Dynamics of the volatile defense of winter "dormant" balsam poplar (Populus balsamifera).

6-Hydroxycylohex-2-en-1-one (6-HCH) has been reported as a major chemical defense of the winter-dormant internodes of balsam poplar (Populus balsamifera) against feeding by herbivores such as the snowshoe hare (Lepus americanus). We report that the concentration of 6-HCH in the fall internodes is triggered by a single hard frost, and then undergoes an exponential decline through volatilization over the winter that results in barely detectable quantities by early spring. We conclude that the role of 6-HCH in the defense of mature balsam poplar is more complex than simply acting as a toxin. Rather, 6-HCH's role as a defensive agent must evolve over the course of the winter from being a co-toxin to a cue for a conditioned flavor aversion (CFA) to finally having no role by late spring.

Papyriferic acid, an antifeedant triterpene from birch trees, inhibits succinate dehydrogenase from liver mitochondria.

Papyriferic acid (PA) is a triterpene that is secreted by glands on twigs of the juvenile ontogenetic phase of resin producing tree birches (e.g., Betula neoalaskana, B. pendula) and that deters browsing by mammals such as the snowshoe hare (Lepus americanus). We investigated the pharmacology of PA as a first step in understanding its antifeedant effect. After oral administration to rats, PA and several metabolites were found in feces but not urine, indicating that little was absorbed systemically. Metabolism involved various combinations of hydrolysis of its acetyl and malonyl ester groups, and hydroxylation of the terpene moiety. The presence of a malonyl group suggested a possible interaction with succinate dehydrogenase (SDH), a mitochondrial enzyme known to be competitively inhibited by malonic acid. The effect of PA on the oxidation of succinate by SDH was examined in mitochondrial preparations from livers of ox, rabbit, and rat. In all three species, PA was a potent inhibitor of SDH. Kinetic analysis indicated that, unlike malonate, PA acted by an uncompetitive mechanism, meaning that it binds to the enzyme-substrate complex. The hydrolysis product of PA, betulafolienetriol oxide, was inactive on SDH. Overall, the evidence suggests that PA acts as the intact molecule and interacts at a site other than the succinate binding site, possibly binding to the ubiquinone sites on complex II. Papyriferic acid was potent (K(iEIS) ranged from 25 to 45 microM in the three species) and selective, as malate dehydrogenase was unaffected. Although rigorous proof will require further experiments, we have a plausible mechanism for the antifeedant effect of PA: inhibition of SDH in gastrointestinal cells decreases mitochondrial energy production resulting in a noxious stimulus, 5-HT release, and sensations of nausea and discomfort. There is evidence that the co-evolution of birches and hares over a large and geographically-diverse area in Northern Europe and America has produced marked differences in the formation of PA by birches, and the tolerance of hares to dietary PA. The present findings on the metabolic fate and biochemical effects of PA provide a rational basis for investigating the mechanisms underlying differences among populations of hares in their tolerance of a PA-rich diet.

Fire drives transcontinental variation in tree birch defense against browsing by snowshoe hares.

Fire has been the dominant disturbance in boreal America since the Pleistocene, resulting in a spatial mosaic in which the most fire occurs in the continental northwest. Spatial variation in snowshoe hare (Lepus americanus) density reflects the fire mosaic. Because fire initiates secondary forest succession, a fire mosaic creates variation in the abundance of early successional plants that snowshoe hares eat in winter, leading to geographic variation in hare density. We hypothesize that fire is the template for a geographic mosaic of natural selection: where fire is greatest and hares are most abundant, hare browsing has most strongly selected juvenile-phase woody plants for defense. We tested the hypothesis at multiple spatial scales using Alaska birch (Betula neoalaskana) and white birch (Betula papyrifera). We also examined five alternative hypotheses for geographic variation in antibrowsing defense. The fire-hare-defense hypothesis was supported at transcontinental, regional, and local scales; alternative hypotheses were rejected. Our results link transcontinental variation in species interactions to an abiotic environmental driver, fire. Intakes of defense toxins by Alaskan hares exceed those by Wisconsin hares, suggesting that the proposed selection mosaic may coincide with a geographic mosaic of coevolution.

Galloyl depsides of tyrosine from young leaves of Inga laurina.

In addition to the free protein amino acid l-tyrosine, the expanding young leaves of Inga laurina accumulate high concentrations of three new depsides, galloyl, m-digalloyl, and m-trigalloyl l-tyrosine (1, 2, and 3). The structures of these compounds were determined on the basis of their spectroscopic properties and through degradation and derivatization experiments. They occur in young leaves at the following dry-weight mass percentages: tyrosine, 10.4%; 1, 3.1%; 2, 5.0%; 3, 1.3%. These concentrations are most consistent with chemical defense during the vulnerable expansion stage of leaf development. Neither free tyrosine nor its galloyl depsides are present in mature leaves.

