Robert J. Grebenok

Insect-induced synthesis of phytoecdysteroids in spinach, Spinacia oleracea

Spinach (Spinacia oleracea) foliage is known to synthesize and accumulate insect molting hormones, predominantly in the form of 20-hydroxyecdysone (20E). We previously demonstrated that root 20E accumulation is increased following root damage. We designed two further experiments to address root responses to both mechanical and insect damage. In plants grown hydroponically, removal of 35% or less of the root mass did not result in changes in root 20E levels. However, removal of 70% of the root mass stimulated 6.0- and 1.5-fold increases in the root and shoot 20E concentrations, respectively. The effects of insect damage on soil-grown plants were investigated by infesting plant roots with black vine weevil (BVW: Otiorhynchus sulcatus) larvae and allowing them to feed for seven days. Decreases in root mass occurred in young plants; however, no changes were detected in mature plants. In all cases, root herbivory resulted in at least a 3.0-fold increase in root 20E concentrations. Our previous experiments implicated jasmonic acid and the analog methyl jasmonate (MJ) in signaling the damage-induced accumulation of root 20E levels. We investigated the activity of other phytohormones and growth regulators (GRs) on the 20E accumulation patterns of young plants as a means of examining the significance of jasmonates in the induction response. Hydroponic additions of MJ (0.5 μM) and the synthetic auxin, 1-naphthaleneacetic acid (NAA; 0.5 μM), resulted in significant increases in root 20E levels. At the concentrations tested, indole-3-acetic acid (IAA), gibberellic acid (GA3), abscisic acid (ABA), and trans-zeatin (Z) had no effects on root 20E concentrations. However, both NAA (0.5–5.0 μM) and Z (5.0 μM) treatments caused increases in the root/shoot dry mass ratios, indicating shifts in resource allocation to the roots. Treatments involving ABA (5.0 μM) and Z (0.5–5.0 μM) caused significant increases in shoot 20E concentrations. No other hormone treatments altered shoot accumulation patterns. The mechanisms underlying the root 20E induction phenomena were investigated through the incorporation of [2-14C]mevalonic acid ([14C]MVA). Within one day, excised roots readily incorporated radioactivity into 20E from [14C]MVA. In intact plants, [14C]MVA absorbed by the roots was rapidly incorporated into root 20E pools following damage and MJ treatments. This implies that the wound-induced root 20E accumulation is the result of increased de novo 20E synthesis in the root.

Damage-Induced Accumulation of Phytoecdysteroids in Spinach: A Rapid Root Response Involving the Octadecanoic Acid Pathway

Some plant defenses are known to be rapidly induced following attack by phytophagous insects. Plant-produced insect molting hormones, termed phytoecdysteroids, are believed to aid plant resistance; however, their dynamics are poorly understood. Using spinach (Spinacia oleracea) as a model system, we examined the inducibility of phytoecdysteroids, primarily 20-hydroxyecdysone (20E), in an effort to characterize potential interactions with herbivorous insects. Rapid phytochemical induction was investigated using damage treatments and applications of defense-related plant-signal analogs, specifically methyl jasmonate (MJ) and methyl salicylate (MSA). Within two days, mechanically damaged roots exhibited two to three fold increases in phytoecdysteroid concentrations. Four days after root damage, small increases in shoot levels were also detectable. Unlike roots, foliar 20E concentrations were unaltered over a range of shoot treatments including insect herbivory (Spodoptera exigua), mechanical damage, and MJ applications. Additions of MJ (12.5–50 μg/liter) to the root systems of hydroponically grown plants stimulated accumulations of root phytoecdysteroids in a dose-dependent manner, similar in magnitude to the response induced by root damage. Under identical conditions, MSA did not affect the accumulation of 20E when added to the hydroponic solutions of undamaged plants. Moreover, MSA inhibited the induction of 20E in wounded roots, but did not interfere with the action of applied MJ. In contrast to mechanical damage, roots did not induce 20E levels when challenged with two different fungal pathogens (Pythium aphanidermatum and Phytophthora capsici).We propose that wound-induced accumulations of 20E are generated in the roots, signaled via endogenous jasmonates, and may confer enhanced resistance against subterranean herbivorous insects.

