Glucosinolate metabolism and its control

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1 eview TED in Plant cience Vol.11 o.2 February 2006 Glucosinolate metabolism and its control C. Douglas Grubb 1,2 and teffen Abel 1 1 Department of Plant ciences, University of California-Davis, ne hields Avenue, Davis, CA 95616, UA 2 Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, UA Glucosinolates and their associated degradation products have long been recognized for their distinctive benefits to human nutrition and plant defense. Because most of the structural genes of glucosinolate metabolism have been identified and functionally characterized in Arabidopsis thaliana, current research increasingly focuses on questions related to the regulation of glucosinolate synthesis, distribution and degradation as well as to the feasibility of engineering customized glucosinolate profiles. ere, we highlight recent progress in glucosinolate research, with particular emphasis on the biosynthetic pathway and its metabolic relationships to auxin homeostasis. We further discuss emerging insight into the signaling networks and regulatory proteins that control glucosinolate accumulation during plant development and in response to environmental challenge. Tame precursors potent products Glucosinolates and their miscellaneous breakdown products, first collectively referred to as mustards oils [1], have long inspired human curiosity. Glucosinolate derivatives not only contribute greatly to the distinctive flavor and aroma of cruciferous vegetables and condiments, but also possess profound biological activities that range from their participation in plant defense and auxin homeostasis to cancer prevention in humans [2 6]. Unlike major classes of plant natural products, glucosinolates comprise a relatively small but diverse group of nitrogenand sulfur-containing secondary metabolites that are largely limited to species of the order Brassicales, which include Brassica crops of economic and nutritional importance as well as the reference plant Arabidopsis thaliana [7,8]. The glucosinolate-defining core structure (the glucone) is derived from select protein amino acids and comprises a b-thioglucosyl residue attached to the original a-carbon to form a sulfated ketoxime (Figure 1). Extensive glucosinolate side-chain modification and amino acid elongation are together responsible for the chemical diversity of the more than 120 reported structures [7]. These hydrophilic, stable compounds are normally sequestered in vacuoles of most plant tissues [9]. Loss of cellular integrity initiates glucosinolate breakdown by myrosinase-catalyzed hydrolysis of the glucosidic bond. Myrosinases are specific Corresponding author: Abel,. (sabel@ucdavis.edu). Available online 9 January 2006 b-thioglucosidases localized in idioblasts (myrosin cells) scattered throughout most tissues of glucosinolateproducing plants [10,11]. Thus, plant injury activates the binary glucosinolate myrosinase system, sometimes called the mustard oil bomb [12,13], leading to the rapid generation of unstable thiohydroximate--sulfate intermediates. ubsequent nonenzymatic elimination of the sulfate group and spontaneous rearrangement of the remaining core structure yields a variety of bioactive products, including isothiocyanates, thiocyanates, nitriles, oxazolidine-2-thiones or epithioalkanes (Figure 1). Chemical conditions such as p, availability of ferrous ions and presence of myrosinase-interacting proteins determine the final composition of the product mix [4,8]. ome degradation products have potent biocidal activities and are likely to contribute to the plant defense arsenal against pathogens and generalist herbivores [14 18]; others are believed to play roles as allelochemicals in mediating plant insect interactions [13,19]. As components of feed and food, products of glucosinolate hydrolysis are of toxicological and pharmacological importance. Although certain glucosinolate derivatives have antinutritional properties [4,7], it is now wellestablished that methionine-derived isothiocyanates can offer substantial protection against cancer [5]. The considerable interest in optimizing glucosinolate content and composition for plant protection and human health, paired with the availability of a powerful model plant system, have made glucosinolates a dynamic area in plant metabolism research. In this review, we will highlight recent progress with particular emphasis on glucosinolate biosynthesis and its regulation. Pathways to glucosinolates Biosynthesis of glucosinolates proceeds in three stages: (i) side-chain elongation of amino acids, (ii) development of the core structure, and (iii) secondary side-chain modifications (Figure 2). Gene identification in Arabidopsis followed by in vitro and in vivo characterization of the gene products have confirmed the tripartite biosynthetic concept derived from early biochemical studies [8,20 22]. The core pathway, common to all glucosinolates, has received the most study in Arabidopsis, and relatively few gaps in our understanding remain. ide-chain elongation and modification strongly influence the bioactivities of glucosinolate breakdown products and continue to be important areas of new research. The evolution and /$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi: /j.tplants

