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The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness

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

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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.

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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.

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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.

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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.

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  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

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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.

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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.

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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).

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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.

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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.

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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.

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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).

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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.

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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

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