Effects of drought stress and nutrient availability on dry matter allocation, phenolic glycosides, and rapid induced resistance of poplar to two lymantriid defoliators.

The growth-differentiation balance hypothesis (GDBH) postulates that variation in resource availability can increase or decrease allocation to secondary metabolism, depending on how growth is affected relative to carbon assimilation. Growth and leaf area of black poplar (Populus nigra) increased substantially in response to increased nutrient availability, while net assimilation rate and photosynthesis were less strongly affected. In response, total phenolic glycoside concentrations declined, which is consistent with GDBH. Drought stress decreased net assimilation rate and photosynthesis as well as growth, while increasing total phenolic glycoside concentrations. This pattern does not follow GDBH, which predicts lower secondary metabolism when resource limitation decreases both growth and carbon assimilation. However, there was a strong negative correlation between growth and total phenolic glycoside concentration consistent with a trade-off between primary and secondary metabolism, a key premise of GDBH. Drought decreased the growth of gypsy moth (Lymantria dispar) larvae but had no effect on whitemarked tussock moth (Orgyia leucostigma). Increased nutrient availability had a positive linear effect on growth of whitemarked tussock moth, but no effect on gypsy moth. Treatment effects on gypsy moth corresponded closely with effects on total phenolic glycosides, whereas effects on whitemarked tussock moth more closely tracked changes in nutritional quality. Localized gypsy moth herbivory elicited rapid induced resistance to gypsy moth, with the effect being independent of water and nutrient availability, but did not affect whitemarked tussock moth, indicating that the effects of biotic and abiotic stress on insect resistance of trees can be species-specific.

Diterpenes from Colophospermum mopane: "missing links" in the biogenesis of 9,13-epoxylabdanes.

Moponeol A (1) and moponeol B (2) were isolated from Colophospermum mopane along with a mixture of their corresponding aldehydes (3 and 4). These substances are primitive diterpenes that we view as the "missing links" in the biosynthesis of the 9,13-epoxylabdanes. The structures of 1 and 2 were elucidated by a combination of spectra (NMR and MS) of the isolates and their mono-p-bromobenzoyl derivatives. The structures of 3 and 4 were confirmed by their ready reduction to 1 and 2. The biosynthetic implications of the stereochemical assignments of these terpenoids are briefly discussed.

Defensive adaptations of Thuja plicata to ungulate browsing: a comparative study between mainland and island populations.

Forests on the Haida Gwaii (HG) archipelago (British Columbia, Canada) evolved for about 10,000 years in the absence of large-mammal browsing. The introduction of black-tailed deer (Odocoileus hemionus sitkensis) from the mainland prior to 1901 provides an opportunity to evaluate changes in the adaptive defensive responses of plants to herbivory. We compared (1) food choice by deer and (2) chemical defence (terpene concentrations) between HG and mainland red cedars (Thuja plicata) using (1) nursery-grown seedlings never exposed to deer, (2) branches from trees that grew before the introduction of deer ("old trees") and (3) saplings exposed to deer herbivory on the mainland and on HG. We used the first two plant categories to test the hypothesis that plants that evolve under low herbivory levels have lower anti-herbivore defences. We used saplings to study the consequences of the dramatic increase in browsing on HG. During food experiments, deer preferred HG seedlings and old tree branches compared to those from the mainland. Total monoterpene concentrations were much higher than diterpene concentrations in all plant categories. Within plant categories, multivariate analysis showed that terpene profiles differed significantly between HG and mainland red cedars: HG seedlings and old trees had lower monoterpene levels. These results suggest that some monoterpenes may be determinants of deer food choice and that the defences of HG plants are less effective than those of mainland plants. The deer used branches from HG and mainland saplings indiscriminately. However, terpene profiles differed significantly between HG and mainland saplings, with multivariate analysis suggesting a higher defensive response in browsed HG saplings. Monoterpene profiles were different in lightly and heavily browsed saplings from HG, suggesting that under the current browsing regime, individuals with the greatest constitutive defences, or with greatest potential for induced defences, grow better and are selected on HG.

Defense of winter-dormant Alaska paper birch against snowshoe hares.

Mature growth-phase internodes of Alaska paper birch (Betula resinifera) are preferred by the snowshoe hare (Lepus americanus) over juvenile growth-phase internodes due to the low food value of the latter. While the mature over juvenile preferencec cannot be explained by the levels of inorganic nutrients or gross chemical fractions (resins or phenols), it can be explained by the striking differences in secondary metabolites of the two growth phases. The principle compound which renders the juvenile phase internodes unpalatable is papyriferic acid, a triterpene which is a demonstrated feeding deterrent to snowshoe hares and which is present in juvenile internodes at concentrations 25 times greater than those in mature internodes.