Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3-to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 and CYP79B3 were both highly induced. Specifically, the indole glucosinolate N-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutant coi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducing mpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.Glucosinolates are amino acid-derived natural plant products that function in the defense against herbivores and microorganisms. Upon tissue disruption, e.g. caused by insect feeding, glucosinolates are hydrolyzed by specific thioglucosidases called myrosinases to produce an array of biologically active compounds, typically isothiocyanates, nitriles, and thiocyanates (for review, see Halkier, 1999;Rask et al., 2000). These compounds function as inhibitors of microbial growth (Mari et al., 1993;Manici et al., 1997), as attractants for specialist insects, and as deterrents of generalist herbivores. For humans, glucosinolates are important as flavor compounds, as cancer-preventive agents, and as biopesticides in agriculture.Glucosinolate biosynthesis is considered a threestep process: First, the amino acid may enter the chain elongation pathway, in which the condensing enzymes MAM1 and MAM-L have recently been identified (de Quiros et al., 2000;Kroymann et al., 2001). Second, the core glucosinolate structure is formed (see below); and t...

Glucosinolates are natural plant products known as flavor compounds, cancer-preventing agents, and biopesticides. We report cloning and characterization of the cytochrome P450 CYP79B2 from Arabidopsis. Heterologous expression of CYP79B2 in Escherichia coli shows that CYP79B2 catalyzes the conversion of tryptophan to indole-3-acetaldoxime. Recombinant CYP79B2 has a K m of 21 M and a V max of 7.78 nmol/h/ml culture. Inhibitor studies show that CYP79B2 is different from a previously described enzyme activity that converts tryptophan to indole-3-acetaldoxime (Ludwig-Mü ller, J., and Hilgenberg, W. (1990) Phytochemistry, 29, 1397-1400). CYP79B2 is wound-inducible and expressed in leaves, stem, flowers, and roots, with the highest expression in roots. Arabidopsis overexpressing CYP79B2 has increased levels of indole glucosinolates, which strongly indicates that CYP79B2 is involved in indole glucosinolate biosynthesis. Our data show that oxime production by CYP79s is not restricted to those amino acids that are precursors for cyanogenic glucosides. Our data are consistent with the hypothesis that indole glucosinolates have evolved from cyanogenesis. Indole-3-acetaldoxime is a precursor of the plant hormone indole-3-acetic acid, which suggests that CYP79B2 might function in biosynthesis of indole-3-acetic acid. Identification of CYP79B2 provides an important tool for modification of the indole glucosinolate content to improve nutritional value and pest resistance.Glucosinolates are natural plant products characterized by having a thioglucose moiety, a sulfonated oxime, and a side chain derived from aliphatic, aromatic, and indole amino acids. Glucosinolates are found in the order Capparales, where they co-occur with the endogenous thioglucosidase myrosinase (for review see Ref. 1). Generally, the glucosinolate-myrosinase system is believed to play an important role in plant defense. In human affairs, glucosinolates are important as flavor compounds, cancer-preventive agents, and biopesticides, for example. There is a strong interest in controlling the level of glucosinolates to improve flavor and nutritional qualities of food crops and to study the physiological role of glucosinolates in plants, e.g. in plant-pest interaction.Glucosinolates are related to cyanogenic glucosides as both groups of natural plant products are derived from amino acids and have oximes as intermediates. Cytochromes P450 of the CYP79 family have been shown to catalyze the conversion of both aliphatic and aromatic amino acids to their corresponding oximes in the biosynthesis of cyanogenic glucosides (2-4). Recently, we have shown that the aromatic amino acid phenylalanine is converted to its oxime by CYP79A2 from Arabidopsis (5). This is in accordance with biochemical data from microsomal enzyme systems isolated from the glucosinolate-producing Sinapis alba, Tropaeolum majus, and Carica papaya (6 -10).The nature of the enzymes catalyzing oxime production in the biosynthesis of glucosinolates has been subject of discussions as independent studies ...