Same Host-Plant, Different Sterols: Variation in Sterol Metabolism in an Insect Herbivore Community

Insects lack the ability to synthesize sterols de novo, which are required as cell membrane inserts and as precursors for steroid hormones. Herbivorous insects typically utilize cholesterol as their primary sterol. However, plants rarely contain cholesterol, and herbivorous insects must, therefore, produce cholesterol by metabolizing plant sterols. Previous studies have shown that insects generally display diversity in phytosterol metabolism. Despite the biological importance of sterols, there has been no investigation of their metabolism in a naturally occurring herbivorous insect community. Therefore, we determined the neutral sterol profile of Solidago altissima L., six taxonomically and ecologically diverse herbivorous insect associates, and the fungal symbiont of one herbivore. Our results demonstrated that S. altissima contained Δ7-sterols (spinasterol, 22-dihydrospinasterol, avenasterol, and 24-epifungisterol), and that 85% of the sterol pool existed in a conjugated form. Despite feeding on a shared host plant, we observed significant variation among herbivores in terms of their qualitative tissue sterol profiles and significant variation in the cholesterol content. Cholesterol was absent in two dipteran gall-formers and present at extremely low levels in a beetle. Cholesterol content was highly variable in three hemipteran phloem feeders; even species of the same genus showed substantial differences in their cholesterol contents. The fungal ectosymbiont of a dipteran gall former contained primarily ergosterol and two ergosterol precursors. The larvae and pupae of the symbiotic gall-former lacked phytosterols, phytosterol metabolites, or cholesterol, instead containing an ergosterol metabolite in addition to unmetabolized ergosterol and erogsterol precursors, thus demonstrating the crucial role that a fungal symbiont plays in their nutritional ecology. These data are discussed in the context of sterol physiology and metabolism in insects, and the potential ecological and evolutionary implications.

Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.

Phytoecdysteroid Turnover in Spinach: Long-term Stability Supports a Plant Defense Hypothesis

Using short (8-day) and long-term (28-day) experiments, we examined the stability of 20-hydroxyecdysone (20E) and the dominant phytosterols synthesized from a pulse of [2-14C]mevalonic acid ([14C]MVA) in hydroponically grown spinach (Spinacia oleracea). In the short-term experiment, plant dry mass and shoot 20E pools steadily increased. Root uptake of [14C]MVA resulted in the stable incorporation of 14C radiolabel into whole plant 20E pools, with no significant changes over time. Levels of free and saponifiable phytosterols increased in the shoots while 14C-labeled shoot phytosterols remained constant. Unexpectedly, both 14C-labeled and unlabeled pools of root phytosterols decreased over time. In the long-term experiment, plant dry mass and shoot 20E levels increased over time, while total 14C-labeled 20E pools remained constant. Both root and shoot phytosterol pools increased over time while the 14C incorporation in these pools remained constant. Together these experiments indicate that 20E in spinach is metabolically stabile and thus shares this characteristic with plant terpenoids of known defensive function. While little is known about phytosterol turnover in plants, our results suggest that phytosterols can indeed exist in a very dynamic state but may also be stable over time.

The physiology of sterol nutrition in the pea aphid Acyrthosiphon pisum

The phloem sap of fava bean (Vicia faba) plants utilized by the pea aphid Acyrthosiphon pisum contains three sterols, cholesterol, stigmasterol and sitosterol, in a 2:2:1 ratio. To investigate the nutritional value of these sterols, pea aphids were reared on chemically-defined diets containing each sterol at 0.1, 1 and 10μgml(-1) with a sterol-free diet as control. Larval growth rate and aphid lifespan did not vary significantly across the diets, indicating that sterol reserves can buffer some performance indices against a shortfall in dietary sterol over at least one generation. However, lifetime reproductive output was depressed in aphids on diets containing stigmasterol or no sterol, relative to diets supplemented with cholesterol or sitosterol. The cholesterol density of embryos in teneral adults was significantly higher than in the total body; and the number and biomass of embryos in aphids on diets with stigmasterol and no sterols were reduced relative to diets with cholesterol or sitosterol, indicating that the reproductive output of the pea aphid can be limited by the amount and composition of dietary sterol. In a complementary RNA-seq analysis of pea aphids reared on plants and diets with different sterol contents, 7.6% of the 17,417 detected gene transcripts were differentially expressed. Transcript abundance of genes with annotated function in sterol utilization did not vary significantly among treatments, suggesting that the metabolic response to dietary sterol may be mediated primarily at the level of enzyme function or metabolite concentration.

