Legumes or parts thereof with reduced quinolizidine alkaloid content

Abstract

The present invention relates to legumes, preferably non-GMO plants, or parts thereof with decreased or low amounts of quinolizidine alkaloids (QAs) in the seeds. In particular, the invention relates to lupin plants having reduced QA transporter activity, methods for obtaining such and for preparing plant products therefrom.

Description

[0001] P7181 PC00

[0002] Legumes or parts thereof with reduced quinolizidine alkaloid content

[0003] Technical field

[0004] The present invention relates to legumes, preferably non-GMO plants, or parts thereof with decreased or low amounts of quinolizidine alkaloids (QAs) in the seeds. In particular, the invention relates to lupin plants having reduced QA transporter activity, methods for obtaining such and for preparing plant products therefrom.

[0005] Background

[0006] The global demand for plant-based protein sources is increasing, and likewise is the demand for methods for sustainable production thereof.

[0007] Lupins (Lupinus spp.) are legumes with large potential as sustainable protein sources, with a protein content in the seeds of up to 44%. Lupin cultivation has recently gained significant attention because lupins are more tolerant to several abiotic stresses than other legumes and have great potential for recovering poor soils. A limitation that hinders the widespread cultivation of lupins is their natural accumulation of bitter and toxic alkaloids of the quinolizidine type (QAs). Low-QA varieties (“sweet” varieties) exist, but their residual alkaloid content is still problematic and as QAs are likely important for plant fitness, their complete abolishment from the plant is not a desirable strategy for crop improvement. The most likely function of QAs is to defend the plant against herbivores, including aphids and snails. Accordingly, sweet varieties have been observed to sustain larger herbivore damage than high-QA (“bitter”) varieties.

[0008] Summary

[0009] Most if not all of the QAs in seeds are synthesized elsewhere in the plant and later transported to the seeds (Otterbach et al., 2019). The invention shows that seed QA levels can be controlled by modulating their transport. Thus, the invention targets the QA transport processes to reduce the accumulation of QAs in the seeds. So far, the molecular basis of QA transport from biosynthetic tissues to the seeds has remained uncharacterised.

[0010] Narrow-leafed lupin (NLL, Lupinus angustifolius) is a cultivated lupin species grown in Europe and Australia for its protein-rich seeds. Hitherto, use of seeds from high-QA (“bitter”) varieties of narrow-leafed lupins, also known as bitter NLLs or bitter lupins, P7181 PC00 have been hampered due to the high amounts of QAs in the green parts of the plant as well as the seeds. Thus, the use of bitter lupins as food requires extensive preprocessing (debittering), as done for example for lupini bean snacks made of bitter white lupin (L. al bus).

[0011] The present invention provides transporter-engineered variants of bitter NLL with seeds possessing a significantly reduced content of several QAs, and having pods with increased levels. This phenotype is desirable, because the low level of QAs in the seeds make debittering easier, if required, and / or render the seeds more eatable, whereas the high levels of QAs in the remainder of the plant render the plant more resistant to insects and other herbivores.

[0012] In particular, among a large pool of candidate genes, the inventors have identified a total of 14 QA transporters, including the purine uptake permeases (PLIPs) LLIP3.1, LUP3.4, LUP3.5, LUP1.3, LUP1.4 and the WALLS ARE THIN-1 (WAT1) protein HH130. Interestingly, the inventors found that single knockouts of QA transporters lead to somewhat reduced QA levels in the seeds of bitter NLL, whereas deletion of both LUP3.1 and LUP3.5 results in a phenotype with significant reduction of the levels of QAs in the seeds and increased levels in the pods, of bitter NLL.

[0013] Thus, the present disclosure provides a solution to maintaining the level of protection of the plant against herbivores conferred by the high QA levels, but at the same time rendering the seeds more eatable, by redistributing seed QAs in the plant.

[0014] In a main aspect, the present disclosure relates to a quinolizidine alkaloid (QA)- containing legume, preferably a lupin plant, or part thereof, wherein said QA-containing legume carries a mutation in at least one gene encoding a QA transport protein.

[0015] Preferably, said QA-containing legume has reduced content of one or more QAs in the seeds, compared to a corresponding QA-containing legume not comprising said mutation.

[0016] In another main aspect, provided is QA-containing legume, preferably a lupin plant, or part thereof, wherein said QA-containing legume carries one or more of: i. a mutation in the LUP3. 1 gene leading to reduced LUP3.1 activity or loss-of- function of LUP3.1; P7181 PC00 ii. a mutation in the LUP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5; and / or iii. a mutation in the HH130 gene.

[0017] Another aspect of the present disclosure relates to a method of reducing or decreasing the QA content in a QA-containing legume or part thereof, comprising modifying the activity of at least one QA transport protein in cells of said QA-containing legume. For example, said QA transport protein may be LLIP3.1 (SEQ ID NO: 1), LLIP3.4 (SEQ ID NO: 23), LUP3.5 (SEQ ID NO: 3), HH130 (SEQ ID NO: 5), LUP1.3 (SEQ ID NO: 7), or LLIP1.4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0018] The QA-containing legume may be a lupin plant, for example of the species narrow- leafed lupin (NLL, Lupinus angustifolius). As described herein above, lupins are known for their high nutritional value, including high protein content of the seeds, and use in food products.

[0019] The present disclosure also relates to a plant product comprising a QA-containing legume or part thereof, described herein. Said plant product may for example be: i. an extract prepared from seeds of said QA-containing legume; ii. flour prepared from the seeds of said QA-containing legume and / or from the extract; iii. kernel flour prepared from the endosperm of the seeds of said QA-containing said legume; iv. hulls prepared from the seeds of said QA-containing legume or part thereof; v. meal prepared from the seeds of said QA-containing legume or part thereof; vi. plant flakes prepared from the seeds of said QA-containing legume; or vii. dry seeds prepared from the seeds of said QA-containing legume.

[0020] Also disclosed herein is a method of producing a plant product, said method comprising the steps of: i. providing seeds of a QA-containing legume as described herein; and ii. processing said seeds into a plant product, wherein the processing preferably comprises or consists of drying said seeds into dry seeds. P7181 PC00

[0021] In other aspects, provided is a plant cell of the QA-containing legume or part thereof described herein, seeds from the QA-containing legume as described herein, and / or seed flour prepared from said seeds.

[0022] Disclosed is also use of a QA-containing legume or part thereof described herein, in a food product, and / or as animal feed.

[0023] Description of Drawings

[0024] Figure 1. QA uptake into Xenopus oocytes mediated by LUP transporters from NLL. Oocytes expressing individual LLIPs were incubated for 1 h in lupin plant extracts and their internal QA contents were analysed by LC-MS. Oocytes injected with water were used as negative controls. Bars represent mean normalized values of three replicate measurements, and error bars represent standard deviations. A sample of the plant extract was also analysed at a dilution that would allow direct comparison to the internal oocyte QA measurements assuming an internal volume of 1 pL per oocyte.

[0025] Figure 2. Hierarchical clustering (HCL) analysis of the 11 NLL LUPs based on their expression patterns in different tissues of biosynthetic organs (seed, pod, and stem). The expression patterns are shown as a heatmap, with the color scale representing transcripts per million (TPM). The dotted box represents selection of LUP3.1 , LUP3.5, and LUP2.1 as possibly involved in the loading of QAs into the pod vasculature for long-distance transport to the seeds.

[0026] Figure 3. Subcellular localisation of selected LUP transporters fused to fluorescent proteins and expressed in Nicotiana benthamiana. Analogous images of a plasma membrane marker (AtPIP2A-CFP) and a tonoplast marker (g-TIP-mCherry) are shown for comparison. For each protein fusion, an image capturing the fluorescence of the fusion partner and the autofluorescence of chlorophyll (left) is contrasted with an image capturing only the fluorescence of the fusion partner (right). In the case of the tonoplast marker, tonoplastic invaginations are visible at positions corresponding to chloroplasts (white arrows).

[0027] Figure 4. Electrophysiological studies with the three selected LUPs. A) Currents recorded by the two-electrode voltage-clamp technique applied to Xenopus oocytes expressing the LUP transporters and exposed to 3 mM lupanine tartrate at pH 5.5 at a P7181 PC00 holding potential of -60 mV. Arrows above the curves indicate the experimental timeline: 1 , perfusion with eKulori buffer; 2, perfusion with lupanine tartrate in eKulori buffer; and 3, perfusion with eKulori to return to baseline. B) Determination of substrate affinity ( M) via saturation curves fitted to the Michaelis-Menten equation at a holding potential of -60 mV. Substrate affinities were also calculated at four other holding potentials, showing only small differences in KM values (Figure 8).

