Plant with low glucosinolate content in seeds

Abstract

The present invention provides a plant of the kind of cruciferae, or a part thereof, having low UMAMIT glucosinolate transporter activity. In particular, the present invention provides a plant of the kind of cruciferae carrying a mutation in a gene encoding a UMAMIT transporter and having a low seed glucosinolate content. In addition, the present invention describes a plant product prepared from the cruciferae plant or part thereof, as well as a method of producing the plant.

Description

Technical Field

[0001] The present invention relates to the field of agricultural products, and more particularly to crops and parts thereof having low UMAMIT glucosinolate transporter activity. Specifically, the present invention relates to Brassicales plants having a mutation in at least one gene encoding an UMAMIT transporter and having seeds with a low concentration of glucosinolates. Seeds of such plants are useful for producing seed products with a naturally low level of glucosinolates, such as seeds or seed cakes. The present invention also relates to a method for producing said Brassicales plants, and products prepared from the plants of the present invention. Background of the Invention

[0003] Plants of the order Brassicales, including the family Brassicaceae (or Cruciferae), contain many cultivars that provide sources of condiments, vegetables, feed crops, and the economically important crops rapeseed (Brassica napus and Brassica campestris or rapa) and mustard (Brassica juncea) for humans.

[0004] Seeds of rapeseed or other Brassica plants can be used as a source of seed oil. After the oil is extracted from the seeds, a protein-rich cake remains. This cake is an ideal animal feed. However, the potential of this source depends on the level of glucosinolates in the remaining seed cake.

[0005] A prominent and characteristic chemical property of Brassicales plants is their high content of glucosinolates (a class of natural plant products derived from amino acids and containing thioglucose and sulfonated oximes). These sulfur-containing secondary metabolites are important because they are cleaved by the hydrolytic enzyme myrosinase (glucosinolate glucohydrolase; EC 3.2.3.1) after plant damage to yield a variety of physiologically active products, such as nitriles, episulfides, oxazolidine-2-thiones, thiocyanates, and isothiocyanates. Glucosinolates can be found in all parts of Brassicales plants and are toxic to mammals, thus playing an important role in the plant's defense against herbivores.

[0006] According to the optimal defense theory, the plant organs with the highest fitness value accumulate the highest levels of defensive compounds to protect against herbivores and pathogens. Thus, the highest glucosinolate concentrations are found in reproductive organs, including seeds, siliques, flowers, and developing inflorescences, followed by young leaves, roots, and fully expanded leaves. Specifically, glucosinolates are known to be transported from maternal tissues to seeds / embryos in plants and accumulate to high levels there, resulting in high levels of defense in the seeds.

[0007] In the 1970s, traditional breeding produced a multilocus-dependent Brassica napus cultivar with reduced glucosinolate content in all parts of the plant, including seeds. This "00" (double-low) variety and its progeny subsequently became the most widely grown rapeseed cultivar in the Northern Hemisphere. However, the continuous selection bottleneck caused by this single source has limited the genetic diversity of future B. napus breeding programs and restricted interspecific hybridization. This has posed a serious problem for B. napus breeders who are working to improve yield and disease resistance through interspecific hybridization and introduce new traits such as drought tolerance.

[0008] Another problem with the "00" variety is that although the glucosinolate content of the seeds is low, it is not completely free of glucosinolates. The pressed seed cake obtained after oil extraction from the "00" variety typically contains less than 18 - 24 micromoles of total glucosinolates (GSL) per gram of dry weight (compared to 120 - 150 micromoles of total GSL per gram in traditional rapeseed meal). When used in compound feeds, the palatability of ruminants sets the allowable level of total GSL to no more than 10 - 15 micromoles per gram of dry weight, which means that if the GSL content of the seed cake is low, then theoretically animal feed could be almost entirely formulated from the "00" pressed seed cake. However, it has recently been found that poultry and pigs are much more sensitive to GSL than ruminants, and GSL levels above 2 - 4 micromoles per gram of dry weight in the feed can severely affect the reproductive efficiency of these animals. A truly "zero GSL" variety would bring significant commercial advantages to animal feed formulators, pressed seed cake producers, and farmers, as it could increase the amount of seed cake that can be included in compound feeds and eliminate the need to continuously monitor the GSL content of their products.

[0009] For humans, the toxic compounds contained in the edible parts of plants reduce their nutritional value. However, reducing antinutritional factors by blocking biosynthetic pathways often comes with an adverse impact on plant fitness, such as increased susceptibility to biotic or abiotic stresses.

[0010] The overall concept of reducing glucosinolates in seeds by modification is known. Modifying the glucosinolate importer gene to result in low total glucosinolate content in plants, including seeds, has been achieved. For example, WO 2012 / 004013 discloses the modification of the glucosinolate importer gene GTR and discloses a 60 - 70% reduction in glucosinolate levels in Brassica rapa and Brassica juncea plants. However, the glucosinolate levels in the obtained seeds are still too high for applications such as in seed cake or meal, e.g., in poultry feed. Additionally, a systemic reduction in glucosinolate content in plants is also undesirable as it may affect their natural defenses.

[0011] Accordingly, there is a significant commercial need for Brassicales plants with a tissue - specific reduction in glucosinolate content, e.g., Brassicales plants having a reduced glucosinolate content only in seeds while maintaining appropriate glucosinolate levels in other tissues. Summary of the Invention

[0012] The inventors have identified members of a Brassicaceae - specific branch of "usually multiple amino acid in - and - out transporters" (UMAMIT) that are essential for the accumulation of glucosinolates in Arabidopsis thaliana seeds. Surprisingly, the inventors found that the main function of UMAMIT29, UMAMIT30, and UMAMIT31 is to export glucosinolates into seeds. The inventors found that single, double, and triple exporter mutants have significantly reduced levels of glucosinolates (up to 95%) in seeds, far lower than previously obtained results. The exporter mutants do not alter the overall distribution of glucosinolates in the plant, so the plant retains a normal plant phenotype, ensuring adequate natural defense against herbivores. The inventors further found that orthologous genes in Brassica napus have glucosinolate transport capabilities similar to those of Arabidopsis.

[0013] The present invention relates to tissue - specific glucosinolate exporters in plants because only the exporters associated with exporting glucosinolates from the mother plant to the seeds are modified. By identifying and modifying these specific glucosinolate exporters, the glucosinolate levels in seeds can be specifically reduced without reducing the glucosinolate levels in the rest of the plant. Thus, the mother plant still maintains a high level of defense.

[0014] Accordingly, the present inventors have solved the problem of providing a Brassicales plant that maintains appropriate glucosinolate levels in other tissues but has a significantly reduced glucosinolate level in its seeds.

[0015] In one aspect, the present invention provides a Brassicaceae plant or a part thereof, wherein the Brassicaceae plant carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO: 1, AtUMAMIT29 shown in SEQ ID NO: 2, AtUMAMIT30 shown in SEQ ID NO: 3, AtUMAMIT31 shown in SEQ ID NO: 4, BnaA09G0714200ZS shown in SEQ ID NO: 5, BnaC05G0010000ZS shown in SEQ ID NO: 6, BnaA01G0222900ZS shown in SEQ ID NO: 7, BnaC01G0283800ZS shown in SEQ ID NO: 8, BnaC03G0332100ZS shown in SEQ ID NO: 9, BnaA09G0692700ZS shown in SEQ ID NO: 10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity, whereby the Brassicaceae plant or a part thereof expresses at least one mutant UMAMIT transporter, wherein the mutant UMAMIT transporter has low or no glucosinolate transporter activity and is expressed at a low level or not expressed.

[0016] In another aspect, the present invention provides a plant product comprising a Brassicaceae plant or a part thereof, or prepared from the seeds of said Brassicaceae plant or a part thereof, wherein said Brassicaceae plant carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, wherein said Brassicaceae plant or a part thereof expresses at least one mutant UMAMIT transporter, whereby said mutant UMAMIT transporter has low or no glucosinolate transporter activity and is expressed at a low level or not expressed.

[0017] In another aspect, the present invention provides a seed cake prepared from the seeds of a Brassicaceae plant, wherein said Brassicaceae plant is as described elsewhere herein.

[0018] In some aspects, the present invention provides a method for producing a plant product comprising a Brassicaceae plant or a part thereof having a low glucosinolate content, said method comprising the steps of:

[0019] a. providing a Brassicaceae plant or a part thereof as described elsewhere herein; and

[0020] b. processing said Brassicaceae plant or a part thereof into a plant product, such as seed oil or seed cake.

[0021] In another aspect, the present invention provides a method for altering the glucosinolate content in a Brassicaceae plant or a part thereof, the method comprising the step of altering the functional activity or expression of at least one UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO: 1, AtUMAMIT29 shown in SEQ ID NO: 2, AtUMAMIT30 shown in SEQ ID NO: 3, AtUMAMIT31 shown in SEQ ID NO: 4, BnaA09G0714200ZS shown in SEQ ID NO: 5, BnaC05G0010000ZS shown in SEQ ID NO: 6, BnaA01G0222900ZS shown in SEQ ID NO: 7, BnaC01G0283800ZS shown in SEQ ID NO: 8, BnaC03G0332100ZS shown in SEQ ID NO: 9, BnaA09G0692700ZS shown in SEQ ID NO: 10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: Identification of key source tissues and transporters for glucosinolate accumulation in Arabidopsis seeds. a. Analysis of glucosinolate content in grafted Arabidopsis seeds. Five-week-old wild-type Col-0 (WT), biosynthesis null mutant myb28 myb29 cyp79b2 cyp79b3 (null), and transporter mutant gtr1 gtr2 (gtr) were stem-grafted to each other at 1 cm above the ground. Seeds were harvested from mature pods of the scions, and total glucosinolate (GLS) content was quantified from pools of 10 seeds (n ≥ 3 plants per group, but 2 plants for the WT / null grafts). b, Total GLS in pools of 10 seeds from Col-0, umamit29-1 (ut29-1), umamit29-2 (ut29-2), and plants complemented with pUT29(6kb)-UT29 (genomic fragment)-mVenus (ut29-1C) at different days after pollination (DAP) (data are representative of three independent lines) (n = 6 plants per group). Four box plots are shown per day. The four box plots represent Col-0, ut29-1, ut29-2, and ut29-1C from left to right. c, Expression of pSUR1::SUR1-mTQ2 (top) and pUT29::UT29-mVenus (bottom) in developing pods collected at different days after pollination (DAP). Inset shows the funiculus and seed at lower magnification. Scale bars: 50 μm (main panel) and 100 μm (inset). a, Data are presented as mean ± s.d. Letters indicate significant differences by non-parametric Kruskal-Wallis test and subsequent Dunn's test (p < 0.05). b, Time-course data were fitted to a linear model, and pairwise comparisons between genotypes were made based on estimated marginal means (EMM). Seed glucosinolate accumulation in Col-0 and ut29-1C was significantly different from ut29-1 and ut29-2 (Tukey-corrected p < 0.0001). Abbreviations: DAP = days after pollination; GLS = glucosinolate; SUR1 = SUPERROOT1; UT = UMAMIT; WT = wild type.

[0023] Figure 2: Biochemical and Biophysical Properties of Arabidopsis UMAMIT29, UMAMIT30, and UMAMIT31. a, Glucosinolate accumulation in Xenopus laevis oocytes expressing UMAMIT29 (UT29) or injected with water (H2O-inj). Oocytes were incubated with 4MTB (64 μM) and I3M (34 μM) in Kulori buffer at pH 5.5 (mean ± s.d., n = 15). b, Membrane currents associated with 2-propenyl glucosinolate (2Prop) uptake in a representative oocyte expressing UMAMIT29 (UT29), voltage-clamped at -60 mV and perfused with 10 mM 2Prop in Kulori buffer for 2 min. c, Charge-coupled stoichiometry in 13 UT29-expressing oocytes (gray) and 10 H2O-injected oocytes (black) during the clamp shown in b was estimated by the correlation between current-time integration and 2Prop uptake. d, Effect of pH on UT29-mediated glucosinolate import. Intracellular 2Prop in oocytes expressing UT29 or injected with H2O (H2O-inj) was quantified after incubation with 1 mM 2Prop at pH 7.4 and pH 5.5 with or without 0.1 mM CCCP for 60 min. e, Effect of extracellular cations on UT29-mediated 2Prop import. Kulori buffer (90 mM NaCl and 1 mM KCl) was replaced with 91 mM choline + Cl - (choline + ) or 91 mM N-methyl-d-glutamine + Cl - (NMDG + ) to replace Kulori buffer (90 mM Na + Cl - and 1 mM K + Cl -) The cations in. After incubation in 5 mM 2Prop at pH 7.4 for 60 minutes, intracellular 2Prop was quantified in oocytes expressing UT29 or injected with H2O. f, Injection-based efflux assay of 2Prop by UT29. Oocytes expressing UT29 (light gray line) or injected with H2O (dark gray line) were injected with 2Prop (initial internal concentration of approximately 2 mM). Efflux activity was measured by quantifying intracellular and extracellular 2Prop contents over time in oocytes incubated in Kulori buffer (pH 7.4) (mean ± s.d., n = 5). g, Uptake of 0.5 mM 4MTB by oocytes expressing UT29 in the presence or absence of excess glutamine at pH 5. h, Uptake of 0.5 mM 4MTB by oocytes expressing UT29 in the presence or absence of excess 2Prop (10 mM). i, j, Influx activity assays of oocytes expressing UT29, -30, and -31 transporters using an equimolar mixture of glucosinolates (GLS) (0.1 mM each) as substrates, where GLS consists of 11 aliphatic glucosinolates and 1 indole glucosinolate. k, l, Glucosinolates effluxed from oocytes expressing UT incubated for 5 hours in Kulori buffer (pH 5.5) after injection with the glucosinolate mixture used in i and j (initial intracellular concentration of each glucosinolate was approximately 50 μM) (mean ± s.d., n = 4). Glucosinolates were divided into aliphatic and indole groups according to their side chains. n, Biological replicates for each experiment, with 3 oocytes pooled in each replicate. Treatments were compared by one-way ANOVA and subsequent Tukey's post hoc HSD test. Bars labeled with different letters indicate significant differences, p < 0.05). Abbreviations: 4MTB = 4-methylthiobutyl glucosinolate; 2Prop = 2-propenyl glucosinolate; CCCP = protonophore carbonyl cyanide m-chlorophenylhydrazone; I3M = indole-3-ylmethyl glucosinolate; GLS = glucosinolate; UT = UMAMIT.

[0024] Figure 3: Seed traits and glucosinolate distribution in UMAMIT29, -30, and -31 mutants. a, b, Total contents of methionine (a) and tryptophan (b)-derived glucosinolates (GLS) in seeds of Arabidopsis wild type Col-0 (Col-0), umamit29-1 (ut29-1), umamit30-1 (ut30-1), and umamit31-1 (ut31-1) single mutants and ut29-1 ut30-2, ut29-1 ut30-3, ut29-1 ut31-2 double mutants, and ut29-1 ut30-3 ut31-5, ut29-1 ut30-2 ut31-3 triple mutants (mean ± s.d., n = 6). c, d, Glucosinolate contents in developing seeds and seedless pods (i.e., valves, replum, and funiculus) of umamit mutants. Glucosinolate contents in seeds (c) and seedless pods (d) at different pod positions, counted from the first pod on the stem. e, Free amino acids in seeds (mean ± s.d., n ≥ 6 per genotype). f, Seed area of individual seeds of different genotypes estimated by quantifying the area profile of individual seeds (n = 250 - 300 per genotype). g, Total weight of 500 seeds per genotype (mean ± s.d., n = 3). h - k, Glucosinolate contents in roots (h, i) and rosette leaves (j, k) of umamit single, double, and triple mutants and ut29-1 ut30-5 gtr1gtr2 gtr3 mutant (mean ± s.d., n ≥ 6 per genotype). a - k, Treatments were compared by one-way ANOVA analysis and subsequent Tukey's post hoc HSD test. Bars marked with different letters indicate significant differences (P < 0.05). Abbreviations: GLS = glucosinolate; UT = UMAMIT; FW = fresh weight.

[0025] Figure 4: Time course of glucosinolate accumulation in developing siliques and cellular localization of UMAMIT29. a, b, Total glucosinolates (GLS) in pools of 10 developing seeds from wild-type Arabidopsis Col-0 at different days after pollination (a) and corresponding seedless developing siliques (including valves, replum, and funiculus) (b) (n = 6 plants per group). c, Cross-sections of siliques expressing pCYP83A1::CYP83A1-mVenus (top) and pCYP83B1::CYP83B1-mVenus (bottom) at the green mature stage. d, Transporters expressed in the funiculus from transcriptome data. Transporter genes with increased expression in the funiculus from the globular stage (gFUN), heart stage (hFUN) to the green mature stage (mgFUN) were selected, resulting in a list of 10 glucosinolate candidate exporter proteins. e, Total amounts of methionine-derived and tryptophan-derived glucosinolates in pools of 10 seedless (i.e., valve of silique including replum and funiculus) siliques from wild-type Col-0 (Col-0), umamit29-1 (ut29-1), umamit29-2 (ut29-2), and plants complemented with pUT29(6kb)-UT29 (genomic fragment)-mVenus (ut29-1C) at different days after pollination (data are representative of three independent lines) (n = 6 plants per group). Four box plots are shown per day. The four box plots represent Col-0, ut29-1, ut29-2, and ut29-1C from left to right, respectively. f - h, Cellular localization of UMAMIT29-mVenus in live funiculi at 8 days after pollination. f, Accumulation of UMAMIT29-mVenus in the funiculus. Note that the seeds have been dissected in this view so that the funiculus can be optimally exposed for high-resolution live imaging. g, Maximum intensity projection of a Z-stack of the funiculus shown in f at higher magnification. h, Single plane of the Z-stack showing Umami-T29-mVenus localized to the plasma membrane. Inset: Magnified view of UMAMIT29-mVenus signal around chloroplasts in pUT29::UT29-mVenus plants. Green: UT29-mVenus, magenta: chlorophyll autofluorescence. Scale bars: A: 250 μm, B and C: 50 μm, C inset: 10 μm.

[0026] Figure 5: Transcriptome data analysis of tissues of developing pods. Tissue-specific transcript enrichment of UMAMIT branch I genes in developing seeds and funiculi. Heatmaps using hierarchical clustering analysis show the relative mRNA levels of UMAMIT family branch I in each sub-region of seeds and funiculi at the globular (g), heart (h), and mid-green (mg) stages of embryo development. Hierarchical clustering analysis was performed using the default settings of Origin software. Abbreviations: EP = embryo proper; SUS = suspensor; PEN = peripheral endosperm; MCE = micropylar endosperm; CZE = chalazal endosperm; CZSC = chalazal seed coat; SC = distal seed coat; FUN = funiculus.

[0027] Figure 6 : Relative expression levels of UMAMIT branch I genes in wild-type Col-0 and ut29-1 ut31-2 mutants (n = 3). Expression levels were normalized against the reference gene actin (At3g18780). Data are mean ± s.d. Data points outside the line (light grey) are significantly differentially expressed in the two genotypes. Student's t-test, two-tailed, (p < 0.05).

[0028] Figure 7: Loci of UMAMIT29-31 and genotypes of umamit29, -30, and -31 mutants generated by T-DNA insertion and CRISPR-based genome editing. a, Schematic of the tandemly linked UMAMIT29, UMAMIT30, and UMAMIT31 loci, with sgRNA sequences used to target UMAMIT31 and UMAMIT30. Different alleles of the umamit30 (ut30) and umamit31 (ut31) mutants were used in this study. The wild-type gene structures or sequences of UMAMIT30 (UT30) and UMAMIT31 (UT31) (upper panel) are shown above the mutant alleles: the sgRNA target sites in UMAMIT31 of wild-type Arabidopsis thaliana (SEQ ID NO:23) and in UMAMIT30 of wild-type Arabidopsis thaliana (SEQ ID NO:27), and the sgRNA sites in the mutants ut31-3 (SEQ ID NO:24), ut31-4 (SEQ ID NO:25), ut31-5 (SEQ ID NO:26), ut30-2 (SEQ ID NO:28), ut30-3 (SEQ ID NO:29), and ut30-4 (SEQ ID NO:30). Sanger sequencing of PCR products demonstrated the detection of DNA fragments flanking the loci targeted by the sgRNA in each mutant allele. b, Transcription levels of UMAMIT29, UMAMIT30, and UMAMIT31 in Col-0 and T-DNA insertion lines ut29-1, ut29-2, ut30-1, ut31-1 were determined by reverse transcription quantitative real-time PCR. Values are means ± s.d. (n = 4, representing 2 independent experiments with 2 biological replicates each). Quantitative real-time RT-PCR data were relative to the ACTIN2 (ACT2) gene (AT3G18780).

[0029] Figure 8 : Glucosinolate distribution in the stems and cauline leaves of umamit29, -30, and -31 mutants. Contents of methionine-derived (Met-derived) (a, c) and tryptophan-derived (Trp-derived) (b, d) glucosinolates (GLS) in the first internode (from the base of the stem to the first node) (a, b) and cauline leaves (c, d) of umamit single, double, and triple mutants and the ut29-1 ut30-5 gtr1 gtr2 gtr3 mutant (means ± s.d., n ≥ 6 for each genotype). a, b, c indicate significant differences in all pairwise comparisons determined by two-way ANOVA and subsequent post hoc Tukey HSD test (p < 0.05).

[0030] Figure 9 : Phylogeny of a part of the UMAMIT family in Malvidae. Selected part of the maximum likelihood inference tree of UMAMIT homologs in 14 species (s.d. < 0.01, best log-likelihood value (-35897.839)): 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 for each branch. The names of the glucosinolate-producing taxa are shown in bold.