SummaryWe report characterization of SUPERROOT1 (SUR1) as the C±S lyase in glucosinolate biosynthesis. This is evidenced by selective metabolite pro®ling of sur1, which is completely devoid of aliphatic and indole glucosinolates. Furthermore, following in vivo feeding with radiolabeled p-hydroxyphenylacetaldoxime to the sur1 mutant, the corresponding C±S lyase substrate accumulated. C±S lyase activity of recombinant SUR1 heterologously expressed in Escherichia coli was demonstrated using the C±S lyase substrate djenkolic acid. The abolishment of glucosinolates in sur1 indicates that the SUR1 function is not redundant and thus SUR1 constitutes a single gene family. This suggests that the`high-auxin' phenotype of sur1 is caused by accumulation of endogenous C±S lyase substrates as well as aldoximes, including indole-3-acetaldoxime (IAOx) that is channeled into the main auxin indole-3-acetic acid (IAA). Thereby, the cause of the`highauxin' phenotype of sur1 mutant resembles that of two other`high-auxin' mutants, superroot2 (sur2) and yucca1. Our ®ndings provide important insight to the critical role IAOx plays in auxin homeostasis as a key branching point between primary and secondary metabolism, and de®ne a framework for further dissection of auxin biosynthesis.

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Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxyindol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.

In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K m value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.Glucosinolates are amino acid-derived natural plant products, containing a thio-Glc moiety and a sulfonate moiety bound to an oxime function. They are implicated in plant-insect and plant-pathogen interactions, and for humans, they have attracted attention as cancer-preventive agents and flavor compounds (for review, see Halkier, 1999;Rask et al., 2000). In recent years, significant advances have been made in our understanding of the biosynthetic pathway of glucosinolates, which can be divided into the chain elongation pathway, the formation of the core structure, i.e. the conversion of precursor amino acid to parent glucosinolate, and secondary modifications of side chain and Glc moiety of the parent glucosinolate (Wittstock and Halkier, 2002). In the biosynthesis of the core structure, cytochromes P450 belonging to the CYP79 family catalyze the first committed step, i.e. the conversion of amino acids to oximes (for review, see . Cytochromes P450 belonging to the CYP83 family have been shown to catalyze the conversion of aromatic oximes (Bak and Feyereisen, 2001;Bak et al., 2001; Hansen et al., 2001b). The remaining pathway leading to the parent glucosinolate proceeds through a thiohydroximic acid that is first glucosylated to give a desulphoglucosinolate that is finally sulfonated.Glucosinolates are r...

SummaryPlant diseases are major contributing factors for crop loss in agriculture. Here, we show that Arabidopsis plants with high levels of novel glucosinolates (GSs) as a result of the introduction of single CYP79 genes exhibit altered disease resistance. Arabidopsis expressing CYP79D2 from cassava accumulated aliphatic isopropyl and methylpropyl GS, and showed enhanced resistance against the bacterial soft-rot pathogen Erwinia carotovora, whereas Arabidopsis expressing the sorghum CYP79A1 or over-expressing the endogenous CYP79A2 accumulated p-hydroxybenzyl or benzyl GS, respectively, and showed increased resistance towards the bacterial pathogen Pseudomonas syringae. In addition to the direct toxic effects of GS breakdown products, increased accumulation of aromatic GSs was shown to stimulate salicylic acid-mediated defenses while suppressing jasmonate-dependent defenses, as manifested in enhanced susceptibility to the fungus Alternaria brassicicola. Arabidopsis with modified GS profiles provide important tools for evaluating the biological effects of individual GSs and thereby show potential as biotechnological tools for the generation of plants with tailor-made disease resistance.

BackgroundThe glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana.ResultsIn the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C 13- and C 19-bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1.ConclusionsScreening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1Thr146Gly and UGT76G1His155Leu, which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0609-1) contains supplementary material, which is available to authorized users.