Publications by authors named "Christoph Crocoll"

35 Publications

VC1 catalyses a key step in the biosynthesis of vicine in faba bean.

Nat Plants 2021 07 5;7(7):923-931. Epub 2021 Jul 5.

Section for Plant Biochemistry and Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark.

Faba bean (Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds. However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in 400 million genetically predisposed individuals. Here, we use gene-to-metabolite correlations, gene mapping and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1. Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients.
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http://dx.doi.org/10.1038/s41477-021-00950-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611347PMC
July 2021

Specialized Metabolites and Valuable Molecules in Crop and Medicinal Plants: The Evolution of Their Use and Strategies for Their Production.

Genes (Basel) 2021 06 18;12(6). Epub 2021 Jun 18.

Department of Agricultural Science, University of Naples Federico II, Via Università 100, 80055 Portici, NA, Italy.

Plants naturally produce a terrific diversity of molecules, which we exploit for promoting our overall well-being. Plants are also green factories. Indeed, they may be exploited to biosynthesize bioactive molecules, proteins, carbohydrates and biopolymers for sustainable and large-scale production. These molecules are easily converted into commodities such as pharmaceuticals, antioxidants, food, feed and biofuels for multiple industrial processes. Novel plant biotechnological, genetics and metabolic insights ensure and increase the applicability of plant-derived compounds in several industrial sectors. In particular, synergy between disciplines, including apparently distant ones such as plant physiology, pharmacology, 'omics sciences, bioinformatics and nanotechnology paves the path to novel applications of the so-called molecular farming. We present an overview of the novel studies recently published regarding these issues in the hope to have brought out all the interesting aspects of these published studies.
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http://dx.doi.org/10.3390/genes12060936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8235196PMC
June 2021

Sugar transporters enable a leaf beetle to accumulate plant defense compounds.

Nat Commun 2021 05 11;12(1):2658. Epub 2021 May 11.

Research Group Sequestration and Detoxification in Insects, Max Planck Institute for Chemical Ecology, Jena, Germany.

Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates glucosinolate defense compounds from Brassicaceae in the hemolymph. Using phylogenetic analyses of coleopteran major facilitator superfamily transporters, we identify a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTRs are predominantly expressed in the excretory system, the Malpighian tubules. Silencing of PaGTRs leads to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays corroborated the selective retention of glucosinolates by Malpighian tubules of P. armoraciae in situ. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion.
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http://dx.doi.org/10.1038/s41467-021-22982-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113468PMC
May 2021

The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness.

Nat Plants 2021 04 11;7(4):468-480. Epub 2021 Mar 11.

Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel.

Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α-tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 resequenced genomes and genotyping a 650-tomato core collection to identify nine bitter-tasting accessions including the 'high tomatine' Peruvian landraces reported in the literature. These 'bitter' accessions contain a deletion in GORKY, a nitrate/peptide family transporter mediating α-tomatine subcellular localization during fruit ripening. GORKY exports α-tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α-tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a notable innovation in the process of tomato fruit domestication and breeding.
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http://dx.doi.org/10.1038/s41477-021-00865-6DOI Listing
April 2021

Engineering and optimization of the 2-phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana.

Plant J 2021 May 22;106(4):978-992. Epub 2021 Mar 22.

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

2-Phenylethylglucosinolate (2PE) derived from homophenylalanine is present in plants of the Brassicales order as a defense compound. It is associated with multiple biological properties, including deterrent effects on pests and antimicrobial and health-promoting functions, due to its hydrolysis product 2-phenylethyl isothiocyanate, which confers 2PE as a potential application in agriculture and industry. In this study, we characterized the putative key genes for 2PE biosynthesis from Barbarea vulgaris W.T. Aiton and demonstrated the feasibility of engineering 2PE production in Nicotiana benthamiana Domin. We used different combinations of genes from B. vulgaris and Arabidopsis thaliana (L.) Heynh. to demonstrate that: (i) BvBCAT4 performed more efficiently than AtBCAT4 in biosynthesis of both homophenylalanine and dihomomethionine; (ii) MAM1 enzymes were critical for the chain-elongated profile, while CYP79F enzymes accepted both chain-elongated methionine and homophenylalanine; (iii) aliphatic but not aromatic core structure pathway catalyzed the 2PE biosynthesis; (iv) a chimeric pathway containing BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in a two-fold increase in 2PE production compared with the B. vulgaris-specific chain elongation pathway; and (v) profiles of chain-elongated products and glucosinolates partially mirrored the profiles in the gene donor plant, but were wider in N. benthamiana than in the native plants. Our study provides a strategy to produce the important homophenylalanine and 2PE in a heterologous host. Furthermore, chimeric engineering of the complex 2PE biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution. The new-to-nature gene combinations have the potential for application in biotechnological and plant breeding.
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http://dx.doi.org/10.1111/tpj.15212DOI Listing
May 2021

