Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness

Nature Plants volume 7, pages 468–480 (2021)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The ‘metabolic shift’ in SGA composition during tomato fruit development and ripening.
Fig. 2: Discovery of the GORKY candidate gene by mapping the ‘bitterness’ trait.
Fig. 3: Altered GORKY activity leads to overaccumulation of α-tomatine in ripe fruit.
Fig. 4: Overexpression of GORKY leads to overaccumulation of SGA pathway intermediates in transgenic plants.
Fig. 5: GORKY is a tonoplast α-tomatine exporter.
Fig. 6: GORKY is required for modulation of bitterness and self-toxicity by relocating SGAs during tomato fruit ripening.

Data availability

The authors confirm that the data described in the manuscript are available from the corresponding author upon reasonable request. The proteomics data are deposited to ProteomeXChange consortium via the PRIDE partner repository (project accession PXD023289). Source data are provided with this paper.

References

  1. Eich, E. Solanaceae and Convolvulaceae: Secondary Metabolites (Springer, 2008).

  2. Friedman, M. Tomato glycoalkaloids: role in the plant and in the diet. J. Agric. Food Chem. 50, 5751–5780 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Friedman, M. Potato glycoalkaloids and metabolites: roles in the plant and in the diet. J. Agric. Food Chem. 54, 8655–8681 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Itkin, M. et al. Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes. Science 341, 175–179 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. Moco, S. et al. Tissue specialization at the metabolite level is perceived during the development of tomato fruit. J. Exp. Bot. 58, 4131–4146 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Itkin, M. et al. Glycoalkaloid Metabolism1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato. Plant Cell 23, 4507–4525 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Iijima, Y. et al. Steroidal glycoalkaloid profiling and structures of glycoalkaloids in wild tomato fruit. Phytochemistry 95, 145–157 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Roddick, J. G. Intracellular distribution of the steroidal glycoalkaloid α-tomatine in Lycopersicon esculentum fruit. Phytochemistry 15, 475–477 (1976).

    Article  CAS  Google Scholar 

  9. Keukens, E. A. J. et al. Glycoalkaloids selectively permeabilize cholesterol containing biomembranes. Biochim. Biophys. Acta Biomembr. 1279, 243–250 (1996).

    Article  Google Scholar 

  10. Blankemeyer, J. T., White, J. B., Stringer, B. K. & Friedman, M. Effect of α-tomatine and tomatidine on membrane potential of frog embryos and active transport of ions in frog skin. Food Chem. Toxicol. 35, 639–646 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Payne, R. M. E. et al. An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole. Nat. Plants 3, 16208 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Takanashi, K. et al. A multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis japonica. Phytochemistry 138, 76–82 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Shitan, N. Secondary metabolites in plants: transport and self-tolerance mechanisms. Biosci. Biotechnol. Biochem. 80, 1283–1293 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Nour-Eldin, H. H. et al. Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters. Nat. Biotechnol. 35, 377–382 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Shitan, N. & Yazaki, K. Dynamism of vacuoles toward survival strategy in plants. Biochim. Biophys. Acta Biomembr. 1862, 183127 (2019).

  16. Jørgensen, M. E., Nour-Eldin, H. H. & Halkier, B. A. Transport of defense compounds from source to sink: lessons learned from glucosinolates. Trends Plant Sci. 20, 508–514 (2015).

    Article  PubMed  CAS  Google Scholar 

  17. Shoji, T. et al. Multidrug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots. Plant Physiol. 149, 708–718 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Morita, M. et al. Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proc. Natl Acad. Sci. USA 106, 2447–2452 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Goodman, C. D., Casati, P. & Walbot, V. A multidrug resistance–associated protein involved in anthocyanin transport in Zea mays. Plant Cell 16, 1812–1826 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Francisco, R. M. et al. ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-glucosides. Plant Cell 25, 1840–1841 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Behrens, C. E., Smith, K. E., Iancu, C. V., Choe, J. & Dean, J. V. Transport of anthocyanins and other flavonoids by the Arabidopsis ATP-binding cassette transporter AtABCC2. Sci. Rep. 9, 437 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Cárdenas, P. D. et al. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nat. Commun. 7, 10654 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Sonawane, P. D. et al. Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum. Proc. Natl Acad. Sci. USA 115, 5419–5428 (2018).

