Heidi M. Appel

Phenolics in ecological interactions: The importance of oxidation

The ecological activities of plant phenolics are diverse and highly variable. Although some variation is attributable to differences in concentration, structure, and evolutionary history of association with target organisms, much of it is unexplained, making it difficult to predict when and where phenolics will be active. I suggest that our understanding is limited by a failure to appreciate the importance of oxidative activation and the conditions that influence it. I summarize examples of oxidative activation of phenolics in ecological interactions, and argue that physicochemical conditions of the environment that control phenolic oxidation generate variation in ecological activity. Finally, I suggest that measurements of oxidative conditions can improve our predictions of phenolic activity and that experiments must be designed with conditions appropriate to the biochemical mode of phenolic action.

Major Signaling Pathways Modulate Arabidopsis Glucosinolate Accumulation and Response to Both Phloem-Feeding and Chewing Insects

Plant responses to enemies are coordinated by several interacting signaling systems. Molecular and genetic studies with mutants and exogenous signal application suggest that jasmonate (JA)-, salicylate (SA)-, and ethylene (ET)-mediated pathways modulate expression of portions of the defense phenotype in Arabidopsis (Arabidopsis thaliana), but have not yet linked these observations directly with plant responses to insect attack. We compared the glucosinolate (GS) profiles of rosette leaves of 4-week-old mutant and transgenic Arabidopsis (Columbia) plants compromised in these three major signaling pathways, and characterized responses by those plants to feeding by two phloem-feeding aphids (generalist Myzus persicae and specialist Brevicoryne brassicae) and one generalist caterpillar species (Spodoptera exigua Hubner). Blocked JA signaling in coronatine-insensitive (coi1) and enhanced expression of SA-signaled disease resistance in hypersensitive response-like (hrl1) mutants reduced constitutive GS concentrations, while blocking SA signaling at the mediator protein npr1 mutant (NPR) increased them. There was no significant impact on constitutive GS contents of blocking ET signaling (at ET resistant [etr1]) or reducing SA concentrations (nahG transgene). We found increased GS accumulation in response to insect feeding, which required functional NPR1 and ETR1 but not COI1 or SA. Insect feeding caused increases primarily in short-chain aliphatic methylsulfinyl GS. By contrast, responses to exogenous JA, a frequent experimental surrogate for insect attack, were characterized by an increase in indolyl GS. Insect performance, measured as population increase or weight increase, was negatively related to GS levels, but we found evidence that other, ET-regulated factors may also be influential. Plant resistance to (consumption by) S. exigua was not related to insect growth because some plant chemistries inhibited growth while others inhibited feeding. These major signaling pathways modulate Arabidopsis GS accumulation and response to both phloem-feeding and chewing insects, often antagonistically; NPR appears to be central to these interactions. Our results indicate that exogenous signal application and plant consumption measures may not provide useful measures of plant responses to actual insect feeding.

THE GROWTH-DEFENSE TRADE-OFF AND HABITAT SPECIALIZATION BY PLANTS IN AMAZONIAN FORESTS

Tropical forests include a diversity of habitats, which has led to specialization in plants. Near Iquitos, in the Peruvian Amazon, nutrient-rich clay forests surround nutrient-poor white-sand forests, each harboring a unique composition of habitat specialist trees. We tested the hypothesis that the combination of impoverished soils and herbivory creates strong natural selection for plant defenses in white-sand forest, while rapid growth is favored in clay forests. Recently, we reported evidence from a reciprocal-transplant experiment that manipulated the presence of herbivores and involved 20 species from six genera, including phylogenetically independent pairs of closely related white-sand and clay specialists. When protected from herbivores, clay specialists exhibited faster growth rates than white-sand specialists in both habitats. But, when unprotected, white-sand specialists outperformed clay specialists in white-sand habitat, and clay specialists outperformed white-sand specialists in clay habitat. Here we test further the hypothesis that the growth defense trade-off contributes to habitat specialization by comparing patterns of growth, herbivory, and defensive traits in these same six genera of white-sand and clay specialists. While the probability of herbivore attack did not differ between the two habitats, an artificial defoliation experiment showed that the impact of herbivory on plant mortality was significantly greater in white-sand forests. We quantified the amount of terpenes, phenolics, leaf toughness, and available foliar protein for the plants in the experiment. Different genera invested in different defensive strategies, and we found strong evidence for phylogenetic constraint in defense type. Overall, however, we found significantly higher total defense investment for white-sand specialists, relative to their clay specialist congeners. Furthermore, herbivore resistance consistently exhibited a significant trade-off against growth rate in each of the six phylogenetically independent species-pairs. These results confirm theoretical predictions that a trade-off exists between growth rate and defense investment, causing white-sand and clay specialists to evolve divergent strategies. We propose that the growth-defense trade-off is universal and provides an important mechanism by which herbivores govern plant distribution patterns across resource gradients.

