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Gene Duplication in the Diversification of Secondary Metabolism: Tandem 2-Oxoglutarate–Dependent Dioxygenases Control Glucosinolate Biosynthesis in Arabidopsis

Daniel J. Kliebenstein, Virginia M. Lambrix, Michael Reichelt, Jonathan Gershenzon, Thomas Mitchell-Olds
Daniel J. Kliebenstein
Max Planck Institute for Chemical Ecology, 07745 Jena, Germany
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Virginia M. Lambrix
Max Planck Institute for Chemical Ecology, 07745 Jena, Germany
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Michael Reichelt
Max Planck Institute for Chemical Ecology, 07745 Jena, Germany
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Jonathan Gershenzon
Max Planck Institute for Chemical Ecology, 07745 Jena, Germany
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Thomas Mitchell-Olds
Max Planck Institute for Chemical Ecology, 07745 Jena, Germany
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  • For correspondence: tmo@ice.mpg.de

Published March 2001. DOI: https://doi.org/10.1105/tpc.13.3.681

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    Figure 1.

    Side Chain Modifications of Methionine-Derived Glucosinolates in Arabidopsis.

    Potential side chain modifications for the elongated methionine derivative C4 dihomomethionine are shown. Steps with natural variation in Arabidopsis are shown in boldface to the right or left of each enzymatic arrow with the name of the corresponding locus.

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    Figure 2.

    HPLC Results of Ler, Cvi, and Col Glucosinolate Profiles.

    Shown are HPLC results monitored at 229 nm of samples from Ler, Cvi, and Col prepared from equal amounts of 2-week-old rosette tissue. The major methionine-derived glucosinolate peaks are labeled for each ecotype. The large peak at ∼18.5 min in Ler and Col is a tryptophan-derived glucosinolate. The large peak at ∼2.5 min is due to solvent mixing from the injected sample. mAu, milliabsorbance units.

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    Figure 3.

    Fine-Scale Map of GS-AOP.

    A fine-scale genomic map of GS-AOP is shown for reference. Horizontal solid lines represent the four BACs used, and the vertical lines represent the positions of the microsatellites on each BAC. The number to the right of each recombinant inbred population name is the number of recombinations between the given marker and the GS-AOP phenotype. The vertical black bar represents the genomic location of the three 2-ODD genes. The three 2-ODD genes and one related 2-ODD pseudogene are labeled AOP1, AOP2, AOP3, and Pseu, respectively. The F9H3 and T5J8 microsatellite markers are separated by ∼300 kb. The dotted line indicates the region between the two microsatellite markers for the Ler × Cvi cross (top line) or the Ler × Col cross (bottom line).

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    Figure 4.

    Ecotype-Specific Expression of the AOP Genes.

    Shown are the ethidium bromide–stained gels from a quantitative RT-PCR experiment with the Ler, Col, and Cvi parental ecotypes. The Ran gene was used as a loading control. Numbers above the gels (1, 0.3, and 0.1) indicate the amounts of cDNA (μL) used for the RT-PCRs.

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    Figure 5.

    Enzymatic Activity of Heterologously Expressed AOP2.

    HPLC results (monitored at 229 nm) of purified desulfoglucosinolates from bacterial extracts containing heterologously expressed AOP2 fusion proteins are shown. All compound identities were confirmed by comparison of both retention times and UV light absorption profiles with those of authentic standards. 3-MSO, 3-methylsulfinylalkyl glucosinolate; 4-MSO, 4-methylsulfinylalkyl glucosinolate.

    (A) Extract from 3-methylsulfinylalkyl glucosinolate treated with uninduced AOP2 bacterial extract.

    (B) Extract from 3-methylsulfinylalkyl glucosinolate treated with AOP2 enzyme.

    (C) Extract from 4-methylsulfinylalkyl glucosinolate treated with uninduced AOP2 bacterial extract.

    (D) Extract from 4-methylsulfinylalkyl glucosinolate treated with AOP2 enzyme.

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    Figure 6.

    Enzymatic Activity of AOP3 Heterologously Expressed in Escherichia coli.

    E. coli extract was assayed with 3-methylsulfinylpropyl glucosinolate, and products were extracted and analyzed by HPLC as described in Methods.

    (A) Sample of authentic 3-hydroxypropyl glucosinolate (monitored at 229 nm).

    (B) Extract from AOP3 enzyme assay with 3-methylsulfinylpropyl glucosinolate as substrate (monitored at 229 nm).

    (C) Diode array spectrum of standard 3-hydroxypropyl desulfoglucosinolate in (A).