    Article  CAS  Google Scholar 

  24. Cárdenas, P. D. et al. Pathways to defense metabolites and evading fruit bitterness in genus Solanum evolved through 2-oxoglutarate-dependent dioxygenases. Nat. Commun. 10, 5169 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Szymański, J. et al. Analysis of wild tomato introgression lines elucidates the genetic basis of transcriptome and metabolome variation underlying fruit traits and pathogen response. Nat. Genet. 52, 1111–1121 (2020).

    Article  PubMed  CAS  Google Scholar 

  26. Iijima, Y. et al. Involvement of ethylene in the accumulation of esculeoside A during fruit ripening of tomato (Solanum lycopersicum). J. Agric. Food Chem. 57, 3247–3252 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Rick, C. M., Uhlig, J. W. & Jones, A. D. High alpha-tomatine content in ripe fruit of Andean Lycopersicon esculentum var. cerasiforme: developmental and genetic aspects. Proc. Natl Acad. Sci. USA 91, 12877–12881 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhu, G. et al. Rewiring of the fruit metabolome in tomato breeding. Cell 172, 249–261 (2018).

    Article  CAS  PubMed  Google Scholar 

  29. Sánchez-Pérez, R. et al. Mutation of a bHLH transcription factor allowed almond domestication. Science 364, 1095–1098 (2019).

    Article  PubMed  CAS  Google Scholar 

  30. Shang, Y. et al. Biosynthesis, regulation, and domestication of bitterness in cucumber. Science 346, 1084–1088 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. Hardigan, M. A. et al. Genome diversity of tuber-bearing Solanum uncovers complex evolutionary history and targets of domestication in the cultivated potato. Proc. Natl Acad. Sci. USA 114, 9999–10008 (2017).

    Article  CAS  Google Scholar 

  32. Johns, T. & Alonso, J. G. Glycoalkaloid change during the domestication of the potato, Solanum section Petota. Euphytica 50, 203–210 (1990).

    Article  CAS  Google Scholar 

  33. The 100 Tomato Genome Sequencing Consortium. Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing. Plant J. 80, 136–148 (2014)

  34. Léran, S. et al. A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci. 19, 5–9 (2014).

    Article  PubMed  CAS  Google Scholar 

  35. Tieman, D. et al. A chemical genetic roadmap to improved tomato flavor. Science 355, 391–394 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Lin, T. et al. Genomic analyses provide insights into the history of tomato breeding. Nat. Genet. 46, 1220 (2014).

    Article  CAS  PubMed  Google Scholar 

  37. Saito, C. et al. A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis. Plant J. 29, 245–255 (2002).

    Article  PubMed  Google Scholar 

  38. Nelson, B. K., Cai, X. & Nebenführ, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Snowden, C. J., Thomas, B., Baxter, C. J., Smith, J. A. C. & Sweetlove, L. J. A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition. Plant J. 81, 651–660 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Xu, L. et al. Identification of vacuolar phosphate efflux transporters in land plants. Nat. Plants 5, 84–94 (2019).

    Article  CAS  PubMed  Google Scholar 

  41. Roddick, J. G. & Drysdale, R. B. Destabilization of liposome membranes by the steroidal glycoalkaloid α-tomatine. Phytochemistry 23, 543–547 (1984).

    Article  CAS  Google Scholar 

  42. Surak, J. G. & Schifanella, A. V. The toxicity of α-tomatine to Tetrahymena pyriformis. Food Cosmet. Toxicol. 17, 61–67 (1979).

    Article  CAS  PubMed  Google Scholar 

  43. Sandrock, R. W. & VanEtten, H. D. Fungal sensitivity to and enzymatic degradation of the phytoanticipin α-tomatine. Phytopathology 88, 137–143 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Milner, S. E. et al. Bioactivities of glycoalkaloids and their aglycones from Solanum species. J. Agric. Food Chem. 59, 3454–3484 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Jiangyan, D., Liming, D., Hao, W., Lihuan, Y. & Gang, G. Preliminary study of action mechanism of tomatine toxicity to Helicoverpa armigera. Acta Bot. Boreal. Occident. Sin. 26, 117–120 (2006).

    Google Scholar 

  46. Hoagland, R. E. Toxicity of tomatine and tomatidine on weeds, crops and phytopathogens fungi. Allelopath. J. 23, 425–436 (2009).

