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First published online March 21, 2008; 10.1104/pp.107.115279 Plant Physiology 147:415-428 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Genotype, Age, Tissue, and Environment Regulate the Structural Outcome of Glucosinolate Activation1,[W],[OA]Genetics Graduate Group and Department of Plant Sciences, University of California, Davis, Davis, California 95616
Glucosinolates are the inert storage form of a two-part phytochemical defense system in which the enzyme myrosinase generates an unstable intermediate that rapidly rearranges into the biologically active product. This rearrangement step generates simple nitriles, epithionitriles, or isothiocyanates, depending on the structure of the parent glucosinolate and the presence of proteins that promote specific structural outcomes. Glucosinolate accumulation and myrosinase activity differ by plant age and tissue type and respond to environmental stimuli such as planting density and herbivory; however, the influence of these factors on the structural outcome of the rearrangement step remains unknown. We show that the structural outcome of glucosinolate activation is controlled by interactions among plant age, planting density, and natural genetic variation in Arabidopsis (Arabidopsis thaliana) rosette leaves using six well-studied accessions. We identified a similarly complex interaction between tissue type and the natural genetic variation present within these accessions. This raises questions about the relative importance of these novel levels of regulation in the evolution of plant defense. Using mutants in the structural specifier and glucosinolate activation genes identified previously in Arabidopsis rosette leaves, we demonstrate the requirement for additional myrosinases and structural specifiers controlling these processes in the roots and seedlings. Finally, we present evidence for a novel EPITHIOSPECIFIER PROTEIN-independent, simple nitrile-specifying activity that promotes the formation of simple nitriles but not epithionitriles from all glucosinolates tested.
Plants synthesize thousands of secondary metabolites, including glucosinolates, alkaloids, terpenoids, and phenylpropanoids. These diverse chemicals often serve as the primary medium for plant-ecosystem interactions, attracting pollinators and seed dispersers and providing defense against herbivores and pathogens (Wink, 1988  ). Many secondary metabolites have demonstrated defense functions as growth inhibitors, toxins, or feeding deterrents for myriad herbivores and pathogens, ranging from phloem-feeding aphids to leaf-chewing lepidopteran larvae (Burow et al., 2006b; Kim and Jander, 2007). However, adapted herbivores have evolved strategies to detoxify or avoid plant defense systems and often use these phytochemicals to locate appropriate plant hosts. For example, several insect species specialize on glucosinolate-containing plants and use particular glucosinolate structures as cues for host recognition, feeding, and oviposition (Giamoustaris and Mithen, 1995; Griffiths et al., 2001; Moyes and Raybould, 2001; Miles et al., 2005). These opposing selective pressures imposed by generalist and specialist herbivores likely favor the structural diversification of defensive metabolites, as plants seek to avoid detection by adapted specialists yet maintain effective defenses against polyphagous herbivores (Wink, 2003; Lankau, 2007).
Population density can impact secondary metabolite defenses as a consequence of specialist herbivores using these metabolites to locate their hosts. A single isolated plant may not produce sufficient volatile metabolite to attract specialist herbivores and therefore would benefit from an emphasis on direct defenses. However, the combined volatiles of numerous plants producing the same metabolites will increase the risk of attracting specialist herbivores, thus favoring structures that are weaker attractants despite providing less effective defense against polyphagous insects (Fig. 1C
). As such, plastic regulation of defensive metabolite structures in response to conspecific density could be advantageous when the defensive metabolite is also a volatile host recognition cue for adapted herbivores (Lankau, 2007
Extrinsic factors controlling herbivore populations may also influence secondary metabolite diversity. For instance, seasonal changes can alter the herbivore community over the course of plant development (Wolda, 1988 ); therefore, selection might favor defensive metabolite systems capable of regulating structural variation over an individual plant's life (Fig. 1A). Because most insect populations are composed of multiple feeding guilds, they may drive metabolic defenses to respond to differences in insects' preferred feeding tissues (Burow et al., 2006b; Kim and Jander, 2007). If metabolites that provide defense against one guild are ineffective against herbivores with different feeding preferences, defensive metabolite systems capable of changing structural variation across tissues would be favored (Fig. 1B). Here, we focus on the structural outcome of glucosinolate activation in Arabidopsis (Arabidopsis thaliana) as a model secondary metabolite system to test whether structural diversity is modulated in response to development, tissue, and population density, consistent with the above hypotheses.
