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Background: Prolonged exposure to UV radiation disrupts the oxidative stress defense systems; in the absence of adequate reactive oxygen species (ROS)‐scavenging mechanisms, the ROS produced cause oxidative damage to DNA, proteins, carbohydrates, and lipids. Thus, ROS elimination can protect against photoaging and possibly skin cancer. We recently isolated two novel phenolic glycosides (citrulluside H and citrulluside T) with antioxidative activity from young watermelon (Citrullus lanatus) fruits. In the present study, we evaluated the protective effect of these compounds against UV‐B–induced photodamage in normal human dermal fibroblasts (NHDFs). Methods: NHDFs were exposed to UV‐B irradiation at 25 mJ/cm2. Matrix metalloproteinase (MMP)‐1 and MMP‐3 mRNA levels were measured by quantitative real‐time PCR. MMP protein expression, as well as MAPK and NF‐κB signaling, was investigated by Western blot analysis. Activation protein‐1 (AP‐1) transcriptional activity was determined using a luciferase reporter assay. Intracellular ROS levels were measured using the CellROX™ Deep Red Reagent. Results: Citrulluside H and citrulluside T significantly downregulated the protein and mRNA expression of MMP‐1 and MMP‐3, which are associated with the onset of skin wrinkle formation. Importantly, inhibition of UV‐B–induced MMP expression in NHDFs by citrulluside H and citrulluside T was primarily due to the attenuation of MAPK/AP‐1 and NF‐κB signaling via scavenging of the generated ROS. Conclusion: Our findings delineated both citrulluside H and citrulluside T as potential agents for alleviating UV‐induced skin photoaging. [ABSTRACT FROM AUTHOR]

Phenolic compounds in pig diets, originating either from feed ingredients or additives, may occur as glycosides, that is conjugated to sugar moieties. Upon ingestion, their bioavailability and functionality depend on hydrolysis of the glycosidic bond by endogenous or microbial glycosidases. Hence, it is essential to map the glycosidase activities towards phenolic glycosides present along gut. Therefore, the activity of three key glycosidases, that is α‐glucosidase (αGLU), β‐glucosidase (βGLU) and β‐galactosidase (βGAL), was quantified in small intestinal mucosa and digesta of piglets at different gastrointestinal sites (stomach, three parts of small intestine, caecum and colon) and at different ages around weaning (10 days before and 0, 2, 5, 14 and 28 days after weaning). Activity assays were performed with p‐nitrophenyl glycosides at neutral pH. The αGLU activities in mucosa and digesta were low (overall means 1.4 and 60 U respectively) as compared to βGLU (15.2 and 199 U) and βGAL (23.4 and 298 U; p <.001). Moreover, αGLU activity in mucosa was unaffected by age. Conversely, βGLU and βGAL activities dropped significantly after weaning. Minimal levels, ranging between 18% and 54% of the pre‐weaning values, were reached at 5 days post‐weaning. Similarly, in small intestinal digesta, reductions from 60% up to 90% were observed for the three enzyme activities on day five post‐weaning as compared to pre‐weaning levels. In caecal contents, activities were lowest at 14 days post‐weaning, while in stomach and colon no clear weaning‐induced effects were observed. Our data suggest that weaning affects the glycosidase activity in mucosa (mainly endogenous origin) and digesta (primarily bacterial origin) with the most pronounced effects occurring 5 days post‐weaning. Moreover, differences in activities exist between different glycosidases and between gut locations. These insights can facilitate the prediction of the fate of existing and newly synthetized glycosides after oral ingestion in piglets. [ABSTRACT FROM AUTHOR]

