| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online October 22, 2008; 10.1104/pp.108.129981 Plant Physiology 148:2096-2108 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Novel 2-Oxoacid-Dependent Dioxygenase Involved in the Formation of the Goiterogenic 2-Hydroxybut-3-enyl Glucosinolate and Generalist Insect Resistance in Arabidopsis[C],[W],[OA]Plant Biochemistry Laboratory, Department of Plant Biology, VKR Centre for Pro-Active Plants, Faculty of Life Sciences, University of Copenhagen, DK–1871 Frederiksberg C, Denmark (B.G.H., B.A.H.); Department of Plant Sciences and Plant Biology Graduate Group, University of California, Davis, California 95616–8746 (R.E.K., J.A.O., V.M.L., D.J.K.); Max Planck Institute for Chemical Ecology, D–07745 Jena, Germany (V.M.L., T.M.-O., J.G., D.J.K.); and Department of Biology, Duke University, Durham, North Carolina 27708 (T.M.-O.)
Glucosinolates are secondary metabolites found almost exclusively in the order Brassicales. They are synthesized from a variety of amino acids and can have numerous side chain modifications that control biological function. We investigated the biosynthesis of 2-hydroxybut-3-enyl glucosinolate, which has biological activities including toxicity to Caenorhabditis elegans, inhibition of seed germination, induction of goiter disease in mammals, and production of bitter flavors in Brassica vegetable crops. Arabidopsis (Arabidopsis thaliana) accessions contain three different patterns of 2-hydroxybut-3-enyl glucosinolate accumulation (present in leaves and seeds, seeds only, or absent) corresponding to three different alleles at a single locus, GSL-OH. Fine-scale mapping of the GSL-OH locus identified a 2-oxoacid-dependent dioxygenase encoded by At2g25450 required for the formation of both 2R- and 2S-2-hydroxybut-3-enyl glucosinolate from the precursor 3-butenyl glucosinolate precursor. Naturally occurring null mutations and T-DNA insertional mutations in At2g25450 exhibit a complete absence of 2-hydroxybut-3-enyl glucosinolate accumulation. Analysis of herbivory by the generalist lepidopteran Trichoplusia ni showed that production of 2-hydroxybut-3-enyl glucosinolate provides increased resistance. These results show that At2g25450 is necessary for the hydroxylation of but-3-enyl glucosinolate to 2-hydroxybut-3-enyl glucosinolate in planta and that this metabolite increases resistance to generalist herbivory.
Plants utilize a suite of defensive compounds to provide resistance to the attacks of other organisms. These secondary metabolites show high levels of variation in terms of both accumulation and structure (Rodman et al., 1981  ; Zangerl and Berenbaum, 1990; Berenbaum and Zangerl, 1998). This variation is believed to result from an arms race between the plant and its biotic attackers such that each evolutionary innovation in plant defense elicits the evolution of a counteradaptation in the biotic attacker. The repetition of this process may lead to the high level of diversity observed in chemical structures and their amounts (Ehrlich and Raven, 1964) and variation in bioactivity of the different structures (Bowers and Puttick, 1988). An alternative option is that the variation may represent a stable cycling of phenotypic classes (Berenbaum and Zangerl, 1998; Zangerl et al., 2008). The concomitant and often correlated variation in both chemical structure and the total amount of a given class of chemical defense complicates the ability to separate the contribution of these two factors to plant protection (Puttick and Bowers, 1988; Mithen et al., 1995; Giamoustaris and Mithen, 1996). The development of precise tools to tease apart chemical structure and content would make an important contribution to the study of plant chemical defense.
An important model system for studying the role of chemical defenses in plants is the glucosinolates, a class of naturally occurring thioglycosides that are found throughout the order Brassicales. These sulfur-rich plant secondary metabolites are synthesized from a variety of protein amino acids (Met, Leu, Ile, Val, Trp, and Phe; Halkier and Du, 1997
Another important determinant of the final biological activity is the structure of the glucosinolate side chain prior to activation. One example of this structural variation from Arabidopsis (Arabidopsis thaliana) is the dihomomethionine-derived methylthioalkyl side chain, which can be modified to form methylthioalkyl, methylsulfinylalkyl, alkenyl, and hydroxyalkenyl glucosinolates (Fig. 1
; Kliebenstein et al., 2001c
The final member of this set of modular genetic loci is the uncloned GSL-OH locus, which controls the production of 2-hydroxybut-3-enyl glucosinolate (Fig. 1; Parkin et al., 1994 ; van Doorn et al., 1999; Kliebenstein et al., 2001c). This glucosinolate arises by oxidation of 3-butenyl glucosinolate to produce either the 2R- or 2S-hydroxylated derivative (Rossiter et al., 1990; Daubos et al., 1998). Arabidopsis contains both the 2R and 2S enantiomers of 2-hydroxybut-3-enyl glucosinolate, whereas Brassica napus makes only the 2R enantiomer and Crambe abyssinica makes only the 2S enantiomer (Daubos et al., 1998; van Doorn et al., 1998). This suggests that there may be enzymatic differences in the production of 2-hydroxybut-3-enyl glucosinolate among different plant species.