A flavin-dependent monooxygenase catalyzes the initial step in cyanogenic glycoside synthesis in ferns.

Commun Biol 2020 09 11;3(1):507. Epub 2020 Sep 11.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark.

Cyanogenic glycosides form part of a binary plant defense system that, upon catabolism, detonates a toxic hydrogen cyanide bomb. In seed plants, the initial step of cyanogenic glycoside biosynthesis-the conversion of an amino acid to the corresponding aldoxime-is catalyzed by a cytochrome P450 from the CYP79 family. An evolutionary conundrum arises, as no CYP79s have been identified in ferns, despite cyanogenic glycoside occurrence in several fern species. Here, we report that a flavin-dependent monooxygenase (fern oxime synthase; FOS1), catalyzes the first step of cyanogenic glycoside biosynthesis in two fern species (Phlebodium aureum and Pteridium aquilinum), demonstrating convergent evolution of biosynthesis across the plant kingdom. The FOS1 sequence from the two species is near identical (98%), despite diversifying 140 MYA. Recombinant FOS1 was isolated as a catalytic active dimer, and in planta, catalyzes formation of an N-hydroxylated primary amino acid; a class of metabolite not previously observed in plants.
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http://dx.doi.org/10.1038/s42003-020-01224-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486406PMC
September 2020

In Substrate Recognition and Tissue- as Well as Plastid Type-Specific Expression Define the Roles of Distinct Small Subunits of Isopropylmalate Isomerase.

Front Plant Sci 2020 16;11:808. Epub 2020 Jun 16.

Institut für Molekulare Botanik, Fakultät für Naturwissenschaften, Universität Ulm, Ulm, Germany.

In , the heterodimeric isopropylmalate isomerase (IPMI) is composed of a single large (IPMI LSU1) and one of three different small subunits (IPMI SSU1 to 3). The function of IPMI is defined by the small subunits. IPMI SSU1 is required for Leu biosynthesis and has previously also been proposed to be involved in the first cycle of Met chain elongation, the first phase of the synthesis of Met-derived glucosinolates. IPMI SSU2 and IPMI SSU3 participate in the Met chain elongation pathway. Here, we investigate the role of the three IPMI SSUs through the analysis of the role of the substrate recognition region spanning five amino acids on the substrate specificity of IPMI SSU1. Furthermore, we analyze in detail the expression pattern of fluorophore-tagged IPMI SSUs throughout plant development. Our study shows that the substrate recognition region that differs between IPMI SSU1 and the other two IMPI SSUs determines the substrate preference of IPMI. Expression of IPMI SSU1 is spatially separated from the expression of IPMI SSU2 and IPMI SSU3, and IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. Our data show a distinct role for IMPI SSU1 in Leu biosynthesis and for IMPI SSU2 and SSU3 in the Met chain elongation pathway.
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http://dx.doi.org/10.3389/fpls.2020.00808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308503PMC
June 2020

Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Metabolites during Sorghum Germination.

Plant Physiol 2020 07 29;183(3):925-942. Epub 2020 Apr 29.