2 90 eview TED in Plant cience Vol.11 o.2 February 2006 Trp Tyr* (α) Damage Glc ecological relevance of glucosinolate variation has recently been reviewed [2]. The core pathway: a puzzle completed? ynthesis of primary glucosinolates is accomplished in five steps and begins with the oxidation of precursor amino acids to aldoximes by side chain-specific cytochrome P450 monooxygenases (cytochromes P450) of the CYP79 family. The Arabidopsis genome encodes seven CYP79s; the specificities of five enzymes are known Glc (1) (2) (3) (4) Phe* Ile* Leu* x,y 3 Vacuole Val* Myrosin cells or myrosin bodies 4 2 Ala* x -C x -C (+) x -C y C 2 -C-(C 2 ) n -C (5) Met* Figure 1. The mustard oil bomb, a binary (glucosinolate myrosinase) chemical defense system. Glucosinolates are synthesized from tryptophan and seven additional protein amino acids (shown on top as three-letter code in blue) or their chain-elongated homoamino acid derivatives (*). In the general glucosinolate structure given below, the constituents derived from the precursor amino acid are shown in blue. Constituents of the common glucone moiety added during synthesis are shown in black. The glucosinolate side chain can be further modified ( x,y ). The major glucosinolates of Arabidopsis thaliana are derived from methionine, tryptophan and phenylalanine. Upon plant injury, glucosinolates released from vacuoles are hydrolyzed by b-thioglucoside glucohydrolases (myrosinases or TGGs) localized in myrosin cells to glucose and unstable thiohydroximate--sulfates (structure not shown). The aglucone intermediates spontaneously eliminate the sulfate group and rearrange to form: isothiocyanates (1) at p 5 8; nitriles and elemental sulfur (2) if guided by epithiospecifier-like proteins, or at p 2 5 and in the presence of Fe 2C ; thiocyanates (3) at p 8; oxazolidine-2-thiones (4) if a hydroxyl function is present on carbon 3 of the glucosinolate; or epithionitriles (5) if a terminal double bond captures the elemental sulfur released during nitrile formation. Most glucosinolate breakdown products have biocidal activities, mainly because their functional groups have an electrophilic carbon center. Arabidopsis gene identifiers for myrosinases and an epithiospecifier protein (EP) are as follows: TGG1 (At5g26000), TGG2 (A5g25980), TGG3 (At5g48375), TGG4 (At1g47600), TGG5 (At1g51470), TGG6 (At1g51490); EP (At1g54040). (Figure 2) [8,20]. The initial oxidation is not necessarily a committed step because the tryptophan-derived aldoxime is also an intermediate in the synthesis of indole-3- acetic acid (IAA) and camalexin [23,24]. egiospecific post-aldoxime enzymes are less specific for the side chain because they transform non-endogenous and even artificial aldoximes into glucosinolates [25]. The aldoximes are further oxidized by cytochromes P450 of the CYP83 family to aci-nitro compounds or nitrile oxides, strong electrophiles that spontaneously react with thiols to form -alkylthiohydroximate conjugates [26 28]. Cysteine is the likely thiol donor in vivo; however, it is not clear whether this conjugation is enzyme-mediated [8,29]. In Arabidopsis, two non-redundant enzymes, CYP83A1 and CYP83B1, oxidize aliphatic and aromatic aldoximes, respectively [30,31]. It remains to be tested whether CYP83s are the only Arabidopsis proteins with this activity. -alkylthiohydroximate conjugates are cleaved by a C- lyase into thiohydroximates, pyruvate and ammonia. Identification of the enzyme responsible was unsuccessful until the similarity between animal C- lyases and the protein encoded by UPET1 (U1) from Arabidopsis was recognized [29]. The sur1 mutant does not produce detectable levels of glucosinolates and accumulates the cysteine conjugate when fed a labeled aldoxime. This indicates that only one C- lysase acts in glucosinolate synthesis, and that this enzyme lacks side chain specificity [29]. Given that thiohydroximates are reactive and unstable compounds [32], the remainder of the core pathway (i.e. glucosylation followed by sulfation) can be considered a detoxification process [20,33]. In Arabidopsis, glucosyltransferase UGT74B1, which is orthologous to Brassica -GT [34], catalyzes thiohydroximate-specific -glucosylation in vitro [35]. Insertional ugt74b1 knockout lines show significantly decreased but not abolished production of aliphatic and indolyl glucosinolates, demonstrating the presence of other enzymes active toward thiohydroximates. A survey of w100 recombinant Family 1 glucosyltransferases of Arabidopsis [33] revealed in vitro activity of additional UGT74 enzymes toward phenylacetothiohydroximic acid (C.D. Grubb, B.J. Zipp, E.K. Lim, D. Bowles and. Abel, unpublished), including UGT74C1, which has recently been proposed to function in the biosynthesis of aliphatic glucosinolates based on hierarchical cluster analysis of gene expression data [36]. The biochemical study of recombinant UGT74C1 and ugt74c1 loss-of-function alleles will address this possibility. The final step in glucone formation is 3 0 phosphoadenosine phosphosulfate-dependent sulfation of desulfoglucosinolates. Three Arabidopsis proteins, AtT5a, AtT5b and AtT5c, catalyze this reaction with a wide variety of desulfoglucosinolate substrates [37]. Although identifying these proteins closed the last gap in the core pathway, characterizing their kinetic parameters and analyzing null mutations remains an important goal. The peripheral reactions: side chain elongation and decoration Precursor amino acid elongation is analogous to the valine-to-leucine conversion and requires five reactions:

