Conservation and clade-specific diversification of pathogen-inducible tryptophan and indole glucosinolate metabolism in Arabidopsis thaliana relatives

? A hallmark of the innate immune system of plants is the biosynthesis of low-molecular-weight compounds referred to as secondary metabolites. Tryptophan-derived branch pathways contribute to the capacity for chemical defense against microbes in Arabidopsis thaliana. ? Here, we investigated phylogenetic patterns of this metabolic pathway in relatives of A. thaliana following inoculation with filamentous fungal pathogens that employ contrasting infection strategies. ? The study revealed unexpected phylogenetic conservation of the pathogen-induced indole glucosinolate (IG) metabolic pathway, including a metabolic shift of IG biosynthesis to 4-methoxyindol-3-ylmethylglucosinolate and IG metabolization. By contrast, indole-3-carboxylic acid and camalexin biosyntheses are clade-specific innovations within this metabolic framework. A Capsella rubella accession was found to be devoid of any IG metabolites and to lack orthologs of two A. thaliana genes needed for 4-methoxyindol-3-ylmethylglucosinolate biosynthesis or hydrolysis. However, C. rubella was found to retain the capacity to deposit callose after treatment with the bacterial flagellin-derived epitope flg22 and pre-invasive resistance against a nonadapted powdery mildew fungus. ? We conclude that pathogen-inducible IG metabolism in the Brassicaceae is evolutionarily ancient, while other tryptophan-derived branch pathways represent relatively recent manifestations of a plant-pathogen arms race. Moreover, at least one Brassicaceae lineage appears to have evolved IG-independent defense signaling and/or output pathway(s).     

Metabolomics for metabolically manipulated plants: effects of tryptophan overproduction

Advances in molecular breeding technologies have enabled manipulation of the concentrations of specific plant components by modifying the genes that play a key role in their production. This has provided new opportunities to enhance the nutritional quality of major crops. However, given that metabolic pathways form a highly integrated network, any alteration in a given biosynthetic pathway is most likely to effect secondary and unpredicted changes in the metabolite profile of other pathways. Metabolomics technologies can contribute to the efficient detection of such unexpected effects caused by genetic modification. This has relevance not only from the perspective of safety evaluations of newly developed crops, but to basic science focused on uncovering hitherto unknown regulatory mechanisms associated with the biosynthesis and catabolism of primary and secondary metabolites in plants. In this review, recent advances in plant metabolic engineering for the overproduction of tryptophan (Trp), one of the essential amino acids, are described. In particular, the efficacy of a transgene OASA1D that encodes a mutant anthranilate synthase (AS) α subunit of rice in specifically elevating levels of Trp without marked secondary effects on the metabolite profile of rice is demonstrated. Related topics, such as regulation of Trp biosynthesis, possible interactions between the biosyntheses of Trp and other aromatic amino acids, and translocation of Trp in are discussed based on findings derived from metabolomic analyses of Trp-overproducing transgenic plants.     

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.     

Glucosinolate metabolism and its control

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. Here, 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.     

Metabolic and evolutionary costs of herbivory defense: systems biology of glucosinolate synthesis

Here, we describe our updated mathematical model of Arabidopsis thaliana Columbia metabolism, which adds the glucosinolates, an important group of secondary metabolites, to the reactions of primary metabolism. In so doing, we also describe the evolutionary origins of the enzymes involved in glucosinolate synthesis. We use this model to address a long-standing question in plant evolutionary biology: whether or not apparently defensive compounds such as glucosinolates are metabolically costly to produce. We use flux balance analysis to estimate the flux through every metabolic reaction in the model both when glucosinolates are synthesized and when they are absent. As a result, we can compare the metabolic costs of cell synthesis with and without these compounds, as well as inferring which reactions have their flux altered by glucosinolate synthesis. We find that glucosinolate production can increase photosynthetic requirements by at least 15% and that this cost is specific to the suite of glucosinolates found in A.?thaliana, with other combinations of glucosinolates being even more costly. These observations suggest that glucosinolates have evolved, and indeed likely continue to evolve, for herbivory defense, since only this interpretation explains the maintenance of such costly traits.     

Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory

Arabidopsis (Arabidopsis thaliana) defenses against herbivores are regulated by the jasmonate (JA) hormonal signaling pathway, which leads to the production of a plethora of defense compounds. Arabidopsis defense compounds include tryptophan-derived metabolites, which limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, we used Arabidopsis mutants disrupted in the synthesis of tryptophan-derived secondary metabolites to identify phytochemicals involved in the defense against T. urticae. We show that of the three tryptophan-dependent pathways found in Arabidopsis, the indole glucosinolate (IG) pathway is necessary and sufficient to assure tryptophan-mediated defense against T. urticae. We demonstrate that all three IGs can limit T. urticae herbivory, but that they must be processed by myrosinases to hinder T. urticae oviposition. Putative IG breakdown products were detected in mite-infested leaves, suggesting in planta processing by myrosinases. Finally, we demonstrate that besides IGs, there are additional JA-regulated defenses that control T. urticae herbivory. Together, our results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple IGs, specific myrosinases, and additional JA-dependent defenses.

