Abstract
Plant membrane transporters controlling metabolite distribution contribute key agronomic traits1,2,3,4,5,6. To eliminate anti-nutritional factors in edible parts of crops, the mutation of importers can block the accumulation of these factors in sink tissues7. However, this often results in a substantially altered distribution pattern within the plant8,9,10,11,12, whereas engineering of exporters may prevent such changes in distribution. In brassicaceous oilseed crops, anti-nutritional glucosinolate defence compounds are translocated to the seeds. However, the molecular targets for export engineering of glucosinolates remain unclear. Here we identify and characterize members of the USUALLY MULTIPLE AMINO ACIDS MOVE IN AND OUT TRANSPORTER (UMAMIT) family—UMAMIT29, UMAMIT30 and UMAMIT31—in Arabidopsis thaliana as glucosinolate exporters with a uniport mechanism. Loss-of-function umamit29 umamit30 umamit31 triple mutants have a very low level of seed glucosinolates, demonstrating a key role for these transporters in translocating glucosinolates into seeds. We propose a model in which the UMAMIT uniporters facilitate glucosinolate efflux from biosynthetic cells along the electrochemical gradient into the apoplast, where the high-affinity H+-coupled glucosinolate importers GLUCOSINOLATE TRANSPORTERS (GTRs) load them into the phloem for translocation to the seeds. Our findings validate the theory that two differently energized transporter types are required for cellular nutrient homeostasis13. The UMAMIT exporters are new molecular targets to improve nutritional value of seeds of brassicaceous oilseed crops without altering the distribution of the defence compounds in the whole plant.
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Data availability
The sequences of proteins were obtained from UniProt (https://www.uniprot.org/), NCBI (https://www.ncbi.nlm.nih.gov/) and Phytozome (https://phytozome-next.jgi.doe.gov/). Data on global mRNA profiling of seeds and funiculus at three seed developmental stages were retrieved in the supporting information of ref. 24. Supporting data are provided in the Supplementary Information (including primers and phylogenetic analysis). Source data are provided with this paper.
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Acknowledgements
We thank A. J. Fuglsang-Madsen, V. Kjær, E. Pupi and J. Skytte Thorsen for laboratory assistance; the support staff at the Center for Advanced Bioimaging and growth facilities at Department of Plant and Environmental Sciences at University of Copenhagen for technical assistance; and I. Dreyer for his comments. We acknowledge the Danish National Research Foundation (DNRF99) for its financial support.
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Contributions
D.X. and B.A.H. conceived the study. D.X. designed the experiments and performed or supervised most of the experiments, including biochemical characterization, mutant generation and in planta characterization, data analysis and bioinformatics analysis. N.C.H.S. and P.H. conducted bio-imaging supervised by A.S. Z.M.B. performed electrophysiology assays. S.R.M. performed part of the grafting experiments. L.L.H. contributed to the time course for glucosinolate seed loading. C.C. performed LC–MS/MS analyses. L.M. contributed to part of the amino acid uptake assays. D.V. contributed to the characterization of UMAMIT29 in oocytes. D.X., H.H.N.-E., Z.M.B., M.E.J. and B.A.H. discussed the data. D.X., H.H.N.-E. and B.A.H. wrote the Article based on a draft provided by D.X. All of the authors commented on the manuscript.
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The subject matter in the manuscript is covered in a patent application (European patent application no. 22207870.1) filed by University of Copenhagen on 16 November 2022. D.X., H.H.N.-E. and B.A.H. are listed as inventors on the patent application. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Time course of glucosinolate accumulation in developing siliques and cellular localization of UMAMIT29.