The production of 2-hydroxybut-3-enyl glucosinolate has diverse biological consequences. It is toxic to insects, bacteria, and nematodes, inhibits the germination of various plant species, and is a major source of bitter flavor in Brassica vegetables (Donkin et al., 1995
To identify the genes required to produce 2-hydroxybut-3-enyl glucosinolate and its resulting biological activity, we have undertaken a genetic, biochemical, and molecular analysis of the production of 2-hydroxybut-3-enyl glucosinolate. We utilized natural variation in gene expression as well as T-DNA mutations within Arabidopsis to show that a novel 2-ODD is required to form 2-hydroxybut-3-enyl glucosinolate. The genetic analysis also suggested that this enzyme determines the total level of aliphatic glucosinolates accumulated and resistance to a generalist insect herbivore. Interestingly, this insect resistance appears to be dependent upon the specific structure of the glucosinolate rather than the total accumulation of glucosinolates, as had been found previously (Kliebenstein et al., 2002
Natural Variation of 2-Hydroxybut-3-enyl Glucosinolate in Arabidopsis
In Brassica, the occurrence of 2-hydroxybut-3-enyl glucosinolate is controlled by the allelic status at the GSL-OH locus and the presence of its precursor, but-3-enyl glucosinolate, whose production is in turn regulated by the GSL-Elong and GSL-AOP loci (Magrath et al., 1994
Neither Landsberg erecta (Ler) nor Cvi accumulates 2-hydroxybut-3-enyl glucosinolate (Kliebenstein et al., 2001b
Further mapping showed that GSL-OH mapped between microsatellite markers: 2 centimorgan (cM) north of T1D16 and 0.3 cM south of F13D4 (Fig. 3 ; Supplemental Table S2). This genomic region contains seven bacterial artificial chromosomes (BACs) and two potential genes that encode enzymes capable of hydroxylating but-3-enyl glucosinolate to its 2-hydroxyl derivative, a cytochrome P450 monooxygenase and a 2-ODD (Fig. 3).
Fine-Scale Mapping of GSL-OH in Cvi x Tacoma To fine-scale map GSL-OH, we generated F2 progeny from a cross between Cvi (nonfunctional GSL-OH) and Tacoma (Tac; fully functional GSL-OH; Supplemental Table S1). These two parents were chosen because they have the same GSL-Elong and GSL-AOP alleles and, as such, only GSL-OH segregates. A total of 192 F2 progeny were analyzed by HPLC for the presence of 2-hydroxybut-3-enyl glucosinolate and genotyped at T1D16 and F13D4. GSL-OH again mapped to a single locus 2.6 cM north of T1D16 and 1.5 cM south of F13D4 (Fig. 3). Additionally, in Cvi x Tac F1 individuals, the full-functional GSL-OH phenotype complemented the seed-functional GSL-OH allele (data not shown). Thus, the full-functional, seed-functional, and null GSL-OH phenotypes are caused by different alleles at a single GSL-OH locus. To further fine-scale map GSL-OH, we generated microsatellite markers on T19L18, F17H15, F13B15, and T22F11. This showed that there was no recombination between GSL-OH and microsatellite F13B15 in a total of 580 F2 progeny (Fig. 3). The F13B15 BAC contains only the 2-ODD candidate gene, At2g25450, suggesting that the enzyme encoded by this gene may have the capacity to catalyze the hydroxylation of but-3-enyl glucosinolate to form 2-hydroxybut-3-enyl glucosinolate.
We sequenced the predicted full-length At2g25450 open reading frame (ORF) from accessions that contained the three GSL-OH variants to determine whether the candidate gene sequence covaried with changes in the accumulation pattern of 2-hydroxybut-3-enyl glucosinolate. The At2g25450 ORF is 1,077 nucleotides long and encodes a protein of 359 amino acids. The cDNAs from accessions with full-functional or seed-functional GSL-OH alleles are nearly identical, with only one to three nucleotide differences among the various copies (Fig. 3). In contrast, the cDNA sequences from accessions with null GSL-OH alleles clearly separated into two groups. Analysis of the At2g25450 cDNA sequences from the GSL-OH null accessions Kondara and Hodja showed that they share a C-to-T substitution, which generates a TAG stop codon. This truncates the protein to 131 amino acids and is probably the reason for the lack of GSL-OH activity in these two accessions. In contrast, the Cvi At2g25450 cDNA encoded an intact ORF that has six nucleotide polymorphisms in comparison with the other sequences, with five of the six polymorphisms being nonsynonymous substitutions (R288-I, G254-E, P218-H, L184-F, and G105-S, with the first amino acid being the consensus and the second being Cvi). Additionally, all five amino acid changes are nonconservative substitutions that could significantly alter the structure of the encoded enzymes. Any of these changes may be sufficient to inactivate At2g25450 and cause the null phenotype in Cvi.
To further corroborate the relationship between At2g25450 and GSL-OH, the expression of At2g25450 in leaves of Arabidopsis accessions was compared with the GSL-OH allele variant present in each accession based on its glucosinolate composition. In all accessions tested, the fully functional GSL-OH allele was associated with high At2g25450 transcript accumulation in the leaf (Fig. 3). In contrast, all accessions with the seed-functional allele had low to nondetectable GSL-OH expression in the leaf (Fig. 3).