Glucosinolates are structurally diverse metabolites produced throughout the Brassicaceae, including the genetic model plant Arabidopsis (Fahey et al., 2001
Further structural diversity is introduced into the glucosinolate system during catabolic glucosinolate activation. The enzyme myrosinase generates an unstable intermediate that subsequently rearranges into the different glucosinolate activation products, with divergent biological activities dependent upon the specific structures produced (Fig. 1D; Louda and Mole, 1991 In this study, we test the role that plant age, growth conditions, and tissue types play in modulating metabolite diversity using the structural outcome of glucosinolate activation in Arabidopsis as our model. We demonstrate that there is quantitative age-dependent regulation of structural outcomes that is modulated by planting density and that exhibits natural genetic variation using six well-characterized accessions. We also detected differential regulation of structural outcomes between roots, flowers, and seedlings. As previous research focused on the structural outcome of glucosinolate activation in mature rosettes, we used previously described mutants in rosette myrosinases and structural specifier genes to demonstrate that glucosinolate activation and the control of structural outcomes in roots and seedlings use distinct myrosinases and structural specifiers, which remain to be identified. This analysis of control over structural variation in the glucosinolate system should aid future studies designed to test whether this variation optimizes plant defenses in response to heterogeneous herbivore populations across different individual and community scales.
Does the Control of Structural Outcomes Show Age-Dependent Regulation? Previous research on the control of structural rearrangement following glucosinolate activation in Arabidopsis has focused primarily on rosette leaves at a single stage in plant development. To determine whether the structural outcome of glucosinolate activation depends on the age of the plant, we determined the proportion of glucosinolate products in each structural class at weekly intervals in rosette leaves in six accessions.
Developmental Trajectory of Simple Nitrile Production in Columbia
Genetic Variation in Simple Nitrile Production To determine whether the developmental trajectory of simple nitrile proportions observed in Col-0 was generalizable, five additional accessions were analyzed (Table II ; Fig. 3A ). Most of these diverse accessions had simple nitrile proportion trajectories similar to those of Col-0 (Fig. 3A). However, there were significant differences between accessions for this trajectory, highlighted by Bayreuth (Bay-0), which had the largest increase in simple nitrile proportions, and Monte Tosso (Mr-0), with the slowest increase during development (Fig. 3A; Table II; accession [ESP] x week). Previous analysis associated genetic polymorphism at ESP with both simple nitrile and epithionitrile formation; however, we did not find a significant effect of ESP genotype on the proportion of simple nitrile structures produced in this collection of accessions. This suggests that the observed simple nitrile production is distinct from ESP-mediated nitrile production and may be controlled by different structural specifier genes (Table II). The trajectory of simple nitrile proportions for endogenous but-3-enyl glucosinolate in Cape Verde Islands (Cvi) and Shahdara (Sha) is nearly identical to that for the exogenous allyl substrate (Supplemental Fig. S1A; Supplemental Table S1).
The Developmental Trajectory of Epithionitrile Production In addition to simple nitriles, the accessions Cvi, Landsberg erecta (Ler), and Sha possess ESP activity leading to the formation of epithionitrile structures from allyl glucosinolate. We tested whether the proportion of epithionitriles produced in these three accessions also depends on plant age (Fig. 3A; Table II). From a low at 3 weeks after germination, epithionitrile proportions increased gradually throughout development until it was the predominant structure produced following allyl or but-3-enyl glucosinolate activation (Fig. 3A; Table II, Supplemental Fig. S1A; Supplemental Table S1). In contrast to simple nitriles, the developmental trajectories for epithionitrile proportions did not vary significantly between Cvi, Ler, and Sha (Table II; accession x week). The proportion of isothiocyanates formed was affected by variation in both plant age and natural genetic diversity, with similar patterns between endogenous and exogenous glucosinolates (Table II; Fig. 3A; Supplemental Fig. S1A). The directionality of the changes agrees with the concept that isothiocyanate production is the default outcome of glucosinolate hydrolysis and is reduced as substrate is diverted into simple nitrile and epithionitrile structures (Fig. 3A).
Structural Outcomes and Rosette Leaf Age
As Arabidopsis in the wild can grow as isolated plants or in more dense populations, the structural outcome of glucosinolate activation might be regulated by the presence of neighboring plants, using either direct signals from the neighboring plants or indirect competition for space, light, or nutrients. To test this, structural outcomes were assayed in plants grown at high and low densities (Fig. 3; Table II).