3 eview TED in Plant cience Vol.11 o.2 February an initial and final transamination, acetyl-coa condensation, isomerization and oxidative decarboxylation [8,20]. Labeling studies have confirmed this pathway [38,39] and methylthioalkylmalate (MAM) synthases, which catalyze the condensation reaction, have been characterized in Arabidopsis and Eruca sativa [40 42]. Predicted plastid targeting signals for MAM gene products, purification of MAM synthase activities from enriched chloroplast preparations, and catalytic properties reminiscent of stromal enzyme regulation by light (basic p optimum, dependence on ATP and divalent metal ions) strongly suggest that methionine side chain elongation occurs in the chloroplast [40,41]. The chainelongated a-keto acid can be transaminated and enter the core pathway, or it can pass through additional elongation cycles that insert up to nine methylene units [7]. Given that the core pathway is proposed to be cytosolic [43,44], chain-elongated a-amino (or a-keto) acids are likely to be exported from the chloroplast. Three partially redundant MAM genes control the variation in side chain length of methionine-derived glucosinolates in Arabidopsis [42,45,46]. econdary modification of the side chain is generally considered to be the final stage in glucosinolate synthesis; however, we note that desulfoglucosinolates could be the true substrates in some cases [13,38,47]. ide chain decorations entail various kinds of oxidations, eliminations, alkylations and esterifications [2,8,20]. Methionine-derived glucosinolates are extensively modified; in Arabidopsis, this structural variety is generated by four polymorphic genetic loci [48]. The substantial natural variation of aliphatic glucosinolates in Arabidopsis has expedited identification of two a-ketoglutarate-dependent dioxygenases, encoded by the tightly linked and duplicated AP2 and AP3 genes, which control production of alkenyl and hydroxyalkyl glucosinolates, respectively [49]. The AP enzymes and their orthologs in Brassica oleracea [50,51] act after the methylthio-to-methylsulfinyl sidechain oxidation [49]. Enzymes responsible for sulfur oxidation as well as for the methoxylations of indolyl glucosinolates remain to be identified, which is likely to be facilitated by comparative QTL mapping [48,49,52,53]. ideways to auxin The indole acetaldoxime junction Genetic screens for developmental aberrations in Arabidopsis identified mutations in several genes of the core pathway that affect both indolyl glucosinolate and IAA accumulation, which points to an interconnecting metabolic grid (Figure 3). The first set of allelic mutants to be isolated, sur1/rty/alf1/hls3, overaccumulate free and conjugated IAA and display characteristic high-auxin seedling phenotypes, such as profuse lateral and adventitious root formation, epinastic cotyledons, or elongated hypocotyls and petioles [54 57]. The underlying genetic lesions inactivate the C- lyase catalyzing the second postaldoxime reaction [29]. A metabolic block of the first postaldoxime step results in a similar IAA increase and associated phenotypes, as reported for loss-of-function cyp83b1 alleles (sur2/rnt1/atr4/red1) [28,58 61]. emarkably, mutational inhibition of the penultimate reaction catalyzed by glucosyltransferase UGT74B1 also leads to elevated levels of free and conjugated IAA, as well as to morphological abnormalities consistent with auxin overproduction [35]. Thus, inhibition of flux through three sequential post-aldoxime reactions is characterized by a decrease in indolyl glucosinolate production and a concomitant increase of total IAA levels. It will be interesting to test whether null alleles of T5a, which encodes the sulfotransferase preferring aromatic desulfoglucosinolate substrates [37], causes a similar deregulation of IAA accumulation. The simplest explanation for the high-auxin phenotypes of plants with blocked post-aldoxime reactions is diversion of excess indole-3-acetaldoxime (IAx) into IAA synthesis [3,62]. This hypothesis is supported by the observation that overexpression of an IAx-forming isoenzyme, CYP79B2, increases both indolyl glucosinolate and IAA levels and results in plants with phenotypes nearly identical to the sur2 mutant [63,64]. Conversely, the cyp79b2 cyp79b3 double mutant lacks indolyl glucosinolates and displays decreased IAA synthesis rates in roots as well as morphological phenotypes consistent with partial IAA deficiency [64,65]. Likewise, overexpression of the IAx-consuming enzyme CYP83B1 results in shorter hypocotyls, loss of apical dominance and elevated indolyl glucosinolate production, suggesting that IAx removal lowers IAA levels [27,28]. Channeling of IAx into indolyl glucosinolate production has been proposed to serve as a regulatory branch point to fine-tune auxin homeostasis [24,28]. This idea is supported by the observation that IAA induces expression of CYP83B1/U2 and UGT74B1, suggesting that genes coding for post-aldoxime enzymes, possibly including U1, are coordinately regulated by an indirect negative feedback loop in IAA synthesis [35,59].A potential physiological function of the IAx branch point in auxin homeostasis is revealed by the red1/cyp83b1 mutation, which was isolated in a screen for mutants suppressing the enhanced de-etiolation response of a phyb-overexpressing line to red light [61]. Interestingly, CYP83B1 is induced by red light in wild type, although in a phyb-independent manner. Thus, light-induced expression of CYP83B1 is proposed to reduce IAA levels by elevating indolyl glucosinolate production, followed by inhibition of hypocotyl elongation and other hallmarks of seedling de-etiolation [61]. A shared IAx pool in auxin and indolyl glucosinolate biosynthesis has recently been questioned by the analysis of gain-of-function yucca mutants, which overexpress a flavin monooxygenase proposed to oxidize tryptamine to -hydroxyl tryptamine in vitro [64,66]. The yucca lines contain w50% more free IAA than the wild type do and display phenotypes characteristic of enhanced auxin synthesis, resembling auxin-overproducing mutants sur1, sur2 and ugt74b1 [66]. esistance of yucca plants to toxic tryptophan analogs suggests that the extra IAA has a tryptophan-dependent source. Given that the YUCCA protein is proposed to function in IAx synthesis, it is surprising that YUCCA-overexpressing plants have slightly reduced indolyl glucosinolate levels relative to wild type [64]. By contrast, overexpression of the prealdoxime enzyme CYP79B2 significantly elevates both

