Publications by authors named "Barbara A Halkier"

33 Publications

Dynamic Modeling of Indole Glucosinolate Hydrolysis and Its Impact on Auxin Signaling.

Front Plant Sci 2018 26;9:550. Epub 2018 Apr 26.

DynaMo Center, Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark.

Plants release chemicals to deter attackers. relies on multiple defense compounds, including indol-3-ylmethyl glucosinolate (I3G), which upon hydrolysis initiated by myrosinase enzymes releases a multitude of bioactive compounds, among others, indole-3-acetonitrile and indole-3-acetoisothiocyanate. The highly unstable isothiocyanate rapidly reacts with other molecules. One of the products, indole-3-carbinol, was reported to inhibit auxin signaling through binding to the TIR1 auxin receptor. On the contrary, the nitrile product of I3G hydrolysis can be converted by nitrilase enzymes to form the primary auxin molecule, indole-3-acetic acid, which activates TIR1. This suggests that auxin signaling is subject to both antagonistic and protagonistic effects of I3G hydrolysis upon attack. We hypothesize that I3G hydrolysis and auxin signaling form an incoherent feedforward loop and we build a mathematical model to examine the regulatory network dynamics. We use molecular docking to investigate the possible antagonistic properties of different I3G hydrolysis products by competitive binding to the TIR1 receptor. Our simulations reveal an uncoupling of auxin concentration and signaling, and we determine that enzyme activity and antagonist binding affinity are key parameters for this uncoupling. The molecular docking predicts that several I3G hydrolysis products strongly antagonize auxin signaling. By comparing a tissue disrupting attack - e.g., by chewing insects or necrotrophic pathogens that causes rapid release of I3G hydrolysis products - to sustained cell-autonomous I3G hydrolysis, e.g., upon infection by biotrophic pathogens, we find that each scenario gives rise to distinct auxin signaling dynamics. This suggests that plants have different defense versus growth strategies depending on the nature of the attack.
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http://dx.doi.org/10.3389/fpls.2018.00550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932361PMC
April 2018

Unravelling Protein-Protein Interaction Networks Linked to Aliphatic and Indole Glucosinolate Biosynthetic Pathways in Arabidopsis.

Front Plant Sci 2017 29;8:2028. Epub 2017 Nov 29.

Department of Plant and Environmental Sciences, Faculty of Science, DynaMo Center, University of Copenhagen, Frederiksberg, Denmark.

Within the cell, biosynthetic pathways are embedded in protein-protein interaction networks. In Arabidopsis, the biosynthetic pathways of aliphatic and indole glucosinolate defense compounds are well-characterized. However, little is known about the spatial orchestration of these enzymes and their interplay with the cellular environment. To address these aspects, we applied two complementary, untargeted approaches-split-ubiquitin yeast 2-hybrid and co-immunoprecipitation screens-to identify proteins interacting with CYP83A1 and CYP83B1, two homologous enzymes specific for aliphatic and indole glucosinolate biosynthesis, respectively. Our analyses reveal distinct functional networks with substantial interconnection among the identified interactors for both pathway-specific markers, and add to our knowledge about how biochemical pathways are connected to cellular processes. Specifically, a group of protein interactors involved in cell death and the hypersensitive response provides a potential link between the glucosinolate defense compounds and defense against biotrophic pathogens, mediated by protein-protein interactions.
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http://dx.doi.org/10.3389/fpls.2017.02028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712850PMC
November 2017

Localization of the glucosinolate biosynthetic enzymes reveals distinct spatial patterns for the biosynthesis of indole and aliphatic glucosinolates.

Physiol Plant 2018 Jun 11;163(2):138-154. Epub 2018 Jan 11.

DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark.

Glucosinolates constitute the primary defense metabolites in Arabidopsis thaliana (Arabidopsis). Indole and aliphatic glucosinolates, biosynthesized from tryptophan and methionine, respectively, are known to serve distinct biological functions. Although all genes in the biosynthetic pathways are identified, and it is known where glucosinolates are stored, it has remained elusive where glucosinolates are produced at the cellular and tissue level. To understand how the spatial organization of the different glucosinolate biosynthetic pathways contributes to their distinct biological functions, we investigated the localization of enzymes of the pathways under constitutive conditions and, for indole glucosinolates, also under induced conditions, by analyzing the spatial distribution of several fluorophore-tagged enzymes at the whole plant and the cellular level. We show that key steps in the biosynthesis of the different types of glucosinolates are localized in distinct cells in separate as well as overlapping vascular tissues. The presence of glucosinolate biosynthetic enzymes in parenchyma cells of the vasculature may assign new defense-related functions to these cell types. The knowledge gained in this study is an important prerequisite for understanding the orchestration of chemical defenses from site of synthesis to site of storage and potential (re)mobilization upon attack.
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http://dx.doi.org/10.1111/ppl.12672DOI Listing
June 2018

Identification of Iridoid Glucoside Transporters in Catharanthus roseus.

