High nitrogen application in maize enhances insecticide tolerance of the polyphagous herbivore Spodoptera litura by induction of detoxification enzymes and intensification of cuticle.

Nitrogen (N) is one of the most intensively used fertilizers in cropping system and could exert a variety of bottom-up effects on the ecological fitness of herbivores. However, the effects of increased N inputs on insect pesticide tolerance have not been comprehensively understood. Bioassays showed that high N (HN) applied to maize plants significantly increased larval tolerance of Spodoptera litura to multiple insecticides. Activities of detoxification enzymes were significantly higher in the larvae fed on maize plants supplied with HN. RNA-seq analysis showed that numerous GST and cuticle-related genes were induced in the larvae fed on HN maize. RT-qPCR analysis further confirmed four GST genes and larval-specific cuticle gene LCP167. Furthermore, when injected with dsRNA specific to GSTe1, GSTs5, and LCP167, the mortality of larvae treated with methomyl was about 3-fold higher than that of dsGFP-injected larvae. Electron microscope observation showed that cuticle of the larvae fed on HN maize was thicker than the medium level of N. These findings suggest that increased application of N fertilizer enhances insecticide tolerance of lepidopteran pests via induction of detoxification enzymes and intensification of cuticle. Thus, overuse of N fertilizer may increase pest insecticide tolerance and usage of chemical insecticides.

Hesperidin as a Species-Specific Modifier of Aphid Behavior.

Hesperidin is a highly bioactive natural flavonoid whose role in ecological interactions is poorly known. In particular, the effects of hesperidin on herbivores are rarely reported. Flavonoids have been considered as prospective biopesticides; therefore, the aim of the present study was to examine the influence of hesperidin on the host plant selection behavior of three aphid (Hemiptera: Aphididae) species: Acyrthosiphon pisum Harrris, Rhopalosiphum padi (L.), and Myzus persicae (Sulz.). The aphid host plants were treated with 0.1% and 0.5% ethanolic solutions of hesperidin. Aphid probing behavior in the no-choice experiment was monitored using electropenetrography and aphid settling on plants in the choice experiment was recorded. The results demonstrated that hesperidin can be applied as a pre-ingestive, ingestive, and post-ingestive deterrent against A. pisum, as an ingestive deterrent against R. padi, and as a post-ingestive deterrent against M. persicae using the relatively low 0.1% concentration. While in A. pisum the deterrent effects of hesperidin were manifested as early as during aphid probing in peripheral plant tissues, in M. persicae, the avoidance of plants was probably the consequence of consuming the hesperidin-containing phloem sap.

Reciprocal mutations of two multifunctional ß-amyrin synthases from Barbarea vulgaris shift α/ß-amyrin ratios.

In the wild cruciferous wintercress (Barbarea vulgaris), ß-amyrin-derived saponins are involved in resistance against insect herbivores like the major agricultural pest diamondback moth (Plutella xylostella). Enzymes belonging to the 2,3-oxidosqualene cyclase family have been identified and characterized in B. vulgaris G-type and P-type plants that differ in their natural habitat, insect resistance and saponin content. Both G-type and P-type plants possess highly similar 2,3-oxidosqualene cyclase enzymes that mainly produce ß-amyrin (Barbarea vulgaris Lupeol synthase 5 G-Type; BvLUP5-G) or α-amyrin (Barbarea vulgaris Lupeol synthase 5 P-Type; BvLUP5-P), respectively. Despite the difference in product formation, the two BvLUP5 enzymes are 98% identical at the amino acid level. This provides a unique opportunity to investigate determinants of product formation, using the B. vulgaris 2,3-oxidosqualene cyclase enzymes as a model for studying amino acid residues that determine differences in product formation. In this study, we identified two amino acid residues at position 121 and 735 that are responsible for the dominant changes in generated product ratios of ß-amyrin and α-amyrin in both BvLUP5 enzymes. These amino acid residues have not previously been highlighted as directly involved in 2,3-oxidosqualene cyclase product specificity. Our results highlight the functional diversity and promiscuity of 2,3-oxidosqualene cyclase enzymes. These enzymes serve as important mediators of metabolic plasticity throughout plant evolution.

(+)-Catechin, epicatechin and epigallocatechin gallate are important inducible defensive compounds against Ectropis grisescens in tea plants.