SUMMARY: The salicinoids are anti‐herbivore phenolic glycosides unique to the Salicaceae (Populus and Salix). They consist of a salicyl alcohol glucoside core, which is usually further acylated with benzoic, cinnamic or phenolic acids. While salicinoid structures are well known, their biosynthesis remains enigmatic. Recently, two enzymes from poplar, salicyl alcohol benzoyl transferase and benzyl alcohol benzoyl transferase, were shown to catalyze the production of salicyl benzoate, a predicted potential intermediate in salicinoid biosynthesis. Here, we used transcriptomics and co‐expression analysis with these two genes to identify two UDP‐glucose‐dependent glycosyltransferases (UGT71L1 and UGT78M1) as candidate enzymes in this pathway. Both recombinant enzymes accepted only salicyl benzoate, salicylaldehyde and 2‐hydroxycinnamic acid as glucose acceptors. Knocking out the UGT71L1 gene by CRISPR/Cas9 in poplar hairy root cultures led to the complete loss of salicortin, tremulacin and tremuloidin, and a partial reduction of salicin content. This demonstrated that UGT71L1 is required for synthesis of the major salicinoids, and suggested that an additional route can lead to salicin. CRISPR/Cas9 knockouts for UGT78M1 were not successful, and its in vivo role thus remains to be determined. Although it has a similar substrate preference and predicted structure as UGT71L1, it appears not to contribute to the synthesis of salicortin, tremulacin and tremuloidin, at least in roots. The demonstration of UGT71L1 as an enzyme of salicinoid biosynthesis will open up new avenues for the elucidation of this pathway. Significance Statement: The salicinoids are bioactive and ecologically significant phenolics glycosides found in poplars and willows. Here, we identify two salicyl benzoate UDP‐glycosyltransferases and use CRISPR/Cas9 knockouts in transgenic poplar hairy roots to demonstrate that one of these enzymes is essential for salicinoid biosynthesis. [ABSTRACT FROM AUTHOR]

Adult and juvenile herbivores of the same species can use divergent feeding strategies, and thus may inhabit and consume different parts of the plant. Because the expression of chemical defences often differs between host plant tissues, this variation may result in disparate performance outcomes for adult and juvenile conspecifics that feed on distinct dietary substrates.The goal of this study was to evaluate how host range may differ between adults and juveniles in a generalist herbivore. We addressed the impacts of among‐ and within‐plant defence variation using the wood‐feeding Asian longhorned beetle (Anoplophora glabripennis) and three host plants having a range of putative resistance.Impacts of host plants on adult and offspring performance were assessed using a series of controlled bioassays. We evaluated adult‐feeding and egg‐laying behaviours in choice and no‐choice experiments using the different hosts, and subsequent offspring establishment. We then evaluated host plant chemical composition related to nutrition and defence.Different plants had strong impacts on adult performance, but these patterns did not extend to effects on offspring. Females were capable of developing eggs when provided Acer rubrum, but not Populus deltoides or Populus tomentosa. Females that produced eggs by feeding on A. rubrum, however, deposited eggs into all three plant species. Larvae hatched and consumed tissues in all three hosts. The differences between adult and juvenile utilization of Populus spp. were reflected in markedly higher salicinoid phenolic concentrations in bark (>2% dw), while wood had trace quantities.Our results demonstrate that plant resistance mechanisms can differentially act upon adult and juvenile life stages of a polyphagous herbivore when there is differential expression of chemical defences among plant tissue types. Anoplophora glabripennis has been a globally successful invader due in part to its broad host range, and our results suggest a mechanism that permits the beetle to exploit marginally resistant plants. This study has implications for how host range differs between insect feeding stages, which is particularly important for invasive, polyphagous species encountering novel food sources. [ABSTRACT FROM AUTHOR]