Plant Cell Physiol 2017 Sep;58(9):1507-1518

DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

Monoterpenoid indole alkaloids (MIAs) are plant defense compounds and high-value pharmaceuticals. Biosynthesis of the universal MIA precursor, secologanin, is organized between internal phloem-associated parenchyma (IPAP) and epidermis cells. Transporters for intercellular transport of proposed mobile pathway intermediates have remained elusive. Screening of an Arabidopsis thaliana transporter library expressed in Xenopus oocytes identified AtNPF2.9 as a putative iridoid glucoside importer. Eight orthologs were identified in Catharanthus roseus, of which three, CrNPF2.4, CrNPF2.5 and CrNPF2.6, were capable of transporting the iridoid glucosides 7-deoxyloganic acid, loganic acid, loganin and secologanin into oocytes. Based on enzyme expression data and transporter specificity, we propose that several enzymes of the biosynthetic pathway are present in both IPAP and epidermis cells, and that the three transporters are responsible for transporting not only loganic acid, as previously proposed, but multiple intermediates. Identification of the iridoid glucoside-transporting CrNPFs is an important step toward understanding the complex orchestration of the seco-iridioid pathway.
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http://dx.doi.org/10.1093/pcp/pcx097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5921532PMC
September 2017

Albugo-imposed changes to tryptophan-derived antimicrobial metabolite biosynthesis may contribute to suppression of non-host resistance to Phytophthora infestans in Arabidopsis thaliana.

BMC Biol 2017 03 20;15(1):20. Epub 2017 Mar 20.

The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, United Kingdom.

Background: Plants are exposed to diverse pathogens and pests, yet most plants are resistant to most plant pathogens. Non-host resistance describes the ability of all members of a plant species to successfully prevent colonization by any given member of a pathogen species. White blister rust caused by Albugo species can overcome non-host resistance and enable secondary infection and reproduction of usually non-virulent pathogens, including the potato late blight pathogen Phytophthora infestans on Arabidopsis thaliana. However, the molecular basis of host defense suppression in this complex plant-microbe interaction is unclear. Here, we investigate specific defense mechanisms in Arabidopsis that are suppressed by Albugo infection.

Results: Gene expression profiling revealed that two species of Albugo upregulate genes associated with tryptophan-derived antimicrobial metabolites in Arabidopsis. Albugo laibachii-infected tissue has altered levels of these metabolites, with lower indol-3-yl methylglucosinolate and higher camalexin accumulation than uninfected tissue. We investigated the contribution of these Albugo-imposed phenotypes to suppression of non-host resistance to P. infestans. Absence of tryptophan-derived antimicrobial compounds enables P. infestans colonization of Arabidopsis, although to a lesser extent than Albugo-infected tissue. A. laibachii also suppresses a subset of genes regulated by salicylic acid; however, salicylic acid plays only a minor role in non-host resistance to P. infestans.

Conclusions: Albugo sp. alter tryptophan-derived metabolites and suppress elements of the responses to salicylic acid in Arabidopsis. Albugo sp. imposed alterations in tryptophan-derived metabolites may play a role in Arabidopsis non-host resistance to P. infestans. Understanding the basis of non-host resistance to pathogens such as P. infestans could assist in development of strategies to elevate food security.
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http://dx.doi.org/10.1186/s12915-017-0360-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358052PMC
March 2017

Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization.

Sci Rep 2016 06 10;6:27766. Epub 2016 Jun 10.

Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true- from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased sensitivity and renders analysis of weak or transient interactions difficult to perform. In this work, we describe the development of reporters that can be chemically induced to dimerize independently of the investigated interactions and thus alleviate these issues. We incorporated our reporters into the widely used split ubiquitin-, bimolecular fluorescence complementation (BiFC)- and Förster resonance energy transfer (FRET)- based methods and investigated different protein-protein interactions in yeast and plants. We demonstrate the functionality of this concept by the analysis of weakly interacting proteins from specialized metabolism in the model plant Arabidopsis thaliana. Our results illustrate that chemically induced dimerization can function as a built-in control for split-based systems that is easily implemented and allows for direct evaluation of functionality.
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http://dx.doi.org/10.1038/srep27766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901268PMC
June 2016

Natural variation in cross-talk between glucosinolates and onset of flowering in Arabidopsis.