[0031] Figure 10 : Maximum likelihood inference tree of UMAMIT genes in Arabidopsis and Brassica napus ZS11 orthologs. The bootstrap (1000 replicates) values for each branch are shown as percentages.

[0032] Figure 11 : Expression profiles of Brassica napus ZS11 UMAMIT genes in pod walls and seeds. Transcriptome data were extracted from the Brassica Expression Database (Brassica EDB). To verify its expression pattern, a BnGTR2 required for seed glucosinolate loading by GWAS analysis was included.

[0033] Figure 12: Cloning (A) and characterization (B) of UMAMIT orthologous genes in Brassica napus Niklas. A) Relative expression of 11 UMAMIT orthologous genes mRNA in different tissues of Niklas (leaves, stems, flowers, pods at three developmental stages: young pods less than 20 DAP, old pods at 30 - 40 DAP, senescent mature pods, and dormant seeds). Expression relative to the actin reference gene (n = 3 plants). Results are representative of two independent experiments. (B), Uptake of 4MTB (4-methylthiobutyl glucosinolate), 2OH-3But (2(R)-2-hydroxy-3-butenyl glucosinolate), and I3M (indol-3-ylmethyl glucosinolate) by oocytes expressing UMAMIT at pH 5 (n = 5 batches, 3 oocytes per batch). Results are representative of three independent experiments.

[0034] Figure 13 : Sequence logo of 51 residues predicted to form the substrate transport cavity based on the amino acid sequences of 96 transporters of UMAMIT branch I. The percentage of conservation of each residue relative to all 96 sequences is shown in the figure. Four of these amino acid residues are 100% conserved and are marked with black dots. The sequence logo was generated by JDet.

[0035] Figure 14 : Uptake of 4MTB, I3M, and BGLS by UMAMIT29 mutant variants with mutations at conserved residues selected within the predicted substrate transport cavity. Different letters indicate significant differences in means (TUKEY HSD test after one-way ANOVA, p < 0.05). Abbreviations: UT, UMAMIT; 4MTB, 4-methylthiobutyl glucosinolate; I3M, indol-3-ylmethyl glucosinolate; BGLS, benzyl glucosinolate.

[0036] Figure 15 : Sequence logo of amino acid residues in the predicted substrate transport cavities of two major transporter clusters of UMAMIT branch I. Differentially conserved residues (asterisks) were estimated using DIVERGE software. Two of the estimated residues from DIVERGE were identical in UMAMIT32 as well as UMAMIT30 and UMAMIT31 and were thus excluded (grey asterisks). The role of 11 residues estimated to be differentially conserved by experimental testing in glucosinolate transporter activity (black asterisks). The sequence logo was generated by JDet. Numbers represent the positions of the residues in UMAMIT29, and the corresponding residues in UMAMIT29, -30, -31, and -32 are visible in Supplementary Table 1. Detailed Description of the Invention

[0038] Definitions

[0039] As used herein, the term "cruciferous plant" refers to plants of the order Brassicales.

[0040] As used herein, the terms "about" and "approximately" in connection with a numerical value preferably mean ±10%, more preferably ±5%, still more preferably ±1%.

[0041] As used herein, the term "Brassicales plant" refers to plants of the order Brassicales. Non-limiting examples of Brassicales plants include plants of the family Brassicaceae such as mustard, Brassica napus (rapeseed), Brassica carinata, Brassica oleracea, and Brassica rapa.

[0042] The "glucosinolate" (abbreviated as "GSL" or "GLS") used in this article refers to an amino acid-derived organic anion of glucosinolate containing a sulfonated aldoxime moiety. Variable side chains depending on the parent amino acid and further side chain modifications confer unique chemical and biological properties on GSL. Approximately 120 different GSLs have been described in the literature, all of which are derived from only 8 different amino acids. The parent amino acid is usually used as a classification criterion. GSLs derived from Ala, Leu, Ile, Val, and Met are called "aliphatic GSLs", GSLs derived from Tyr and Phe are called "aromatic GSLs", and GSLs derived from Trp are called "indole GSLs". The great diversity of GSL types is caused by various modifications on the side chains of the parent amino acids. In particular, methionine undergoes a wide range of conversions. The main aliphatic GSLs in crucifers have side chains in the form of chain extensions derived from Met, such as aliphatic thio-GSLs: 3-methylthiopropyl (3-MTP)-, 4-methylthiobutyl (4-MTB)-, 5-methylthiopentyl (5-MTP)-, 6-methylthiohexyl (6-MTH)-, 7-methylthioheptyl (7-MTH)-, and 8-methylthiooctyl (8-MTO)-GSLs; aliphatic sulfinyl-GSLs: 3-methylsulfinylpropyl (3-MSP)-, 4-methylsulfinylbutyl (4-MSB)-, 5-methylsulfinylpentyl (5-MSP)-, 6-methylsulfinylhexyl (6-MSH)-, 7-methylsulfinylheptyl (7-MSH)-, and 8-methylsulfinyloctyl (8-MSO)-GSLs; aliphatic hydroxy-GSLs: 3-hydroxypropyl (3-OHP)- and 4-hydroxybutyl (4-OHB)-GSLs; aliphatic benzoyloxy-GSLs: 3-benzoyloxypropyl (3-BZOP)- and 4-benzoyloxybutyl (4-BZOB)-GSLs, and aliphatic alkenyl-GSLs: 2-propenyl (2-P)- and 3-butenyl (3-B)-GSLs. The main aromatic GSLs in crucifers have side chains derived from Phe, such as aromatic GSLs: 2-phenylethyl (2-PE)-GSLs. There are also smaller amounts of GSLs with indole side chains derived from Trp, such as indole-GSLs: indole-3-ylmethyl (i3M)-GSLs. GSLs coexist with the GSL-specific thioglucosidase myrosinase in plants. This enzyme is physically separated from GSLs in plants but comes into contact with its substrate after tissue disruption. The resulting hydrolysis products consist of one free glucose and one aglycone molecule per GSL molecule. The aglycone is unstable and readily rearranges into isothiocyanates, nitriles, thiocyanates, and other more or less toxic compounds. Depending on the side chain of the parent amino acid, these hydrolysis products confer the actual biological activity of GSLs, while intact GSLs are considered an inactive storage form.

[0043] The "glucosinolate content" of a plant or plant part as used herein refers to the total amount of GSLs, including aliphatic, aromatic, and indolic GSLs, regardless of the type of GSL. Thus, the "total GSL content" or "GSL content" of a plant or plant part refers to the total content of GSLs in that plant or plant part, expressed on a molecular (nmol / g or μmol / g) basis (rather than on a weight (mg / kg) basis), because the molecular weights of GSLs vary significantly depending on the size of their side chains. The accumulation of GSLs varies depending on tissue and developmental stage. Young leaves and reproductive tissues (e.g., pods and seeds) contain the highest concentrations, while senescent leaves contain the lowest concentrations of GSLs. Intermediate concentrations are found in "large" organs such as roots, leaves, and stems. In addition, there are significant differences in the composition of the GSL profiles in different organs. In vegetative tissues, the GSL content consists of indolic and aliphatic GSLs, with no aromatic GSLs present. In pods and seeds, small amounts of aromatic and indolic GSLs are found, and the remaining GSL content consists entirely of aliphatic GSLs.

[0044] "Glucosinolate transporter activity" refers to the ability of a protein to transport glucosinolates across a cell membrane. Glucosinolate transporter activity can be measured, for example, using the Xenopus oocyte assay described in Example 1. Glucosinolate transporter activity can be associated with one or more different glucosinolates or groups of glucosinolates. For example, methionine-derived and tryptophan-derived glucosinolates represent aliphatic and aromatic glucosinolates, respectively.

[0045] For GSL transport, "low or no" glucosinolate transporter activity or "loss of function" refers to a decrease in activity compared to the wild-type unmutated glucosinolate transporter, for example, using the Xenopus oocyte assay of Example 1 or by studying the GSL content in the seeds of mutant plants. "Low" activity refers to 50% or less, preferably 40% or less, more preferably 30% or less, more preferably 20% or less, more preferably 10% or less of the GLS transporter activity compared to the wild type. "No" GLS transporter activity or "loss of function" refers to a decrease in glucosinolate transporter activity to 5% or less, for example 3% or less, for example 1% or less of the wild-type transporter activity.

[0046] A "decrease in total GSL content" or "increase in total GSL content" of a plant or plant part measured by the method of the present invention is measured relative to the total GSL content of a reference plant or plant part having a similar genetic background. The total GSL content can be measured by any suitable method. Methods for quantifying total GSL content and determining the GSL composition of plant material are well known in the art and include, but are not limited to: HPLC-UV desulfurization method, involving HPLC analysis of a methanol extract desulfurized and eluted from a sephadex anion exchange column, as described, for example, by Hansen et al. (2007, Plant J. 50(5):902-910); MALDI-TOF mass spectrometry of intact GSLs, such as Botting et al. (2002, J. Agric. Food Chem. 50(5):983–988); near-infrared reflectance spectroscopy, as described by Font et al. (2005, J. Agric. Sci. 143:65–73); methods for generating spectrophotometrically active degradation products, as summarized in Clarke (2010, Anal. Methods 2:310-325); HPLC mass spectrometry of intact glucosinolates, as described by Rochfort et al. (2008, Phytochemistry 69:1671).

[0047] Whenever the "plant" of the present invention is referred to, it should be understood that, unless otherwise specified, plant parts, plant progeny retaining the parental distinguishing characteristics (especially the glucosinolate content of a specific plant part), such as seeds obtained by selfing or hybridization, such as hybrid seeds (obtained by crossing two inbred parental lines), hybrid plants and their derived plant parts are also covered herein.

[0048] As used herein, "plant part" refers to any part of a plant, including plant cells, plant tissues, plant organs, pods or seed pods, seeds, detached parts such as roots, leaves, flowers, pollen, etc.

[0049] As used herein, "wild type" refers to the most common form of a plant or gene in nature. A "wild type plant" refers to a plant having the most common phenotype of such plants in a natural population. A "wild type allele" refers to an allele of a gene required to produce a wild type phenotype. In contrast, a "mutant plant" refers to a plant having a different rare phenotype of such plants in a natural population or a plant produced by artificial intervention (such as mutagenesis), and a "mutant allele" refers to an allele of a gene required to produce a mutant phenotype.

[0050] A homolog or functional homolog can be any polypeptide that has at least some sequence identity with a reference polypeptide and retains at least one aspect of the original function. In this context, a functional homolog of the UMAMIT transporter refers to a polypeptide that has at least some sequence identity with the UMAMIT transporter or a fragment thereof and is capable of functioning as a glucosinolate transporter similar to the UMAMIT transporter.

[0051] As used herein, the term "sequence identity" refers to the relatedness between two amino acid sequences or two nucleotide sequences, i.e., the candidate sequence (e.g., mutant sequence) and the reference sequence (e.g., wild-type sequence) based on their pairwise alignment. For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is implemented by the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or higher (obtainable from https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are: a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of 30BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0052] (Number of identical residues x 100) / (length of the alignment - total number of gaps in the alignment)

[0053] The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence (e.g., a natural variant or a haplotype of SEQ ID NO:1) corresponds to a given position in SEQ ID NO:1 (the reference sequence). For example, if a natural variant has two additional amino acids at the N-terminus, then the 70th position in the natural variant will correspond to the 68th position in SEQ ID NO:1.

[0054] For the purposes of the present invention, sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), which is implemented by the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or higher. The parameters used are: a gap open penalty of 10, a gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0055] (Number of identical deoxynucleotides x 100) / (length of the alignment - total number of gaps in the alignment).

[0056] As is generally understood in the art, the terms "corresponding sequence", "corresponding region" or "corresponding residue" refer to a region or residue on a second amino acid or nucleotide sequence that occupies the same (i.e., equivalent) position as a region or residue on a first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparison purposes. Thus, the residue at the first position in the first peptide sequence does not necessarily correspond to the residue at the same first position in the second peptide sequence, but may correspond to the residue at the second position in the second peptide sequence that best aligns with the residue at the first position in the first peptide sequence when the first and second peptide sequences are optimally aligned. The alignment can be performed by any method known in the art, for example, by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is implemented by the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or higher. The parameters used can be a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix.

[0057] As used herein, the term "missense mutation" refers to a mutation in a nucleotide sequence that causes an amino acid in the polypeptide encoded by the nucleotide sequence to be changed to another amino acid.

[0058] "Mutation" includes deletions, insertions, substitutions, transversions, and point mutations in the coding region and / or non-coding region of a gene. The deletion can be a deletion of the entire gene or a deletion of a part of the gene. A point mutation can involve a change in a single base pair and can result in a premature stop codon, a frameshift mutation, a splice site mutation, or an amino acid substitution. A gene that contains a mutation as compared to the wild-type gene can be referred to as a "mutant gene". In the present invention, a mutant gene typically encodes a polypeptide having a different sequence from the wild-type gene, and the polypeptide can be referred to as a "mutant polypeptide". A mutant polypeptide can contain an amino acid substitution. For example, such a substitution can be described, for example, as "the amino acid XXX at position n has been replaced by the amino acid YYY", where XXX describes the amino acid at a specific position (n) of the wild-type polypeptide and YYY describes the amino acid at the same position in the mutant polypeptide when the two genes are aligned.

[0059] The terms "UMAMIT transporter" and "UMAMIT exporter" are used interchangeably herein.

[0060] A Brassicaceae plant comprising a mutation in a gene encoding an UMAMIT transporter

[0061] The present invention relates to a Brassicaceae plant or a part thereof, and products and methods for producing them, wherein the Brassicaceae plant carries a mutation in a gene encoding an UMAMIT transporter, such as any of the mutations in the gene encoding an UMAMIT transporter described herein.

[0062] Accordingly, one aspect of the present invention provides a Brassicaceae plant or a part thereof, wherein the Brassicaceae plant carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO: 1, AtUMAMIT29 shown in SEQ ID NO: 2, AtUMAMIT30 shown in SEQ ID NO: 3, AtUMAMIT31 shown in SEQ ID NO: 4, BnaA09G0714200ZS shown in SEQ ID NO: 5, BnaC05G0010000ZS shown in SEQ ID NO: 6, BnaA01G0222900ZS shown in SEQ ID NO: 7, BnaC01G0283800ZS shown in SEQ ID NO: 8, BnaC03G0332100ZS shown in SEQ ID NO: 9, BnaA09G0692700ZS shown in SEQ ID NO: 10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith, whereby the Brassicaceae plant or a part thereof expresses at least one mutant UMAMIT transporter, wherein the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level or not expressed.

[0063] The glucosinolate transporter activity of the UMAMIT transporter can be measured by any method known in the art. In a preferred embodiment, the glucosinolate transporter activity of the UMAMIT transporter is measured using the methods described in Examples 1 and 2 herein.

[0064] In some embodiments, the Brassicales plant carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity therewith, whereby the Brassicales plant or a part thereof expresses at least one mutant UMAMIT transporter, wherein the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level or not expressed.

[0065] In some embodiments, Brassicaceae plants carry a mutation in the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or in a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0066] In some embodiments, Brassicaceae plants carry a mutation in the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or in a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0067] In some embodiments, Brassicaceae plants carry a mutation in the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or in a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0068] In some embodiments, the Brassicaceae plant carries a mutation in the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0069] In some embodiments, the Brassicaceae plant carries a mutation in the UMAMIT transporter BnaA09G0714200ZS shown in SEQ ID NO:5 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0070] In some embodiments, a Brassicaceae plant carries a mutation in the UMAMIT transporter BnaC05G0010000ZS shown in SEQ ID NO:6 or in a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0071] In some embodiments, a Brassicaceae plant carries a mutation in the UMAMIT transporter BnaA01G0222900ZS shown in SEQ ID NO:7 or in a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0072] In some embodiments, the Brassicaceae plant carries a mutation in the UMAMIT transporter BnaC01G0283800ZS shown in SEQ ID NO:8 or in a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0073] In some embodiments, the Brassicaceae plant carries a mutation in the UMAMIT transporter BnaC03G0332100ZS shown in SEQ ID NO:9 or in a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0074] In some embodiments, the Brassicaceae plant carries a mutation in the UMAMIT transporter BnaA09G0692700ZS shown in SEQ ID NO:10 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0075] The Brassicaceae plant of the present invention may contain mutations in more than one gene encoding the UMAMIT transporter.

[0076] Thus, in some embodiments, a Brassicaceae plant or a part thereof carries a mutation in at least two genes encoding a UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0077] Thus, in some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a gene encoding the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0078] In some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, and a gene encoding the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0079] In some embodiments, a Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a gene encoding the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0080] In some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, and a gene encoding the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0081] In some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or its respective functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a gene encoding the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or its respective functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0082] In some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or its respective functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a gene encoding the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or its respective functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0083] In some embodiments, a Brassicaceae plant or a part thereof carries a mutation in at least three genes encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0084] Thus, in some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, a gene encoding the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a gene encoding the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0085] In some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or its respective functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, a gene encoding the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or its respective functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a gene encoding the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or its respective functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0086] In some embodiments, a Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, a gene encoding the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a gene encoding the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0087] In some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity; a gene encoding the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity; and a gene encoding the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0088] In some embodiments, a Brassicaceae plant or a part thereof carries a mutation in at least four genes encoding a UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.

[0089] Thus, in some embodiments, the Brassicaceae plant or a part thereof carries a mutation in a gene encoding the UMAMIT transporter AtUMAMIT28 shown in SEQ ID NO:1 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto; a gene encoding the UMAMIT transporter AtUMAMIT29 shown in SEQ ID NO:2 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto; a gene encoding the UMAMIT transporter AtUMAMIT30 shown in SEQ ID NO:3 or a respective functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto,A gene encoding the UMAMIT transporter AtUMAMIT31 shown in SEQ ID NO:4 or its respective functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0090] Several orthologs that can be used for the purposes of the present invention are listed in Tables 1-4 below: AtUMAMIT28 (SEQ ID NO:1), AtUMAMIT29 (SEQ ID NO:2), AtUMAMIT30 (SEQ ID NO:3), AtUMAMIT31 (SEQ ID NO:4).

[0091] The orthologs listed below can be obtained from the Brassica napus pan-genome database (website: http: / / cbi.hzau.edu.cn / cgi-bin / bnapus / search (Song et al., 2021)) using the identifiers listed in the following table. The same identifier can be used to access both the genomic sequence and the encoded protein.

[0092] Table 1 - Available Brassicaceae orthologs of AtUmamiT28

[0093]

[0094]

[0095]

[0096] Table 2 - Available Brassicaceae orthologs of AtUmamiT29

[0097]

[0098]

[0099] Table 3 - Available Brassicaceae orthologs of AtUmamiT30

[0100]

[0101]

[0102]

[0103]

[0104] Table 4 - Available Brassicaceae orthologs of AtUmamiT31

[0105]

[0106]

[0107]

[0108] In some embodiments, therefore, there is provided a Brassicaceae plant or a part thereof carrying a mutation in a gene encoding a UMAMIT transporter selected from the group consisting of: BnaA09G0679100GG, BnaA09G0679000GG, BnaC05G0008000GG, BnaA09G0647600GG, BnaA09G0647700GG, BnaA10G0008000GG, BnaA09G0623500NO, BnaA09G0623600NO, BnaC05G0007600NO, Bnascaffold2891G0004400NO, BnaA09G0624000NO, BnaC08G0554500QU, BnaA09G0664600QU, BnaC08G0554400QU, BnaA10G0008300QU, Bnascaffold2503G0022000QU, BnaA09G0664700QU, BnaA09G0724700SL, BnaA09G0689800SL, BnaA09G0724800SL, BnaC05G0007600SL, BnaA09G0689900SL, Bnascaffold3068G0013800SL, BnaA09G0577200TA, BnaA09G0628700TA, BnaC05G0007500TA, BnaA09G0577100TA, BnaA10G0008300TA, BnaA09G0628800TA, BnaA09G0658500WE, BnaA09G0658600WE, BnaC05G0009100WE, BnaA10G0009300WE, BnaA09G0659000WE, BnaA09G0692800ZS, BnaA09G0692700ZS, BnaA09G0714200ZS, BnaA09G0714300ZS, BnaA10G0008500ZS, BnaC05G0010000ZS, BnaA09G0634900ZY, BnaA09G0666500ZY, Bnascaffold4696G0013500ZY, BnaC05G0007300ZY, BnaA09G0635000ZY, BnaA09G0666400ZY, BolC5g28871H, BolC8g52858H, BraA10g42362Z, BraA09g42228Z, and BraA09g42227Z.

[0109] In some embodiments, provided is a Brassicaceae plant or a part thereof carrying a mutation in a gene encoding a UMAMIT transporter selected from the group consisting of: BnaC02G0303900GG, BnaA02G0239900GG, BnaC02G0303800GG, BnaC02G0289800NO, BnaA02G0248400NO, BnaA02G0248300NO, BnaC02G0286300QU, BnaA02G0247800QU, BnaA02G0247700QU, BnaC02G0286400QU, BnaC02G0230500SL, BnaA02G0281700SL, BnaC02G0230600SL, BnaC02G0254900TA, BnaA02G0214900TA, BnaC02G0255000TA, BnaC02G0326100WE, BnaA02G0260700WE, BnaA02G0260600WE, BnaC02G0326200WE, BnaC02G0337200ZS, BnaA02G0251300ZS, BnaC02G0337100ZS, BnaA02G0251200ZS, BnaA02G0278400ZY, BnaA02G0278500ZY, BnaC02G0162700ZY, BnaC02G0162600ZY, BolC2g09864H, and BraA02g07429Z.

