Morten Egevang Jørgensen

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

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.

The Arabidopsis NPF3 protein is a GA transporter

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.

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

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.

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

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.

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

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.

Origin and evolution of transporter substrate specificity within the NPF family

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 Brassicales 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 Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, 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.

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

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.

Functional analysis of a triplet deletion in the gene encoding the sodium glucose transporter 3, a potential risk factor for ADHD

Sodium-glucose transporters (SGLT) belong to the solute carrier 5 family, which is characterized by sodium dependent transport of sugars and other solutes. In contrast, the human SGLT3 (hSGLT3) isoform, encoded by SLC5A4, acts as a glucose sensor that does not transport sugar but induces membrane depolarization by Na+ currents upon ligand binding. Whole-exome sequencing (WES) of several extended pedigrees with high density of attention-deficit/hyperactivity disorder (ADHD) identified a triplet ATG deletion in SLC5A4 leading to a single amino acid loss (ΔM500) in the hSGLT3 protein imperfectly co-segregating with the clinical phenotype of ADHD. Since mutations in homologous domains of hSGLT1 and hSGLT2 were found to affect intestinal and renal function, respectively, we analyzed the functional properties of hSGLT3[wt] and [ΔM500] by voltage clamp and current clamp recordings from cRNA-injected Xenopus laevis oocytes. The cation conductance of hSGLT3[wt] was activated by application of glucose or the specific agonist 1-desoxynojirimycin (DNJ) as revealed by inward currents in the voltage clamp configuration and cell depolarization in the current clamp mode. Almost no currents and changes in membrane potential were observed when glucose or DNJ were applied to hSGLT3[ΔM500]-injected oocytes, demonstrating a loss of function by this amino acid deletion in hSGLT3. To monitor membrane targeting of wt and mutant hSGLT3, fusion constructs with YFP were generated, heterologously expressed in Xenopus laevis oocytes and analyzed for membrane fluorescence by confocal microscopy. In comparison to hSGLT3[wt] the fluorescent signal of mutant [ΔM500] was reduced by 43% indicating that the mutant phenotype might mainly result from inaccurate membrane targeting. As revealed by homology modeling, residue M500 is located in TM11 suggesting that in addition to the core structure (TM1-TM10) of the transporter, the surrounding TMs are equally crucial for transport/sensor function. In conclusion, our findings indicate that the deletion [ΔM500] in hSGLT3 inhibits membrane targeting and thus largely disrupts glucose-induced sodium conductance, which may, in interaction with other ADHD risk-related gene variants, influence the risk for ADHD in deletion carriers.

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

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.