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

Abstract

In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value1, as demonstrated in the plant model Arabidopsis, in which the major defence compounds, glucosinolates2, are translocated to seeds on maturation3. 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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Figure 1: Identification of Arabidopsis transporters with glucosinolate uptake activity.
Figure 2: Biophysical and biochemical transport properties of GTRs.
Figure 3: Glucosinolate analysis of gtr1 and gtr2 knockout mutants.
Figure 4: Localization of GTR1 and GTR2.

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Acknowledgements

We thank M. Palmgren, A. Schulz and D. Kliebenstein for comments on the manuscript and H. K. S. Jepsen, L. B. Møller and A. Kraus for technical assistance. We thank D. Klærke and technicians T. K. Soland, B. L. Christensen, M. Olsen and C. Derrer for providing X. laevis oocytes. We thank the Salk, Stanford, Plant Gene Expression Center (SSP) Consortium and the RIKEN Genome Science Center for providing full-length cDNAs. Imaging data were achieved at the Center for Advanced Bioimaging, University of Copenhagen. H.H.N-.E. was supported by the Danish Research Council for Technology and Production (FTP) grant 09-065827/274-08-0354. M.B. was supported by a Marie Curie fellowship (grant PIEF-GA-2008-221236). I.D. was supported by a Marie-Curie Career Integration Grant of the European Union (FP7-PEOPLE-2011-CIG No. 303674 (Regopoc)). D.G. was supported by the Deutsche Forschungsgemeinschaft grant GE2195/1-1 and R.H. by Deutsche Forschungsgemeinschaft grants within FOR 1061. B.A.H. is partner of The VKR Research Centre for Pro-Active Plants funded by the Villum Kann Rasmussen Foundation. Funding for DynaMo Center of Excellence is provided by the Danish National Research Foundation.

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Authors and Affiliations

Authors

Contributions

H.H.N-.E. identified the GTRs, designed the study, performed the phylogenetic analyses, contributed to biochemical characterization, data analysis, confocal localization experiments and analyses of metabolite profiling data and wrote the paper. T.G.A. performed the confocal localization experiments, generated and characterized glucosinolate in the gtr knockout mutants and contributed to biochemical characterization, study design and preparation of the manuscript. M.B. purified glucosinolates necessary for biochemical characterization and contributed to study design, data analysis and preparation of the manuscript. S.R.M. generated and analysed GTR-promoter-β-glucuronidase lines, contributed to confocal localization experiments and performed germination experiments. M.E.J. performed metabolite profiling of seeds and analysed data and performed seed morphology and yield analyses. C.E.O. performed LC–MS analyses for the transporter library screen and for metabolite profiling. I.D. performed theoretical discussion on GTR kinetics. R.H. contributed to the study design and data analyses. D.G. performed the biophysical characterization of the GTR transport mechanism and contributed to the theoretical discussion on GTR kinetics, study design and preparation of the manuscript. B.A.H. contributed to and supervised the study design and contributed to data analyses and preparation of the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Barbara Ann Halkier.

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Competing interests

The subject matter of this publication forms the basis of a patent application USSN 61/362,390 and EP10075299.7.

Supplementary information

Supplementary Information 1

This file contains Supplementary Figures 1, 3-7 and legend for Supplementary Figure 2 (see separate file for Supplementary Figure 2), additional references, Supplementary Materials and Methods, Supplementary References, Supplementary Table 1, a Supplementary Discussion and full legends for Supplementary Movies 1-4. (PDF 1294 kb)

Supplementary Information 2

This file contains Supplementary Figure 2 (see Supplementary Information file for legend). (PDF 2312 kb)

Supplementary Movie 1

Representative movie of z-stack spanning 27.0 μm of five-week-old leaves from stable Arabidopsis lines transformed with pGTR1(2kb)-GTR1(genomic fragment)-YFP(venus)-GTR1_3’UTR(0.3kb) (seven independent lines), showing plasma membrane localization of GTR1. (MOV 1303 kb)

Supplementary Movie 2

This movie shows background YFP fluorescence in non-transformed plants. (MOV 737 kb)

Supplementary Movie 3

Representative movie of a z-stack spanning 16.4 μm of five-week-old leaves from stable Arabidopsis lines transformed with pGTR2(2kb)-GTR2 (genomic fragment)-mOrange-GTR2_3'UTR(0.3kb) (22 independent lines), showing plasma membrane localization of GTR2. (MOV 862 kb)

Supplementary Movie 4

This movie shows background mOrange fluorescence in non-transformed plants. (MOV 275 kb)

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Nour-Eldin, H., Andersen, T., Burow, M. et al. NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488, 531–534 (2012). https://doi.org/10.1038/nature11285

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