[0028] Figure 5. QA chemotypes of the individual LLIP3.1 (panel A) and LLIP3.5 (panel B) knockouts in their homozygous state (horn, light grey bars) in comparison to wildtype siblings (WT, dark grey bars). Each panels shows the QA chemotypes of pods at 30 days after anthesis (30 DAA), seeds at 30 DAA, and mature seeds, respectively. Bars represent mean, normalized QA amounts (peak areas normalized by internal standard), and error bars represent standard deviations of three biological replicates (n=3) as analysed by LC-MS. Asterisks indicate statistically significant differences on a Student’s t-test (*p< 0.05; **p < 0.01).

[0029] Figure 6. QA chemotypes of the LUP3.1 / LUP3.5 double knockout in the fully homozygous state (horn, light grey bars) in comparison to a control [wildtype for LLIP3.1 and heterozygous for LLIP3.5 (WT / Het), dark grey bars]. The different graphs show the QA chemotypes of leaves at 30 days after anthesis (30 DAA), pods at 30 DAA, and mature seeds. Bars represent mean, normalized QA amounts (peak areas normalized by internal standard), and error bars represent standard deviations of 3 biological replicates as analysed by LC-MS. Asterisks indicate statistically significant differences on a Student’s t-test (*p< 0.05; **p < 0.01).

[0030] Figure 7. QA chemotypes of the single (LLIP3.1 and LLIP3.5) and double (LUP3.1xLUP3.5) knockout mutants in comparison to a wildtype control (WT). The different panels show the QA chemotypes in leaves at 30 days after anthesis (DAA) (panel A), pods at 30 DAA (panel B), and mature seeds (panel C). For each panel, the three graphs show the contents of lupanine, angustifoline, and 13-hydroxylupanine. Bars represent mean, normalized QA amounts (peak areas normalized by internal standard) as analysed by LC-MS. Error bars represent standard deviations of either 4-8 replicates (panels A and B) or 4-5 replicates (panel C). Asterisks indicate statistically significant differences on a Student’s t-test (*p< 0.05; **p < 0.01). P7181 PC00

[0031] Figure 8. Voltage dependency of substrate affinity ( M) for LLIP3.1, LLIP3.5 and LLIP2.1 measured against lupanine. Each data point is the result of fitting a saturation curve on the Michaelis-Menten equation at the given holding potential. For each saturation curve, the tested concentrations of lupanine tartrate were the same as described in Figure 4B.

[0032] Figure 9. Heat map illustrating the LCM- and RNASeq-based expression patterns of 16 candidate QA exporter genes (HH001-HH016) and two QA biosynthetic genes (LDC and CAO). HH001 L (renamed herein as LLIP1.3) and HH001S represent the same candidate (HH001) but with an alternative start codon. Rows represent candidate genes, and columns represent NLL tissues. The color key indicates the expression level in transcripts per million (TPM), with white indicating the lowest and dark indicates the highest expression.

[0033] Figure 10. Schematic workflow of the diffusion-based export assay used for the identification of QA exporters.

[0034] Figure 11. Identification of a QA exporter (HH001L, renamed herein as LLIP1.3) using a diffusion-based export screen. Panels A and B show the screening of 16 candidates in 4 pools, while panels C and D show the testing of individual candidates from pool 2.9. Panels A and C show the content of lupanine and angustifoline in Xenopus oocytes after the QA-loading step (O-#1 , Oocytes #1 in Fig. 10) in comparison to the same content after the subsequent incubation in clean buffer (O-#2, Oocytes #2 in Fig. 10). Bars show the mean normalized peak areas relative to the internal standard (caffeine), with error bars representing the standard deviation of four replicates. Asterisks show significant differences (*P< 0.05, **P< 0.01). Panels B and D show the contents of lupanine and angustifoline in the clean buffer after incubation with QA- loaded oocytes (Medium #2 in Fig. 10). Bars show the normalized peak area relative to the internal standard (caffeine; single replicate). A replicate of the export assay with HH001L (renamed herein as LLIP1.3) including four replicates for the QA measurements on the clean buffer is shown in Figure 14.

[0035] Figure 12. Diffusion-based QA export assays for selected PLIPs from NLL. Panel A shows the content of lupanine and angustifoline in cRNA-injected Xenopus oocytes after the QA-loading step (O-#1 , Oocytes #1) in comparison to the same content after P7181 PC00 the subsequent incubation in clean buffer (O-#2, Oocytes #2). Panel B shows the content of lupanine and angustifoline in the clean buffer after incubation with QA- loaded oocytes (Media #2). For both panels, bars show mean normalized peak areas relative to the internal standard (caffeine), with error bars representing the standard deviation of 4 replicates. Asterisks show significant differences (*P< 0.05, **P< 0.01).

[0036] Figure 13. Tissue-specific expression of the QA exporters LLIP1.3 and LLIP1.4 in comparison to the biosynthesis genes LDC and CAO. Rows represent genes, and columns represent NLL tissues. The color key indicates the expression level in transcripts per million (TPM), with white indicating the lowest and dark indicates the highest expression.

[0037] Figure 14. Replicate of the QA export assay with HH001L (renamed herein as LLIP1.3) shown in Figures 11C and 11D. Panel A shows the content of lupanine and angustifoline in Xenopus oocytes after the QA-loading step (O-#1, Oocytes #1 in Fig. 10) in comparison to the same content after the subsequent incubation in clean buffer (O-#2, Oocytes #2 in Fig. 10). Panel B shows the contents of lupanine and angustifoline in the clean buffer after incubation with QA-loaded oocytes (Medium #2 in Fig. 10). In both panels, bars show mean normalized peak areas relative to the internal standard (caffeine), with error bars representing the standard deviation from 4 replicates. Asterisks show significant differences (*P< 0.05, **P< 0.01).

[0038] Figure 15. Schematic diagram showing a generic set of membrane transporters (question marks) involved in transferring phloem-imported nutrients from seed coats to embryos (e.g. cotyledons) in developing grain legume seeds. Adapted from Zhang et al., 2007.

[0039] Figure 16. Seed chemotype of the LLIP3.4 Q171 stop knockout mutant. The graphs show a comparison of seed QA levels between the homozygous mutant (Hom, dark grey bars) and a wildtype sibling (WT, light grey bars) in either embryo (left graph) or seed coat tissues (right graph). Bars represent mean normalized peak areas derived from LC-MS analysis. Error bars represent the standard deviation of 3 replicates. Asterisks indicate significant differences (*p<0.05, **p<0.01). P7181 PC00

[0040] Figure 17. Seed chemotype of the LLIP3.4 W330stop knockout mutant. The graphs show a comparison of seed QA levels between the homozygous mutant (Hom, dark grey bars) and a wildtype sibling (WT, light grey bars) in either embryo (left graph) or seed coat tissues (right graph). Bars represent mean normalized peak areas derived from LC-MS analysis. Error bars represent the standard deviation of 3 replicates. Asterisks indicate significant differences (*p<0.05, **p<0.01).

[0041] Figure 18. Identification of a novel QA importer of the IIMAMIT family (HH130 (Lup017053.1)) upon screening of 180 candidate seed transporters. Panel A shows the identification of Pool 4.3 as a cRNA pool able to confer QA uptake ability in Xenopus oocytes. Panel B shows the testing of the five individual cRNAs from Pool 4.3 in the same system. Graphs show the content of individual QAs in oocytes expressing candidate transporters after a 1-hour incubation in lupin plant extract. For comparison, the QA content in water-injected oocytes (mock) is shown. Bars represent the mean normalized LC-MS peak areas relative to the internal standard (caffeine), with error bars representing the standard deviation of 3-4 replicates. Asterisks show significant differences (*P< 0.05, **P< 0.01).

[0042] Figure 19. Tissue-specific expression pattern of HH130 (Lup017053.1) in comparison to the embryo specific QA importer LLIP3.4, the two phloem loaders LLIP3.1 and LLIP3.5, and the two QA exporters LLIP1.3 and LLIP1.4, including expression in embryo and seed coat. The heat map indicates gene expression levels in different NNL tissues in transcripts per million (TPM), with white indicating the lowest and dark indicating the highest expression. The boxes highlight similarities in expression indicating possible redundancy.

[0043] Detailed description

[0044] Definitions

[0045] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.