Limitations of folin assays of foliar phenolics in ecological studies

We examined the response of the widely used Folin-Denis assay to purified tannins from 16 woody plant species and to three commercial polyphenol preparations often used as standards. The reagents response to these chemical mixtures differed significantly among sources (tree species, commercial preparations) and sampling dates, even though the mixtures contained the same total dry weight of tannins. Response to commercial standards usually did not resemble response to actual plant tannin and produced estimates that differed from actual concentrations by as much as twofold. Species-based and seasonal differences in polyphenol composition are evidently responsible for these variable results. Reagents that depend on redox reactions, such as the Folin-Denis, do not produce reliable absolute or relative quantification of phenolics when different species or samples from different dates are compared, and use of commercial standards does not resolve this problem.

Significance of metabolic load in the evolution of host specificity of Manduca sexta

The existence of elevated metabolic loads associated with the detoxification of plant allelochemicals has been proposed to be an important selective force in the evo- lution of dietary specialization of herbivorous insects. In this study we have examined the effects of one host plant toxin (nicotine) and three nonhost plant toxins (xanthotoxin, precocene II, and canavanine) on the growth and energy metabolism of the tobacco horn- worm, Manduca sexta (Lepidoptera: Sphingidae). Although M. sexta has three mechanisms for reducing the toxic effects of nicotine, nicotine has a dose-dependent growth-inhibiting effect on third-instar larvae at dietary concentrations ranging from 0.25 to 8.0%. Thus, herbivory by adapted species, as well as nonadapted species, can potentially select for higher levels of defensive chemicals in plants. At higher concentrations, where the effects of nicotine are most pronounced, growth re- duction is associated with reduced consumption (which reduces the amount of food assim- ilated) and an increase in the duration of the instar (which increases the amount of the assimilate pool that must be allocated to maintenance metabolism), thus leaving less assimilate for allocation to growth. The respiration rates of larvae on diets containing up to 1% nicotine are the same as the respiration rates of larvae on nicotine-free diets, while the respiration rates of larvae on diets containing 4 to 8% nicotine are lower than those of larvae on the nicotine-free diet. Thus, the processing of nicotine does not impose a sig- nificant metabolic cost on M. sexta larvae, and it is invalid to infer increased metabolic costs of detoxification from the observation of concomitant decreases in growth rate (GR or RGR) and the efficiency of conversion of digested food (ECD) of larvae on toxin- containing diets. In a manner similar to nicotine, the growth-reducing effects of the nonhost plant toxins (xanthotoxin, precocene II, and canavanine) can be explained on the basis of their effects on consumption and the duration of the third instar. In no case is growth reduction a consequence of an increase in metabolic rate. Instead, reductions in growth result from the diversion of assimilate from growth to energy metabolism mandated by a longer instar, and from decreases in the size of the assimilate pool resulting from decreases in consumption and/or assimilation. Thus, the effects of allelochemicals on the energy budget of M. sexta result from their activity as metabolic toxins and feeding deterrents reducing rates of growth and consumption, and not from their diversion of energy from growth to allelochemical processing. We conclude that (1) the processing of host plant allelochemicals does not impose a significant energy demand on Manduca sexta and (2) the energy costs of allelochemical processing are unlikely either to constrain the expansion or to drive the contraction of the host plant range of M. sexta. Therefore, we suggest that the concept of metabolic load is not a useful one in understanding the effects of plant allelochemicals on the growth and efficiency of food utilization or in explaining the evolution of dietary specialization of lepidopteran herbivores.

Flexible resource allocation during plant defense responses

Plants are organisms composed of modules connected by xylem and phloem transport streams. Attack by both insects and pathogens elicits sometimes rapid defense responses in the attacked module. We have also known for some time that proteins are often reallocated away from pathogen-infected tissues, while the same infection sites may draw carbohydrates to them. This has been interpreted as a tug of war in which the plant withdraws critical resources to block microbial growth while the microbes attempt to acquire more resources. Sink-source regulated transport among modules of critical resources, particularly carbon and nitrogen, is also altered in response to attack. Insects and jasmonate can increase local sink strength, drawing carbohydrates that support defense production. Shortly after attack, carbohydrates may also be drawn to the root. The rate and direction of movement of photosynthate or signals in phloem in response to attack is subject to constraints that include branching, degree of connection among tissues, distance between sources and sinks, proximity, strength, and number of competing sinks, and phloem loading/unloading regulators. Movement of materials (e.g., amino acids, signals) to or from attack sites in xylem is less well understood but is partly driven by transpiration. The root is an influential sink and may regulate sink-source interactions and transport above and below ground as well as between the plant and the rhizosphere and nearby, connected plants. Research on resource translocation in response to pathogens or herbivores has focused on biochemical mechanisms; whole-plant research is needed to determine which, if any, of these plant behaviors actually influence plant fitness.

