Glucosinolates (GLS) are a group of secondary metabolites found almost exclusively in Brassicaceae (Agerbirk and Olsen, 2012). GLS are nitrogen and sulfur rich compounds, forming a two-component defense system (mustard oil bomb) with myrosinases against herbivores and microorganisms (Bones and Rossiter, 1996; Rask et al., 2000). Upon tissue damage by herbivores, the mustard oil bomb comes into action, where myrosinases hydrolyze GLS into different degradation products that are toxic to the enemy (Rask et al., 2000; Chen and Andreasson, 2001). Apart from plant defense, some of the GLS degradation products are physiologically significant in plant nutrition and growth regulation (Hull et al., 2000; Rask et al., 2000; Kutz et al., 2002; Grubb et al., 2004; Katz et al., 2015). GLS breakdown products are part of human consumption and health. For instance, some of the GLS metabolites give characteristic flavors to Brassica vegetables (cabbage, cauliflower, broccoli, etc.) and condiments (mustard, horseradish, wasabi, etc.); and some others such as sulforaphane, indole-3-carbinol and phenethyl isothiocyanate act as cancer-preventive agents (Zhang et al., 1994; Hecht, 2000; Nakajima et al., 2001; Keck and Finley, 2004; Hayes et al., 2008). Moreover, Brassica crops are used for crop rotation and/or biofumigation as certain GLS metabolites exhibit natural biopesticide properties (Gimsing and Kirkegaard, 2009).

Glucosinolates are evolutionarily younger and evolved from cyanogenic glucosides. Cyanogenic glucosides are widespread in planta, whereas GLS are restricted to the order Capparales and the genus Drypetes of Euphorbiaceae (Johnson et al., 2009). Cyanogenic glucosides and GLS do not coexist in plants, except for one species, Carica papaya that produces both phenylalanine-derived cyanogenic glucosides and GLS (Bennett et al., 1997). Evolutionary link between cyanogenic glucosides and GLS is supported by having similarities in their biosynthesis such as amino acids as precursors and CYTOCHROME P450s (CYPs) as aldoxime metabolizing enzymes (Bak et al., 1998, 2001; Hansen et al., 2001a; Naur et al., 2003).

Glucosinolates (GLS) are characterized by having a thioglucose moiety, a sulfonated oxime, and a side-chain derived from aliphatic, aromatic, or indole amino acids (Figure 1). Currently, more than 130 different GLS structures have been identified (Agerbirk and Olsen, 2015). GLS are biosynthesized from amino acids and stored in the vacuoles of specific laticifer-like sulfur-rich cells called S-cells localized in the phloem cap along the vasculature and the leaf margins (Koroleva et al., 2010). GLS hydrolyzing myrosinases are localized in myrosin cells and are spatially separated from GLS (Thangstad et al., 1991; Xue et al., 1993; Andreasson et al., 2001; Husebye et al., 2002). Biosynthesis and long-distance transport of GLS are critical for spatio-temporal distribution of the GLS in plants (Nour-Eldin et al., 2012; Andersen and Halkier, 2014; Jørgensen et al., 2015). In Arabidopsis, transport of GLS compounds is mediated by transporter proteins GTR1/NPF2.10 and GTR2/NPF2.11 (Nour-Eldin et al., 2012).

Several lines of evidence suggest that indole GLS are metabolically linked to auxin homeostasis. Interruption of GLS metabolism leads to severe defects in plant growth and development similar to high-auxin phenotypes (Boerjan et al., 1995; Mikkelsen et al., 2000, 2004; Bak and Feyereisen, 2001; Hansen et al., 2001b; Reintanz et al., 2001; Chen et al., 2003; Tantikanjana et al., 2004; Skirycz et al., 2006; Ueda et al., 2006). The high-auxin phenotypes of GLS mutants were an effect of blocking the indole GLS pathway downstream of indole-3-acetaldoxime (IAOx), which resulted in overflow of IAOx to indole-3-acetic acid (IAA) (Halkier and Gershenzon, 2006; Nafisi et al., 2006). In this review, we discuss current knowledge on potential involvement of GLS especially indole GLS metabolism in auxin homeostasis.

The typical chemical structure of GLS consists of β-D-glucopyranose residue linked via a sulfur atom to a (Z)-N-hydroximinosulfate ester plus a precursor amino acid derived R group (Figure 1). Based on the precursor amino acid and the types of modification to the variable R group, GLS can be classified into aromatic (phenylalanine or tyrosine), aliphatic (alanine, leucine, isoleucine, methionine, or valine), and indole GLS [tryptophan (TRP)] (Fahey et al., 2001; Agerbirk and Olsen, 2012). A list and structures of GLS can be found in an excellent review (Clarke, 2010).