[0046] The terms “some embodiments” and “other embodiments” can include one, or more than one embodiment. P7181 PC00

[0047] The term "transport protein" as used herein refers to a protein or a polypeptide, which is capable of assisting and / or facilitating movement of one or more compounds between at least two different tissues of a QA-containing legume. The transported compound(s) may be ion(s), small molecule(s), and / or macromolecule(s), however, in relation to the present disclosure the transported compound(s) are preferably QA(s). The transport is typically assisted across a cell membrane. Transport proteins typically assist and / or facilitate movement of one or more compounds by facilitated diffusion, active transport, osmosis, or reverse diffusion. The location of a transport protein may dictate what compounds, it assists and / or facilitates transport of simply due to the availability of said compounds and / or the substrate affinity of said transport protein. The transport protein may either be unidirectional or bidirectional with respect to direction of the transport across the cell membrane of the same or different tissues. For bidirectional transporters, the physiological direction of transport may be dictated by the electrochemical gradient of the transported compound, such as the transported QA. A transport protein comprises at least one domain embedded in a physiological membrane, such as at least one transmembrane domain or part of the protein. Further, unless context dictates otherwise, as used herein " transport protein " includes protein fragments that retain the relevant transport activity, and may include artificial proteins synthesized to retain the relevant transport activity. Transport protein may also be referred to herein as transporting protein, transporter, permease, channel, carrier protein, or membrane transporter, and the terms are used interchangeably.

[0048] The term "polypeptide" as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length. As is known to those skilled in the art, polypeptides may be processed and / or modified. The terms polypeptide and protein are used interchangeably herein.

[0049] The term "functional homologue" of an amino acid sequence, refers to a polypeptide comprising said amino acid sequence with the proviso that one or more amino acids are substituted, deleted, added, and / or inserted, and which polypeptide has (qualitatively) the same functionality. Preferably, a functional homologue shares at least at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said amino acid sequence. A functional homologue of a polypeptide preferably has essentially the same P7181 PC00 or similar functionality as the polypeptide it is a functional homologue of. A functional homologue of a mutant polypeptide with loss-of-function similarly also has the same loss-of-function.

[0050] The term "mutant protein" means a protein with one or more mutations compared to a protein occurring in nature. In some embodiments, the mutant protein has a sequence that differs from that of all proteins occurring in nature. In preferred embodiments, the mutant protein is a protein fragment that contains at the most about any of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 contiguous amino acids from a full-length protein.

[0051] The term "functional homologue" of a nucleic acid sequence, refers to a polynucleotide comprising said nucleic acid sequence with the proviso that one or more nucleotides are substituted, deleted, added, and / or inserted, and which polynucleotide encodes the same polypeptide. Preferably, a functional homologue shares at least at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said nucleic acid sequence.

[0052] The terms “mutant gene” or “mutated gene” as used herein refers to a gene carrying at least one mutation such that the mutant gene is incapable of directing the efficient expression of a full-length, fully functional gene product. An endogenous gene can be mutated in the sense of the present invention when the endogenous gene comprises one or more mutations, such as: (a) a "missense mutation", which is a change in the nucleic acid sequence that results in the substitution of an amino acid for another amino acid; (b) a "nonsense mutation" or "STOP mutation", which is a change in the nucleic acid sequence that results in the introduction of a premature STOP codon and, thus, premature termination of translation (resulting in a truncated protein); plant genes contain the translation stop codons "TGA" (UGA in RNA), "TAA" (UAA in RNA) and "TAG" (UAG in RNA); thus any nucleotide substitution, insertion, deletion which results in one of these codons in the mature mRNA in the reading frame will terminate translation, (c) an "insertion mutation" of one or more nucleotides, due to one or more nucleotides having been added in the coding sequence of the nucleic acid; (d) a "deletion mutation" of one or more nucleotides, due to one or more nucleotides having been deleted in the coding sequence of the nucleic acid; (e) a "frameshift mutation", P7181 PC00 resulting in the nucleic acid sequence being translated in a different frame downstream of the mutation. A frameshift mutation can have various causes, such as the insertion, deletion or duplication of one or more nucleotides. As already mentioned, it is desired that the mutation(s) in the endogenous gene preferably result in a mutant protein comprising significantly reduced or no biological activity in vivo or in the production of no protein. Any mutation which results in a protein comprising at least one amino acid insertion, deletion and / or substitution relative to the wildtype protein can in principle lead to significantly reduced or no biological activity. It is, however, understood that mutations in certain parts of the protein are more likely to result in a reduced function, such as activity, of the mutant LLIP3.1 , LLIP3.5, HH130, LLIP1.3, or LLIP1.4 protein, such as mutations leading to truncated proteins, whereby significant portions of the functional domains are lacking.

[0053] The term “sequence identity” as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wildtype sequence) based on their pairwise alignment. For 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. Mo / . Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0054] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0055] The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position in a reference sequence.

[0056] For purposes of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS P7181 PC00 package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0057] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment).

[0058] The term “legume” herein refers to a plant of the botanical family Fabaceae or Leguminosae. Many legumes are grown as crops for harvest and consumption of their edible seeds. Legume crops may be referred to as pulses, if grown and harvested for their dry seeds. Lupins, including both sweet and bitter lupins, are legumes.

[0059] According to the definition of legume, a “QA-containing legume” herein refers to a legume that produces and contains one or more quinolizidine alkaloids (QAs). The QA(s) may be stored in the seeds of said QA-containing legume, be stored in specific tissues, and / or distributed throughout the legume (plant). The content and distribution of QA(s) may vary throughout the lifespan of the legume. Different QA-containing legumes may produce and / or store different QAs. For example, the QA profile of a QA- containing legume typically vary between legumes of different clades, different species of the same family, and different varieties of the same species. Lupins, such as narrow- leafed lupin (NLL), are QA-containing legumes. Depending on the lupin species, the QA profile and content of different QAs may vary. For example, so-called sweet lupins are generally producing and containing less QAs than bitter lupins. Other legume species, than lupins, of the Genisteae (Genistoid) clade containing QAs are also considered QA-containing legumes according to the definition herein. For example, the golden chain tree (Laburnum anagyroides) accumulates the QA cytisine and is considered a QA-containing legume.

[0060] The term “mature seeds” refers to seeds of a QA-containing legume, such as lupin seeds, being ready for harvest. Typically, lupin seeds are considered to be mature when the seeds have a suitable dryness. Mature seeds typically contain less than 30% water, typically less than 20% of water, preferably in the range of 8 to 30% water. % provided as w / w of the total seed. Mature seeds, with respect to lupins, may also be P7181 PC00 characterised by coloration, which can be white, yellow, grey, or another color depending on the lupin species and / or variety, whereas immature seeds typically are green. Mature seeds may also be characterised by being harder and more form that immature seeds.

[0061] The terms “dry seeds” and “dried seeds” as used herein refers to seeds of a QA- containing legume, such as lupin seeds, having a moisture content of the most 20 %, preferably at the most 15 %, for example in the range of 10-12 %. Typically, mature seeds are harvested, following which the moisture content is reduced to prevent spoilage during long term storage. Other terms for dry seeds may be dry grains or pulses, the latter is typically used for dry grains for human consumption.

[0062] The term “vasculature” herein refers to conducting tissue of plants, including legumes. The different vascular tissues within a plant constitute the vascular tissue system of said plant. Two of the major components of the plant vasculature are the phloem and xylem, each transporting fluid(s) and nutrients within the plant. The vasculature may also herein be referred to as vascular tissue and the terms are used interchangeably.

[0063] The term “reduced activity” when used in relation to a mutated / truncated protein, such as a mutated / truncated QA-transport protein, may refer to a reduction in activity of said protein of at least 50% compared to a corresponding protein that is not mutated / truncated, preferably when cultivated, such as grown, in similar or identical conditions, such as at least 60%, such as at least 70% such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% reduction in activity compared to a corresponding protein that is not mutated / truncated, preferably when cultivated, such as grown, in similar or identical conditions. The term may in some embodiments refer to complete loss of activity of the mutated / truncated protein compared to a corresponding protein that is not mutated / truncated, preferably when cultivated, such as grown, in similar or identical conditions.

[0064] QA-containing legumes with transporter mutations

[0065] In a main aspect, the present disclosure concerns a QA-containing legume or part thereof, wherein said QA-containing legume carries a mutation in at least one gene encoding a QA transport protein, preferably wherein said QA-containing legume has reduced content of one or more QAs in the seeds, compared to a corresponding QA- P7181 PC00 containing legume not comprising said mutation. The QA content may be reduced in either or both of the seed coat and / or seed embryo of seeds of said QA-containing legume.

