Natural Variation in the Glucosinolate Hydrolysis Products Formed in Various Arabidopsis Ecotypes.
All ecotypes produced primarily isothiocyanates or nitriles. Depicted are GC-FID chromatograms representing each of the major glucosinolate types and each of the classes of hydrolysis products.
(A) Col-0: methylsulfinylalkyl glucosinolates, isothiocyanates.
(B) Col × Ler RIL CS 1907: methylsulfinylalkyl glucosinolates, nitriles (similar profiles were seen in Bn-0 and Da-0).
(C) Da(1)-12: hydroxyalkyl glucosinolates, isothiocyanates.
(D) Ler: hydroxyalkyl glucosinolates, nitriles.
(E) Mr-0: alkenyl glucosinolates, isothiocyanates.
(F) Ei-2: alkenyl (allyl) glucosinolate, nitriles.
(G) Tac: alkenyl (3-butenyl and 2-hydroxyl-3-butenyl) glucosinolates, nitriles.
A complete list of surveyed ecotypes is given in Methods. In comparing the nomenclature of glucosinolates and isothiocyanates with their corresponding nitriles, the numbering system must be adjusted because the nitrile carbon atom is counted as part of the alkyl chain. IS, internal standard, n-propylisothiocyanate; 3MSOP-ITC, 3-methylsulfinylpropyl isothiocyanate; 4MSOB-ITC, 4-methylsulfinylbutyl isothiocyanate; 4MSOB-NIT, 4-methylsulfinylbutyl nitrile (hydrolysis product of 3-methylsulfinylpropyl glucosinolate); 5MSOP-NIT, 5-methylsulfinylpentyl nitrile (hydrolysis product of 4-methylsulfinylbutyl glucosinolate); 3OHP-ITC, 3-hydroxypropyl isothiocyanate; 4OHB-NIT, 4-hydroxybutyl nitrile (hydrolysis product of 3-hydroxypropyl glucosinolate); Allyl-ITC, allyl isothiocyanate; 3BUT-NIT, 3-butenyl nitrile (hydrolysis product of allyl glucosinolate); EB-NIT, 3,4-epithiobutyl nitrile (hydrolysis product of allyl glucosinolate); EP-NIT, 4,5-epithiopentyl nitrile (hydrolysis product of 3-butenyl glucosinolate); and HEP-NIT, (3R)-3-hydroxy-4,5-epithiopentyl nitrile (hydrolysis product of (2R)-2-hydroxy-3-butenyl glucosinolate, both diastereomers of this compound are present).

Amino Acid Sequence of Arabidopsis ESP and Some Sequence Relationships.
(A) Amino acid sequence of Arabidopsis ESP. Underlined are regions corresponding to peptides of B. napus ESP protein sequenced by Bernardi et al. (2000).
(B) Dendrogram of Arabidopsis ESP in relation to B. napus ESP and other Arabidopsis sequences.

Reactions Catalyzed by Arabidopsis ESP Expressed in E. coli.
Glucosinolates with terminal alkene functions were converted to epithionitriles, and other glucosinolates were converted to simple nitriles. When comparing the nomenclature of glucosinolates with their corresponding nitriles, the numbering system must be adjusted because the nitrile carbon atom is counted as part of the alkyl chain.

Isothiocyanates and Nitriles as Deterrents of T. ni Herbivory.
In this graph, the percentage of nitrile formed (of total glucosinolate hydrolysis products) in 56 Col × Ler RILs is plotted against the percentage of rosette leaves damaged by T. ni in a 48-hr feeding assay. The results indicate that isothiocyanates are more efficient than are nitriles in deterring T. ni herbivory.

Model for Roles of Myrosinase and Arabidopsis ESP in the Hydrolysis of Glucosinolates to Nitriles.
Myrosinase hydrolyzes the thioglucose linkage, which leads to an unstable aglycone that in the absence of other factors can spontaneously rearrange to form an isothiocyanate. However, in the presence of ESP, the aglycone is converted to a nitrile by cleavage of the C–S bond. When a terminal olefin is present in the glucosinolate side chain, the sulfur atom is recaptured as an episulfide, thus forming a three-membered thiirane ring. n = 1 or 2.

Major Hydrolysis Products of Plant Glucosinolates.
In this investigation, we describe an Arabidopsis protein that promotes the formation of both nitriles and epithionitriles upon plant damage. R, variable side chain; n = 1 or 2.

QTLs Regulating Glucosinolate Hydrolysis and T. ni Herbivory in 96 Col × Ler.
(A) and (B) QTLs are found on chromosomes 1 (A) and 3 (B). The solid lines represent the lod scores for the amount of nitriles formed as a percentage of the total glucosinolate hydrolysis products; the dashed line represents the lod scores for the percentage of rosette leaf area damaged by T. ni. The horizontal line is the 0.05 significance level obtained after 500 permutations of the markers and data.
(C) Mean percentage of nitriles formed in Col × Ler RIL from each of four allelic classes determined by presence of alleles from either Col or Ler at the nga280 locus on chromosome 1 and the AthChib locus on chromosome 3.

Expression of Arabidopsis ESP Is Correlated with the Formation of Nitriles as Major Glucosinolate Hydrolysis Products in the Col × Ler RIL.
Shown are ethidium bromide–stained gels of semi-quantitative RT-PCR from one plant of each of the four allelic classes shown in Figure 3C. Equal amounts of cDNA were used for loading in each lane. The nga280 and AthChib allelic states are shown above the corresponding lane. Arabidopsis ESP expression correlates closely with Ler status at nga280. Relative to the Col allele, the Ler allele at nga280 increases the percentage of nitriles formed by nearly 10-fold. RAN is a loading control. The numbers at the head of each column indicate the Col × Ler RIL used.

Dendrogram of Arabidopsis ESP Genomic Sequences from Seven Ecotypes.
The tree was generated with midpoint rooting, and all branches have >65% bootstrap support. Ecotypes Ru-0, Da(1)-12, Col-0, and Mr-0 hydrolyze glucosinolates to isothiocyanates and thus do not have a functional ESP. Ru-0, Da(1)-12, and Col-0 do not express the ESP gene; Mr-0 does, but it has a deletion in the ORF.

Effects of Individual Glucosinolate Hydrolysis Products on T. ni Herbivory.
T. ni larvae were allowed to feed on 168 F2 families of Da(1)-12 × Ei-2 that were classified according to the predominant glucosinolate breakdown product formed per line. 4OBP-NIT, 4-hydroxylbutyl nitrile; 3OHP-ITC, 3-hydroxypropyl isothiocyanate; EB-NIT, 3,4-epithiobutyl nitrile; Allyl-ITC, allyl isothiocyanate. The error bars indicate ±se.