The Biosynthesis of Glucosinolates, Insights, Inconsistencies, and Unknowns PDF

Annual Plant Reviews (2019) 2, 1–31 http://onlinelibrary.wiley.
com
doi: 10.1002/9781119312994.apr0708
THE BIOSYNTHESIS OF
GLUCOSINOLATES: INSIGHTS,
INCONSISTENCIES, AND
UNKNOWNS
Luke Bell
School of Agriculture Policy & Development, University of Reading, Reading, UK
Abstract: Glucosinolates (GSLs) and their hydrolysis products (GHPs) are of

great interest within the scientific community. Isothiocyanates (ITCs) are one
type of hydrolysis product that have efficacy against some forms of cancer and
prevention of some neurodegenerative disorders. They can also have potent effects
upon pests and diseases of crops and give rise to the characteristic tastes and
flavours of many commonly consumed Brassicales crops. The genetic mechanisms
underlying GSL biosynthesis and regulation of GHPs are well elucidated in
the model organism Arabidopsis thaliana, and research over the last fifteen years
has also greatly expanded knowledge within Brassica spp. There are, of course,
many hundreds of GSL-producing species within the Brassicales order, with
potentially very different and/or advanced mechanisms of GSL biosynthesis,
metabolism, and catabolism. This article summarises the current understanding of
GSL biosynthesis and highlights areas of exploration beyond the ‘Arabidopsis and
Brassica bubble’ of GSL research. Novel GSLs, and the gene encoding proteins that
regulate their biosynthetic pathways, could greatly enhance our knowledge of
Brassicales phytochemistry, evolution, and natural history if given more attention.
Keywords: isothiocyanates, glucosinolate hydrolysis products, myrosinase, Bras-

sicales, Brassicaceae
1 Introduction
Glucosinolates (GSLs) are sulfur-containing compounds produced by

the Brassicales order of plants (Biosynthesis of Cyanogenic Glycosides,
Glucosinolates and Nonprotein Amino Acids; Biosynthesis of Cyanogenic
Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

© 2019 John Wiley & Sons, Ltd. Published 2019 by John Wiley & Sons, Ltd.
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H H2
H2C C C C N
S
Epithionitrile
P
ES
HO R S C N
H2O Glucose TFP
Thiocyanate
O OH R C SH pH7
R S OH R N C S
C Myrosinase − pH4
NOSO3 /NSP Isothiocyanate
HO
NOSO3−
R C N
Glucosinolate Nitrile
H
H2C C C CH2
H
O NH
C
S
Oxazolidine-thione
Figure 1 Glucosinolate (GSL) molecules are sulfur-containing secondary metabolites

produced by Brassicales species, primarily as a means of defence. Myrosinase enzymes in
the presence of water hydrolyse GSLs, to produce glucose and various hydrolysis
products derived from the variable side chain (R). Initial hydrolysis products are unstable
and undergo a Lossen-type rearrangement whereby sulfate is lost. The resulting
hydrolysis product forms an isothiocyanate (ITC) at neutral pH. Nitriles and epithionitriles
can form from specific GSLs via conversion by an enzyme cofactor (epithionitrile specifier
protein, ESP; nitrile specifier protein, NSP). Some GSLs can also form thiocyanates via
thiocyanate forming protein (TFP), or undergo spontaneous rearrangements to form
oxazolidine-thiones or ITC tautomers.
Glycosides, Glucosinolates and Non-Protein Amino Acids). Three distinct

‘types’ are known and grouped according to the structure of variable side
chain moieties: aliphatic, indolic, and aromatic. Within each of these ‘types’
numerous conformations exist, particularly for aliphatic GSLs. To date, over
130 different GSLs have been identified as naturally occurring, with several
others hypothesised and yet to be observed. For an excellent review of GSL
structures in an evolutionary context refer to Agerbirk and Olsen (2012).
GSLs are hydrolysed by myrosinase enzymes to produce a wide variety
of breakdown products, such as isothiocyanates (ITCs), nitriles, epithion-
itriles, thiocyanates, and oxazolidine-thiones (Figure 1). The structures
and diversity of these hydrolysis products are largely dependent upon the
variable side chain of each individual GSL species, and their formation can
be influenced by the presence of specifier proteins, pH, and the presence of
metal ions, to name but a few.
There has been significant interest in the understanding of GSLs and their
genetic control for over 30 years, and for a variety of reasons. These include
optimisation through breeding and agronomy (Grubb and Abel, 2006; Kang

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The Biosynthesis of Glucosinolates: Insights, Inconsistencies, and Unknowns
et al., 2006; Jakopic et al., 2016), to enhance health-related benefits (associated

with hydrolysis products such as ITCs; Haughn et al., 1991; Faulkner et al.,
1998; Gross et al., 2000; Agudo et al., 2008; Verkerk et al., 2009; Mikkelsen
et al., 2010; Guo et al., 2014; Tortorella et al., 2015; Villarreal-Garcia et al., 2016;
Sivapalan et al., 2018; Thomas et al., 2018; New Medical Applications of Plant
Secondary Metabolites (From APR Volume 39)), reduction/removal within
seed meals to reduce adverse health effects in ruminants (Mithen, 2001; Sodhi
et al., 2002; Harper et al., 2012; Michael and VanBuren, 2015; He et al., 2018),
and as a form of pest control through biofumigation (Hossain et al., 2014).
An approach often referred to as metabolic engineering (Kastell et al., 2015;
Kumar et al., 2017) is also an area of intense study with the goal of manip-
ulating plant tissues to produced desired GSL types and quantities. This is
particularly of interest in Brassica oleracea varieties and Brassica napus oilseed
cultivars, as well as in vitro cultures (Variyar et al., 2014; Kastell et al., 2015).
GSLs are synthesised within the vascular tissue of plants and specifically
within the endoplasmic reticulum of cells. These are then typically trans-
ported to localised vacuoles throughout the plant and stored together with
ascorbic acid, which is thought to modulate myrosinase activity (Variyar
et al., 2014).
This article will give a summary of the insights gained into the biosynthesis
of these compounds, but also highlight areas where clarity is needed through
further experimentation. In addition, the unknown genes and mechanisms of
novel GSL synthesis in crops such as radish and rucola will be discussed.
2 Insights into Glucosinolate Biosynthesis and Control
2.1 Gene Identification and Functionality

As with other biosynthetic pathways, genes associated with GSL synthesis
have been identified using combinations of molecular techniques, such as
gene and transcript sequencing, identification of quantitative trait loci (QTL),
and the creation of genetic and physical maps through use of mapping popu-
lations and genetic markers (Mithen and Campos, 1996). The recombination
of genes is then determined, and molecular markers specific to genes and
their function developed, either for analytical or breeding purposes. Such
methods were employed in the initial stages of GSL biosynthesis gene iden-
tifications in Arabidopsis thaliana and have provided a means of targeting spe-
cific traits for further study (Hall et al., 2001; Kliebenstein et al., 2002). Typ-
ically, knockout mutants have been generated to scrutinise and verify the
function and coexpression of GSL biosynthesis genes and perform evalua-
tions of the resulting phenotypes (Grubb et al., 2002).
Arabidopsis contains more than 50 genes related to GSL biosynthesis (The
Arabidopsis Thaliana Genome: Towards a Complete Physical Map), Eruca