VILLUM Research Center for Plant Plasticity, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg 1871, Denmark

Dhurrin is the most abundant cyanogenic glucoside found in sorghum ( where it plays a key role in chemical defense by releasing toxic hydrogen cyanide upon tissue disruption. Besides this well-established function, there is strong evidence that dhurrin plays additional roles, e.g. as a transport and storage form of nitrogen, released via endogenous recycling pathways. However, knowledge about how, when and why dhurrin is endogenously metabolized is limited. We combined targeted metabolite profiling with matrix-assisted laser desorption/ionization-mass spectrometry imaging to investigate accumulation of dhurrin, its recycling products and key general metabolites in four different sorghum lines during 72 h of grain imbibition, germination and early seedling development, as well as the spatial distribution of these metabolites in two of the lines. Little or no dhurrin or recycling products were present in the dry grain, but their de novo biosynthesis started immediately after water uptake. Dhurrin accumulation increased rapidly within the first 24 h in parallel with an increase in free amino acids, a key event in seed germination. The trajectories and final concentrations of dhurrin, the recycling products and free amino acids reached within the experimental period were dependent on genotype. Matrix-assisted laser desorption/ionization-mass spectrometry imaging demonstrated that dhurrin primarily accumulated in the germinating embryo, confirming its function in protecting the emerging tissue against herbivory. The dhurrin recycling products, however, were mainly located in the scutellum and/or pericarp/seed coat region, suggesting unknown key functions in germination.
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http://dx.doi.org/10.1104/pp.19.01357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333723PMC
July 2020

De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew.

Plant Cell Environ 2020 06 26;43(6):1571-1583. Epub 2020 Apr 26.

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

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.
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http://dx.doi.org/10.1111/pce.13766DOI Listing
June 2020

Characterization of Arabidopsis CYP79C1 and CYP79C2 by Glucosinolate Pathway Engineering in Shows Substrate Specificity Toward a Range of Aliphatic and Aromatic Amino Acids.

Front Plant Sci 2020 14;11:57. Epub 2020 Feb 14.

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

Glucosinolates (GLSs) are amino acid-derived defense compounds characteristic of the Brassicales order. Cytochromes P450s of the CYP79 family are the entry point into the biosynthetic pathway of the GLS core structure and catalyze the conversion of amino acids to oximes. In , CYP79A2, CYP79B2, CYP79B3, CYP79F1, and CYP79F2 have been functionally characterized and are responsible for the biosynthesis of phenylalanine-, tryptophan-, and methionine-derived GLSs, respectively. However, the substrate(s) for CYP79C1 and CYP79C2 were unknown. Here, we investigated the function of CYP79C1 and CYP79C2 by transiently co-expressing the genes together with three sets of remaining genes required for GLS biosynthesis in . Co-expression of with either the aliphatic or aromatic core structure pathways resulted in the production of primarily leucine-derived 2-methylpropyl GLS and phenylalanine-derived benzyl GLS, along with minor amounts of GLSs from isoleucine, tryptophan, and tyrosine. Co-expression of displayed minor amounts of GLSs from valine, leucine, isoleucine, and phenylalanine with the aliphatic core structure pathway, and similar GLS profile (except the GLS from valine) with the aromatic core structure pathway. Additionally, we co-expressed and with the chain elongation and aliphatic core structure pathways. With the chain elongation pathway, CYP79C2 still mainly produced 2-methylpropyl GLS derived from leucine, accompanied by GLSs derived from isoleucine and from chain-elongated mono- and dihomoleucine, but not from phenylalanine. However, co-expression of only resulted in GLSs derived from chain-elongated amino acid substrates, dihomoleucine and dihomomethionine, when the chain elongation pathway was present. This shows that CYP79 activity depends on the specific pathways co-expressed and availability of amino acid precursors, and that description of GLS core structure pathways as "aliphatic" and "aromatic" pathways is not suitable, especially in an engineering context. This is the first characterization of members of the CYP79C family. Co-expression of CYP79 enzymes with engineered GLS pathways in is a valuable tool for simultaneous testing of substrate specificity against multiple amino acids.
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http://dx.doi.org/10.3389/fpls.2020.00057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033466PMC
February 2020

The γ-hydroxybutyric acid (GHB) analogue NCS-382 is a substrate for both monocarboxylate transporters subtypes 1 and 4.