HPLC analysis of leaves from accessions with the fully functional allele identified heritable differences in the ratio of but-3-enyl glucosinolate to 2-hydroxybut-3-enyl glucosinolate (Kliebenstein et al., 2001c
At2g25450 Expression and GSL-OH Enzyme Activity At2g25450 is expressed in some accessions that do not contain but-3-enyl glucosinolate, the substrate for the encoded enzyme (Fig. 3). For example, the Ei-2 accession does not accumulate any but-3-enyl or 2-hydroxybut-3-enyl glucosinolate (Fig. 5A ). However, after incubating Ei-2 leaves with extract containing but-3-enyl glucosinolate (Fig. 5B), the leaves took up the but-3-enyl glucosinolate precursor and converted it into both enantiomeric forms of 2-hydroxybut-3-enyl glucosinolate in the same ratio as observed previously for accessions producing 2-hydroxybut-3-enyl glucosinolate (Fig. 5C). This shows that At2g25450 expression and but-3-enyl glucosinolate-2-hydroxylase activity are found in accessions that do not contain the precursor glucosinolate. Utilizing this substrate-feeding protocol, we tested the correlation between At2g25450 expression and in vivo but-3-enyl glucosinolate-2-hydroxylase activity in 12 accessions that do not naturally contain the substrate. The absence of the precursor in these plants allowed for a simultaneous direct measurement of but-3-enyl glucosinolate uptake and its subsequent conversion to 2-hydroxybut-3-enyl glucosinolate. We observed a positive correlation between the accumulation of At2g25450 mRNA and but-3-enyl glucosinolate-2-hydroxylase activity (Fig. 5D; n = 11, r = 0.913, P < 0.0001).
T-DNA Mutation in At2g25450 Abolishes Glucosinolate-2-hydroxylase Activity
To further confirm the causal relationship between At2g25450 and 2-hydroxylase activity, we obtained a homozygous T-DNA insertion mutant in the Columbia (Col-0) background (SALK_089807). The T-DNA insertion is located in the second exon of At2g25450 and abolishes At2g25450 expression (Supplemental Fig. S1). While the standard Arabidopsis reference accession for T-DNA knockouts, Col-0, does not contain 2-hydroxybut-3-enyl glucosinolate due to a lack of its precursor but-3-enyl glucosinolate, we relied on the fact that introduction of a functional AOP2 into Col-0 causes the accumulation of both but-3-enyl glucosinolate and 2-hydroxybut-3-enyl glucosinolate (Li and Quiros, 2003
As observed previously, introduction of a functional AOP2 into Col-0 results in an elevated total aliphatic glucosinolate level (Fig. 6; Wentzell et al., 2007 ). Interestingly, combining AOP2 expression in Col-0 with an At2g25450 T-DNA mutation led to a further increase in aliphatic glucosinolate accumulation. This suggests that At2g25450, in addition to producing 2-hydroxybut-3-enyl glucosinolate, also affects aliphatic glucosinolate accumulation. As this effect requires a functional AOP2 gene, it suggests that this repressive function is not an inherent property of the At2g25450 transcript or protein. Instead, it is possible that the 2-hydroxybut-3-enyl glucosinolate product may feedback regulate the biosynthetic pathway or that At2g25450 directly influences the inductive capacity of AOP2 (Fig. 6; Wentzell et al., 2007). These results confirm that At2g25450 is the GSL-OH locus and likely encodes the enzyme responsible for the conversion of but-3-enyl to 2-hydroxybut-3-enyl glucosinolate, which, interestingly, also affects the regulation of the aliphatic glucosinolate pathway.
Arabidopsis resistance against generalist lepidopteran herbivory has been consistently shown to be controlled by increasing glucosinolate content or activation (Kliebenstein et al., 2002
GSL-OH Promoter Variation and Expression To identify the polymorphisms responsible for differential gene expression, we sequenced 4.9 kb of the At2g25450 genomic region from the 12 accessions that differ in At2g25450 expression levels (Fig. 5). The sequenced region extended from the stop codon of the upstream gene to the start codon of the downstream gene and is expected to include all regulatory elements. Sequence comparison identified a unique 120-bp motif that is repeated five times in accessions with low leaf GSL-OH alleles and four times in full-functional alleles (Fig. 8 ; Supplemental Table S3). Two motifs overlap with the putative transcription start and stop sites, while the others are in the promoter region (Fig. 8). BLAST analysis against the complete Arabidopsis genomic sequence showed that this motif is not found elsewhere. The difference in motif number between the expression classes is caused by a deletion removing part of two intact motifs and the intervening DNA to produce one intact motif in the high-expression accessions (Fig. 8). The one accession with undetectable leaf expression, Ler, has two separate deletions that remove two-thirds of the two motifs flanking the ORF. Because the sequence extended from the upstream gene to the downstream gene in all accessions, this eliminates the possibility of a tandemly duplicated At2g25450. Thus, the three different variant classes are probably due to differential expression of At2g25450 caused by polymorphisms altering these unique motifs.