    Google Scholar 

  47. Iijima, Y. et al. Metabolite annotations based on the integration of mass spectral information. Plant J. 54, 949–962 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Yamanaka, T. et al. C22 isomerization in α-tomatine-to-esculeoside A conversion during tomato ripening is driven by C27 hydroxylation of triterpenoidal skeleton. J. Agric. Food Chem. 57, 3786–3791 (2009).

    Article  CAS  PubMed  Google Scholar 

  49. Xu, D. et al. GTR-mediated radial import directs accumulation of defensive glucosinolates to sulfur-rich cells in the phloem cap of Arabidopsis inflorescence stem. Mol. Plant 12, 1474–1484 (2019).

    Article  CAS  PubMed  Google Scholar 

  50. Xu, D. et al. Rhizosecretion of stele-synthesized glucosinolates and their catabolites requires GTR-mediated import in Arabidopsis. J. Exp. Bot. 68, 3205–3214 (2016).

    PubMed Central  Google Scholar 

  51. Nour-Eldin, H. H. et al. NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488, 531–534 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Grunewald, S. et al. The tapetal major facilitator NPF2.8 is required for accumulation of flavonol glycosides on the pollen surface in Arabidopsis thaliana. Plant Cell 32, 1727–1748 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Jørgensen, M. E. et al. Origin and evolution of transporter substrate specificity within the NPF family. eLife 6, e19466 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Carqueijeiro, I., Noronha, H., Duarte, P., Gerós, H. & Sottomayor, M. Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport. Plant Physiol. 162, 1486–1496 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Roytrakul, S. & Verpoorte, R. Role of vacuolar transporter proteins in plant secondary metabolism: Catharanthus roseus cell culture. Phytochem. Rev. 6, 383–396 (2007).

    Article  CAS  Google Scholar 

  56. Sarrion-Perdigones, A., Palaci, J., Granell, A. & Orzaez, D. Design and construction of multigenic constructs for plant biotechnology using the GoldenBraid cloning strategy. Methods Mol. Biol. 1116, 133–151 (2014).

    Article  CAS  PubMed  Google Scholar 

  57. Vazquez-Vilar, M. et al. A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods 12, 10 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Orzaez, D. et al. A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation. Plant Physiol. 150, 1122–1134 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Liu, Y., Schiff, M. & Dinesh-Kumar, S. P. Virus-induced gene silencing in tomato. Plant J. 31, 777–786 (2002).

    Article  CAS  PubMed  Google Scholar 

  60. Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R. & Siuzdak, G. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal. Chem. 78, 779–787 (2006).

    Article  CAS  PubMed  Google Scholar 

  61. Kuhl, C., Tautenhahn, R., Böttcher, C., Larson, T. R. & Neumann, S. CAMERA: an integrated strategy for compound spectra extraction and annotation of liquid chromatography/mass spectrometry data sets. Anal. Chem. 84, 283–289 (2012).

    Article  CAS  PubMed  Google Scholar 

  62. Tang, Y., Horikoshi, M. & Li, W. ggfortify: unified interface to visualize statistical result of popular R packages. R J. 8, 478–489 (2016).

  63. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Expósito-Rodríguez, M., Borges, A. A., Borges-Pérez, A. & Pérez, J. A. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol. 8, 131 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

    Article  CAS  PubMed  Google Scholar 

  66. Leuzinger, K. et al. Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J. Vis. Exp. 77, e50521 (2013).

    Google Scholar 

  67. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  68. Shatil-Cohen, A. et al. Measuring the osmotic water permeability coefficient (Pf) of spherical cells: isolated plant protoplasts as an example. J. Vis. Exp. 92, e51652 (2014).

    Google Scholar 

  69. Snowden, C. J., Thomas, B., Baxter, C. J., Smith, J. A. C. & Sweetlove, L. J. Isolation of tonoplast vesicles from tomato fruit pericarp. Bio-Protoc. 5, e1686 (2015).

    Article  PubMed  Google Scholar 

  70. Szymanski, J. et al. Label-free deep shotgun proteomics reveals protein dynamics during tomato fruit tissues development. Plant J. 90, 396–417 (2017).

