Publications by authors named "Hussam Hassan Nour-Eldin"

24 Publications

  • Page 1 of 1

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

mGWAS Uncovers Gln-Glucosinolate Seed-Specific Interaction and its Role in Metabolic Homeostasis.

Plant Physiol 2020 06 21;183(2):483-500. Epub 2020 Apr 21.

Division of Biological Sciences, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211

Gln is a key player in plant metabolism. It is one of the major free amino acids that is transported into the developing seed and is central for nitrogen metabolism. However, Gln natural variation and its regulation and interaction with other metabolic processes in seeds remain poorly understood. To investigate the latter, we performed a metabolic genome-wide association study (mGWAS) of Gln-related traits measured from the dry seeds of the Arabidopsis () diversity panel using all potential ratios between Gln and the other members of the Glu family as traits. This semicombinatorial approach yielded multiple candidate genes that, upon further analysis, revealed an unexpected association between the aliphatic glucosinolates (GLS) and the Gln-related traits. This finding was confirmed by an independent quantitative trait loci mapping and statistical analysis of the relationships between the Gln-related traits and the presence of specific GLS in seeds. Moreover, an analysis of Arabidopsis mutants lacking GLS showed an extensive seed-specific impact on Gln levels and composition that manifested early in seed development. The elimination of GLS in seeds was associated with a large effect on seed nitrogen and sulfur homeostasis, which conceivably led to the Gln response. This finding indicates that both Gln and GLS play key roles in shaping the seed metabolic homeostasis. It also implies that select secondary metabolites might have key functions in primary seed metabolism. Finally, our study shows that an mGWAS performed on dry seeds can uncover key metabolic interactions that occur early in seed development.
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http://dx.doi.org/10.1104/pp.20.00039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271782PMC
June 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

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

A transportome-scale amiRNA-based screen identifies redundant roles of Arabidopsis ABCB6 and ABCB20 in auxin transport.

Nat Commun 2018 10 11;9(1):4204. Epub 2018 Oct 11.

School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, 69978, Israel.

Transport of signaling molecules is of major importance for regulating plant growth, development, and responses to the environment. A prime example is the spatial-distribution of auxin, which is regulated via transporters to govern developmental patterning. A critical limitation in our ability to identify transporters by forward genetic screens is their potential functional redundancy. Here, we overcome part of this functional redundancy via a transportome, multi-targeted forward-genetic screen using artificial-microRNAs (amiRNAs). We generate a library of 3000 plant lines expressing 1777 amiRNAs, designed to target closely homologous genes within subclades of transporter families and identify, genotype and quantitatively phenotype, 80 lines showing reproducible shoot growth phenotypes. Within this population, we discover and characterize a strong redundant role for the unstudied ABCB6 and ABCB20 genes in auxin transport and response. The unique multi-targeted lines generated in this study could serve as a genetic resource that is expected to reveal additional transporters.
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http://dx.doi.org/10.1038/s41467-018-06410-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182007PMC
October 2018

Advances in methods for identification and characterization of plant transporter function.

J Exp Bot 2017 07;68(15):4045-4056

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

Transport proteins are crucial for cellular function at all levels. Numerous importers and exporters facilitate transport of a diverse array of metabolites and ions intra- and intercellularly. Identification of transporter function is essential for understanding biological processes at both the cellular and organismal level. Assignment of a functional role to individual transporter proteins or to identify a transporter with a given substrate specificity has notoriously been challenging. Recently, major advances have been achieved in function-driven screens, phenotype-driven screens, and in silico-based approaches. In this review, we highlight examples that illustrate how new technology and tools have advanced identification and characterization of plant transporter functions.
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http://dx.doi.org/10.1093/jxb/erx140DOI Listing
July 2017

Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters.

Nat Biotechnol 2017 04 13;35(4):377-382. Epub 2017 Mar 13.