Several lines of evidence strongly support the identification of At2g25450 as the causal gene for the GSL-OH QTL. First, fine-scale mapping of 2-hydroxybut-3-enyl glucosinolate production in several Arabidopsis populations identified At2g25450 as the best candidate gene with the appropriate enzymatic activity to convert but-3-enyl to 2-hydroxybut-3-enyl glucosinolate (Figs. 2 and 3). Second, association experiments using Arabidopsis accessions showed a nearly total congruence between At2g25450 expression and the production of 2-hydroxybut-3-enyl glucosinolate from both endogenous and exogenous precursor (Figs. 4 and 5). Furthermore, examination of existing genomic sequence polymorphism data indicates that At2g25450 does not have any linkage disequilibrium with its neighboring genes and hence varies independently of these genes in the accessions. This shows that this association between GSL-OH and sequence and expression variation in the accessions is centered on the At2g25450 gene itself and not on any proximal loci (Borevitz et al., 2007 ; Clark et al., 2007). Furthermore, three accessions with nonfunctional GSL-OH alleles have loss-of-function mutations in At2g25450. Finally, an independent T-DNA knockout in At2g25450 abolishes all measurable GSL-OH activity. In combination, these multiple and independent genetic lines of evidence show that At2g25450 is the GSL-OH QTL.
At2g25450 (GSL-OH) belongs to the 2-ODD gene superfamily, a group that has at least 100 members in Arabidopsis, but none of the closely related Arabidopsis 2-ODDs have any defined biochemical functions (Supplemental Fig. S2). 2-ODDs are nonmembranous dioxygenases with activities similar to those of the membrane-bound cytochrome P450 monooxygenases, but usually requiring Fe2+ ions and a 2-oxoacid such as ascorbate or oxoglutarate (De Carolis and De Luca, 1993
Total aliphatic glucosinolate content is often used as a variable to explain insect resistance without including information about the specific structures. Our data show that the different aliphatic glucosinolate structures are not functionally equivalent as herbivory deterrents in the Col-0 background (Fig. 7). The methylsulfinylalkyl glucosinolates were the most effective deterrents, with the hydroxyalkenyl glucosinolates being more effective than their alkenyl precursors. This is in spite of lines with methylsulfinylalkyl glucosinolates having the lowest levels of total aliphatic glucosinolates (Fig. 6). Thus, At2g25450, in addition to controlling hydroxyalkenyl glucosinolate production, also affects resistance to insect herbivory. The increased generalist lepidopteran resistance of hydroxyalkenyl glucosinolates over simple alkenyl glucosinolates may explain why most Arabidopsis accessions with alkenyl glucosinolates produce them as the hydroxyalkenyl form (Kliebenstein et al., 2001c
This observation of alkenyl glucosinolates being a less effective generalist lepidopteran defense, specifically against T. ni, than methylsulfinylalkyl glucosinolates is in contradiction to the fact that half of the natural Arabidopsis accessions make alkenyl glucosinolates (Kliebenstein et al., 2001c
Numerous accessions lacking the but-3-enyl glucosinolate precursor nevertheless had detectable At2g25450 mRNA and measurable in vivo 2-hydroxylase activity (Fig. 8). If this enzyme has no natural substrate in these plants, one would expect the encoding gene to rapidly lose its function over the course of evolutionary time. Why then is GSL-OH activity found in lines lacking the necessary precursor? One possibility is that the levels of out-crossing and levels in Arabidopsis are sufficient to maintain a functional GSL-OH even in lines lacking precursor. An alternative is that the enzyme is multifunctional and required for another biosynthetic reaction. However, this second reaction, if it exists, must not be critical for basic growth and development, because the accessions Kondara and Hodja, which have premature stop codons, and the T-DNA insertion line are all viable (Fig. 9). Furthermore, this second reaction cannot affect insect herbivory or glucosinolate content in the absence of the but-3-enyl precursor (Figs. 6 and 7). The presence of enzymes lacking precursors has interesting implications for the manipulation of secondary metabolic pathways. It has been proposed to utilize transgenic technologies to introduce new biosynthetic capacities into plants. However, the presence of hidden enzyme activities makes it difficult to predict the final compound that may accumulate from a given transgenic modification. As more and more genes of secondary metabolism are studied, it will be interesting to see what proportion of these encode enzyme activities that are hidden by the lack of an endogenous substrate.
The stereoselectivity of but-3-enyl glucosinolate-2-hydroxylase varies among species within the Brassicales. In Arabidopsis, both the 2R and 2S enantiomers of 2-hydroxybut-3-enyl glucosinolate are produced in a constant ratio (2R:2S) of 1:3. While some enzyme reactions lead to a racemic mixture of products, it is unusual to find a reaction that yields a mixture of two enantiomers in a fixed, nonequivalent ratio. In contrast, Brassica napus and Arabis alpina make only the 2R enantiomer of 2-hydroxybut-3-enyl glucosinolate, while Crambe abyssinica makes only the 2S enantiomer (Daubos et al., 1998
The isolation of the gene responsible for converting but-3-enyl glucosinolate to 2-hydroxybut-3-enyl glucosinolate provides a key molecular tool for studying the function and formation of 2-hydroxybut-3-enyl glucosinolate. The GSL-OH gene creates new opportunities for the modification of glucosinolate-containing agricultural species. 2-Hydroxybut-3-enyl glucosinolate is goiterogenic because the hydroxyl substituent promotes the formation of the cyclic 5-vinyl-oxazolidine-2-thione (Fenwick et al., 1983
Accession Analysis All Arabidopsis (Arabidopsis thaliana) plant lines were obtained from the Arabidopsis Stock Center. Ten plants were grown in 3.25- x 3.25- x 2.25-inch pots at 18 pots to a flat for 3 weeks in a standard soil-vermiculite mixture at 26°C. They were placed 10 inches from four 60-W cool-white GE bulbs and four 60-W wide-spectrum bulbs in a 16-h-light/8-h-dark photoperiod. This study utilized a collection of 37 accessions, as listed in Supplemental Table S1.