4 92 eview TED in Plant cience Vol.11 o.2 February 2006 (a) ide chain elongation α α (C 2 ) n Transamination 2 α-kga Glu α-keto acid 2. Condensation Acetyl-CoA CoA 2-Alkylmalate MAM1, MAM2, MAML (Met) 3. Isomerization 3-Alkylmalate Unknown 4. xidative decarboxylation AD + AD C 2 omoketo acid Unknown 5. Transamination Glu α-kga 2 omoamino acid (b) Glucone formation α α Glc (C 2 ) n (C 2 ) n xidation 2 2ADP 2 2 2ADP + C 2, 3 2 Aldoxime CYP79F1 (Met 1 6 ) CYP79B2 (Trp) CYP79A2 (Phe) CYP79F2 (Met 5,6 ) CYP79B3 (Trp) 2. (i) xidation ADP ADP aci-itro compound CYP83A1 (Met) CYP83B1 (Trp, Phe) (ii) Conjugation + Cys 2 (Cys) -Alkyl thiohydroximate pontaneous? GT? 3. C- cleavage (Cys) 2 Pyruvate, 3 Thiohydroximate C- Lyase 4. Glucosylation UDP-Glc UDP Glc Desulfoglucosinolate UGT74B1 5. ulfation Glc PAP PAP Glc 3 Glucosinolate T5a (Trp, Phe) T5b (Met) T5c (Met) (c) ide chain modification α Glc (C 2 ) n 3 α Glc (C 2 ) n x 3 xidation of Met-derived glucosinolates 3C 3C (C 2 ) n (C 2 ) n α-kga, 2 uccinate, C 2 α-kga, 2 2 C C C 2 (C 2 ) n-2 (C 2 ) n-1 Alkenyl-G ydroxy-g AP2 AP3 uccinate, C 2 TED in Plant cience

5 eview TED in Plant cience Vol.11 o.2 February indolyl glucosinolate and IAA levels [63,64]. It has therefore been argued that IAx derived from a hypothetical YUCCA pathway is exclusively shunted into IAA synthesis [64]. Although the YUCCA-catalyzed reaction remains to be fully integrated into auxin metabolism [62,67,68], there is an alternative explanation for the dominant yucca phenotype that is compatible with the hypothesis of a common IAx pool contributing to both pathways. It has been reported that tryptamine binds tightly to the active site of CYP83B1 (K s w18 mm) and competitively inhibits IAx oxidation [27,28]. Primary amines bind to cytochrome P450 enzymes by positioning the electron lone pair of the amino group in close vicinity to the heme iron [27]. Thus, -hydroxyl tryptamine is perhaps even more potent as a competitive inhibitor of CYP83B1 than tryptamine because its hydroxylated amino group mimics the oxime group of the IAx substrate more closely. If so, -hydroxyl tryptamine produced by YUCCA in vivo would inhibit CYP83B1 and result in lower indolyl glucosinolate production and in the diversion of excess IAx into IAA synthesis, as described for yucca and sur2 seedlings [28,59,64]. The reported reduction of indolyl glucosinolates in tryptamine-treated wild-type plants is consistent with such a scenario [28]. owever, as suggested by the floozy mutant in petunia [68], this observation does not rule out a glucosinolate core pathway-independent route to IAA via YUCCA. Less charted terrain The precise reactions from IAx to IAA remain to be elucidated and could proceed via indole-3-acetaldehyde (IAAld) or indole-3-acetonitrile (IA) given that both compounds mimic IAA application [67]. Enzymes that produce IA or IAAld from IAx have not been isolated, and the evidence for a role for nitrilases and aldehyde oxidases in IAA synthesis is not compelling [62,67]. Three nitrilases in Arabidopsis (IT1 IT3) convert IA to indole-3-acetamide (IAM) and IAA; however, their specific activities for IA hydrolysis are low and K m values unexpectedly high (7 30 mm), arguing against a function in the IAx IAA conversion [3,28,69,70]. Although the nit1 mutation confers resistance to exogenous IA, it fails to suppress the high-auxin phenotype of sur2, which would be expected if IA is directly derived from IAx [28]. By contrast, IA levels tend to follow indolyl glucosinolate levels in Arabidopsis mutants [3]. In addition, both the sur1 and sur2 mutant show higher IAA and lower IA synthesis rates in roots than the wild type do, consistent with their high-auxin and low-indolyl glucosinolate chemotypes [65]. Thus, in the Brassicaceae, IA is likely to be derived from myrosinase-mediated indole-3-methyl glucosinolate turnover in vivo (Figure 3). itrilases can hydrolyze IA to IAA under certain physiological or pathological conditions, such as seed development and germination, sulfur deficiency, or during the manifestation of the clubroot disease [69 72]. Given the catalytic properties of IT1 IT3, controlled in vivo indolyl glucosinolate turnover is likely to be the ratelimiting step in this more circuitous route to IAA. Formation of IAAld in the IAx IAA conversion is suggested by the sur2/cyp83b1 mutant, which overaccumulates both IAA and IAAld [59], as well as by the sur1 mutant, which expresses higher activities of an aldehyde oxidase capable of converting IAAld to IAA [73]. Although glucosinolate synthesis mutants have significantly contributed to our current understanding of tryptophan-dependent auxin biosynthesis in Arabidopsis, exploring the IAx IAA route continues to be a major challenge. It is noteworthy that loss-of-function mutations in CYP79F1 (bushy/supershoot) not only abolish the formation of short-chain methionine-derived glucosinolates but also increase the levels of IAA and cytokinin by unknown mechanisms [43,74,75]. eciprocal control of the aliphatic and indolyl branches of glucosinolate synthesis (discussed below) is likely to affect auxin and consequently cytokinin homeostasis. Furthermore, given that 80% of the synthesized methionine is converted to -adenosyl methionine, which is the second most widely used cofactor after ATP [76], the possible formation of various -adenosyl (homo)-methionines and the metabolic consequences of their accumulation in cyp79f1 and cyp79f2 mutants (e.g. altered polyamine synthesis [77]) is an unexplored area deserving attention. Controlling glucosinolates ystemic distribution Glucosinolate profiles have been systematically monitored in Arabidopsis during plant development and