[0066] In preferred embodiments, said QA transport protein is LLIP3.1 , LLIP3.4, LLIP3.5, HH130, LLIP1.3, or LLIP1.4. Preferably, said mutation is one or more of: i. a mutation in the LUP3. 1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1; ii. a mutation in the LUP3.4 gene leading to reduced LLIP3.4 activity or loss-of- function of LLIP3.4; iii. a mutation in the LUP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5; iv. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; v. a mutation in the LUP1.3 gene leading to a reduced LLIP1.3 activity or loss of function of LLIP1.3; and / or vi. a mutation in the LUP1.4 gene leading to reduced LLIP1.4 activity or loss of function of LLIP1.4.

[0067] In some embodiments, said QA transport protein is LLIP3.1 , LLIP3.5, HH130, LLIP1.3, or LLIP1.4, and said mutation is one or more of: i. a mutation in the LUP3. 1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1; ii. a mutation in the LUP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5; and / or iii. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; or said mutation is one or more of: iv. a mutation in the LUP1.3 gene leading to a reduced LLIP1.3 activity or loss of function of LLIP1.3; and / or v. a mutation in the LUP1.4 gene leading to reduced LLIP1.4 activity or loss of function of LLIP1.4.

[0068] In another main aspect, the present disclosure regards a QA-containing legume or part thereof, wherein said QA-containing legume carries one or more of: P7181 PC00 i. a mutation in the LLIP1.1 gene leading to reduced LLIP1.1 activity or loss-of- function of LLIP1.1 , preferably wherein the LLIP1.1 gene is a gene encoding LLIP1.1 of SEQ ID NO: 13 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP1.1 gene comprises the coding sequence as set forth in SEQ ID NO: 14 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 14; ii. a mutation in the LLIP1.2 gene leading to reduced LLIP1.2 activity or loss-of- function of LLIP1.2, preferably wherein the LLIP1.2 gene is a gene encoding LLIP1.2 of SEQ ID NO: 15 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP1.2 gene comprises the coding sequence as set forth in SEQ ID NO: 16 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 16; iii. a mutation in the LLIP1 .3 gene leading to reduced LLIP1.3 activity or loss-of- function of LLIP1.3, preferably wherein the LLIP1.3 gene is a gene encoding LLIP1 .3 of SEQ ID NO: 7 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP1.3 gene comprises the coding sequence as set forth in SEQ ID NO: 8 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 8; iv. a mutation in the LLIP1 .4 gene leading to reduced LLIP1.4 activity or loss-of- function of LLIP1.4, preferably wherein the LLIP1.4 gene is a gene encoding LLIP1 .4 of SEQ ID NO: 9 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP1.4 gene comprises the coding sequence as set forth in SEQ ID NO: 10 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 10; v. a mutation in the LLIP2.1 gene leading to reduced LLIP2.1 activity or loss-of- function of LLIP2.1 , preferably wherein the LLIP2.1 gene is a gene encoding LLIP2.1 of SEQ ID NO: 21 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP2.1 gene comprises the coding sequence as set forth in SEQ ID NO: 22 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 22; P7181 PC00 vi. a mutation in the LLIP2.2 gene leading to reduced LLIP2.2 activity or loss-of- function of LLIP2.2, preferably wherein the LLIP2.2 gene is a gene encoding LLIP2.2 of SEQ ID NO: 27 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP2.2 gene comprises the coding sequence as set forth in SEQ ID NO: 28 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 28; vii. a mutation in the LLIP3.1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1 , preferably wherein the LLIP3.1 gene is a gene encoding LLIP3.1 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP3.1 gene comprises the coding sequence as set forth in SEQ ID NO: 2 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 2; viii. a mutation in the LLIP3.2 gene leading to reduced LLIP3.2 activity or loss-of- function of LLIP3.2, preferably wherein the LLIP3.2 gene is a gene encoding LLIP3.2 of SEQ ID NO: 11 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP3.2 gene comprises the coding sequence as set forth in SEQ ID NO: 12 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 12; ix. a mutation in the LLIP3.3 gene leading to reduced LLIP3.3 activity or loss-of- function of LLIP3.3, preferably wherein the LLIP3.3 gene is a gene encoding LLIP3.3 of SEQ ID NO: 25 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP3.3 gene comprises the coding sequence as set forth in SEQ ID NO: 26 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 26; x. a mutation in the LLIP3.4 gene leading to reduced LLIP3.4 activity or loss-of- function of LLIP3.4, preferably wherein the LLIP3.4 gene is a gene encoding LLIP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP3.4 gene comprises the coding sequence as set forth in SEQ ID NO: 24 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 24; P7181 PC00 xi. a mutation in the LLIP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5, preferably wherein the LLIP3.5 gene is a gene encoding LLIP3.5 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP3.5 gene comprises the coding sequence as set forth in SEQ ID NO: 4 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 4; xii. a mutation in the LLIP5.1 gene leading to reduced LLIP5.1 activity or loss-of- function of LLIP5.1 , preferably wherein the LLIP5.1 gene is a gene encoding LLIP5.1 of SEQ ID NO: 17 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP5.1 gene comprises the coding sequence as set forth in SEQ ID NO: 18 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 18; xiii. a mutation in the LLIP4.1 gene leading to reduced LLIP4.1 activity or loss-of- function of LLIP4.1 , preferably wherein the LLIP4.1 gene is a gene encoding LLIP4.1 of SEQ ID NO: 19 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the LLIP4.1 gene comprises the coding sequence as set forth in SEQ ID NO: 20 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 20; and / or xiv. a mutation in the HH130 gene leading to reduced HH130 activity or loss-of- function of HH130, preferably wherein the HH130 gene is a gene encoding HH130 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the HH130 gene comprises the coding sequence as set forth in SEQ ID NO: 6 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 6.

[0069] The coding sequence is the part of the genomic sequence encoding a polypeptide, and corresponds to the cDNA sequence.

[0070] In another main aspect, the present disclosure concerns a QA-containing legume or part thereof, wherein said QA-containing legume carries one or more of: P7181 PC00 i. a mutation in the LUP3. 1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1 ; ii. a mutation in the LUP3.4 gene leading to reduced LLIP3.4 activity or loss-of- function of LLIP3.4; iii. a mutation in the LUP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5; and / or iv. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; and / or wherein said QA-containing legume carries one or more of: v. a mutation in the LUP1.3 gene leading to a reduced LLIP1.3 activity or loss of function of LLIP1.3; and / or vi. a mutation in the LUP1.4 gene leading to reduced LLIP1.4 activity or loss of function of LLIP1.4.

[0071] In preferred embodiments, the QA-containing legume is a lupin plant. The skilled person will appreciate that the amino acid sequence of LLIP3.1 , LLIP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , and LLIP5.1 , and / or the nucleic acid sequence of the gene encoding any one of LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LLIP3.3, LLIP3.4, LLIP4.1 , and LLIP5.1 , may vary amongst different lupin species. The mutation(s) of the lupin plants of the present disclosure are mutation(s) compared to the wildtype sequence of LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , and LUP5.1 , and / or the LUP3.1 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene, the LUP1.4 gene, the LUP1. 1 gene, the LUP1.2 gene, the LUP2. 1 gene, the LUP2.2 gene, the LUP3.2 gene, the LUP3.3 gene, the LUP3.4 gene, the LUP4. 1 gene, and the LUP5. 1 gene, in the particular lupin species.

[0072] The activity of any one of LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , and LUP5.1 , or functional homologues thereof, may be a transporter activity. Thus, LLIP3.1 , LLIP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , and / or LLIP5.1 , or functional homologues thereof, may be a QA transport protein. In other words, LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , P7181 PC00

[0073] LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , and LUP5.1, or functional homologues thereof, may be a QA transporter, for example lupin QA transporters.

[0074] QA transporter and / or QA transport protein implies that the protein is capable of assisting and / or facilitating movement of one or more QAs between different tissues of a QA-containing legume. In other words, QA transporters and / or QA transport proteins are capable of assisting and / or facilitating transport of one or more QAs between at least two different tissues of a QA-containing legume. Preferably, the transport proteins of the present disclosure are QA transport proteins, and more specifically lupin QA transport proteins capable of assisting and / or facilitating movement of at least one or more of lupanine, angustifoline, and / or 13-hydroxylupanine.