Eur J Pharm Sci 2020 Feb 20;143:105203. Epub 2019 Dec 20.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

The small-molecule ligand (E)-2-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is an analogue of γ-hydroxybutyric acid (GHB) and is widely used for probing the brain-specific GHB high-affinity binding sites. To reach these, brain uptake is imperative, and it is therefore important to understand the molecular mechanisms of NCS-382 transport in order to direct in vivo studies. In this study, we hypothesized that NCS-382 is a substrate for the monocarboxylate transporter subtype 1 (MCT1) which is known to mediate blood-brain barrier (BBB) permeation of GHB. For this purpose, we investigated NCS-382 uptake by MCT subtypes endogenously expressed in tsA201 and MDA-MB-231 cell lines in assays of radioligand-based competition and fluorescence-based intracellular pH measurements. To further verify the results, we measured NCS-382 uptake by means of mass spectrometry in Xenopus laevis oocytes heterologously expressing MCT subtypes. As expected, we found that NCS-382 is a substrate for MCT1 with half-maximal effective concentrations in the low millimolar range. Surprisingly, NCS-382 also showed substrate activity at MCT4 as well as uptake in water-injected oocytes, suggesting a component of passive diffusion. In conclusion, transport of NCS-382 across membranes differs from GHB as it also involves MCT4 and/or passive diffusion. This should be taken into consideration when designing pharmacological studies with this compound and its closely related analogues. The combination of MCT assays used here exemplifies a setup that may be suitable for a reliable characterization of MCT ligands in general.
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http://dx.doi.org/10.1016/j.ejps.2019.105203DOI Listing
February 2020

Metabolic engineering of Synechocystis sp. PCC 6803 for the production of aromatic amino acids and derived phenylpropanoids.

Metab Eng 2020 01 10;57:129-139. Epub 2019 Nov 10.

Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark. Electronic address:

In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, these organisms do not directly compete with agricultural resources. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. Amino acids are traditionally produced by extraction from protein hydrolysates, chemical synthesis or fermentation pathways using heterotrophic microorganisms. In this work we demonstrate for the first time the efficient overproduction of phenylalanine and tyrosine from CO in a Synechocystis sp. PCC 6803 strain heterologously expressing the feedback-inhibition-resistant AroG and TyrA enzymes from E. coli. Production titers reached 904 ± 53 mg/gDW (580 ± 34 mg/L) of phenylalanine and 64 ± 3.7 mg/gDW (41 ± 2.3 mg/L) of tyrosine after 10 days of photoautotrophic growth. We estimate that the production of the two amino acids corresponds to 56% of the total fixed carbon. Phenylalanine and tyrosine are the precursors for phenylpropanoids, thus, we tested the functionality of several phenylpropanoid biosynthetic enzymes in the generated cyanobacterium strains and successfully achieved the production of 470 ± 70 mg/gDW (207 mg/L) of p-coumaric acid, 267 ± 31 mg/gDW (114 mg/L) of cinnamic acid and 47.4 ± 13.9 mg/gDW (12.6 mg/L) of caffeic acid after 6 days of photoautotrophic growth. All compounds were secreted to the growth medium. Our work enlarges the repertoire and yield of heterologous chemicals produced by Synechocystis and contributes to extend the molecular knowledge about this cyanobacterium.
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http://dx.doi.org/10.1016/j.ymben.2019.11.002DOI Listing
January 2020

An Optimized Screen Reduces the Number of GA Transporters and Provides Insights Into Nitrate Transporter 1/Peptide Transporter Family Substrate Determinants.

Front Plant Sci 2019 3;10:1106. Epub 2019 Oct 3.

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

Based on recent in vitro data, a relatively large number of the plant nitrate transporter 1/peptide transporter family (NPF) proteins have been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes, wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities toward organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis suggests distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed.
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http://dx.doi.org/10.3389/fpls.2019.01106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785635PMC
October 2019

GTR-Mediated Radial Import Directs Accumulation of Defensive Glucosinolates to Sulfur-Rich Cells in the Phloem Cap of Arabidopsis Inflorescence Stem.

Mol Plant 2019 11 29;12(11):1474-1484. Epub 2019 Jun 29.