Front Plant Sci 2015 8;6:697. Epub 2015 Sep 8.

Department of Plant and Environmental Sciences, Faculty of Science, DNRF Center DynaMo, University of Copenhagen Frederiksberg, Denmark ; Department of Plant and Environmental Sciences, Faculty of Science, Copenhagen Plant Science Centre, University of Copenhagen Frederiksberg, Denmark.

Naturally variable regulatory networks control different biological processes including reproduction and defense. This variation within regulatory networks enables plants to optimize defense and reproduction in different environments. In this study we investigate the ability of two enzyme-encoding genes in the glucosinolate pathway, AOP2 and AOP3, to affect glucosinolate accumulation and flowering time. We have introduced the two highly similar enzymes into two different AOP (null) accessions, Col-0 and Cph-0, and found that the genes differ in their ability to affect glucosinolate levels and flowering time across the accessions. This indicated that the different glucosinolates produced by AOP2 and AOP3 serve specific regulatory roles in controlling these phenotypes. While the changes in glucosinolate levels were similar in both accessions, the effect on flowering time was dependent on the genetic background pointing to natural variation in cross-talk between defense chemistry and onset of flowering. This variation likely reflects an adaptation to survival in different environments.
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http://dx.doi.org/10.3389/fpls.2015.00697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561820PMC
October 2015

The Glucosinolate Biosynthetic Gene AOP2 Mediates Feed-back Regulation of Jasmonic Acid Signaling in Arabidopsis.

Mol Plant 2015 Aug 7;8(8):1201-12. Epub 2015 Mar 7.

DynaMo Center of Excellence, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark; Department of Plant Sciences, University of California at Davis, Davis, CA 95616, USA.

Survival in changing and challenging environments requires an organism to efficiently obtain and use its resources. Due to their sessile nature, it is particularly critical for plants to dynamically optimize their metabolism. In plant primary metabolism, metabolic fine-tuning involves feed-back mechanisms whereby the output of a pathway controls its input to generate a precise and robust response to environmental changes. By contrast, few studies have addressed the potential for feed-back regulation of secondary metabolism. In Arabidopsis, accumulation of the defense compounds glucosinolates has previously been linked to genetic variation in the glucosinolate biosynthetic gene AOP2. AOP2 expression can increase the transcript levels of two known regulators (MYB28 and MYB29) of the pathway, suggesting that AOP2 plays a role in positive feed-back regulation controlling glucosinolate biosynthesis. We generated mutants affecting AOP2, MYB28/29, or both. Transcriptome analysis of these mutants identified a so far unrecognized link between AOP2 and jasmonic acid (JA) signaling independent of MYB28 and MYB29. Thus, AOP2 is part of a regulatory feed-back loop linking glucosinolate biosynthesis and JA signaling and thereby allows the glucosinolate pathway to influence JA sensitivity. The discovery of this regulatory feed-back loop provides insight into how plants optimize the use of resources for defensive metabolites.
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http://dx.doi.org/10.1016/j.molp.2015.03.001DOI Listing
August 2015

Arabidopsis gulliver1/SUPERROOT2-7 identifies a metabolic basis for auxin and brassinosteroid synergy.

Plant J 2014 Dec;80(5):797-808

School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, 151-747, Korea.

Phytohormone homeostasis is essential for proper growth and development of plants. To understand the growth mechanisms mediated by hormonal levels, we isolated a gulliver1 (gul1) mutant that had tall stature in the presence of both brassinazole and the light. The gul1 phenotype depended on functional BR biosynthesis; the genetic introduction of dwarf4, a BR biosynthetic mutation, masked the long hypocotyl phenotype of gul1. Furthermore, BR biosynthesis was dramatically enhanced, such that the level of 22-hydroxy campesterol was 5.8-fold greater in gul1. Molecular cloning revealed that gul1 was a missense mutation, resulting in a glycine to arginine change at amino acid 116 in SUPERROOT2 (CYP83B1), which converts indole acetaldoxime to an S-alkyl thiohydroximate adduct in the indole glucosinolate pathway. Auxin metabolite profiling coupled with quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of auxin biosynthetic genes revealed that gul1/sur2-7 activated multiple alternative branches of tryptophan-dependent auxin biosynthetic pathways. Furthermore, exogenous treatment of gul1/sur2-7 with BRs caused adventitious roots from hypocotyls, indicative of an increased response to BRs relative to wild-type. Different from severe alleles of sur2, gul1/sur2-7 lacked 'high-auxin' phenotypes that include stunted growth and callus-like disintegration of hypocotyl tissues. The auxin level in gul1/sur2-7 was only 1.6-fold greater than in the wild-type, whereas it was 4.2-fold in a severe allele like sur2-8. Differences in auxin content may account for the range of phenotypes observed among the sur2 alleles. This unusual allele provides long-sought evidence for a synergistic interaction between auxin and BRs in promoting growth in Arabidopsis at the level of their biosynthetic enzymes.
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http://dx.doi.org/10.1111/tpj.12678DOI Listing
December 2014

USER-derived cloning methods and their primer design.

Methods Mol Biol 2014 ;1116:59-72

DynaMo Center of Excellence, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.

Uracil excision-based cloning through USER™ (Uracil-Specific Excision Reagent) is an efficient ligase-free cloning technique that comprises USER cloning, USER fusion, and USER cassette-free (UCF) USER fusion. These USER-derived cloning techniques enable seamless assembly of multiple DNA fragments in one construct. Though governed by a few simple rules primer design for USER-based fusion of PCR fragments can prove time-consuming for inexperienced users. The Primer Help for USER (PHUSER) software is an easy-to-use primer design tool for USER-based methods. In this chapter, we present a PHUSER software protocol for designing primers for USER-derived cloning techniques.
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http://dx.doi.org/10.1007/978-1-62703-764-8_5DOI Listing
September 2014

A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants.

Trends Plant Sci 2014 Jan 18;19(1):5-9. Epub 2013 Sep 18.

Biochimie et Physiologie Moléculaire des Plantes, UMR CNRS/INRA/UM2/SupAgro, Institut de Biologie Intégrative des Plantes 'Claude Grignon', Place Viala, 34060 Montpellier, France. Electronic address:

Members of the plant NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family display protein sequence homology with the SLC15/PepT/PTR/POT family of peptide transporters in animals. In comparison to their animal and bacterial counterparts, these plant proteins transport a wide variety of substrates: nitrate, peptides, amino acids, dicarboxylates, glucosinolates, IAA, and ABA. The phylogenetic relationship of the members of the NRT1/PTR family in 31 fully sequenced plant genomes allowed the identification of unambiguous clades, defining eight subfamilies. The phylogenetic tree was used to determine a unified nomenclature of this family named NPF, for NRT1/PTR FAMILY. We propose that the members should be named accordingly: NPFX.Y, where X denotes the subfamily and Y the individual member within the species.
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http://dx.doi.org/10.1016/j.tplants.2013.08.008DOI Listing
January 2014

De novo genetic engineering of the camalexin biosynthetic pathway.

J Biotechnol 2013 Sep 3;167(3):296-301. Epub 2013 Jul 3.

Center for Dynamic Molecular Interactions, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.

Camalexin is a tryptophan-derived phytoalexin that is induced in the model plant Arabidopsis thaliana upon pathogen attack. Only few genes in the biosynthetic pathway of camalexin remain unidentified, however, investigation of candidate genes for these steps has proven particularly difficult partly because of redundancy in the genome of Arabidopsis. Here we describe metabolic engineering of the camalexin biosynthetic pathway in the transient Nicotiana benthamiana expression system. Camalexin accumulated in levels corresponding to what is seen in induced Arabidopsis thaliana. We have used this system to evaluate candidate genes suggested to be involved in the camalexin pathway. This has provided biochemical evidence for CYP71A12 conducting same reaction as CYP71A13 in the pathway. We discuss the prospects of using metabolic engineering of camalexin, both with respect to engineering plant defense and as a tool for screening yet unidentified candidate genes in the camalexin pathway.
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http://dx.doi.org/10.1016/j.jbiotec.2013.06.013DOI Listing
September 2013

Engineering of glucosinolate biosynthesis: candidate gene identification and validation.

Methods Enzymol 2012 ;515:291-313

Center for Dynamic Molecular Interactions, Department of Plant Biology and Biotechnology, Molecular Plant Biology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej, Frederiksberg C, Copenhagen, Denmark.

The diverse biological roles of glucosinolates as plant defense metabolites and anticancer compounds have spurred a strong interest in their biosynthetic pathways. Since the completion of the Arabidopsis genome, functional genomics approaches have enabled significant progress on the elucidation of glucosinolate biosynthesis, although in planta validation of candidate gene function often is hampered by time-consuming generation of knockout and overexpression lines in Arabidopsis. To better exploit the increasing amount of data available from genomic sequencing, microarray database and RNAseq, time-efficient methods for identification and validation of candidate genes are needed. This chapter covers the methodology we are using for gene discovery in glucosinolate engineering, namely, guilt-by-association-based in silico methods and fast proof-of-function screens by transient expression in Nicotiana benthamiana. Moreover, the lessons learned in the rapid, transient tobacco system are readily translated to our robust, versatile yeast expression platform, where additional genes critical for large-scale microbial production of glucosinolates can be identified. We anticipate that the methodology presented here will be beneficial to elucidate and engineer other plant biosynthetic pathways.
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http://dx.doi.org/10.1016/B978-0-12-394290-6.00020-3DOI Listing
February 2013

Genes involved in the evolution of herbivory by a leaf-mining, Drosophilid fly.

Genome Biol Evol 2012 19;4(9):900-16. Epub 2012 Jul 19.

Department of Ecology and Evolutionary Biology, University of Arizona, AZ, USA.

Herbivorous insects are among the most successful radiations of life. However, we know little about the processes underpinning the evolution of herbivory. We examined the evolution of herbivory in the fly, Scaptomyza flava, whose larvae are leaf miners on species of Brassicaceae, including the widely studied reference plant, Arabidopsis thaliana (Arabidopsis). Scaptomyza flava is phylogenetically nested within the paraphyletic genus Drosophila, and the whole genome sequences available for 12 species of Drosophila facilitated phylogenetic analysis and assembly of a transcriptome for S. flava. A time-calibrated phylogeny indicated that leaf mining in Scaptomyza evolved between 6 and 16 million years ago. Feeding assays showed that biosynthesis of glucosinolates, the major class of antiherbivore chemical defense compounds in mustard leaves, was upregulated by S. flava larval feeding. The presence of glucosinolates in wild-type (WT) Arabidopsis plants reduced S. flava larval weight gain and increased egg-adult development time relative to flies reared in glucosinolate knockout (GKO) plants. An analysis of gene expression differences in 5-day-old larvae reared on WT versus GKO plants showed a total of 341 transcripts that were differentially regulated by glucosinolate uptake in larval S. flava. Of these, approximately a third corresponded to homologs of Drosophila melanogaster genes associated with starvation, dietary toxin-, heat-, oxidation-, and aging-related stress. The upregulated transcripts exhibited elevated rates of protein evolution compared with unregulated transcripts. The remaining differentially regulated transcripts also contained a higher proportion of novel genes than the unregulated transcripts. Thus, the transition to herbivory in Scaptomyza appears to be coupled with the evolution of novel genes and the co-option of conserved stress-related genes.
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http://dx.doi.org/10.1093/gbe/evs063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516228PMC
January 2013

Engineering of benzylglucosinolate in tobacco provides proof-of-concept for dead-end trap crops genetically modified to attract Plutella xylostella (diamondback moth).

Plant Biotechnol J 2012 May 19;10(4):435-42. Epub 2012 Jan 19.

Section for Molecular Plant Biology, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark.

Glucosinolates are biologically active natural products characteristic of crucifers, including oilseed rape, cabbage vegetables and the model plant Arabidopsis thaliana. Crucifer-specialist insect herbivores, like the economically important pest Plutella xylostella (diamondback moth), frequently use glucosinolates as oviposition stimuli. This suggests that the transfer of a glucosinolate biosynthetic pathway to a non-crucifer would stimulate oviposition on an otherwise non-attractive plant. Here, we demonstrate that stable genetic transfer of the six-step benzylglucosinolate pathway from A. thaliana to Nicotiana tabacum (tobacco) results in the production of benzylglucosinolate without causing morphological alterations. Benzylglucosinolate-producing tobacco plants were more attractive for oviposition by female P. xylostella moths than wild-type tobacco plants. As newly hatched P. xylostella larvae were unable to survive on tobacco, these results represent a proof-of-concept strategy for rendering non-host plants attractive for oviposition by specialist herbivores with the long-term goal of generating efficient dead-end trap crops for agriculturally important pests.
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http://dx.doi.org/10.1111/j.1467-7652.2011.00680.xDOI Listing
May 2012

Modulation of sulfur metabolism enables efficient glucosinolate engineering.

BMC Biotechnol 2011 Jan 31;11:12. Epub 2011 Jan 31.

Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Background: Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1), resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization.

Results: To optimize heterologous glucosinolate production, we have in this study performed a comparative metabolite analysis of BGLS-producing N. benthamiana leaves in the presence or absence of GGP1. The analysis revealed that the increased BGLS levels in the presence of GGP1 were accompanied by a high accumulation of the last intermediate, desulfoBGLS, and a derivative thereof. This evidenced a bottleneck in the last step of the pathway, the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to desulfoBGLS by the sulfotransferase AtSOT16. While substitution of AtSOT16 with alternative sulfotransferases did not alleviate the bottleneck, experiments with the three genes involved in the formation and recycling of PAPS showed that co-expression of adenosine 5'-phosphosulfate kinase 2 (APK2) alone reduced the accumulation of desulfoBGLS and its derivative by more than 98% and increased BGLS accumulation 16-fold.

Conclusion: Adjusting sulfur metabolism by directing sulfur from primary to secondary metabolism leads to a remarkable improvement in BGLS accumulation and thereby represents an important step towards a clean and efficient production of glucosinolates in heterologous hosts. Our study emphasizes the importance of considering co-substrates and their biological nature in metabolic engineering projects.
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http://dx.doi.org/10.1186/1472-6750-11-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042935PMC
January 2011

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

J Exp Bot 2011 Jan 15;62(3):1337-46. Epub 2010 Nov 15.

Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Research Centre for Pro-Active Plants, DK-1871 Frederiksberg C, Denmark.

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.
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http://dx.doi.org/10.1093/jxb/erq369DOI Listing
January 2011

Differential effects of indole and aliphatic glucosinolates on lepidopteran herbivores.

J Chem Ecol 2010 Aug 9;36(8):905-13. Epub 2010 Jul 9.

Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Braunschweig, Germany.

Glucosinolates are a diverse group of defensive secondary metabolites that is characteristic of the Brassicales. Arabidopsis thaliana (L.) Heynh. (Brassicaceae) lines with mutations that greatly reduce abundance of indole glucosinolates (cyp79B2 cyp79B3), aliphatic glucosinolates (myb28 myb29), or both (cyp79B2 cyp79B3 myb28 myb29) make it possible to test the in vivo defensive function of these two major glucosinolate classes. In experiments with Lepidoptera that are not crucifer-feeding specialists, aliphatic and indole glucosinolates had an additive effect on Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) larval growth, whereas Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) and Manduca sexta (L.) (Lepidoptera: Sphingidae) were affected only by the absence of aliphatic glucosinolates. In the case of two crucifer-feeding specialists, Pieris rapae (L.) (Lepidoptera: Pieridae) and Plutella xylostella (L.) (Lepidoptera: Plutellidae), there were no major changes in larval performance due to decreased aliphatic and/or indole glucosinolate content. Nevertheless, choice tests show that aliphatic and indole glucosinolates act in an additive manner to promote larval feeding of both species and P. rapae oviposition. Together, these results support the hypothesis that a diversity of glucosinolates is required to limit the growth of multiple insect herbivores.
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http://dx.doi.org/10.1007/s10886-010-9825-zDOI Listing
August 2010

USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.

Methods Mol Biol 2010 ;643:185-200

Department of Plant Biology and Biotechnology, and VKR Centre Pro-Active Plants, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark.

The explosive development of the field of molecular biology has led to the need for simpler and more efficient cloning techniques. These requirements are elegantly met by the ligation-free cloning technique called USER cloning. USER cloning is suitable not only for everyday and high-throughput cloning but also for the one-step construction of complex DNA constructs, which can be achieved in a variant called USER fusion. In this chapter, we present a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.
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http://dx.doi.org/10.1007/978-1-60761-723-5_13DOI Listing
September 2010

Biosynthesis of glucosinolates--gene discovery and beyond.

Trends Plant Sci 2010 May 19;15(5):283-90. Epub 2010 Mar 19.

Plant Biochemistry Laboratory, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

Glucosinolates are sulfur-rich secondary metabolites characteristic of the Brassicales order with important biological and economic roles in plant defense and human nutrition. Application of systems biology tools continues to identify genes involved in the biosynthesis of glucosinolates. Recent progress includes genes in all three phases of the pathway, i.e. side-chain elongation of precursor amino acids, formation of the core glucosinolate structure and side-chain decoration. Major breakthroughs include the ability to produce glucosinolates in Nicotiana benthamiana, the finding that specific glucosinolates play a key role in Arabidopsis innate immune response, and a better understanding of the link between primary sulfur metabolism and glucosinolate biosynthesis.
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http://dx.doi.org/10.1016/j.tplants.2010.02.005DOI Listing
May 2010

Regulatory networks of glucosinolates shape Arabidopsis thaliana fitness.

Curr Opin Plant Biol 2010 Jun 11;13(3):348-53. Epub 2010 Mar 11.

Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark.

Systems biology approaches address higher levels of complex, but dynamic metabolic regulatory networks utilizing single accessions of a species. This contrasts with the likelihood that plants utilize genetic diversity of both individual genes and regulatory networks as a solution to surviving in a complex environment. This would require systems biology to begin a more inclusive search for 'all' networks within a species. In this review, we will highlight how natural genetic diversity within particularly aliphatic glucosinolates in Arabidopsis thaliana and related species has resulted in highly complex, dynamic regulatory networks enabling the plant to adapt to a highly changing environment. We will discuss how this diversity is essential for the fitness performance of A. thaliana.
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http://dx.doi.org/10.1016/j.pbi.2010.02.002DOI Listing
June 2010

Non-volatile intact indole glucosinolates are host recognition cues for ovipositing Plutella xylostella.

J Chem Ecol 2009 Dec;35(12):1427-36

Boyce Thompson Institute for Plant Biology, Ithaca, NY 14853, USA.

The diamondback moth (Plutella xylostella), a crucifer-specialist pest, has been documented to employ glucosinolates as host recognition cues for oviposition. Through the use of mutant Arabidopsis thaliana plants, we investigated the role of specific classes of glucosinolates in the signaling of oviposition by P. xylostella in vivo. Indole glucosinolate production in A. thaliana was found to be crucial in attracting oviposition. Additionally, indole glucosinolates functioned as oviposition cues only when in their intact form. 4-Methoxy-indol-3-ylmethylglucosinolate was implicated as an especially strong oviposition attractant in vitro, suggesting that indole glucosinolate secondary structure may play a role in P. xylostella host recognition as well. Aliphatic glucosinolate-derived breakdown products were found to attract P. xylostella, but only after damage or in the absence of indole glucosinolates. Furthermore, mutant plants lacking both intact indole glucosinolates and aliphatic glucosinolate breakdown products exhibited decreased oviposition attractiveness beyond that of the progenitor mutants lacking either component of the glucosinolate-myrosinase system. Therefore, we conclude that nonvolatile indole glucosinolates and volatile aliphatic glucosinolate breakdown products both appear to play important roles as host recognition cues for P. xylostella oviposition.
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http://dx.doi.org/10.1007/s10886-009-9723-4DOI Listing
December 2009

A novel 2-oxoacid-dependent dioxygenase involved in the formation of the goiterogenic 2-hydroxybut-3-enyl glucosinolate and generalist insect resistance in Arabidopsis,.

Plant Physiol 2008 Dec 22;148(4):2096-108. Epub 2008 Oct 22.

Plant Biochemistry Laboratory, Department of Plant Biology, VKR Centre for Pro-Active Plants, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark.

Glucosinolates are secondary metabolites found almost exclusively in the order Brassicales. They are synthesized from a variety of amino acids and can have numerous side chain modifications that control biological function. We investigated the biosynthesis of 2-hydroxybut-3-enyl glucosinolate, which has biological activities including toxicity to Caenorhabditis elegans, inhibition of seed germination, induction of goiter disease in mammals, and production of bitter flavors in Brassica vegetable crops. Arabidopsis (Arabidopsis thaliana) accessions contain three different patterns of 2-hydroxybut-3-enyl glucosinolate accumulation (present in leaves and seeds, seeds only, or absent) corresponding to three different alleles at a single locus, GSL-OH. Fine-scale mapping of the GSL-OH locus identified a 2-oxoacid-dependent dioxygenase encoded by At2g25450 required for the formation of both 2R- and 2S-2-hydroxybut-3-enyl glucosinolate from the precursor 3-butenyl glucosinolate precursor. Naturally occurring null mutations and T-DNA insertional mutations in At2g25450 exhibit a complete absence of 2-hydroxybut-3-enyl glucosinolate accumulation. Analysis of herbivory by the generalist lepidopteran Trichoplusia ni showed that production of 2-hydroxybut-3-enyl glucosinolate provides increased resistance. These results show that At2g25450 is necessary for the hydroxylation of but-3-enyl glucosinolate to 2-hydroxybut-3-enyl glucosinolate in planta and that this metabolite increases resistance to generalist herbivory.
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http://dx.doi.org/10.1104/pp.108.129981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2593654PMC
December 2008

Determination of the absolute configuration of the glucosinolate methyl sulfoxide group reveals a stereospecific biosynthesis of the side chain.

Phytochemistry 2008 Nov 20;69(15):2737-42. Epub 2008 Oct 20.

Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Strasse 8, D-07745 Jena, Germany.

Glucosinolates are plant metabolites containing an anionic nitrogeneous thioglucosidic core structure and a structurally diverse amino acid-derived side chain, which after hydrolysis by thioglucohydrolases (myrosinases) afford biological active degradation products such as nitriles and isothiocyanates. Structural diversity in glucosinolates is partially due to enzymatic modifications occurring on the preformed core structure, like the recently described oxidation of sulfides to sulfoxides catalyzed by a flavin monooxygenase identified in Arabidopsis thaliana. The enzyme product, 4-methylsulfinylbutylglucosinolate, bears a chiral sulfoxide group in its side chain. We have analyzed the epimeric purity of 4-methylsulfinylbutylglucosinolate by NMR methods using a chiral lanthanide shift reagent. The absolute configuration of the sulfoxide group has been established by comparing the 1H NMR spectra of the two sulfoximine diastereomers of natural 4-methylsulfinylbutylglucosinolate. According to our data, 4-methylsulfinylbutylglucosinolate isolated from broccoli and A. thaliana is a pure epimer and its sulfoxide group has the RS configuration. The product of the A. thaliana flavin monooxygenase has these same properties demonstrating that the enzyme is stereospecific and supporting its involvement in glucosinolate side chain formation.
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http://dx.doi.org/10.1016/j.phytochem.2008.09.008DOI Listing
November 2008

Identifying the molecular basis of QTLs: eQTLs add a new dimension.

Trends Plant Sci 2008 Feb 11;13(2):72-7. Epub 2008 Feb 11.

Department of Plant Biology, University of Copenhagen, Copenhagen, Denmark.

Natural genetic variation within plant species is at the core of plant science ranging from agriculture to evolution. Whereas much progress has been made in mapping quantitative trait loci (QTLs) controlling this natural variation, the elucidation of the underlying molecular mechanisms has remained a bottleneck. Recent systems biology tools have significantly shortened the time required to proceed from a mapped locus to testing of candidate genes. These tools enable research on natural variation to move from simple reductionistic studies focused on individual genes to integrative studies connecting molecular variation at multiple loci with physiological consequences. This review focuses on recent examples that demonstrate how expression QTL data can be used for gene discovery and exploited to untangle complex regulatory networks.
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http://dx.doi.org/10.1016/j.tplants.2007.11.008DOI Listing
February 2008

Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis.

Plant Cell 2007 Jun 15;19(6):2039-52. Epub 2007 Jun 15.

Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology, Faculty of Life Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark.

Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola.
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http://dx.doi.org/10.1105/tpc.107.051383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1955726PMC
June 2007

Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis.

Plant J 2007 Jun 25;50(5):902-10. Epub 2007 Apr 25.

Plant Biochemistry Laboratory, Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark.

The cancer-preventive activity of cruciferous vegetables is commonly attributed to isothiocyanates resulting from the breakdown of the natural products glucosinolates (GSLs). Sulforaphane, the isothiocyanate derived from 4-methylsulfinylbutyl GSL, is thought to be the major agent conferring cancer-preventive properties, whereas the isothiocyanate of 4-methylthiobutyl GSL does not have the same activity. We report the identification of an Arabidopsis flavin-monooxygenase (FMO) enzyme, FMO(GS-OX1), which catalyzes the conversion of methylthioalkyl GSLs into methylsulfinylalkyl GSLs. This is evidenced by biochemical characterization of the recombinant protein, and analyses of the GSL content in FMO(GS-OX1) overexpression lines and an FMO(GS-OX1) knock-out mutant of Arabidopsis. The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs, with an approximately fivefold increase in 4-methylsulfinylbutyl GSL in seeds. Identification of FMO(GS-OX1) provides a molecular tool for breeding of Brassica vegetable crops with increased levels of this important GSL, which has implications for production of functional foods enriched with the cancer-preventive sulforaphane.
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http://dx.doi.org/10.1111/j.1365-313X.2007.03101.xDOI Listing
June 2007

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products.

Nucleic Acids Res 2007 27;35(7):e55. Epub 2007 Mar 27.

Plant Biochemistry Laboratory, Institute of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Denmark.

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5' end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3' overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.
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http://dx.doi.org/10.1093/nar/gkm106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1874642PMC
June 2007

Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes.

Plant Methods 2006 Oct 27;2:17. Epub 2006 Oct 27.

Plant Biochemistry Laboratory, Center for Molecular Plant Physiology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

Background: We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector.

Results: Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized.

Conclusion: Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.
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http://dx.doi.org/10.1186/1746-4811-2-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1637106PMC
October 2006

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments.

Nucleic Acids Res 2006 25;34(18):e122. Epub 2006 Sep 25.

Plant Biochemistry Laboratory, Center for Molecular Plant Physiology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.
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http://dx.doi.org/10.1093/nar/gkl635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635280PMC
December 2006
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