offered aspen, a forb (Lathyrus pauciflorus), and a grass (Bromus inermis) collected from the aspen understory. Animals supplemented with high protein or tannins showed greater intake of aspen leaves than animals supplemented with bark or the control diet (P < 0.05), likely because some condensed tannins have a positive effect on protein nutrition and protein aids in PSC detoxification. Overall, animals supplemented with bark showed the lowest aspen intake, suggesting PSC in bark and aspen leaves had additive inhibitory effects on intake. In summary, these results suggest that not only the concentration but also the types and proportions of nutrients and chemical defenses available in the plant community influence aspen use by herbivores.     

Root Chemistry in Populus tremuloides: Effects of Soil Nutrients,Defoliation, and Genotype

Although genetic, environmental, and G x E effects on aboveground phytochemistry have been well documented in trembling aspen (Populus tremuloides), little work has focused on the same factors affecting tissues underground. Belowground plant defenses are likely important mediators of root-feeding herbivores that can strongly influence plant fitness. We used a common garden of potted aspen trees to explore the individual and interactive effects of soil nutrient availability, foliar damage, genotype, and their interactions, on concentrations of phytochemicals in aspen roots. Our common garden experiment employed 12 aspen genotypes that were planted into either low- or high-nutrient soil environments. Half of the trees were subjected to defoliation for two successive years, while the others were protected from damage. At the end of the growing season after the second defoliation, we harvested the trees to obtain root samples for which we assessed levels of phenolic glycosides, condensed tannins, nitrogen, and starch. Phenolic glycosides were most affected by genotype, while the other root phytochemicals were most responsive to soil nutrient conditions. The effects of defoliation were observed in interaction with soil nutrient environment and/or genotype. Interestingly, the effect of defoliation on phenolic glycosides was mediated by soil nutrients, whereas the effect of defoliation on condensed tannins was observed in concert with effects of both soil nutrients and genotype. Comparison of data from this study with an earlier, related study revealed that concentrations of phenolic glycosides and condensed tannins are lower in roots than leaves, and less responsive to defoliation. That soil nutrient environment affects root phytochemical concentrations is not unexpected given the intimate association of roots and soil, but the complex interactions between soil nutrients, aboveground damage, and genotype, and their effects on root phytochemistry, are intriguing. Variation in root chemistry could have wide-reaching effects on soil microbial communities, nutrient cycling, and herbivores. Additionally, the response of phytochemicals to damage across organs can link different, spatially separated herbivores as they use different parts of the same plant resource.     

Phenolics in black oak bark and leaves

Catechin, quercitrin, robinin, quercetin 3-methyl ether, scopoletin, cholorogenic acid, several leucoanthocyanins, and condensed and hydrolyzable tannins were identified in bark and leaves ofQuercus velutina Lamarck. The concentrations of most phenolics in leaves increased as the growing season progressed, whereas those of most phenolics in bark remained essentially unchanged. Qualitative differences in bark and leaf phenolics among different trees were negligible.     

Leaf Ontogeny Influences Leaf Phenolics and the Efficacy of Genetically Expressed Bacillus thuringiensis cry1A(a) d-Endotoxin in Hybrid Poplar Against Gypsy Moth

We tested the hypothesis that ontogenetic variation in leaf chemistry could affect the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin, and thus provide spatial variation in (1) foliage protection and (2) selective pressures that could delay the resistance of folivores. Our model consisted of clonal hybrid Populus plants (NC5339). Consumption of foliage and relative growth rates of gypsy moth, Lymantria dispar (L.) increased, and phenolic glycoside concentrations decreased, as leaves from transformed plants containing the cry1A(a) d-endotoxin and nontransformed plants matured from leaf plastochron index (LPI) 1–6. Feeding and growth rates were negatively correlated with phenolic glycosides in both transformed and nontransformed foliage. The presence of the B. thuringiensis d-endotoxin was at most, additive to the effect of the phenolic glycosides. Feeding and growth rates were positively correlated with condensed tannins in transformed foliage, but there was no relationship with condensed tannins in nontransformed foliage. The results indicate that the presence of foliar allelochemicals of poplar can enhance the effectiveness of genetically expressed B. thuringiensis d-endotoxin against gypsy moth larvae. However, the spatial variation in gypsy moth performance in response to the combination of foliar allelochemicals and d-endotoxin was not greater than the effect of ontogenetic variation in foliar allelochemicals alone. These results suggest that for this important pest, foliage protection may be obtained without genetically engineered defenses, and instead, by relying on ontogenetic and clonal variation in allelochemicals. The benefits of combining novel resistance mechanisms with natural ones will depend upon the specific folivore's adaptation to natural resistance mechanisms, such as allelochemicals. Moreover, some of the greatest benefits from transgenic resistance may arise from the need to protect trees from multiple pests, some of which may not be deterred by, or may even prefer, allelochemicals that confer protection from a few species.     