Density Modulates Simple Nitrile Production in Col-0
Genetic Variation in Developmental Trajectories in Response to Planting Density Epithionitrile proportions were also affected by planting density, with high density accelerating the age-dependent increase in exogenous and endogenous epithionitrile proportions in both Cvi and Sha (Tables I and II; Figs. 2 and 3; Supplemental Fig. S1; Supplemental Tables S1 and S2). In contrast, simple nitrile proportions decline over time in these accessions at elevated density. The observation of distinct trajectories for simple nitrile and epithionitrile proportions also supports the idea that simple nitrile formation following glucosinolate activation is not an exclusively ESP-mediated process.
Structural Outcomes and the Onset of Bolting
The preferences of different insect guilds for specific tissues could lead to tissue-specific regulation of the structural outcome of glucosinolate activation. To determine whether structural outcomes vary between plant tissues, we compared rosettes, flowers, cauline leaves, seedlings, and roots in these accessions.
Structural Outcomes in Cauline Leaves and Florets
Structural Outcomes in Seedlings Arabidopsis seedlings have a different glucosinolate composition than mature tissues, and we proceeded to test whether they also produce different structures following glucosinolate activation (Petersen et al., 2002 ; Brown et al., 2003). All accessions had high simple nitrile proportions at 3 and 7 d after germination, with higher levels of simple nitrile structures at 7 d than at any age of rosette leaf (Fig. 3). Simple nitrile proportions had a statistically significant accession (ESP) x day interaction, indicating differences in the temporal control of simple nitrile formation in seedlings, with some accessions showing a rapid increase in simple nitrile proportions and others showing little to no change (Fig. 5A; Table III
). Similar to the rosette, ESP did not affect simple nitrile proportions in seedlings (Table III).
Epithionitrile proportions were low in 3-d-old seedlings and increased from 3 to 7 d in Ler and Cvi but not in Sha (Fig. 5B; Table III). Endogenous glucosinolate structural outcomes exhibited the same trends observed for allyl glucosinolate in Col-0, Cvi, and Sha seedlings, although the magnitude of simple nitrile proportions for 4-methylsulfinylbutyl glucosinolate was considerably lower (Supplemental Fig. S4). Col-0 and Ler seedlings tested using the exogenous benzyl glucosinolate showed the same trend of high simple nitrile proportions at 3 and 7 d (data not shown). These data suggest that juvenile Arabidopsis seedlings tend to form more simple nitrile structures relative to mature rosettes. It is possible that this may play a role in the turnover of endogenous glucosinolates during seed germination (Petersen et al., 2002 ), as the less toxic simple nitrile structures are more readily metabolized.
Glucosinolate Activation in Roots
Do Known Structural Specifier Genes Control Structural Outcomes in Roots and Seedlings? All previous genetic and biochemical analyses of the structural outcome of glucosinolate activation in Arabidopsis focused on mature rosette tissue. However, the previous data show that each tissue produces a different mixture of glucosinolate activation products. Therefore, we tested the known rosette leaf structural specifier genes and myrosinases for a role in glucosinolate activation in root and seedling tissues.
ESP-Independent Simple Nitrile Formation
Structural Outcomes in ESM1 Mutants ESM1 is another glucosinolate structural specifier gene, which inhibits simple nitrile formation in rosettes with functional ESM1 (Zhang et al., 2006 ). ESM1 knockout seedlings had elevated simple nitriles relative to the parental Col-0 at 7 d after germination (P = 0.004) but not at 3 d, indicating that ESM1 does not affect simple nitrile proportions until after day 3 (Fig. 7A; Supplemental Fig. S5). In contrast, simple nitrile proportions were unchanged in the roots of the ESM1 knockout (Fig. 7C). The ESM1 overexpression line was created in a recombinant inbred line, CS1995, derived from a cross between Col-0 and Ler (Zhang et al., 2006). No significant changes in simple nitrile or epithionitrile proportions were detected between CS1995 and 35S::ESM1 lines in either seedlings or roots (Fig. 7; Supplemental Fig. S5). This contrasts with the rosettes, in which both the ESM1 knockout and 35S lines alter simple nitrile proportions (Zhang et al., 2006). Thus, ESM1 does not inhibit the unidentified simple nitrile specifier activity in Col-0 seedlings or roots.