[0110] In some embodiments, provided is a Brassicaceae plant or a part thereof carrying a mutation in a gene encoding a UMAMIT transporter, wherein the UMAMIT transporter is selected from the group consisting of: BnaC03G0326100GG, BnaA03G0226900GG, BnaA01G0215700GG, BnaC01G0262700GG, BnaA03G0226800GG, BnaC01G0263000GG, BnaC03G0325800GG, BnaC03G0326000GG, BnaC02G0304200GG, BnaA02G0240100GG, BnaC03G0251900NO, BnaA03G0272800NO, BnaA01G0189100NO, BnaA02G0248600NO, BnaC03G0251700NO, BnaC02G0290200NO, BnaA03G0272700NO, BnaC05G0297600NO, BnaC05G0297300NO, BnaC03G0252000NO, BnaC03G0317800QU, BnaC03G0317900QU, BnaC03G0317600QU, BnaA03G0263200QU, BnaC01G0188500QU, BnaA03G0262800QU, BnaA01G0168400QU, BnaC01G0188900QU, BnaC02G0286800QU, BnaA03G0263100QU, BnaA02G0248000QU, BnaC03G0209800SL, BnaA01G0193300SL, BnaA03G0197300SL, BnaC02G0231000SL, BnaA02G0281500SL, BnaC01G0253100SL, BnaC03G0210100SL, BnaA03G0269600TA, BnaA03G0269800TA, BnaA02G0215100TA, BnaA03G0269900TA, BnaC02G0255300TA, BnaC01G0212600TA, BnaC01G0213100TA, BnaC03G0292000TA, BnaC03G0291900TA, BnaA01G0144700TA, BnaC03G0291700TA, BnaC03G0265500WE, BnaC01G0224300WE, BnaA03G0285900WE, BnaA02G0260400WE, BnaC02G0326500WE,BnaC03G0265600WE, BnaA01G0142400WE, BnaC03G0265300WE, BnaA03G0286100WE, BnaC01G0224600WE, BnaA03G0286000WE, BnaA01G0222900ZS, BnaC03G0331600ZS, BnaC03G0332100ZS, BnaA02G0251500ZS, BnaC03G0332000ZS, BnaC02G0337600ZS, BnaA03G0275500ZS, BnaA03G0275200ZS, BnaC01G0283800ZS, BnaC01G0283500ZS, BnaA03G0275300ZS, BnaC02G0163100ZY, BnaA03G0295200ZY, BnaC03G0268300ZY, BnaC03G0268600ZY, BnaC03G0268500ZY, BnaC01G0208900ZY, BnaA03G0295000ZY, BnaA03G0295100ZY, BnaC01G0209300ZY, BnaA01G0220000ZY, BnaA02G0278700ZY, BolC3g16132H, BolC2g09869H, BolC3g16128H, BolC3g16133H, BolC1g03106H, BolC1g03101H, BraA03g12335Z, BraA03g12337Z, BraA02g07432Z and BraA01g02441Z.

[0111] In some embodiments, provided is a Brassicaceae plant or a part thereof carrying a mutation in a gene encoding a UMAMIT transporter, the UMAMIT transporter being selected from the group consisting of: BnaA02G0240000GG, BnaC02G0304100GG, Bnascaffold1465G0001500GG, BnaA09G0014000GG, BnaC02G0290100NO, BnaA09G0016000NO, BnaA02G0248500NO, BnaC09G0009500QU, BnaA02G0247900QU, BnaA09G0011000QU, BnaC02G0286700QU, BnaA09G0009300SL, Bnascaffold966G0010300SL, BnaA02G0281600SL, BnaC02G0230900SL, BnaA02G0215000TA, BnaC09G0008700TA, BnaA09G0005700TA, BnaC02G0255200TA, BnaC02G0326400WE, BnaC09G0008200WE, BnaA02G0260500WE, BnaA09G0011300WE, BnaC09G0002100ZS, BnaC02G0337500ZS, Bnascaffold0025G0022100ZS, BnaA02G0251400ZS, BnaA09G0019300ZS, Bnascaffold0025G0022000ZS, BnaC09G0001300ZY, BnaC02G0163000ZY, BnaA02G0278600ZY, BnaA09G0018200ZY, BolC9g53044H, BolC2g09868H, BraA02g07431Z, and BraA09g35773Z.

[0112] Preferably, the mutation of the present invention alters the activity and / or expression of the encoded UMAMIT transporter.

[0113] In some embodiments, the mutant UMAMIT transporter is a mutant glucosinolate transporter having reduced glucosinolate transporter activity compared to the wild-type protein. In some embodiments, the mutant gene encodes a mutant glucosinolate transporter having reduced glucosinolate transporter activity compared to the wild-type protein.

[0114] Thus, in some embodiments, the reduction is at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% reduction in glucosinolate transporter activity of the mutant glucosinolate transporter compared to the wild-type protein. The glucosinolate transporter activity of the UMAMIT transporter can be measured by any method known in the art. In a preferred embodiment, the glucosinolate transporter activity of the UMAMIT transporter is measured using the methods described in Examples 1 and 2 herein.

[0115] In some embodiments, the mutant gene has a reduced expression of the encoded glucosinolate transporter compared to the wild-type gene.

[0116] Thus, in some embodiments, the reduced expression is at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% reduction in the expression of the mutant gene compared to the wild-type gene. The reduced expression can be measured using methods well known in the art, such as by quantitative reverse transcription PCR (RT-qPCR). In a preferred embodiment, the reduced expression is measured using the methods described in Examples 1 and 2 herein.

[0117] The reduced gene expression and / or reduced transporter activity of the encoded UMAMIT transporter results in a reduction in glucosinolate content in specific parts (such as seeds) of cruciferous plants.

[0118] Thus, in some embodiments, when grown and prepared under the same conditions, the seeds of the Brassicaceae plant or part thereof have a reduced glucosinolate content as compared to Brassicaceae plants or parts thereof that are identical in other genotypic respects but do not contain a mutation in any of the genes. In some embodiments, the reduction in glucosinolate content is a reduction of at least 50%, such as at least 51%, such as at least 52%, such as at least 53%, such as at least 54%, such as at least 55%, such as at least 56%, such as at least 57%, such as at least 58%, such as at least 59%, such as at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.

[0119] In some embodiments, a reduced gene expression of the encoded UMAMIT transporter and / or a reduced transporter activity do not have a substantial effect on the size of the seeds.

[0120] Thus, in some embodiments, when grown and prepared under the same conditions, the seeds of the Brassicaceae plant or part thereof having a reduced glucosinolate content are of substantially the same size as the seeds of Brassicaceae plants or parts thereof that are identical in other genotypic respects but do not contain any of the mutations in the UMAMIT exporter proteins defined herein.

[0121] In some embodiments, the reduced gene expression and / or reduced transporter activity of the encoded UMAMIT transporter do not have a substantial effect on the dry weight of the seeds. However, in other embodiments, when grown and prepared under the same conditions, the seeds of Brassicaceae plants or parts thereof having a reduced glucosinolate content have a slightly lower dry weight compared to the seeds of Brassicaceae plants or parts thereof that are otherwise identical in genotype but do not contain any of the mutations in the UMAMIT exporter proteins defined herein. In some embodiments, when grown and prepared under the same conditions, the dry weight of the seeds of Brassicaceae plants or parts thereof having a reduced glucosinolate content is no more than 7%, such as no more than 6%, such as no more than 5%, such as no more than 4%, such as no more than 3%, such as no more than 2%, such as no more than 1% lower than the dry weight of the seeds of Brassicaceae plants or parts thereof that are otherwise identical in genotype but do not contain any of the mutations in the UMAMIT exporter proteins defined herein.

[0122] In some embodiments, the dry weight of the seeds of Brassicaceae plants or parts thereof having a reduced glucosinolate content is 1% to 7% lower, such as 2% to 6% lower, such as 3% to 5% lower, such as 4% to 6% lower, than the dry weight of the seeds of Brassicaceae plants or parts thereof that are otherwise identical in genotype but do not contain any of the mutations in the UMAMIT exporter proteins defined herein.

[0123] The Brassicaceae plants according to the invention can have a reduced glucosinolate level in a specific tissue (e.g., seeds), while having a wild-type or near-wild-type level in other tissues.

[0124] Thus, in some embodiments, when grown and prepared under the same conditions, the vegetative tissues of the Brassicaceae plants or parts thereof have a glucosinolate content that is substantially the same as that of Brassicaceae plants or parts thereof that are otherwise identical in genotype but do not contain the mutations in any of the said genes.

[0125] In some embodiments, the Brassicaceae plants according to the invention have a reduced glucosinolate level in the seeds, while having a wild-type or near-wild-type glucosinolate level in other tissues of the plants. In some embodiments, the glucosinolate concentration in the seeds is less than 18 micromoles per gram of dry weight of the seeds, such as less than 17 micromoles per gram of dry weight, such as less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such as less than 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per gram of dry weight, such as less than 3 micromoles per gram of dry weight, such as less than 2 micromoles per gram of dry weight, such as less than 1 micromole per gram of dry weight.

[0126] In some embodiments, the glucosinolate concentration in the seeds is less than 100 nanomoles per gram of dry weight of the seeds, such as less than 50 nanomoles per gram of dry weight, such as less than 25 nanomoles per gram of dry weight, such as less than 10 nanomoles per gram of dry weight, such as less than 1 nanomole per gram of dry weight, such as less than 0.5 nanomole per gram of dry weight, such as less than 0.05 nanomole per gram of dry weight, such as less than 0.005 nanomole per gram of dry weight.

[0127] In some embodiments, the seeds do not have a measurable glucosinolate level.

[0128] In some embodiments, the Brassicaceae plants are oilseed crops. In some embodiments, the Brassicaceae plants are protein crops. In some embodiments, the Brassicaceae plants are oilseed and protein crops.

[0129] In some embodiments, the Brassicaceae plants are Brassicaceae oilseed crops. In some embodiments, the Brassicaceae plants are Brassicaceae protein crops. In some embodiments, the Brassicaceae plants are Brassicaceae oilseed and protein crops.

[0130] Oilseed seeds and / or protein crops, such as Brassicaceae oilseed seeds and / or protein crops, include but are not limited to Brassica, Camelina, Crambe, Eruca, Raphanus, Lepidium, and Thlaspi.

[0131] In some embodiments, the plant belongs to the Brassicaceae family. In some embodiments, the plant belongs to the Brassica genus. In some embodiments, the plant is selected from mustard (B. juncea), rapeseed (Brassica napus), Ethiopian mustard, B. oleracea, turnip. In other embodiments, the plant belongs to the Lepidium genus, such as the L. campestre plant. In some embodiments, the plant belongs to the Camelina genus. In some embodiments, the plant belongs to the Crambe genus. In some embodiments, the plant belongs to the Eruca genus. In some embodiments, the plant belongs to the Raphanus genus. In some embodiments, the plant belongs to the Thlaspi genus.

[0132] In some embodiments, the Brassicales plants of the present invention are not obtained only by a method that is essentially a biological method. The offspring of Brassicales plants obtained by a technical method are considered herein not to be obtained only by a method that is essentially a biological method because their parent plants are obtained by a technical method.

[0133] In one embodiment, the Brassicales plant carries one or more mutations as disclosed elsewhere herein, wherein the one or more mutations are induced by chemical and / or physical agents.

[0134] In one embodiment, the plant is prepared by a method comprising an induced mutagenesis step, or the plant is the offspring of a plant prepared by a method comprising an induced mutagenesis step.

[0135] Thus, the Brassicales plant can be a plant prepared by a method comprising the following steps or the offspring of a plant prepared by a method comprising the following steps:

[0136] · Mutagenize a Brassicales plant or a part thereof, for example using a chemical mutagen such as NaN3

[0137] · Select a Brassicales plant carrying any mutation as disclosed elsewhere herein.

[0138] Available mutations

[0139] The mutations according to the present invention can result in an alteration of the amino acid sequence of the gene encoding the UMAMIT transporter. Thus, in a preferred embodiment, the mutation is a non-synonymous mutation.

[0140] In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is an insertion. In some embodiments, the mutation is a deletion. In some embodiments, the mutation is a frameshift mutation.

[0141] In some embodiments, the mutation is within the promoter region of the gene. In some embodiments, the mutation is within the coding region of the gene. In some embodiments, the mutation is within the exon region of the gene. In some embodiments, the mutation is within the non-coding region of the gene. In some embodiments, the mutation is within the intron region of the gene. In some embodiments, the mutation is within the termination sequence of the gene.

[0142] In some embodiments, the mutation is a loss-of-function mutation.

[0143] In some embodiments, the mutation is an insertion of a transfer DNA (T-DNA) sequence. In some embodiments, the inserted sequence is pROK2 as shown in SEQ ID NO:21.

[0144] In some embodiments, the Brassicaceae plant comprises one or more mutations in one or more genes encoding a UMAMIT transporter. In some embodiments, the Brassicaceae plant comprises at least two mutations in one or more genes encoding a UMAMIT transporter, wherein the mutations are independently selected from the mutations described herein.

[0145] The Brassicaceae plant may carry one or more amino acid mutations in the UMAMIT transporter that eliminate the glucosinolate transport activity of these transporters but still retain any other regulatory or enzymatic functions thereof. In some embodiments, the one or more amino acid mutations are in the AtUMAMIT29 polypeptide as shown in SEQ ID NO:2 or in a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% or such as at least 95% sequence identity thereto, and the Brassicaceae plant expresses the mutant AtUMAMIT29 polypeptide or a functional homolog thereof having the corresponding mutation.

[0146] Specifically, one or more available mutations for reducing or eliminating the glucosinolate transport activity of AtUMAMIT29 are

[0147] i. Substitution of amino acid 27 (valine (V)) of SEQ ID NO:2 with phenylalanine (F);

[0148] ii. Substitution of amino acid 86 (methionine (M)) of SEQ ID NO:2 with valine (V);

[0149] iii. Substitution of amino acid 109 (leucine (L)) of SEQ ID NO:2 with valine (V);

[0150] iv. The amino acid 263 (glutamine (Q)) of SEQ ID NO:2 is substituted with serine (S);

[0151] v. The amino acid 267 (threonine (T)) of SEQ ID NO:2 is substituted with tyrosine (Y);

[0152] vi. The amino acid 44 (arginine (R)) of SEQ ID NO:2 is substituted with alanine (A);

[0153] vii. The amino acid 200 (tryptophan (W)) of SEQ ID NO:2 is substituted with alanine (A);

[0154] viii. The amino acid 204 (glutamine (Q)) of SEQ ID NO:2 is substituted with alanine (A).

[0155] In some embodiments, Brassicaceae plants express a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% or such as at least 95% sequence identity to AtUMAMIT29 as shown in SEQ ID NO:2. In such embodiments, the sequence of the functional homolog is first optimally aligned with the AtUMAMIT29 sequence shown in SEQ ID NO:2, for example using the Needleman-Wunsch algorithm, to identify the corresponding amino acids, and then one or more of the following mutations (corresponding mutations) are made to reduce or eliminate the glucosinolate transport activity of the functional homolog of AtUMAMIT29:

[0156] a. The valine corresponding to valine at position 27 of SEQ ID NO:2 is substituted with phenylalanine (V27F);

[0157] b. The methionine corresponding to methionine at position 86 of SEQ ID NO:2 is substituted with valine (M86V);

[0158] c. The leucine corresponding to leucine at position 109 of SEQ ID NO:2 is substituted with valine (L109V);

[0159] d. The glutamine corresponding to glutamine at position 263 of SEQ ID NO:2 is substituted with serine (Q263S);

[0160] e. The threonine corresponding to threonine at position 267 of SEQ ID NO:2 is substituted with tyrosine (T267Y);

[0161] f. The arginine corresponding to arginine at position 44 of SEQ ID NO:2 is substituted with alanine (R44A);

[0162] g. The tryptophan corresponding to the 200th position of SEQ ID NO:2 is substituted with alanine (W200A);

[0163] h. The glutamine corresponding to the 204th position of SEQ ID NO:2 is substituted with alanine (Q204A).

[0164] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO:12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is AtUMAMIT29 as shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutant AtUMAMIT29 polypeptide contains a substitution of amino acid 27 (valine (V)) of SEQ ID NO:2 with phenylalanine (F), or the mutant functional homolog of AtUMAMIT29 contains a substitution of valine (V) corresponding to amino acid 27 of SEQ ID NO:2 with phenylalanine (F). Compared with the corresponding wild-type UMAMIT29 polypeptide, the mutant polypeptide may have at most 20% average total glucosinolate import activity, such as at most 15%, such as at most 10%, such as at most 5%.

[0165] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO:12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 86 (methionine (M)) of SEQ ID NO:2 with valine (V), or the mutant functional homolog of AtUMAMIT29 comprises a substitution of methionine (M) corresponding to methionine of amino acid 86 of SEQ ID NO:2 with valine (V). Compared to the corresponding wild-type UMAMIT29 polypeptide, the mutant polypeptide may have at most 20% average total glucosinolate import activity, such as at most 15%, such as at most 10%, such as at most 5%.

[0166] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO:12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutated gene encodes a mutated AtUMAMIT29 polypeptide, wherein the mutated AtUMAMIT29 is AtUMAMIT29 as shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutated AtUMAMIT29 polypeptide comprises a substitution of leucine (L) at amino acid 109 of SEQ ID NO:2 with valine (V), or the mutated functional homolog of AtUMAMIT29 comprises a substitution of leucine (L) corresponding to leucine at amino acid 109 of SEQ ID NO:2 with valine (V). Compared to the corresponding wild-type UMAMIT29 polypeptide, the mutant polypeptide may have at most 20% of the average total glucosinolate import activity, such as at most 15%, such as at most 10%, such as at most 5%.

[0167] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO: 2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutant AtUMAMIT29 polypeptide comprises a substitution of glutamine (Q) at amino acid 263 of SEQ ID NO: 2 with serine (S), or the mutant functional homolog of AtUMAMIT29 comprises a substitution of glutamine (Q) corresponding to glutamine at amino acid 263 of SEQ ID NO: 2 with serine (S). Compared to the corresponding wild-type UMAMIT29 polypeptide, the mutant polypeptide may have at most 30% of the average total glucosinolate import activity, such as at most 25%, such as at most 20%, such as at most 15%, such as at most 10%, such as at most 5%.

[0168] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO:12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 267 (threonine (T)) of SEQ ID NO:2 with tyrosine (Y), or the mutant functional homolog of AtUMAMIT29 comprises a substitution of the threonine (T) corresponding to amino acid 267 of SEQ ID NO:2 with tyrosine (Y). Compared to the corresponding wild-type UMAMIT29 polypeptide, the mutant polypeptide may have at most 15% of the average total glucosinolate import activity, such as at most 10%, such as at most 5%.

[0169] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO:12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 44 (arginine (R)) of SEQ ID NO:2 with alanine (A), or the mutant functional homolog of AtUMAMIT29 comprises a substitution of arginine (R) corresponding to arginine of amino acid 44 of SEQ ID NO:2 with alanine (A). Compared to the corresponding wild-type UMAMIT29 polypeptide, the mutant polypeptide may have at most 20% average total glucosinolate import activity, such as at most 15%, such as at most 10%, such as at most 5%.

[0170] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO:12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 200 (tryptophan (W)) of SEQ ID NO:2 with alanine (A), or the mutant functional homolog of AtUMAMIT29 comprises a substitution of tryptophan (W) corresponding to amino acid 200 of SEQ ID NO:2 with alanine (A).

[0171] In some embodiments, the Brassicaceae plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO:12) or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, except that the mutant AtUMAMIT29 polypeptide comprises a substitution of glutamine (Q) at amino acid 204 of SEQ ID NO:2 with alanine (A), or the mutant functional homolog of AtUMAMIT29 comprises a substitution of glutamine (Q) corresponding to glutamine at amino acid 204 of SEQ ID NO:2 with alanine (A).

[0172] In some embodiments, the Brassicaceae plant or a part thereof thus carries a mutation in the AtUMAMIT29 gene as shown in SEQ ID NO: 12 or a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, and wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO: 2 or a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity, except that the mutant polypeptide comprises a substitution selected from V27F, M86V, L109V, Q263S>T267Y, R44A, W200A, and Q204A or a corresponding substitution relative to the AtUMAMIT29 shown in SEQ ID NO: 2.

[0173] The Brassicaceae plant or a part thereof may also express a mutant AtUMAMIT29 polypeptide or a homolog thereof carrying more than one of said substitutions. In some embodiments, the Brassicaceae plant or a part thereof thus carries a mutation in the AtUMAMIT29 gene as shown in SEQ ID NO: 12 or a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, and wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO: 2 or a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity, except that the mutant polypeptide comprises one or more substitutions selected from V27F, M86V, L109V, Q263S, T267Y, R44A, W200A, and Q204A or one or more corresponding substitutions relative to the AtUMAMIT29 shown in SEQ ID NO: 2.

[0174] In addition to one or more mutations in one or more genes encoding UMAMIT transporters described herein, the Brassicaceae plants of the present invention may further comprise one or more additional mutations in one or more additional genes.

[0175] Specific examples of Brassicaceae plants comprising a mutation in a gene encoding a UMAMIT transporter

[0176] In some embodiments, the plant contains the T-DNA insertion (Vector_pAC106) of SEQ ID NO:22 in the first intron of AtUMAMIT29 (SEQ ID NO:12) or in the corresponding intron of its functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto. The Met-derived glucosinolate (GLS) content of Arabidopsis seeds having this T-DNA insertion is about 34.74 ± 9.04 nmol mg -1 , and the Trp-derived GLS content is about 1.21 ± 0.75 nmol mg -1 .

[0177] In some embodiments, the plant contains the T-DNA insertion (Vector_pAC106) of SEQ ID NO:22 in the fourth exon of AtUMAMIT29 (SEQ ID NO:12) or in the corresponding exon of its functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.