    Article  CAS  PubMed  Google Scholar 

  71. Wiśniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Article  CAS  PubMed  Google Scholar 

  72. de Brito Francisco, R. & Martinoia, E. The vacuolar transportome of plant specialized metabolites. Plant Cell Physiol. 59, 1326–1336 (2018).

    PubMed  Google Scholar 

  73. Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).

    Article  CAS  PubMed  Google Scholar 

  74. Deutsch, E. W. et al. The ProteomeXchange consortium in 2020: enabling ‘big data’ approaches in proteomics. Nucleic Acids Res. 48, D1145–D1152 (2020).

    CAS  PubMed  Google Scholar 

  75. Nour-Eldin, H. H., Hansen, B. G., Norholm, M. H. H., Jensen, J. K. & Halkier, B. A. Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res. 34, e122 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  76. Jozwiak, A. et al. Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery. Nat. Chem. Biol. 16, 740–748 (2020).

    Article  CAS  PubMed  Google Scholar 

  77. Dong, Y., Li, B. & Aharoni, A. More than pictures: when MS imaging meets histology. Trends Plant Sci. 21, 686–698 (2016).

    Article  CAS  PubMed  Google Scholar 

  78. Dong, Y. et al. Sample preparation for mass spectrometry imaging of plant tissues: a review. Front. Plant Sci. 7, 60 (2016).

    PubMed  PubMed Central  Google Scholar 

  79. Feldberg, L., Dong, Y., Heinig, U., Rogachev, I. & Aharoni, A. DLEMMA-MS-Imaging for identification of spatially localized metabolites and metabolic network map reconstruction. Anal. Chem. 90, 10231–10238 (2018).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Adelis Foundation, Leona M. and Harry B. Helmsley Charitable Trust, Jeanne and Joseph Nissim Foundation for Life Sciences, Tom and Sondra Rykoff Family Foundation Research and the Raymond Burton Plant Genome Research Fund for supporting the A.A. laboratory activity. The research was partially funded by the Israel Science Foundation personal grant to A.A. (ISF grant no. 1805/15). A.A. is the incumbent of the P. J. Cohn Professorial Chair. Optical imaging using the SP8-STED Leica microscope was made possible thanks to the de Picciotto Cancer Cell Observatory unit in memory of W. and R. Lesser, at the Moross Integrated Cancer Center Life Science Core Facilities, Weizmann Institute of Science. Additional support for this work was provided by Danish National Research Foundation grant no. DNRF99 (D.V., C.C. and H.H.N.-E.), Danish Council for Independent Research FTP grant nos DFF6111–00331 (S.K.L.), Human Frontier Science Program grant no. RGY0075/2015 (H.H.N.-E.). We thank technician L. Svenningsen (PLEN, Copenhagen University) for excellent technical assistance. We thank R. Tadmor-Levi for taking part in the bitter accessions characterization. We would like to acknowledge R. Bronstein, Y. Eshed and Z. Lippman for InDel marker development. We thank A. Jozwiak and I. Lichtarev for their advice on figure design.

Author information

Author notes

  1. These authors contributed equally: Yana Kazachkova, Itay Zemach, Asaph Aharoni.

Authors and Affiliations

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

    Yana Kazachkova, Sayantan Panda, Samuel Bocobza, Andrii Vainer, Ilana Rogachev, Yonghui Dong, Shifra Ben-Dor, Simon Michaeli & Asaph Aharoni

  2. Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot, Israel

    Itay Zemach & Dani Zamir

  3. Gilat Research Center, Agricultural Research Organization, Rishon Lezion, Israel

    Sayantan Panda

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

    Dorottya Veres, Christa Kanstrup, Sophie Konstanze Lambertz, Christoph Crocoll & Hussam Hassan Nour-Eldin

