Effects of long-term UV-exposure and plant sex on the leaf phenoloxidase activities and phenolic concentrations of Salix myrsinifolia (Salisb.).

The accumulation of flavonoids on the leaf surface is a well-characterized protective mechanism against UV-B radiation. Other protective mechanisms, such as the induction of antioxidative enzymes and peroxidase-mediated lignification may also be important. The effects of UV-B radiation have mainly been considered in short-term studies, whereas ecologically more relevant long-term field studies are still rare. Here we examined the effects of long-term exposure to enhanced UV-B radiation on the activities of two antioxidative enzymes, polyphenol oxidase (PPO; EC 1.10.2.2 and EC 1.14.18.1) and guaiacol peroxidase (POD; EC 1.11.1.7), as well as the phenolic concentrations in two sexes of the dioecious species, Salix myrsinifolia. After three consecutive growth seasons with enhanced UV-B radiation, we found that PPO activity was decreased by UV radiation in male plants, which might explain their lower UV-B tolerance when compared to female plants. In addition, male plants had higher specific activity than did female plants under ambient conditions, supporting the idea that males of S. myrsinifolia are generally more growth-oriented than females. By contrast, neither UV treatment nor sex had significant effects on the POD activities of willows. Gender differences in the concentrations of phenolic compounds are in line with the general concept that males are less well defended than females. We suggest that the inability to increase PPO and POD activity, along with lower accumulation of UV-B absorbing compounds under UV-B exposure, might be one of the reasons why males had thinner leaves and were less tolerant of UV-B than were females.

Asynchrony between Host Plant and Insects-Defoliator within a Tritrophic System: The Role of Herbivore Innate Immunity.

The effects of asynchrony in the phenology of spring-feeding insect-defoliators and their host plants on insects' fitness, as well as the importance of this effect for the population dynamics of outbreaking species of insects, is a widespread and well-documented phenomenon. However, the spreading of this phenomenon through the food chain, and especially those mechanisms operating this spreading, are still unclear. In this paper, we study the effect of seasonally declined leafquality (estimated in terms of phenolics and nitrogen content) on herbivore fitness, immune parameters and resistance against pathogen by using the silver birch Betula pendula--gypsy moth Lymantria dispar--nucleopolyhedrovirus as the tritrophic system. We show that a phenological mismatch induced by the delay in the emergence of gypsy moth larvae and following feeding on mature leaves has negative effects on the female pupal weight, on the rate of larval development and on the activity of phenoloxidase in the plasma of haemolymph. In addition, the larval susceptibility to exogenous nucleopolyhydrovirus infection as well as covert virus activation were both enhanced due to the phenological mismatch. The observed effects of phenological mismatch on insect-baculovirus interaction may partially explain the strong and fast fluctuations in the population dynamics of the gypsy moth that is often observed in the studied part of the defoliator area. This study also reveals some indirect mechanisms of effect related to host plant quality, which operate through the insect innate immune status and affect resistance to both exogenous and endogenous virus.

Effects of overproduction of condensed tannins and elevated temperature on chemical and ecological traits of genetically modified hybrid aspens (Populus tremula × P. tremuloides).

Gene transfer techniques offer new possibilities to study regulation of phenolic pathways and the defensive role of phenolics. Hybrid aspen lines (Populus tremula × tremuloides) that overexpress the PtMYB134 transcription factor were used to study the effects of condensed tannin production on plant physiology and plant defenses. The MYB134 protein activates all the known genes of the biosynthetic pathway for condensed tannins (CTs), so overexpression of MYB134 was expected to increase CT concentration in all tissues of the plants. Two out of three MYB134 overexpression lines (46 and 54) accumulated high levels of CTs and (+)-catechin, with a concomitant decrease in the levels of salicylates, but one transgenic line, MYB 61, failed to overproduce CTs. The concentrations of phenolic compounds generally were lower in the aspen leaves grown under elevated temperature than in those grown under ambient temperature. A specialist leaf beetle, Phratora vitellinae (Coleoptera: Chrysomelidae), was chosen to examine how over-expression of MYB134 and elevated temperature affect the food choice of a beetle adapted to feed on leaves rich in salicylates but containing little CT. Specialist beetles preferred the leaves grown at ambient temperatures possibly because these leaves had higher concentrations of salicylates, which are feeding stimulants. Beetles also preferred MYB line 61, which contained a normal level of CT but a slightly elevated level of salicylates. Our results show that transgenic plants are powerful tools, but that enhancing one secondary pathway may lead to unexpected effects on other pathways, and thus impact characteristics such as plant resistance against herbivores, especially under changing climatic conditions.