vary significantly between tissues and organs [78,79]. Consistent with a prominent function in plant defense, the highest glucosinolate concentrations are found in reproductive organs, including seeds, siliques, flowers and developing inflorescences, followed by young leaves, Figure 2. tages of glucosinolate biosynthesis. (a) ide chain elongation of precursor amino acids is initiated by a transamination reaction, followed by condensation of the resulting a-keto acid with acetyl-coa to form a 2-alkylmalate derivative. In methionine side chain elongation, methylthioalkylmalate synthases (MAM1, MAM2, MAML) catalyze this reaction [40,46]. The condensation and the following two steps, isomerization and oxidative decarboxylation, are conceptually analogous to the first three reactions of the citric acid cycle, in which oxaloacetate is extended by one methylene unit to a-ketoglutarate. ere, a a-keto acid is elongated by one carbon, which can either be transaminated to the homoamino acid or pass through additional elongation cycles that insert up to nine methylene units. (b) Amino acids and chain-elongated homoamino acids enter the core pathway by oxidation to aldoximes catalyzed by cytochrome P450 monooxygenases (cytochrome P450) of the CYP79 family [8,20]. Inthe second step, aldoximes are oxidized by enzymes of the CYP83 family to reactive aci-nitro or nitrile oxide intermediates (only the structure of the aci-nitro compound is shown; the nitrile oxide is formed by its dehydration). These strong electrophiles can react spontaneously with either cysteine or, perhaps catalyzed by glutathione -transferase (GT), with glutathione [8,20]. Cleavage of the -alkylthiohydroximate conjugate by a C- lyase produces thiohydroximates [29], which are glucosylated by UGT74B1, a UDPglucose-dependent glucosyltransferase, to desulfo-glucosinolates [35]. ulfation concludes the synthesis of primary glucosinolates [37]. (c) Glucosinolate side chains can be extensively modified by oxidation, elimination, akylation or esterification. Two side-chain modifications of methionine-derived glucosinolates have been studied in some detail, catalyzed by a-ketoglutarate dioxygenases AP2 and AP3 [49]. Characterized enzymes of the glucosinolate pathway in Arabidopsis thaliana are listed on the right with their amino acid substrate specificities or preferences given in parenthesis. Arabidopsis gene identifiers are as follows: MAM1 (At5g23010), MAML (At5g23020); CYP79F1 (At1g16410), CYP79F2 (At1g16400); CYP79B2 (At4g39950), CYP79B3 (At2g22330); CYP79A2 (At5g05260), CYP83A1 (At4g13770); CYP83B1 (At4g31500); C- lyase (At2g20610); UGT74B1 (At1g24100); T5a (At1g74100), T5b (At1g74090), T5c (At1g18590); AP2 (At4g03060), AP3 (At4g03050). Bioinformatics approaches [36,87] suggest additional genes with putative functions in glucosinolate synthesis: GT (At3g03190, At1g78370); C- lyase (At5g36160); UGT74C1 (At2g31790).

6 94 eview TED in Plant cience Vol.11 o.2 February 2006 Chorismate YUCCA 2 2 Tryptophan Tryptamine -hydroxy tryptamine AT1 CYP79B2 CYP79B3 IAx -Alkylthiohydroximate sur1 Thiohydroximate UGT74B1 (Cys) C- Lyase CYP83B1 sur2/red1/atr4 IAA IA IT1 IT2 IT3 C ugt74b1 Glc Desulfoglucosinolate Glucosinolate 3 Glc TGGs 3 Thiohydroximate--sulfate TED in Plant cience Figure 3. egulation of glucosinolate biosynthesis and its intersection with auxin metabolism in Arabidopsis thaliana. Tryptophan (Trp) is the precursor to indolyl glucosinolates (pathway on the left) and to indole-3-acetic acid (IAA), the principal auxin (reactions on the right). everal genetic studies implicate indole-3-acetaldoxime (IAx) as a key branch point between glucosinolate and auxin synthesis. Inhibition of post-aldoxime reactions by loss-of-function mutations (red bars) and overexpression of the pre-aldoxime enzyme CYP79B2 lead to IAA overproduction, presumably via elevated IAx levels [29,35,54,58,59,63,64]. owever, increased consumption of IAx by overexpression of CYP83B1 or decreased IAx production in the cyp79b2 cyp79b3 double mutant reduces free auxin concentrations [27,28,64]. The relevance of the IAx branch point for fine-tuning IAA levels via diverting flux into the indolyl glucosinolate branch is corroborated by auxin-responsive induction of CYP83B1 and UGT74B1 (green broken arrows) [35,59]. The precise reactions from IAx to IAA are presently unknown (light-grey arrows). An alternative Trp-dependent route to IAA via IAx is proposed to involve hydroxylation of tryptamine by YUCCA, a flavin monooxygenase [66]; however, the YUCCA reaction remains to be integrated into the auxin synthesis grid (light-grey arrows). YUCCA overexpressor lines show high auxin levels but low indolyl glucosinolate concentrations, implying separate IAx pools in glucosinolate and auxin synthesis (yellow box). owever, the yuccad pheno- and chemotypes can be explained by competitive inhibition of CYP83B1 by tryptamine [27,28] and -hydroxy tryptamine (red, broken inhibition arrow), which would mimic sur2 mutants characterized by high auxin but low indolyl glucosinolate concentrations. on-defense related, endogenous indolyl glucosinolate turnover, possibly catalyzed by myrosinases (TGGs), can produce indole-3-acetonitrile (IA), which can be hydrolyzed by nitrilases (IT) to IAA. Although operation of this pathway has been established, its physiological relevance remains to be demonstrated [69 71]. A genetic screen for altered tryptophan regulation (atr) mutants identified AT1, a MYB transcription factor that activates CYP79B2, CYP79B3, CYP83B1 and genes in Trp biosynthesis (blue broken arrows) [81,93]. Interestingly, atr4 is allelic to sur2, and its analysis revealed feedback inhibitions of AT1 expression by intermediates of indolyl glucosinolate synthesis (black, broken inhibition arrow) [60].