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

In the phloem cap region of Arabidopsis plants, sulfur-rich cells (S-cells) accumulate >100 mM glucosinolates (GLS), but are biosynthetically inactive. The source and route of S-cell-bound GLS remain elusive. In this study, using single-cell sampling and scanning electron microscopy with energy-dispersive X-ray analysis we show that two GLS importers, NPF2.10/GTR1 and NPF2.11/GTR2, are critical for GLS accumulation in S-cells, although they are not localized in the S-cells. Comparison of GLS levels in S-cells in multiple combinations of homo- and heterografts of gtr1 gtr2, biosynthetic null mutant and wild-type plants indicate that S-cells accumulate GLS via symplasmic connections either directly from neighboring biosynthetic cells or indirectly to non-neighboring cells expressing GTR1/2. Distinct sources and transport routes exist for different types of GLS, and vary depending on the position of S-cells in the inflorescence stem. Based on these findings, we propose a model illustrating the GLS transport routes either directly from biosynthetic cells or via GTR-mediated import from apoplastic space radially into a symplasmic domain, wherein the S-cells are the ultimate sink. Similarly, we observed accumulation of the cyanogenic glucoside defensive compounds in high-turgor cells in the phloem cap of Lotus japonicus, suggesting that storage of defensive compounds in high-turgor cells may be a general mechanism for chemical protection of the phloem cap.
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http://dx.doi.org/10.1016/j.molp.2019.06.008DOI Listing
November 2019

Changing substrate specificity and iteration of amino acid chain elongation in glucosinolate biosynthesis through targeted mutagenesis of methylthioalkylmalate synthase 1.

Biosci Rep 2019 07 2;39(7). Epub 2019 Jul 2.

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

Methylthioalkylmalate synthases catalyse the committing step of amino acid chain elongation in glucosinolate biosynthesis. As such, this group of enzymes plays an important role in determining the glucosinolate composition of Brassicaceae species, including Based on protein structure modelling of MAM1 from and analysis of 57 MAM sequences from Brassicaceae species, we identified four polymorphic residues likely to interact with the 2-oxo acid substrate. Through site-directed mutagenesis, the natural variation in these residues and the effect on product composition were investigated. Fifteen MAM1 variants as well as the native MAM1 and MAM3 from were characterised by heterologous expression of the glucosinolate chain elongation pathway in Detected products derived from leucine, methionine or phenylalanine were elongated with up to six methylene groups. Product profile and accumulation were changed in 14 of the variants, demonstrating the relevance of the identified residues. The majority of the single amino acid substitutions decreased the length of methionine-derived products, while approximately half of the substitutions increased the phenylalanine-derived products. Combining two substitutions enabled the MAM1 variant to increase the number of elongation rounds of methionine from three to four. Notably, characterisation of the native MAMs indicated that MAM1 and not MAM3 is responsible for homophenylalanine production. This hypothesis was confirmed by glucosinolate analysis in and mutants of .
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http://dx.doi.org/10.1042/BSR20190446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603273PMC
July 2019

Deletion of biosynthetic genes, specific SNP patterns and differences in transcript accumulation cause variation in hydroxynitrile glucoside content in barley cultivars.

Sci Rep 2019 04 5;9(1):5730. Epub 2019 Apr 5.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark.

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides, potentially involved in alleviating pathogen and environmental stresses. These compounds include the cyanogenic glucoside epiheterodendrin. The biosynthetic genes are clustered. Total hydroxynitrile glucoside contents were previously shown to vary from zero to more than 10,000 nmoles g in different barley lines. To elucidate the cause of this variation, the biosynthetic genes from the high-level producer cv. Mentor, the medium-level producer cv. Pallas, and the zero-level producer cv. Emir were investigated. In cv. Emir, a major deletion in the genome spanning most of the hydroxynitrile glucoside biosynthetic gene cluster was identified and explains the complete absence of hydroxynitrile glucosides in this cultivar. The transcript levels of the biosynthetic genes were significantly higher in the high-level producer cv. Mentor compared to the medium-level producer cv. Pallas, indicating transcriptional regulation as a contributor to the variation in hydroxynitrile glucoside levels. A correlation between distinct single nucleotide polymorphism (SNP) patterns in the biosynthetic gene cluster and the hydroxynitrile glucoside levels in 227 barley lines was identified. It is remarkable that in spite of the demonstrated presence of a multitude of SNPs and differences in transcript levels, the ratio between the five hydroxynitrile glucosides is maintained across all the analysed barley lines. This implies the involvement of a stably assembled multienzyme complex.
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http://dx.doi.org/10.1038/s41598-019-41884-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450869PMC
April 2019

De novo production of benzyl glucosinolate in Escherichia coli.

Metab Eng 2019 07 1;54:24-34. Epub 2019 Mar 1.

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

Microbial production of plant specialised metabolites is challenging as the biosynthetic pathways are often complex and can contain enzymes, which function is not supported in traditional production hosts. Glucosinolates are specialised metabolites of strong commercial interest due to their health-promoting effects. In this work, we engineered the production of benzyl glucosinolate in Escherichia coli. We systematically optimised the production levels by first screening different expression strains and by modification of growth conditions and media compositions. This resulted in production from undetectable to approximately 4.1 μM benzyl glucosinolate, but also approximately 3.7 μM of desulfo-benzyl glucosinolate, the final intermediate of this pathway. Additional optimisation of pathway flux through entry point cytochrome P450 enzymes and PAPS-dependent sulfotransferase increased the production additionally 5-fold to 20.3 μM (equivalent to 8.3 mg/L) benzyl glucosinolate.
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http://dx.doi.org/10.1016/j.ymben.2019.02.004DOI Listing
July 2019

Biotechnological approaches in glucosinolate production.

J Integr Plant Biol 2018 Dec 1;60(12):1231-1248. Epub 2018 Oct 1.

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

Glucosinolates (GLSs) are sulfur-rich, amino acid-derived defense compounds characteristic of the Brassicales order. In the past, GLSs were mostly known as anti-nutritional factors in fodder, biopesticides in agriculture, and flavors in condiments such as mustard. However, in recent times, GLSs have received increased attention as promoters of human health. This has spurred intensive research towards generating rich sources of health-promoting GLSs. We provide a comprehensive overview of the biotechnological approaches applied to reach this goal. This includes optimization of GLS production and composition in native, GLS-producing plants, including hairy root and cell cultures thereof, as well as synthetic biology approaches in heterologous hosts, such as tobacco and the microbial organisms Escherichia coli and Saccharomyces cerevisiae. The progress using these different approaches is discussed.
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http://dx.doi.org/10.1111/jipb.12705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585788PMC
December 2018

Glutathione transferases catalyze recycling of auto-toxic cyanogenic glucosides in sorghum.

Plant J 2018 06 19;94(6):1109-1125. Epub 2018 May 19.

VILLUM Research Center for Plant Plasticity, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, 1871, Denmark.

Cyanogenic glucosides are nitrogen-containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin-derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant-specific lambda class (GSTLs) to produce p-hydroxyphenyl acetonitrile. This is further metabolized to p-hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p-glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co-localized with expression of the genes encoding the p-hydroxyphenyl acetonitrile-metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue.
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http://dx.doi.org/10.1111/tpj.13923DOI Listing
June 2018

Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus.

Plant J 2018 03 14;93(5):943-958. Epub 2018 Feb 14.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.

Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species.
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http://dx.doi.org/10.1111/tpj.13822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838521PMC
March 2018

Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata.

J Nat Prod 2017 10 4;80(10):2692-2698. Epub 2017 Oct 4.

Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia , Adelaide, South Australia 5000, Australia.

The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.
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http://dx.doi.org/10.1021/acs.jnatprod.7b00437DOI Listing
October 2017

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways.

Microb Cell Fact 2017 Aug 15;16(1):140. Epub 2017 Aug 15.

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

Background: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria.

Results: In this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites.

Conclusions: There is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to 'traditional' metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.
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http://dx.doi.org/10.1186/s12934-017-0757-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556357PMC
August 2017

Biological activity and LC-MS/MS profiling of extracts from the Australian medicinal plant Acacia ligulata (Fabaceae).

Nat Prod Res 2018 Mar 21;32(5):576-581. Epub 2017 Apr 21.

a School of Pharmacy and Medical Sciences, Quality Use of Medicines and Pharmacy Research Centre , Sansom Institute for Health Research, University of South Australia , Adelaide , Australia.

Acacia ligulata A.Cunn. ex Benth. (Fabaceae: Mimosoideae) is a native Australian plant used traditionally by Australian Aboriginal groups. This study was undertaken to investigate the bioactivity of A. ligulata extracts and to evaluate their chemical composition. Potential antibacterial, cytotoxic and enzyme inhibitory effects relevant to traditional medicinal and food uses of the species were examined and LC-MS/MS was performed to investigate the chemical composition. Antibacterial activity was observed for bark and leaf extracts with an MIC for the bark extract of 62.5 μg/mL against Streptococcus pyogenes. Pod extracts showed cytotoxic effects against cancer cells, with the highest activity against melanoma SK-MEL28 cells with IC values between 40.8 and 80.6 μg/mL. Further, the leaf and pod extracts also inhibited α-amylase EC-3.2.1.1 and α-glucosidase EC-3.2.1.20 with IC values between 9.7-34.8 and 12.6-64.3 μg/mL, respectively. The LC-MS/MS profiling indicated that several different saponins were present in the active extracts.
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http://dx.doi.org/10.1080/14786419.2017.1318383DOI Listing
March 2018

Foliar-applied ethephon enhances the content of anthocyanin of black carrot roots (Daucus carota ssp. sativus var. atrorubens Alef.).

BMC Plant Biol 2017 04 4;17(1):70. Epub 2017 Apr 4.

Section for Crop Sciences, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Hoejbakkegaard Alle 9-13, 2630, Taastrup, Denmark.

Background: Black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) constitute a valuable source of anthocyanins, which are used as natural red, blue and purple food colourants. Anthocyanins and phenolic compounds are specialised metabolites, accumulation of which often requires elicitors, which act as molecular signals in plant stress responses. In the present study, ethephon, an ethylene-generating compound was explored as enhancer of anthocyanin and phenolic contents during growth of 'Deep Purple' black carrots. The effects of ethephon on several parameters were investigated, and the expression of biosynthetic anthocyanin genes was studied during growth and anthocyanin accumulation.

Results: Roots of ethephon-treated carrot plants exhibited an increase in anthocyanin content of approximately 25%, with values ranging from 2.25 to 3.10 mg g fresh weight, compared with values ranging from 1.50 to 1.90 mg g fresh weight in untreated roots. The most rapid accumulation rate for anthocyanins, phenolic compounds, soluble solids and dry matter was observed between 10 and 13 weeks after sowing in both untreated and ethephon-treated carrots. The differences in anthocyanin contents between untreated and treated carrots increased for several weeks after the ethephon treatment was terminated. Five cyanidin-based anthocyanin forms were identified, with variable relative abundance values detected during root growth. Overall, the expression of the anthocyanin biosynthetic genes analysed (PAL1, PAL3, F3H1, DFR1, LDOX2) increased in response to ethephon treatment, as did the expression of the MYB1 transcription factor, which is associated with activation of the phenylpropanoid pathway under stress conditions. In addition, a correlation was proposed between ethylene and sugar contents and the induction of anthocyanin synthesis.

Conclusions: This study presents a novel method for enhancing anthocyanin content in black carrots. This finding is of economic importance as increased pigment concentration per unit of biomass implies improved profitability parameters in food colour production. We provide new insight into the accumulation patterns of the different cyanidin-based anthocyanins and phenolic compounds during root growth. Moreover, we show that enhanced anthocyanin content in ethephon-treated carrots is accompanied by increased expression of anthocyanin biosynthetic genes.
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http://dx.doi.org/10.1186/s12870-017-1021-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381149PMC
April 2017

Tissue-specific gene-expression patterns of genes associated with thymol/carvacrol biosynthesis in thyme (Thymus vulgaris L.) and their differential changes upon treatment with abiotic elicitors.

Plant Physiol Biochem 2017 Jun 25;115:152-162. Epub 2017 Mar 25.

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

Thyme (Thymus vulgaris L.) is known to produce a variety of phenolic monoterpenes such as thymol and carvacrol. Thymol and carvacrol are health-promoting, biocide and antitoxin compounds and have been considered as the main constituents of essential oils in T. vulgaris. To improve our understanding of the regulation of monoterpene biosynthesis in thyme, the expression of genes related to thymol and carvacrol biosynthesis in different tissues and in response to abiotic elicitors was analyzed. Methyl jasmonate (MeJA), salicylic acid (SA), trans-cinnamic acid (tCA) and UV-C irradiation were applied to T. vulgare leaves and transcript levels of early (DXR) and late (TvTPS1, CYP71D178 and CYP71D180) biosynthetic genes of thymol and carvacrol were measured. The results showed that early step and late step genes in thymol/carvacrol biosynthesis are differentially regulated. DXR was not found to be exclusively expressed in glandular trichomes; in contrast, biosynthetic genes including γ-terpinene synthase (TvTPS1) and two cytochrome P450s, CYP71D178 and CYP71D180, were preferentially expressed in glandular secretory trichomes. The high expression of late biosynthetic genes in glandular trichomes, which also contain the highest concentration of thymol and carvacrol, suggests that glandular trichomes are the structure in which thymol/carvacrol biosynthesis and accumulation occur. Our results indicate that in addition to abiotic elicitors, developmental and spatial factors also play a key role in the biosynthesis of thymol and carvacrol, most likely relating to glandular trichome density and/or activity. Hence optimization of these factors could be considered as a useful strategy to achieve high yield of valuable compounds in T. vulgare or other closely related plant species.
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http://dx.doi.org/10.1016/j.plaphy.2017.03.016DOI Listing
June 2017