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

The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds, but translation of loss-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60-70% in two different Brassica species (Brassica rapa and Brassica juncea). Reduction in seed glucosinolates was stably inherited over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed glucosinolate content in other oilseed crops, such as Camelina sativa or Crambe abyssinica.
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http://dx.doi.org/10.1038/nbt.3823DOI Listing
April 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

An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

Nat Plants 2017 01 13;3:16208. Epub 2017 Jan 13.

The John Innes Centre, Department of Biological Chemistry, Norwich Research Park, Norwich NR4 7UK, UK.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.
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http://dx.doi.org/10.1038/nplants.2016.208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238941PMC
January 2017

Rhizosecretion of stele-synthesized glucosinolates and their catabolites requires GTR-mediated import in Arabidopsis.

J Exp Bot 2017 06;68(12):3205-3214

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

Casparian strip-generated apoplastic barriers not only control the radial flow of both water and ions but may also constitute a hindrance for the rhizosecretion of stele-synthesized phytochemicals. Here, we establish root-synthesized glucosinolates (GLS) are in Arabidopsis as a model to study the transport routes of plant-derived metabolites from the site of synthesis to the rhizosphere. Analysing the expression of GLS synthetic genes in the root indicate that the stele is the major site for the synthesis of aliphatic GLS, whereas indole GLS can be synthesized in both the stele and the cortex. Sampling root exudates from the wild type and the double mutant of the GLS importers GTR1 and GTR2 show that GTR-mediated retention of stele-synthesized GLS is a prerequisite for the exudation of both intact GLS and their catabolites into the rhizosphere. The expression of the GTRs inside the stele, combined with the previous observation that GLS are exported from biosynthetic cells, suggest three possible routes of stele-synthesized aliphatic GLS after their synthesis: (i) GTR-dependent import to cells symplastically connected to the cortical cells and the rhizosphere; (ii) GTR-independent transport via the xylem to the shoot; and (iii) GTR-dependent import to GLS-degrading myrosin cells at the cortex. The study suggests a previously undiscovered role of the import process in the rhizosecretion of root-synthesized phytochemicals.
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http://dx.doi.org/10.1093/jxb/erw355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853541PMC
June 2017

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

A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

Methods Mol Biol 2016 ;1405:99-107

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

Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.
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http://dx.doi.org/10.1007/978-1-4939-3393-8_10DOI Listing
November 2016

Collection of Apoplastic Fluids from Arabidopsis thaliana Leaves.

Methods Mol Biol 2016 ;1405:35-42

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

The leaf apoplast comprises the extracellular continuum outside cell membranes. A broad range of processes take place in the apoplast, including intercellular signaling, metabolite transport, and plant-microbe interactions. To study these processes, it is essential to analyze the metabolite content in apoplastic fluids. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves. Here, methods to collect apoplastic washing fluid and guttation fluid from Arabidopsis thaliana leaves are described.
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http://dx.doi.org/10.1007/978-1-4939-3393-8_4DOI Listing
November 2016

A Functional EXXEK Motif is Essential for Proton Coupling and Active Glucosinolate Transport by NPF2.11.

Plant Cell Physiol 2015 Dec 6;56(12):2340-50. Epub 2015 Oct 6.

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

The proton-dependent oligopeptide transporter (POT/PTR) family shares a highly conserved E1X1X2E2RFXYY (E1X1X2E2R) motif across all kingdoms of life. This motif is suggested to have a role in proton coupling and active transport in bacterial homologs. For the plant POT/PTR family, also known as the NRT1/PTR family (NPF), little is known about the role of the E1X1X2E2R motif. Moreover, nothing is known about the role of the X1 and X2 residues within the E1X1X2E2R motif. We used NPF2.11-a proton-coupled glucosinolate (GLS) symporter from Arabidopsis thaliana-to investigate the role of the E1X1X2E2K motif variant in a plant NPF transporter. Using liquid chromatography-mass spectrometry (LC-MS)-based uptake assays and two-electrode voltage clamp (TEVC) electrophysiology, we demonstrate an essential role for the E1X1X2E2K motif for accumulation of substrate by NPF2.11. Our data suggest that the highly conserved E1, E2 and K residues are involved in translocation of protons, as has been proposed for the E1X1X2E2R motif in bacteria. Furthermore, we show that the two residues X1 and X2 in the E1X1X2E2[K/R] motif are conserved as uncharged amino acids in POT/PTRs from bacteria to mammals and that introducing a positive or negative charge in either position hampers the ability to overaccumulate substrate relative to the assay medium. We hypothesize that introducing a charge at X1 and X2 interferes with the function of the conserved glutamate and lysine residues of the E1X1X2E2K motif and affects the mechanism behind proton coupling.
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http://dx.doi.org/10.1093/pcp/pcv145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675897PMC
December 2015