7 eview TED in Plant cience Vol.11 o.2 February the root system and fully expanded leaves. Exceptionally high concentrations are found in the sulfur-rich -cells of the flower stalk [80], which occur in the proximity of myrosin cells between the phloem and endodermis [11]. istochemical analysis of transgenic lines expressing transcriptional fusions between the b-glucuronidase reporter and several promoters of core pathway (CYP79s, UGT74B1) and regulatory (AT1, IQD1) genes has revealed that system-wide biosynthetic capacity is often closely associated with vascular tissues [35,43,44,63,65,74,75,81,82]. Tracer studies have demonstrated de novo synthesis in siliques and phloem transport of glucosinolates from mature and senescing leaves to inflorescences and developing fruits [79,83]. Active and specific glucosinolate uptake into Brassica leaf protoplasts cells is mediated by a proton-coupled symporter [84]. owever, the mechanisms of phloem loading and unloading are unknown. It is possible that myrosinase-resistant desulfoglucosinolates are substrates for phloem loading given that both glucosinolates and desulfoglucosinolates share physicochemical properties compatible with longdistance transport [13,38,47,85]. ignaling networks In addition to the developmental stage, several biotic and abiotic factors modulate leaf and seed glucosinolate profiles, such as pathogen challenge, herbivore damage, mechanical wounding or altered mineral nutrition [8,18,86 88]. A combination of metabolite and transcript profiling has revealed coordinated repression of most glucosinolate pathway genes in response to sulfate limitation [87]. An integrated bioinformatics approach has identified known core pathway genes and predicted additional enzymes with roles in glucosinolate biosynthesis (Figure 2) [87]. Although it is unknown how sulfate availability regulates expression of glucosinolate pathway genes, it is well established that pathogen- and herbivoryinduced glucosinolate production and pathway gene expression are mediated, at least partially, by jasmonates, salicylic acid and ethylene, the major plant hormones associated with specific and broad-spectrum defense responses [14,18,52,86,89,90]. Jasmonate treatment, which stimulates responses to insect attack and necrotrophic pathogens, leads to increased concentrations of both indolyl and specific aliphatic glucosinolates [14,86,90], possibly via multiple signaling pathways [52]. Analysis of mutants defective in hormone synthesis or signaling has revealed regulation of specific indolyl glucosinolate production by salicylic acid as well as complex interactions of jasmonate and salicylate signaling in differential glucosinolate accumulation [52,86,90 92]. uclear proteins AT1, a MYB transcription factor identified in a screen for altered tryptophan regulation (atr) mutants controls production of indolyl glucosinolates [60,93]. A dominant overexpression allele, atr1d, confers elevated expression of tryptophan synthesis and glucosinolate core pathway (CYP79B2, CYP79B3 and CYP83B1) genes. verexpression of AT1 does not alter aliphatic glucosinolate content but instead causes a tenfold higher accumulation of indolyl glucosinolates relative to wild type, as well as a doubling of IAA levels [93]. educed expression of core pathway genes and lower accumulation of indolyl glucosinolates are evident in the recessive atr1-2 mutant. The atr1d allele enhances the tendency of the cyp83b1/sur2 mutant to form adventitious roots, whereas atr1-2 suppresses its high-auxin phenotype, indicating that AT1 regulates auxin metabolism via the IAx intermediate [60,93]. Intricate feedback regulation of the indolyl glucosinolate branch is revealed by atr4, which is allelic to cyp83b1. The atr4 mutant contains increased levels of AT1, CYP79B2 and CYP83B1 transcripts, suggesting feedback inhibition of AT1 expression by indolyl glucosinolates or their intermediates (Figure 3) [60]. This hypothesis is further supported by suppressed CYP79B2, CYP79B3 and CYP83B1 transcript levels in the atr1-2 cyp83b1 double mutant, and by enhanced expression of these genes and AT1 in the cyp79b2 cyp79b3 double mutant, which is deficient in indolyl glucosinolates. Feedback regulation of AT1 expression is likely to involve complex signaling circuits given that AT1 transcript levels are differentially altered by hormone signaling mutations and by the application of various plant hormones [60,93]. Although AT1 specifically activates genes related to tryptophan and indolyl glucosinolate synthesis, other nuclear regulators affecting glucosinolate homeostasis are likely to serve broader functions. These include IQD1, a calmodulin-binding protein [82], and TFL2, a chromodomain protein involved in controlling heterochromatin structure [94,95]. Gain- and loss-of function iqd1 alleles correlate with increased and decreased glucosinolate accumulation, respectively, and overexpression of IQD1 stimulates plant resistance to generalist insects and to Botrytis cinerea, a necrotrophic fungus [82] (M. Levy and. Abel, unpublished). IQD1 encodes a basic nuclear protein that modulates expression of several glucosinolate pathway genes. A plant-specific domain containing a unique arrangement of multiple calmodulin retention motifs, the IQ domain, is a hallmark of IQD1 and 32 related proteins in Arabidopsis. Although IQD1 expression is not appreciably regulated by plant defense hormones, it is moderately induced by mechanical stimuli. This suggests that IQD1 integrates early wound- and pathogen-induced changes of calcium signatures to stimulate and fine-tune an array of coordinated defense responses, including upregulation of glucosinolate production [82]. Biosynthetic control Analysis of 39 Arabidopsis accessions has revealed considerable variation in glucosinolate composition and total content, which is particularly apparent for methioninederived glucosinolates in leaves and for indolyl compounds in seeds [48]. Interestingly, the AP2/AP3 locus has significant control over the production of methioninederived glucosinolates. Absence of methylsulfinylalkyl side chain modification in AP2/AP3 null accessions results in considerably lower accumulation of methioninederived glucosinolates than in accessions with one functional AP allele, which has been explained by