Plant phloem sterol content: forms, putative functions, and implications for phloem-feeding insects

All eukaryotes contain sterols, which serve as structural components in cell membranes, and as precursors for important hormones. Plant vegetative tissues are known to contain mixtures of sterols, but very little is known about the sterol composition of phloem. Plants are food for many animals, but plant-feeding arthropods (including phloem-feeding insets) are unique among animals in that they have lost the ability to synthesize sterols, and must therefore acquire these essential nutrients from their food, or via endosymbionts. Our paper starts by providing a very brief overview of variation in plant sterol content, and how different sterols can affect insect herbivores, including those specializing on phloem. We then describe an experiment, where we bulk collected phloem sap exudate from bean and tobacco, and analyzed its sterol content. This approach revealed two significant observations concerning phloem sterols. First, the phloem exudate from each plant was found to contain sterols in three different fractions – free sterols, sterols conjugated to lipids (acylated), and sterols conjugated to carbohydrates (glycosylated). Second, for both plants, cholesterol was identified as the dominant sterol in each phloem exudate fraction; the remaining sterols in each fraction were a mixture of common phytosterols. We discuss our phloem exudate sterol profiles in a plant physiology/biochemistry context, and how it relates to the nutritional physiology/ecology of phloem-feeding insects. We close by proposing important next steps that will advance our knowledge concerning plant phloem sterol biology, and how phloem-sterol content might affect phloem-feeding insects.

A Dietary Test of Putative Deleterious Sterols for the Aphid Myzus persicae

The aphid Myzus persicae displays high mortality on tobacco plants bearing a transgene which results in the accumulation of the ketosteroids cholestan-3-one and cholest-4-en-3-one in the phloem sap. To test whether the ketosteroids are the basis of the plant resistance to the aphids, M. persicae were reared on chemically-defined diets with different steroid contents at 0.1–10 µg ml−1. Relative to sterol-free diet and dietary supplements of the two ketosteroids and two phytosterols, dietary cholesterol significantly extended aphid lifespan and increased fecundity at one or more dietary concentrations tested. Median lifespan was 50% lower on the diet supplemented with cholest-4-en-3-one than on the cholesterol-supplemented diet. Aphid feeding rate did not vary significantly across the treatments, indicative of no anti-feedant effect of any sterol/steroid. Aphids reared on diets containing equal amounts of cholesterol and cholest-4-en-3-one showed fecundity equivalent to aphids on diets containing only cholesterol. Aphids were reared on diets that reproduced the relative steroid abundance in the phloem sap of the control and modified tobacco plants, and their performance on the two diet formulations was broadly equivalent. We conclude that, at the concentrations tested, plant ketosteroids support weaker aphid performance than cholesterol, but do not cause acute toxicity to the aphids. In plants, the ketosteroids may act synergistically with plant factors absent from artificial diets but are unlikely to be solely responsible for resistance of modified tobacco plants.

Diet micronutrient balance matters: How the ratio of dietary sterols/steroids affects development, growth and reproduction in two lepidopteran insects.

Insects lack the ability to synthesize sterols de novo so they acquire this essential nutrient from their food. Cholesterol is the dominant sterol found in most insects, but in plant vegetative tissue it makes up only a small fraction of the total sterol profile. Instead, plants mostly contain phytosterols; plant-feeding insects generate the majority of their cholesterol by metabolizing phytosterols. However, not all phytosterols are readily converted to cholesterol, and some are even deleterious when ingested above a threshold level. In a recent study we showed that caterpillars reared on tobacco accumulating novel sterols/steroids exhibited reduced performance, even when suitable sterols were present. In the current study we examined how the dominant sterols (cholesterol and stigmasterol) and steroids (cholestanol and cholestanone) typical of the modified tobacco plants affected two insect herbivores (Heliothis virescens and Helicoverpa zea). The sterols/steroids were incorporated into synthetic diets singly, as well as in various combinations, ratios and amounts. For each insect species, a range of performance values was recorded for two generations, with the eggs from the 1st-generation adults as the source of neonates for the 2nd-generation. Performance on the novel steroids (cholestanol and cholestanone) was extremely poor compared to suitable sterols (cholesterol and stigmasterol). Additionally, performance tended to decrease as the ratio of the novel dietary steroids increased. We discuss how the balance of different dietary sterols/steroids affected our two caterpillar species, relate this back to recent studies on sterol/steroid metabolism in these two species, and consider the potential application of sterol/steroid modification in crops.