Plant produced insect molting hormones, termed phytoecdysteroids (PEs), are thought to function as plant defenses against insects by acting as either feeding deterrents or through developmental disruption. In spinach (Spinacia oleracea), 20-hydroxyecdysone (20E) concentrations in the roots rapidly increase following root damage, root herbivory, or methyl jasmonate (MJ) applications. In this inducible system, we investigated the plant defense hypothesis by examining interactions of roots, 20E concentrations, and larvae of the dark-winged fungus gnat (Bradysia impatiens). Root herbivory by B. impatiens larvae resulted in a 4.0- to 6.6-fold increase in root 20E concentrations. In paired-choice tests, increases in dietary 20E stimulated B. impatiens feeding deterrency. B. impatiens larvae preferred control diets, low in 20E, to those constructed from induced roots and those amended with 20E (25 to 50 micro g/g wet mass). When confined to 20E-treated diets, concentrations as low as 5 micro g/g (wet mass) resulted in significantly reduced B. impatiens survivorship compared to controls. The induction of root 20E levels with MJ resulted in a 2.1-fold increase in 20E levels and a 50% reduction in B. impatiens larval establishment. In a paired-choice arena, untreated control roots were damaged significantly more by B. impatiens larvae than MJ-induced roots that contained 3-fold greater 20E levels. Based on dietary preference tests, the 20E concentrations present in the MJ-induced roots (28 micro g/g wet mass) were sufficient to explain this reduction in herbivory. Interactions between spinach roots and B. impatiens larvae demonstrate that PEs can act as inducible defenses and provide protection against insect herbivory.

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 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.

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-13C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway.

Whether or not a plant can recover its investment of resources in a chemical defense is central to the “mobile-immobile” metabolite dichotomy of the resource availability theory. Biochemical measures of metabolite turnover have been used to estimate this trait, but they do not address the ecological question of resource recovery. Numerous studies have found that many Nicotiana species, which normally produce the nitrogen-intensive defense metabolite, nicotine, can rapidly take up and metabolize exogenously administered nicotine from hydroponic solutions. However, Baldwin et al. (1994) found no evidence for turnover of endogenously produced nicotine in pulse-chase experiments using 15NO3 as the biosynthetic precursor in N. sylvestris. Given that the capacity to metabolize nicotine exists, we asked (1) whether N. sylvestris could metabolize exogenously fed nicotine and sustain growth under nitrogen-limited conditions and (2) whether leaf damage alters the plants' ability to use nicotine as a nitrogen source. We fed plants with sufficient nicotine in hydroponic culture to increase their nitrogen pools by 70% at the time of nicotine feeding; in 6–10 consecutive harvests over 28–35 days, we measured the biomass of roots, leaves and stems, and the total nitrogen pools of these plant parts as well as the pools of nicotine, nornicotine and myosmine of these plant parts in undamaged nicotinefed and control plants and finally, in a separate experiment, in nicotine-fed damaged and undamaged plants. Nicotine feeding increased nicotine pools by 1.2 times, which was not sufficient to significantly increase total nitrogen pools at the end of the experiment. Nicotine-fed plants rapidly demethylated their acquired nicotine pools to nornicotine, but did not process the alkaloid pool further than myosmine over the duration of the experiment. Leaf damage significantly increased the nicotine pool, but did not significantly alter the processing of the exogenously acquired nicotine. We conclude that N. sylvestris does not recover the nitrogen invested in nicotine even under nitrogen-limited growth, that the rapid metabolism of exogenously introduced nicotine is likely a detoxification pathway, and that these plants are homeostatic with regard to their nicotine pools.