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sativa over 60 (Bell et al., unpublished data), and B. oleracea and Brassica
rapa contain over 200 (Liu et al., 2017). The vast majority of these genes are
orthologous to those in Arabidopsis, and it may be that species-specific genes
remain undiscovered (see Table 1 for a list and description of the major
genes involved in GSL biosynthesis). E. sativa and Diplotaxis tenuifolia, for
example, have unique GSL profiles composed of several compounds not
present in other genera of the Brassicales. These include dimeric aliphatic
GSLs (dimeric 4-mercaptobutyl-GSL and diglucothiobeinin), the monomeric
4-mercaptobutyl-GSL (glucosativin), and glucorucolamine – a GSL that has
an amine side chain. Other genera such as Barbarea spp., Cardamine spp., and
Raphanus spp. also contain a large diversity of novel compounds not found
in Arabidopsis spp. or Brassica spp. (Barillari et al., 2005; Olsen et al., 2016).
Clearly, there are novel genes encoding proteins that regulate the synthesis of
these compounds, as no doubt there are for every other Brassicales species.
It is only recently, however, that other crops and wild relatives are being
explored for such novelties.
2.2 Transcriptional Regulation and Biosynthesis

It is now well established that GSL biosynthesis can be affected as a result of
both biotic and abiotic stress and is inherently tied to the defensive strategy of
Brassicaceae plants (Fenwick and Heaney, 1983). At the start of the defensive
pathway that leads to the initiation of GSL biosynthesis, are jasmonate-ZIM
domain proteins (JAZs; Cao et al., 2016), methyl jasmonate (MeJA; Wiesner
et al., 2014), ethylene (ET; Kuhn et al., 2017), and salicylic acid (SA; Kastell
et al., 2015), that act as interactors with numerous downstream transcription
factors (TFs; The Jasmonate Cascade and the Complexity of Induced Defence
Against Herbivore Attack)). Jasmonic acid (JA) also acts synergistically with
glucose to modulate the accumulation of GSLs (Guo et al., 2013; Figure 2).
Jasmonates interact with MYC2, the ‘master regulator’ of MYB TFs (Chini
et al., 2016; Zhang et al., 2018) involved with aliphatic (MYB28, MYB29, and
MYB76) and indolic (MYB34, MYB51, and MYB122) GSL biosynthesis (Bjork-
man et al., 2011). Other TFs are also thought to influence indole GSL accumu-
lation, such as Dof1 and IQD1 (Levy et al., 2005; Variyar et al., 2014). Indole
GSL biosynthesis is also closely related to the synthesis of camalexin and
auxin in Brassicales (Nafisi et al., 2007).
The roles of each set of MYB TFs are partially redundant; however, subtle
differences and contributions to the synthesis of GSLs have been observed.
For example, MYB28 is the general driver of aliphatic GSL synthesis (in Ara-
bidopsis & Brassica ssp.) whereas MYB29 responds more specifically to jas-
monate signals, which further contributes to the level of aliphatic GSLs (Hirai
et al., 2007).
The most basic elements of GSL synthesis are well understood, and it is
generally assumed that the core synthetic pathway is shared between genera

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Table 1 Major glucosinolate biosynthesis genes and transcription factors.
Gene name Synonym(s) UniProt description NCBI Gene ID
Transcription
factors
SLIM1 Sulfur Limitation 1; Probable transcription factor that may be involved in the 843708
Ethylene-Insensitive3-like 3 ethylene response pathwaya
(EIL3)
MYC2 BHLH6; EN38; JAI1; JIN1; RAP1 Transcriptional activator. Common transcription factor of light, 840158
abscisic acid (ABA), and jasmonic acid (JA) signaling
pathways. With MYC3 and MYC4, controls additively subsets
of JA-dependent responses. In cooperation with MYB2 is
involved in the regulation of ABA-inducible genes under
drought stress conditions. Can form complexes with all
5
known glucosinolate-related MYBs to regulate glucosinolate
biosynthesis. Binds to the MYC recognition site
(5′ -CACATG-3′ ), and to the G-box (5′ -CACNTG-3′ ) and
Z-box (5′ -ATACGTGT-3′ ) of promoters. Binds directly to the
promoters of the transcription factors PLETHORA1 (PLT1) and

PLT2 and represses their expression. Negative regulator of
blue light-mediated photomorphogenic growth and blue-
and far-red-light regulated gene expression. Activates
multiple TIFY/JAZ promoters. Positive regulator of lateral root
formation. Regulates sesquiterpene biosynthesis. Subjected to
proteasome-dependent proteolysis. The presence of the
destruction element (DE) involved in turnover is required for

the function to regulate gene transcription.
(continued overleaf )
Table 1 (continued)
IQD1 IQ-Domain 1; F11F8.30 Modulates expression of glucosinolate pathway genes. May 820128
associate with nucleic acids and regulate gene expression at
the transcriptional or post-transcriptional level. Recruits
KLCR1 and calmodulin proteins to microtubules, thus being a
potential scaffold in cellular signaling and trafficking.
MYB28 HAG1; PMG1; MFB13.22 Major regulator of short-chained aliphatic glucosinolates (GLSs) 836263
biosynthesis. Together with MYB29/HAG3 and MYB76/HAG2,
promotes aliphatic glucosinolate biosynthesis but represses
indolic glucosinolate biosynthesis. Prevents insect
performance (e.g. lepidopteran insect Mamestra brassicae and
Spodoptera exigua) by promoting glucosinolates.
MYB29 HAG3; PMG2; MBK20.15 Plays a minor rheostat role in aliphatic glucosinolates (GLSs) 830662
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biosynthesis, mostly short chained. Together with
MYB28/HAG1 and MYB76/HAG2, promotes aliphatic
glucosinolate biosynthesis but represses indolic glucosinolate
biosynthesis. Prevents insect performance (e.g. lepidopteran
insect Mamestra brassicae) by promoting glucosinolates.

MYB76 HAG2; MBK20.16 Plays a role in determining the spatial distribution of aliphatic 830663
glucosinolates (AGLSs) within the leaf, mostly short chained.
Together with MYB28/HAG1 and MYB29/HAG3, promotes
aliphatic glucosinolate biosynthesis and represses indolic
glucosinolate biosynthesis, but could not activate
aliphatic-GSL biosynthesis on its own.
MYB34 ATR1; MSL3.10 Transcription factor involved in tryptophan gene activation and 836210
in indole-3-acetic acid (IAA) and indolic glucosinolates (IG)
biosynthesis. Acts as a direct transcriptional activator of both
Trp synthesis genes and Trp secondary metabolism genes.
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MYB51 HIG1; F25I16.9 Transcription factor positively regulating indolic glucosinolate 838438
biosynthetic pathway genes.
MYB122 F2P9.5 Transcription factor involved in glucosinolates biosynthesis. 843748
Aliphatic
glucosinolate
biosynthesis
BCAT4 Methionine aminotransferase; Converts 2-oxo acids to branched-chain amino acids. Shows 821508
MMB12_16 activity with L-Leu, LIle and L-Val as amino donors and
alpha-keto-glutarate as an amino acceptor, but no activity for
D-isomers of Leu, Ile, Val, Asp, Glu or Ala. Acts on methionine
and its derivatives and the corresponding 2-oxo acids.
Catalyzes the initial deamination of methionine to
4-methylthio-2-oxobutyrate as well as the transamination of
other typical intermediates of the methionine chain
elongation pathway.
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MAM1 Methylthioalkylmalate synthase Determines the side chain length of aliphatic glucosinolate 832365
1; IMS3, IPMS, AT2, MAM-L, structures. Catalyzes exclusively the condensation reactions of
MAML both the first and second methionine carbon chain
elongation.