Methods and pitfalls of extracting condensed tannins and other phenolics from plants: Insights from investigations onEucalyptus leaves

Optimal conditions for extraction of tannins and other phenolics from tree foliage and their subsequent storage rarely have been investigated. We investigated methods of drying leaves, optimal solvents, and the effects of light and temperature on the extractability and stability of condensed tannins (proanthocyanidins) and total phenolics from leaves ofEucalyptus trees. Aqueous acetone was a better solvent than aqueous methanol for condensed tannins and total phenolics, but condensed tannins were less stable in aqueous acetone than aqueous methanol. Stability of condensed tannins also was decreased substantially by room temperature versus 4°C and by exposure to indirect sunlight, although the assay for total phenolics was unaffected. For quantitative estimation of condensed tannins, extraction with 50% acetone was better than methods of direct analysis of leaf tissue. The highest estimates of total condensed tannins were obtained by exhaustive extraction with 50% acetone followed by direct analysis of the residue. Lyophilization of fresh leaf increased yield of condensed tannin (although usually by less than 10%). Lyophilization and subsequent storage of extracts had little effect on assays for condensed tannins or total phenolics.     

Interactions between Bacteria And Aspen Defense Chemicals at the Phyllosphere – Herbivore Interface

Plant- and insect-associated microorganisms encounter a diversity of allelochemicals, and require mechanisms for contending with these often deleterious and broadly-acting compounds. Trembling aspen, Populus tremuloides, contains two principal groups of defenses, phenolic glycosides (salicinoids) and condensed tannins, which differentially affect the folivorous gypsy moth, Lymantria dispar, and its gut symbionts. The bacteria genus Acinetobacter is frequently associated with both aspen foliage and gypsy moth consuming that tissue, and one isolate, Acinetobacter sp. R7-1, previously has been shown to metabolize phenolic glycosides. In this study, we aimed to characterize further interactions between this Acinetobacter isolate and aspen secondary metabolites. We assessed bacterial carbon utilization and growth in response to different concentrations of phenolic glycosides and condensed tannins. We also tested if enzyme inhibitors reduce bacterial growth and catabolism of phenolic glycosides. Acinetobacter sp. R7-1 utilized condensed tannins but not phenolic glycosides or glucose as carbon sources. Growth in nutrient-rich medium was increased by condensed tannins, but reduced by phenolic glycosides. Addition of the P450 enzyme inhibitor piperonyl butoxide increased the effects of phenolic glycosides on Acinetobacter sp. R7-1. In contrast, the esterase inhibitor S,S,S,-tributyl-phosphorotrithioate did not affect phenolic glycoside inhibition of bacterial growth. Degradation of phenolic glycosides by Acinetobacter sp. R7-1 appears to alleviate the cytotoxicity of these compounds, rather than provide an energy source. Our results further suggest this bacterium utilizes additional, complementary mechanisms to degrade antimicrobial phytochemicals. Collectively, these results provide insight into mechanisms by which microorganisms contend with their environment within the context of plant-herbivore interactions.     

Phytochemical Variation in Quaking Aspen: Effects on Gypsy Moth Susceptibility to Nuclear Polyhedrosis Virus

The performance of gypsy moths (Lymantria dispar) feeding on quaking aspen (Populus tremuloides) is strongly influenced by host foliar chemistry and susceptibility to a nuclear polyhedrosis virus (LdNPV), but the relationship of susceptibility to chemistry is poorly understood. We investigated the effects of genetic and resource-mediated variation in phytochemistry on viral pathogenicity. Trees were grown in pots in a common garden. Disks were punched from aspen leaves, inoculated with LdNPV and fed to third instars. Additional leaves were analyzed for levels of nitrogen, starch, phenolic glycosides, and condensed tannins. Despite marked variation among trees in levels of phenolic glycosides and tannins, we observed minimal variation in larval susceptibility to LdNPV. Viral pathogenicity was only weakly (inversely) correlated with tannin concentrations in one of two experiments. These results suggest that differential defoliation of aspen by gypsy moths in the field is due to the direct effects of host chemistry on larval performance rather than to the indirect effects of host chemistry on efficacy of this natural enemy.     