Overbrowsing by ungulates is a major cause of poor aspen stand regeneration across North America and Eurasia. In general, factors driving ungulate browser preferences include concentrations of plant secondary compounds and the nutritional composition (non-structural carbohydrates, protein and minerals) of foliage. While each of these phytochemical factors has been shown to independently influence ungulate preference, the relative impact of each factor is unknown, as no study to date has examined them simultaneously., Plant fitness depends not only on the capacity of plants to resist browsing, but also on their capacity to tolerate browsing once it has occurred. Little is known of aspen tolerance to browsing, which inflicts a different form of damage than insect herbivory., We employed multiple aspen genotypes, replicate trees of which were subjected to different soil nutrient treatments, to investigate: (i) the effects of aspen genotype, nutrient treatment and genotype × nutrient interactions on susceptibility to browsing by white-tailed deer, (ii) the phytochemical basis for the patterns observed in (i), and (iii) the effects of genotype, soil nutrients and their interaction on short-term tolerance to deer browsing., Aspen genotypes varied markedly in their vernal susceptibility to deer browsing. Genetic variation in early season levels of non-structural carbohydrates (sugars), protein and multiple macro- and trace minerals had the strongest influence on tree susceptibility to browsing. In contrast, levels of phytochemical defences had minimal effects, although the range of levels expressed in this study was small. Soil nutrient availability did not significantly influence deer preference., The extent of browsing affected post-browse tolerance across genotypes. Soil nutrient treatment had little differential effect on tolerance, and, for the most part, genotypes did not display differential tolerance to browsing, regardless of which soil nutrient treatment they experienced., Synthesis. Genetic variation for susceptibility to browsing indicates that ungulate browsers have the potential to be agents of selection in aspen populations. In contrast with previous studies in aspen highlighting the importance of phytochemical defences in shaping preferences of browsing mammals, our results indicate that the nutritional composition of foliage (sugars, protein and mineral concentrations) can have sizable effects on preference. The observed lack of influence of soil nutrient availability on tree browsing tolerance contrasts with predictions of the limiting resource model, the prevailing model for plant tolerance. [ABSTRACT FROM AUTHOR]

1 Trembling aspen Populus tremuloides Michaux is an important forest species in the Great Lakes region and displays tremendous genetic variation in foliar chemistry. Elevated carbon dioxide (CO2) and ozone (O3) may also influence phytochemistry and thereby alter the performance of insect herbivores such as the aspen leaf beetle Chrysomela crotchi Brown. 2 The present study aimed to relate genetic- and atmospheric-based variation in aspen phytochemistry to C. crotchi performance (larval development time, adult mass, survivorship). The experiment was conducted at the Aspen Free-Air CO2 Enrichment (FACE) site in northern Wisconsin. Beetles were reared on three aspen genotypes under elevated CO2 and/or O3. Leaves were collected to determine chemical characteristics. 3 The foliage exhibited significant variation in nitrogen, condensed tannins and phenolic glycosides among genotypes. CO2 and O3, however, had little effect on phytochemistry. Nonetheless, elevated CO2 decreased beetle performance on one aspen genotype and had inconsistent effects on beetles reared on two other genotypes. Elevated O3 decreased beetle performance, especially for beetles reared on an O3-sensitive genotype. Regression analyses indicated that phenolic glycosides and nitrogen explain a substantial amount (27–45%) of the variation in herbivore performance. 4 By contrast to the negative effects that are typically observed with generalist herbivores, aspen leaf beetles appear to benefit from phenolic glycosides, chemical components that are largely genetically-determined in aspen. The results obtained in the present study indicate that host genetic variation and atmospheric concentrations of greenhouse gases will be important factors in the performance of specialist herbivores, such as C. crotchi, in future climates. [ABSTRACT FROM AUTHOR]

Expression of foliar secondary compounds and extrafloral nectaries (EFNs) within the same leaves may be incompatible if secondary compounds repel beneficial insects that might otherwise be attracted to EFNs. This study examined the within-plant distributions of phenolic glycosides and EFNs in trembling aspen, Populus tremuloides, and their relationships to herbivore damage. Populus tremuloides expresses extrafloral nectaries (EFNs) on a subset of its leaves. We studied short and tall naturally occurring aspen ramets across multiple sites in interior Alaska. Contrary to our expectations, foliar phenolic glycoside concentrations were approximately 30% greater on leaves bearing EFNs than on leaves without EFNs. The mean concentration of foliar phenolic glycosides in short ramets was nine times that in tall ramets. Phenolic glycoside concentration was negatively related to leaf mining damage by Phyllocnistis populiella (Lepidoptera; Gracilliadae) at concentrations greater than 27 mg/g, whereas the presence of EFNs was unrelated to mining damage. The positive association of chemical defensive compounds and EFNs in leaves suggests that, for species with variation in EFN expression, negative correlations between herbivory and EFN expression may arise indirectly from associated effects of other, correlated types of defense. [ABSTRACT FROM AUTHOR]