Phosphorylation at serine 52 and 635 does not alter the transport properties of glucosinolate transporter AtGTR1.

Plant Signal Behav 2016 ;11(2):e1071751

a Center for Dynamic Molecular Interactions (DynaMo); University of Copenhagen ; Frederiksberg , Denmark.

Little is known about how plants regulate transporters of defense compounds. In A. thaliana, glucosinolates are transported between tissues by NPF2.10 (AtGTR1) and NPF2.11 (AtGTR2). Mining of the PhosPhat4.0 database showed two cytosol exposed phosphorylation sites for AtGTR1 and one membrane-buried phosphorylation site for AtGTR2. In this study, we investigate whether mutation of the two potential regulatory sites of AtGTR1 affected transport of glucosinolates in Xenopus oocytes. Characterization of AtGTR1 phosphorylation mutants showed that phosphorylation of AtGTR1 - at the two reported phosphorylation sites - is not directly involved in regulating AtGTR1 transport activity. We hypothesize a role for AtGTR1-phosphorylation in regulating protein-protein interactions.
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http://dx.doi.org/10.1080/15592324.2015.1071751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4883914PMC
December 2016

Transport of defense compounds from source to sink: lessons learned from glucosinolates.

Trends Plant Sci 2015 Aug 12;20(8):508-14. Epub 2015 May 12.

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

Plants synthesize a plethora of defense compounds crucial for their survival in a challenging and changing environment. Transport processes are important for shaping the distribution pattern of defense compounds, albeit focus hitherto has been mostly on their biosynthetic pathways. A recent identification of two glucosinolate transporters represents a breakthrough in our understanding of glucosinolate transport in Arabidopsis and has advanced knowledge in transport of defense compounds. In this review, we discuss the role of the glucosinolate transporters in establishing dynamic glucosinolate distribution patterns and source-sink relations. We focus on lessons learned from glucosinolate transport that may apply to transport of other defense compounds and discuss future avenues in the emerging field of defense compound transport.
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http://dx.doi.org/10.1016/j.tplants.2015.04.006DOI Listing
August 2015

An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas.

Biotechnol J 2015 Mar 13;10(3):473-9. Epub 2014 Oct 13.

Division of Biological Sciences, California Center for Algal Biotechnology, University of California San Diego, La Jolla, CA, USA.

The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains.
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http://dx.doi.org/10.1002/biot.201400172DOI Listing
March 2015

Elucidating the role of transport processes in leaf glucosinolate distribution.

Plant Physiol 2014 Nov 10;166(3):1450-62. Epub 2014 Sep 10.

Danish National Research Foundation Center for Dynamic Molecular Interactions (S.R.M., H.H.N.-E., B.A.H.) and Department of Plant and Environmental Sciences, Faculty of Science (S.R.M., C.E.O., H.H.N.-E., B.A.H.), University of Copenhagen, DK-1871 Frederiksberg C, Denmark