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

    Yangjie Hu & Eilon Shani

Authors
  1. Yana Kazachkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Itay Zemach
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sayantan Panda
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Samuel Bocobza
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrii Vainer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ilana Rogachev
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yonghui Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shifra Ben-Dor
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dorottya Veres
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Christa Kanstrup
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sophie Konstanze Lambertz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Christoph Crocoll
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yangjie Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Eilon Shani
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Simon Michaeli
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Hussam Hassan Nour-Eldin
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Dani Zamir
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Asaph Aharoni
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.K. designed experiments, produced transgenic plants, performed SGA profiling, did proteomics analysis and wrote the manuscript. I.Z. carried out the ‘bitterness locus’ mapping and identified ‘bitter lines’. S.B. and I.Z. identified the deletion. S.P. and A.V. performed gene expression analysis. Y.D. performed MALDI imaging. I.R. performed α-tomatine quantification. Y.H. and E.S. carried out protoplast localization experiments. S.M. and Y.K. carried out localization experiments in N. benthamiana. D.V., S.K.L., C.C., C.K. and H.H.N.-E. performed transporter assays. S.B.-D. designed real-time PCR primers and annotated genomic region, flanking GORKY. D.Z. provided ‘bitter’ lines and supervised the mapping experiments. A.A. planned and supervised the study. Y.H., S.M., E.S., D.V., C.C. and H.H.N.-E. supported the study with their expertise and contributed to writing selected sections of the manuscript.

Corresponding author

Correspondence to Asaph Aharoni.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Erich Grotewold, Jie Luo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Steroidal glycoalkaloid (SGA) metabolism during tomato fruit development and ripening.

Cholesterol serves as a precursor for SGA biosynthesis. In tomato, cholesterol is further modified through oxidation, hydroxylation, transamination, acetylation and glycosylation to form SGAs. Dashed arrows represent multiple biosynthetic reactions whereas solid arrows represent a single step. Ac: Acetoxy; Glc: Glucose; Gal: Galactose; Xyl: Xylose.

Extended Data Fig. 2 Tomato accessions harbouring a deletion in GORKY accumulate α-tomatine and its downstream derivatives in ripe fruit.

Levels of α-tomatine and downstream SGAs detected in red fruit are shown for nine bitter-tasting accessions as compared to WT fruit. Bar plots represent mean ± SE (n=3 for EA03055 and EA05978 and n=4 for the other accessions), asterisks indicate significant changes compared to WT samples as calculated by unpaired two-tailed t-test (*P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001). LC–qTOF was employed for SGA profiling.

Source data

Extended Data Fig. 3 Phylogenetic analysis of GORKY and other NPF transporters.

Classes of NPF transporters (NPF1–8) are indicated in colour in accordance with Léran et al.34 Sequences of characterized specialized metabolite transporters from Arabidopsis (At GTR1, At GTR2 and At FST1), Carica papaya (GTR1-l and GTR2-l) and Catharanthus roseus (Cat NPF2.9) were added to the analysis. Red dot points to the GORKY subclade including its related proteins investigated here (that is St NPF1.3; St NPF1.4 and St NPF1.6) and other proteins putatively transporting SAs and/or SGAs in the other Solanaceae species. [S. lycopersicum, (Sl)], cultivated potato [S. tuberosum, (St)], wild tomato [S. pennellii, (Sp)], Solanum pimpinellifolium (Spim), Capsicum annuum (Ca), cultivated eggplant [S. melongena, (Sm)], Nicotiana benthamiana (Nb) and Nicotiana tabacum (Nt). Amino acid sequences used in the phylogenetic analysis are provided in Supplementary Dataset 2.

Extended Data Fig. 4 Tissue-specific expression and α–tomatine uptake of GORKY and its related proteins NPF1.3, NPF1.4 and NPF1.6 in S. lycopersicum.

Quantitative Real Time-PCR (qRT-PCR) analysis was used to study gene expression of GORKY (c)and its homologues, NPF1.3 (a), NPF1.4 (b) and NPF1.6 (d). TIP41 was used as a reference gene. Bars represent mean ± SE (n=3 for mature green (MG), breaker (BR) and red ripe (RR) tomato fruit samples and n=4 for leaf and flower bud (FL) samples), normalized by average gene expression levels of the respective gene in the leaf tissue. The expression levels of NPF1.6 were notably lower than of the reference gene. Asterisks indicate a significant difference from leaf samples calculated by unpaired two-tailed t-test (*P value < 0.05; **P value < 0.01; ***P value < 0.001; P value < 0.0001). FL, flower buds; MG, mature green; BR, breaker; RR, red ripe fruit. e, Alpha-tomatine uptake of GORKY and it’s close homologues. The bar in the plot represents mean ± SE (n = 6, with 5 pooled oocytes each, from two different experiments, pooled after normalization), normalized to the measured media levels. Asterisks indicate a significant difference compared to mock, calculated by the unpaired two-tailed t-test (*P value < 0.05; ***P value < 0.001).