Genetic and environmental factors behind foliar chemistry of the mature mountain birch.

Previous studies of mountain birch (Betula pubescens spp. czerepanovii) repeatedly have found differences between individual trees in herbivory-related traits, but rarely have yielded estimates of the additive genetic variation of these traits or of their relationship to habitat. We used thirty-year-old birch half-sibs in a northern common garden to estimate the effect of genetics and local microhabitat on resistance-related traits. Genetic estimates of foliar chemistry have been studied only rarely with trees as old as these. Moth performance (Epirrita autumnata), rust (Melampsoridium betulinum) incidence levels, and the general level of natural herbivory damage to individual trees were used as direct measures of birch resistance. Chemical resistance-related traits in plant chemistry included 15 individual phenolics, 16 amino acids, and phenoloxidase activities in the foliage. We also followed birch phenology and growth. Our results show that the genotype of the birch was the most important determinant of phenolic composition and phenoloxidase activity, but that amino acid levels were best explained by the microhabitat of the birch. We also found that the phenology of the birch had a high heritability, although its variation was low. Our results reveal rich genetic variation in birch chemistry.

Boron nutrition affects the carbon metabolism of silver birch seedlings.

Boron (B) is an essential micronutrient whose deficiency is common both in agriculture and in silviculture. Boron deficiency impairs the growth of plants and affects many metabolic processes like carbohydrate metabolism. Boron deficiency and also excess B may decrease the sink demand by decreasing the growth and sugar transport which may lead to the accumulation of carbohydrates and down-regulation of photosynthesis. In this study, we investigated the effects of B nutrition on the soluble and storage carbohydrate concentrations of summer leaves and autumn buds in a deciduous tree species, Betula pendula Roth. In addition, we investigated the changes in the pools of condensed tannins between summer and autumn harvests. One-year-old birch seedlings were fertilized with a complete nutrient solution containing three different levels of B: 0, 30 and 100% of the standard level for complete nutrient solution. Half of the seedlings were harvested after summer period and another half when leaves abscised. The highest B fertilization level (B100) caused an accumulation of starch and a decrease in the concentrations of hexoses (glucose and fructose) in summer leaves, whereas in the B0 seedlings, hexoses (mainly glucose) accumulated and starch decreased. These changes in carbohydrate concentrations might be related to the changes in the sink demand since the autumn growth was the smallest for the B100 seedlings and largest for the B30 seedlings that did not accumulate carbohydrates. The autumn buds of B30 seedlings contained the lowest levels of glucose, glycerol, raffinose and total polyols, which was probably due to the dilution effect of the deposition of other substances like phenols. Condensed tannins accumulated in high amounts in the birch stems during the hardening of seedlings and the largest accumulation was detected in the B30 treatment. Our results suggest that B nutrition of birch seedlings affects the carbohydrate and phenol metabolism and may play an important role in the hardening process of the seedlings.

Boron fertilization enhances the induced defense of silver birch.