Samples were extracted and analyzed by HPLC as described previously (Kliebenstein et al., 2001c
DNA was isolated with the 96-well DNA prep described previously. The primers listed in Supplemental Table S2 were utilized for fine-scale mapping in both the Ler x Cvi and Cvi x Tac populations. Five microliters of the 96-well DNA prep was added to 20 µL of PCR mixture (2.5 mM MgCl2, 200 pM primers, and 0.5 units of Taq) and run with the following cycle program (95°C for 3 min; 40 cycles of 95°C for 20 s, 56°C for 20 s, and 72°C for 1 s; 72°C for 3 min; and 4°C final). The microsatellites were then scored on 4% agarose.
Glucosinolates were extracted and analyzed from 5 mg of seeds from each Ler x Cvi RIL to phenotype the GSL-OH activity. Scoring of the Ler x Cvi RILs was done by analyzing the seeds for the presence of the 2R- and 2S-2-hydroxybut-3-enyl glucosinolate as well as for the benzylated derivatives 2R- and 2S-2-benzyloxy-but-3-enyl glucosinolate. Lines that produced these glucosinolates were given a score of 1. Lines that did not contain any of the detectable products were given a score of 0. QTL mapping was done with QTL Cartographer using composite interval mapping and 1,000 permutations to estimate the 0.05 significance threshold (Basten et al., 1999 For the Cvi x Tac mapping experiment, homozygous Cvi and Tac were crossed and the resulting F1 progeny were selfed to generate an F2 population. A total of 192 Cvi x Tac F2 plants were simultaneously scored for the T1D16 and F13D4 microsatellites (Supplemental Table S2) and the GSL-OH phenotype by HPLC. They were phenotyped by planting a single F2 seed per cell in a 96-well flat. The seeds were allowed to germinate and grown for 4 weeks under a 9-h-light/15-h-dark photoperiod. After 4 weeks, two to three leaves were harvested for glucosinolate extraction and HPLC analysis, and one to two leaves were taken for DNA purification. After confirming that the GSL-OH locus was between T1D16 and F13D4, an additional 388 F2 progeny were genotyped with these microsatellites to identify recombinant progeny. All recombinant progeny were then scored for the T19L18#2, T22F11, and F17H15 microsatellites (Supplemental Table S2), and their HPLC phenotypes were scored in the F3 generation.
But-3-enyl glucosinolate was obtained from 30 g of freeze-dried Cvi and extracted with 200 mL of methanol for 4 h. Cvi contains but-3-enyl glucosinolate and has no detectable accumulation of 2-hydroxybut-3-enyl glucosinolate (Kliebenstein et al., 2001c
Total RNA was isolated from approximately 100 mg of leaf tissue utilizing the Trizol reagent. Approximately 1 µg of total RNA was utilized for cDNA synthesis as described (Frohman et al., 1988
cDNA was generated as described previously. Primers for the RAN cDNA were utilized to standardize for the amount of cDNA present in each of the samples. Each accession utilized in this study was first sequenced as described above. This sequence was then used to generate the GSOHRT-F and GSOHRT-R primers, which are perfectly homologous to all of the accessions tested (Supplemental Table S2). The GSOH and RAN primers were then used to amplify specific cDNAs from 10, 1, and 0.1 µL of starting cDNA. The resulting products were then separated on a gel and detected with an Eagle Eye and ethidium bromide. The resulting files were transferred to a phosphoimager, which was used in densitometric mode to measure the cDNA produced. Each accession was measured in triplicate.
A T-DNA mutation in the second exon of At2g25450, SALK_089807, was obtained from the Arabidopsis Biological Resource Center. This was then crossed three independent times to Col-0 containing a functional AOP2 transgene (Wentzell et al., 2007
Numerous attempts at expressing the protein in Escherichia coli or Saccharomyces cerevisiae using multiple vectors and systems were unsuccessful, potentially from the in vitro protein being insoluble. Additionally, the presence of two direct repeats at the beginning and end of the protein sequence led to the requirement for recombination-suppressed E. coli, but in the absence of such, an Agrobacterium tumefaciens complementation construct would be quickly destroyed in the bacteria prior to introduction into the plant. As such, we were unable to conduct in planta complementation attempts with the resources available.