Antimicrobial Activity of Polyphenols Mediates Plant-Herbivore Interactions

The view that polyphenols have as their main function defending plants against herbivores has only equivocal experimental support. Polyphenol action against plant pathogenic microbes, however, is well substantiated in the literature, and this substantiation extends to nonpathogenic microbes as well. It is not surprising then that polyphenols are being shown to have significant activity against microbes that are either symbionts or pathogens of insect herbivores. We suggest that any influence of plant polyphenols on herbivores may often arise indirectly via action on herbivore-associated microbes, rather than by acting directly on the herbivore.

The influence of host plant on gut conditions of gypsy moth (Lymantria dispar) caterpillars

Abstract We examined the pH and E h (redox potential) of the lumen of the digestive tract of gypsy moth caterpillars on six different host plants to understand the physiochemical conditions under which plant nutrients are extracted and allelochemicals are detoxified. To identify potential plant characteristics associated with the gut conditions, we determined the plant pH, E h , pH buffering capacity, and level of reducing phenolic groups for each host. We found (1) host plants differ in specific traits such as leaf pH, pH buffering capacity, E h , and the level of phenolic reducing groups; (2) the pH of the midgut is alkaline and independent of host plant, but the pH of the foregut and hindgut is acid to neutral and host plant dependent; (3) the E h of foregut, midgut, and hindgut are oxidizing and determined by both plant and insect traits; and (4) the plant trait that best predicts midgut E h is the level of phenolic reducing groups. We argue that the alkaline and oxidizing midgut conditions of gypsy moth caterpillars improve nutrient digestion and influence the activity of host plant allelochemicals, especially foliar phenolics.

PhenoPhyte: a flexible affordable method to quantify 2D phenotypes from imagery.

BackgroundAccurate characterization of complex plant phenotypes is critical to assigning biological functions to genes through forward or reverse genetics. It can also be vital in determining the effect of a treatment, genotype, or environmental condition on plant growth or susceptibility to insects or pathogens. Although techniques for characterizing complex phenotypes have been developed, most are not cost effective or are too imprecise or subjective to reliably differentiate subtler differences in complex traits like growth, color change, or disease resistance.ResultsWe designed an inexpensive imaging protocol that facilitates automatic quantification of two-dimensional visual phenotypes using computer vision and image processing algorithms applied to standard digital images. The protocol allows for non-destructive imaging of plants in the laboratory and field and can be used in suboptimal imaging conditions due to automated color and scale normalization. We designed the web-based tool PhenoPhyte for processing images adhering to this protocol and demonstrate its ability to measure a variety of two-dimensional traits (such as growth, leaf area, and herbivory) using images from several species (Arabidopsis thaliana and Brassica rapa). We then provide a more complicated example for measuring disease resistance of Zea mays to Southern Leaf Blight.ConclusionsPhenoPhyte is a new cost-effective web-application for semi-automated quantification of two-dimensional traits from digital imagery using an easy imaging protocol. This tool’s usefulness is demonstrated for a variety of traits in multiple species. We show that digital phenotyping can reduce human subjectivity in trait quantification, thereby increasing accuracy and improving precision, which are crucial for differentiating and quantifying subtle phenotypic variation and understanding gene function and/or treatment effects.

Temporal Changes in Allocation and Partitioning of New Carbon as 11C Elicited by Simulated Herbivory Suggest that Roots Shape Aboveground Responses in Arabidopsis

Summary: Radiotracer studies reveal how roots can regulate a plants defensive responses to insect herbivory. Using the short-lived isotope 11C (t1/2 = 20.4 min) as 11CO2, we captured temporal changes in whole-plant carbon movement and partitioning of recently fixed carbon into primary and secondary metabolites in a time course (2, 6, and 24 h) following simulated herbivory with the well-known defense elicitor methyl jasmonate (MeJA) to young leaves of Arabidopsis (Arabidopsis thaliana). Both 11CO2 fixation and 11C-photosynthate export from the labeled source leaf increased rapidly (2 h) following MeJA treatment relative to controls, with preferential allocation of radiolabeled resources belowground. At the same time, 11C-photosynthate remaining in the aboveground sink tissues showed preferential allocation to MeJA-treated, young leaves, where it was incorporated into 11C-cinnamic acid. By 24 h, resource allocation toward roots returned to control levels, while allocation to the young leaves increased. This corresponded to an increase in invertase activity and the accumulation of phenolic compounds, particularly anthocyanins, in young leaves. Induction of phenolics was suppressed in sucrose transporter mutant plants (suc2-1), indicating that this phenomenon may be controlled, in part, by phloem loading at source leaves. However, when plant roots were chilled to 5°C to disrupt carbon flow between above- and belowground tissues, source leaves failed to allocate resources belowground or toward damaged leaves following wounding and MeJA treatment to young leaves, suggesting that roots may play an integral role in controlling how plants respond defensively aboveground.