MAM2 Methylthioalkylmalate synthase Catalyzes only the first methionine chain elongation cycle. –
2
MAM3 Methylthioalkylmalate synthase Determines the side chain length of aliphatic glucosinolate 832366
3; IMS2; IPMS; IT1; MAM-L structures. Accepts all the omega-methylthio-2-oxoalkanoic
acids needed to form the known C3 to C8 glucosinolates.
Also able to convert pyruvate to citramalate, 2-oxoisovalerate
to isopropylmalate, 4-methyl-2-oxopentanoate and
5-methyl-2-oxohexanoate for Leu-derived glucosinolates,

3-methyl-2-oxopentanoate for Ile-derived glucosinolates and
phenylpyruvate to phenylethyl glucosinolate.
Table 1 (continued)
BCAT3 Branched-chain-amino-acid Converts 2-oxo acids to branched-chain amino acids. Acts on 824130
aminotransferase 3; leucine, isoleucine and valine. Also involved in methionine
T16K5.30 chain elongation cycle of aliphatic glucosinolate formation.
Catalyzes the conversion of 5-methylthiopentyl-2-oxo and
6-methylthiohexyl-2-oxo acids to their respective Met
derivatives, homomethionine and dihomo-methionine,
respectively.
CYP79F1 Dihomomethionine Catalyzes the conversion of the short-chain elongated 838211
N-hydroxylase; BUS1; SPS1; methionines di-,tri-, and tetrahomomethionine to their
F3O9.21 respective aldoximes 5-methylthiopentanaldoxime,
6-methylthiohexanaldoxime, and 7-methylheptanaldoxime.
CYP79F2 Hexahomomethionine Catalyzes the conversion of the long-chain elongated 838210
N-hydroxylase; F3O9.20 methionines penta- and hexahomomethionine to their
8
corresponding aldoximes 8-methylthiooctanaldoxime and
9-methylthiononanaldoxime.
CYP83A1 Cytochrome P450 83A1; Involved in the metabolism of aliphatic and aromatic oximes. 827011
CYP83; REF2; F18A5.160 Involved in the biosynthesis of both short-chain and
long-chain aliphatic glucosinolates

GSTF11 Glutathione S-transferase F11; May be involved in the conjugation of reduced glutathione to a 821227
GSTF6; T17B22.12 wide number of exogenous and endogenous hydrophobic
electrophiles and have a detoxification role against certain
herbicides.
GSTU20 Glutathione S-transferase U20; Exhibits glutathione-dependent thiol transferase activities. Can 844173
FIP1;F3F9.11 use glutathione (GSH) and 1-chloro-2,4-dinitrobenzene
(CDNB) as substrates. Involved in the regulation of far-red
light influence on development. Regulator of the interplay
between light and JA signaling by increasing JAR1/FIN219
efficiency. Maybe involved in gravitropic signal transduction
(Probable).
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SUR1 S-alkyl-thiohydroximate lyase; C-S lyase involved in glucosinolate biosynthesis. Converts 816585
ALF1; HLS3; RTY; RTY1 S-(alkylacetohydroximoyl)-L-cysteine to thiohydroximate.
Functions in auxin homeostasis. Probably required for
glucosinolate activation in response to pathogens.
UGT74C1 UDP-glycosyltransferase 74C1; – 817736
F20M17.17
SOT18 Cytosolic sulfotransferase; Sulfotransferase that utilizes 3′ -phospho-5′ -adenylyl sulfate 843749
ST5B, F2P9.4 (PAPS) as sulfonate donor to catalyze the sulfate conjugation
of desulfo-glucosinolates (dsGSs), the final step in the
biosynthesis of the glucosinolate core structure. Preferred
substrates are the long-chain desulfo-glucosinolates,
7-methylthioheptyl and 8-methylthiooctyl, derived from
methionine.
SOT17 Cytosolic sulfotransferase 17; Sulfotransferase that utilizes 3′ -phospho-5′ -adenylyl sulfate 838440
ST5C, F25I16.7; F25I16_11 (PAPS) as sulfonate donor to catalyze the sulfate conjugation
9
biosynthesis of the glucosinolate core structure. Substrate
preference is desulfo-benzyl
glucosinolate > desulfo-6-methylthiohexyl glucosinolate.

Increased specific activity with increasing chain length of
desulfo-glucosinolate derived from methionine.
FMO GS-OX1/ FMO3 Catalyzes the conversion of methylthioalkyl glucosinolates into 842897/842551/
2/3/4/5 methylsulfinylalkyl glucosinolates. Able to S-oxygenate both 842553/842554/
desulfo- and intact 4-methylthiobutyl glucosinolates, but no 837766
activity with methionine, dihomomethionine or
5-methylthiopentaldoxime.
AOP1/2 AOP1.1; T4I9.5 2-oxoglutarate-dependent dioxygenase involved in 828100

glucosinolates biosynthesis. Catalyzes the conversion of
methylsulfinylalkyl glucosinolates to alkenyl glucosinolates.
Table 1 (continued)
Indolic
glucosinolate
biosynthesis
ASA1 Anthranilate synthase alpha; Part of a heterotetrameric complex that catalyzes the two-step 830457
AMT1; JDL1;TRP5; WEI2 biosynthesis of anthranilate, an intermediate in the
biosynthesis of L-tryptophan. In the first step, the
glutamine-binding beta subunit of anthranilate synthase (AS)
provides the glutamine amidotransferase activity which
generates ammonia as a substrate that, along with
chorismate, is used in the second step, catalyzed by the large
alpha subunit of AS to produce anthranilate. Plays an
important regulatory role in auxin production via the
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tryptophan-dependent biosynthetic pathway.
TSB1 Tryptophan synthase beta chain The beta subunit is responsible for the synthesis of L-tryptophan 835571
1; MBG8.7 from indole and L-serine.
CYP79B2 Tryptophan N-monooxygenase Converts tryptophan to indole-3-acetaldoxime, a precursor for 830154
1; T5J17.120 tryptophan-derived glucosinolates and indole-3-acetic acid