a,b, Total glucosinolates (GLS) from a pool of 10 developing seeds (a) and in the corresponding developing silique without seeds (including silique valves, repla and funiculi) (b) from wild-type Arabidopsis Col-0 at different days after pollination (n = 6 plants per group). c, Cross section of siliques expressing pCYP83A1::CYP83A1-mVenus (top) and pCYP83B1::CYP83B1-mVenus (bottom) at mature green stage. d, Funiculus-expressed transporters from transcriptomics data24. Transporter genes were selected that showed an increase in expression from heart stage to mature green stage, resulting in a list of glucosinolate candidate exporters. e, Total methionine- and tryptophan-derived glucosinolates from developing siliques without seeds (i.e. silique valves including septa and funiculi) from wild-type Col-0 (n = 63), umamit29-1(ut29-1, (n = 58)), umamit29-2 (ut29-2, (n = 62)) and plants complemented with pUT29(6kb)-UT29 (genomic fragment)-mVenus (ut29-1C, (n = 55)) (data are representative of three independent lines) at different days after pollination (DAP). (n = total harvested siliques number per genotype). For box plot, the centre line indicates the median, the box limits denote the lower and upper quartiles, the dots indicate individual data points, the centre square indicates the mean, and the whiskers denote the highest and lowest data points. f–h, Cellular localization of UMAMIT29-mVenus at day 8 after pollination in living funiculi. f, UMAMIT29-mVenus accumulation in the funiculus. Note that the seed is detached in this view, thus exposing the funiculus optimally for live imaging at high resolution. g, Maximum intensity projection of a Z-stack through the funiculus shown in panel f at larger magnification. h, Single plane of the Z-stack showing that Umami-T29-mVenus is localized at the plasma membrane. Insert: Magnification of UMAMIT29-mVenus signal surrounding the chloroplasts in pUT29::UT29-mVenus plants. Green: UT29-mVenus, magenta: chlorophyll autofluorescence. Scale bars: c: 50 µm, f: 250 µm, g and h: 50 µm, h insert: 10 µm.
Extended Data Fig. 2 Biochemical and biophysical characterization of UMAMIT29 in Xenopus oocytes.
a, 4-methylthiobutyl glucosinolate (4MTB) uptake over time in oocytes expressing UMAMIT29 (UT29) or injected with H2O (H2O-inj). Oocytes were incubated for indicated times in 1 mM 4MTB (pH 5.5) and the content was determined in individual oocytes by LC-MS. Data are representative of two independent experiments. b, Current-voltage relationships for an UT29-expressing (UT29) and a H2O-injected (H2O-inj) oocyte incubated in Kulori solution with (grey) and without (red) 10 mM 2Propenyl glucosinolate (2Prop) at pH 5.5. c, Effect of pH and extracellular Cl− on UT29-mediated import to oocytes incubated in 1 mM 2Prop for 60 min at pH 5.5 gluconate (Kulori buffer containing 90 mM Na+ gluconate instead of 90 mM NaCl), pH 5.5 Cl− and pH 7.4 Cl− (normal Kulori buffer with 95 mM Cl−). d–f, Membrane potential of oocytes measured in response to the substitution of anions and cations in Kulori buffer. d, pH 5.5 gluconate, pH 5.5 Cl− and pH 7.4 Cl−. e, 100 µM protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added into normal Kulori buffer at pH 5.5. f, 91 mM choline+ Cl− (Choline+) or 91 mM N-methyl-d-glutamine+ Cl− (NMDG+) were used to substitute 90 mM Na+Cl− and 1 mM K+Cl− in Kulori buffer, as the sole monovalent ions. Data are mean ± s.d. of individual data points (c,d,e,f) collected from at least two independent experiments or mean ± s.d. of three technical replicates from one representative experiment (b). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test (c–f). Bars labelled with different letters are significantly different, p < 0.05).
Extended Data Fig. 3 Effect of pH on export of glucosinolates by UMAMIT29.