The lines generated using the At2g25450 T-DNA mutation and the functional AOP2 transgene were used for assaying resistance to T. ni herbivory. At least 10 plants for each of the four genotypic classes were obtained per cross. Each plant was in an individual pot, and these pots were physically separated to prevent larval movement. T. ni eggs were obtained from Benzon Research and hatched on artificial medium. After 48 h, two first instar larvae were placed on each plant, and herbivory was measured every 24 h by visual measurements whereby leaf area removed was estimated using a ruled grid. The entire experiment was replicated twice. Herbivory at each time point was independently analyzed via ANOVA using a general linear model. In this model, yrgc denotes the herbivory in square centimeters on Arabidopsis genotype g from cross c in replicate r. The ANOVA model for the glucosinolate accumulation is yrgc = µ + Gg + Cc + Rr +
The following materials are available in the online version of this article.
Received September 17, 2008; accepted October 17, 2008; published October 22, 2008.
1 These authors contributed equally to the article.  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).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[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.108.129981 * Corresponding author; e-mail kliebenstein{at}ucdavis.edu.
Alonso-Blanco C, Peeters AJM, Koornneef M, Lister C, Dean C, van den Bosch N, Pot J, Kuiper MTR (1998) Development of an AFLP based linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and construction of a Ler/Cvi recombinant inbred line population. Plant J 14: 259–271[CrossRef][Web of Science][Medline] Angelini L, Lazzeri L, Galletti S, Cozzani A, Macchia M, Palmieri S (1998) Antigerminative activity of three glucosinolate-derived products generated by myrosinase hydrolysis. Seed Science and Technology 26: 771–779 Barth C, Jander G (2006) Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense. Plant J 46: 549–562[CrossRef][Web of Science][Medline] Basten CJ, Weir BS, Zeng ZB (1999) QTL Cartographer, Version 1.13. Department of Statistics, North Carolina State University, Raleigh, NC Beekwilder J, van Leeuwen W, van Dam NM, Bertossi M, Grandi V, Mizzi L, Soloviev M, Szabados L, Molthoff JW, Schipper B, et al (2008) The impact of the absence of aliphatic glucosinolates on insect herbivory in Arabidopsis. PLoS ONE 3: e2068 Benderoth M, Textor S, Windsor AJ, Mitchell-Olds T, Gershenzon J, Kroymann J (2006) Positive selection driving diversification in plant secondary metabolism. Proc Natl Acad Sci USA 103: 9118–9123 Berenbaum MR, Zangerl AR (1998) Chemical phenotype matching between a plant and its insect herbivore. Proc Natl Acad Sci USA 95: 13743–13748 Bones AM, Rossiter JT (1996) The myrosinase-glucosinolate system, its organisation and biochemistry. Physiol Plant 97: 194–208[CrossRef] Bones AM, Thangstad OP, Haugen OA, Espevik T (1991) Fate of myrosin cells: characterization of monoclonal antibodies against myrosinase. J Exp Bot 42: 1541–1549 Borevitz JO, Hazen SP, Michael TP, Morris GP, Baxter IR, Hu TT, Chen H, Werner JD, Nordborg M, Salt DE, et al (2007) Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana. Proc Natl Acad Sci USA 104: 12057–12062 Bowers MD, Puttick GM (1988) Response of generalist and specialist insects to qualitative allelochemical variation. J Chem Ecol 14: 319–334[CrossRef][Web of Science] Burow M, Müller R, Gershenzon J, Wittstock U (2006) Altered glucosinolate hydrolysis in genetically engineered Arabidopsis thaliana and its influence on the larval development of Spodoptera littoralis. J Chem Ecol 32: 2333–2349[CrossRef][Medline] Clark RM, Schweikert G, Toomajian C, Ossowski S, Zeller G, Shinn P, Warthmann N, Hu TT, Fu G, Hinds DA, et al (2007) Common sequence polymorphisms shaping genetic diversity in Arabidopsis thaliana. Science 317: 338–342 Daubos P, Grumel V, Iori R, Leoni O, Palmieri S, Rollin P (1998) Crambe abyssinica meal as starting material for the production of enantiomerically pure fine chemicals. Ind Crops Prod 7: 187–193 Daxenbichler ME, Spencer GF, Carlson DG, Rose GB, Brinkler AM, Powell RG (1991) Glucosinolate composition of seeds from 297 species of wild plants. Phytochemistry 30: 2623–2638[CrossRef] De Carolis E, De Luca V (1993) Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus. J Biol Chem 268: 5504–5511 de Vos M, Kriksunov KL, Jander G (2008) Indole-3-acetonitrile production from indole glucosinolates deters oviposition by Pieris rapae. Plant Physiol 146: 916–926 Donkin SG, Eiteman MA, Williams PL (1995) Toxicity of glucosinolates and their enzymatic decomposition products to Caenorhabditis elegans. J