Boron (B) deficiency is a common micronutrient deficiency that has been reported to affect the phenolic metabolism of plants. Thus, it may play a role in defense against herbivorous animals. However, the role of B in a plant's resistance to herbivores has not received any particular attention from researchers. In this study, we tested the effects of B nutrition 1) on the biochemical and mechanical defenses of birches and the growth of seedlings, and 2) the resistance of seedlings to larvae of the autumnal moth, Epirrita autumnata. Boron fertilization improved the resistance of birch, which was shown as reduced pupal weight of the herbivore. However, B fertilized trees suffered from heavier defoliation than unfertilized ones due to compensation feeding of larvae. The growth of the seedlings was diminished, and several biochemical changes occurred in leaves of herbivore seedlings, and B also played a role in these changes. Polyphenoloxidases (PPOs) and peroxidases (PODs) and their substrates, chlorogenic acids, were induced by herbivory in B fertilized seedlings but not in unfertilized seedlings. The lower pupal weights and increased consumption of the herbivores were probably linked to the plants' phenoloxidase-mediated production of reactive quinones, which decrease the nutritive value. Herbivory upon new stems led to an increase in the number of resin glands that provide defense against mammalian herbivores. Herbivory also had a substantially negative effect on B concentration in leaves of B fertilized seedlings. We postulate that B nutrition of trees may play a significant role in the induced defense of birches.

Effects of elevated ultraviolet-B radiation on a plant-herbivore interaction.

Enhanced ultraviolet-B (UV-B) radiation may have multiple effects on both plants and animals and affect plant-herbivore interactions directly and indirectly by inducing changes in host plant quality. In this study, we examined combined effects of UV-B and herbivory on the defence of the mountain birch (Betula pubescens ssp. czerepanovii) and also the effects of enhanced UV-B radiation on a geometrid with an outbreak cycle: the autumnal moth (Epirrita autumnata). We established an experiment mimicking ozone depletion of 30% (a relevant level when simulating ozone depletion above Northern Lapland). Both arctic species responded only slightly to the enhanced level of UV-B radiation, which may indicate that these species are already adapted to a broader range of UV-B radiation. UV-B exposure slightly induced the accumulation of myricetin glycosides but had no significant effect on the contents of quercetin or kaempferol derivatives. Mountain birch seedlings responded more efficiently to herbivory wounding than to enhanced UV-B exposure. Herbivory induced the activities of foliar oxidases that had earlier been shown to impair both feeding and growth of moth larvae. In contrast, the contents of foliar phenolics did not show the same response in different clones, except for a decrease in the contents of tannin precursors. The induction of foliar phenoloxidase activities is a specific defence response of mountain birches against insect herbivory. To conclude, our results do not support the hypothesis that the outbreak cycle of the autumnal moth can be explained by the cycles of solar activity and UV-B.

Foliar oxidases as mediators of the rapidly induced resistance of mountain birch against Epirrita autumnata.

Induced resistance of the mountain birch against its main defoliator Epirrita autumnata is a well-characterized phenomenon. The causal mechanism for this induced deterioration, however, has not been unequivocally explained, and no individual compound or group of traditional defensive compounds has been shown to explain the phenomenon. Phenolic compounds are the main secondary metabolites in mountain birch leaves, and the biological activity of phenolics usually depends on their oxidation. In this study, we found that the activity of polyphenoloxidases (PPOs), enzymes that oxidize o-diphenols to o-diquinones, was induced in trees with introduced larvae, and bioassays showed that both growth and consumption rates of larvae were reduced in damaged trees. PPO activity was negatively associated with both larval growth and consumption rates in trees with bagged larvae, but not in control trees. Our results suggest that the oxidation of phenolics by PPOs may be a causal explanation for the rapidly induced resistance of mountain birch against E. autumnata. This finding also helps to explain why correlations between insect performance and phenolics (without measuring indices explaining their oxidation) may not produce consistent results.

Immunological memory of mountain birches: effects of phenolics on performance of the autumnal moth depend on herbivory history of trees.