Source data

Extended Data Fig. 5 Knockout mutation in GORKY has a major impact on the SGA profile of ripe fruit.

Generation of loss-of-function GORKY lines through CRISPR/Cas9 gene editing. a, Position of the four guide RNAs targeting the third GORKY gene exon are indicated. b, Examples of mutations generated by editing are depicted below. PAM sequence is indicated in red; target sequence is depicted in blue and the adjacent nucleotides are shown in black. Dots represent the deletions. c, The levels of α-tomatine and its hydroxy- and acetoxy-SGA derivatives are significantly increased in gorky ripe fruit. Three independent T1 generation transformants were analyzed (#1, #2 and #3). Bars represent mean ± SE (n=3 for WT, n=4 for line #1 and #3, n=7 for line #2); asterisks indicate significant changes compared to WT (non-transformed) fruit samples as calculated by two-tailed unpaired t-test (*P value < 0.05; **P value < 0.01;***P value < 0.001; ****P value < 0.0001). LC-qTOF was employed for SGA analysis.

Source data

Extended Data Fig. 6 Overexpression of GORKY in tomato leaves leads to the accumulation of downstream hydroxy- and acetoxy-derivatives including esculeoside A.

Esculeoside A, typically detected merely in the breaker and ripe fruit, accumulates in GORKY-Ox leaf tissues. Bars represent mean ± SE (n=3 for WT, n=5 for line #1 and #3, n=4 for line #2), asterisks indicate significant changes compared to WT (non-transformed) samples as calculated by two-tailed unpaired t-test (*P value < 0.05; **P value < 0.01). LC-qTOF was employed for SGA profiling analysis.

Source data

Extended Data Fig. 7 Tomato plants overexpressing GORKY on EA07290 background (GORKY-Ox) display severe morphological phenotypes.

a, non-transformed EA07290 plants; b, GORKY-Ox (in the EA07290 background). c, Leaves of non-transformed EA07290 and GORKY-Ox plants.

Extended Data Fig. 8 Overexpression of GORKY in the bitter accession EA07290 background restores the SGA profile to red, ripe fruit.

The levels of α-tomatine and dehydrotomatine are significantly decreasing in GORKY-Ox (in the EA07290 background fruit). On the other hand, levels of esculeoside A and dehydroesculeoside A, characteristic to WT red, ripe fruit, dramatically increase. SGA levels for plants from two independent tomato transgenic T1 lines are depicted (#1 and #2). In line #1 the levels of hydroxy-and acetoxytomatine are significantly decreased, while the level of acetoxy-hydroxytomatine is increased. In both lines, the levels of di-hydroxytomatine are increased. Bars represent mean ± SE (n=4 for WT, n=3 for lines #1, #2), asterisks indicate significant changes compared to WT (non-transformed EA07290) samples as calculated by unpaired two-tailed t-test (*P value < 0.05; **P value < 0.01;***P value < 0.001; ****P value < 0.0001). LC-qTOF was employed for SGA analysis.

Source data

Extended Data Fig. 9 Alteration of GORKY activity in tomato fruit does not affect GAME gene expression.

Quantitative Real Time-PCR (qRT-PCR) analysis of GAME1, GAME5 and GAME4 transcript levels in ripe fruit tissues of plants overexpressing GORKY (independent transformants #1, #2 and #3) and knockout lines (mutant alleles #1, #2 and # 3) with WT tomato fruits as control. TIP41 was used as a reference gene. Bars represent mean ± SE (n=3 for WT, n=5 for gorky line #2; n=4 for other lines), normalized by average GORKY expression levels in WT fruit tissue. Asterisks indicate a significant difference from tomato fruit samples calculated by unpaired two-tailed t-test (*P value =0.0142).

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1, 3 and 4 and Datasets 1 and 2.

Reporting Summary

Supplementary Table 2

Proteins enriched in tonoplast membrane fraction.

Supplementary Dataset 3

Shotgun proteomics of tonoplast-enriched membrane fractions from wild-type tomato fruit.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kazachkova, Y., Zemach, I., Panda, S. et al. The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness. Nat. Plants 7, 468–480 (2021). https://doi.org/10.1038/s41477-021-00865-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-021-00865-6

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing