Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana (L.) Heynh. glucosinolate mutants and the development of clubroot disease

Mutants and wild type plants of Arabidopsis thaliana were analysed for differences in glucosinolate accumulation patterns, indole-3-acetic acid (IAA) biosynthesis and phenotype. A previously identified series of mutants, termed TU, with altered glucosinolate patterns was used in this study. Only the line TU8 was affected in shoot phenotype (shorter stems, altered branching pattern). Synthesis of IAA and metabolism were not much affected in the TU8 mutant during seedling development, although the content of free IAA peaked earlier in TU8 during plant development than in the wild type. Indole glucosinolates and IAA may, however, be involved in the development of clubroot disease caused by the obligate biotrophic fungus Plasmodiophora brassicae since the TU3 line had a lower infection rate than the wild type, and lines TU3 and TU8 showed decreased symptom development. The decline in clubroot formation was accompanied by a reduced number of fungal structures within the root cortex and slower development of the fungus. Indole glucosinolates were lower in infected roots of TU3 and TU8 than in control roots of these lines, whereas in wild-type plants the differences were not as prominent. Free IAA and indole-3-acetonitrile (IAN) were increased in infected roots of the wild type and mutants with normal clubroot symptoms, whereas they were reduced in infected roots of mutants TU3 and TU8. These results indicate a role for indole glucosinolates and IAN/IAA in relation to symptom development in clubroot disease. Received: 23 July 1998 / Accepted: 12 January 1999     

Biochemical Genetics of Plant Secondary Metabolites in Arabidopsis thaliana: The Glucosinolates

Mutants of Arabidopsis thaliana with a glucosinolate content different from wild type were isolated by screening a mutagenized population of plants. Six mutants were detected out of a population of 1200 screened. One of these mutants, TU1, was analyzed in detail. Leaf and seed tissues of line TU1 lack or have reduced amounts of many of the aliphatic glucosinolates found in the wild type due to a recessive allele, gsm1, of a single nuclear gene, GSM1. The seed phenotype is inherited as a maternal effect suggesting that the embryo is dependent on the maternal tissue for its glucosinolates. Experiments involving feeding of (14)C-labeled intermediates suggested that the gsm1 allele results in a metabolic block which decreases the availability of several amino acid substrates required for glucosinolate biosynthesis: 2-amino-6-methylthiohexanoic acid, 2-amino-7-methylthioheptanoic acid, and 2-amino-8-methylthiooctanoic acid. The mutation does not result in any obvious changes in morphology or growth rate. A pathway for the biosynthesis of glucosinolates in A. thaliana is proposed.     

Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis by defense signaling pathways

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.     

Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. Despite this importance, little is known about the regulation of secondary metabolite accumulation. We are studying the regulation of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate how secondary metabolism is controlled. We utilized Ler and Cvi, two ecotypes of Arabidopsis that have striking differences in both the types and amounts of glucosinolates that accumulate in the seeds and leaves. QTL analysis identified six loci determining total aliphatic glucosinolate accumulation, six loci controlling total indolic glucosinolate concentration, and three loci regulating benzylic glucosinolate levels. Our results show that two of the loci controlling total aliphatic glucosinolates map to biosynthetic loci that interact epistatically to regulate aliphatic glucosinolate accumulation. In addition to the six loci regulating total indolic glucosinolate concentration, mapping of QTL for the individual indolic glucosinolates identified five additional loci that were specific to subsets of the indolic glucosinolates. These data show that there are a large number of variable loci controlling glucosinolate accumulation in Arabidopsis thaliana.     

The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes

The Arabidopsis ref2 mutant was identified in a screen for plants having altered fluorescence under UV light. Characterization of the ref2 mutants showed that they contained reduced levels of a number of phenylpropanoid pathway-derived products: sinapoylmalate in leaves, sinapoylcholine in seeds, and syringyl lignin in stems. Surprisingly, positional cloning of the REF2 locus revealed that it encodes CYP83A1, a cytochrome P450 sharing a high degree of similarity to CYP83B1, an enzyme involved in glucosinolate biosynthesis. Upon further investigation, ref2 mutants were found to have reduced levels of all aliphatic glucosinolates and increased levels of indole-derived glucosinolates in their leaves. These results show that CYP83A1 is involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates and suggest a novel metabolic link between glucosinolate biosynthesis, a secondary biosynthetic pathway found only in plants in the order Capparales, and phenylpropanoid metabolism, a pathway found in all plants and considered essential to the survival of terrestrial plant species.     

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.     

Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate

Cruciferous plants produce a wide variety of glucosinolates as a protection against herbivores and pathogens. However, very little is known about the importance of individual glucosinolates in plant defense and the regulation of their production in response to herbivory. When Myzus persicae (green peach aphid) feeds on Arabidopsis aliphatic glucosinolates pass through the aphid gut intact, but indole glucosinolates are mostly degraded. Although aphid feeding causes an overall decrease in Arabidopsis glucosinolate content, the production of 4-methoxyindol-3-ylmethylglucosinolate is induced. This altered glucosinolate profile is not a systemic plant response, but is limited to the area in which aphids are feeding. Aphid feeding on detached leaves causes a similar change in the glucosinolate profile, demonstrating that glucosinolate transport is not required for the observed changes. Salicylate-mediated signaling has been implicated in other plant responses to aphid feeding. However, analysis of eds5, pad4, npr1 and NahG transgenic Arabidopsis, which are compromised in this pathway, demonstrated that aphid-induced changes in the indole glucosinolate profile were unaffected. The addition of purified indol-3-ylmethylglucosinolate to the petioles of cyp79B2 cyp79B3 mutant leaves, which do not produce indole glucosinolates, showed that this glucosinolate serves as a precursor for the aphid-induced synthesis of 4-methoxyindol-3-ylmethylglucosinolate. In artificial diets, 4-methoxyindol-3-ylmethylglucosinolate is a significantly greater aphid deterrent in the absence of myrosinase than its metabolic precursor indol-3-ylmethylglucosinolate. Together, these results demonstrate that, in response to aphid feeding, Arabidopsis plants convert one indole glucosinolate to another that provides a greater defensive benefit.     