Glucosinolate Activation and Structural Outcomes in TGG1/2 Mutants
The variable biological activities of the different glucosinolate activation product structures, and evidence of complex regulatory patterns in glucosinolate accumulation and myrosinase activity (Petersen et al., 2002 ; Brown et al., 2003; Barth and Jander, 2006), led us to hypothesize that structural outcomes may be subject to diverse developmental and environmental regulatory inputs. To test this, we assayed the structural outcome of glucosinolate activation in rosette leaves throughout development in six accessions grown at two different planting densities and in seedlings, flowers, cauline leaves, and roots. Our results reveal previously unknown levels of regulation in structural outcomes, dependent on plant age, tissue, and planting density. Microarray transcript profiling has shown that genes involved in glucosinolate activation and controlling structural outcomes respond to a variety of stimuli, including herbivory, fertilizer, and light regimen (Reymond et al., 2004; Hirai et al., 2005; Maruyama-Nakashita et al., 2006; Dombrecht et al., 2007). However, it was not tested directly whether this gene regulation results in age-, tissue-, or environment-dependent regulation of structural outcomes. Recently, the leaf myrosinases and ESP were also shown to have distinct patterns of regulation between specific plant tissues that varied among Arabidopsis accessions (Barth and Jander, 2006; Burow et al., 2007). Assuming that the primary role of the glucosinolate system is plant defense, regulation of structural outcomes might allow plants to optimize their defenses in response to changing risks of herbivory.
Striking differences in the structural outcome of glucosinolate activation were observed over the course of plant development and among tissues tested. All accessions produced primarily isothiocyanates in the flowers and high levels of simple nitriles in the roots and seedlings (Figs. 5–7
Significant natural variation was identified among the six Arabidopsis accessions tested in tissue-, age-, and density-dependent regulation of the structural outcomes of glucosinolate activation. Nearly every tissue or condition tested showed evidence for control by natural genetic variation (Figs. 3–7
To date, ESP is the only known structural specifier protein in plants capable of increasing the production of epithionitriles from alkenyl glucosinolates and simple nitriles from all other substrates (Tookey, 1973
We have described several patterns of age- and tissue-dependent regulation of the structural outcome of glucosinolate activation and interactions with natural variation, setting the stage for detailed molecular studies to resolve the basis of this variation. These novel regulatory inputs indicate that Arabidopsis regulates the mixture of activation product structures over tissue, development, and planting density. However, the ecological roles of many of these defensive metabolites remain undefined, requiring further experiments to understand the ecological significance of these results. In particular, the biological role of simple nitrile activation products remains unclear, as simple nitrile structures are generally less toxic and provide less effective defense against polyphagous herbivores when compared directly with the corresponding isothiocyanates (Nastruzzi et al., 2000 ; Jander et al., 2001; Lambrix et al., 2001; Barth and Jander, 2006; Zhang et al., 2006). We hypothesize that increased proportions of simple nitrile and epithionitrile products reduce the risk of detection by adapted herbivores or provide indirect defense by attracting predators and parasites of adapted herbivores. The use of knockout and overexpression mutants with altered regulation of structural outcomes of glucosinolate activation will clarify the ecological significance of these results.
Glucosinolate Activation Assay
The structural outcome of glucosinolate activation was assayed using a modified version of the previously published protocol (Lambrix et al., 2001 Structural outcomes are reported as percentages of simple nitrile, epithionitrile, or isothiocyanate products for a particular glucosinolate. By dividing the absolute amount of a particular structure by the sum of all possible products, the effects of myrosinase activity and differences in the biosynthesis and accumulation of the endogenous substrates are canceled, since they affect both the numerator and denominator equally. This assay is not a quantitative measure of total myrosinase activity, both because three of the accessions used in this study produce allyl glucosinolate and, therefore, the substrate concentration is not constant and because this assay reaches saturation for some samples.