In Arabidopsis (Arabidopsis thaliana), a strategy to defend its leaves against herbivores is to accumulate glucosinolates along the midrib and at the margin. Although it is generally assumed that glucosinolates are synthesized along the vasculature in an Arabidopsis leaf, thereby suggesting that the margin accumulation is established through transport, little is known about these transport processes. Here, we show through leaf apoplastic fluid analysis and glucosinolate feeding experiments that two glucosinolate transporters, GTR1 and GTR2, essential for long-distance transport of glucosinolates in Arabidopsis, also play key roles in glucosinolate allocation within a mature leaf by effectively importing apoplastically localized glucosinolates into appropriate cells. Detection of glucosinolates in root xylem sap unambiguously shows that this transport route is involved in root-to-shoot glucosinolate allocation. Detailed leaf dissections show that in the absence of GTR1 and GTR2 transport activity, glucosinolates accumulate predominantly in leaf margins and leaf tips. Furthermore, we show that glucosinolates accumulate in the leaf abaxial epidermis in a GTR-independent manner. Based on our results, we propose a model for how glucosinolates accumulate in the leaf margin and epidermis, which includes symplasmic movement through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in these peripheral cell layers.
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http://dx.doi.org/10.1104/pp.114.246249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226354PMC
November 2014

Integration of biosynthesis and long-distance transport establish organ-specific glucosinolate profiles in vegetative Arabidopsis.

Plant Cell 2013 Aug 30;25(8):3133-45. Epub 2013 Aug 30.

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

Although it is essential for plant survival to synthesize and transport defense compounds, little is known about the coordination of these processes. Here, we investigate the above- and belowground source-sink relationship of the defense compounds glucosinolates in vegetative Arabidopsis thaliana. In vivo feeding experiments demonstrate that the glucosinolate transporters1 and 2 (GTR1 and GTR2), which are essential for accumulation of glucosinolates in seeds, are likely to also be involved in bidirectional distribution of glucosinolates between the roots and rosettes, indicating phloem and xylem as their transport pathways. Grafting of wild-type, biosynthetic, and transport mutants show that both the rosette and roots are able to synthesize aliphatic and indole glucosinolates. While rosettes constitute the major source and storage site for short-chained aliphatic glucosinolates, long-chained aliphatic glucosinolates are synthesized both in roots and rosettes with roots as the major storage site. Our grafting experiments thus indicate that in vegetative Arabidopsis, GTR1 and GTR2 are involved in bidirectional long-distance transport of aliphatic but not indole glucosinolates. Our data further suggest that the distinct rosette and root glucosinolate profiles in Arabidopsis are shaped by long-distance transport and spatially separated biosynthesis, suggesting that integration of these processes is critical for plant fitness in complex natural environments.
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http://dx.doi.org/10.1105/tpc.113.110890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784604PMC
August 2013

The emerging field of transport engineering of plant specialized metabolites.

Curr Opin Biotechnol 2013 Apr 4;24(2):263-70. Epub 2012 Oct 4.

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

From a biotechnological perspective transport processes represent attractive targets for modulation of metabolite levels and are the foundation for the emerging field of transport engineering. Potential applications of transport engineering include control of metabolite accumulation in a tissue-specific manner in crop plants as well as increased yields of commercially valuable compounds produced in synthetic biology approaches. Within specialized metabolism, recent advances include identification of not only vacuolar but now also plasma membrane-localized transporters and neo-functionalization of members of primary metabolite transporter families to include specific roles in transport of specialized metabolites. As glucosinolates are specialized metabolites of the model plant Arabidopsis, glucosinolate transport processes emerge as a model system for studying transport of specialized metabolites.
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http://dx.doi.org/10.1016/j.copbio.2012.09.006DOI Listing
April 2013

NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds.

Nature 2012 Aug;488(7412):531-4

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

In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value, as demonstrated in the plant model Arabidopsis, in which the major defence compounds, glucosinolates, are translocated to seeds on maturation. The molecular basis for long-distance transport of glucosinolates and other defence compounds, however, remains unknown. Here we identify and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters. The gtr1 gtr2 double mutant did not accumulate glucosinolates in seeds and had more than tenfold over-accumulation in source tissues such as leaves and silique walls, indicating that both plasma membrane-localized transporters are essential for long-distance transport of glucosinolates. We propose that GTR1 and GTR2 control the loading of glucosinolates from the apoplasm into the phloem. Identification of the glucosinolate transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.
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http://dx.doi.org/10.1038/nature11285DOI Listing
August 2012
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