[0178] In some embodiments, the plant contains the T-DNA insertion (Vector_pAC106) of SEQ ID NO:22 in the first exon of AtUMAMIT30 (SEQ ID NO:13) or in the corresponding exon of its functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity. The Met-derived GLS content of Arabidopsis seeds with this T-DNA insertion is about 128.76 ± 15.80 nmol mg -1 , and the Trp-derived GLS content is about 2.13 ± 0.42 nmol mg -1 .

[0179] In some embodiments, the plant contains the T-DNA insertion (Vector_pAC106) of SEQ ID NO:22 in the first exon of AtUMAMIT31 (SEQ ID NO:13) or in the corresponding exon of its functional homolog having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity. The Met-derived GLS content of Arabidopsis seeds with this T-DNA insertion is about 154.83 ± 7.01 nmol mg -1 , and the Trp-derived GLS content is about 0.64 ± 0.43 nmol mg -1 .

[0180] In some embodiments, the plant contains the T-DNA insertion (pROK2) of SEQ ID NO: 21 in the corresponding introns of all alleles of the first intron of AtUMAMIT29 (SEQ ID NO: 12) or its functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, and contains an 11-nucleotide deletion at positions 1048-1059 of all alleles of AtUMAMIT30 (SEQ ID NO: 13) or its functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity. The Met-derived GLS content of Arabidopsis seeds with this T-DNA insertion and this deletion is about 12.97 ± 1.25 nmol mg -1 , and the Trp-derived GLS content is about 2.18 ± 0.07 nmol mg -1 .

[0181] In some embodiments, the plant contains the T-DNA insertion (pROK2) of SEQ ID NO:21 in the corresponding introns of all alleles of the first intron of AtUMAMIT29 (SEQ ID NO:12) or its functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity. It contains an 11-nucleotide deletion at positions 1048-1059 of both alleles of AtUMAMIT30 (SEQ ID NO:13) or at the corresponding positions of all alleles of its functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity. It has a 3-nucleotide deletion at positions 57-59 of both alleles of AtUMAMIT31 (SEQ ID NO:14) or at the corresponding positions of all alleles of its functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.The Met-derived GLS content of Arabidopsis seeds with this T-DNA insertion and these deletions was approximately 10.94 ± 0.62 nmol mg. -1 , and the Trp-derived GLS content was approximately 0.13 ± 0.01 nmol mg -1 .

[0182] In some embodiments, the plant contains the T-DNA insertion (pROK2) of SEQ ID NO:21 in the corresponding introns of all alleles of its functional homologs that have at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to the first intron of AtUMAMIT29 (SEQ ID NO:12). It contains a 9-nucleotide deletion at positions 1044-1052 of all alleles of AtUMAMIT30 (SEQ ID NO:13) or at corresponding positions of its functional homologs that have at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity. It contains a 4-nucleotide insertion at positions 57-59 of all alleles of AtUMAMIT31 (SEQ ID NO:14) or at corresponding positions of its functional homologs that have at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.The Met-derived GLS content of Arabidopsis seeds with the T-DNA insertion, the deletion, and the insertion was approximately 10.94 ± 0.62 nmol mg. -1 , and the Trp-derived GLS content was approximately 0.13 ± 0.01 nmol mg -1 .

[0183] In some embodiments, the plant contains the T-DNA insertion (pROK2) of SEQ ID NO:21 in the corresponding introns of all alleles of its functional homologs that have at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity with the first intron of AtUMAMIT29 (SEQ ID NO:12). The plant contains an insertion of 1 nucleotide at position 1051 of all alleles of its functional homologs that have at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity with AtUMAMIT30 (SEQ ID NO:13). The plant contains a deletion of 12 nucleotides at positions 60-71 of all alleles of its functional homologs that have at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity with AtUMAMIT31 (SEQ ID NO:14).The Met-derived GLS content of Arabidopsis seeds with the T-DNA insertion, the insertion, and the deletion is about 17.56 ± 0.89 nmol mg. -1 , and the Trp-derived GLS content is about 0.14 ± 0.01 nmol mg -1 .

[0184] Plant products and methods for their production

[0185] The present invention also provides plant products prepared from cruciferous plants (such as any cruciferous plant or part thereof described herein) carrying a mutation in the gene encoding the UMAMIT transporter of the present invention.

[0186] In some embodiments, the plant part is a seed.

[0187] Accordingly, in one aspect, there is provided a plant product comprising a Brassicales plant or a part thereof, or prepared from the seeds of said Brassicales plant or a part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologues having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, whereby said Brassicales plant or a part thereof expresses at least one mutant UMAMIT transporter, wherein said mutant UMAMIT transporter has low or no glucosinolate transporter activity and is expressed at a low level or not expressed.

[0188] In some embodiments, the Brassicales plant or a part thereof is as defined elsewhere herein.

[0189] Examples of plant products available according to the present invention include products prepared from the seeds of plants, such as seed oil, seed cake and seed meal.

[0190] Accordingly, in some embodiments, the plant product is made from seeds. In some embodiments, the plant product is an oil, such as seed oil. In some embodiments, the plant product is seed cake. In some embodiments, the plant product is seed meal.

[0191] On the one hand, there is provided a seed cake prepared from the seeds of a plant of the order Brassicales, wherein the plant of the order Brassicales is as described elsewhere herein.

[0192] Thus, in some embodiments, there is provided a seed cake prepared from the seeds of a plant of the order Brassicales, wherein the plant of the order Brassicales carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity therewith, whereby the plant of the order Brassicales or a part thereof expresses at least one mutant UMAMIT transporter, wherein the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level, or is not expressed.

[0193] In some embodiments, the seeds contain less than 18 micromoles of glucosinolate per gram of dry weight of the seeds, such as less than 17 micromoles per gram of dry weight, such as less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such as less than 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per gram of dry weight, such as less than 3 micromoles per gram of dry weight, such as less than 2 micromoles per gram of dry weight, such as less than 1 micromole per gram of dry weight.

[0194] In some embodiments, when cultivated and prepared under the same conditions, the seeds contain a glucosinolate concentration that is at most 15%, such as at most 14%, such as at most 13%, such as at most 12%, such as at most 11%, such as at most 10%, such as at most 9%, such as at most 8%, such as at most 7%, such as at most 6%, such as at most 5%, such as at most 5%, such as at most 3%, such as at most 2%, or such as at most 1% of the glucosinolate concentration in seeds prepared from plants that are otherwise identical to the Brassicales plant in genotype but do not contain a mutation in any of the genes.

[0195] In some embodiments, when cultivated and prepared under the same conditions, the seed cake prepared from the Brassicales plant contains a glucosinolate concentration that is at most 15%, such as at most 14%, such as at most 13%, such as at most 12%, such as at most 11%, such as at most 10%, such as at most 9%, such as at most 8%, such as at most 7%, such as at most 6%, such as at most 5%, such as at most 5%, such as at most 3%, such as at most 2%, or such as at most 1% of the glucosinolate concentration in the seed cake prepared from seeds of plants that are otherwise identical to the Brassicales plant in genotype but do not contain a mutation in any of the genes.

[0196] In some embodiments, the plant seed product contains glucosinolate at a concentration of at most 30 micromoles per gram of dry weight of the plant seed product, such as at most 25 micromoles per gram of dry weight, such as at most 20 micromoles per gram of dry weight, such as at most 15 micromoles per gram of dry weight, such as at most 14 micromoles per gram of dry weight, such as at most 13 micromoles per gram of dry weight, such as at most 12 micromoles per gram of dry weight, such as at most 11 micromoles per gram of dry weight, such as at most 10 micromoles per gram of dry weight, such as at most 9 micromoles per gram of dry weight, such as at most 8 micromoles per gram of dry weight, such as at most 7 micromoles per gram of dry weight, such as at most 6 micromoles per gram of dry weight, such as at most 5 micromoles per gram of dry weight, such as at most 4 micromoles per gram of dry weight, such as at most 3 micromoles per gram of dry weight, such as at most 2 micromoles per gram of dry weight, or such as at most 1 micromole per gram of dry weight.

[0197] In some embodiments, the plant seed product contains glucosinolate at a concentration of at most 1.5 mmol per kilogram of the plant product, such as at most 1.25 mmol per kilogram of the plant product, such as at most 1 mmol per kilogram of the plant product, at most 0.75 mmol per kilogram of the plant product, such as at most 0.5 mmol per kilogram of the plant product, such as at most 0.25 mmol per kilogram of the plant product, such as at most 0.1 mmol per kilogram of the plant product.

[0198] In some embodiments, the plant seed product does not contain a measurable concentration of glucosinolate. Methods for measuring glucosinolate content are well known in the art and are described in the "Definitions" section herein.

[0199] In some aspects, there is also provided a method for producing a plant product comprising a Brassicales plant or a part thereof having a low glucosinolate content, the method comprising the steps

[0200] a. providing a Brassicales plant or a part thereof as described elsewhere herein; and

[0201] b. processing the Brassicales plant or a part thereof into a plant product, such as seed oil or seed cake.

[0202] In some embodiments, there is thus provided a method for producing a plant product comprising a Brassicales plant or a part thereof having a low glucosinolate content, the method comprising the following steps:

[0203] a. Provide a Brassicaceae plant or a part thereof, wherein the Brassicaceae plant or the part thereof carries a mutation in at least one gene encoding a UMAMIT transporter, and the UMAMIT transporter is selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity, whereby the Brassicaceae plant or the part thereof expresses at least one mutant UMAMIT transporter, and the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level, or is not expressed; and

[0204] sequence identity, whereby the Brassicaceae plant or the part thereof expresses at least one mutant UMAMIT transporter, and the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level, or is not expressed; and

[0205] b. Process the Brassicaceae plant or the part thereof into a plant product, such as seed oil or seed cake.

[0206] The plant seed products described herein can be particularly used as animal feed and fodder. Glucosinolates are undesirable substances in animal feed and fodder as they can cause growth retardation, reduced performance (decreased milk and egg production), impaired reproductive activity, and impaired liver and kidney function. For compound feeds, the total GSL level tolerated by the palatability of ruminants is set at no more than 10 - 15 micromoles per gram of dry weight. However, poultry and pigs are more sensitive to GSL levels than ruminants, and GSL levels exceeding 2 - 4 micromoles per gram of dry weight in the feed can severely affect the reproductive efficiency of these animals. Additionally, for rapeseed meal or press cake, it is recommended to limit the total glucosinolate content in the feed for monogastric animals to 1 - 1.5 mmol per kilogram of feed, and the glucosinolate concentration in the feed for young animals should be even lower (Alexander et al., 2008).

[0207] Accordingly, in some embodiments, there is provided an animal feed comprising a plant product prepared from the seeds or parts thereof of a plant of the order Brassicales, wherein the plant of the order Brassicales carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby the plant of the order Brassicales or its parts express at least one mutant UMAMIT transporter, wherein the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level, or is not expressed.

[0208] In some embodiments, there is provided a plant seed product prepared from a plant of the order Brassicales or its parts as described herein for use in animal feed.

[0209] The plant product can be defined as described in the section "Plant Products and Methods for Their Production" herein. In a preferred embodiment, the plant product is prepared from seeds. In a more preferred embodiment, the plant product is seed cake, seed oil, or seed meal.

[0210] Method for altering the glucosinolate content of a Brassicaceae plant or a part thereof

[0211] The Brassicaceae plant or a part thereof of the present invention carrying a mutation in the gene encoding a UMAMIT transporter and having a reduced glucosinolate content can be prepared in any useful manner.

[0212] In one aspect, therefore, there is provided a method for altering the glucosinolate content in a Brassicaceae plant or a part thereof, the method comprising the step of altering the functional activity or expression of at least one UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0213] The step of altering the functional activity of at least one UMAMIT transporter can be accomplished using any available method known in the art.

[0214] In some embodiments, the step of altering the functional activity is carried out by nuclease-based gene editing. In some embodiments, the step of altering the functional activity is carried out by CRISPR / Cas9 gene editing. In some embodiments, the step of altering the functional activity is carried out by gene targeting. In some embodiments, the step of altering the functional activity is carried out by transposon mutagenesis. In some embodiments, the step of altering the functional activity is carried out by transfer DNA-induced insertion. In some embodiments, the step of altering the functional activity is carried out by gene knockdown. In some embodiments, the step of altering the functional activity is carried out by RNA interference.

[0215] In some embodiments, the step of altering the functional activity is carried out by random mutagenesis. In some embodiments, the step of altering the functional activity is carried out using a non-GMO method. In some embodiments, the method further comprises one or more plant crosses.

[0216] Crucifer plants or parts thereof according to the invention, which carry a mutation in the gene encoding the UMAMIT transporter and have a reduced glucosinolate content, can be prepared in any available manner.

[0217] For example, crucifer plants according to the invention can be prepared by a method comprising the following steps:

[0218] a. Providing seeds of a crucifer plant;

[0219] b. Randomly mutating the seeds of the crucifer plant,

[0220] c. Selecting seeds or parts thereof of a crucifer plant carrying a mutant gene encoding a mutant UMAMIT transporter polypeptide, the mutant UMAMIT transporter polypeptide carrying a mutation that results in the mutant UMAMIT transporter having low or no glucosinolate transporter activity, being expressed at a low level, or not being expressed.

[0221] In some embodiments, the step of altering the functional activity further comprises one or more steps of propagating the crucifer plant or parts thereof to obtain multiple crucifer plants or parts thereof each carrying the mutation.

[0222] In particular, crucifer plants carrying a specific mutation in the gene encoding the UMAMIT transporter can be prepared and identified substantially as described in international patent application WO 2018 / 001884, using primers and probes designed to identify the mutation in the gene.

[0223] It is also possible to use various site-directed mutagenesis methods to prepare Brassicaceae plants carrying mutations in the genes encoding UMAMIT transporters. For example, the method can be based on the sequence of the coding sequence of the gene encoding the UMAMIT transporter, such as a gene comprising a sequence according to any one of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In one embodiment, any one of CRISPR, TALEN, zinc fingers, meganucleases, and DNA-cleaving antibiotics described in WO 2017 / 138986 is used to prepare Brassicaceae plants. In one embodiment, the CRISPR / Cas9 technology, such as using an RNA-guided Cas9 nuclease, is used to prepare Brassicaceae plants. This can be carried out as described in Lawrenson et al., Genome Biology (2015) 16:258; DOI 10.1186 / s13059-015-0826-7, except that the single guide RNA sequence is designed based on the gene sequence of the gene encoding the UMAMIT transporter. In one embodiment, a combination of TALEN and CRISPR / Cas9 technologies, such as using an RNA-guided Cas9 nuclease, is used to prepare Brassicaceae plants. This can be carried out as described in the method of Holme et al., 2017, except that the TALEN and the single guide RNA sequence are designed based on the gene sequences provided herein.

[0224] In one embodiment, a combination of homologous directed repair, DNA-cleaving nucleases, and donor DNA fragments is used to prepare Brassicaceae plants. This technique can be carried out as described in the method of Sun et al., 2016, except that the DNA-cleaving nuclease is designed based on the gene sequences provided herein.

[0225] In one embodiment of the present invention, the aim is to provide agronomically useful Brassicaceae plants carrying mutations in the gene encoding the UMAMIT transporter. In addition to the mutations in the gene encoding the UMAMIT transporter, other factors can be considered in generating commercial Brassicaceae plant varieties that can be used to prepare products from their seeds, such as seed yield and size, and other parameters related to seed quality and composition. Since many (if not all) of the relevant traits have been shown to be genetically controlled, the present invention also provides modern, homozygous, high-yielding cultivars that can be prepared by crossing with the Brassicaceae plants disclosed in the present application. Skilled Brassicaceae breeders will be able to screen and cultivate Brassicaceae plants and cross them with other Brassicaceae plants to obtain excellent cultivars. Alternatively, breeders can use the plants of the present invention for further mutagenesis to generate new cultivars that carry additional mutations in addition to the mutations in the gene encoding the UMAMIT transporter.

[0226] The present invention also includes Brassicaceae plants carrying mutations in the gene encoding the UMAMIT transporter prepared by plant breeding methods including self-crossing, backcrossing, population crossing and other methods. The present invention can combine with the backcrossing method to introduce the mutation of the mutant gene encoding the UMAMIT transporter into another cultivar.

[0227] A method for accelerating the plant breeding process includes applying tissue culture and regeneration techniques for initial proliferation of the generated mutants. Thus, another aspect of the present invention is to provide cells that generate Brassicaceae plants carrying one or more mutations in the gene encoding the UMAMIT transporter after growth and differentiation. For example, breeding can include traditional hybridization, preparation of fertile anther-derived plants or use of microspore culture. Examples

[0228] Example 1 - Glucosinolate export by UMAMIT is crucial for seed accumulation

[0229] According to the optimal defense theory, the plant organs with the highest fitness values (such as seeds and tubers) accumulate the highest levels of defense compounds for protection against herbivores and pathogens. However, for humans, these usually toxic defense compounds significantly reduce the nutritional value of these edible tissues. However, reducing antinutritional factors by blocking the biosynthetic pathway is accompanied by an adverse effect on plant fitness. Since defense compounds are usually transferred to edible tissues, eliminating transporters along the source-to-sink pathway provides a strategy to reduce the toxic compounds in the edible part while maintaining the defense ability of other tissues. Loss-of-function mutations in the import and export proteins located on the plasma membrane can have significantly different effects on the distribution pattern within the whole plant. For example, in Arabidopsis, H +Mutations in the coupled glucosinolate transporter (GTR, which functions as a protein importing glucosinolate defense compounds from the apoplast) not only result in the elimination of glucosinolates accumulated in seeds (without de novo synthesis), but also lead to a major change in the distribution pattern of glucosinolates throughout the plant. The latter is due to the flow of apoplast-accumulated glucosinolates along the transpiration stream to distant organs. In contrast, blocking the mechanism by which defense compounds are exported to the apoplast before being imported into the symplasm can provide a strategy for eliminating the accumulation of anti-nutritional compounds in sink tissues without affecting defense distribution. However, due to the general lack of knowledge about the exporters of specific metabolites, the potential of engineering exporters remains largely unexplored. In this article, we identified and characterized members of the usually multiple amino acid in and out transporters (UMAMIT) family, UMAMIT29, -30, and -31, as glucosinolate exporters, and showed that mutants of these exporters can specifically inhibit the accumulation of glucosinolates in Arabidopsis thaliana seeds without altering the defense distribution in other parts of the plant.

[0230] Materials and Methods

[0231] Plant materials and growth conditions

[0232] Arabidopsis thaliana ecotype Columbia-0 (Col-0) (N7000) gtr1 gtr2 7 and gtr1 gtr2 gtr3 9 (gtr1, line SAIL_801_G03; gtr2, line SAIL_20_B07; gtr3, line GK-099B01) have been previously described. T-DNA insertion mutants umamit29-1 (Salk_133129C), umamit29-2 (GK-007H08), umamit30-1 (SALK_146977C), and umamit31-1 (GK-266E08) were all ordered from the European Arabidopsis Stock Centre NASC (Nottingham). The lack of expression of the target genes was confirmed by qRT-PCR (Extended Data Figure 8b). Other mutants were generated by CRISPR-Cas9-mediated genome editing. The ut29 ut31 and ut29 ut30 double mutants were generated by introducing single-guide RNAs (sgRNAs) / Cas9 targeting UMAMIT31 and UMAMIT30, respectively, into the umamit29-1 mutant. The umamit29 umamit30 umamit31 triple mutant was generated by co-introducing the sgRNA targeting UMAMIT30 and the sgRNA targeting UMAMIT31 into umamit29-1. The gtr1 gtr2 gtr3 umamit29 umamit30 quintuple mutant was generated by introducing the sgRNA targeting UMAMIT30 into gtr1 gtr2 gtr3 umamit29-1. To genotype the transgenic lines, rosette leaves were collected from each plant and genomic DNA was isolated using the CTAB method. PCR amplification was performed using primers flanking the sgRNA target sites, and mutations in the target genes were identified using Sanger sequencing. PCR was carried out using Phusion DNA polymerase. All primers were synthesized by TAG Copenhagen A / S.

[0233] For microscopy, plants expressing pCYP83A1 (2 kb 5′ regulatory sequence): CYP83A1 (coding sequence)-mVenus 47 , pCYP83B1 (2 kb 5′ regulatory sequence): CYP83B1 (coding sequence)-mVenus 47 , pSUR1 (2 kb 5′ regulatory sequence): SUR1 (coding sequence)-mTurquoise2 and pUMAMIT29 (5 kb 5′ regulatory sequence): UMAMIT29 (genomic sequence)-mVenus were grown under long-day conditions. Different genotypes were planted side by side for metabolite extraction and bioimaging sample collection, with light conditions: 16 h, 21 °C; dark: 8 h, 21 °C; humidity: 55%; light intensity: 100–140 μmol m -2 s -1 .

[0234] Plasmid construction

[0235] The genomic fragment of UMAMIT29 (from 4220 bp upstream of the start codon to the stop codon TGA) was amplified from the genomic DNA of Arabidopsis Col-0 and cloned into USER TMIn the expression cassette, the expression cassette was inserted into the open pFastRedU-mVenus plant expression vector. sgRNA was designed using CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The sgRNA was amplified from the expression cassette using primers containing BsaI recognition sites. The PCR fragments were cloned into pKIR1.1R by GoldenGate cloning technology and digested with AarI for multiplex gene editing. The final binary vector was transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation.