Exported 2Propenyl glucosinolate (2Prop) (a), 4-methylthiobutyl glucosinolate (4MTB) (b) and indolyl-3-methyl glucosinolate (I3M) (c) levels were quantified in UMAMIT29-expressing oocytes (UT29, mean ± s.d. n = 10) or H2O-injected oocytes (mean ± s.d. n = 5) incubated at pH 5.5 or pH 7.4 for 4 h after injection of an equimolar mixture of glucosinolates (initial intracellular concentrations of individual glucosinolates are 50 µM). Glucosinolate export by UT29 at pH 5.5 and pH 7.4 was compared by one-way ANOVA analysis followed by Tukey’s post-hoc HSD test. Bars labelled with different letters are significantly different (P < 0.05).
Extended Data Fig. 4 Analysis of import and export of 2Prop and 13C,15N-isotope-labelled glutamine by UMAMIT29-expressing Xenopus oocytes.
a, Measured concentration of 13C,15N-isotope-labelled glutamine in the media for uptake assay (single measurement). b, Endogenous glutamine (Gln) in H2O-injected (H2O-inj) and UT29-expressing (UT29) oocytes after incubation with 0.4 mM, 0.8 mM, 2 mM and 10 mM labelled glutamine at pH 5.5 for 1 h. c, Intracellular level of 13C,15N-isotope-labelled glutamine in H2O-injected and UT29-expressing oocytes after incubation with 0.4 mM, 0.8 mM, 2 mM and 10 mM labelled glutamine at pH 5.5 for 1 h. d–i, Injection-based export assay using a mixture of 10 mM 13C,15N-isotope-labelled glutamine and 10 mM 2Propenyl glucosinolate (2Prop). Intracellular 13C,15N-isotope-labelled glutamine (d) and exported 13C,15N-isotope-labelled glutamine (e) and 2Prop (f) was determined by LC-MS analysis. Export of 2Prop but not glutamine by UMAMIT29 was observed. g, Level of exported endogenous glutamine in media in the assay. The dashed line in d marks the amount of injected substrate. Data are mean ± s.d. of individual data points collected from at least two independent experiments. *,**,*** show p value of student T-test, one tale, is less than 0.05, 0.01, 0.001 respectively.
Extended Data Fig. 5 Analysis of import and export of 2Prop and 13C,15N-isotope-labelled glutamate by UMAMIT29-expressing Xenopus oocytes.
a–d, Glutamate (Glu) and 2Propenyl glucosinolate (2Prop) import assays. a, Concentration of 13C,15N-isotope-labelled glutamate in the media for uptake assay. b, Endogenous glutamate level in H2O-injected and UMAMIT29-expressing oocytes (UT29). c,d, Intracellular level of 2Prop (c) and 13C,15N-isotope-labelled glutamate (d) in H2O-injected and UT29-expressing oocytes after incubation with substrate at pH 5.5 for 1 h. e–h, Injection-based export assay using a mixture of 30 mM 13C,15N-isotope-labelled glutamate and 30 mM 2Prop. Quantification of intracellular 13C,15N-isotope-labelled glutamate (Glu) (e) and 13C,15N-isotope-labelled aspartate (Asp) (f) at t = 0 and t = 3 h and exported 13C,15N-isotope-labelled glutamate (g) and 2Prop (h) after 3 h. Export of 2Prop but not glutamate by UMAMIT29 was observed. i, Level of exported endogenous glutamate in media in export assays. The dashed line in e marks the amount of injected substrate. Data are mean ± s.d. of individual data points collected from at least two independent experiments. *,**,*** show p value of student T-test, one tale, is less than 0.05, 0.01, 0.001, respectively.
Extended Data Fig. 6 Tissue-specific transcript enrichment of UMAMIT Clade I genes in developing seeds and funiculi.
Robust Multichip Average (RMA) normalized GeneChip data were retrieved from transcriptomic profiling of subregions of the seeds and the funiculus when the embryo enters at the globular (g), heart (h) and mature green (mg) stages24. a, Heatmap generated from standardized expression levels of each UMAMIT genes across different tissues followed by hierarchically clustering showing the enrichment of each gene in particular subregions of the seeds. b, Split heatmap generated from the non-standardized data reflecting the differences between the expression levels of each UMAMIT gene in different tissues. Abbreviation: EP = embryo proper; SUS = suspensor; PEN = peripheral endosperm; MCE = micropylar endosperm; CZE = chalazal endosperm; CZSC = chalazal seed coat; SC = distal seed coat; FUN = funiculus.