Plants have been suggested to have an immunological memory comparable to animals. The evidence for this, however, is scarce. In our study with the mountain birch -- Epirrita autumnata system, we demonstrated that birches exposed as long as 5 yr to feeding of E. autumnata larvae (delayed induced resistance, DIR), responded more strongly to a new challenge than trees without an herbivory history. Pupal weights remained lower, and the duration of the larval period was prolonged in the DIR trees, although immunity, measured as an encapsulation rate, was not affected. We further demonstrated that the effects of birch phenolics on performance of E. autumnata were different in the exposed (DIR) trees from naive control trees, although we found only one significant change in chemistry. The quercetin:kaemferol ratio was increased in DIR trees, suggesting that herbivory caused oxidative stress in birches. In DIR trees, phenolics, especially hydrolyzable tannins (HTs), affected pupal weights negatively, whereas in control trees, the effects were either nonsignificant or positive. HTs also prolonged the duration of the larval period of females, whereas peroxidase (POD) activity prolonged that of males. We suggest that the causal explanation for the induced resistance was an enhanced oxidation of phenolic compounds from the DIR trees in the larval digestive tract. Phenolic oxidation produces semiquinones, quinones, free radicals, and ROS, which may have toxic, antinutritive, and/or repellent properties against herbivores.

Foliar phenolics are differently associated with Epirrita autumnata growth and immunocompetence.

The quality of available food may affect insect herbivores directly (via growth and survivorship) and/or indirectly (by modifying insect vulnerability to parasitoids and pathogens). We examined the relationship between different phenolic compounds, belonging to various phenolic groups, in Betula pubescens spp. czerepanovii (mountain birch) foliage and the larval performance of the geometrid Epirrita autumnata (autumnal moth). Direct effects on insect performance were described by pupal weight, developmental rate, and survivorship; indirect effects were described by the encapsulation rate of an implant inserted into the insect hemocoel, a commonly used way to describe insect immune defense. We found profound differences in the effects of different phenolic categories: several individual hydrolyzable tannins were associated positively with larval performance but negatively with level of immune defense, whereas flavonoid glycosides were inversely related to larval survival but showed no association with the larvae immune defense.

Temperature as a modifier of plant-herbivore interaction.

Temperature directly affects the growth, survival, and development rates of poikilothermic insect herbivores; it may also have an important indirect impact, via the activities of plant defensive enzymes. The effects of wounding birch leaves and temperature on the growth and development rates of a Lepidopteran moth, Epirrita autumnata, were studied. We also examined the activities of a mountain birch (Betula pubescesns spp. czerepanovii) defensive enzymes, specifically the polyphenoloxidases (PPOs), in relation to temperature and wounding. The optimal temperature for early instars in terms of survival and developmental rates was between +15 and 20 degrees C. Wounding treatment had different effects on birch PPO activity depending on the temperature: at +12 degrees C, wounding decreased the activity, suggesting induced amelioration at that temperature, whereas at +25 degrees C, wounding increased the activity, suggesting induced resistance. However, larval growth was retarded slightly, but significantly, on the leaves of wounded twigs at both temperatures. Both PPO activity and larval growth rates were affected within 12 h, indicating the existence of a transcription- and translation-independent defense system in birch leaves. We suggest that underlying the increase in PPO activity and the decrease in larval growth rate may be H2O2, which has been shown to accumulate in response to wounding. Our results also provide a possible biological mechanism for the hypothesis that low temperatures promote the success of E. autumnata and other Lepidopteran larvae via decreased defensive enzyme activities of host plants at lower temperatures.

Defoliating insect immune defense interacts with induced plant defense during a population outbreak.

During population outbreaks, top-down and bottom-up factors are unable to control defoliator numbers. To our knowledge, details of biotic interactions leading to increased population density have not been studied during real population outbreaks. We experimentally assessed the strength of plant defenses and of insect immunocompetence, assumed to contribute to active insect resistance against parasitoids and pathogens, in the geometrid Epirrita autumnata during a steep increase in population density. We demonstrated rapid (same-season) induced resistance in the foliage of its host, mountain birch. The response was systemic, spreading throughout the tree, and retarded larval growth rate by approximately 10%. On the other hand, no direct delayed carry-over effects were found in the next season in larval growth rate, mortality, or pupal mass. Larval damage to a tree during the previous year, however, significantly (by approximately 13%) accelerated the advance of the immune response (measured as melanization of an implant inserted into the pupal hemocoel). The encapsulation rate correlated positively with larval mortality in trees in which larvae had been introduced the previous year, but not in control trees. Both of these observations suggest that induced plant defense was associated with an increased insect immunocompetence during the population increase.