Genetic control of natural variation in Arabidopsis glucosinolate accumulation

Glucosinolates are biologically active secondary metabolites of the Brassicaceae and related plant families that influence plant/insect interactions. Specific glucosinolates can act as feeding deterrents or stimulants, depending upon the insect species. Hence, natural selection might favor the presence of diverse glucosinolate profiles within a given species. We determined quantitative and qualitative variation in glucosinolates in the leaves and seeds of 39 Arabidopsis ecotypes. We identified 34 different glucosinolates, of which the majority are chain-elongated compounds derived from methionine. Polymorphism at only five loci was sufficient to generate 14 qualitatitvely different leaf glucosinolate profiles. Thus, there appears to be a modular genetic system regulating glucosinolate profiles in Arabidopsis. This system allows the rapid generation of new glucosinolate combinations in response to changing herbivory or other selective pressures. In addition to the qualitative variation in glucosinolate profiles, we found a nearly 20-fold difference in the quantity of total aliphatic glucosinolates and were able to identify a single locus that controls nearly three-quarters of this variation.     

Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.     

Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana

The glucosinolate content of various organs of the model plant Arabidopsis thaliana (L.) Heynh., Columbia (Col-0) ecotype, was analyzed at different stages during its life cycle. Significant differences were noted among organs in both glucosinolate concentration and composition. Dormant and germinating seeds had the highest concentration (2.5-3.3% by dry weight), followed by inflorescences, siliques (fruits), leaves and roots. While aliphatic glucosinolates predominated in most organs, indole glucosinolates made up nearly half of the total composition in roots and late-stage rosette leaves. Seeds had a very distinctive glucosinolate composition. They possessed much higher concentrations of several types of aliphatic glucosinolates than other organs, including methylthioalkyl and, hydroxyalkyl glucosinolates and compounds with benzoate esters than other organs. From a developmental perspective, older leaves had lower glucosinolate concentrations than younger leaves, but this was not due to decreasing concentrations in individual leaves with age (glucosinolate concentration was stable during leaf expansion). Rather, leaves initiated earlier in development simply had much lower rates of glucosinolate accumulation per dry weight gain throughout their lifetimes. During seed germination and leaf senescence, there were significant declines in glucosinolate concentration. The physiological and ecological significance of these findings is briefly discussed.     

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis

Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular compartmentation of FMO(GS-OX1) was also detected by transiently expressing a FMO(GS-OX1)-yellow fluorescent protein (YFP) fusion protein in tobacco leaves. The results showed that FMO(GS-OX1-5) were expressed basically in vascular tissues, especially in phloem cells, like other glucosinolate biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other glucosinolate biosynthetic genes. It is suggested that the spatial expression pattern of FMO(GS-OX1-5) determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. FMO(GS-OX1)-YFP fusion protein analysis identified FMO(GS-OX1) as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalized aliphatic glucosinolate biosynthetic pathway is discussed.     

Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana

Glucosinolates are secondary metabolites involved in pathogen and insect defense of cruciferous plants. Although seeds and vegetative tissue often have very different glucosinolate profiles, few genetic factors that determine seed glucosinolate accumulation have been identified. An HPLC-based screen of 5500 mutagenized Arabidopsis thaliana lines produced 33 glucosinolate mutants, of which 21 have seed-specific changes. Five of these mutant lines, representing three genetic loci, are compromised in the biosynthesis of benzoyloxyglucosinolates, which are only found in seeds and young seedlings of A. thaliana. Genetic mapping and analysis of T-DNA insertions in candidate genes identified BZO1 (At1g65880), which encodes an enzyme with benzoyl-CoA ligase activity, as being required for the accumulation of benzoyloxyglucosinolates. Long-chain aliphatic glucosinolates are elevated in bzo1 mutants, suggesting substrate competition for the common short-chain aliphatic glucosinolate precursors. Whereas bzo1 mutations have seed-specific effects on benzoyloxyglucosinolate accumulation, the relative abundance of 3-benzoyloxypropyl- and 4-benzoyloxybutylglucosinolates depends on the maternal genotype.     

Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum

Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate‐derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen‐challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long‐chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate‐derived isothiocyanates was dependent on side chain elongation and modification, with 8‐methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short‐chained aliphatic glucosinolates.     

Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana

The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 Å resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2′-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2′-methylthio)ethylmalate ∼100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.     

Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross-react with other metabolites. These methylsulfinylalkyl glucosinolate specific antibodies have prospective use in multiple applications such as ELISA, co-immunoprecipitation and immunolocalization of glucosinolates.