Extended Data Fig. 7 Genomic loci of UMAMIT29-31 and genotypes of umamit29, −30 and −31 mutants by T-DNA insertion and CRISPR-based genome editing.
a, The sgRNA sequence used to target UMAMIT31 and UMAMIT30 in a schematic representation of tandemly-linked UMAMIT29 UMAMIT30 and UMAMIT31 genomic loci. Different alleles of umamit30 (ut30) mutants and umamit31 (ut31) mutants were used in the study. Wild-type gene structures or sequences of UMAMIT30 (UT30) and UMAMIT31 (UT31) (top) are shown above the mutant alleles. Sanger sequencing of the PCR products denoting the detection of the DNA fragment flanking the loci targeted by sgRNAs in each mutant allele are shown. b, Transcript levels of UMAMIT29, UMAMIT30 and UMAMIT31 in Col-0 and T-DNA insertion lines ut29-1, ut29-2, ut30-1, ut31-1 as determined by quantitative real-time PCR with reverse transcription. The relative fold gene expression of samples was calculated with 2–∆∆Ct method. Values are mean ± s.d. (n = 4, representing 2 independent experiments with 2 biological repeats each). Quantitative real-time RT–PCR data are relative to ACTIN2(ACT2) gene (AT3G18780). Primers used in b and c are listed in Supplementary File. 3. c, Relative expression levels of UMAMIT clade I genes in wild-type Col-0 versus ut29-1 ut31-2 mutants (n = 3). Expression levels were normalized against the reference gene actin (At3g18780). Data are means ± s.d. Data point outside the lines (in red) are significantly differentially expressed in two genotypes. Student T-test, two tale, (p < 0.05).
Extended Data Fig. 8 Distribution of glucosinolates in stem and cauline leaves of umamit29, −30 and −31 mutants.
Content of methionine-derived (Met-derived) (a,c) and tryptophan-derived (Trp-derived) (b,d) glucosinolates (GLS) in the first internode (from base of the stem to the first node) (a,b) and the cauline leaves (c,d) of Col-0 (n = 6), umamit single (n = 7, 3, 8 for ut29-1, ut30-1, ut31-1, respectively), double (n = 16) and triple mutants (n = 7), gtr1 gtr2 gtr3 mutants (n = 6) as well as of ut29-1 ut30-5 gtr1 gtr2 gtr3 mutants (n = 5) (mean ± s.d.). a, b, c indicate significant differences determined by two-way ANOVA followed by post-hoc Tukey’s HSD test for all pairwise comparisons (p < 0.05).
Extended Data Fig. 9 Phylogeny of UMAMIT family from 14 plant species.
a, Selected phylogeny of UMAMIT family in Malvidae. Names of glucosinolate-producing taxa are shown in bold. b, Maximum-likelihood inferred tree (s.d. <0.01, optimal log-likelihood value (−35897.839)) of UMAMIT homologues from 14 species: Gossypium hirsutum, Theobroma cacao, Carica papaya, Arabidopsis thaliana, Brassica rapa, Glycine max, Manihot esculenta, Solanum lycopersicum, Zea mays, Vitis vinifera, Oryza sativa japonica, Eutrema salsugineum, Capsella rubella, and Citrus clementina. RAxML generated bootstrap values are shown for each branch. Branches of glucosinolate-producing taxa are shown in colour. Parameters for phylogenetic analysis are shown in Supplementary File. 1.
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Xu, D., Sanden, N.C.H., Hansen, L.L. et al. Export of defensive glucosinolates is key for their accumulation in seeds. Nature 617, 132–138 (2023). https://doi.org/10.1038/s41586-023-05969-x
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DOI: https://doi.org/10.1038/s41586-023-05969-x
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