(IAA). Involved in the biosynthetic pathway to
4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN), a cyanogenic
metabolite required for inducible pathogen defense
CYP79B3 Tryptophan N-monooxygenase Converts tryptophan to indole-3-acetaldoxime, a precursor for 816765
2; T26C19.1 tryptophan derived glucosinolates and indole-3-acetic acid
(IAA).
CYP71A13 Indoleacetaldoxime Involved in the biosynthesis of the indole-derived phytoalexin 817628
dehydratase; T11J7.16 camalexin. Catalyzes the conversion of indole-3-acetaldoxime
to indole-3-acetonitrile. Required for resistance to A.
brassicicola and B. cinerea.
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CYP83B1 Cytochrome P450 83B1; ATR4; Involved in the metabolism of aromatic oximes. Catalyzes the 829277
RED1; RNT1; RUNT1; SUR2 oxime metabolizing step in indole glucosinolate biosynthesis
by converting indole-3-acetaldoxime into
indole-3-S-alkyl-thiohydroximate. Probably required for
glucosinolate activation in response to pathogens. Functions
in auxin homeostasis because indole-3-acetaldoxime also
serves as a precursor for auxin biosynthesis. Specifically
metabolizes (E)-phydroxyphenylacetaldoxime into an
S-alkylthiohydroximate
GSTF9 Glutathione S-transferase F9; In vitro, possesses glutathione S-transferase activity toward 817636
GLUTTR; GSTF7; F7F1.7 1-chloro-2,4-dinitrobenzene (CDNB) and benzyl
isothiocyanate (BITC), and glutathione peroxidase activity
toward cumene hydroperoxide and linoleic
acid-13-hydroperoxide. May be involved in the conjugation
of reduced glutathione to a wide number of exogenous and
endogenous hydrophobic electrophiles and have a
detoxification role against certain herbicides.
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GSTF10 Glutathione S-transferase 10; In vitro, possesses glutathione S-transferase activity toward 817637
ERD13; GSTF4; F7F1.8 1-chloro-2,4-dinitrobenzene (CDNB) and benzyl
isothiocyanate (BITC). May be involved in the conjugation of
reduced glutathione to a wide number of exogenous and
endogenous hydrophobic electrophiles and have a

detoxification role against certain herbicides.
UGT74B1 UDP-glycosyltransferase 74B1; Involved in the biosynthesis of glucosinolate. In in vitro assay, 839022
F3I6.2 may use phenylacetothiohydroximate (PATH), but not
phenylacetic acid (PAA), indole-3-acetic acid (IAA) or salicylic
acid (SA) as substrate. Specific for the thiohydroximate
functional group and does not glucosylate the carboxylate
group or a hydroxyl group.

SOT16 Cytosolic sulfotransferase 16; Sulfotransferase that utilizes 3′ -phospho-5′ -adenylyl sulfate 843750
CORI-7; ST5A; F2P9.3 (PAPS) as sulfonate donor to catalyze the sulfate conjugation
biosynthesis of the glucosinolate core structure.
Table 1 (continued)
CYP81F2 Cytochrome P450 81F2; IGM1; Involved in indole glucosinolate biosynthesis. Catalyzes 835828
MJB24.3 hydroxylation reactions of the glucosinolate indole ring.
Converts indol-3-yl-methylglucosinolate (I3M) to
4-hydroxy-indol-3-ylmethylglucosinolate (4OH-I3M) and/or
1-hydroxy-indol-3-yl-methylglucosinolate (1OH-I3M)
intermediates. These hydroxy intermediates are converted to
4-methoxyindol-3-yl-methylglucosinolate (4MO-I3M) and
1-methoxy-indol-3-ylmethylglucosinolate (1MO-I3M) by
indole glucosinolate methyltransferase 1 and 2 (IGMT1 and
IGMT2). Contributes to defense against the green peach
aphid (Myzus persicae), a generalist phloemfeeding
herbivore. Required for the biosynthesis of antifungal indole
glucosinolate metabolites. Required for the
pathogen-induced accumulation of 4MOI3M, which in turn
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is activated by the atypical BGLU26/PEN2 myrosinase
(PubMed:19095900). Required for the biosynthesis of
Trp-derived anti-fungal compounds and non-host resistance
to the necrotrophic fungal pathogen Plectosphaerella

cucumerina. Required for resistance to the non-adapted
fungal pathogen Colletotrichum gloeosporioides.
IGMT1/2/3/4/ 5 Indole glucosinolate Involved in indole glucosinolate biosynthesis. Catalyzes 838706/838708/
O-methyltransferase methoxylationreactions of the glucosinolate indole ring. 838707/838709/
Converts the hydroxy intermediates 844013
4-hydroxy-indol-3-yl-methylglucosinolate (4OH-I3M) and
1-hydroxy-indol-3-ylmethylglucosinolate (1OH-I3M) to
4-methoxy-indol-3-yl-methylglucosinolate (4MO-I3M) and
1-methoxy-indol-3-ylmethylglucosinolate (1MO-I3M),
respectively.
a = has a different function in the context of sulfur limitation.
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Sulfate (environmental)
SULTR
Sulfate (cellular)
MTA spermine ATPS
APS
SPMS Primary S- Secondary
metabolism APR APK S-metabolism
dcSAM spermidine Sulfite PAPS

SOT
MTA SiR
Sulfide
SPDS
OAS Sulfated
compounds
(GSLs: see
dcSAM putrescine Methionine Cysteine b)
PAP
Translation
of proteins
SAM SAL1
GCL/GSH
γ -EC AMP
GSHS
ASC H2O2
NADPH GSH
ROS
GR
GPX DHAR
APX2
GSSG
NADP+ GSH:GSSG ratio
MDHAR
MDHA 2H2O
(a) DHA
Figure 2 The sulfur metabolism (a) and glucosinolate (GSL) biosynthesis (b) pathways
of Brassicaceae plants. Source: (a) Adapted with permission from Chan et al. (2013). ©
Springer Nature and (b) Adapted with permission from Gigolashvili et al. (2009) ©
Elsevier.
of the Brassicales (Olsen et al., 2016). GSLs themselves are derived from the
amino acids methionine (aliphatic), tryptophan (indolic), phenylalanine (aro-
matic), and the sugar glucose (Hanschen et al., 2014). Other amino acids such
as valine, leucine, isoleucine can also be utilised in some species (Olsen et al.,
2016). The assembly of the core structure can be divided into three stages: (i)
chain-elongation and addition of methylene groups, (ii) formation of the core
GSL structure, and (iii) secondary modifications of the side chain (hydroxy-
lation, methylation, oxidation, or desaturation in most cases; Mithen, 2001;

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Methyl jasmonate
Wounding
Sulfate deficiency
SLIM1 MYC2 IQD1
Glucose
Salicylic acid
Pathogens
MYB28
MYB51
MYB29
MYB34 MYB122
MYB76
Methionine Chorismate
BCAT4
ASA1
α-keto-acid Indole
MAM1 TSB1
MAM2
MAM3 Tryptophzan
BCAT3
Chain-elongated methionine CYP79B2
CYP79B3
CYP79F1
CYP79F2
CYP71A13
Aldoxime Indole 3-acetonitrile IAA
NIT2
CYP83A1 CYP83B1 NIT3
Nitrile-oxide
GSTF11 GSTF9
GSTU20 GSTF10
S-alkyl-thiohydroximate
SUR1 SUR1 Myrosinase (TGG)
Thiohydroximate
UGT 74C1 UGT 74B1
NSP
Desulfo-glucosinolate
SOT18 SOT16 Indole hydrolysis Nitrile
SOT17 products hydrolysis products
CYP81F2
FMO GS-OX Isothiocyanate
AOP1 Indolic glucosinolates hydrolysis products
AOP2 Aliphatic glucosinolates
GRS1
Figure 2 Continued
Wittstock and Halkier, 2002; Bjorkman et al., 2011; Sun et al., 2011; Hanschen
et al., 2014; Variyar et al., 2014; Tortorella et al., 2015).
Chain elongation occurs by the deamination of the amino acid to a 2-oxo
acid, and methylene group addition. This process of elongation can continue
to extend the carbon chain (and thereby give rise to different GSLs) by

14
up to nine methylene groups (Olsen et al., 2016); or the 2-oxo acid can be
reaminated to the amino acid. The elongated amino acid is then converted
to an aldoxime by cytochrome P450 oxygenases (e.g. CYP79F1, CYP79F2,
aliphatic; CYP79B2, CYP79B3, indolic; Wittstock and Halkier, 2002; Wiesner
et al., 2013; Hanschen et al., 2014; Kastell et al., 2015; Aziz et al., 2016).
The aldoxime is then further modified into a nitrile oxide by CYP83A1/B1
(Smolen and Bender, 2002) and converted to an S-alkyl-thiohydroximate
by glutathione-S-transferases (GSTs). SUR1 then converts this to a thiohy-
droximate and UGT74B1/C1 to a desulfo-GSL to complete the core structure
(Mithen, 2001). Subsequently, SOT enzymes perform sulfation, and the side
chain is modified by additional enzymes according to the precursor amino
acid structure (Kliebenstein et al., 2001; Mithen, 2001).
There are three groups of SOTs (SOT16, SOT17, and SOT18), and there
is a diverse range of copy numbers between species (Kopriva et al., 2016).
The activity of these enzymes is thought to follow a diurnal rhythm, being
up-regulated in light and down-regulated in darkness. Interestingly, SOTs
appear to be regulated by the HY5 TF, not MYBs as with other GSL synthesis
genes. In fact, HY5 is known to repress GSL-related MYBs to varying degrees.
Indolic TFs such as MYB51 and MYB34 are down-regulated during darkness,
thus repressing indolic GSL biosynthesis, whereas aliphatic MYB expression
remains relatively unaffected (Huseby et al., 2013).
2.3 Influences of Abiotic Stress

Abiotic stress (or lack thereof) influences GSL profiles of Brassicales (The
Molecular Networks of Abiotic Stress Signaling). Factors such as light (Qian
et al., 2016; Neugart et al., 2018a), temperature (Schreiner et al., 2002; Kissen
et al., 2016), hypoxia (Guo et al., 2016), and water status (Bjorkman et al.,
2011) have varying effects upon the types and amounts of specific GSLs syn-
thesised. It is also known that there are significant changes to GSL profiles
and concentrations postharvest (Villarreal-Garcia et al., 2016), and this has
significant implications for the potential health benefits of Brassicales crops.
Light, and particularly exposure to UV-B radiation, is thought to play a key
role in the expression of genes related to secondary metabolites, such as the
UVR8 receptor (Hanschen et al., 2014). If sufficient enough in intensity, UV-B
radiation acts as a stressor, inducing changes in growth, development, and
morphology of plants, as well as the population of phytochemicals. The phe-
nomenon has also been observed in terpenoid and flavonoid gene expression
patterns (Flavonoids) and is indicative of a generalised response to this form
of abiotic stress (Variyar et al., 2014).
Ambient growth temperature also plays a role in modulating GSL biosyn-
thesis, and large numbers of related genes are differentially expressed
between 9 and 21 ∘ C in Arabidopsis. Aliphatic GSL biosynthesis gene expres-
sion patterns are particularly elevated at lower temperatures in some

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cultivars, with marked increases observed in MYB28 and MYB29 TFs.

Secondary modification genes such as FMOGS-OX1 and FMOGS-OX4 are also
up-regulated, and AOP2 and MAM3 down-regulated, indicating a shift in
GSL profile composition. Indolic GSL-related genes, however, generally
show the opposite trend; being down-regulated at colder temperatures
and increasing with higher. This includes MYB51, the predominant regu-
lator of indolic GSL biosynthesis, as well as P450 cytochromes CYP79B3
and CYP83B1, that catalyse the initial steps of core structure formation
(Kissen et al., 2016; Figure 2). It is uncertain how such changes in profile
or composition improve cold temperature tolerance, or whether this is
a generalised response to abiotic triggers and/or suboptimal growing
conditions.
2.4 The Relationship Between Indolic Glucosinolates, Auxins,

and Camalexin
The regulation of indolic GSL biosynthesis is separate and distinct from
aliphatic. Indolic GSL biosynthesis has been noted for its association with
that of the auxin, indole-3-acetic acid (IAA) (Bones and Rossiter, 1996; Barlier
et al., 2000; van Dam et al., 2009; Auxin as an Intercellular Signal; Auxin
Metabolism and Signaling). Hydrolysis of glucobrassicin by myrosinase
generates sulfate and indole-3-acetonitrile (IAN), which can be converted
to IAA by nitrilase enzymes (e.g. NIT2 and NIT3; Bones and Rossiter, 1996;
Barlier et al., 2000; Cao et al., 2016). In this fashion, indolic GSLs may act
as precursors to auxins and form an important part of plant-signalling,
development, and sulfur homeostasis in Brassicaceae species (Bennett et al.,
1995; Kim et al., 2004; Jakovljevi et al., 2013).
The pathway is also closely linked with camalexin biosynthesis, another
sulfur-containing defence metabolite. Indole-3-acetaldoxime (IAOx) func-
tions at branch points between indole GSL, auxin, and camalexin synthesis,
and P450 cytochromes CYP83B1 also function as part of the latter compound
pathway (Smolen and Bender, 2002; Hoecker et al., 2004). IAN is also a key
intermediate for camalexin synthesis, and so hydrolysis of indolic GSLs
may also be involved as a donor to this process. The exact interaction and
codependence of these pathways are, however, unclear (Grubb and Abel,
2006; Nafisi et al., 2007).
2.5 Interactions and Relationships with Sulfur Metabolism

GSLs are sulfur-containing compounds and are intrinsically linked with
S-metabolism and regulation (Figure 2; Control of Sulfur Uptake, Assimi-
lation and Metabolism). Experimental evidence consistently shows that S
fertilisation induces the biosynthesis of GSLs (Neugart et al., 2018a) and
that MYB28 is explicitly involved in this process (Traka et al., 2013). S is

16
utilised in both primary and secondary metabolism, and the regulation of its
homeostasis at any given point in a plants’ life cycle will inherently impact
upon the amount devoted to GSL synthesis. Under heavy metal stress,
for example, genes associated with GSL biosynthesis are down-regulated,
and organic S is shunted to support the synthesis of key detoxification
compounds such as glutathione (GSH) and phytochelatins (PCs). Evidence
suggests that the enzymes utilised in each of these respective processes are
under strict genetic control, and there is significant crosstalk between the
respective pathways with some enzymes performing dual roles within each
(Jakovljevi et al., 2013).
In times of S deficiency, genes such as SLIM1 and SDI1 are known to influ-
ence and down-regulate MYB28 and MYB29, and thus reduce the amount
of sulfur dedicated to the synthesis of aliphatic GSLs (Aghajanzadeh et al.,
2014; Henríquez-Valencia et al., 2018). Likewise, MYB34 is also known to be
down-regulated in roots of Arabidopsis, and therefore reduces synthesis of
indolic GSLs in these tissues.
It is thought that GSLs may be remobilised under S limitation rather than
newly synthesised. The simultaneous overexpression of myrosinases may,
in turn, be responsible for the reduction in GSLs as they are hydrolysed
(Maruyama-Nakashita et al., 2003); possibly as a means of freeing up
stored sulfate for further catabolism. Frerigmann and Gigolashvili (2014),
however, speculated that MYB51 and MYB122 are subject to a ‘low indole
GSL signal’ regulatory feedback loop, as their expression has been observed
to increase under S-limitation. The exact molecular mechanisms governing
these processes are largely unknown (Kopriva et al., 2016).
GSLs are thought to be an important form of sulfur storage in Brassicales,
as well as acting as a reservoir for plant defence. They are, however, likely to
come at a high metabolic cost, and as such this might explain why such down-
regulation occurs in times of nutrient deficiency. The corresponding upregu-
lation of myrosinase genes would also suggest that GSLs are catabolised to
free up such reservoirs of sulfur and sustain primary S metabolism (Kopriva
et al., 2016).
The six major GSL MYB TFs are also known to regulate enzymes such as
ATP-sulfurylase (ATP-S). ATP-S catalyses the activation of inorganic SO4 2– to
create adenosine-5′ -phosphosulfate (APS) and reduced S2– (sulfide), which
is incorporated into cysteine. This amino acid then donates or is a precursor
to, numerous S-containing compounds such as methionine and GSH. This
indicates that GSL-related MYBs influence both primary and secondary
S-metabolism genes to varying degrees, influencing the supply of S for the
purposes of GSL synthesis (Kopriva et al., 2016).
ATP-S and adenosine 5′ phosphosulfate kinases (APKs) are locally coex-
pressed with GSL biosynthesis genes within the leaf margins and vasculature;
typically within chloroplasts (ATPS1, -3, -4, APK1, and APK2). APS is phos-
phorylated by APK to form 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS;

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Aziz et al., 2016), a sulfate donor essential for the sulfation of desulfo-GSLs by
SOTs (Henríquez-Valencia et al., 2018). Many other compounds can be syn-
thesised from this pathway, such as brassinosteroids, sulfojasmonates, and
sulfoflavonoids (Kopriva et al., 2015). The close physical proximity of expres-
sion between active genes and biosynthesis is further indication that there is
a close relationship between S-metabolism and secondary metabolite com-
pounds, facilitating rapid synthesis in response to stress (Kopriva et al., 2016;
Wisecaver et al., 2017).
Other phytonutrients such as nitrogen and selenium also have an impact
upon GSL biosynthesis (Neugart et al., 2018b). While the effects of these
will not be discussed in detail here, many other interacting factors between
micronutrients and phytohormones exist and remain to be fully explored
and elucidated (Marino et al., 2016).
2.6 Evolutionary Function(s) of the Glucosinolate-myrosinase

System
For over 40 years, it has been generally agreed within the scientific literature
that the GSL-myrosinase system is primarily a means of defence against
pests and disease (Modes of Action of Defensive Secondary Metabolites;
Introduction: Biochemistry, Physiology and Ecological Functions of Sec-
ondary Metabolites) but may also function as a means of regulating growth
(Francisco et al., 2016) and influence the onset of flowering (Jensen et al.,
2015). Indole GSLs are essential for the formation of callose deposits in
response to microbial infection (Clay et al., 2009), and synthesis is increased
upon wounding (Verkerk et al., 2001). Production of indole GSL hydrolysis
products can also deter ovipositing by Pieris rapae (de Vos et al., 2008). Other
pests, such as turnip root fly (Delia floralis), are also known to cause increases
in foliar aliphatic GSL concentrations (Birch et al., 1992), and GSL content
is known to affect the fitness of green peach aphid (Myzus persicae; Madsen
et al., 2015).
It is thought that gene mutations related to cyanogenic glucosides occurred
approximately 85–90 million years ago for indolic GSLs, and between 60 and
65 million years ago for aliphatic GSLs (Edger et al., 2015). Cyanogenic glu-
cosides are widely utilised throughout the plant kingdom, and their modi-
fication has led to the diversity of GSL compounds seen in modern species
(Mithen, 2001; Stauber et al., 2012). The CYP79 family of genes, for example,
is present throughout the plant kingdom, but in distantly related species such
as poplar (Populus trichocarpa), these are primarily involved in the synthesis
of volatile aldoximes. The CYP79 genes present within the Brassicales have
evolved to utilise such aldoximes for the subsequent synthesis of GSLs. Sim-
ilar modifications have occurred in other plant families, such as the synthesis
of furanocoumarins in the Apiaceae (Edger et al., 2015).

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3 Inconsistencies and Unknowns
3.1 Biotic Stress and Symbioses – Two Sides of the Same Coin?
The assumption that high GSL-containing plants are more resistant to her-
bivory or certain diseases does not always withstand scrutiny, though the
weight of evidence falls strongly on the side of GSLs and GHPs acting broadly
as deterrents. Studies in Arabidopsis have found pest-specific GSL biosynthe-
sis (Rohr et al., 2009), whereas others have shown conflicting results and are
suggestive of adaptation on the part of some insects and pathogens to these
chemical defences.
Insect feeding studies in Arabidopsis have found genetic loci for resistance
between ecotypes that were not explained by differences in GSL concen-
trations or composition (Jander et al., 2001). This suggests, firstly, that the
GSL-myrosinase system is not effective at uniformly dissuading all pest
species; and secondly, that the GSL-myrosinase system is only one of several
that plants can employ to deter herbivory. This is also evidenced in experi-
ments conducted on P. rapae, M. persicae, Brevicoryne brassicae, and Spodoptera
exigua, where P. rapae was the only pest not to elicit an increase in aliphatic
GSL content (Mewis et al., 2006), suggesting pest specific responses by
plants. That being said, experiments with transgenic Arabidopsis plants have
shown that P. rapae is able to effectively detoxify glucosinalbin hydrolysis
products (Muller et al., 2003; Agerbirk et al., 2007) and so may have evolved
mechanisms to circumvent such recognition by the plant.
Experiments using Arabidopsis myc2 mutants have shown a clear suscep-
tibility to herbivory by Spodoptera littoralis, as leaves are almost completely
devoid of GSLs. Similar observations were seen by Beekweelder et al. (2008)
where Mamestra brassicae larval weights were 2.6-fold higher on plants
devoid of aliphatic GSLs (caused by a double knockout mutation in myb28
and myb29). GSL related genes are significantly up-regulated after herbivore
attack (Hopkins et al., 2009), though many studies focus solely on these
without observing secondary metabolite changes more broadly. This often
leads to the conclusion that GSLs are specifically up-regulated, though this
may not be the case. The presence or absence of specific GSL biosynthesis
genes such as MAM2 can confer specialised resistance to some herbivores.
This is the case in Arabidopsis Ler ecotypes, but not Col, as AtMAM2 is only
found in the former, and confers increased resistance to S. exigua (Koornneef
et al., 2004).
There is also mounting evidence that bacterial and fungal species play a
role in eliciting and promoting GSL biosynthesis. The rhizobacterium Bacillus
amyloliquefaciens, for example, has been shown to elicit GSL biosynthesis
by transcriptionally activating sulfur assimilation genes in Arabidopsis,
which leads to increased sulfur uptake and accumulation (Aziz et al., 2016).
Clearly, there are potentially many such relationships such as this that have

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developed over the course of evolutionary time and are only just being
elucidated.
These experiments suggest that the ‘evolutionary arms race’ is ongoing
and that GSL profiles are likely under high selection pressure from herbivory
and fungal/bacterial symbioses. Events such as gene and genome duplica-
tions have historically induced exchanges in competitive advantage between
insects/diseases and plants (Edger et al., 2015), and it would be extremely
insightful to understand how such interactions are driving the modern evo-
lution of wild Brassicales species.
Numerous other classes of defence compound are produced by even
relatively simple species like Arabidopsis. Flavonoids and terpenoids are
also known for eliciting similar effects against pests (Phukan et al., 2016). It
is sometimes tempting for researchers to focus solely on a single aspect of
plant defence (such as GSLs), but it is highly likely that no single pathway or
compound abundance will be responsible for determining a plant’s overall
fitness (Rasmann et al., 2015). There is undoubtedly a synergy between
defensive responses, yet the understanding of these relationships remains
minimal, partly due to a somewhat siloed approach to scientific research.
3.2 Species-Specific Glucosinolates – Missing Pieces of the Puzzle?

As highlighted previously (Section 2.2), the core GSL biosynthesis pathway
is well established in Arabidopsis, and the genes giving rise to some specific
GSLs have been elucidated (Kliebenstein et al., 2001). It would be erroneous
to say, however, that the pathway is well elucidated and understood in other
more complex crops and wild species (save perhaps B. oleracea). Almost all
of what is known about GSL biosynthesis in these ‘higher’ plants stems from
homology with Arabidopsis, but it contains only a small percentage of the pos-
sible GSLs discovered to date (Bjorkman et al., 2011). This, therefore, means
that genes responsible for the biosynthesis of glucorucolamine, glucolimnan-
thin, or other more ‘exotic’ GSLs, are completely absent from the literature,
and any development in our understanding of these compounds and their
specific functions must come from novel gene discovery in other species.
Relatively little work is presently being conducted outside of the Arabidop-
sis and Brassica ‘bubble’. Research conducted comparing the genomes of Ara-
bidopsis with B. rapa found 102 orthologous genes in the latter species related
to the GSL biosynthesis pathway (Wang et al., 2011; Comparative Genomics).
This approach is clearly powerful for identifying candidate genes of interest
between related species but does little to identify new genes with different
functions. Whilst the majority of ‘core’ GSL biosynthesis genes are highly con-
served (there is 84.3% sequence homology between Arabidopsis and B. rapa,
for example) there are still substantial differences between species sequences
for chain elongation and secondary modification genes (79.9%; Wang et al.,
2011).

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One of the only recent examples of novel GSL gene discovery outside of
Arabidopsis and Brassica was the identification of glucoraphasatin synthase 1
(GRS1) in radish (Raphanus sativus) by Kakizaki et al. (2017). In their paper, a
series of experiments using mutant radish plants and transgenic Arabidopsis
showed that GRS1 modifies glucoerucin to form glucoraphasatin; a GSL
unique to the genus Raphanus. GRS1 is a 2-oxoglutarate-dependent dioxy-
genase and is responsible for the desaturation of the glucoerucin side chain.
The authors also hypothesised that the proteins encoded by FMOGS-OX genes
then further modify glucoraphasatin to produce glucoraphenin (another
unique GSL present in Raphanus spp.) in the same fashion that glucoraphanin
is produced from glucoerucin. Indeed, relatively low concentrations of both
these latter GSLs can be found in radish cultivars. Kakizaki et al. (2017)
hypothesised that the function of GRS1 evolved from similar genes within
a Brassica-Raphanus ancestor between 15.6 and 28.3 million years ago, but
that the novel function for producing glucoraphasatin was generated only
in the Raphanus lineage after divergence with Brassica spp. It is likely that
other unique enzymes evolved in other Brassicaceae genera such as Eruca
and Diplotaxis, and this, in turn, has given rise to their distinct and complex
GSL profiles.
Another area for exploration is the presence of multiple GSL biosyn-
thesis gene copies in ‘higher’ Brassicales and their relative expression
patterns and functions. This is not routinely accounted for in experimental
literature (particularly qRT-PCR experiments) but may explain some of
the subtle differences in GSL profiles observed within species (such as
aliphatic-indole-aromatic ratios). Some gene copies within the pathway
may be redundant in function, however, this has yet to be widely tested.
Work conducted on the four copies of BjMYB28 in B. juncea, for example,
has shown differential expression in a tissue-specific manner, and control
aliphatic GSL biosynthesis across the life cycle of the plant (Augustine
et al., 2013). Conversely, some GSL-related genes may be independently
expressed and serve novel purposes and specificities that have yet to be
fully recognised or understood. The same could be said of myrosinases, as
there are often numerous copies of TGG1 and TGG2 present with distinct
expression patterns (Pan et al., 2014) that may serve specific functions in
different tissues or ontogenic stages. The evolutionary cost/benefit of having
different and multiple copies of GSL-myrosinase related genes are not well
known at the present time.
3.3 Glucosinolate and Hydrolysis Product Diversity – Evolutionary

Specificity?
Not only is there a large diversity of GSL compounds and profiles present
within the order Brassicales, but there is also an even greater diversity of GSL

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hydrolysis products (GHPs; Wittstock, 2011; Wittstock et al., 2016). GHPs are
poorly characterised and quantified within the literature, owing to a number
of factors associated with their chemical structure. Many are of low molecular
weight, are extremely volatile, and are highly unstable or reactive, meaning
they may only exist for a few minutes or hours in some cases. These charac-
teristics make them difficult to isolate in a pure form as they degrade quickly,
and often occur in only very small quantities.
GHPs can be divided into six main classes and are formed as a result
of a combination of myrosinase hydrolysis, coenzyme modification, and
environmental conditions: ITCs, thiocyanates (via thiocyanate specifier
protein; TSP), nitriles (via nitrile-specifier protein; NSP), epithionitriles (via
epithionitrile specifier protein; ESP), indoles, and ascorbigens (Kuchernig
et al., 2012). Other products are produced when the ITCs are unstable
and are formed via spontaneous rearrangement to lower energy states or
tautomers. Examples include oxazolidine-2-thiones (formed from GSLs such
as progoitrin and glucobarbarin), oxazolidine-2-one (glucobarbarin, but pos-
sibly enzyme controlled; Agerbirk et al., 2018), and 1,3-thiazepane-2-thione
(glucosativin; Fechner et al., 2018).
With such possible diversity present within the chemistry of these com-
pounds, there is likely an even greater amount of genetic diversity present
that regulates and/or directs the formation of specific GHPs in different
circumstances. This is most evident between varieties of B. oleracea. Cabbage,
kale, broccoli, cauliflower, and Brussels sprout cultivars are all members
of this species, however, GSL profiles can be markedly different; and even
within varieties (Rosa and Rodrigues, 2001), cultivars display differing
propensities for ITC formation over nitriles, for example. These varieties
have been formed as a result of artificial selection for structural and flavour
characteristics over centuries, yet there is presently no satisfactory genetic
or transcriptional explanation as to why broccoli contains predominantly
glucoraphanin and why cabbage does not; or why nitrile formation is greater
in some cultivars of broccoli than others. This is also reflected in the GSL
compositions of minor species such as Diplotaxis spp. and Eruca sativa.
These crops have a large phytochemical diversity, and some cultivars of
which accumulate greater concentrations of aromatic GSLs but others do not
(D’Antuono et al., 2008).
There is also very little knowledge about the specific functions of individ-
ual GSLs and their respective GHPs. The diversity of these compounds would
suggest a high degree of selection pressure in different ecological niches; how-
ever, it is relatively unexplored territory from a research perspective. Some
compounds are quite clearly deterrents, producing pungent and sometimes
foul aromas that can also act as irritants to certain species. Others, however,
apparently have little or no perceptible taste, flavour or aroma, and so their
mechanism of action may be entirely different. Whether specific GSLs act in

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specific circumstances, or whether there is synergy between all GSLs in a

plant’s profile is similarly unknown. Some species contain only two to three
GSL compounds, whereas others may contain dozens. Some contain predom-
inantly aliphatic GSLs, others aromatic. Some produce predominantly nitriles
or epithionitriles, rather than ITCs (Eckardt, 2001).
This begs the question: why have plants within the order Brassicales diver-
sified in this way? All suffer from disease, herbivory and viral infections, and
so it does not seem apparent that one strategy for GSL biosynthesis or one
GSL profile is ‘better’ than another from this perspective. It is likely that we
are still unaware of significant influencing factors surrounding the evolution
and coevolution of GSLs, as well as their diversity and function in planta (Ras-
mann et al., 2015).
As has been done with disease resistance traits (Koornneef et al., 2004),
utilisation of naturally occurring genetic variation present in germplasm
collections would be an ideal resource for elucidating genomic differences
between varieties of B. oleracea, or for any given species with sufficiently large
geographical diversity (Alonso-Blanco and Koornneef, 2000). At present,
our level of understanding of these differences is only just extending to
the polymorphism level of individual genes. The next challenge will be to
understand how polymorphisms interact across multiple genes to determine
the final GSL and GHP profile of a plant.
3.4 Glucosinolate Turnover and Ontogenic Changes

It is well known that GSL profiles are dynamic and change according to plant
ontogenic stages (e.g. during germination, vegetative growth, flowering,
seed set, and senescence; Magrath and Mithen, 1993; Rangkadilok et al.,
2002; Bonte et al., 2017) as well as tissue type (van Dam et al., 2009), but
the signalling mechanisms and transcriptomic changes that regulate this
process have not been well studied outside of Arabidopsis (Brown et al., 2003).
Myrosinases and nitrilases (NIT1, NIT2, NIT3) are known to have a role in
GSL turnover (Agerbirk et al., 2008), but again, it is unclear how the activity
of these enzymes is regulated in order to modulate GSL concentration in
planta under different environmental conditions and ontogenic stages.
There is evidence within the literature of disparity between GSL biosynthe-
sis gene activity and observed GSL concentrations. For example, the addition
of nitrogen (ammonium) to B. rapa ssp. chinensis typically reduces transcript
levels for indolic GSL biosynthesis genes, however, concentrations of indolic
GSLs within the same tissue were observed to increase (Neugart et al., 2018b).
Conversely, in broccoli, it has been observed that gene expression levels are
high after germination and GSL concentrations decrease (Gao et al., 2014).
The reasoning for this in the literature is that there is a rapid degradation
of GSLs during sprout development as a means of defence; however, this

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hypothesis has not been tested explicitly. Clearly, there are still mechanisms
relating to GSL synthesis, storage, breakdown, and turnover of which we
are not fully aware, and require further study in the context of abiotic stress
response.
3.5 Aromatic Glucosinolate Biosynthesis – The Neglected

Pathway?
Indole and aliphatic GSLs and their biosynthesis have featured most promi-
nently in the literature to date, however, comparatively little is known specif-
ically about the aromatic synthesis pathway (Kang et al., 2006). It is generally
assumed that the processes involved in aromatic GSL core structure forma-
tion and elongation are similar to that of indole compounds (Kliebenstein
et al., 2001); however, it is uncertain if there are specific genes involved in
differentiating this process. It is generally agreed that aromatic GSLs such as
glucosinalbin are synthesised from tryptophan, and others such as gluconas-
turtiin from phenylalanine (Agerbirk et al., 2008).
Specific MYB TFs have been identified in regulating aliphatic and indolic
GSL biosynthesis, but any that may govern aromatic compounds in this way
have so far remained elusive. Some authors have speculated that MYB28
may function as the controlling TF in species where aromatic compounds
are present in high concentrations (Zhang et al., 2016). There is, however, no
direct evidence for this, as the function of these TFs and other genes has not
been established in the same way as for Arabidopsis.
4 Summary
While the core GSL synthesis pathway is now relatively well elucidated, there
is still much to be learned regarding regulation and control. The specific func-
tions of different GSLs in different crops are unclear, as are the evolutionary
pressures that have led to the selection of such a diverse array of side chain
structures and hydrolysis products. Artificial selection of B. oleracea varieties
has demonstrated that GSL profiles are malleable, and can theoretically be
targeted for improvement of quality traits such as health-related benefits,
taste and flavour, as well as improved disease resistance. While Arabidopsis
serves well for testing the underlying mechanisms of GSL biosynthesis on
a relatively basic level, it is likely that novel genes have evolved in ‘higher’
Brassicales that may not necessarily have analogous functions with the model
organism. There is a need to explore more deeply outside of the model organ-
ism and crop ‘bubble’ to identify novel genes and utilise such knowledge for
crop improvement.

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Annual Plant Reviews (2019) 2, 1–31 http://onlinelibrary.wiley.

Abstract: Glucosinolates (GSLs) and their hydrolysis products (GHPs) are of

Keywords: isothiocyanates, glucosinolate hydrolysis products, myrosinase, Bras-

Glucosinolates (GSLs) are sulfur-containing compounds produced by

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Figure 1 Glucosinolate (GSL) molecules are sulfur-containing secondary metabolites

Glycosides, Glucosinolates and Non-Protein Amino Acids). Three distinct

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

et al., 2006; Jakopic et al., 2016), to enhance health-related benefits (associated

2 Insights into Glucosinolate Biosynthesis and Control

2.1 Gene Identiﬁcation and Functionality

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

2.2 Transcriptional Regulation and Biosynthesis

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Gene name Synonym(s) UniProt description NCBI Gene ID

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

destruction element (DE) involved in turnover is required for

Gene name Synonym(s) UniProt description NCBI Gene ID

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

5-methyl-2-oxohexanoate for Leu-derived glucosinolates,

Gene name Synonym(s) UniProt description NCBI Gene ID

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

AOP1/2 AOP1.1; T4I9.5 2-oxoglutarate-dependent dioxygenase involved in 828100

Gene name Synonym(s) UniProt description NCBI Gene ID

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

group or a hydroxyl group.

Gene name Synonym(s) UniProt description NCBI Gene ID

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

MTA spermine ATPS

dcSAM spermidine Sulfite PAPS

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

SLIM1 MYC2 IQD1

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

2.3 Inﬂuences of Abiotic Stress

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

cultivars, with marked increases observed in MYB28 and MYB29 TFs.

2.4 The Relationship Between Indolic Glucosinolates, Auxins,

2.5 Interactions and Relationships with Sulfur Metabolism

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

2.6 Evolutionary Function(s) of the Glucosinolate-myrosinase

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

3 Inconsistencies and Unknowns

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

3.2 Species-Speciﬁc Glucosinolates – Missing Pieces of the Puzzle?

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

3.3 Glucosinolate and Hydrolysis Product Diversity – Evolutionary

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

specific circumstances, or whether there is synergy between all GSLs in a

3.4 Glucosinolate Turnover and Ontogenic Changes

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

3.5 Aromatic Glucosinolate Biosynthesis – The Neglected

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.

Annual Plant Reviews Online, Volume 2. Edited by Jeremy Roberts.