[0075] LUP3.1

[0076] With respect to the present disclosure, the wildtype LLIP3.1 gene may be any gene encoding LLIP3.1 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto. It may be preferred that said wildtype LLIP3.1 comprises the amino acids, which are conserved amongst LLIP3.1 of different lupin species. The wildtype LLIP3.1 gene of NLL is a gene encoding LLIP3.1 of SEQ ID NO: 1 or a functional homologue thereof having at least 70%, preferably at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. Preferably, the wildtype LLIP3.1 gene of NLL is a gene encoding LUP3.1 of SEQ ID NO: 1. In some embodiments, the wildtype LUP3.1 gene comprises the coding sequence provided herein as SEQ ID NO: 2. In other words, in some embodiments the gene encoding LUP3.1 consists of or comprises the sequence set forth in SEQ ID NO: 2 or a functional homologue thereof having at least 70% sequence identity thereto.

[0077] Thus, in some embodiments the LUP3. 1 gene is a gene encoding LUP3.1 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments, the LUP3. 1 gene comprises the coding sequence as set forth in SEQ ID NO: 2 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 2.

[0078] LUP3.1 as well as functional homologues thereof preferably have QA import protein activity. In particular, LUP3.1 as well as functional homologues thereof preferably have P7181 PC00

[0079] QA import protein activity comparable to the QA import protein activity of LLIP3.1 of SEQ ID NO: 1. Preferably, said QA import protein activity comprises import of one or more QAs into the vasculature, for example pod vasculature, of a QA-containing legume, such as of a lupin plant. Said one or more QAs may be lupanine, 13- hydroxylupanine and esters thereof, and / or angustifoline.

[0080] For lupin plants, the activity of LLIP3.1 is further detailed in Example 1 and / or Example

[0081] 2 herein below. Thus, when expressed in the pod vasculature of lupin plants, such as NLLs, LLIP3.1 and functional homologues thereof may assist or be capable of assisting import of one or more QAs into the pod vasculature. Without being bound by theory, LLIP3.1 as well as functional homologues thereof may have QA export protein activity. Such export protein activity is further detailed in Example 2 herein below.

[0082] LU P3.1 -facilitated QA import into the vasculature may subsequently be followed by transport into seeds. This may facilitate the accumulation of said one or more QAs in seeds of a QA-containing legume. Without being bound by theory, LUP3.1 may also possess QA export activity, and the physiological role of LUP3.1 may depend on the context, i.e. where it is expressed and / or the physiological context. Thus, without being bound by theory, if or when expressed in the seed coat of lupin plants, such as NLLs, LUP3.1 and functional homologues thereof may assist or be capable of assisting export of one or more QAs from the seed coat to other tissues.

[0083] LUP3.5

[0084] With respect to the present disclosure, the wildtype LUP3.5 gene may be any gene encoding LUP3.5 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto. It may be preferred that said wildtype LUP3.5 comprises the amino acids, which are conserved amongst LUP3.5 of different lupin species. The wildtype LUP3.5 gene of NLL is a gene encoding LUP3.5 of SEQ ID NO:

[0085] 3 or a functional homologue thereof having at least 70%, preferably at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. Preferably, the wildtype LUP3.5 gene of NLL is a gene encoding LUP3.5 of SEQ ID NO: 3. In some embodiments, the wildtype LUP3.5 gene comprises the coding sequence provided herein as SEQ ID NO: 4. In other words, in some embodiments the gene encoding LUP3.5 consists of or comprises the sequence set P7181 PC00 forth in SEQ ID NO: 4 or a functional homologue thereof having at least 70% sequence identity thereto.

[0086] Thus, in some embodiments the LUP3.5 gene is a gene encoding LLIP3.5 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments, the LUP3.5 gene comprises the coding sequence as set forth in SEQ ID NO: 4 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 4.

[0087] LLIP3.5 as well as functional homologues thereof preferably have QA import protein activity. In particular, LLIP3.5 as well as functional homologues thereof preferably have QA import protein activity comparable to the QA import protein activity of LLIP3.5 of SEQ ID NO: 3. Preferably, said QA import protein activity comprises import of one or more QAs into the vasculature, for example pod vasculature, of a QA-containing legume, such as of a lupin plant. Said one or more QAs may be lupanine, 13- hydroxylupanine and esters thereof, and / or angustifoline.

[0088] For lupin plants, the activity of LLIP3.5 is further detailed in Example 1 and / or Example 2 herein below. Thus, when expressed in the pod vasculature of lupin plants, such as NLLs, LLIP3.5 and functional homologues thereof may assist or be capable of assisting import of one or more QAs into the pod vasculature. Without being bound by theory, LLIP3.5 as well as functional homologues thereof may have QA export protein activity. Said QA export protein activity of LLIP3.5 and functional homologues thereof may comprise export of one or more QAs from the seeds, such as the seed coats, of a QA- containing legume, for example a lupin plant. Export protein activity of LLIP3.5 is further detailed in Example 2 herein below.

[0089] LUP3.5-facilitated QA import into the vasculature may subsequently be followed by transport into seeds. This may facilitate accumulation of said one or more QAs in seeds of a QA-containing legume. Without being bound by theory, LLIP3.5 may also possess QA export activity, and the physiological role of LLIP3.5 may depend on the context, i.e. where it is expressed and / or the physiological context. Thus, without being bound by theory, if or when expressed in the seed coat of lupin plants, such as NLLs, LUP3.5 and functional homologues thereof may assist or be capable of assisting export of one or more QAs from the seed coat to other tissues. P7181 PC00

[0090] LLIP3.4

[0091] With respect to the present disclosure, the wildtype LLIP3.4 gene may be any gene encoding LLIP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto. It may be preferred that said wildtype LLIP3.4 comprises the amino acids, which are conserved amongst LLIP3.4 of different lupin species. The wildtype LLIP3.4 gene of NLL is a gene encoding LLIP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70%, preferably at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. Preferably, the wildtype LLIP3.4 gene of NLL is a gene encoding LUP3.4 of SEQ ID NO: 23. In some embodiments, the wildtype LUP3.4 gene comprises the coding sequence provided herein as SEQ ID NO: 24. In other words, in some embodiments the gene encoding LUP3.4 consists of or comprises the sequence as set forth in SEQ ID NO: 24 or a functional homologue thereof having at least 70% sequence identity thereto.

[0092] Thus, in some embodiments the LUP3.4 gene is a gene encoding LUP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments, the LUP3.4 gene comprises the coding sequence as set forth in SEQ ID NO: 24 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 24.

[0093] LUP3.4 as well as functional homologues thereof preferably have QA import protein activity. In particular, LUP3.4 as well as functional homologues thereof preferably have QA import protein activity comparable to the QA import protein activity of LUP3.4 of SEQ ID NO: 23. Without being bound by theory, said QA import protein activity of LUP3.4 and functional homologues thereof may comprise import of one or more QAs into seeds, such as seed embryos, of QA-containing legumes, such as of lupin plants. This may enable accumulation of said one or more QAs in seeds of a QA-containing legume. Said one or more QAs may be lupanine, 13-hydroxylupanine and esters thereof, and / or angustifoline.

[0094] For lupin plants, this activity of LUP3.4 is further detailed in Example 3 herein below.

[0095] Thus, when expressed in the seeds of lupin plants, such as NLLs, LUP3.4 and functional homologues thereof, may assist or be capable of assisting import of one or P7181 PC00 more QAs into seeds, such as seed embryos, of a QA-containing legume, such as of lupin plants.

[0096] HH130

[0097] With respect to the present disclosure, the wildtype HH130 gene may be any gene encoding HH130 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto. It may be preferred that said wildtype HH130 comprises the amino acids, which are conserved amongst HH130 of different lupin species. The wildtype HH130 gene of NLL is a gene encoding HH130 of SEQ ID NO: 5 or a functional homologue thereof having at least 70%, preferably at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. Preferably, the wildtype HH130 gene of NLL is a gene encoding HH130 of SEQ ID NO: 5. In some embodiments, the wildtype HH130 gene comprises the coding sequence provided herein as SEQ ID NO: 6. In other words, in some embodiments the gene encoding HH130 consists of or comprises the sequence as set forth in SEQ ID NO: 6 or a functional homologue thereof having at least 70% sequence identity thereto.

[0098] Thus, in some embodiments the HH130 gene is a gene encoding HH130 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments, the HH130 gene comprises the coding sequence as set forth in SEQ ID NO: 6 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 6.