The tea plant, Camellia sinensis (L.) O. Kuntze, is an economically important, perennial woody plant rich in catechins. Although catechins have been reported to play an important role in plant defences against microbes, their roles in the defence of tea plants against herbivores remain unknown. In this study, we allowed the larvae of Ectropis grisescens, a leaf-feeding pest, to feed on the plants, and alternatively, we wounded the plants and then treated them with E. grisescens oral secretions (WOS). Both approaches triggered jasmonic acid-, ethylene- and auxin-mediated signalling pathways; as a result, plants accumulated three catechin compounds: (+)-catechin, epicatechin and epigallocatechin. Not only was the mass of E. grisescens larvae fed on plants previously infested with E. grisescens or treated with WOS significantly lower than that of larvae fed on controls, but also artificial diet supplemented with epicatechin, (+)-catechin or epigallocatechin gallate reduced larval growth rates. In addition, the exogenous application of jasmonic acid, ethylene or auxin induced the biosynthesis of the three catechins, which, in turn, enhanced the resistance of tea plants to E. grisescens, leading to the coordination of the three signalling pathways. Our results suggest that the three catechins play an important role in the defences of tea plants against E. grisescens.

The Ca2+ Channel CNGC19 Regulates Arabidopsis Defense Against Spodoptera Herbivory.

Cellular calcium elevation is an important signal used by plants for recognition and signaling of environmental stress. Perception of the generalist insect, Spodoptera litura, by Arabidopsis (Arabidopsis thaliana) activates cytosolic Ca2+ elevation, which triggers downstream defense. However, not all the Ca2+ channels generating the signal have been identified, nor are their modes of action known. We report on a rapidly activated, leaf vasculature- and plasma membrane-localized, CYCLIC NUCLEOTIDE GATED CHANNEL19 (CNGC19), which activates herbivory-induced Ca2+ flux and plant defense. Loss of CNGC19 function results in decreased herbivory defense. The cngc19 mutant shows aberrant and attenuated intravascular Ca2+ fluxes. CNGC19 is a Ca2+-permeable channel, as hyperpolarization of CNGC19-expressing Xenopus oocytes in the presence of both cyclic adenosine monophosphate and Ca2+ results in Ca2+ influx. Breakdown of Ca2+-based defense in cngc19 mutants leads to a decrease in herbivory-induced jasmonoyl-l-isoleucine biosynthesis and expression of JA responsive genes. The cngc19 mutants are deficient in aliphatic glucosinolate accumulation and hyperaccumulate its precursor, methionine. CNGC19 modulates aliphatic glucosinolate biosynthesis in tandem with BRANCHED-CHAIN AMINO ACID TRANSAMINASE4, which is involved in the chain elongation pathway of Met-derived glucosinolates. Furthermore, CNGC19 interacts with herbivory-induced CALMODULIN2 in planta. Together, our work reveals a key mechanistic role for the Ca2+ channel CNGC19 in the recognition of herbivory and the activation of defense signaling.

MYC2-Activated TRICHOME BIREFRINGENCE-LIKE37 Acetylates Cell Walls and Enhances Herbivore Resistance.

O-Acetylation of polysaccharides predominantly modifies plant cell walls by changing the physicochemical properties and, consequently, the structure and function of the cell wall. Expression regulation and specific function of cell wall-acetylating enzymes remain to be fully understood. In this report, we cloned a previously identified stunted growth mutant named sucrose uncoupled1 (sun1) in Arabidopsis (Arabidopsis thaliana). SUN1 encodes a member of the TRICHOME BIREFRINGEN-LIKE family, AtTBL37 AtTBL37 is highly expressed in fast-growing plant tissues and encodes a Golgi apparatus-localized protein that regulates secondary cell wall thickening and acetylation. In sun1, jasmonate signaling and expression of downstream chemical defense genes, including VEGETATIVE STORAGE PROTEIN1 and BRANCHED-CHAIN AMINOTRANSFERASE4, are increased but, unexpectedly, sun1 is more susceptible to insect feeding. The central transcription factor in jasmonate signaling, MYC2, binds to and induces AtTBL37 expression. MYC2 also promotes the expression of many other TBLs Moreover, MYC activity enhances cell wall acetylation. Overexpression of AtTBL37 in the myc2-2 background reduces herbivore feeding. Our study highlights the role of O-acetylation in controlling plant cell wall properties, plant development, and herbivore defense.

Spinescence and Total Phenolic Content Do Not Influence Diet Preference of a Critically Endangered Megaherbivore, but the Mix of Compounds Does.