1 The present study assessed the relationship between clonally variable rates of defoliation in trembling aspen ( Populus tremuloides Michx.) and two potential resistance traits: defensive chemistry and leaf phenology. 2 In 2001, coincident with a major outbreak of the forest tent caterpillar ( Malacosoma disstria Hubner) in the northcentral U.S.A., we monitored defoliation rates, phytochemical composition, and foliar development in 30 clones of trembling aspen. Leaf chemistry was also assessed in re-flushed leaves and 2 years post-outbreak. 3 Early in the season, differences in defoliation among clones were substantial but, by mid-June, all clones were completely defoliated. Leaf nitrogen, condensed tannins, and phenolic glycosides varied among clones but did not relate to defoliation levels. Budbreak phenology differed by 3 weeks among clones and clones that broke bud early or late relative to forest tent caterpillar eclosion experienced reduced rates of defoliation. 4 Defoliation led to increased tannins and slight decreases in phenolic glycoside concentrations in damaged leaf remnants, but to moderately decreased tannins and a six-fold increase in phenolic glycosides in reflushed leaves. This shift in chemical composition may significantly affect late season herbivores. 5 These results suggest that aspen chemical resistance mechanisms are ineffective during intense episodic eruptions of outbreak folivores such as the forest tent caterpillar. Variable budbreak phenology may lead to differential susceptibility during less intense outbreak years and, at peak forest tent caterpillar population densities, mechanisms affording tolerance are probably more important than chemical defences. [ABSTRACT FROM AUTHOR]

With the emerging field of community genetics, it is important to quantify the key mechanisms that link genetics and community structure. We studied cottonwoods in common gardens and in natural stands and examined the potential for plant chemistry to be a primary mechanism linking plant genetics and arthropod communities. If plant chemistry drives the relationship between plant genetics and arthropod community structure, then several predictions followed. We would find (i) the strongest correlation between plant genetic composition and chemical composition; (ii) an intermediate correlation between plant chemical composition and arthropod community composition; and (iii) the weakest relationship between plant genetic composition and arthropod community composition. Our results supported our first prediction: plant genetics and chemistry had the strongest correlation in the common garden and the wild. Our results largely supported our second prediction, but varied across space, seasonally, and according to arthropod feeding group. Plant chemistry played a larger role in structuring common garden arthropod communities relative to wild communities, free-living arthropods relative to leaf and stem modifiers, and early-season relative to late-season arthropods. Our results did not support our last prediction, as host plant genetics was at least as tightly linked to arthropod community structure as plant chemistry, if not more so. Our results demonstrate the consistency of the relationship between plant genetics and biodiversity. Additionally, plant chemistry can be an important mechanism by which plant genetics affects arthropod community composition, but other genetic-based factors are likely involved that remain to be measured. [ABSTRACT FROM AUTHOR]

• Here, the growth–differentiation balance hypothesis (GDBH) was tested by quantifying temporal variation in the relative growth rate (RGR), net assimilation rate (NAR), and phenylpropanoid concentrations of two willow species ( Salix sericea and Salix eriocephala) across five fertility levels. • Initially, RGR increased and total phenylpropanoids declined (although every individual phenolic did not) as fertility increased, but NAR was unaffected. Subsequently, NAR and phenylpropanoids declined in the low fertility treatment, generating a quadratic response of secondary metabolism across the nutrient gradient. As above- and below-ground growth rates equilibrated, NAR and phenylpropanoids increased in the low fertility treatment, re-establishing a negative linear effect of fertility on secondary metabolism. • A transient quadratic response of secondary metabolism is predicted when GDBH is integrated with models of optimal phenotypic plasticity, occurring when low NAR imposes carbon constraints on secondary metabolism in low nutrient environments. Once plants acclimate to nutrient limitation, the equilibrium allocation state is predicted to be a negative correlation between growth and secondary metabolism. • Although both willow species generally responded according to GDBH, the complexity observed suggests that prediction of the effects of nutrient availability on secondary metabolism (and other plastic responses) in specific cases requires a priori knowledge of the physiological status of the plant and soil nutrient availability. [ABSTRACT FROM AUTHOR]