[0099] HH130 as well as functional homologues thereof preferably have QA import protein activity. In particular, HH130 as well as functional homologues thereof preferably have QA import protein activity comparable to the QA import protein activity of HH130 of SEQ ID NO: 5. Without being bound by theory, said QA import protein activity of HH130 and functional homologues thereof may comprise import of one or more QAs into seeds, such as seed embryos, of QA-containing legumes, such as of lupin plants. This may enable accumulation of said one or more QAs in seeds of a QA-containing legume. Said one or more QAs may be lupanine, 13-hydroxylupanine and esters thereof, and / or angustifoline. The seed embryo, also known as filial embryo, is typically surrounded by the seed coat, also known as maternal seed coat. Without being bound by theory, said QA import protein activity of HH130 and functional homologues thereof P7181 PC00 may also comprise import of one or more QAs into the vasculature, such as the pod vasculature, of QA-containing legumes, such as of lupin plants.

[0100] For lupin plants, this activity of HH130 is further detailed in Example 3 herein below. Thus, when expressed in the seeds of lupin plants, such as NLLs, HH130 and functional homologues thereof, may assist or be capable of assisting import of one or more QAs into seeds, such as seed embryos, of a QA-containing legume, such as of lupin plants. HH130 and functional homologues thereof, may assist or be capable of assisting import of one or more QAs into the vasculature, such as the pod vasculature, of a QA-containing legume, such as of lupin plants.

[0101] Activity of LUP3.1, LUP3.4, LUP3.5, and HH130

[0102] As detailed herein above, the activity of each of wildtype LLIP3.1, LLIP3.4, LLIP3.5, HH130, and functional homologues thereof, may be an import activity, preferably QA import activity. The activity of wildtype LLIP3.1, LLIP3.5 and functional homologues thereof may also comprise export activity, preferably QA export activity. This implies, that each of LLIP3.1 , LLIP3.4, LLIP3.5, and HH130 may be capable of assisting and / or facilitating movement of one or more QAs between at least two different tissues of said QA-containing legume.

[0103] QA import activity implies, that the QA importer, i.e. QA import protein, is capable of assisting and / or facilitating import of one or more QAs into the same tissue wherein it is expressed. QA export activity implies, that the QA exporter, i.e. QA export protein, is capable of assisting and / or facilitating export of one or more QAs out of the same tissue wherein it is expressed.

[0104] In some embodiments, the LLIP3.1 activity and / or the LLIP3.5 activity is QA import protein activity. Preferably said QA import protein activity of each of LLIP3.1 and / or LLIP3.5, and functional homologues thereof, comprises import of said one or more QAs into the vasculature, such as the pod vasculature, of said QA-containing legume. In other embodiments, the HH130 activity is QA import protein activity. Preferably said QA import protein activity of HH130 and functional homologues thereof comprises import of said one or more QAs into the seeds, such as into the seed embryos, of said QA- containing legume. Said QA import protein activity of HH130 and functional P7181 PC00 homologues thereof preferably comprises import of said one or more QAs into the vasculature, such as the pod vasculature, of said QA-containing legume.

[0105] The activity of LLIP3.1 and / or LLIP3.5, as well as of functional homologues of any one of LLIP3.1 and LLIP3.5, may be dictated either by the tissue wherein it is expressed, and / or the level (e.g. concentration or content) and / or distribution of QAs. Thus, without being bound by theory said LLIP3.1 and / or LLIP3.5, and functional homologues of any of the two, may be a bidirectional transport protein. In other words, LLIP3.1 and / or LLIP3.5 may have bidirectional transport activity depending on the physiological context they are within.

[0106] Thus, in other embodiments, the LLIP3.1 activity and / or the LLIP3.5 activity is QA export protein activity. Without being bound by theory, said QA export protein activity of LLIP3.5 and functional homologues thereof may comprise export of said one or more QAs from the seed, such as from the seed coat, of said QA-containing legume.

[0107] In preferred embodiments, said QA-containing legume is a lupin plant, such as of the species NLL. Said one or more QAs may be lupanine, 13-hydroxylupanine and esters thereof, and / or angustifoline.

[0108] Thus, in some embodiments, the LLIP3.1 activity, the LLIP3.4 activity, the LLIP3.5 activity, and / or the HH130 activity is QA import protein activity and said one or more QAs is at least one of lupanine, 13-hydroxylupanine and esters thereof, and / or angustifoline. Thus, in some embodiments, the LLIP3.1 activity is lupanine import activity, 13-hydroxylupanine import activity, and / or angustifoline import activity, preferably into the vasculature of said QA-containing legume. Thus, in some embodiments, the LLIP3.4 activity is lupanine import activity, 13-hydroxylupanine import activity, and / or angustifoline import activity, such as import activity into the seeds, such as the seed embryos, of said QA-containing legume. In other embodiments, the LLIP3.5 activity is lupanine import activity, 13-hydroxylupanine import activity, and / or angustifoline import activity, preferably into the vasculature of said QA-containing legume. In other embodiments, the HH130 activity is lupanine import activity, 13-hydroxylupanine import activity, and / or angustifoline import activity, preferably into the vasculature and / or seed embryo of said QA-containing legume. In some embodiments, the LLIP3.1 activity and / or the LLIP3.5 activity is QA export protein P7181 PC00 activity and said one or more QAs is at least one of lupanine, 13-hydroxylupanine and esters thereof, and / or angustifoline. Thus, in some embodiments, the LLIP3.1 activity is lupanine export activity, 13-hydroxylupanine export activity, and / or angustifoline export activity. In other embodiments, the LLIP3.5 activity is lupanine export activity, 13- hydroxylupanine export activity, and / or angustifoline export activity, such as export activity from the seeds, such as the seed coats.

[0109] It follows, that modification of the activity of one or more of LLIP3.1 , LLIP3.4, LLIP3.5, and / or HH130, or functional homologues thereof, may impact the distribution and / or accumulation of one or more QAs in a QA-containing legume. The associated phenotype of said modification, may depend on the targeted transport protein and / or the extent of the modification, e.g. elimination of most of the activity or partial reduction of said activity. Useful modification are described herein elsewhere in the section “Mutation in gene encoding QA transport protein”.

[0110] LUP1.3

[0111] With respect to the present disclosure, the wildtype LUP1.3 gene may be any gene encoding LUP1.3 of SEQ ID NO: 7 or a functional homologue thereof having at least 70% sequence identity thereto. It may be preferred that said wildtype LUP1.3 comprises the amino acids, which are conserved amongst LUP1.3 of different lupin species. The wildtype LUP1.3 gene of NLL is a gene encoding LUP1.3 of SEQ ID NO: 7 or a functional homologue thereof having at least 70%, preferably at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. Preferably, the wildtype LUP1.3 gene of NLL is a gene encoding LUP1.3 of SEQ ID NO: 7. In some embodiments, the wildtype LUP1.3 gene comprises the coding sequence provided herein as SEQ ID NO: 8. In other words, in some embodiments the gene encoding LUP1.3 consists of or comprises the sequence set forth in SEQ ID NO: 8 or a functional homologue thereof having at least 70% sequence identity thereto.

[0112] Thus, in some embodiments the LUP1.3 gene is a gene encoding LUP1.3 of SEQ ID NO: 7 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments, the LUP1.3 gene comprises the coding sequence as set forth in SEQ ID NO: 8 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 8. P7181 PC00

[0113] LLIP1.3 as well as functional homologues thereof preferably have QA export protein activity. In particular, LLIP1.3 as well as functional homologues thereof preferably have QA export protein activity comparable to the QA export protein activity of LLIP1.3 of SEQ ID NO: 7. Said QA export protein activity may comprise export of one or more QAs from QA source tissue, such as biosynthetic tissue of said QAs, of said QA- containing legume. QA source tissue, such as biosynthetic tissue of said QAs, may be, but is not limited to, the epidermis of leaves, pods, and / or stems of said QA-containing legume. Said QA export protein activity comprises export of one or more QAs from the seed coat of seeds of said QA-containing legume, preferably for subsequent import into the seed embryo.

[0114] LUP1.4

[0115] With respect to the present disclosure, the wildtype LLIP1.4 gene may be any gene encoding LLIP1.4 of SEQ ID NO: 9 or a functional homologue thereof having at least 70% sequence identity thereto. It may be preferred that said wildtype LLIP1.4 comprises the amino acids, which are conserved amongst LLIP1.4 of different lupin species. The wildtype LLIP1.4 gene of NLL is a gene encoding LLIP1.4 of SEQ ID NO: 9 or a functional homologue thereof having at least 70%, preferably at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. Preferably, the wildtype LLIP1.4 gene of NLL is a gene encoding LUP1.4 of SEQ ID NO: 9. In some embodiments, the wildtype LUP1.4 gene comprises the coding sequence provided herein as SEQ ID NO: 10. In other words, in some embodiments the gene encoding LUP1.4 consists of or comprises the sequence as set forth in SEQ ID NO: 10 or a functional homologue thereof having at least 70% sequence identity thereto.

