Swords into plowshares? Nicotiana sylvestris does not use nicotine as a nitrogen source under nitrogen-limited growth

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.     

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

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. Received: 24 April 1996 / Accepted: 18 July 1996     

Interactions between Spinacia oleracea and Bradysia impatiens: a role for phytoecdysteroids

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.     

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.     

Preference of cabbage white butterflies and honey bees for nectar that contains amino acids

Summary 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.     

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.