1. Many Salicaceae species naturally form hybrid swarms with parental and hybrid taxa that differ in secondary chemical profile and in resistance to herbivores. Theoretically, the differential mortality in the seedling stage can lead to changes in trait expression and alter subsequent interactions between plants and herbivores. This study examines whether herbivory by the generalist slug Arion subfuscus, which causes extensive mortality in young willow seedlings, causes shifts in (a) the foliar chemistry of F2 willow hybrids ( Salix sericea and Salix eriocephala), and (b) the subsequent susceptibility to Japanese Beetles, Popillia japonica. 2. In 2001, two populations of F2 seedlings were generated: those that survived slug herbivory (80–90% of seedlings placed in the field were killed by the slugs) were designated as S-plants, whereas C-plants (controls) experienced no mortality. 3. Common garden experiments with cuttings from these populations, in 2001 and 2002, revealed extensive variation in the phenolic chemistry of F2 hybrids, but revealed no significant difference between S- and C-plants, although the levels of foliar nutrients, proteins and nitrogen tended to be higher in S-plants. 4. Concentrations of salicortin and 2′-cinnamoylsalicortin explained 55 and 38% of the the variation in leaf damage caused by Japanese beetles, and secondary chemistry was highly correlated within replicate clones (salicortin R2= 0.85, 2-cinnamoylsalicortin R2= 0.77, condensed tannins R2= 0.68). 5. Interestingly, Japanese beetle damage and condensed tannins were positively correlated within the S-plants, but not in the C-plants, suggesting that slugs had selected for plants with a positive relationship between tannins and P. japonica damage. This is unlikely to be a consequence of a preference for tannins, but is suggested to be related to the elevated nutrient levels in the S-plants, perhaps in combination with the complex-binding properties of tannins. 6. The damage was highly correlated within replicate clones and a model choice analysis suggested that Japanese beetle damage may be explained by four factors: concentrations of salicortin, condensed tannins, and nitrogen, as well as the specific leaf area (thick leaves were damaged less). [ABSTRACT FROM AUTHOR]

• Salicin-based phenolic glycosides, hydroxycinnamate derivatives and flavonoid-derived condensed tannins comprise up to one-third of Populus leaf dry mass. Genes regulating the abundance and chemical diversity of these substances have not been comprehensively analysed in tree species exhibiting this metabolically demanding level of phenolic metabolism. • Here, shikimate-phenylpropanoid pathway genes thought to give rise to these phenolic products were annotated from the Populus genome, their expression assessed by semiquantitative or quantitative reverse transcription polymerase chain reaction (PCR), and metabolic evidence for function presented. • Unlike Arabidopsis, Populus leaves accumulate an array of hydroxycinnamoyl-quinate esters, which is consistent with broadened function of the expanded hydroxycinnamoyl-CoA transferase gene family. Greater flavonoid pathway diversity is also represented, and flavonoid gene families are larger. Consistent with expanded pathway function, most of these genes were upregulated during wound-stimulated condensed tannin synthesis in leaves. • The suite of Populus genes regulating phenylpropanoid product accumulation should have important application in managing phenolic carbon pools in relation to climate change and global carbon cycling. [ABSTRACT FROM AUTHOR]

• Costs of defense are thought to maintain genetic variations in the expression of defense within plant populations. As with many plant species, aspen exhibits considerable variation in allocation to secondary metabolites. This study examined the independent and interactive effects of genotype, soil fertility and belowground competition on defensive chemistry and growth in trembling aspen ( Populus tremuloides). • Four aspen genotypes were grown with high and low soil fertility, and with and without root competition. Physiological, morphological and allocational determinants of growth were measured to identify growth–defense tradeoffs. • Nutrient limitation and competition decreased growth, leaf mass ratio, leaf nitrogen concentration and photosynthesis, and increased root : shoot ratio and leaf condensed tannin concentrations. The competition treatment also resulted in increased leaf phenolic glycoside (PG) concentrations. • Aspen growth was negatively correlated with PG concentrations under low fertility with competition. The relationship between growth and its major determinants was also negatively related to foliar condensed tannins expressed as a proportion of tree mass, indicating an additional indirect cost of allocation to secondary metabolites. [ABSTRACT FROM AUTHOR]