Three IGs and specific myrosinases help protect Arabidopsis thaliana against herbivory by the two-spotted spider mite T. urticae.     

Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2–fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.     

Lessons learned from metabolic engineering of cyanogenic glucosides

Plants produce a plethora of secondary metabolites which constitute a wealth of potential pharmaceuticals, pro-vitamins, flavours, fragrances, colorants and toxins as well as a source of natural pesticides. Many of these valuable compounds are only synthesized in exotic plant species or in concentrations too low to facilitate commercialization. In some cases their presence constitutes a health hazard and renders the crops unsuitable for consumption. Metabolic engineering is a powerful tool to alter and ameliorate the secondary metabolite composition of crop plants and gain new desired traits. The interplay of a multitude of biosynthetic pathways and the possibility of metabolic cross-talk combined with an incomplete understanding of the regulation of these pathways, explain why metabolic engineering of plant secondary metabolism is still in its infancy and subject to much trial and error. Cyanogenic glucosides are ancient defense compounds that release toxic HCN upon tissue disruption caused e.g. by chewing insects. The committed steps of the cyanogenic glucoside biosynthetic pathway are encoded by three genes. This unique genetic simplicity and the availability of the corresponding cDNAs have given cyanogenic glucosides pioneering status in metabolic engineering of plant secondary metabolism. In this review, lessons learned from metabolic engineering of cyanogenic glucosides in Arabidopsis thaliana (thale cress), Nicotiana tabacum cv Xanthi (tobacco), Manihot esculenta Crantz (cassava) and Lotus japonicus (bird’s foot trefoil) are presented. The importance of metabolic channelling of toxic intermediates as mediated by metabolon formation in avoiding unintended metabolic cross-talk and unwanted pleiotropic effects is emphasized. Likewise, the potential of metabolic engineering of plant secondary metabolism as a tool to elucidate, for example, the impact of secondary metabolites on plant–insect interactions is demonstrated.     

Regulation of plant glucosinolate metabolism

Glucosinolates and their degradation products are known to play important roles in plant interaction with herbivores and micro-organisms. In addition, they are important for human life. For example, some degradation products are flavor compounds and some exhibit anticarcinogenic properties. Recent years have seen great progress made in the understanding of glucosinolate biosynthesis in Arabidopsis thaliana. The core glucosinolate biosynthetic pathway has been revealed using biochemical and reverse genetics approaches. Future research needs to focus on questions related to regulation and control of glucosinolate metabolism. Here we review current status of studies on the regulation of glucosinolate metabolism at different levels, and highlight future research towards elucidating the signaling and metabolic network that control glucosinolate metabolism.     

Characterization of metabolite quantitative trait loci and metabolic networks that control glucosinolate concentration in the seeds and leaves of Brassica napus

? Glucosinolates are a major class of secondary metabolites found in the Brassicaceae, whose degradation products are proving to be increasingly important for human health and in crop protection. ? The genetic and metabolic basis of glucosinolate accumulation was dissected through analysis of total glucosinolate concentration and its individual components in both leaves and seeds of a doubled-haploid (DH) mapping population of oilseed rape/canola (Brassica napus). ? The quantitative trait loci (QTL) that had an effect on glucosinolate concentration in either or both of the organs were integrated, resulting in 105 metabolite QTL (mQTL). Pairwise correlations between individual glucosinolates and prior knowledge of the metabolic pathways involved in the biosynthesis of different glucosinolates allowed us to predict the function of genes underlying the mQTL. Moreover, this information allowed us to construct an advanced metabolic network and associated epistatic interactions responsible for the glucosinolate composition in both leaves and seeds of B. napus. ? A number of previously unknown potential regulatory relationships involved in glucosinolate synthesis were identified and this study illustrates how genetic variation can affect a biochemical pathway.     

Brassinosteroids fine-tune secondary and primary sulfur metabolism through BZR1-mediated transcriptional regulation

For adaptation to ever-changing environments,plants have evolved elaborate metabolic systems coupled to a regulatory network for optimal growth and defense. Regulation of plant secondary metabolic pathways such as glucosinolates(GSLs) by defense phytohormones in response to different stresses and nutrient deficiency has been intensively investigated, while how growth-promoting hormone balances plant secondary and primary metabolism has been largely unexplored. Here, we found that growth-promotin...     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.