Leaf damage by herbivores inNicotiana sylvestris Spegazzini and Comes (Solanaceae) produces a damage signal that dramatically increasesde novo nicotine synthesis in the roots. The increased synthesis leads to increases in whole-plant nicotine pools, which in turn make plants more resistant to further herbivore attack. Because signal production and the response to the signal occur in widely separated tissues, the speed with which different damage signals exit a damaged leaf can be studied. We propose that electrical damage signals should exit a leaf faster (less than 60 min) than chemical damage signals. Excision of a leaf induces a smaller increase in nicotine production than does puncture damage, so we examined our proposition by excising previously punctured leaves at 1, 60, and 960 min after leaf puncture and quantifying the induced whole-plant nicotine pools six days later when the induced nicotine production had reached a maximum. Significant induced nicotine production occurred only if punctured leaves were excised more than 1 hr after puncture, which is consistent with the characteristics of a slow-moving chemical signal rather than a fast-moving electrical signal. We explore the nature of the chemical signal and demonstrate that additions of 90µg or more of methyl jasmonate (MJ) in an aqueous solution to the roots of hydroponically grown plants inducede novo nicotine synthesis from(15)NO3 in a manner similar to that induced by leaf damage. We examine the hypothesis that jasmonic acid (JA) functions in the transfer of the damage signal from shoot to root. Using GC-MS techniques to quantify whole-plant JA pools, we demonstrate that leaf damage rapidly (<0.5 hr) increases shoot JA pools and, more slowly (<2 hr), root JA pools. JA levels subsequently decay to levels found in undamaged plants within 24 hr and 10 hr for shoots and roots, respectively. The addition of sufficient quantities (186µg) of MJ in a lanolin paste to leaves from hydroponically grown plants significantly increased endogenous root JA pools and increasedde novo nicotine synthesis in these plants. However, the addition of 93µg or less of MJ did not significantly increase endogenous root JA pools and did not significantly affectde novo nicotine synthesis. We propose that wounding increases shoot JA pools, which either directly through transport or indirectly through a systemin-like signal increase root JA pools, which, in turn, stimulate root nicotine synthesis and increase whole-plant nicotine pools.

We performed field tests of alkaloid induction in Nicotiana attenuata plants growing in southwestern Utah with mimicry of the two major types of damage inflicted by invertebrate and vertebrate herbivores: leaf damage and stalk removal, respectively. In undamaged plants, seasonal increases in leaf nicotine content occurred at a rate of 0.046% leaf dry mass/day. Leaf damage doubled the accumulation rate to 0.086-0.138% leaf dry mass/day, while stalk removal resulted in a quadrupling of the accumulation rate to 0.206% leaf dry mass/day. These damage-induced increases in nicotine accumulation are significantly larger than between-plant and phenological variations. Leaf damage to the nornicotine-(N. repanda and N. trigonophylla) and anabasine-accumulating (N. glauca) Nicotiana species native to North America resulted in 1.5- to 5-fold increases in their principal leaf alkaloid pools. We conclude that alkaloid induction is not limited to nicotine-accumulating Nicotiana species and that herbivores feeding on previously damaged plants are likely to encounter tissues with alkaloid titers significantly higher than those of undamaged plants.

Amino acids occur in most floral nectars but their role in pollinator attraction is relatively unstudied. Nectars of butterfly-pollinated flower tend to have higher concentrations of amino acids than do flowers pollinated by bees and many other animals, suggesting that amino acids are important attractants of butterflies to flowers. In order to determine whether amino acids are important in attracting butterflies and bees, we tested the preference of cabbage white butterflies (Pieris rapae) and honey bees (Apis mellifera) by allowing them to feed from artificial flowers containing sugar-only or sugar-amino acid mimics ofLantana camara nectar. Honey bees and female cabbage white butterflies consumed more sugar-amino acid nectar than sugar-only nectar. In addition, female cabbage white butterflies visited artificial flowers containing sugar-amino acid nectars more frequently than flowers containing sugar-only nectars; honey bees spent more time consuming the sugar-amino acid nectar. Male cabbage white butterflies did not discriminate between the two nectars. These results support the hypothesis that the amino acids of nectar contribute to pollinator attraction and/or feeding.