Abstract We investigated the effects of soil nutrient and water availability on the growth and chemistry of the silky willow (Salix sericea Marshall), and on the performance of the imported willow leaf beetle (Plagiodera versicolora Laichartig). Our major aims were to determine whether there are nutrient–water interactions on plant traits and whether this leads to parallel interactions for herbivore performance. We used a 2 × 3 fully factorial design, which consisted of high and low nutrient treatments crossed with dry, field capacity, and flooded water treatments. We found that nutrient additions increased plant growth, but only in field capacity and flooded conditions (nutrient–water interaction). Leaf nitrogen content also depended on the interaction between soil nutrients and water: nutrient addition resulted in a larger increase in foliar nitrogen in the field capacity treatment than in the flooded and dry treatments. Of the two phenolic glycosides measured, salicortin and 2′-cinnamoylsalicortin, only one was affected by the treatments. 2′-cinnamoylsalicortin concentration was lower in the high nutrient–dry treatment compared with the other treatments. In contrast to plant responses, there were no interactions found for larval or pupal weight or development time. Nutrient addition led to an increase in female pupal weight, and foliar N was positively correlated with female pupal weight and negatively correlated with female development time. In addition, leaf water was positively correlated with female development time. The lack of interactions for insect performance may stem from the small absolute differences in foliar nitrogen content associated with the interaction between the nutrients and water. Taken together, our results suggest that nutrient–water interactions influence plant traits that are potentially important for insect performance (leaf nitrogen and water), but these interactions do not produce... [ABSTRACT FROM AUTHOR]

Abstract. Herbivorous beetles were sampled in central Slovakia and in the Tatra Mountains of northern Slovakia from seven different Salix species which are partly characterized by smooth leaves containing phenolic glycosides and partly by hairy leaves containing tannins. The collection included about 8000 individuals representing 129 species. Of the 129 species, 77 species are able to use the willows as hosts; the remaining 52 ‘tourist’ species comprise less than 3% of the collected individuals. The data on species richness and abundance did not generally support the ‘feeding specialization’ hypothesis. The proportion of specialized (= monophagous and oligophagous) beetles feeding on willows of both morphological and biochemical groups was in the same range. Host plants of the two groups could support high diversity and high evenness values, even when leaf characteristics and plant chemicals largely influenced species assemblage. The region in which the willow trees grow had a considerable impact on host plant use. Generalist beetle species predominated in central Slovakia. By contrast, in the Tatra Mountains, specialist feeders which are able to use phenolic glycosides to their advantage were predominant. The number of species and the total density of individuals collected from willows containing phenolic glycosides (S. fragilis and S. purpurea ) did not usually vary between the two regions. In contrast, fewer species and individuals were found in the Tatra Mountains when they settled on willow species containing tannins (S. caprea and S. cinerea ). Also, the phylogenetic status of host plants affected species assemblages. In central Slovakia willow species of the subgenus Vetrix (S. purpurea, S. caprea and S. cinerea ) generally showed a higher beetle diversity (Hs ) than species of the subgenus Salix (S. fragilis, S. alba and S. triandra ), although both subgenera comprise species of both morphological and biochemical... [ABSTRACT FROM AUTHOR]

Summary1. This study investigated how phytochemical variation among clones of quaking aspen Populus tremuloides, growing in a common habitat, affects the growth and fecundity of a model herbivore. 2. Gypsy moth Lymantria dispar larvae were reared from egg hatch to pupation on 10 aspen clones in the field or on excised foliage in the laboratory. Foliage was collected from each clone, and concentrations of phenolic glycosides, condensed tannins, nitrogen, and water were determined. 3. Herbivore fitness parameters and aspen phytochemical concentrations varied significantly among clones. In both the field and laboratory, larvae reared on clones containing high concentrations of phenolic glycosides exhibited prolonged developmental times and reduced pupal weights and fecundity. Herbivore performance parameters were also related positively to foliar nitrogen concentrations in the laboratory. Food consumption, but neither growth nor reproductive parameters, were related positively to condensed tannin concentrations. 4. In this study, foliar concentrations of phenolic glycosides were implicated as a significant determinant of food quality for gypsy moths, consistent with results of previous laboratory experiments. Additionally, this study documents a case in which host plant variation at a local level influences the performance and possibly the distribution and abundance of an important herbivore. [ABSTRACT FROM AUTHOR]