In contrast to understanding spinescence in savanna woody species, little is known about the functions of plant secondary metabolites (PSM). Negative effects of PSMs on individual animal performance potentially translate into negative effects on herbivore population growth. Hence, understanding PSM functions is important for the conservation of savanna megafauna. We tested the view that black rhinoceros (Diceros bicornis) diet preference is not affected by spinescence or total phenolic abundance. We hypothesized that the composition of phenolic mixtures, however, would affect preference. Furthermore, we tested our data from 71 woody species for a trade-off between structural and chemical defenses. Spinescence type, and spinescence generally, did not deter black rhino feeding. Using eco-metabolomic data, we found that total abundance of phenolics did not affect preference, but mixture composition did and that the probability of spinescence trading off against phenolics depended on the mixture. We note that our study was restricted to black rhino and that diet preferences of other mammal herbivores might be influenced by subtle differences in phenolic mixtures. However, our results did support a previous, more detailed study of phenolic profiles of six species showing the same patterns in relation to preference generalised across mammal herbivore species in savannas. Our results represent substantial advancement in the understanding of the roles of PSMs, especially flavonoid compounds, in the functioning of savanna ecosystems, and highlight the need to dig deeper into broad groups of traits such as spinescence or total phenolics to improve understanding of woody plant defenses in savannas.

Species-Specific Induction of Plant Volatiles by Two Aphid Species in Apple: Real Time Measurement of Plant Emission and Attraction of Lacewings in the Wind Tunnel.

Upon damage by herbivores, plants release herbivory-induced plant volatiles (HIPVs). To find their prey, the pest's natural enemies need to be fine-tuned to the composition of these volatiles. Whereas standard methods can be used in the identification and quantitation of HIPVs, more recently introduced techniques such as PTR-ToF-MS provide temporal patterns of the volatile release and detect additional compounds. In this study, we compared the volatile profile of apple trees infested with two aphid species, the green apple aphid Aphis pomi, and the rosy apple aphid Dysaphis plantaginea, by CLSA-GC-MS complemented by PTR-ToF-MS. Compounds commonly released in conjunction with both species include nonanal, decanal, methyl salicylate, geranyl acetone, (Z)-3-hexenyl acetate, (Z)-3-hexenyl butanoate, (Z)-3-hexenyl 2-methyl-butanoate, (E)-ß-caryophyllene, ß-bourbonene and (Z)-3-hexenyl benzoate. In addition, benzaldehyde and (E)-ß-farnesene were exclusively associated with A. pomi, whereas linalool, (E)-4,8-dimethyl-1,3,7-nonatriene were exclusively associated with D. plantaginea. PTR-ToF-MS additionally detected acetic acid (AA) and 2-phenylethanol (PET) in the blends of both trees attacked by aphid species. In the wind tunnel, the aphid predator, Chrysoperla carnea (Stephens), responded strongly to a blend of AA and PET, much stronger than to AA or PET alone. The addition of common and species-specific HIPVs did not increase the response to the binary blend of AA and PET. In our setup, two host-associated volatiles AA + PET appeared sufficient in the attraction of C. carnea. Our results also show the importance of combining complementary methods to decipher the odor profile associated with plants under pest attack and identify behaviourally active components for predators.

Zea mays Volatiles that Influence Oviposition and Feeding Behaviors of Spodoptera frugiperda.

Fall armyworm (Spodoptera frugiperda) is a major global pest of many crops, including maize (Zea mays). This insect is known to use host plant-derived volatile organic compounds to locate suitable hosts during both its adult and larval stages, yet the function of individual compounds remains mostly enigmatic. In this study, we use a combination of volatile profiling, electrophysiological assays, pair-wise choice behavioral assays, and chemical supplementation treatments to identify and assess specific compounds from maize that influence S. frugiperda host location. Our findings reveal that methyl salicylate and (E)-alpha-bergamotene are oviposition attractants for adult moths but do not impact larval behavior. While geranyl acetate can act as an oviposition attractant or repellent depending on the host volatile context and (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) is an oviposition deterrent. These compounds can also be attractive to the larvae when applied to specific maize inbreds. These data show that S. frugiperda uses different plant volatile cues for host location in its adult and larval stage and that the background volatile context that specific volatiles are perceived in, alters their impact as behavioral cues.

Water Deficiency and Induced Defense Against a Generalist Insect Herbivore in Desert and Mediterranean Populations of Eruca sativa.