[0116] Thus, in some embodiments the LUP1.4 gene is a gene encoding LUP1.4 of SEQ ID NO: 9 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments, the LUP1.4 gene comprises the coding sequence as set forth in SEQ ID NO: 10 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 10.

[0117] LUP1.4 as well as functional homologues thereof preferably have QA export protein activity. In particular, LUP1.4 as well as functional homologues thereof preferably have P7181 PC00

[0118] QA export protein activity comparable to the QA export protein activity of LLIP1 .4 of SEQ ID NO: 9. Said QA export protein activity may comprise export of one or more QAs from QA source tissue, such as biosynthetic tissue of said QAs, of said QA- containing legume. QA source tissue, such as biosynthetic tissue of said QAs, may be, but is not limited to, the epidermis of leaves, pods, and / or stems of said QA-containing legume. Said QA export protein activity comprises export of one or more QAs from the seed coat of seeds of said QA-containing legume, preferably for subsequent import into the seed embryo.

[0119] Activity of LUP1.3 and LUP1.4

[0120] As detailed herein above, the activity of each of wildtype LLIP1.3, LLIP1.4, and functional homologues thereof, may be an export activity, preferably QA export activity. This implies, that each of LLIP1.3 and LLIP1.4 is capable of assisting transport, such as facilitating transport, of at least one QA between at least two different tissues of said QA-containing legume.

[0121] QA export activity implies, that the QA exporter, i.e. QA export protein, is capable of assisting and / or facilitating export of one or more QAs from the same tissue wherein it is expressed.

[0122] Thus, in some embodiments, the LLIP1.3 activity and / or the LLIP1.4 activity is QA export protein activity. Preferably, said QA export protein activity may comprise export of one or more QAs from QA source tissue, such as biosynthetic tissue of said QAs, of said QA-containing legume. QA source tissue, such as biosynthetic tissue of said QAs, may be, but is not limited to, the epidermis of leaves, pods, and / or stems of said QA- containing legume. Said QA export protein activity comprises export of one or more QAs from the seed coat of seeds of said QA-containing legume, preferably for subsequent import into the seed embryo.

[0123] In preferred embodiments, said QA-containing legume is a lupin plant, such as of the species NLL. Said one or more QAs may be lupanine, 13-hydroxylupanine and esters thereof, and / or angustifoline.

[0124] In other embodiments, the LLIP1.3 activity and / or the LLIP1.4 activity is QA export protein activity and wherein said QA is at least one of lupanine, 13-hydroxylupanine P7181 PC00 and esters thereof, and angustifoline. Thus, in some embodiments, the LLIP1.3 activity is lupanine export activity, 13-hydroxylupanine export activity, and / or angustifoline export activity. In other embodiments, the LLIP1.4 activity is lupanine export activity, 13-hydroxylupanine export activity, and / or angustifoline export activity.

[0125] It follows, that modification of the activity of one or more of LLIP1.3 and / or LLIP1.4, or functional homologues thereof, impact the distribution and / or accumulation of one or more QAs in said QA-containing legume. In preferred embodiments, the activity is modified of one or more of LLIP3.1 , LLIP3.5, HH130, LLIP1.3, and / or LLIP1.4, or functional homologues thereof. The associated phenotype of said modification, depends on the targeted transport protein and / or the extent of the modification, e.g. elimination of most of the activity or partial reduction of said activity. Useful modification are described herein elsewhere in the section “Mutation in gene encoding QA transport protein”.

[0126] QA transporter mutation combinations

[0127] The QA-containing legume of the present disclosure carries at least one mutation in at least one gene encoding LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1, LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , and LUP5.1 , preferably at least one gene encoding at least one of LUP3.1, LUP3.4, LUP3.5, HH130, LUP1.3, and LUP1.4. This implies, that the legume may carry multiple mutations, for example mutations in at least two different genes encoding either LUP3.1, LUP3.4, LUP3.5, and HH130, or mutations in both of LUP1.3, and LUP1.4. In the present section, different legume designs are provided for obtaining low level or zero QAs in the legume seeds, such as lupin seeds without any or low levels of QAs.

[0128] In some embodiments, the present disclosure concerns a QA-containing legume carrying a mutation in the LUP3. 1 gene, said mutation leading to reduced LUP3.1 activity or loss-of-function of LUP3.1, and a mutation in the LUP3.5 gene, said mutation leading to reduced LUP3.5 activity or loss-of-function of LUP3.5.

[0129] Thus, in some embodiments the QA-containing legume of the present disclosure, such as the lupin plant, carries P7181 PC00 a mutation in the LUP3. 1 gene comprising the coding sequence as set forth in SEQ ID NO: 2, and a mutation in the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0130] For example, in some embodiments the QA-containing legume of the present disclosure is a lupin plant carrying a mutation in the LUP3. 1 gene and in the LUP3.5 gene, wherein the mutated LUP3. 1 gene is LUP3.1_G360A(Trp120Stop) of SEQ ID NO: 72 and the mutated LUP3.5 gene is LUP3.5_G138A(Trp46Stop) of SEQ ID NO: 75 or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0131] In other embodiments, the present disclosure regards a QA-containing legume carrying a mutation in the LUP3. 1 gene, said mutation leading to reduced LLIP3.1 activity or loss-of-function of LLIP3.1, a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5, and a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130.

[0132] Thus, in some embodiments the QA-containing legume of the present disclosure, such as the lupin plant, carries a mutation in the LUP3. 1 gene comprising the coding sequence as set forth in SEQ ID NO: 2, a mutation in the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4, and a mutation in the HH130 gene comprising the coding sequence as set forth in SEQ ID NO: 6, or a functional homologue of any one of the aforementioned at least 70% sequence identity thereto.

[0133] For example, in some embodiments the QA-containing legume of the present disclosure is a lupin plant carrying a mutation in the LUP3. 1 gene, the LUP3.5 gene P7181 PC00 and the HH130 gene, wherein the mutated LUP3. 1 gene is

[0134] LUP3. 1_G360A(Trp120Stop) of SEQ ID NO: 72, the mutated LUP3.5 gene is LUP3.5_G138A(Trp46Stop) of SEQ ID NO: 75, and the mutated HH130 gene is HH130_G603A(W201stop) of SEQ ID NO: 76, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0135] The QA-containing legume carrying a mutation in one or more of the genes encoding LLIP3.1 , LLIP3.5 and / or HH130 may have reduced QA import protein activity compared to a corresponding QA-containing legume not comprising said mutation(s), preferably when cultivated, such as grown, in similar or identical conditions.

[0136] In yet other embodiments, the present disclosure concerns a QA-containing legume carrying a mutation in the LUP1.3 gene, said mutation leading to reduced LLIP1.3 activity or loss-of-function of LLIP1.3, and a mutation in the LUP1.4 gene, said mutation leading to reduced LLIP1.4 activity or loss-of-function of LLIP1.4.

[0137] Thus, in some embodiments the QA-containing legume of the present disclosure, such as the lupin plant, carries a mutation in the LUP1.3 gene comprising the coding sequence as set forth in SEQ ID NO: 8 and a mutation in the LUP1.4 gene comprising the coding sequence as set forth in SEQ ID NO: 10, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0138] The QA-containing legume carrying a mutation in one or more of the genes encoding LLIP1.3 and / or LLIP1.4 may have reduced QA export protein activity compared to a corresponding QA-containing legume not comprising said mutation(s), preferably when cultivated, such as grown, in similar or identical conditions.

[0139] For reducing the content of QAs in seeds of a QA-containing legume, it may be useful to reduce the QA transport activity in the seed coat of said QA-containing legume. For example, QA-containing legumes with reduced or essentially no QA transport activity, P7181 PC00 preferably no QA export activity, in the seed coat may produce seeds having embryos with reduced QA content, i.e. low QA content or containing no QAs. QA-containing legumes with reduced QA transport activity, such as QA export activity, in the seed coat may be obtained by reducing the activity of LLIP3.5 and LLIP1.4, or functional homologues thereof.

[0140] In yet other embodiments, the present disclosure regards a QA-containing legume carrying a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5, and a mutation in the LUP1.4 gene, said mutation leading to reduced LLIP1.4 activity or loss-of-function of LLIP1.4.