Nematol 27: 258–262[Medline] Ehrlich PR, Raven PH (1964) Butterflies and plants: a study in coevolution. Evolution Int J Org Evolution 18: 586–608[CrossRef][Web of Science] Fahey JW, Zalcmann AT, Talalay P (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56: 5–51[CrossRef][Web of Science][Medline] Fenwick GR, Heaney RK, Mullin WJ (1983) Glucosinolates and their breakdown products in food and food plants. Crit Rev Food Sci Nutr 18: 123–201[Medline] Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002 Giamoustaris A, Mithen R (1996) Genetics of aliphatic glucosinolates. IV. Side-chain modification in Brassica oleracea. Theor Appl Genet 93: 1006–1010[CrossRef][Web of Science] Gigolashvili T, Yatusevich R, Berger B, Müller C, Flügge UI (2007) The R2R3-MYB transcription factor HAG1/MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana. Plant J 51: 247–261[CrossRef][Web of Science][Medline] Greer MA (1956) Isolation from rutabaga seed of progoitrin, the precursor of the naturally occurring antithyroid compound, goitrin (L-5-vinyl-2-thiooxazolidone). J Am Chem Soc 78: 1260–1261 Greer MA, Deeney JM (1959) Antithyroid activity elicited by the ingestion of pure progoitrin, a naturally occurring thioglycoside of the turnip family. J Clin Invest 38: 1465–1474[CrossRef][Web of Science][Medline] Halkier BA, Du L (1997) The biosynthesis of glucosinolates. Trends Plant Sci 2: 425–431[CrossRef] Halkier BA, Gershenzon J (2006) Biology and biochemistry of glucosinolates. Annu Rev Plant Biol 57: 303–333[CrossRef][Medline] Hansen BG, Kliebenstein DJ, Halkier BA (2007) Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis. Plant J 50: 902–910[CrossRef][Web of Science][Medline] Haughn GW, Davin L, Giblin M, Underhill EW (1991) Biochemical genetics of plant secondary metabolites in Arabidopsis thaliana: the glucosinolates. Plant Physiol 97: 217–226 Hirai M, Sugiyama K, Sawada Y, Tohge T, Obayashi T, Suzuki A, Ryoichi A, Sakurai N, Suzuki H, Aoki K, et al (2007) Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis. Proc Natl Acad Sci USA 104: 6478–6483 Jonczyk R, Schmidt H, Osterrieder A, Fiesselmann A, Schullehner K, Haslbeck M, Sicker D, Hofmann D, Yalpani N, Simmons C, et al (2008) Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: characterization of Bx6 and Bx7. Plant Physiol 146: 1053–1063 Kelly PJ, Bones A, Rossiter JT (1998) Sub-cellular immunolocalization of the glucosinolate sinigrin in seedlings of Brassica juncea. Planta 206: 370–377[CrossRef][Web of Science][Medline] Kim JH, Jander G (2007) Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate. Plant J 49: 1008–1019[Medline] Kliebenstein D, Lambrix V, Reichelt M, Gershenzon J, Mitchell-Olds T (2001a) Gene duplication and the diversification of secondary metabolism: side chain modification of glucosinolates in Arabidopsis thaliana. Plant Cell 13: 681–693 Kliebenstein DJ, Gershenzon J, Mitchell-Olds T (2001b) Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds. Genetics 159: 359–370 Kliebenstein DJ, Kroymann J, Brown P, Figuth A, Pedersen D, Gershenzon J, Mitchell-Olds T (2001c) Genetic control of natural variation in Arabidopsis thaliana glucosinolate accumulation. Plant Physiol 126: 811–825 Kliebenstein DJ, Pedersen D, Mitchell-Olds T (2002) Comparative analysis of insect resistance QTL and QTL controlling the myrosinase/glucosinolate system in Arabidopsis thaliana. Genetics 161: 325–332 Koch M, Haubold B, Mitchell-Olds T (2001) Molecular systematics of the Brassicaceae: evidence from coding plastidic matK and nuclear Chs sequences. Am J Bot 88: 534–544 Koroleva OA, Davies A, Deeken R, Thorpe MR, Tomos AD, Hedrich R (2000) Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk. Plant Physiol 124: 599–608 Kroymann J, Textor S, Tokuhisa JG, Falk KL, Bartram S, Gershenzon J, Mitchell-Olds T (2001) A gene controlling variation in Arabidopsis glucosinolate composition is part of the methionine chain elongation pathway. Plant Physiol 127: 1077–1088 Lambrix V, Reichelt M, Mitchell-Olds T, Kliebenstein D, Gershenzon J (2001) The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. Plant Cell 13: 2793–2807 Lander E, Abrahamson J, Barlow A, Daly M, Lincoln S, Newburg L, Green P (1987) Mapmaker a computer package for constructing genetic-linkage maps. Cytogenet Cell Genet 46: 642 Lankau RA (2007) Specialist and generalist herbivores exert opposing selection on a chemical defense. New Phytol 175: 176–184[CrossRef][Web of Science][Medline] Li G, Quiros CF (2003) In planta side-chain glucosinolate modification in Arabidopsis by introduction of dioxygenase Brassica homolog BoGSL-ALK. Theor Appl Genet 106: 1116–1121[Medline] Magrath R, Bano F, Morgner M, Parkin I, Sharpe