Wound-induced oxidative responses in mountain birch leaves.

AIMS: The aim of the study was to examine oxidative responses in subarctic mountain birch, Betula pubescens subsp. czerepanovii, induced by herbivory and manual wounding. METHODS: Herbivory-induced changes in polyphenoloxidase, peroxidase and catalase activities in birch leaves were determined. A cytochemical dye, 3,3-diaminobenzidine, was used for the in situ and in vivo detection of H2O2 accumulation as a response to herbivory and wounding. To localize peroxidase activity in leaves, 10 mm H2O2 was applied to the dye reagent. KEY RESULTS: Feeding by autumnal moth, Epirrita autumnata, larvae caused an induction in polyphenoloxidase and peroxidase activities within 24 h, and a concomitant decrease in the activity of antioxidative catalases in wounded leaves. Wounding also induced H2O2 accumulation, which may have both direct and indirect defensive properties against herbivores. Wound sites and guard cells showed a high level of peroxidase activity, which may efficiently restrict invasion by micro-organisms. CONCLUSION: Birch oxidases together with their substrates may form an important front line in defence against herbivores and pathogens.

Trade-off between synthesis of salicylates and growth of micropropagated Salix pentandra.

We studied the relationship between biosynthesis of salicylates, the main chemical defenses in willow and growth of Salix pentandra by cultivating plants in the presence of 2-aminoindan-2-phosphonic acid (AIP), a powerful inhibitor of phenylalanine ammonia-lyase (PAL: EC 4.3.1.5.). AIP inhibited efficiently, though not totally, the endogenous synthesis of salicylates. This inhibition markedly increased plant growth. Exogenous application of the precursors of salicylates, benzoic acid (BA), salicylic acid (SA), and helicin, increased the levels of several individual salicylates, but decreased the growth of plants cultivated in the presence of AIP. These results suggest a trade-off between plant growth and the synthesis of salicylates in S. pentandra. Phenylalanine, which accumulated in response to inhibitor treatment, but was decreased by precursor treatments, may be a common and limiting precursor for both plant growth and salicylate synthesis. The biosynthesis of salicin is suggested to proceed mainly via benzoyl-glucose, an intermediate in the synthesis of salicylic acid. Salicin is the most obvious precursor of more substituted salicylates, salicortin, acetylsalicortin, and tremulacin. In addition, we found that the salicylate pools of mature plant parts of S. pentandra were not subject to turnover, implying that the maintenance of salicylates does not demand high resources of plants, although their initial construction is costly.

In vitro degradation of willow salicylates.

Salicylates are defensive compounds against a great variety of generalist herbivores. Salicortin and its derivatives are labile compounds that degrade immediately when cell compartmentalization is ruptured, producing a 6-hydroxy-2-cyclohexenone (6-HCH) moiety that is a strong antifeeding cue. We studied the in vitro degradation of willow salicylates in the presence and absence of foliar enzymes at acidic, neutral, and alkaline pHs. Higher substituted salicylates were degraded in the absence of foliar enzymes at alkaline pH and in the presence of foliar enzymes at all three pHs. Salicin and its diglucoside, on the other hand, were degraded only in the presence of foliar enzymes at acidic pH, probably by beta-glucosidase activity. The main degradation products of higher substituted salicylates were salicin, 6-HCH, and catechol in both the absence and presence of enzymes, suggesting that the production of 6-HCH and catechol do not necessarily demand enzymatic activity. We propose that the degradation of salicylates begins with the cleavage of a 1-hydroxy-6-oxo-2-cyclohexen-1-carbonyl moiety by foliar esterases and/or alkaline condition. This moiety is decarboxylated in nonenzymatic reaction to an anion of 2-hydroxy-3-cyclohexenone, which is tautomerized to the enol form and further to the keto form, 6-HCH. Hydroxyketone can be also oxidized to catechol, a substrate of polyphenol oxidases.