8 96 eview TED in Plant cience Vol.11 o.2 February 2006 differential feedback regulation of glucosinolate biosynthesis by its various end products [48,49]. Interdependent metabolic control of the aliphatic and indolyl glucosinolate branches is suggested by several mutations that block or restrict synthesis of one class of glucosinolates but cause a compensatory increase in the other class [31,43,64,74,75]. These observations suggest homeostatic control of glucosinolate synthesis, which could be achieved by reciprocal negative feedback regulation between both branches using glucosinolate intermediates or end products as inhibitors [31]. An alternative explanation is competition among the cytochrome P450 monooxygenases of the core pathway for electrons to reduce one atom of the dioxygen substrate to water. These electrons are provided by ADP via ADP:cytochrome P450 reductase. The total supply of ADP in non-photosynthetic conditions is controlled by flux through the oxidative pentose phosphate pathway. Limited ADP supply could therefore be responsible for the interdependence of aliphatic and indolyl glucosinolate biosynthesis because inhibition of one branch would increase ADP availability for the other, resulting in some degree of total glucosinolate maintenance. In addition, our proposed limiting electron hypothesis explains the impact of the side chain elongation locus (MAM) on total glucosinolate accumulation. Arabidopsis accessions synthesizing aliphatic glucosinolates principally from dihomo-methionine accumulate w20% more total glucosinolates than accessions using mainly homomethionine as the amino acid precursor (calculated from data in [48]). The additional round of side chain elongation yields an extra AD in reactions analogous to the citric acid cycle, which could be converted to ADP via the malate dehydrogenase and malic enzyme reactions and shuttled to the cytosol. Thus, side chain elongation can provide ADP independently of the pentose phosphate pathway and thereby increase total glucosinolate production. Finally, the limiting electron model is consistent with available information on the regulation of genes encoding glucose-6-phosphate dehydrogenase, which catalyzes the first and committed step of the pentose phosphate pathway, and ADP:cytochrome P450 reductase. Genes coding for both enzymes are induced by wounding, pathogen challenge and other stresses [96,97], conditions known to increase production of secondary metabolites with protective functions, including glucosinolates. Metabolic channeling via multienzyme complex (metabolon) formation is proposed to minimize negative flux interference, to contain toxic intermediates, and to facilitate effective synthesis of specific natural products, as recently discussed for terpenoids, phenylpropanoids, alkaloids and cyanogenic glucosides [98]. Direct evidence for glucosinolate metabolons is not yet available. owever, overexpression of sorghum CYP79A1 in Arabidopsis, which catalyzes aldoxime formation from tyrosine in the biosynthesis of dhurrin, a cyanogenic glucoside in sorghum, leads to the accumulation of large quantities of p-hydroxybenzylglucosinolate in Arabidopsis [99]. This suggests recruitment of CYP79A1 into a glucosinolatededicated metabolon to encapsulate the reactive aldoxime intermediate and functional interaction with Arabidopsis post-aldoxime enzymes [98,99]. Glucosinolate core pathway enzymes are likely to assemble into multienzyme complexes on the cytosolic surface of the E, which could include enzymes of IAA synthesis in the case of indolyl glucosinolates (Figure 4). Glucosinolate turnover Defense-induced glucosinolate breakdown is activated by tissue damage and myrosinase-initiated hydrolysis. Variation of myrosinase isoenzymes, encoded by a small gene family in Arabidopsis [100], and myrosinase-interacting proteins, such as epithiospecifier proteins, as well as polymorphic genetic loci determining glucosinolate side chain structure, provide a modular system for generating a multitude of breakdown products with diverse ecological roles in plant insect interactions [2,16,100,101]. ondefensive, in vivo glucosinolate turnover is suggested by dynamic changes of glucosinolate content and composition during seed germination and early plant development, which inversely correlate with myrosinase expression [78,79]. Isothiocyanates have been identified as in vivo catabolites in glucosinolate feeding studies [102] and as major volatiles after challenging Arabidopsis with oxidative stress [103]. It is interesting to note that methionine-derived isothiocyanates effectively activate a set of mammalian genes coding for detoxification enzymes (e.g. glutathione -transferases) via redox signaling, which is one reason for the cancer-preventive properties of Brassica-based diets [5]. Given that redox signaling plays