Specific Polyphenols and Tannins are Associated with Defense Against Insect Herbivores in the Tropical Oak Quercus oleoides

The role of plant polyphenols as defenses against insect herbivores is controversial. We combined correlative field studies across three geographic regions (Northern Mexico, Southern Mexico, and Costa Rica) with induction experiments under controlled conditions to search for candidate compounds that might play a defensive role in the foliage of the tropical oak, Quercus oleoides. We quantified leaf damage caused by four herbivore guilds (chewers, skeletonizers, leaf miners, and gall forming insects) and analyzed the content of 18 polyphenols (including hydrolyzable tannins, flavan-3-ols, and flavonol glycosides) in the same set of leaves using high performance liquid chromatography and mass spectrometry. Foliar damage ranged from two to eight percent per region, and nearly 90% of all the damage was caused by chewing herbivores. Damage due to chewing herbivores was positively correlated with acutissimin B, catechin, and catechin dimer, and damage by mining herbivores was positively correlated with mongolinin A. By contrast, gall presence was negatively correlated with vescalagin and acutissimin B. By using redundancy analysis, we searched for the combinations of polyphenols that were associated to natural herbivory: the combination of mongolinin A and acutissimin B had the highest association to herbivory. In a common garden experiment with oak saplings, artificial damage increased the content of acutissimin B, mongolinin A, and vescalagin, whereas the content of catechin decreased. Specific polyphenols, either individually or in combination, rather than total polyphenols, were associated with standing leaf damage in this tropical oak. Future studies aimed at understanding the ecological role of polyphenols can use similar correlative studies to identify candidate compounds that could be used individually and in biologically meaningful combinations in tests with herbivores and pathogens.     

Effects of large mammalian herbivores and ant symbionts on condensed tannins of Acacia drepanolobium in Kenya

Condensed tannins have been considered to be important inducible defenses against mammalian herbivory. We tested for differences in condensed tannin defenses in Acacia drepanolobium in Kenya over two years among different large mammalian herbivore treatments [total exclusion, antelope only, and megaherbivore (elephants and giraffes) + antelope] and with four different ant symbiont species on the trees. We predicted that (1) condensed tannin concentrations would be lowest in the mammal treatment with the lowest level of herbivory (total exclusion), (2) trees occupied by mutualist ants that protect the trees most aggressively would have lower levels of tannins, and (3) if chemical defense production is costly, there would be a trade-off between tannin concentrations, growth, and mechanical defenses. Mean tannin concentrations increased from total exclusion treatments to wildlife-only treatments to megaherbivore + antelope treatments. In 1997, condensed tannin concentrations were significantly lower in trees occupied by the ant Crematogaster nigriceps, the only ant species that actively removed axillary buds. Contrary to our prediction, trees occupied by ant species that protect the trees more aggressively against mammalian herbivores did not have lower overall levels of condensed tannins. There was no consistent evidence of a trade-off between tannin concentrations and growth rate, but there was a positive correlation between mean thorn length and mean tannin concentrations across species of ant inhabitants and across herbivore treatments in 1997. Contrary to our expectation, trees had higher tannin concentrations in the upper parts of the canopy where there is little herbivory by mammals.     

Preserving leaves for tannin and phenolic glycoside analyses: A comparison of methods using three willow taxa

Members of the Salicaceae often produce phenolic glycosides and condensed tannins. There is much debate on the best method for the preservation of leaf material prior to chemical analysis. Published results indicate freeze-drying, a method commonly used for tannin analysis, may be inappropriate for phenolic glycosides, unless done in a manner to prevent thawing during the drying process. Another commonly employed method, air-drying, is appropriate for phenolic glycosides but inappropriate for condensed tannins. I present evidence using willow leaves that demonstrates that: (1) leaves freeze-dried in external flasks without temperature control contain lower concentrations of phenolic glycosides (salicortin and 2-cinnamoylsalicortin), (2) air-dried leaves have reduced concentrations of condensed tannins, while (3) vacuum-dried fresh leaves have high concentrations of both phenolic glycosides and condensed tannins. Freeze-drying caused salicortin and 2-cinnamoyl salicortin concentrations to drop by 20 mg/g and 4 mg/g, respectively. Salicin, a product of salicortin and 2-cinnamoyl salicortin degradation, is absent in vacuum-dried leaves, present in air-dried leaves and very high in freeze-dried leaves. Thus, the presence of salicin in this system is an artifact of the preservation technique. Condensed tannin concentrations dropped nearly 20 mg/g when leaves were air-dried. Thus, vacuum-drying fresh leaves allows researchers to quantify phenolic glycosides and condensed tannins from the same leaf material.     

Copaifera langsdorffii Bark as a Source of Chemicals: Structural and Chemical Characterization

The chemical composition and the anatomy of Copaifera langsdorffii bark are reported here for the first time by studying trees grown in a native forest area in the Amazon region, Brazil. The bark is thin, dark reddish brown, and exfoliates in irregular flakes. It is very dense, showing highly lignified cells and abundant sclereids, and cellular fillings of phenolic nature. It includes a poorly developed rhytidome and a periderm with thin- and thick-walled phellem cells. The mean chemical composition was: ash 3.7%, total extractives 21.3%, mainly corresponding to polar compounds soluble in ethanol and water, suberin 0.8%, and lignin 36.6%. The polysaccharides showed a predominance of glucose and xylose (66.4% and 23.5% of total monosaccharides, respectively). The ethanol-water bark extract had a high content in phenolics: total phenolics 589.2 mg gallic acid/g extract, flavonoids 441.9 mg catechin/g extract, and tannins 54.8 mg catechin/g extract. The antioxidant activity was high, comparable to known antioxidant reference compounds: 720.3 mg Trolox per g of extract or 92.1 mg Trolox per g of bark. After bark grinding, the finest fraction was enriched in polar extractives (40.6%). C. langsdorffii bark is a potential source of functional extractives, therefore representing a valorization of the residual bark obtained during the industrial tree processing for timber.     

Transgenic upregulation of the condensed tannin pathway in poplar leads to a dramatic shift in leaf palatability for two tree-feeding Lepidoptera

Transgenic hybrid aspen (Populus tremula x tremuloides) overexpressing the MYB134 tannin regulatory gene show dramatically enhanced condensed tannin (proanthocyanidin) levels, as well as shifts in other phenolic metabolites. A series of insect bioassays with forest tent caterpillars (Malacosoma disstria) and gypsy moth (Lymantria dispar) caterpillars was carried out to determine how this metabolic shift affects food preference and performance of generalist tree-feeding lepidopterans. Both species showed a distinct preference for the high-tannin MYB134 overexpressor plants, and L. dispar performance was enhanced relative to controls. L. dispar reached greater pupal weight and showed reduced time to pupation when reared on the MYB134 overexpressing poplar. These results were unexpected since enhanced condensed tannin levels were predicted to act as feeding deterrents. However, the data may be explained by the observed decrease in the salicinoids (phenolic glycosides) salicortin and tremulacin that accompanied the upregulation of the condensed tannins in the transgenics. We conclude that for these two lepidopteran species, condensed tannin levels are unlikely to be a major determinant of caterpillar food preference or performance. However, our experiments show that overexpression of a single regulatory gene in transgenic aspen can have a significant impact on herbivorous insects.     

Phenolic Compounds in Red Oak and Sugar Maple Leaves Have Prooxidant Activities in the Midgut Fluids of <Emphasis Type="Italic">Malacosoma disstria</Emphasis> and <Emphasis Type="Italic">Orgyia leucostigma</Emphasis> Caterpillars

Phenolic compounds are generally believed to be key components of the oxidative defenses of plants against pathogens and herbivores. However, phenolic oxidation in the gut fluids of insect herbivores has rarely been demonstrated, and some phenolics could act as antioxidants rather than prooxidants. We compared the overall activities of the phenolic compounds in red oak (Quercus rubra) and sugar maple (Acer saccharum) leaves in the midgut fluids of two caterpillar species, Malacosoma disstria (phenolic-sensitive) and Orgyia leucostigma (phenolic-tolerant). Three hypotheses were examined: (1) ingested sugar maple leaves produce higher levels of semiquinone radicals (from phenolic oxidation) in caterpillar midgut fluids than do red oak leaves; (2) O. leucostigma maintains lower levels of phenolic oxidation in its midgut fluids than does M. disstria; and (3) phenolic compounds in tree leaves have overall prooxidant activities in the midgut fluids of caterpillars. Sugar maple leaves had significantly lower ascorbate:phenolic ratios than did red oak leaves, suggesting that phenolics in maple would oxidize more readily than those in oak. As expected, semiquinone radicals were at higher steady-state levels in the midgut fluids of both caterpillar species when they fed on sugar maple than on red oak, consistent with the first hypothesis. Higher semiquinone radical levels were also found in M. disstria than in O. leucostigma, consistent with the second hypothesis. Finally, semiquinone radical formation was positively associated with two markers of oxidation (protein carbonyls and total peroxides). These results suggest that the complex mixtures of phenolics in red oak and sugar maple leaves have overall prooxidant activities in the midgut fluids of M. disstria and O. leucostigma caterpillars. We conclude that the oxidative defenses of trees vary substantially between species, with those in sugar maple leaves being especially active, even in phenolic-tolerant herbivore species.     

Reserves Accumulated in Non-Photosynthetic Organs during the Previous Growing Season Drive Plant Defenses and Growth in Aspen in the Subsequent Growing Season

Plants store non-structural carbohydrates (NSC), nitrogen (N), as well as other macro and micronutrients, in their stems and roots; the role of these stored reserves in plant growth and defense under herbivory pressure is poorly understood, particularly in trees. Trembling aspen (Populus tremuloides) seedlings with different NSC and N reserves accumulated during the previous growing season were generated in the greenhouse. Based on NSC and N contents, seedlings were assigned to one of three reserve statuses: Low N–Low NSC, High N–Medium NSC, or High N–High NSC. In the subsequent growing season, half of the seedlings in each reserve status was subjected to defoliation by forest tent caterpillar (Malacosoma disstria) while the other half was left untreated. Following defoliation, the effect of reserves was measured on foliar chemistry (N, NSC) and caterpillar performance (larval development). Due to their importance in herbivore feeding, we also quantified concentrations of phenolic glycoside compounds in foliage. Seedlings in Low N-Low NSC reserve status contained higher amounts of induced phenolic glycosides, grew little, and supported fewer caterpillars. In contrast, aspen seedlings in High N-Medium or High NSC reserve statuses contained lower amounts of induced phenolic glycosides, grew faster, and some of the caterpillars which fed on these seedlings developed up to their fourth instar. Furthermore, multiple regression analysis indicated that foliar phenolic glycoside concentration was related to reserve chemistry (NSC, N). Overall, these results demonstrate that reserves accumulated during the previous growing season can influence tree defense and growth in the subsequent growing season. Additionally, our study concluded that the NSC/N ratio of reserves in the previous growing season represents a better measure of resources available for use in defense and growth than the foliar NSC/N ratios.     

Phenolic Responses of Mountain Crowberry (Empetrum nigrum ssp. hermaphroditum) to Global Climate Change are Compound Specific and Depend on Grazing by Reindeer (Rangifer tarandus)

Mountain crowberry (Empetrum nigrum ssp. hermaphroditum) is a keystone species in northern ecosystems and exerts important ecosystem-level effects through high concentrations of phenolic metabolites. It has not been investigated how crowberry phenolics will respond to global climate change. In the tundra, grazing by reindeer (Rangifer tarandus) affects vegetation and soil nutrient availability, but almost nothing is known about the interactions between grazing and global climate change on plant phenolics. We performed a factorial warming and fertilization experiment in a tundra ecosystem under light grazing and heavy grazing and analyzed individual foliar phenolics and crowberry abundance. Crowberry was more abundant under light grazing than heavy grazing. Although phenolic concentrations did not differ between grazing intensities, responses of crowberry abundance and phenolic concentrations to warming varied significantly depending on grazing intensity. Under light grazing, warming increased crowberry abundance and the concentration of stilbenes, but decreased e.g., the concentrations of flavonols, condensed tannins, and batatasin-III, resulting in no change in total phenolics. Under heavy grazing, warming did not affect crowberry abundance, and induced a weak but consistent decrease among the different phenolic compound groups, resulting in a net decrease in total phenolics. Our results show that the different phenolic compound groups may show varying or even opposing responses to warming in the tundra at different levels of grazing intensity. Even when plant phenolic concentrations do not directly respond to grazing, grazers may have a key control over plant responses to changes in the abiotic environment, reflecting multiple adaptive purposes of plant phenolics and complex interactions between the biotic and the abiotic factors.     

Foliar phenolics of nebraska sandhills prairie graminoids: Between-years,seasonal, and interspecific variation

Because of their potential as antiherbivore defenses, plant phenolics elicit considerable attention. We made quantitative and qualitative analyses of phenolics, alkaloids, cyanogenic glycosides, and saponins in the dominant graminoids of a Nebraska Sandhills prairie. We examined the foliage of seven species:Agropyron smithii Rydb.,Andropogon hallii Hack.,Andropogon scoparius Michx.,Bouteloua gracilis (H.B.K) Lag. ex Griffiths,Calamovilfa longifolia (Hook.) Scribn.,Carex heliophila Mack., andStipa comata Trin & Rupr. Their leaves contain low levels of phenolics that vary significantly among species. A more detailed examination of the three species with the highest levels of phenolics showed among-year, seasonal, and spatial heterogeneity in the levels of total phenolics. In all seven species, the majority of the specific phenolics present have the Chromatographic properties of phenylpropanoids and are likely to be present as sugar-linked derivatives such as free glycosides or cell wall-bound phenolics. These species do not contain condensed tannins. The absence of other common kinds of secondary metabolites indicates that these graminoids are unlikely to have significant chemical defenses, at least in terms of substances likely to be active against mammalian herbivores. In this, they exemplify the situation thought typical for prairie graminoids.     

Tannin Composition Affects the Oxidative Activities of Tree Leaves

We examined whether tannin composition plays an important role in explaining the oxidative activities of tree leaves of Acer saccharum (sugar maple) and Quercus rubra (red oak). Sugar maple leaves contained substantial amounts of ellagitannins, condensed tannins, and galloyl glucoses, whereas red oak leaves contained almost exclusively condensed tannins. Oxidative activities of the crude phenolics from both species, and the phenolic fractions from sugar maple, were measured with electron paramagnetic resonance (EPR) spectrometry and UV-visible spectrophotometry. The two assays produced similar results: (1) sugar maple phenolics produced larger semiquinone radical concentrations,and higher semiquinone decay rates and browning rates than did red oak phenolics;(2) ellagitannin levels were positively associated with the three measures of oxidative activity; and (3) condensed tannin and galloyl glucose levels were negatively associated with these measures. The negative relationship between condensed tannin levels and oxidative activity resulted from the antioxidant effects of condensed tannins on hydrolyzable tannins; several purified condensed tannins significantly decreased the concentrations of semiquinone radicals and browning rates of pedunculagin (an ellagitannin) and pentagalloyl glucose. As expected, whole-leaf extracts from sugar maple produced elevated levels of semiquinone radicals, but none were observed in red oak extracts when the two species were compared with an EPR time-course assay. We conclude that the oxidative activities of tree leaves may be affected by tannin composition, and that the prooxidant activity of ellagitannins may be decreased by co-occurring condensed tannins.     

Divergence in Structure and Activity of Phenolic Defenses in Young Leaves of Two Co-Occurring <Emphasis Type="Italic">Inga</Emphasis> Species

The leaves of tropical forest trees are most likely to suffer herbivore damage during the period of expansion. Herbivore selection on young leaves has given rise to a variety of leaf developmental strategies and age-specific chemical defense modes. We are studying correlations between leaf developmental types and chemical defenses in the Neotropical genus Inga. We have characterized defense metabolites in Inga goldmanii and Inga umbellifera, two species that co-occur in the lowland moist forest of Panama. These congeners have markedly different young-leaf developmental phenotypes but suffer approximately equal rates of herbivory. Bioassays of whole and fractionated leaf extracts using larvae of Heliothis virescens show that I. goldmanii chemical defenses are nearly three times more inhibitory than those of I. umbellifera. In both species, most of the inhibitory activity resides in complex mixtures of monomeric and polymeric flavan-3-ols. This group comprises >30% of young leaf dry weight in both I. goldmanii and I. umbellifera. The species’ phenolic chemistry differs markedly, however, both in the structure of the monomeric units and in the distribution of polymer sizes. The differences in chemical structure have pronounced effects on their bioactivities, with I. goldmanii flavans being twice as inhibitory to H. virescens larvae as I. umbellifera flavans, and more than three times more efficient at protein binding. Given the extraordinarily high polyphenol concentrations that are found in the young leaves of these species, protein precipitation could be an important mechanism of growth inhibition. Nevertheless, our data show that another mode of phenolic action, possibly oxidative stress, occurs simultaneously.