Origin and evolution of transporter substrate specificity within the NPF family.

Elife 2017 03 3;6. Epub 2017 Mar 3.

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

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the . Biochemical characterization of orthologs along the phylogenetic lineage from cassava to suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.
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http://dx.doi.org/10.7554/eLife.19466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336358PMC
March 2017

CB5C affects the glucosinolate profile in Arabidopsis thaliana.

Plant Signal Behav 2016 08;11(8):e1160189

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

Cytochrome b5 (CB5) proteins are small heme-binding proteins, that influence cytochrome P450 activity. While only one CB5 isoform is found in mammals, higher plants have several isoforms of these proteins. The roles of the many CB5 isoforms in plants remain unknown. We hypothesized that CB5 proteins support the cytochrome P450 enzymes of plant specialized metabolism and found CB5C from Arabidopsis thaliana to co-express with glucosinolate biosynthetic genes. We characterized the glucosinolate profiles of 2 T-DNA insertion mutants of CB5C, and found that long-chained aliphatic glucosinolates were reduced in one of the mutant lines - a phenotype that was exaggerated upon methyl-jasmonate treatment. These results support the hypothesis, that CB5C influences glucosinolate biosynthesis, however, the mode of action remains unknown. Furthermore, the mutants differed in their biomass response to methyl jasmonate treatment. Thereby, our results highlight the varying effects of T-DNA insertion sites, as the 2 analyzed alleles show different phenotypes.
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http://dx.doi.org/10.1080/15592324.2016.1160189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5022417PMC
August 2016

The Arabidopsis NPF3 protein is a GA transporter.

Nat Commun 2016 May 3;7:11486. Epub 2016 May 3.

Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv 69978, Israel.

Gibberellins (GAs) are plant hormones that promote a wide range of developmental processes. While GA signalling is well understood, little is known about how GA is transported or how GA distribution is regulated. Here we utilize fluorescently labelled GAs (GA-Fl) to screen for Arabidopsis mutants deficient in GA transport. We show that the NPF3 transporter efficiently transports GA across cell membranes in vitro and GA-Fl in vivo. NPF3 is expressed in root endodermis and repressed by GA. NPF3 is targeted to the plasma membrane and subject to rapid BFA-dependent recycling. We show that abscisic acid (ABA), an antagonist of GA, is also transported by NPF3 in vitro. ABA promotes NPF3 expression and GA-Fl uptake in plants. On the basis of these results, we propose that GA distribution and activity in Arabidopsis is partly regulated by NPF3 acting as an influx carrier and that GA-ABA interaction may occur at the level of transport.
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http://dx.doi.org/10.1038/ncomms11486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857387PMC
May 2016

Analysis and Quantification of Glucosinolates.

Curr Protoc Plant Biol 2016 Mar;1(2):385-409

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

Recent advances in liquid chromatography and mass spectrometry have made it possible to increase the throughput of phytochemical analyses at high sensitivity. These improvements have made it more feasible to monitor metabolic processes at the metabolite level. Glucosinolates, the primary defense compounds in the model plant Arabidopsis thaliana, in particular, have gained increasing attention as model compounds for quantitative genetics and metabolic regulation. Depending on the plant species, tissue, glucosinolate content, complexity of the glucosinolate profile, and, most importantly, the overall purpose of the experiment, different choices need to be made regarding the methods of extraction and analysis. In this chapter, we describe different approaches for the analysis of glucosinolates and highlight advantages, disadvantages, and technical pitfalls. © 2016 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cppb.20027DOI Listing
March 2016
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