[0236] Grafting of Arabidopsis thaliana inflorescence stems

[0237] Grafting of Arabidopsis thaliana inflorescence stems was performed according to the previously described method (Nisar et al., 2012) with slight modifications. When the primary inflorescence meristem reached a height of 5 cm above the rosette leaves, plants with similar flower stem thickness were selected from more than 30 plants of each genotype. The inflorescence-bearing stem (scion) was removed from the rootstock by a horizontal cut (leaving 1 - 2 cm of the stem on the rootstock), and a small drop of water was immediately placed on the rootstock to prevent it from drying. The scion was cut horizontally and placed in a petri dish filled with water to prevent air from entering the vascular bundles. When both the scion and the rootstock were ready, a V-shaped wedge was created on the rootstock using a 0.5 cm long midline incision along the length of the rootstock. The scion was quickly cut into a long V-shaped wedge under water and inserted into the incision of the rootstock. The graft junction was fixed with a silicone tube and wrapped with paraffin film (1 cm above and below the silicone tube) to maintain hydraulic expansion and prevent drying. The grafted plants were covered with a transparent plastic film for 7 days, and the humidity was maintained at 70%. After 7 days, 20% of the plastic film was gradually removed each day, and the plants were watered regularly to acclimatize the plants to normal growth conditions within a week. Seeds for glucosinolate extraction were harvested from senescent pods of 8-week-old grafted plants.

[0238] Labeling flowers to analyze protein localization and glucosinolates in developing pods

[0239] Flowers with visible white petals were defined as the start of pollination and marked with a sewing thread. To quantify the glucosinolate content and profile of developing pods, pods were collected starting from 5 days after pollination. After collection, the pods were quickly frozen in liquid nitrogen and freeze-dried, and then dissected into seeds and seedless pods for glucosinolate analysis. For protein localization in developing pods of SUR1-mTurquoise2 (Goedhart et al., 2012) plants, developing seeds were exposed by removing one pod valve, and then the seeds were mounted in perfluorodecalin (Sigma) for in vivo imaging. For CYP83A1-mVenus and CYP83B1-mVenus plants, 30-μm sections of pods at the green-ripe stage were generated using a vibratome (Leica, Germany) for in vivo imaging. For UMAMIT29-mVenus plants, the pods were fixed and cleared using the ClearSee protocol (Kurihara et al. 2015). Briefly, the pods were fixed in a vacuum in a PBS (pH 6.7) solution containing 4% (w / v) paraformaldehyde (Sigma) with 0.1% (v / v) SR2200 cell wall dye (Renaissance Chemicals) for 60 minutes. For young pods (<16 days after pollination), a slit was made on two pod valves before fixation to facilitate the entry of the fixative. For older pods, the valves were already cracked, allowing the fixative to enter. The fixed pods were washed twice in PBS and then transferred to ClearSee solution (10% (w / v) xylitol, 15% (w / v) sodium deoxycholate, 25% (w / v) urea). The pods were cleared for at least 1 month and the ClearSee solution was changed 3 times. On the day of imaging, one pod valve was gently removed and the cleared specimen was mounted in ClearSee for fixation for imaging. All images were acquired on a Leica SP5-X confocal scanning laser microscope (Leica microsystems). mTurquoise2 was excited at 458 nm and emitted at 468 - 500 nm, while mVenus was excited at 514 nm and the emission was recorded at 525 - 560 nm.

[0240] Plant RNA Extraction and Quantitative PCR

[0241] Total RNA was extracted from a pool of three pods at positions 5, 6, and 7, starting from the first pod on the stem of each plant, using the spectrum Plant Total RNA Kit (Sigma). The extracted RNA was treated with DnaseI (AMPDK1-1KT, Sigma-Aldrich) for DNase treatment. 1 μg of RNA was reverse-transcribed into cDNA using the iScript cDNA Synthesis Kit (#1708891, Bio-Rad). To quantify the expression of glucosinolate biosynthesis genes, qRT-PCR was performed using the DyNAmo Flash SYBRGreen qPCR Kit (F-415L, Thermo Scientific) and gene-specific primers. All values were normalized to the transcriptional level of the ACTIN2 gene (AT3G18780).

[0242] Biochemical Characterization of UMAMIT in Xenopus laevis Oocytes

[0243] A linear DNA template for in vitro transcription was generated by PCR from the pNB1u plasmid using Phusion High-Fidelity DNA Polymerase (NEB). The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen). Capped cRNA was synthesized in vitro using the mMessage mMachine T7 Kit (Ambion). The concentration of the synthesized cRNA for each transporter gene was normalized to 500 ng·μL -1 , aliquoted, and stored at -20 °C. For expression in Xenopus laevis oocytes, a maximum of one cRNA thawing cycle was performed.

[0244] Xenopus laevis oocytes were purchased from Ecocyte Bioscience (Germany). 50.6 nL of cRNA (500 ng μL -1 ) was injected into the oocytes using a Drummond NANOJECT II (Drummond scientific company, Broomall 116 Pennsylvania). The injected oocytes were incubated at 16 °C in HEPES-based Kulori buffer (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES pH 7.4) supplemented with gentamicin (100 μg mL -1 ) for 3 days. For the control injected with H2O, 50.6 nL of nuclease-free water (Ambion) was injected instead of cRNA.

[0245] The uptake assay of Xenopus laevis oocytes was performed as described previously ( (et al., 2017), with some modifications. Three days after injection of cRNA, the oocytes were pre-incubated in 5 mL of Kulori buffer (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM MES pH 5.0) for 5 minutes and then incubated with Kulori buffer containing the substrate for a given time. The oocytes were washed 5 times with Milli-Q water (20 mL each time) and homogenized with 80% methanol (containing internal standard). Subsequently, the oocyte extract was centrifuged at 12,000 x g for 10 minutes at 4 °C. The supernatant was diluted with water and filtered through a 0.22 μm filter plate (MSGVN2250, Merck Millipore), and then analyzed by LC-MS / MS as described below.

[0246] For the output analysis, 48 hours after injection of cRNA, 23 nL of 2Prop (100 mM) or a glucosinolate mixture (the concentration of individual glucosinolates in the mixture was 2 mM) was injected into the oocytes to achieve initial intracellular concentrations of approximately 2.2 mM or 50 μM, respectively. After washing once in 5 mL of Kulori buffer (pH 7.4), some oocytes were harvested for the 0-time sample, and the remaining oocytes were incubated in Kulori buffer (pH 7.4) in a 96-well U-bottom microtiter plate (Greiner Bio-One) (3 oocytes per well, containing 100 μL of buffer). The oocytes and the external medium were harvested at different time points. The harvested oocytes were washed 5 times in Milli-Q water (20 mL each time) and homogenized with 80% methanol. 10 μL of the external medium was taken for quantification of glucosinolates in the medium. Extraction, filtration, and LC-MS / MS analysis were as described below. For the input and output assays, the glucosinolate and amino acid concentrations in the oocytes were calculated based on the estimated volume of 1 μL of cytoplasmic oocytes.

[0247] Two-electrode voltage-clamp electrophysiology

[0248] Using the two-electrode voltage-clamp technique (TEVC) on an automated Roboocyte2 (Multichannel Systems, Reutlingen, Germany), the 2Prop-induced current was recorded in oocytes expressing UMAMIT29. The electrodes were backfilled with a mixture of 3M KCl and 1.5M acetate. The electrode resistance was 280 - 1000 kΩ. To test the electrogenicity of 2Prop input mediated by UMAMIT29, oocytes expressing UMAMIT29 were clamped at a membrane potential of -60 mV and continuously perfused with MES-based eKulori buffer (2 mM LaCl3, 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM MES pH 5.5). Currents were recorded under continuous perfusion in the absence and presence of 10 mM 2Prop.

[0249] For the current-voltage (IV) relationship, before and after adding 10 mM 2Prop, the membrane potential of the oocytes was stepped from -80 mV to 0 mV in 100 msec increments of 20 mV while recording the current.

[0250] To verify that 2Prop was being imported into oocytes expressing UMAMIT29 during electrophysiological measurements, the oocytes were clamped at -60 mV and currents were recorded while continuously perfusing with eKulori buffer (pH 5.5) containing 10 mM 2Prop for 2 minutes. Subsequently, the oocytes were washed three times and homogenized in 50% methanol. The preparation of oocyte samples was as described above. The 2Prop content in individual oocytes was quantified by LC-MS / MS.

[0251] To measure the change in membrane potential in response to changes in monovalent anions (Cl - ) and cations (Na + ) or in response to the addition of Kulori buffer containing the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), un-injected oocytes were incubated in various uptake buffers for 5 minutes and then the membrane potential was recorded using the TEVC technique on a Roboocyte2 (Multichannel Systems, Reutlingen, Germany).

[0252] The glucosinolates were analyzed by LC-MS / MS

[0253] Typically, for obtaining the lowest matrix effect and for improving the sensitivity of LC-MS / MS targeted quantification, the individual quantification of glucosinolates (i.e., glucosinolates derived from plant materials) is performed in the form of desulfo-glucosinolates. To analyze glucosinolates and / or amino acids in Xenopus oocytes in transport assays, we quantified intact glucosinolates and glucosinolate-amino acid mixtures using an external standard curve.

[0254] To analyze glucosinolates as desulfo-glucosinolates, chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was carried out on a Kinetex 1.7u XB-C18 column (100x 2.1mm, 1.7μm, Phenomenex, Torrance, CA, USA). Mobile phases A and B were aqueous formic acid (0.05%) and acetonitrile (supplemented with 0.05% formic acid), respectively. The elution conditions were as follows: 0 - 0.5 min, 2% B; 0.5 - 1.2 min, 2 - 30% B; 1.2 - 2.0 min 30 - 100% B, 2.0 - 2.5 min 100% B, 2.5 - 2.6 min 100 - 2% B and 2.6 - 4 min 2% B. The flow rate of the mobile phase was 400 μl min -1 . The column temperature was maintained at 40 °C. Liquid chromatography was coupled with an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ionization source (ESI) operating in positive ionization mode. Instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at +3500 V. The cone temperature was set at 300 °C, the cone gas was set at 20 psi. The heated probe temperature was set at 400 °C, the probe gas flow was set at 40 psi. The nebulizing gas was set at 60 psi, and the collision gas was set at 1.5 mTorr. Nitrogen was used as the probe and nebulizing gas, and argon was used as the collision gas. The active exhaust was continuously on. Multiple reaction monitoring (MRM) was used to monitor the analyte parent ion → product ion transitions. Detailed mass transition values can be found in (Jensen et al., 2015; Crocoll et al., 2016a). Both the Q1 and Q3 quadrupoles were maintained at unit resolution. Data acquisition and processing were performed using Bruker MS Workstation software (version 8.2.1, Bruker, Bremen, Germany). The linearity of ionization efficiency was verified by analyzing dilution series. p-Hydroxybenzyl or 2-Prop glucosinolate was used as the internal standard.

[0255] Chromatographic analysis for the determination of free amino acids in plant materials and intact glucosinolates with or without resolved amino acids was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Plant and oocyte extracts were prepared in 85% methanol. Supernatants were collected after centrifugation at 12,000 x g for 10 min. The supernatant samples were mixed with 13 C, 15 N-labeled amino acids (algal amino acids 13 C, 15 N, Isotec, Miamisburg, USA) in a 1:10 ratio at a concentration of 1 μg mL -1 . The diluted samples were filtered ([[]] 0.22 μm PVDF filter (Merck Millipore, Tullagreen, Ireland) and used directly for LC-MS analysis. The analysis was carried out as previously described (Mirza et al., 2016) with the following modifications. Briefly, chromatographic analysis was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was carried out on a Zorbax Eclipse XDB-C18 column (100 x 3.0 mm, 1.8 μm, Agilent Technologies, Germany). Aqueous formic acid (0.05%) and acetonitrile (supplemented with 0.05% formic acid) were used as mobile phases A and B, respectively. The elution conditions were as follows: 3% B from 0 - 1.2 min; 3 - 65% B from 1.2 - 4.3 min; 65 - 100% B from 4.3 - 4.4 min; 100% B from 4.4 - 4.9 min, 100 - 3% B from 4.9 - 5.0 min and 3% B from 5.0 - 6.0 min. The flow rate of the mobile phase was 500 μL*min -1, the column temperature was maintained at 40 °C. Liquid chromatography was coupled with an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ionization source (ESI). The instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at 3000 V in positive ion mode. The cone temperature was set at 300 °C and the cone gas flow was set at 20 psi. The heated probe temperature was set at 400 °C and the probe gas flow was set at 50 psi. The nebulizing gas was set at 60 psi and the collision gas was set at 1.6 mTorr. Nitrogen was used as the cone gas and nebulizing gas, and argon was used as the collision gas. Multiple reaction monitoring (MRM) was used to monitor the analyte molecular ion → fragment ion transitions: the same method as that for Arg and Lys was selected for the MRM of amino acids. Both the Q1 and Q3 quadrupoles were maintained at unit resolution. Bruker MS Workstation software (version 8.2.1, Bruker, Bremen, Germany) was used for data acquisition and processing. Except for tryptophan (which was quantified with 13 C, 15 N-Phe), various amino acids in the sample were quantified by the corresponding 13 C, 15 N-labeled amino acid internal standards. Tryptophan was quantified with 13 C, 15 N-Phe, with a response factor of 0.42 used. Asparagine and glutamine were quantified with 13 C, 15 N-Asp and 13 C, 15 N-Glu, with response factors of 1.0 and 0.36 used respectively. Glucosinolates were quantified by an external dilution series, and the linearity of the ionization efficiency was verified by analyzing the dilution series.

[0256] Molecular phylogenetic analysis was performed by the maximum likelihood method

[0257] Using Arabidopsis UMAMIT29 as a bait, protein sequences (253 amino acid sequences) of 14 plant taxa were exported from NCBI Blast: 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. MUSCLE 58 was used to generate a sequence alignment, and BMGE 59 was used for trimming. The IQ-TREE web server ( http: / / iqtree.cibiv.univie.ac.at / ) was used for maximum likelihood analysis. The tree with the optimal log-likelihood (-35897.839) is shown ( Figure 9 ). Bootstrap values are located at each node and were calculated based on 1000 replicates. The resulting tree was visualized using iTOL.

[0258] Statistical analysis

[0259] The sample size was not predetermined using statistical methods. The researchers were informed of the allocation in both the experiment and the assessment. Excel (Microsoft), Origin2021 software (OriginLab), or Rstudio were used for data analysis and plotting. Comparisons between two groups were performed using a two-tailed Student's t-test or a Mann-Whitney U-test, depending on the homogeneity of variance and the normality of residuals. One-way ANOVA was used to compare three or more groups, followed by a post hoc multiple comparison test. P-values were calculated by comparing models, and a decrease in the factor level indicated a significant difference between groups. The data were plotted as graphs as much as possible, and the sample sizes were indicated in the corresponding legends.

[0260] Results

[0261] Source tissues of seed glucosinolates

[0262] Due to the lack of understanding of the synthesis sites of seed-bound glucosinolates, it has been difficult to date to use the transcriptome from source tissues to find output gene candidates. Therefore, we first set out to determine the source tissues that produce seed-bound glucosinolates. In Arabidopsis thaliana, glucosinolates have been detected in phloem sap, and it has been shown that exogenously applied p-hydroxybenzyl glucosinolate is transferred from rosette leaves or roots to seeds. This suggests that rosette leaves and roots may contribute to seed glucosinolate content. To test this hypothesis, we grafted floral tissues (i.e., the upper stem with flowers) of the biosynthetic null mutant myb28 myb29 cyp79b2 cyp79b3 as scions onto wild-type vegetative tissues (i.e., the lower stem with rosette leaves and roots) as rootstocks. Less than 6% of the glucosinolate levels in seeds of wild-type self-grafts were detected in seeds of biosynthetic null scions grafted onto wild-type rootstocks, while reciprocal grafting of wild-type scions onto biosynthetic null rootstocks accumulated glucosinolate levels in seeds identical to those of wild-type self-grafts ( Figure 1 a). In addition, we performed reciprocal grafting with the gtr1 gtr2 mutant, which has previously been shown to have very low levels of glucosinolates in seeds. Glucosinolates were not detected in seeds of gtr1 gtr2 scions grafted onto wild-type rootstocks, while reciprocal grafting of wild-type floral tissues onto gtr1 gtr2 mutant rootstocks accumulated glucosinolate levels identical to those of wild-type self-grafts ( Figure 1 a). Collectively, these data indicate that vegetative tissues are not the main source of seed glucosinolates in Arabidopsis, and de novo synthesis and transport within reproductive tissues are sufficient to supply glucosinolates to seeds.

[0263] To determine the main sites within reproductive tissues that produce seed-bound glucosinolates, we investigated the correlation between seed glucosinolate accumulation and the presence of CYP83A1 and CYP83B1, which represent biosynthetic markers for the production of aliphatic methionine-derived glucosinolates and indole tryptophan-derived glucosinolates by cells, respectively. Time-course experiments on developing wild-type Arabidopsis Col-0 seeds showed that glucosinolate accumulation in seeds began on day 8 after pollination (when the embryo entered the green ripe stage), and seeds continued to accumulate glucosinolates thereafter throughout seed development ( Figure 4 a). Notably, both CYP83A1 and CYP83B1 biosynthetic markers were present in the funiculus at the green ripe stage (when glucosinolate accumulation began). High expression of glucosinolate biosynthesis and supporting genes in the funiculus confirmed this. In addition, our analysis showed that CYP83A1 accumulated to significantly higher levels in the funiculus than CYP83B1 ( Figure 4c), which is highly consistent with the fact that aliphatic glucosinolates account for the majority (>90%) of the total glucosinolates in Arabidopsis seeds. Since the funiculus is the only vascular connection between the septum of the pod and the seed, it is considered to be part of the long-distance transport highway. Our results strongly suggest that the funiculus is also a major hub for the production and export of seed-bound glucosinolates. The glucosinolate levels in the valve of the pod with the funiculus and septum (hereinafter referred to as the pod valve) remained constant from the green ripe stage until the valve began to senesce ( Figure 4 b). This indicates that the pod valve and the funiculus continuously produce and export glucosinolates, which are ultimately transported to the seeds.

[0264] UMAMIT29 is crucial for seed loading

[0265] Based on the proposed role of the funiculus in seed glucosinolate production, we selected the gene encoding the transmembrane protein with the highest relative expression in the funiculus at the green ripe stage ( Figure 4 d), and measured the glucosinolate levels in the seeds of the corresponding knockout mutants. Compared with the wild type, the total glucosinolate levels in the seeds of two independent mutant alleles of UMAMIT29 were generally reduced by 80% ( Figure 1 b), while the glucosinolate levels in the mature pod valves were not significantly reduced ( Figure 4 e). Complementation of the mutant phenotype with UMAMIT29 tagged with the yellow fluorescent protein mVenus indicated that UMAMIT29 is essential for glucosinolate accumulation in seeds ( Figure 1 b).

[0266] We analyzed the cellular localization of the mVenus-tagged UMAMIT29 fusion protein relative to the mTurquoise2 (mTQ2) fusion protein of the biosynthetic marker enzyme SUPERROOT1 (SUR1, common for both aliphatic and indolic glucosinolates) in the pod during seed development. Confocal Z-stack images showed that SUR1-mTQ2 accumulated to high levels in all cells of the funiculus in the pods with embryos at the green ripe stage and during subsequent development ( Figure 1 c). At the green ripe stage, UMAMIT29-mVenus was present in the plasma membranes of cortical cells and cells near the xylem vessels in the funiculus, as well as in the outer integument and chalazal seed coat ( Figure 1 c, Figure 4 f-h). The co-localization of UMAMIT29 with the common glucosinolate biosynthetic marker SUR1, combined with its localization to the plasma membranes of vascular border cells, suggests that UMAMIT29 can function as a glucosinolate exporter in source tissues such as the funiculus.

[0267] UMAMITs Involved in Glucosinolate Transport

[0268] Next, we biochemically characterized UMAMIT29 in Xenopus laevis oocytes. At pH 5.5 (mimicking the acidic apoplast), oocytes expressing UMAMIT29 imported 4-methylthiobutyl glucosinolate (4MTB) and indole-3-ylmethyl glucosinolate (I3M), the major methionine-derived and tryptophan-derived glucosinolates in Arabidopsis seeds, while control oocytes injected with H2O did not ( Figure 2 a). Two-electrode voltage-clamp (TEVC) electrophysiology showed that a positive current was induced in UMAMIT29-expressing oocytes clamped at -60 mV using 10 mM of the standard 2-propenyl glucosinolate (2Prop), while no current was induced in oocytes injected with H2O ( Figure 2 b). Thus, UMAMIT29-mediated glucosinolate import led to a net inward movement of negative charge. As organic anions, glucosinolates carry one negative charge per molecule. Therefore, we observed that the magnitude of the positive current measured by TEVC was proportional to the amount of 2Prop imported. By quantifying the total amount of positive charge induced by 2Prop and the number of 2Prop molecules imported in a single UMAMIT29-expressing oocyte over 2 min, we estimated the stoichiometry between the net charge moving across the membrane and the 2Prop molecules imported to be 0.90 ± 0.23 ( Figure 2 c), indicating that UMAMIT29-mediated 2Prop import follows a uniport mechanism. Notably, the intracellular concentration of 2Prop imported by UMAMIT29 reached a peak within 60 min of incubation and was ~30% of the extracellular concentration ( Figure 2 a). This finding supports the uniport mechanism, where glucosinolate import down its concentration gradient is hindered by the strength of the oocyte's negative membrane potential.

[0269] To further investigate the transport mechanism, we performed a series of experiments in which the external glucosinolate concentration was kept constant and glucosinolate import was monitored at varying membrane potentials. First, current-voltage (I-V) curves (-80 to 0 mV) on UMAMIT29-expressing oocytes perfused with 10 mM 2Prop showed an increase in the positive 2Prop-induced current upon depolarization of the oocyte membrane potential, i.e., a decrease in the opposing negative membrane potential led to an increase in glucosinolate influx. In addition, we measured an increase in 2Prop uptake by UMAMIT29 in oocytes where the membrane potential was depolarized by exchanging 95% of the Cl - in the external buffer with gluconate or by the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the uptake medium at pH 5.5 ( Figure 2d). Instead, we found a reduced uptake of 2Prop in oocytes, where the cations in the uptake buffer (i.e., K + and Na + ) were replaced with the quaternary ammonium choline + or the secondary amine N-methyl-d-glucamine + (NMDG + ), hyperpolarizing the membrane potential ( Figure 2 e). Collectively, these results suggest that the UMAMIT29-mediated uptake of monovalent glucosinolate anions follows a uniport mechanism controlled by the transmembrane electrochemical gradient.

[0270] To test whether UMAMIT29 can facilitate the export of glucosinolates, we directly injected 2Prop into oocytes expressing UMAMIT29 and control oocytes (intracellular 2Prop concentration was approximately 2 mM). In oocytes expressing UMAMIT29, the intracellular 2Prop level decreased over time, accompanied by an increase in 2Prop in the extracellular medium ( Figure 2 f), while control oocytes did not show export activity. Notably, the export (and import) of 2Prop by UMAMIT29 was enhanced when the external medium pH was 5.5 (mimicking acidic apoplast) compared to pH 7.4 ( Figure 2 d), indicating that protons have a regulatory effect on UMAMIT29 activity.

[0271] The name of the UMAMIT family reflects that family members were previously characterized as amino acid facilitators. We studied whether UMAMIT29 can transport amino acids by exposing oocytes expressing UMAMIT29 to 13 C, 15 N isotope-labeled glutamine and glutamate (well-known UMAMIT substrates 27 ) at concentration ranges of 0.4 - 10 mM (glutamine) and 2 - 20 mM (glutamate) respectively, generating an inward gradient relative to the endogenous amino acid concentration. No amino acid gradient led to the accumulation of detectable levels of isotope-labeled amino acids in oocytes. Upon injecting 13 C, 15In the output assay of N-isotope-labeled glutamine or glutamate, no output from oocytes expressing UMAMIT29 into the medium was detected. In contrast, we observed the formation of intracellular isotope-labeled aspartate in both oocytes expressing UMAMIT29 and control oocytes, accompanied by a significant decrease in isotope-labeled glutamine and glutamate in the oocytes. This indicates that a portion of the injected isotope-labeled amino acids is metabolically converted within the oocytes and thus cannot be utilized by UMAMIT29. Since we could measure the transport of glucosinolates but not amino acids by UMAMIT29, we investigated whether external amino acids would affect the input of glucosinolates. We found that up to 1000-fold excess of glutamine (100 mM) did not inhibit the uptake of 4MTB. In contrast, 4MTB uptake was completely outcompeted by 10-fold excess of 2Prop ( Figure 2 g, h). The data indicate that the input and output of glucosinolates mediated by UMAMIT29 are not affected by glutamine and glutamate at concentrations within the physiological range.

[0272] Arabidopsis UMAMIT29 belongs to branch I of the UMAMIT family, which consists of seven members: UMAMIT26 to -32. Since UMAMIT30 and -31 are additional UMAMIT genes expressed together with UMAMIT29 in the funiculus at the green-ripe stage ( Figure 5 ), their input activities in oocytes were screened using an equimolar mixture of 11 aliphatic glucosinolates and 1 indole glucosinolate at 100 μM. Oocytes expressing UMAMIT29 and UMAMIT30 accumulated both types of glucosinolates, while oocytes expressing UMAMIT31 preferred indole glucosinolates over aliphatic glucosinolates ( Figure 2 i, j). Five hours after injecting an equimolar mixture of aliphatic and indole glucosinolates into the oocytes (intracellular concentration of each glucosinolate was approximately 50 μM), the output activities of UMAMIT29-31 were demonstrated by monitoring the release of glucosinolates into the medium ( Figure 2 k, i). Similar to the substrate preference observed in the input assay, UMAMIT31 preferred to output indole glucosinolates over aliphatic glucosinolates, while UMAMIT29 and UMAMIT30 showed broad substrate specificity ( Figure 2 k, i). Our results indicate that not only UMAMIT29 but also UMAMIT30 and UMAMIT31 are glucosinolate uniporters that can passively facilitate the efflux of glucosinolates from the cytoplasm (e.g., the cytoplasm of source cells) to the apoplast along the electrochemical gradient.

[0273] Role of UMAMIT in seed loading

[0274] UMAMIT30 and UMAMIT31 can export glucosinolates in Xenopus oocytes, indicating that they may act together with UMAMIT29 to export glucosinolates to seeds. Analysis of existing data from the funiculus transcriptome atlas showed that UMAMIT31 was highly and specifically expressed in the funiculi of developing pods with embryos at the green ripe stage, while the expression of UMAMIT30 was generally low ( Figure 5 ). Consistent with the expression patterns and specificities, seeds of the umamit30 single mutant had wild-type glucosinolate profiles and levels, while the indole-type glucosinolates in the umamit31 mutant were specifically reduced by approximately 50% ( Figure 3 a, b). Glucosinolate analysis of seeds from the tandem UMAMIT29 and UMAMIT31 double mutant showed that the levels of aliphatic glucosinolates in umamit29 umamit31 were the same as those in the umamit29 single mutant (reduced by approximately 80% of the wild-type level), while the levels of indole glucosinolates in umamit29 umamit31 seeds were reduced to approximately 5% of the wild-type level ( Figure 3 b). The remaining aliphatic glucosinolates in the umamit29 umamit31 mutant suggest that in the absence of these two UMAMIT transporters, different transporters are responsible for glucosinolate transport. Notably, in the umamit29 umamit31 mutant, UMAMIT30 transcripts were upregulated by more than 4-fold in pods ( Figure 6 ), indicating that UMAMIT30 was induced to express in this mutant to transport glucosinolates to seeds in the absence of UMAMIT29 and UMAMIT31. In fact, compared with the umamit29 single mutant, the levels of aliphatic glucosinolates in seeds of the umamit29 umamit30 double mutant were further reduced by approximately 50% ( Figure 3 a, b, Figure 7 ). The total glucosinolate level in seeds of the umamit29umamit30umamit31 triple mutant was approximately 5% of the wild-type level ( Figure 3 a, b, Figure 7 ). In contrast, the glucosinolate levels in the developing pod valves of the umamit29 umamit30 mutant were comparable to those of the wild-type ( Figure 3d). The total free amino acid levels in the seeds of the umamit29 and umamit31 single mutants and the umamit29 umamit31 double mutant were not significantly different from those of the wild type, while the seeds of the umamit30 single mutant, the umamit29 umamit30 double mutant, and the umamit29 umamit30 umamit31 triple mutant showed increased total free amino acid levels ( Figure 3 e). The seed size and total seed weight of the umamit29 umamit30 double mutant and the umamit29 umamit30 umamit31 triple mutant were both reduced compared to the wild type levels. ( Figure 3 f and g). In summary, the substrate preferences of UMAMIT29, -30, and -31 measured in oocytes were consistent with the significant reduction of specific glucosinolates in the seeds of different umamit mutants, which supports a key role of these UMAMITs in transporting glucosinolates into the seeds.

[0275] Next, to evaluate the transport engineering potential of the UMAMIT exporter proteins, we investigated the effect of eliminating the three UMAMIT exporter proteins on glucosinolate accumulation in other organs (i.e., roots, rosette leaves, stems, and cauline leaves). The total glucosinolate levels in the roots of the single, double, or triple mutants of UMAMIT29, -30, and -31 were comparable to those of the wild type, while the glucosinolate levels in the roots of the gtr1 gtr2 gtr3 mutant were significantly reduced. Figure 3 h-k). Similarly, the total glucosinolate levels in the rosette leaves, cauline leaves, and stems of the umamit single and multiple mutants were comparable to those of the wild type, while strong over-accumulation of glucosinolates was observed in the rosette leaves and cauline leaves of the gtr1 gtr2 gtr3 mutant. Figure 3 h-k, Figure 8 ). Interestingly, the altered glucosinolate distribution pattern in the gtr1 gtr2 gtr3 mutant was completely restored to the wild type level in the umamit29 umamit30 gtr1 gtr2 gtr3 quintuple mutant. Figure 3 h-k, Figure 8 ). In planta data support that UMAMIT29, -30, and -31 function as glucosinolate exporter proteins and are potential molecular targets for seed-specific glucosinolate elimination through transport engineering.

[0276] Discussion

[0277] In this study, we identified UMAMIT29, UMAMIT30, and UMAMIT31 as glucosinolate exporters that play a key role in glucosinolate accumulation in seeds, as seeds of the umamit29 umamit30 umamit31 exporter mutants had less than 6% of the glucosinolate levels of the wild type. We previously identified the active high-affinity H + coupled glucosinolate importers (GTR1, GTR2, and GTR3) and showed that the elimination of seed glucosinolates in the gtr1 gtr2 gtr3 triple mutant was accompanied by an altered glucosinolate accumulation profile in the rest of the plant. Suppression of this phenotype in the umamit29 umamit30 gtr1 gtr2 gtr3 quintuple mutant provides genetic evidence that UMAMIT functions as an exporter to deliver glucosinolates to the apoplast.

[0278] Biochemical and biophysical characterization of UMAMIT29-31 in oocytes indicated that glucosinolates are exported passively along an electrochemical gradient. In Arabidopsis, the plasma membrane potential under physiological conditions ranges from -100 to -150 mV, and UMAMIT29-31 is expected to facilitate the efficient export of monovalent anionic glucosinolates from biosynthetic cells to the acidic apoplast. A recent theoretical study hypothesized that nutrient homeostasis mechanisms aimed at achieving a desired cytoplasmic concentration of a given nutrient require the involvement of at least two different energized transporter types. The presence of passive UMAMIT exporters and H + coupled GTR importers meets this requirement. Thus, H + coupled high-affinity GTR importers deplete glucosinolates in the apoplast, generating a strong outward chemical gradient (from the cytoplasm to the apoplast) that, together with the strong negative membrane potential, promotes the efficient export of glucosinolates via the passive uniport mechanism of UMAMIT29-31. Interestingly, glucosinolate exporters, as well as sugar exporters (Sugars Will Eventually be Exported Transporters, SWEETs) and amino acid exporters (UMAMITs), are all uniporters, suggesting the existence of a general passive uniport mechanism for exporting compounds from the symplast in source tissues.

[0279] UMAMIT29-31 belongs to the UMAMIT clade I proteins (UMAMIT26-32), where UMAMIT26-31 are Brassicaceae-specific ( Figure 9)。This highlights the potential for transporter families involved in primary metabolism to have evolved substrate specificities for specialized metabolites, as recently demonstrated in the NPF family. Similarly, the identification of UMAMIT as a glucosinolate exporter suggests that the UMAMIT family may represent a new family of exporters for specialized metabolites. Interestingly, the substrate specificities of three UMAMIT exporters (where UMAMIT29 and -30 have broad substrate specificities, while UMAMIT31 is specific for indole glucosinolates) are reflected in three GTR importers, where GTR1 and GTR2 have broad substrate specificities, while GTR3 prefers indole glucosinolates, suggesting co-evolution of substrate preferences between these different transporter families. Previous studies of UMAMIT amino acid export activity using Xenopus laevis oocytes and Saccharomyces cerevisiae cells have reported different results, which may reflect the difficulty in measuring transporter activity for primary metabolites that are co-metabolized by the heterologous host expressing the transporter. Minor changes in the levels of free amino acids in seeds of various umamit mutants and myb28myb29 (MYB28 and 29 are major regulators of aliphatic glucosinolates) and gtr1 gtr2 mutants41 are worthy of future studies to investigate the relationship between glucosinolates and free amino acids in seeds.

[0280] Based on the overlapping presence of UMAMIT29, -30, and -31 with glucosinolate biosynthesis marker enzymes in the funicles at the green-ripe stage, we propose that the funicle is the major source tissue of glucosinolates, from which UMAMIT29, -30, and -31 export de novo synthesized seed-bound glucosinolates to the apoplast, followed by secondary activation of high-affinity H + coupled GTRs that import them into the phloem, by which glucosinolates are transferred to the seeds. Overall, because transporter mutants of primary metabolites (such as sucrose and amino acids, whose importers and exporters are known) are usually pleiotropic, the understanding of the seed loading process is hampered by the lack of tools. This is generally not the case for specialized metabolite transporters. Glucosinolate UMAMIT exporters and GTR importers are essential for seed accumulation and provide powerful molecular tools to dissect the process of seed loading into the embryo.

[0281] Transport engineering, which changes metabolite distribution patterns by altering transporter gene expression, has made progress in improving agronomic traits (Moore et al., 2015; Krattinger et al., 2016; Kim et al., 2021). This article shows that the umamit29 umamit30 umamit31 exporter mutants do not change the overall distribution of glucosinolates in plants, in contrast to the reduction (roots, stems) or strong over-accumulation (rosette leaves, cauline leaves, pod valves) in the gtr1 gtr2 gtr3 glucosinolate importer mutants. Therefore, from an agricultural perspective, UMAMIT glucosinolate exporters have high biotechnological potential as molecular breeding targets for eliminating antinutritional glucosinolates in the seed meal of cruciferous oilseed crops through transport engineering without affecting glucosinolate defenses in the rest of the plant.

[0282] Example 2 - Glucosinolate transport activity of Brassica napus UMAMIT transporters

[0283] Seeds of the model plant Arabidopsis thaliana and Brassica crops accumulate glucosinolates produced and exported by maternal tissues. In Arabidopsis, AtUMAMIT28, AtUMAMIT29, AtUMAMIT30, and AtUMAMIT31 from the clade I UMAMIT family have been characterized as bidirectional glucosinolate facilitators. Recent data show that AtUMAMIT29, AtUMAMIT30, and AtUMAMIT31 are essential for glucosinolate accumulation in seeds, with AtUMAMIT29 playing a major role. We selected the AtUMAMIT28, AtUMAMIT29, AtUMAMIT30, and AtUMAMIT31 glucosinolate facilitators as the main targets for translating the umamit loss-of-function phenotype from Arabidopsis to Brassica crops.

[0284] The initial work focused on reducing the glucosinolate content in the seeds of Brassica napus (B. napus, AACC, 2n = 38). Brassica napus is an allopolyploid species, the result of a natural hybridization between two diploid ancestors, turnip (AA, 2n = 20) and B. oleracea (CC, 2n = 18), approximately 7,500 years ago. During evolution, domestication, and breeding practices, genome duplications, chromosomal rearrangements, and deletion events have led to significant changes in the copy number and gene structure of Brassica napus cultivars (including UMAMIT orthologs).

[0285] Materials and Methods

[0286] Identification of Arabidopsis clade I UMAMIT orthologs.

[0287] Using the AtUMAMIT26 - 32 amino acids as query sequences, query the BnPIR: Brassica napus pan - genomic information resource (http: / / cbi.hzau.edu.cn / bnapus / ) to identify UMAMIT orthologs in Brassica napus.

[0288] Brassica UMAMIT nomenclature

[0289] Brassica UMAMIT orthologs are named according to their sequence identity with the corresponding Arabidopsis orthologs and their sub - genomic associations, as shown below: Bn represents the plant species Brassica napus; UMAMIT26 - 32 indicates which Arabidopsis UMAMIT gene the gene is an ortholog of. A and C represent the sub - genomic positions according to the U - triangle (A represents the position in the AA genome derived from turnip, and C represents the position in the CC genome derived from B. oleracea). 1, 2, 3, and 4 represent the amino acid sequence homology with the Arabidopsis orthologs. The genes are numbered according to the decrease in amino acid sequence homology, where 1 represents the highest identity level.

[0290] Phylogenetic tree

[0291] Align the sequences using CLASWX with a gap - opening penalty of - 2.9, a gap - extension of 0, and a hydrophobicity multiplier of 1.2. Manually adjust poorly aligned regions. Infer the evolutionary history using the maximum - likelihood method and the JTT - based matrix model. The tree with the highest log - likelihood value (-9060.10) is shown. The percentage of trees in which the associated taxa cluster together is shown next to the branches. The initial tree for the heuristic search is automatically obtained by applying the neighbor - joining algorithm and the BioNJ algorithm to the pairwise distance matrix estimated using the JTT model and then selecting the topology with a higher log - likelihood value. A discrete Gamma distribution is used to model the differences in evolutionary rates between sites (5 categories (+G, parameter = 1.5532)). The rate - variation model allows some sites to remain invariant during evolution ([+I], 0.84% of sites). The tree is drawn to scale, and the branch lengths are measured in the number of substitutions per site. The evolutionary analysis is conducted in MEGA X.

[0292] Cloning

[0293] Total RNA was extracted from different tissues of Brassica napus at different developmental stages, and cDNA synthesis was performed using the Superscript III First-Strand Synthesis kit (Invitrogen) and oligo(dT) primers. The full-length cDNA sequences of 11 BnUMAMIT genes were amplified from the cDNA by PCR; due to the high sequence similarity, the primers were placed in the more variable UTR region. The PCR fragments were cloned into pNB1u and verified by sequencing.

[0294] Expression in Xenopus laevis oocytes

[0295] According to the manufacturer's instructions, a linear DNA template for in vitro transcription was generated from the pNB1u plasmid by PCR using Phusion high-fidelity DNA polymerase (NEB). The PCR products were purified using the QIAquick PCR Purification kit (Qiagen). Capped cRNA was synthesized in vitro using the mMessage mMachine T7 kit (Ambion). The synthesized cRNA concentration of each transporter gene was normalized to 500 ng / μl, aliquoted, and stored at -20 °C. For expression in Xenopus laevis oocytes, at most one cRNA thawing cycle was used.

[0296] Xenopus laevis oocytes were purchased from Ecocyte Bioscience (Germany). 50.6 nL of cRNA was injected into the oocytes using a Drummond NANOJECT II (Drummond scientific company, Broomall 116, Pennsylvania). The cRNA (500 ng / μl) was injected into the oocytes. The injected oocytes were incubated in HEPES-based Kulori buffer (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES pH 7.4) supplemented with gentamicin (100 μg / mL) at 16 °C for three days. For the control injected with mock, 50.6 nL of nuclease-free water (Ambion) was injected instead of cRNA.

[0297] The Xenopus laevis oocyte uptake assay was performed essentially as previously described with some modifications. Three days after injection of cRNA, the oocytes were pre-incubated for 5 minutes in 5 ml of kulori buffer (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM MES pH 5.0), and then incubated with kulori buffer containing the substrate (100 μM glucosinolate) for a given time. The oocytes were washed 5 times with Milli-Q water (20 ml each time) and homogenized with 80% methanol (containing internal standard). Subsequently, the oocyte extract was centrifuged at 12,000 g for 10 minutes at 4 °C. The supernatant was diluted with water and filtered through a 0.22 μm filter plate 131 (MSGVN2250, Merck Millipore), and then analyzed by LC-MS / MS as described below.

[0298] Desulfo-glucosinolate analysis by LC-MS / TripleQuad

[0299] The samples were diluted 10-fold with deionized water and then analyzed by liquid chromatography-mass spectrometry. Chromatographic analysis was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was carried out on a Kinetex 1.7 uXB-C18 column (100 x 2.1 mm, 1.7 μm, Phenomenex, Torrance, CA, USA). Mobile phases A and B were aqueous formic acid (0.05%) and acetonitrile (supplemented with 0.05% formic acid), respectively. The elution conditions were as follows: 0 - 0.5 minutes, 2% B; 0.5 - 1.2 minutes, 2 - 30% B; 1.2 - 2.0 minutes 30 - 100% B, 2.0 - 2.5 minutes 100% B, 2.5 - 2.6 minutes, 100 - 2% B and 2.6 - 4 minutes 2% B. The mobile phase flow rate was 400 μl min -1。The column temperature was maintained at 40 °C. Liquid chromatography was coupled with an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ionization source (ESI) operating in positive ionization mode. Instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at +3500 V. The cone temperature was set at 300 °C and the cone gas was set at 20. The heated probe temperature was set at 400 °C and the probe gas flow was set at 40 psi. The nebulizer gas was set at 60 psi and the collision gas was set at 1.5 mTorr. Nitrogen was used as the probe and nebulizer gas, and argon was used as the collision gas. Active exhaust was continuously turned on. Multiple reaction monitoring (MRM) was used to monitor the analyte parent ion → product ion transitions. Detailed mass transition values can be found in (Jensen et al., 2015; Crocoll et al., 2016a). Both the Q1 and Q3 quadrupoles were maintained at unit resolution. Data acquisition and processing were performed using Bruker MS Workstation software (version 8.2.1, Bruker, Bremen, Germany). The linearity of ionization efficiency was verified by analyzing dilution series. p-Hydroxybenzyl or 2-propenyl glucosinolate was used as the internal standard.

[0300] Results

[0301] Phylogenetic analysis of the sequence of the well-assembled ZS11 reference genome (Song et al., 2020) indicated that it encodes 4 UMAMIT28, 4 UMAMIT29, 11 AtUMAMIT30, and 6 AtUMAMIT31 orthologs ( Figure 10 ). We were unable to assign Arabidopsis orthologs to BnaA09G0692800ZS, BnaA09G0714300ZS, BnaC02G0337200ZS, and BnaA02G0251300ZS, which clustered in the outer layer of the UMAMIT26-29 subclade, distinct from the UMAMIT30 and UMAMIT31 subclades. We did not identify homologs of AtUMAMIT26 and AtUMAMIT27 from Brassica napus.

[0302] Of the 27 UMAMITs in ZS11, four annotated genes (BnaC02G0337200ZS, BnaA02G0251300ZS, Bnacaffold0025G0022000ZS, and Bnascaffold0025G0022100ZS) were excluded from further analysis because they had mutations that led to the emergence of premature stop codons, resulting in very short proteins.

[0303] To focus on BnaUMAMIT-expressing genes most likely to play a role in the seed loading of glucosinolates, we mined Brassica EDB ( transcriptome data in (https: / / brassica.biodb.org / ) and analyzed 23 UMAMIT orthologs in ZS11 Expression in seeds and pod valves of cultivars ( Figure 11 ).

[0304] Transcript analysis of the expression of each UMAMIT-expressing gene in different tissues and developmental stages showed that BnUMAMIT28A1, BnUMAMIT28C1, BnUMAMIT30A2, and BnUMAMIT30C2 had the highest expression in the pod walls during the pod-setting and maturity stages, while BnUmamiT30C1, BnUmamiT28C1, and BnUmamiT28A2 were highly expressed in seeds ( Figure 11 ). In contrast, transcripts corresponding to (BnUmamiT29C1, BnUmamiT29A1, BnUmamiT30C5, BnUmamiT30A3, BnUmamiT30C6, BnUmamiT30C4, BnUmamiT30A1, BnUmamiT30A4, BnUmamiT30C3, BnUmamiT30A5, BnUmamiT31C2, BnUmamiT31C1, BnUmamiT31A1, BnUmamiT31A2, BnUmamiT28A3, BnUmamiT32A2, BnUmamiT32C3, BnUmamiT32C1, BnUmamiT32A1, BnUmamiT32C2, BnUmamiT32A3) were hardly detected in seeds and pod walls.

[0305] Based on the above in silico analysis of UMAMIT expression in the ZS11 cultivar, we set out to clone UMAMITs enriched in pods and / or seeds and detect their glucosinolate transport activity. In the high-glucosinolate 0 variant Niklas, we isolated cDNA from leaves, stems, young pods, old pods, mature pods, and dry seeds. Eleven UMAMIT ortholog-encoding sequences were cloned using gene-specific primers ( Figure 12 A). Five of them (BnUMAMIT28C1N, BnUMAMIT30A2N, BnUMAMIT30C2N, BnUmamiT30C1N, and BnUmamiT28A2N) were paralogous genes of ZS11 genes and were most highly expressed in reproductive organs (pod walls and seeds) ( Figure 11)。We did not detect transcripts of paralogs of pod-expressed BnUMAMIT28A1ZS in Niklas, but identified BnUMAMIT28A3N, whose transcripts were not shown in the ZS11 database.

[0306] Next, we tested the glucosinolate transport activity of Niklas orthologs by heterologous expression in Xenopus oocytes. Interestingly, among the 11 cloned UMAMIT genes, 6 UMAMITs (BnUMAMIT28A3N, BnUMAMIT28C1N, BnUMAMIT30A2N, BnUMAMIT30C2N, BnUmamiT30C1N, and BnUmamiT28A2N) were highly expressed in the pods and / or seeds of Niklas and ZS11, and they transported glucosinolates into oocytes with different activities and substrate specificities ( Figure 12 B), while the remaining 5 orthologs did not show glucosinolate uptake. Among the six functional transporters, BnUmamiT28A2N, BnUmamiT28A3N, and BnUmamiT30A2N preferred to take up 4MTB and I3M in the form of an equimolar mixture rather than the plaintiff's echinacoside 2OH-3-but. In contrast, BnUmamiT28C1N transported all glucosinolates (the plaintiff's echinacoside 4MTB, I3M) into oocytes without a particular preference. The six BnUmamiT glucosinolate transporters expressed in reproductive tissues were targets for mutagenesis, and BnUmamiT28C1N was the key target because 2OH-3-but echinacoside is the main antinutritional glucosinolate in Niklas seeds.

[0307] Example 3 - Reducing Glucosinolate Levels in Brassica napus Seeds by Mutating UMAMIT Glucosinolate Export Proteins

[0308] Multiple umamit mutants were generated in Brassica napus var. 0 Niklas using DNA-free genome editing technology targeting all six UMAMIT orthologs (i.e., BnUMAMIT28A3N, BnUMAMIT28C1N, BnUMAMIT30A2N, BnUMAMIT30C2N, BnUmamiT30C1N, and BnUmamiT28A2N). Alternatively, we will use EMS-mediated mutagenesis technology to construct a TILLING population in Niklas and screen for single loss-of-function mutants of each ortholog. Multiple mutants will be generated by successive generations of crossing and mutation stacking.

[0309] Step 1: Use the RNP complex in protoplasts to screen for highly efficient gRNAs targeting the UMAMIT gene

[0310] Design multiple candidate gRNAs to target regions conserved among all six UMAMIT orthologs for introducing indel mutations, and test their efficiency together with Cas9 by PEG-mediated transformation of RNP complexes into protoplasts. The gRNAs are prepared by in vitro transcription, and the Cas9 protein will be purified from cell extracts of an Escherichia coli strain expressing Cas9 (March et al., 1989). We will prepare protoplasts from 14-day-old cotyledons with high regeneration capacity. Harvest 200 μL containing 2×10 5 / mL protoplasts, and transfect with gRNA, Cas9 protein, Lipofectamine TM 3000 and Plus agent TM transfection reagent, as well as polyethylene glycol (PEG) 4000. We use capillary electrophoresis-based analysis of fluorescently labeled PCR products to evaluate the efficiency of gRNAs by the amplicon indel detection analysis (IDAA) method.

[0311] Step 2: Regenerate UMAMIT mutant strains from protoplasts

[0312] The tissue regeneration efficiency of genome-edited protoplasts depends on the cultivar and is relatively low. Therefore, we compared and tested multiple existing protocols for regenerating whole plants from transfected rapeseed protoplasts to develop a method for the Niklas cultivar. The most efficient gRNAs targeting UMAMIT orthologs (from Step 2) are transformed into rapeseed protoplasts as RNP complexes together with Cas9. Calli are regenerated from single protoplasts by hormone induction containing 2,4-D, BAP, and NAA, and then treated with NAA, GA3, and 2iP to induce shoots and subsequent roots. Seedlings will be screened on a large scale using the gene-specific primers generated in Step 1 and the IDAA method (see above) to successfully identify genome-edited UMAMIT orthologs. Genome editing will be confirmed by Sanger sequencing. The genome-edited UMAMIT mutant seedlings will be transplanted into soil and grown in a climate chamber at 22 °C with 16 h light / 8 h dark until seed maturity. We will analyze the levels and profiles of glucosinolates in the obtained homozygous UMAMIT mutant lines to evaluate the effect of genome editing.

[0313] Example 4 - Identification of key amino acid residues in AtUMAMIT29 for transporting glucosinolates

[0314] Materials and methods

[0315] Mining the Amino Acid Sequences of UMAMIT Homologs

[0316] The amino acid sequences of the UMAMIT clade I (UMAMIT26 - 32) were obtained from TAIR of Arabidopsis thaliana( https: / / www.arabidopsis.org ), BnPIR of Brassica napus ZS11 (http: / / cbi.hzau.edu.cn / bnapus / index.php) (Song et al., 2020), Genoscope of Brassica rapa Z1 ((Belser et al., 2018), https: / / www.genoscope.cns.fr / externe / plants / index.html ), Brassica oleracea HDEM, phytozome 13 of Manihot esculenta (https: / / phytozome - next.jgi.doe.gov) and NCBI of Physcomitrella patens. Except for MaCap / 01 (NCBI ID: XP_023632698.1), MaCar / 15 (NCBI ID: XP_021905388.1) and MaCar / 16 (NCBI ID: XP_021887563.1) which were exported from NCBI, the sequences of other plant species were those of (Zhao et al., 2021).

[0317] Multiple Sequence Alignment and Phylogenetic Analysis

[0318] The sequences of 27 plant species were aligned in MEGA X (https: / / www.megasoftware.net / ) (Kumar et al., 2018) using MUSCLE with default settings (Edgar, 2004). Sequences containing indels within any helix were removed, except for AsSol / 42, FaMed / 43 and roots. The final alignment contained 97 sequences. Sequence identities were made using JDet (http: / / csbg.cnb.csic.es / JDet / ) (Muth et al., 2012). A phylogenetic tree was generated in MEGA X using the neighbor - joining method (1000 bootstraps) and annotated in iTOL (Letunic and Bork, 2021).

[0319] Diverge 3.0 beta 1 was used to estimate differentially conserved amino acids

[0320] DIVERGE 3.0 beta 1 (hereinafter referred to as DIVERGE) was used to identify differentially conserved amino acids (https: / / github.com / xungulab / diverge) (Gu et al., 2013). A multiple sequence alignment containing 97 sequences and its corresponding neighbor-joining phylogenetic tree (see above) were loaded into the program. The estimation of cluster-specific functional divergence (corresponding to the amount of differential conservation) was calculated based on the algorithm in Gu et al., (2013), and the final scores are shown in Table 5 below.

[0321] Table 5 - Amino acid residues predicted to constitute the substrate transport cavity of Arabidopsis UMAMIT29 and UMAMIT32.

[0322]

[0323]

[0324] Blackened residues represent 11 differentially conserved amino acids identified in the molecular dynamics simulation analysis. NA: Not obtained. * Residues with the same residues in UT30 / 31 and UT32 with a score ≠ 0.

[0325] Protein Modeling and Analysis

[0326] The structures of Arabidopsis UMAMIT29 (Uniprot ID: Q9M131) and AtUMAMIT32 (Uniprot ID: Q9LI65) were modeled using RaptorX (http: / / raptorx.uchicago.edu / ContactMap / ) (Ma et al., 2015; Wang et al., 2017; Wang et al., 2016) and AlphaFold (https: / / alphafold.ebi.ac.uk / ) (Senior et al., 2020; Jumper et al., 2021). The protein models were drawn by the PyMOL Molecular Graphics System version 2.4 LLC (https: / / pymol.org / 2 / ). The residues constituting the active sites of UMAMIT29 and UMAMIT32 were determined using the CAVER webtool v1.0 ( https: / / loschmidt.chemi.muni.cz / caverweb / )(Stourac et al., 2019), and then manually inspected and slightly modified in PyMOL 2.4.

[0327] Generation of UMAMIT29 Mutant Variants

[0328] DNA fragments of UT29_UT32-11 (i.e., UMAMIT29 with 11 differential conserved residues from UMAMIT32) and UT29_clade1-3cons (i.e., UMAMIT29 with 3 UMAMIT conserved residues mutated to alanine) were both purchased from Twist Bioscience. UMAMIT29 single-residue mutant variants were generated by USER cloning. The pNB1u plasmid was PCR amplified using Phusion High-Fidelity DNA Polymerase (NEB), and the PCR products were purified using Cycle Pure Kit (Omega Bio-tek) to obtain linear DNA templates for in vitro transcription. In vitro transcription of the template DNA was performed using the mMessage mMachine TM T7 Transcription Kit (InVitrogen). The RNA transcripts (about 600 ng / μL) were aliquoted into 10 μL per tube and stored at -18 °C until use.

[0329] Measurement of Transport Activity in Xenopus Oocytes

[0330] Xu et al. (2016) described the transport assay using Xenopus oocytes. Briefly, defolliculated Xenopus oocytes (stage V or VI) were ordered from Ecocyte Bioscience and the Department of Drug Design and Pharmacology, University of Copenhagen. 50 nL of RNA (about 600 ng / μL) was injected into the oocytes using Nanoject II (Drummond Scientific). For the mock (negative control), 50 nL of sterile Milli- H2O was injected into the oocytes. The injected oocytes were incubated in Kulori buffer pH 7.4 (5 mM MES, 90 mM NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2) supplemented with gentamicin (100 μg / mL) at 16 °C for three days before the assay.

[0331] The experiment was conducted as follows: First, the oocytes were pre-incubated in Kulori buffer pH 5 (5 mM MES, 90 mM NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2) without substrate for 5 minutes. Then the oocytes were incubated in Kulori buffer pH 5 (5 mM MES, 90 mM NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2) supplemented with 4-methylthiobutyl glucosinolate (4MTB), indol-3-ylmethyl glucosinolate (I3M), and benzyl glucosinolate (BGLS) (200 μM each) for 1 hour. After 1 hour, the oocytes were washed in five Petri dishes containing Milli- H2O. The oocytes in the last Petri dish were aliquoted into tubes, three oocytes per tube. The residual water in each tube was removed, and the oocytes were homogenized in 50 μL of 50% internal standard solution containing 1.25 μM sinigrin (62.5 pmol per sample). The tubes were placed at -18 °C overnight.

[0332] Sample Preparation for LC / MS Analysis

[0333] Glucosinolates were extracted and quantified in the form of desulfo-glucosinolates as previously described (Crocoll et al., 2016b). Briefly, 45 μL of DEAE-Sephadex A-25 was loaded into a 96-well filter plate (0.45 μM) using a MultiScreen Column Loader (Merck Millipore). 300 μL of H2O was added to each well, and the plate was incubated at room temperature for 3 - 4 hours or overnight in the refrigerator. A vacuum of 2 - 4 seconds was applied using a vacuum manifold to remove the excess H2O. After centrifugation at >20,000 xg for >15 minutes at 4 °C on ice, all the supernatants were added to the DEAE-sephadex, and a vacuum of 2 - 4 seconds was applied. Then the wells were washed twice with 100 μL of 70% methanol and twice with 100 μL of H2O using a 2 - 4 second vacuum. In the final washing step, the plate was briefly centrifuged at 5900 RPM. 20 μL of sulfatase was added to each well, and the plate was incubated overnight at room temperature. The desulfo-glucosinolates were eluted with 90 μL of H2O at 5900 RPM. The plate was placed at -18 °C until LC / MS analysis.

[0334] Data Analysis and Statistics

[0335] Plotting and statistics were performed using R Studio (version 2021.09.0+351). The statistical methods were as follows: First, a one-way ANOVA test was performed on the compounds against RNA, and then the Tukey Honestly Significant Difference (Tukey HSD) test was used for multiple pairwise comparisons of the means of each RNA (P<0.05).

[0336] Results

[0337] Substrate Translocation Cavity of UMAMIT29

[0338] Since there is currently no experimentally determined structure for any UMAMIT family member, we used the de novo protein modeling tool RaptorX (Ma et al., 2015; Wang et al., 2016; Wang et al., 2017) to generate a model of the putative closed conformation of UMAMIT29. Based on our model, we proposed a substrate transport cavity of UMAMIT29 defined by helices 1-4 and 6-9, and selected 51 residues as putative substrate binding sites according to solvent accessibility. We hypothesized that the transport cavity contains highly conserved residues crucial for transport activity. To test this, we aligned 97 protein sequences consisting of UMAMIT clade I homologs from 27 plant species and created a sequence logo containing the 51 residues identified in the structural analysis. Four residues, R44, G82, W200, and Q204, within the binding cavity were 100% conserved in all sequences of the multiple sequence alignment ( Figure 13 ). This indicates that these residues are crucial for the functional or structural characteristics of the transporter. When R44, W200, and Q204 in UMAMIT29 (UMAMIT29#3CON) were mutated to alanine separately, the import activities of aliphatic 4-methylthiobutyl glucosinolate (4MTB) and benzyl glucosinolate (BGLS) were reduced by more than 80%, and the import activity of indole-3-ylmethyl glucosinolate (I3M) was reduced by approximately 50% ( Figure 14 ). Interestingly, changing only R44 (UMAMIT29#R44A) reduced the transport activities of all tested glucosinolates by 88-98% ( Figure 14 ). Identification of residues within the predicted substrate transport cavity crucial for glucosinolate transport activity supports our model.

[0339] Key Residues for Glucosinolate Transport Activity of UMAMIT29

[0340] Phylogenetic analysis of the above 97 sequences showed that homologs of the glucosinolate-transporting UMAMIT proteins specific to Brassicaceae and the non-glucosinolate-transporting UMAMIT proteins not specific to Brassicaceae were divided into two distinct clusters. We hypothesized that the differentially conserved residues in the protein-binding cavities located within these two clusters could reveal the positions determining specificity.

[0341] Analysis of the sequence identities of 51 predicted substrate-transporting cavity residues between the two clusters identified 13 differentially conserved residues ( Figure 15 ; Table 5). Among these 13 positions, two amino acid residues were identical between the non-glucosinolate-transporting UMAMIT32 and the glucosinolate-transporting UMAMIT30 and -31 and were thus not considered further.

[0342] Subsequently, by generating 11 UMAMIT29 mutant variants in which each of the 11 residues was individually changed to the corresponding residue of UMAMIT32, we set out to investigate the contribution of these 11 differentially conserved residues to glucosinolate transport activity. The glucosinolate substrate specificities of the mutant variants were compared with those of UMAMIT29, which has a broad substrate specificity for methionine-derived aliphatic glucosinolates and tryptophan-derived indole glucosinolates. The mutant variants were expressed in Xenopus oocytes, and the uptake of a mixture of three glucosinolates (i.e., the aliphatic 4-methylthiobutyl glucosinolate (4MTB), the indole-3-ylmethyl glucosinolate (I3M), and the benzyl glucosinolate (BGLS)) was tested. Eight out of the 11 mutant variants showed reduced transport activity for all three glucosinolates (see Table 6 below).

[0343] Table 6 - Glucosinolate uptake activities of mutant variants of UMAMIT29.

[0344]

[0345]

[0346] 1 Mean ± SD

[0347] UT: UMAMIT; 4MTB: 4-methylthiobutyl glucosinolate; I3M: indole-3-ylmethyl glucosinolate; BGLS: benzyl glucosinolate

[0348] Among the eight mutant variants, UMAMIT29#V27F, -M86V, -L109V, Q263S, and -T267Y showed the most significant reduction in activity, with the total glucosinolate input being approximately 75 - 97% lower than that of wild-type UMAMIT29 when expressed in oocytes (Table 6).

[0349] Although the total glucosinolate input activity of all these mutant variants was significantly reduced, UMAMIT29#M86V, -L109V, -Q263S, and -T267Y were located near the center of the cavity, while UMAMIT29#V27F was located more distally at the end of helix I in the computational protein model.

[0350] Compared with the wild type, the 4MTB and BGLS inputs of UMAMIT29#L197W were reduced, while the level of input I3M was not affected. The I3M and BGLS inputs of UMAMIT29#S289I were reduced, while the level of input 4MTB was similar to that of the wild type (Table 6). UMAMIT29#M201F (as the only mutant variant) showed no reduction in any glucosinolate, but an increase in 4MTB (Table 6).

[0351] Since we only replaced one amino acid residue in UMAMIT29 with the corresponding amino acid residue in the homolog UMAMIT32, we expected the UMAMIT29 mutant variants to be functionally expressed. The observations supported this, that is, compared with the wild type, the UMAMIT29#S289I variant showed a specific increase in 4MTB input and a reduction of more than 40% in I3M and BGLS inputs; the 4MTB input of the UMAMIT29#L197W variant was reduced, but the I3M and BGLS levels were similar, while the I3M increased. The altered glucosinolate substrate specificities of these UMAMIT29 mutant variants indicate that these residues are crucial for glucosinolate binding.

[0352] Item

[0353] 1. A Brassicaceae plant or a part thereof, wherein the Brassicaceae plant carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith, whereby the Brassicaceae plant or a part thereof expresses at least one mutant UMAMIT transporter, wherein the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level or not expressed.

[0354] 2. The Brassicaceae plant or a part thereof according to item 1, wherein the mutant UMAMIT transporter is a mutant glucosinolate transporter having reduced glucosinolate transporter activity compared to the wild-type protein, and wherein the reduction is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as 100% reduction in glucosinolate transporter activity of the mutant glucosinolate transporter compared to the wild-type protein.

[0355] 3. The Brassicaceae plant or a part thereof according to any one of the preceding items, wherein the mutant gene has reduced expression encoding a glucosinolate transporter compared to the wild-type gene, and wherein the reduced expression is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% reduction in expression of the mutant gene compared to the wild-type gene.

[0356] 4. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein when cultivated and prepared under the same conditions, the seeds of the Brassicaceae plant or the part thereof have a reduced glucosinolate content compared to Brassicaceae plants or parts thereof that are identical in other genotypic aspects but do not contain a mutation in any of said genes, and wherein the reduction in glucosinolate content is at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% or such as at least 95%.

[0357] 5. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein when cultivated and prepared under the same conditions, the vegetative tissue of the Brassicaceae plant or the part thereof has a substantially identical glucosinolate content compared to Brassicaceae plants or parts thereof that are identical in other genotypic aspects but do not contain a mutation in any of said genes.

[0358] 6. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the mutation is selected from the group consisting of: missense mutations, frameshift mutations, insertions and deletions.

[0359] 7. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the plant belongs to the genus Brassica or Lepidium, such as wherein the plant is a mustard, rapeseed (Brassica napus), Ethiopian mustard, B. oleracea, turnip or L. campestre plant.

[0360] 8. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the part is a seed.

[0361] 9. According to claim 0, wherein the glucosinolate concentration of the seeds is less than 18 micromoles per gram of dry weight of the seeds, such as less than 17 micromoles per gram of dry weight of the seeds, such as less than 16 micromoles per gram of dry weight of the seeds, such as less than 15 micromoles per gram of dry weight of the seeds, such as less than 14 micromoles per gram of dry weight of the seeds, such as less than 13 micromoles per gram of dry weight of the seeds, such as less than 12 micromoles per gram of dry weight of the seeds, such as less than 11 micromoles per gram of dry weight of the seeds, such as less than 10 micromoles per gram of dry weight of the seeds, such as less than 9 micromoles per gram of dry weight of the seeds, such as less than 8 micromoles per gram of dry weight of the seeds, such as less than 7 micromoles per gram of dry weight of the seeds, such as less than 6 micromoles per gram of dry weight of the seeds, such as less than 5 micromoles per gram of dry weight of the seeds, such as less than 4 micromoles per gram of dry weight of the seeds, such as less than 3 micromoles per gram of dry weight of the seeds, such as less than 2 micromoles per gram of dry weight of the seeds, such as less than 1 micromole per gram of dry weight of the seeds.

[0362] 10. A plant product, which comprises a plant of the order Brassicales or a part thereof, or is prepared from the seeds of the plant of the order Brassicales or a part thereof, wherein the plant of the order Brassicales carries a mutation in at least one gene encoding a UMAMIT transporter, and the UMAMIT transporter is selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith, whereby the plant of the order Brassicales or a part thereof expresses at least one mutant UMAMIT transporter, and the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level or is not expressed.

[0363] 11. According to claim 0, wherein the plant product is prepared from seeds, for example, wherein the plant product is oil, seed cake or seed meal.

[0364] 12. A seed cake prepared from the seeds of a plant of the order Brassicales, wherein the plant of the order Brassicales is as defined in claim 1 to 0.

[0365] 13. A method for altering the glucosinolate content in a Brassicaceae plant or a part thereof, the method comprising the step of altering the functional activity or expression of at least one UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.

[0366] 14. The method according to item 13, wherein the step of altering the functional activity of the at least one UMAMIT transporter is effected by: nuclease-based gene editing, such as by CRISPR / Cas9 gene editing, by random mutagenesis, by gene targeting, by transposon mutagenesis, by transfer DNA-induced insertion, or by gene knockdown, such as by RNA interference.

[0367] 15. A method for producing a plant product comprising a Brassicaceae plant or a part thereof having a low glucosinolate content, the method comprising the steps of:

[0368] a. providing a Brassicaceae plant or a part thereof according to any one of items 1 to 9; and

[0369] b. processing the Brassicaceae plant or a part thereof into a plant product, such as seed oil or seed cake.

[0370] Sequence summary

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[0427] References

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Claims

1. A plant of the order Brassicales or a part thereof, wherein the Brassicales plant is an oilseed and / or protein crop, and wherein the plant carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO: 1, AtUMAMIT29 shown in SEQ ID NO: 2, AtUMAMIT30 shown in SEQ ID NO: 3, AtUMAMIT31 shown in SEQ ID NO: 4, BnaA09G0714200ZS shown in SEQ ID NO: 5, BnaC05G0010000ZS shown in SEQ ID NO: 6, BnaA01G0222900ZS shown in SEQ ID NO: 7, BnaC01G0283800ZS shown in SEQ ID NO: 8, BnaC03G0332100ZS shown in SEQ ID NO: 9, BnaA09G0692700ZS shown in SEQ ID NO: 10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby the Brassicales plant or part thereof expresses at least one mutant UMAMIT transporter, wherein the mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level or not expressed.

2. The Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the Brassicaceae plant carries mutations in at least two genes encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO: 1, AtUMAMIT29 shown in SEQ ID NO: 2, AtUMAMIT30 shown in SEQ ID NO: 3, AtUMAMIT31 shown in SEQ ID NO: 4, BnaA09G0714200ZS shown in SEQ ID NO: 5, BnaC05G0010000ZS shown in SEQ ID NO: 6, BnaA01G0222900ZS shown in SEQ ID NO: 7, BnaC01G0283800ZS shown in SEQ ID NO: 8, BnaC03G0332100ZS shown in SEQ ID NO: 9, BnaA09G0692700ZS shown in SEQ ID NO: 10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.

3. The Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the Brassicaceae plant carries mutations in at least three genes encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO: 1, AtUMAMIT29 shown in SEQ ID NO: 2, AtUMAMIT30 shown in SEQ ID NO: 3, AtUMAMIT31 shown in SEQ ID NO: 4, BnaA09G0714200ZS shown in SEQ ID NO: 5, BnaC05G0010000ZS shown in SEQ ID NO: 6, BnaA01G0222900ZS shown in SEQ ID NO: 7, BnaC01G0283800ZS shown in SEQ ID NO: 8, BnaC03G0332100ZS shown in SEQ ID NO: 9, BnaA09G0692700ZS shown in SEQ ID NO: 10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.

4. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein the Brassicaceae plant carries mutations in at least four genes encoding UMAMIT transporters selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.

5. The Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the UMAMIT transporter is selected from the group consisting of: BnaA09G0679100GG, BnaA09G0679000GG, BnaC05G0008000GG, BnaA09G0647600GG, BnaA09G0647700GG, BnaA10G0008000GG, BnaA09G0623500NO, BnaA09G0623600NO, BnaC05G0007600NO, Bnascaffold2891G0004400NO, BnaA09G0624000NO, BnaC08G0554500QU, BnaA09G0664600QU, BnaC08G0554400QU, BnaA10G0008300QU, Bnascaffold2503G0022000QU, BnaA09G0664700QU, BnaA09G0724700SL, BnaA09G0689800SL, BnaA09G0724800SL, BnaC05G0007600SL, BnaA09G0689900SL, Bnascaffold3068G0013800SL, BnaA09G0577200TA, BnaA09G0628700TA, BnaC05G0007500TA, BnaA09G0577100TA, BnaA10G0008300TA, BnaA09G0628800TA, BnaA09G0658500WE, BnaA09G0658600WE, BnaC05G0009100WE, BnaA10G0009300WE, BnaA09G0659000WE, BnaA09G0692800ZS, BnaA09G0692700ZS, BnaA09G0714200ZS, BnaA09G0714300ZS, BnaA10G0008500ZS, BnaC05G0010000ZS, BnaA09G0634900ZY, BnaA09G0666500ZY, Bnascaffold4696G0013500ZY, BnaC05G0007300ZY, BnaA09G0635000ZY, BnaA09G0666400ZY, BolC5g28871H, BolC8g52858H, BraA10g42362Z, BraA09g42228Z, and BraA09g42227Z.

6. The Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the UMAMIT transporter is selected from the group consisting of: BnaC02G0303900GG, BnaA02G0239900GG, BnaC02G0303800GG, BnaC02G0289800NO, BnaA02G0248400NO, BnaA02G0248300NO, BnaC02G0286300QU, BnaA02G0247800QU, BnaA02G0247700QU, BnaC02G0286400QU, BnaC02G0230500SL, BnaA02G0281700SL, BnaC02G0230600SL, BnaC02G0254900TA, BnaA02G0214900TA, BnaC02G0255000TA, BnaC02G0326100WE, BnaA02G0260700WE, BnaA02G0260600WE, BnaC02G0326200WE, BnaC02G0337200ZS, BnaA02G0251300ZS, BnaC02G0337100ZS, BnaA02G0251200ZS, BnaA02G0278400ZY, BnaA02G0278500ZY, BnaC02G0162700ZY, BnaC02G0162600ZY, BolC2g09864H, and BraA02g07429Z.

7. The Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the UMAMIT transporter is selected from the group consisting of: BnaC03G0326100GG, BnaA03G0226900GG, BnaA01G0215700GG, BnaC01G0262700GG, BnaA03G0226800GG, BnaC01G0263000GG, BnaC03G0325800GG, BnaC03G0326000GG, BnaC02G0304200GG, BnaA02G0240100GG, BnaC03G0251900NO, BnaA03G0272800NO, BnaA01G0189100NO, BnaA02G0248600NO, BnaC03G0251700NO, BnaC02G0290200NO, BnaA03G0272700NO, BnaC05G0297600NO, BnaC05G0297300NO, BnaC03G0252000NO, BnaC03G0317800QU, BnaC03G0317900QU, BnaC03G0317600QU, BnaA03G0263200QU, BnaC01G0188500QU, BnaA03G0262800QU, BnaA01G0168400QU, BnaC01G0188900QU, BnaC02G0286800QU, BnaA03G0263100QU, BnaA02G0248000QU, BnaC03G0209800SL, BnaA01G0193300SL, BnaA03G0197300SL, BnaC02G0231000SL, BnaA02G0281500SL, BnaC01G0253100SL, BnaC03G0210100SL, BnaA03G0269600TA, BnaA03G0269800TA, BnaA02G0215100TA, BnaA03G0269900TA, BnaC02G0255300TA, BnaC01G0212600TA, BnaC01G0213100TA, BnaC03G0292000TA, BnaC03G0291900TA, BnaA01G0144700TA, BnaC03G0291700TA, BnaC03G0265500WE, BnaC01G0224300WE, BnaA03G0285900WE, BnaA02G0260400WE, BnaC02G0326500WE, BnaC03G0265600WE,BnaA01G0142400WE, BnaC03G0265300WE, BnaA03G0286100WE, BnaC01G0224600WE, BnaA03G0286000WE, BnaA01G0222900ZS, BnaC03G0331600ZS, BnaC03G0332100ZS, BnaA02G0251500ZS, BnaC03G0332000ZS, BnaC02G0337600ZS, BnaA03G0275500ZS, BnaA03G0275200ZS, BnaC01G0283800ZS, BnaC01G0283500ZS, BnaA03G0275300ZS, BnaC02G0163100ZY, BnaA03G0295200ZY, BnaC03G0268300ZY, BnaC03G0268600ZY, BnaC03G0268500ZY, BnaC01G0208900ZY, BnaA03G0295000ZY, BnaA03G0295100ZY, BnaC01G0209300ZY, BnaA01G0220000ZY, BnaA02G0278700ZY, BolC3g16132H, BolC2g09869H, BolC3g16128H, BolC3g16133H, BolC1g03106H, BolC1g03101H, BraA03g12335Z, BraA03g12337Z, BraA02g07432Z and BraA01g02441Z., 8. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein the UMAMIT transporter is selected from the group consisting of: BnaA02G0240000GG, BnaC02G0304100GG, Bnascaffold1465G0001500GG, BnaA09G0014000GG, BnaC02G0290100NO, BnaA09G0016000NO, BnaA02G0248500NO, BnaC09G0009500QU, BnaA02G0247900QU, BnaA09G0011000QU, BnaC02G0286700QU, BnaA09G0009300SL, Bnascaffold966G0010300SL, BnaA02G0281600SL, BnaC02G0230900SL, BnaA02G0215000TA, BnaC09G0008700TA, BnaA09G0005700TA, BnaC02G0255200TA, BnaC02G0326400WE, BnaC09G0008200WE, BnaA02G0260500WE, BnaA09G0011300WE, BnaC09G0002100ZS, BnaC02G0337500ZS, Bnascaffold0025G0022100ZS, BnaA02G0251400ZS, BnaA09G0019300ZS, Bnascaffold0025G0022000ZS, BnaC09G0001300ZY, BnaC02G0163000ZY, BnaA02G0278600ZY, BnaA09G0018200ZY, BolC9g53044H, BolC2g09868H, BraA02g07431Z, and BraA09g35773Z.

9. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein the mutant UMAMIT transporter is a mutant glucosinolate transporter having reduced glucosinolate transporter activity compared to the wild-type protein.

10. The Brassicaceae plant or part thereof according to claim 9, wherein the reduction is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as 100% reduction in glucosinolate transporter activity of the mutant glucosinolate transporter compared to the wild-type protein.

11. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein the mutant gene has reduced expression of the encoded glucosinolate transporter compared to the wild-type gene.

12. The Brassicaceae plant or part thereof according to claim 11, wherein the reduced expression is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% reduction in the expression of the mutant gene compared to the wild-type gene.

13. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein when cultivated and prepared under the same conditions, the seeds of the Brassicaceae plant or part thereof have a reduced glucosinolate content compared to Brassicaceae plants or parts thereof that are identical in other genotypic aspects but do not contain a mutation in any of the said genes.

14. The Brassicaceae plant or part thereof according to claim 13, wherein the reduction in the glucosinolate content is at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% or such as at least 95%.

15. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein when cultivated and prepared under the same conditions, the vegetative tissue of the Brassicaceae plant or part thereof has a glucosinolate content that is substantially the same as that of Brassicaceae plants or parts thereof that are identical in other genotypic aspects but do not contain a mutation in any of the said genes.

16. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein the mutation is selected from the group consisting of: missense mutations, insertions or deletions.

17. The Brassicaceae plant or part thereof according to any one of the preceding claims, wherein the Brassicaceae plant or part thereof carries a mutation in the AtUMAMIT29 gene shown in SEQ ID NO:12 or a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, wherein the mutant gene encodes a mutant AtUMAMIT29 polypeptide, wherein the mutant AtUMAMIT29 is the AtUMAMIT29 shown in SEQ ID NO:2 or a functional homolog thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, except that the mutant polypeptide comprises one or more substitutions selected from the group consisting of: a. Valine corresponding to valine at position 27 in SEQ ID NO:2 is replaced by phenylalanine (V27F); b. Methionine corresponding to methionine at position 86 in SEQ ID NO:2 is replaced by valine (M86V); c. Leucine corresponding to leucine at position 109 in SEQ ID NO:2 is replaced by valine (L109V); d. Glutamine corresponding to glutamine at position 263 in SEQ ID NO:2 is replaced by serine (Q263S); e. The threonine corresponding to threonine at position 267 in SEQ ID NO:2 is replaced by tyrosine (T267Y); f. The arginine corresponding to arginine at position 44 in SEQ ID NO:2 is replaced by alanine (R44A); g. The tryptophan corresponding to tryptophan at position 200 in SEQ ID NO:2 is replaced by alanine (W200A); and h. The glutamine corresponding to glutamine at position 204 in SEQ ID NO:2 is replaced by alanine (Q204A).

18. A Brassicaceae plant or a part thereof according to any one of claims 1 to 16, wherein the mutation is a frameshift mutation.

19. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the mutation is located within the promoter region, coding region such as exon region, non-coding region such as intron region and / or termination sequence of the gene.

20. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the mutation is a loss-of-function mutation.

21. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the mutation is an insertion of a T-DNA sequence, such as pROK2 shown in SEQ ID NO:21 or Vector_pAC106 shown in SEQ ID NO:

22.

22. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the plant belongs to the family Brassicaceae.

23. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the plant belongs to a genus selected from the group consisting of Brassica, Camelina, Crambe, Eruca, Lepidium and Thlaspi.

24. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the plant is a B. juncea, B. napus (rapeseed), B. carinata, B. oleracea, B. rapa or L. campestre plant.

25. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the part is a seed.

26. A Brassicaceae plant or a part thereof according to claim 25, wherein when the plant is cultivated and prepared under the same conditions, the seeds have substantially the same size as the seeds of a Brassicaceae plant or a part thereof that is otherwise identical in genotype but does not contain a mutation in any of the transporters defined in claims 1-8.

27. A Brassicaceae plant or a part thereof according to any one of claims 25 to 26, wherein the glucosinolate concentration in the seeds is less than 18 micromoles per gram of dry weight of the seeds, for example less than 17 micromoles per gram of dry weight of the seeds, for example less than 16 micromoles per gram of dry weight of the seeds, for example less than 15 micromoles per gram of dry weight of the seeds, for example less than 14 micromoles per gram of dry weight of the seeds, for example less than 13 micromoles per gram of dry weight of the seeds, for example less than 12 micromoles per gram of dry weight of the seeds, for example less than 11 micromoles per gram of dry weight of the seeds, for example less than 10 micromoles per gram of dry weight of the seeds, for example less than 9 micromoles per gram of dry weight of the seeds, for example less than 8 micromoles per gram of dry weight of the seeds, for example less than 7 micromoles per gram of dry weight of the seeds, for example less than 6 micromoles per gram of dry weight of the seeds, for example less than 5 micromoles, for example less than 4 micromoles per gram of dry weight of the seeds, for example less than 3 micromoles per gram of dry weight of the seeds, for example less than 2 micromoles per gram of dry weight of the seeds, for example less than 1 micromole per gram of dry weight of the seeds.

28. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the plant is not obtained only by a method that is essentially biological.

29. A Brassicaceae plant or a part thereof according to any one of the preceding claims, wherein the plant is an oilseed crop of the Brassicaceae family and / or a protein crop of the Brassicaceae family.

30. A plant product comprising a plant of the order Brassicales or a part thereof, or prepared from the seeds of said plant of the order Brassicales or a part thereof, wherein said plant of the order Brassicales carries a mutation in at least one gene encoding a UMAMIT transporter selected from the group consisting of AtUMAMIT28 shown in SEQ ID NO:1, AtUMAMIT29 shown in SEQ ID NO:2, AtUMAMIT30 shown in SEQ ID NO:3, AtUMAMIT31 shown in SEQ ID NO:4, BnaA09G0714200ZS shown in SEQ ID NO:5, BnaC05G0010000ZS shown in SEQ ID NO:6, BnaA01G0222900ZS shown in SEQ ID NO:7, BnaC01G0283800ZS shown in SEQ ID NO:8, BnaC03G0332100ZS shown in SEQ ID NO:9, BnaA09G0692700ZS shown in SEQ ID NO:10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith, whereby said plant of the order Brassicales or a part thereof expresses at least one mutant UMAMIT transporter, wherein said mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at a low level or not expressed.

31. The plant product according to claim 30, wherein the plant of the order Brassicales or a part thereof is as defined in any one of claims 1 to 29.

32. The plant product according to any one of claims 30 to 31, wherein the plant product is an oil, such as seed oil.

33. The plant product according to any one of claims 30 to 31, wherein the plant product is a seed cake or a seed meal.

34. A plant product according to any one of claims 30 to 33, wherein the plant product is prepared from seeds, optionally wherein the glucosinolate concentration of the seeds is less than 18 micromoles per gram of dry weight of the seeds, such as less than 17 micromoles per gram of dry weight of the seeds, such as less than 16 micromoles per gram of dry weight of the seeds, such as less than 15 micromoles per gram of dry weight of the seeds, such as less than 14 micromoles per gram of dry weight of the seeds, such as less than 13 micromoles per gram of dry weight of the seeds, such as less than 12 micromoles per gram of dry weight of the seeds, such as less than 11 micromoles per gram of dry weight of the seeds, such as less than 10 micromoles per gram of dry weight of the seeds, such as less than 9 micromoles per gram of dry weight of the seeds, such as less than 8 micromoles per gram of dry weight of the seeds, such as less than 7 micromoles per gram of dry weight of the seeds, such as less than 6 micromoles per gram of dry weight of the seeds, such as less than 5 micromoles per gram of dry weight of the seeds, such as less than 4 micromoles per gram of dry weight of the seeds, such as less than 3 micromoles per gram of dry weight of the seeds, such as less than 2 micromoles per gram of dry weight of the seeds, such as less than 1 micromole per gram of dry weight of the seeds.

35. A plant product according to claim 34, wherein the plant product contains glucosinolates at a concentration of at most 30 micromoles per gram of dry weight of the plant seed product, such as at most 25 micromoles per gram of dry weight of the plant seed product, such as at most 20 micromoles per gram of dry weight of the plant seed product, such as at most 15 micromoles per gram of dry weight of the plant seed product, such as at most 14 micromoles per gram of dry weight of the plant seed product, such as at most 13 micromoles per gram of dry weight of the plant seed product, such as at most 12 micromoles per gram of dry weight of the plant seed product, such as at most 11 micromoles per gram of dry weight of the plant seed product, such as at most 10 micromoles per gram of dry weight of the plant seed product, such as at most 9 micromoles per gram of dry weight of the plant seed product, such as at most 8 micromoles per gram of dry weight of the plant seed product, such as at most 7 micromoles per gram of dry weight of the plant seed product, such as at most 6 micromoles per gram of dry weight of the plant seed product, such as at most 5 micromoles per gram of dry weight of the plant seed product, such as at most 5 micromoles per gram of dry weight of the plant seed product, such as at most 3 micromoles per gram of dry weight of the plant seed product, such as at most 2 micromoles per gram of dry weight of the plant seed product, or such as at most 1 micromole per gram of dry weight of the plant seed product.

36. A plant product according to claim 34, for use as animal feed.

37. A seed cake prepared from the seeds of a plant of the order Brassicales, wherein the plant of the order Brassicales is as defined in any one of claims 1 to 29.

38. An animal feed comprising the plant product according to claim 34 or the seed cake according to claim 37.

39. The seed cake according to claim 37, wherein when cultivated and prepared under the same conditions, the glucosinolate concentration in the seed cake prepared from the Brassicales plant is at most 15%, such as at most 10%, or such as at most 5% of the glucosinolate concentration in the seed cake prepared from the seeds of a plant that is otherwise identical to the Brassicales plant in other genotypic aspects but does not contain a mutation in any of said genes.

40. A method for altering the glucosinolate content in a Brassicales plant or a part thereof, the method comprising the step of altering the functional activity or expression of at least one UMAMIT transporter selected from the group consisting of: AtUMAMIT28 shown in SEQ ID NO: 1, AtUMAMIT29 shown in SEQ ID NO: 2, AtUMAMIT30 shown in SEQ ID NO: 3, AtUMAMIT31 shown in SEQ ID NO: 4, BnaA09G0714200ZS shown in SEQ ID NO: 5, BnaC05G0010000ZS shown in SEQ ID NO: 6, BnaA01G0222900ZS shown in SEQ ID NO: 7, BnaC01G0283800ZS shown in SEQ ID NO: 8, BnaC03G0332100ZS shown in SEQ ID NO: 9, BnaA09G0692700ZS shown in SEQ ID NO: 10, and their respective functional homologs having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.

41. The method according to claim 40, wherein the step of altering the functional activity of the at least one UMAMIT transporter is effected by: nuclease-based gene editing, such as by CRISPR / Cas9 gene editing, by random mutagenesis, by gene targeting, by transposon mutagenesis, by transfer DNA-induced insertion, or by gene knockdown, such as by RNA interference.

42. A method for producing a plant product comprising a Brassicales plant or a part thereof having a low glucosinolate content, the method comprising the following steps: a. providing a Brassicales plant or a part thereof according to any one of claims 1 to 29; and b. processing the Brassicales plant or a part thereof into a plant product, such as seed oil or seed cake.

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