Glucosinolates biosynthetic pathway comprises three steps (Figure 2): amino acid chain-elongation, core structure formation, and secondary modifications (Chen and Andreasson, 2001; Halkier and Gershenzon, 2006). In the amino acid chain-elongation step, certain aliphatic and aromatic amino acids are elongated by insertion of methylene groups into their side chains. These reactions are mediated by the METHYLTHIOALKYLMALATE SYNTHASE (MAM) 1-3 and MAM-like (MAML) genes (Kroymann et al., 2001; Textor et al., 2007). The amino acid moiety of either chain elongated or not, is converted to a core GLS structure in a series of reactions. During this process, amino acids are converted to their corresponding aldoximes by CYP79s (Figure 2). CYP79A2 catalyzes phenylalanine (Wittstock and Halkier, 2000), CYP79F1 and CYP79F2 metabolize chain-elongated methionine (Hansen et al., 2001b; Chen et al., 2003), whereas CYP79B2/B3 convert TRP (Hull et al., 2000) to their aldoximes. The enzyme catalyzing homophenylalanine is unknown. Aldoximes are metabolized to S-alkylthiohydroximates by CYP83s. Methionine-derived aldoximes are catalyzed by CYP83A1, whereas aromatic- and indole-acetaldoximes are catalyzed by CYP83B1/SUPERROOT2 (SUR2) (Bak and Feyereisen, 2001; Hemm et al., 2003). S-alkylthiohydroximates are cleaved into thiohydroximates by a C-S lyase/SUPERROOT1 (SUR1) (Mikkelsen et al., 2004), followed by S-GLUCOSYLTRANSFERASE (UGT) mediated glycosylation (Petersen et al., 2001). Finally, sulfonation of the desulfo-GLS is carried out by sulfotransferases (Piotrowski et al., 2004). Later, GLS core structure undergoes several secondary modifications at the side chain and glucose moiety (Hopkins et al., 2009). Side chain of aliphatic GLS is modified by oxygenation, hydroxylation, alkenylation, and benzoylation, whereas side chain of indole GLS is modified by hydroxylation and methoxylation (Sønderby et al., 2010b).

Camalexin is a major phytoalexin found in specific group of Cruciferous plants including Arabidopsis (Glawischnig, 2007; Rauhut and Glawischnig, 2009; Bednarek et al., 2011). Camalexins are synthesized in response to fungal pathogens and play positive role in their resistance (Pedras et al., 2011). In camalexin biosynthetic pathway, CYP71A13/12 convert IAOx into indole-3-acetonitrile (IAN) (Nafisi et al., 2007; Müller et al., 2015) which is then oxidized and conjugated to glutathione by glutathione-S-transferase GSTF6 to produce GSH (IAN) (Su et al., 2011). This GSH (IAN) is metabolized to Cys(IAN) by γ-glutamyl peptidases GGP1 and GGP3 (Geu-Flores et al., 2011) which is metabolized to camalexin by CYP71B15/PAD3 (Zhou et al., 1999; Schuhegger et al., 2006; Böttcher et al., 2009).

IAA is proposed to be biosynthesized from two pathways, TRP-independent and TRP-dependent pathway (Woodward and Bartel, 2005; Chandler, 2009; Normanly, 2010). TRP-dependent IAA biosynthesis is considered as the main route of IAA biosynthesis in plants (Figure 3).

Some of the GLS mutants were isolated from the genetic screens aimed to identify genes involved in auxin homeostasis in Arabidopsis. For instance, sur1 was isolated in a screen designed to identify mutants with high-auxin phenotypes including small and epinastic cotyledons, an elongated hypocotyl, excess adventitious and lateral roots, and a reduced number of leaves (Boerjan et al., 1995). The different alleles of sur1, alf1, rty, and hsl3, which encodes a C-S lyase, identified in independent root morphology screens, also showed high-auxin related abnormal root morphology (Celenza et al., 1995; King et al., 1995; Lehman et al., 1996). Later, sur2 and rnt1, loss-of-function mutants of CYP83B1, were found to display the phenotypes similar to sur1 (Delarue et al., 1998; Winkler et al., 1998; Barlier et al., 2000; Bak et al., 2001). UGT74B1 encodes a UDP-glucose:thiohydroximate S-glycosyltransferase. Insertional mutations in UGT74B1 resulted in phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light grown plants (Grubb et al., 2004).

The GLS mutants with high-auxin phenotypes were found to have altered levels of IAA along with impaired GLS content. In sur1/rty, free and conjugated IAA levels were over accumulated (Boerjan et al., 1995; King et al., 1995) with undetectable levels of all types of GLS (Mikkelsen et al., 2004). Similarly, the mutants of UGT74B1 having excess free and conjugated IAA were associated with reduction in all types of GLS compared to their control plants (Grubb et al., 2004). Indole GLS production was reduced to 50% in sur2 plants (Bak et al., 2001) compared to wild-type, whereas free IAA levels were increased at all developmental stages tested (Delarue et al., 1998). cyp79f1/bushy1/sps mutants showed extremely bushy phenotype (Reintanz et al., 2001; Tantikanjana et al., 2001). cyp79f1 mutant showed decreased levels of short-chain derived GLS but increased levels of indole-3-ylmethyl-GLS, IAA (Reintanz et al., 2001), and cytokinin (Tantikanjana et al., 2001). Because the cytokinin responsive reporter ARR5::uidA and auxin responsive reporter DR5::uidA in the cyp79f1 mutant showed that increased levels of cytokinin, but not auxin, correlate well with a root-specific expression pattern, the bushy phenotype might be caused by increased level of cytokinin (Tantikanjana et al., 2004). Both auxin and cytokinin can influence the hormone levels of each other. Increased cytokinin levels in cyp79f1 might induce accumulation of auxin. Alternatively, increased indole GLS production in cyp79f1 likely increased IAA biosynthesis (Mikkelsen et al., 2003). CYP79B2/B3 were up-regulated in stressed plants, resulting in increased indole GLS and IAA (Mikkelsen et al., 2003). Therefore, it is also possible that sps/cyp79f1 mutants were stressed because of the perturbation of cytokinin homeostasis, which in turn up-regulates CYP79B2/B3 genes (Tantikanjana et al., 2004).

The altered IAA levels found in the GLS mutants were proposed to be synthesized from IAOx (Hull et al., 2000), a common precursor for indole GLS, camalexin, and IAA (Figure 3). The CYP71 clade genes SUR2/CYP83B1 and CYP71A13/12 channel IAOx into biosynthesis of indole GLS and camalexin, respectively. SUR2 catalyzes IAOx into an indole-3-S-alkyl-thiohydroximate and is subsequently metabolized to indole GLS (Bak et al., 2001).

Indole-3-acetaldoxime channeling into production of either IAA, or secondary metabolites indole GLS or camalexin must be tightly controlled. In CYP79B2 overexpressing plants, elevated IAOx has been found to be channeled into biosynthesis of indole GLS (Mikkelsen et al., 2000) and IAA (Zhao et al., 2002). In response to the increased production of IAA in CYP79B2 overexpressors, transcripts of IAA-inducible genes including IAA/AUX, SAUR, and GH3s were induced (Zhao et al., 2002). Consistently, disruption of CYP79B2/B3 abolished production of indole GLS, and affected rate of IAA biosynthesis in the mutants (Zhao et al., 2002). CYP79B2 was shown to highly express in response to silver nitrate treatment that induces camalexin synthesis. Consistently, cyp79B2 single and cyp79B2/B3 double mutants were unable to synthesize camalexin under induced conditions (Glawischnig et al., 2004; Ljung et al., 2005). In the absence of pathogen attack, IAN levels were not altered in cyp71A13 knockout mutants (Sugawara et al., 2009), indicating fine-tuned regulation of IAOx metabolic channeling into the corresponding pathways. Loss-of-function mutations of SUR2 restricted IAOx flux into biosynthesis of indole GLS, resulting in decreased indole GLS production (Bak et al., 2001) and increased free IAA levels (Delarue et al., 1998). In agreement with the elevated free IAA levels, sur2 disruption induced transcription of early auxin responsive genes such as Aux/IAAs and GH3s (Mikkelsen et al., 2009; Morant et al., 2010). Conversely overexpression of SUR2 led to increased production of indole GLS (Bak et al., 2001). It was shown that TAM can competitively inhibit SUR2 that resulted in conversion of IAOx into IAA (Bak and Feyereisen, 2001). These studies indicate that IAOx plays important roles in plant development and defense responses as a branch point for biosynthesis of indole GLS, camalexin, and IAA.

Differential activation of IAOx pathway resulted in altered auxin homeostasis in post-acetaldoxime mutants or overexpressors of CYP79B2 (Nafisi et al., 2006). For instance, in sur2, increased endogenous IAA levels were associated with up-regulation of CYP79B2, IAA conjugation genes such as GH3s (Morant et al., 2010), and subsequent accumulation of IAA catabolites such as IAA-aspartate and oxindole-3-acetic acid (Barlier et al., 2000). Consistently, IAA-leucine resistant 1-like family of amidohydrolases ILL1 and ILL2 which release IAA from amide conjugates were down-regulated indicating that increased IAA levels were catalyzed to irreversible conjugates in sur2 plants (Morant et al., 2010). Similar to sur2 plants, overexpression of CYP79B2 induced expression of auxin responsive genes and increased accumulation of IAN (Zhao et al., 2002). It appears that impaired aliphatic GLS production indirectly affects production of IAOx derived indole GLS and IAA levels, as demonstrated by upregulation of CYP79B2/B3 genes in CYP79F1 co-suppressed plants (Hansen et al., 200lb), and increased accumulation of indole-3-ylmethyl-GLS, IAA in cyp79f1 mutant (Reintanz et al., 2001). Nevertheless, the increased IAA levels in cyp79f1 mutants may not be responsible for the bushy phenotype.

It was shown that cyp79b2/b3 double mutants displayed wild-type like IAA levels under normal growth conditions, but showed a modest decrease of free IAA levels under high temperature (Zhao et al., 2002; Sugawara et al., 2009). Overexpression of CYP79B2 significantly elevated levels of indole GLS and IAN but with normal IAA levels (Zhao et al., 2002). It was suggested that CYP79B2/B3 were primarily responsible for production of secondary metabolites GLS and camalexin (Glawischnig et al., 2004). These observations question whether IAOx pathway can contribute to basal IAA production, and its role in regulating plant growth and development.

Besides this, potential involvement of IAOx pathway in certain circumstances is well documented. It has been proposed that root growth under sulfur starvation is initiated by extra IAA produced from IAN (Kutz et al., 2002). IAA produced from IAOx and IPA-pathways has been shown to involve in PIF4 mediated hypocotyl elongation in response to high temperature (Franklin et al., 2011). The expression of TAA1 and CYP79B2 genes were induced in response to high temperature, however, their expression was greatly reduced in pif4-101 mutants (Franklin et al., 2011). Similarly, IAOx and IPA pathways were shown to be hyperactive during high temperature induced microsporogenesis as demonstrated by tremendous increase of transcripts of NIT2 and TAA1 (Rodríguez-Sanz et al., 2015). Recently, it has been demonstrated that miR10515 promotes IAA biosynthesis via IAOx pathway under high temperature by suppressing SUR1 (Kong et al., 2015). Overexpression of miR10515 partially phenocopied sur1 phenotype with repressed SUR1 expression and elevated IAA concentration. NIT3 expression was strongly induced or repressed in the miR10515 overexpressing and silenced plants, respectively (Kong et al., 2015). Low auxin phenotype of cyp79b2/b3 double mutants appeared only in high temperature grown plants (Zhao et al., 2002). These reports suggest that IAOx pathway may provide extra auxin in response to environmental stresses.

It was reported that upregulation of the IAOx pathway can compensate defects in the IPA pathway (Stepanova et al., 2008). The elevated IAA in sur2 plants had attenuated the meristem maintenance and lateral root formation defects of TAA1 and TAR2 double mutants wei8 tar2. Additionally, dwarf phenotypes of sur2/rty1 were alleviated in wei8 tar2 sur2 and wei8 tar2 rty1 genetic backgrounds suggesting an existence of a functional overlap between the IPA- and IAOx-dependent routes of auxin biosynthesis (Stepanova et al., 2008). Developmental defects in ASA1/WEI2 mutants were suppressed by excess IAA levels accumulated in sur1 and sur2 mutants, respectively (Stepanova et al., 2005). These findings indicate that IAOx pathway may be operative under normal growth conditions as well; however, further studies are needed to confirm this idea. Regardless of how significant this pathway is in controlling plant growth and development, endogenous IAOx and its metabolizing enzymes CYP79B2/B3 were found only in GLS plants. Thus, IAOx pathway is considered as a species-specific pathway (Sugawara et al., 2009).

Biosynthetic pathways of indole GLS, camalexin and IAA are metabolically connected by their common metabolic intermediate IAOx. Disruption of indole GLS production leads to altered auxin homeostasis via differential activation of IAOx pathway. Physiological role of NITs in IAA biosynthesis is so far not conclusive. Our current knowledge on IAOx pathway suggests that this pathway is likely operative under special circumstances. Identification of the enzyme responsible for conversion of IAOx to IAA, and its functional characterization under normal and induced conditions would help us to better understand the role of this pathway in plant development.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.