A, Lister C, Dean C, Turner J, Ludiate D, Mithen R (1994) Genetics of aliphatic glucosinolates. I. Side chain elongation in Brassica napus and Arabidopsis thaliana. Heredity 72: 290–299[CrossRef][Web of Science] Manici LM, Lazzeri L, Baruzzi G, Leoni O, Galletti S, Palmieri S (2000) Suppressive activity of some glucosinolate enzyme degradation products on Pythium irregulare and Rhizoctonia solani in sterile soil. Pest Manag Sci 56: 921–926 Mithen R (1992) Leaf glucosinolate profiles and their relationships to pest and disease resistance in oilseed rape. Euphytica 63: 71–83[CrossRef] Mithen R, Clarke J, Lister C, Dean C (1995) Genetics of aliphatic glucosinolates. III. Side-chain structure of aliphatic glucosinolates in Arabidopsis thaliana. Heredity 74: 210–215 Mithen R, Toroser D (1995) Biochemical genetics of aliphatic glucosinolates in Brassica and Arabidopsis. In Seminar Series 56. Society for Experimental Biology. Cambridge University Press, Cambridge, UK, pp 261–275 Mithen RF, Lewis BG, Heaney RK, Fenwick GR (1987) Resistance of leaves of Brassica species to Leptosphaeria maculans. Trans Br Mycol Soc 88: 525–531 Nho CW, Jeffery E (2004) Crambene, a bioactive nitrile derived from glucosinolate hydrolysis, acts via the antioxidant response element to upregulate quinone reductase alone or synergistically with indole-3-carbinol. Toxicol Appl Pharmacol 198: 40–48[CrossRef][Medline] Parkin I, Magrath R, Keith D, Sharpe A, Mithen R, Lydiate D (1994) Genetics of aliphatic glucosinolates. II. Hydroxylation of alkenyl glucosinolates in Brassica napus. Heredity 72: 594–598 Pearson AW, Greenwood NM, Butler EJ, Fenwick GR (1983) Biochemical changes in layer and broiler chickens when fed on a high-glucosinolate rapeseed meal. Br Poult Sci 24: 417–427[Medline] Peterson CJ, Cosse A, Coats JR (2000) Insecticidal components in the meal of Crambe abyssinica. Journal of Agricultural and Urban Entomology 17: 27–35 Prescott AG (1993) A dilemma of dioxygenases (or where biochemistry and molecular-biology fail to meet). J Exp Bot 44: 849–861 Puttick GM, Bowers MD (1988) Effect of qualitative and quantitative variation in allelochemicals on a generalist insect: iridoid glycosides and the southern armyworm. J Chem Ecol 14: 335–351[CrossRef][Web of Science] Reichelt M, Brown PD, Schneider B, Oldham NJ, Stauber E, Tokuhisa J, Kliebenstein DJ, Mitchell-Olds T, Gershenzon J (2002) Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana. Photochemistry 59: 663–671 Rodman JE, Kruckeberg AR, Alshehbaz IA (1981) Chemotaxonomic diversity and complexity in seed glucosinolates of Caulanthus and Streptanthus (Cruciferae). Syst Bot 6: 197–222 Rossiter JT, James DC, Atkins N (1990) Biosynthesis of 2-hydroxy-3-butenylglucosinolate and 3-butenylglucosinolate in Brassica napus. Phytochemistry 29: 2509–2512[CrossRef][Web of Science] Sønderby IE, Hansen BG, Bjarnholt N, Ticconi C, Halkier BA, Kliebenstein DJ (2007) A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates. PLoS ONE 2: e1322[CrossRef] Tierens KJ, Thomma B, Brower M, Schmidt J, Kistner K, Porzel A, Mauch-Mani B, Cammue B, Broekaert W (2001) Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens. Plant Physiol 125: 1688–1699 van Doorn HE, van der Kruk GC, van Holst GJ, Raaijmakers-Ruijs N, Postma E, Groeneweg B, Jongen WHF (1998) The glucosinolates sinigrin and progoitrin are important determinants for taste preference and bitterness of brussels sprouts. J Sci Food Agric 78: 30–38[CrossRef][Web of Science] van Doorn JE, van der Kruk GC, van Holst GJ, Schoofs M, Broer JB, de Nijs JJM (1999) Quantitative inheritance of the progoitrin and sinigrin content in brussels sprouts. Euphytica 108: 41–52 Wentzell AM, Rowe HC, Hansen BG, Ticconi C, Halkier BA, Kliebenstein DJ (2007) Linking metabolic QTL with network and cis-eQTL controlling biosynthetic pathways. PLOS Genetics 3: e162 Windsor AJ, Reichelt M, Figuth A, Svatos A, Kroymann J, Kliebenstein DJ, Gershenzon J, Mitchell-Olds T (2005) Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae). Photochemistry 66: 1321–1333 Zangerl AR, Berenbaum MR (1990) Furanocoumarin induction in wild parsnip: genetics and populational variation. Ecology 71: 1933–1940[CrossRef][Web of Science] Zangerl AR, Stanley MC, Berenbaum MR (2008) Selection for chemical trait remixing in an invasive weed after reassociation with a coevolved specialist. Proc Natl Acad Sci USA 105: 4547–4552 Zhang ZY, Ober JA, Kliebenstein DJ (2006) The gene controlling the quantitative trait locus EPITHIOSPECIFIER MODIFIER1 alters glucosinolate hydrolysis and insect resistance in Arabidopsis. Plant Cell 18: 1524–1536 This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
rgc, where 