In natural and agricultural ecosystems, plants are often simultaneously or sequentially exposed to combinations of stressors. Here we tested whether limited water availability (LWA) affects plant response to insect herbivory using two populations of Eruca sativa from desert and Mediterranean habitats that differ in their induced defenses. Considering that such differences evolved as responses to biotic and possibly abiotic stress factors, the two populations offered an opportunity to study ecological aspects in plant response to combined stresses. Analysis of chemical defense mechanisms showed that LWA significantly induced total glucosinolate concentrations in the Mediterranean plants, but their concentrations were reduced in the desert plants. However, LWA, with and without subsequent jasmonate elicitation, significantly induced the expression of proteinase inhibitor in the desert plants. Results of a no-choice feeding experiment showed that LWA significantly increased desert plant resistance to Spodoptera littoralis larvae, whereas it did not affect the relatively strong basal resistance of the Mediterranean plants. LWA and subsequent jasmonate elicitation increased resistance against the generalist insect in Mediterranean plants, possibly due to both increased proteinase inhibitor expression and glucosinolate accumulation. The effect of LWA on the expression of genes involved in phytohormone signaling, abscisic acid (ABA-1) and jasmonic acid (AOC1), and the jasmonate responsive PDF1.2, suggested the involvement of abscisic acid in the regulation of defense mechanisms in the two populations. Our results indicate that specific genotypic responses should be considered when estimating general patterns in plant response to herbivory under water deficiency conditions.

Factors Affecting Foliar Oregonin and Condensed Tannin in Red Alder (Alnus rubra Bong.): Phytochemicals Implicated In Defense Against Western Tent Caterpillar (Malacosoma californicum Packard).

Past work shows a significant negative correlation between foliar oregonin concentration and western tent caterpillar (Malacosoma californicum Packard) feeding on red alder (Alnus rubra Bong.). Above an oregonin threshold of 20% leaf dry weight, little feeding by caterpillars is observed. Concentrations of defensive chemicals are influenced by plant genotype, environmental conditions, insect feeding, and the interactions of these factors. Our objective was to measure the effects of nitrogen (N) availability and wounding on foliar oregonin and condensed tannin concentrations in red alder genotypes. One-year-old seedlings from 100 half-sib red alder families were treated with two levels of ammonium nitrate (NH4NO3) for two growing seasons in a common garden. In the second year, leaves from 50 families from the fertilization experiment were used in a bioassay feeding experiment to determine the effects of N fertilization and genotype on WTC damage, and to identify a subset of 20 families with a range of damage to analyze for phytochemical composition. In separate experiments, wound-induction treatments were conducted outdoors and, in a greenhouse using the N treated trees in their third and fourth year, respectively. Foliar condensed tannin, oregonin and N concentrations were measured and ranked among the plant genotypes, and between the two N treatments and two wounding treatments. Results showed that oregonin and condensed tannin concentrations varied among the alder genotypes. Leaf N concentration was negatively correlated with concentration of oregonin. Neither of the measured phenolic compounds responded to wounding. The results suggest that red alder foliar oregonin and condensed tannin are likely constitutive defenses that are largely determined by genotype, and that the negative correlation of defense compounds with plant internal N status holds in this N-fixing tree.

Maize Endochitinase Expression in Response to Fall Armyworm Herbivory.

A large percentage of crop loss is due to insect damage, especially caterpillar damage. Plant chitinases are considered excellent candidates to combat these insects since they can degrade chitin in peritrophic matrix (PM), an important protective structure in caterpillar midgut. Compared to chemical insecticides, chitinases could improve host plant resistance and be both economically and environmentally advantageous. The focus of this research was to find chitinase candidates that could improve plant resistance by effectively limiting caterpillar damage. Five classes of endochitinase (I-V) genes were characterized in the maize genome, and we isolated and cloned four chitinase genes (chitinase A, chitinase B, chitinase I, and PRm3) present in two maize (Zea mays L.) inbred lines Mp708 and Tx601, with different levels of resistance to caterpillar pests. We also investigated the expression of these maize chitinases in response to fall armyworm (Spodoptera frugiperda, FAW) attack. The results indicated that both chitinase transcript abundance and enzymatic activity increased in response to FAW feeding and mechanical wounding. Furthermore, chitinases retained activity inside the caterpillar midgut and enzymatic activity was detected in the food bolus and frass. When examined under scanning electron microscopy, PMs from Tx601-fed caterpillars showed structural damage when compared to diet controls. Analysis of chitinase transcript abundance after caterpillar feeding and proteomic analysis of maize leaf trichomes in the two inbreds implicated chitinase PRm3 found in Tx601 as a potential insecticidal protein.

Triterpenes induced by young apple fruits in response to herbivore attack.