Commercially available myrosinase purified from Sinapis alba (Sigma-Aldrich catalog no. T4528) was assayed for structural outcomes in the absence of plant tissues using allyl glucosinolate as the substrate. To control for abiotic factors influencing nonenzymatic production of simple nitrile and epithionitrile product structures, activation was assayed in the presence of MS medium and soil with and without S. alba myrosinase (Tookey, 1973
Six accessions of Arabidopsis were analyzed to test for natural genetic variation in the regulation of structural outcomes of glucosinolate activation. The accessions Bay-0, Col-0, Cvi, Ler, and Sha were selected because they are the parents of three large recombinant inbred line populations (Lister and Dean, 1993
Mr-0, Cvi, and Sha produce allyl glucosinolate, while Cvi and Sha also accumulate but-3-enyl glucosinolate in rosette leaves (Kliebenstein et al., 2001c
Rearrangement outcomes were surveyed at weekly intervals in the rosette leaves of plants grown alone and with numerous conspecific neighbors. Plants were maintained at 25°C in a controlled-environment chamber with a 12-h light cycle under 150 µE fluorescent lighting. Seeds were cold stratified at 4°C for 48 h, then germinated and grown in Premier ProMix potting soil. Plants were watered and fertilized by subirrigation at weekly intervals. The low-density treatment consisted of a single plant per 49-cm2 pot. The high-density plants were maintained at approximately 100 plants per 49-cm2 pot, ensuring extensive contact and crowding between adjacent plants throughout the experiment. The high-density plants were maintained in the same chamber and grown at the same time as the low-density plants. Rosette leaves were assayed beginning at 2 and 3 weeks for the high- and low-density treatments, respectively. Pots were removed from the growth chamber, and rosette tissue was harvested in three independent biological replicates sampled from separate pots for each time point, accession, and planting density. All replicates were grown in independent randomized complete blocks. At weeks 3 and 4 in the low-density treatment and at weeks 2 through 5 at high density, rosette leaves from multiple plants were pooled to obtain sufficient tissue for each replicate. At least three leaves were collected from each individual included in every replicate for all pooled samples. Trajectories were ended after 7 weeks for Ler and after 8 weeks for Col-0 and Mr-0 in the low-density treatment, and the high-density treatment of Ler was not included due to limited germination. Wounded plants were never returned to the growth chamber, and at no point during this experiment were the plants subjected to visible pests. To test for an association between structural outcomes and leaf age, three leaves were sampled from 8-week-old plants of each accession and assayed individually. These plants were maintained at low density under conditions identical to the developmental trajectory and never subjected to visible pests. The youngest leaf was sampled from the innermost whorl and not yet fully expanded. A healthy, fully expanded leaf was sampled from the middle whorl of the plant. The oldest leaf was sampled from the outermost whorl and was beginning to senesce, with approximately 10% of the leaf area yellow. This was repeated using four plants of each accession grown simultaneously.
Structural outcomes were assayed in the flowers and cauline leaves using four plants of each accession grown at low densities in 49-cm2 pots and maintained in the same chambers and conditions as the developmental trajectory and free of pests throughout their life cycle. When the primary inflorescence reached 20 cm in height, all flowers from a single plant were collected and pooled to obtain sufficient tissue. Cauline and rosette leaves from these plants were assayed simultaneously for these comparisons. This provided four independent biological replicates per tissue per accession. Root analysis required the growth of each accession on vertically oriented plates of MS medium (Gibco-BRL). Seeds were surface sterilized and cold stratified to ensure synchronous germination and maintained at 20°C under 160 µE fluorescent lighting. Root tissue from approximately 100 plants was pooled for each replicate, and three independent biological replicates taken from separate plates were assayed for each accession. Care was taken to exclude shoot tissue and any MS medium. Structural outcomes were also assayed using exogenous benzyl glucosinolate in Ler and Col-0. To control for the possibility that the presence of root chemical or ion concentrations were altering structural outcomes, boiled roots were incubated for 5 min with S. alba myrosinase and then assayed for structural outcomes. For seedlings, each accession was germinated and grown on MS medium under conditions identical to those for the roots. At 3 and 7 d after germination, seedlings were assayed for structural outcomes. Approximately 50 seedlings were pooled for each sample, with three independent biological replicates sampled from different plates for each accession and time point. Care was taken to sample the aerial portions of the seedlings. All plates were free of pests and contamination by visual examination.
We obtained previously described mutants in ESP, ESM1, and the myrosinases TGG1 and TGG2, the known glucosinolate activation and structural specifier genes identified in the rosettes, to determine whether they also function in roots and seedlings (Lambrix et al., 2001
Although all three structural outcomes are contingent on one another, they each describe biochemically distinct outcomes, with potentially different regulatory patterns; thus, each was analyzed separately. Simple nitrile and isothiocyanate proportions were analyzed using a general linear mixed model, with density (high or low) and ESP genotype (functional or nonfunctional) as fixed factors, accession as a random factor nested within ESP, and age (in weeks) as a repeated measure to account for sampling these genotypes multiple times throughout development. Since epithionitrile formation was confined exclusively to accessions with functional ESP, analysis of epithionitrile proportions was restricted to Cvi, Ler, and Sha, and ESP genotype was dropped from the model. All interactions between factors were also tested (Table II). Since wounding can alter structural outcomes, sampled plants were discarded after each assay. Thus, repeated sampling occurred at the level of accession rather than in individual plants. For each trait, we also investigated whether endogenous allyl glucosinolate synthesis in Cvi, Sha, and Mr-0 and the mass of tissue sampled affected structural outcomes by including each as a covariate in preliminary analyses. Neither had significant effects for any trait; thus, both were dropped from the final models.