a role in plant defense, regulated glucosinolate turnover and isothiocyanate production in vivo should be a promising field of study. Future prospects The era of structural gene discovery in glucosinolate research, greatly aided by a combination of molecular, genetic and genomic approaches in Arabidopsis, has passed its peak. Early biochemical models of glucosinolate synthesis, which provided the first guidance in this area of research [21,22], have largely been confirmed [8]. Current research increasingly focuses on glucosinolate transport and turnover, on regulatory mechanisms of glucosinolate biosynthesis, and on the feasibility of customizing glucosinolate profiles by molecular breeding [104,105] and transgenic approaches [99,101]. The mechanisms responsible for system-wide distribution and cellular transport of glucosinolates, or their desulfo derivatives, are presently unknown, which is also true for defenseindependent glucosinolate catabolism in vivo and its physiological relevance. A thorough understanding of glucosinolate pathway regulation will not only require the study of pathway gene expression in response to internal and external factors and their corresponding signaling networks, but also will have to address neoclassic questions of enzyme biochemistry, such as subcellular localization of enzymes, intracellular trafficking and channeling of intermediates, metabolon organization and flux control, regulation of enzyme activity by effectors and covalent modification, or protein structure function relationships as recently reported for myrosinase

9 eview TED in Plant cience Vol.11 o.2 February Chloroplast Met side-chain elongation, Trp synthesis YUCCA Phenylpropanoid synthesis Met n Trp IAx T5b, T5c UGT74B1 CYP79F1/ CYP79F2 Alx CYP83A1 C- Lyase GT? CYP79B2/ CYP79B3 IAx CYP83B1 UGT74B1 C- Lyase T5a GT? IAA Camalexin synthesis AG DG DG IG E AG IG Vacuole DG Phloem transport DG AG IG TED in Plant cience Figure 4. ubcellular organization of glucosinolate and auxin synthesis pathways in Arabidopsis thaliana. In contrast to other natural product pathways in plants, there is only indirect evidence for a glucosinolate metabolon [98]. Precursor amino acid synthesis and side chain elongation take place in the chloroplasts [40,41]. Methionine- and tryptophan-derived metabolites are depicted by brown and red circles, respectively. The oxidation reactions of the glucosinolate core pathway, catalyzed by CYP79 and CYP83 enzymes occur at the E-cytosol interface, as demonstrated by the targeting of CYP79F1- and CYP79F2-reporter protein fusions to the E (blue ellipsoid) [43]. Early biochemical studies in Brassica showed that the glucosyl- and sulfotransferases are soluble enzymes [21,22]. Multienzyme complex (metabolon) formation of glucosinolate pathway enzymes (light-grey ellipsoids) is suggested by channeling of aldoxime intermediates. If flux is disrupted by genetic lesions, metabolic interference by aldoximes is a consequence, as reported for inhibition of phenylpropanoid synthesis by aliphatic aldoximes (Alx) [31] and for elevated indole-3-acetic acid (IAA) production owing to accumulation of indole-3-acetaldoxime (IAx) [59]. The conversion of IAx to IAA (dark-grey ellipsoids) is presently unknown but could proceed in physical association with an indolyl glucosinolate metabolon. IAA synthesis via YUCCA (depicted by a second set of dark-grey ellipsoids) is thought to use a possibly separate IAx pool [64,66], as indicated by the red dotted line. IAx is also a precursor to camalexin, a phytoalexin in Arabidopsis [23,24]. Aliphatic and indolyl glucosinolates (AG and IG) are transported into the vacuoles or exported to the phloem by unknown proteins and mechanisms (white ellipsoids). Desulfo-glucosinolates (DG) have been proposed as myrosinaseresistant phloem transport forms [13,38,47,85], which could be exported by similar mechanisms as the corresponding glucosinalates (indicated by red and brown broken lines). [106,107]. ur knowledge of enzyme regulation in glucosinolate synthesis is rudimentary at best. Inhibition of CYP83B1 by tryptamine, a possible intermediate in auxin synthesis, and interference between the glucosinolate and other metabolic pathways, which is indicated by the accumulation of aldoxime intermediates in glucosinolate mutants, point to such biochemical circuits [27,28,31]. Precise understanding of glucosinolate enzymology and metabolons will be necessary for the successful alteration of glucosinolate profiles by metabolic engineering without violating the principle of substantial equivalence [98]. A proof of concept and an important step toward the general acceptance of genetically modified plants has recently been provided by introducing the dhurrin pathway of

10 98 eview TED in Plant cience Vol.11 o.2 February 2006 sorghum into Arabidopsis. emarkably, Arabidopsis lines transgenic for the entire pathway accumulate high amounts of the cyanogenic glucoside without inadvertent effects on plant morphology, metabolites and the transcriptome. owever, Arabidopsis plants with an incomplete dhurrin pathway are stunted and show significant alterations in general gene expression and metabolite levels, probably because of the accumulation of toxic intermediates that cannot be incorporated into glucosinolate synthesis [98,99]. Thus, metabolic engineering of customized glucosinolate profiles for enhanced plant protection and for the design of functional foods in longterm, nutritional cancer-prevention strategies is a realistic prospect. 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