Apples Malus domestica, known as a rich source of triterpene acids, induced more variety and quantity of triterpene acids in response to herbivory or mechanical damage. There were 3 major induced compounds: pomaceic acid and euscaphic acid, both of which are known apple triterpene acids, and 2α,19α-dihydroxy-3-oxours-12-en-28-oic acid (named eriobotoric acid), which was first identified in apples. In this study, the 3 compounds' induction curves after damage, varietal differences in induction amounts, and physiological roles against pest insects were further investigated. Eriobotoric acid showed clear antifeedant activity against lepidopteran insect Spodoptera litura but not against apple pests.

Exogenous nitrogen enhances poplar resistance to leaf herbivory and pathogen infection after exposure to soil cadmium stress.

Elemental defense hypothesis suggests that toxic metals accumulated in plant tissues could enhance plant defense against herbivores and pathogens. Since over-accumulation of metals in plant organs will pose negative effects on plant health, it is necessary to find a way to alleviate metal-induced toxicity in plants while keeping or even improving plant resistance. Exogenous nitrogen (N) application was reported to have such alleviation effect while stimulating metal accumulation in plant tissues. In this study, we examined whether soil N addition in three different doses to a poplar species under cadmium (Cd) stress can simultaneously improve plant growth and resistance to four herbivorous insects and a leaf pathogen. The results showed that N application to Cd-amended soil prominently enhanced plant growth and leaf Cd accumulation. While N addition in three doses all remarkably reduced herbivore growth than control plants, only the highest N dose exerted stronger inhibition than the sole Cd-treated plants. In the paired-choice experiment, plants supplied with the highest N dose showed an enhanced deterrent effect on herbivore preference than plants exposed to sole Cd. Furthermore, plant resistance to the leaf pathogen infection was strongly enhanced as the levels of N addition increased. Leaf sugar and three main defensive chemicals were not affected by N application implied that such enhanced effect of N on plant resistance was due to increased leaf Cd accumulation. Our results suggested that the application of exogenous N over a certain amount could enhance the resistance of Cd-treated plants to leaf herbivory and pathogen infection.

Functional Studies of Plant Latex as a Rich Source of Bioactive Compounds: Focus on Proteins and Alkaloids.

Latex, a sticky emulsion produced by specialized cells called laticifers, is a crucial part of a plant's defense system against herbivory and pathogens. It consists of a broad spectrum of active compounds, which are beneficial not only for plants, but for human health as well, enough to mention the use of morphine or codeine from poppy latex. Here, we reviewed latex's general role in plant physiology and the significance of particular compounds (alkaloids and proteins) to its defense system with the example of Chelidonium majus L. from the poppy family. We further attempt to present latex chemicals used so far in medicine and then focus on functional studies of proteins and other compounds with potential pharmacological activities using modern techniques such as CRISPR/Cas9 gene editing. Despite the centuries-old tradition of using latex-bearing plants in therapies, there are still a lot of promising molecules waiting to be explored.

Inefficient weapon-the role of plant secondary metabolites in cotton defence against the boll weevil.

MAIN CONCLUSION: Cotton genotypes displayed similar volatile organic compound (VOC) profiles, but major differences in terpenoid aldehyde (TA) content. The differences in VOC production were minor among genotypes, but these differences are crucial for boll weevil attraction. Weevils did not display any preference in feeding behaviour towards cotton genotypes, suggesting physiological adaptation to cope with cotton chemical defence mechanisms. Plant cultivar selection for resistance to herbivore pests is an effective, environmentally safe and inexpensive method to implement in integrated pest management programmes. In this study, we evaluated seven cotton genotypes with respect to the production of volatile organic compounds (VOCs) and non-volatile compounds [terpenoid aldehydes (TAs)], and the attraction and feeding preference of adult boll weevils. Chemical analyses of VOCs from BRS-293, BRS-Rubi, CNPA TB-15, CNPA TB-85, CNPA TB-90, Delta Opal, and Empire Glandless showed that there were few qualitative and quantitative differences across the range of genotypes. In contrast, major differences in TA content were observed, with CNPA TB-15 and CNPA TB-85 producing higher levels of TAs compared to the other genotypes. Our results showed that boll weevil attraction to cotton genotypes varied, suggesting that the ratios and quantities of emitted cotton VOCs are important for host location. However, boll weevil feeding behaviour was neither positively nor negatively influenced by the terpenoid content (non-volatile compounds) of cotton genotypes. The results in this study suggest that boll weevils have adapted physiologically to cope with cotton chemical defence mechanisms.