As a result of excluding the high-density treatment of the accession Ler, and because the high-density treatment had enough tissue to be sampled starting at 2 weeks, as opposed to 3 weeks for the low density treatment, these data sets were unbalanced. Additionally, structural proportions were approximately log-normally distributed. To account for these departures from standard assumptions, the trajectory data were analyzed using the GLIMMIX procedure in SAS. This procedure minimizes the potential biases introduced by unbalanced and nonnormal data by using restricted penalized likelihood to fit the model and estimate random effects (Wolfinger and O'Connell, 1993 To determine whether age, density, and their interaction have similar effects on exogenous and endogenous glucosinolate structural outcomes in Col-0, Cvi, and Sha, data for each substrate were analyzed within an accession according to the following model: proportion = age + density + age x density. The results obtained for each substrate were compared within each accession to determine whether the significance and directionality of each term were the same. Finally, to determine whether structural outcomes varied with leaf age within individual plants, differently aged leaves within a single rosette were compared within each accession using paired t tests. All post hoc comparisons were performed using Tukey adjustments.
Differences in structural outcomes between flowers, cauline leaves, and rosette leaves within an accession were analyzed using paired t tests within an accession. Root structural outcome data were compared between accessions, and within Col-0 and Ler between the benzyl and allyl glucosinolate substrates, using two-tailed heteroscedastic t tests, which allow for unequal sample variances. Post hoc comparisons were performed using Tukey adjustments. Seedling structural outcome data were also analyzed using the GLIMMIX procedure. Each structural outcome was analyzed separately for the effects of days after germination, accession, ESP genotype (either functional or nonfunctional), and all possible interactions (Table III). Accession was nested within ESP genotype for simple nitrile and isothiocyanate proportions. For epithionitrile proportions, only the data from Cvi, Ler, and Sha were analyzed, and ESP genotype was removed from the model. Days were treated as repeated measures of the accessions. Post hoc comparisons were performed using Tukey adjustments. Mutants in the known structural specifier and myrosinase genes identified in the rosettes were tested for their effects on structural outcomes using two-tailed heteroscedastic t tests. ESP overexpression and ESP knockout lines were compared with Col-0, as were the ESM1 knockout and the TGG1/2 double knockout lines. The ESM1 overexpression line was compared with the recombinant inbred line CS1995.
The following materials are available in the online version of this article.
We thank Beverly Ajie (Center for Population Biology, University of California, Davis [UC Davis]) for assistance with the statistical analysis of the developmental data and many helpful discussions. We also thank Heather Rowe (Genetics Graduate Group, UC Davis) for critical review of the manuscript and Dr. Joshua Chang Mell (Genetics Graduate Group, UC Davis) for numerous helpful discussions. We are extremely grateful to Dr. Andrew Waterhouse and Mauri Anderson (Department of Viticulture and Enology, UC Davis) for the use of their gas chromatograph. Seeds of the TGG1/2 double knockout line were kindly provided by Dr. Georg Jander (Cornell University). Seeds of the 35S::ESP line were kindly provided by Dr. Ute Wittstock (Technische Universität Braunschweig). Benzyl glucosinolate was a generous gift of Dr. Steffen Abel (UC Davis). Received December 20, 2007; accepted March 16, 2008; published March 21, 2008.
1 This work was supported by the National Science Foundation (grant nos. DBI 0642481 and MCB 0323759 to D.J.K.; Dissertation Improvement Grant DEB 0608516 to A.M.W.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Daniel J. Kliebenstein (kliebenstein{at}ucdavis.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.115279 * Corresponding author; e-mail kliebenstein{at}ucdavis.edu.
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), Col-0 (
) Mr-0 (
), Cvi (
), Ler (*), and Sha (
). Significant changes between days within an accession are indicated by dashed lines. A, Percentage of simple nitrile in seedlings of all accessions. B, Percentage of epithionitrile in Cvi, Ler, and Sha seedlings. C, Percentage of isothiocyanate in seedlings of all accessions.