    (D) Diode array spectrum of 3-hydroxypropyl desulfoglucosinolate produced by AOP3 in (B).

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    Figure 7.

    Tree of AOP cDNAs from Ler, Cvi, and Col.

    Cladogram of the relations of AOP1, AOP2, and AOP3 from the Ler, Cvi, and Col reference ecotypes. Either experimental cDNA sequences or predicted cDNA sequences from genomic sequences were used for the alignments shown. The scale was reduced between 0.02 and 0.16, and midpoint branching was used for this tree. Each sequence is listed with the ecotype from which it was obtained. AOP1 A. lyrata was obtained from A. lyrata.

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    Figure 8.

    Putative Enzymatic Reactions for the 2-ODD Genes in the GS-AOP Region.

    The molecular structures and putative enzymatic reactions catalyzed by the AOP2 and AOP3 enzymes are shown. Side chains of up to eight carbon atoms (n = 6) are known in Arabidopsis. Heterologous expression studies demonstrated the conversion of methylsulfinylalkyl glucosinolates to their alkenyl and hydroxyalkyl derivatives for three-carbon-atom side chains (propyl; n = 1). For glucosinolates with four-carbon-atom side chains (butyl; n = 2), only the methylsulfinylalkyl-to-alkenyl conversion was demonstrated.

Tables

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    Table 1.

    Genetic Association of GS-AOP Phenotype with AOP Expressiona

    AOP2
    EcotypeGS-AOPExpbFxnlcAOP3 ExpAOP1.1 ExpAOP1.2 Exp
    Cal-0ALK++–+–
    Cnt-1ALK++–+–
    KondaraALK++–+–
    CviALK++–+–
    Ema-1ALK++–+–
    HodjaALK++–+–
    Kas-1ALK++–+–
    Mrk-0ALK++–+–
    SorboALK++–+–
    Su-0ALK++–+–
    TacALK++–+–
    Yo-0ALK++–+–
    Per-1Null+––+–
    ColNull+––+–
    Bl-1OHP–+–+
    Ka-0OHP–+–+
    LerOHP–+–+
    Lip-0OHP–+–+
    PetOHP–+–+
    Pi-0OHP–+–+
    Wei-0OHP–+–+
    • ↵a (+), the presence of mRNA expression and/or functional protein; (–), the absence of mRNA expression or nonfunctional protein.

    • ↵b Exp, expression.

    • ↵c The sequence contains no premature stop codons or frameshift mutations.

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    Table 2.

    AOP Primers

    NameRepeatSequenceLerColCvi
    F9H3-FTATTGCCAAAATTTCTGTAGCA126130122
    F9H3-RCTCGACGACTTGTTGTTGGT
    T5J8-F1GAAGCCAAGACGCAGAAGAAGAG125150225
    T5J8-R1TCTCATTATTCCCCACAATGC
    F4C21-FTAGCGCTTCATCTAGTTACGCTTT165171167
    F4C21-RCCCGGACTGAACCAAACTAA
    T5J8-F2TACGATCATCGGTGTTCACCTT160125140
    T5J8-R2GAAAATAAATCGTCATATGGTGTACTG
    AOP1-FATGGATTCAGACTTTGTTCCT
    AOP1-RAAAGGCAGCGAAAGCATGG
    AOP2-FATGGGTTCATGCAGTCTTCA
    AOP2-RTGCTTCGGAGACGGCACAAT
    AOP3-FATGGGTTCATGCAGTCCTC
    AOP3-RTTTCCCAGCAGAGACGCC
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Gene Duplication in the Diversification of Secondary Metabolism: Tandem 2-Oxoglutarate–Dependent Dioxygenases Control Glucosinolate Biosynthesis in Arabidopsis
Daniel J. Kliebenstein, Virginia M. Lambrix, Michael Reichelt, Jonathan Gershenzon, Thomas Mitchell-Olds
The Plant Cell Mar 2001, 13 (3) 681-693; DOI: 10.1105/tpc.13.3.681

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Gene Duplication in the Diversification of Secondary Metabolism: Tandem 2-Oxoglutarate–Dependent Dioxygenases Control Glucosinolate Biosynthesis in Arabidopsis
Daniel J. Kliebenstein, Virginia M. Lambrix, Michael Reichelt, Jonathan Gershenzon, Thomas Mitchell-Olds
The Plant Cell Mar 2001, 13 (3) 681-693; DOI: 10.1105/tpc.13.3.681
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The Plant Cell Online: 13 (3)
The Plant Cell
Vol. 13, Issue 3
Mar 2001
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