Volatiles and Tannins in Pistacia lentiscus and Their Role in Browsing Behavior of Goats (Capra hircus).

Goat herding is an important tool in the ecologically sound management of Mediterranean shrublands and woodlands, although effective levels of woody biomass removal by the goats is neither guaranteed nor easy to predict. Preliminary observations indicated that one reason for this may be poor understanding of plant-herbivore interactions that operate intraspecifically at the local spatial scale. We asked, whether goats show intraspecific preferences among neighboring plants when foraging a small local population of Pistacia lentiscus, a dominant tall shrub. First, we characterized and quantified the profile of stored and emitted volatile organic compounds (VOCs) and the PEG-binding capacity of tannins (a proxy for protein binding capacity) in the foliage of P. lentiscus shrubs, sampled within an area of 0.9 ha. We then tested goat preference between pairs of these shrubs that differed in chemical composition. Almost all sampled P. lentiscus shrubs were allocated to one of two distinct VOC chemotypes: one dominated by germacrene D and limonene (designated chemotype L) and the other by germacrene D and α-pinene (chemotype P). In contrast, continuous moderate variability was found in the binding capacity of tannins in the foliage. Goats showed preference for shrubs of chemotype L over those of chemotype P, and their preference was negatively correlated with the binding capacity of tannins. Possible influences of VOCs on goat preference that may explain the observed patterns are discussed in the light of possible context-dependent interpretation of plant VOC signals by large mammalian herbivores.

Less Is More: a Mutation in the Chemical Defense Pathway of Erysimum cheiranthoides (Brassicaceae) Reduces Total Cardenolide Abundance but Increases Resistance to Insect Herbivores.

Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hübner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.

Impact of Seasonal and Temperature-Dependent Variation in Root Defense Metabolites on Herbivore Preference in Taraxacum officinale.

Plants experience seasonal fluctuations in abiotic and biotic factors such as herbivore attack rates. If and how root defense expression co-varies with seasonal fluctuations in abiotic factors and root herbivore attack rates is not well understood. Here, we evaluated seasonal changes in defensive root latex chemistry of Taraxacum officinale plants in the field and correlated the changes with seasonal fluctuations in abiotic factors and damage potential by Melolontha melolontha, a major natural enemy of T. officinale. We then explored the causality and consequences of these relationships under controlled conditions. The concentration of the defensive sesquiterpene lactone taraxinic acid ß-D glucopyranosyl ester (TA-G) varied substantially over the year and was most strongly correlated to mean monthly temperature. Both temperature and TA-G levels were correlated with annual fluctuations in potential M. melolontha damage. Under controlled conditions, plants grown under high temperature produced more TA-G and were less attractive for M. melolontha. However, temperature-dependent M. melolontha feeding preferences were not significantly altered in TA-G deficient transgenic lines. Our results suggest that fluctuations in temperature leads to variation in the production of a root defensive metabolites that co-varies with expected attack of a major root herbivore. Temperature-dependent herbivore preference, however, is likely to be modulated by other phenotypic alterations.

Phytochemical Shift from Condensed Tannins to Flavonoids in Transgenic Betula pendula Decreases Consumption and Growth but Improves Growth Efficiency of Epirrita autumnata Larvae.

Despite active research, antiherbivore activity of specific plant phenolics remains largely unresolved. We constructed silver birch (Betula pendula) lines with modified phenolic metabolism to study the effects of foliar flavonoids and condensed tannins on consumption and growth of larvae of a generalist herbivore, the autumnal moth (Epirrita autumnata). We conducted a feeding experiment using birch lines in which expression of dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin reductase (ANR) had been decreased by RNA interference. Modification-specific effects on plant phenolics, nutrients and phenotype, and on larval consumption and growth were analyzed using uni- and multivariate methods. Inhibiting DFR expression increased the concentration of flavonoids at the expense of condensed tannins, and silencing DFR and ANR decreased leaf and plant size. E. autumnata larvae consumed on average 82% less of DFRi plants than of unmodified controls, suggesting that flavonoids or glandular trichomes deter larval feeding. However, larval growth efficiency was highest on low-tannin DFRi plants, indicating that condensed tannins (or their monomers) are physiologically more harmful than non-tannin flavonoids for E. autumnata larvae. Our results show that genetic manipulation of the flavonoid pathway in plants can effectively be used to produce altered phenolic profiles required for elucidating the roles of low-molecular weight phenolics and condensed tannins in plant-herbivore relationships, and suggest that phenolic secondary metabolites participate in regulation of plant growth.