[0141] Thus, in some embodiments the QA-containing legume of the present disclosure, such as the lupin plant, carries a mutation in the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4 and a mutation in the LUP1.4 gene comprising the coding sequence as set forth in SEQ ID NO: 10, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0142] For reducing the content of QAs in seeds of a QA-containing legume, it may be useful to reduce the QA transport activity in the seed embryo of said QA-containing legume. In other words, it may be useful to reduce the import of QAs into the seed embryo of the QA-containing legume. For example, QA-containing legumes with reduced or essentially no QA transport activity, preferably no QA import activity, in the seed embryo may produce seeds having embryos with reduced QA content, i.e. low QA content or containing no QAs. QA-containing legumes with reduced QA transport activity, such as QA import activity, in the seed embryo may be obtained by reducing the activity of LLIP3.4 and HH130 or functional homologues thereof.

[0143] In other embodiments, the present disclosure concerns a QA-containing legume carrying P7181 PC00 a mutation in the LUP3.4 gene, said mutation leading to reduced LLIP3.4 activity or loss-of-function of LLIP3.4, and a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130.

[0144] Thus, in some embodiments the QA-containing legume of the present disclosure, such as the lupin plant, carries a mutation in the LUP3.4 gene comprising the coding sequence as set forth in SEQ ID NO: 24, and a mutation in the HH130 gene comprising the coding sequence as set forth in SEQ ID NO: 6, or a functional homologue of any one of the aforementioned at least 70% sequence identity thereto.

[0145] For example, in some embodiments the QA-containing legume of the present disclosure is a lupin plant carrying a mutation in the LUP3.4 gene and in the HH130 gene, wherein the mutated LUP3.4 gene is LUP3.4_C511T(Q171stop) of SEQ ID NO: 73 and the mutated HH130 gene is HH130_G603A(W201stop) of SEQ ID NO: 76, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0146] In other embodiments the QA-containing legume of the present disclosure is a lupin plant carrying a mutation in the LUP3.4 gene and in the HH130 gene, wherein the mutated LUP3.4 gene is LUP3.4_G990A(W330stop) of SEQ ID NO: 74 and the mutated HH130 gene is HH130_G603A(W201stop) of SEQ ID NO: 76, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0147] Mutation in gene encoding QA transport protein

[0148] The mutations disclosed herein in this section are mutations in the genes encoding LUP3.1 , LUP3.4, LUP3.5, LUP1.3, LUP1.4, and / or HH130 compared to the wildtype genes, or compared to functional homologues of the wildtype genes in any QA- containing legume, such as in any lupin species. Preferably, the mutation in any one of the genes is a mutation afflicting one more or more of the amino acids, which are conserved amongst LLIP3.1 of different lupin species, LLIP3.4 of different lupin species, P7181 PC00

[0149] LLIP3.5 of different lupin species, LLIP1.3 of different lupin species, LLIP1.4 of different lupin species, and / or HH130 of different lupin species, or of functional homologues thereof.

[0150] The definition of the position of an amino acid in relation to a polypeptide of the present disclosure is in general made to SEQ ID NO: 1 as reference sequence for LLIP3.1 , to SEQ ID NO: 23 as reference sequence for LLIP3.4, to SEQ ID NO: 3 as reference sequence for LLIP3.5, to SEQ ID NO: 5 as reference sequence for HH130, to SEQ ID NO: 7 as reference sequence for LLIP1.3, and to SEQ ID NO: 9 as reference sequence for LLIP1.4, but it is understood that the sequence of wildtype LLIP3.1, LLIP3.4, LLIP3.5, HH130, LLIP1.3, and LUP1.4 from other lupin species or varieties may differ to some extent from the polypeptide sequences of SEQ ID NO: 1 , SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively. Thus, is it to be understood that following sequence alignment between another wildtype LLIP3.1 , LUP3.4, LUP3.5, HH130, LUP1.3, and LUP1.4, and the reference polypeptides of SEQ ID NO: 1 , SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively, an amino acid corresponds to position X of SEQ ID NO: 1 , SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively, if it aligns to the same position.

[0151] The mutation(s) of the lupin plants of the present disclosure are mutation(s) compared to the wildtype sequence of LUP3.1, LUP3.4, LUP3.5, LUP1.3, LUP1.4, and / or HH130, and / or the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene, and / or the LUP1.4 gene, in the particular lupin species.

[0152] In some embodiments of the present disclosure, the mutation in the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene is one of the following mutations: i. a mutation leading to a premature stop codon; ii. a mutation in a splice site; iii. a frame-shift mutation; iv. deletion of the entire LUP3. 1 gene, the LUP3.4 gene, LUP3.5 gene, HH130 gene, LUP1.3 gene, and / or LUP1.4 gene or a part thereof; P7181 PC00 v. a mutation in the active site of LLIP3.1, LLIP3.4, LLIP3.5, HH130, LLIP1.3 and / or LUP1.4 leading to reduced or abolished LUP3.1, LUP3.4, LUP3.5, HH130, LLIP1.3 and / or LLIP1.4 activity or; vi. a mutation in the promoter region regulating transcription of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene leading to reduced or abolished transcription or translation.

[0153] In some embodiments of the present disclosure, the mutation in the LUP3. 1 gene is a premature stop codon. Accordingly, in some embodiments, the mutant LLIP3.1 gene encodes a mutant LLIP3.1 protein, wherein said mutant LLIP3.1 is truncated. Typically, truncated LLIP3.1 comprises the N-terminal part of LLIP3.1 , but lacks C-terminal parts. Truncated LLIP3.1 may for example be caused by a premature stop codon. It is also possible that mutant LLIP3.1 comprises the N-terminal part of LLIP3.1 , lacks the C- terminal part, but comprise another C-terminus, which does not derive from LLIP3.1. This may for example be the case if the mutation is a mutation in a splice site, causing aberrant splicing, or if the mutation is a frame shift mutation.

[0154] In embodiments wherein the the mutation in the LUP3. 1 gene is a premature stop codon, said premature stop codon may be positioned at any one of codons 1 to 120 of the LUP3.1 gene. Codons are herein numbered according to which amino acid they encode, i.e. the codon encoding the first amino acid of LLIP3.1 is codon 1, the codon encoding amino acid 10 is codon 10, and so forth. This numbering also applies to codons of LLIP3.4, LLIP3.5, HH130, LLIP1.3, and LLIP1.4. In some embodiments, said mutation is a premature stop codon positioned at codon 120 of the LUP3.1 gene, for example of a LUP3.1 gene encoding SEQ ID NO: 1 and / or the LUP3.1 gene comprising the coding sequence as set forth in SEQ ID NO: 2 and / or a LUP3.1 gene of SEQ ID NO: 72 but having a guanine (G) in place of the adenine (A) at nucleotide 3330 of SEQ ID NO: 72, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. For example, the mutated LUP3. 1 gene may be LUP3. 1_G360A(Trp120Stop) of SEQ ID NO: 72 or a functional homologue thereof having at least 70% sequence identity thereto, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. Thus, said mutant gene may encode a truncated version of LLIP3.1 consisting of amino acids 1 to 120 of SEQ ID NO: 1. In preferred embodiments, said mutant gene encodes a truncated version of LLIP3.1 P7181 PC00 consisting of amino acids 1 to 119 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto. For example, in some embodiments the QA-containing legume, such as the lupin plant, carries a guanine (G) to adenine (A) mutation at a position corresponding to nucleotide 360 of SEQ ID NO: 2 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments of the present disclosure, the QA-containing legume is a NLL carrying a guanine to adenine mutation at position 360 of SEQ ID NO: 2 or a functional homologue thereof having at least 70% sequence identity thereto.

[0155] In other embodiments the mutation in the LUP3.1 gene results in a gene encoding a mutated protein, such as mutant LLIP3.1 , lacking at least the 237 most C-terminal amino acids of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto. Thus, in some embodiments mutant LLIP3.1 may comprise the N-terminal part of LLIP3.1 or a functional homologue thereof having at least 70% sequence identity thereto, but lack some or all of the C-terminal part. Preferably, said mutant LLIP3.1 lacks at least the 50 most C-terminal amino acids, such as at least the 100 most C terminal amino acids, such as at least the 150 most C-terminal amino acids, such as at least the 200 most C-terminal amino acids, such as at least the 237 most C-terminal amino acids of wildtype LUP3.1 , such as LLIP3.1 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto.

[0156] In some embodiments of the present disclosure, the mutation in the LUP3.4 gene is a premature stop codon. Accordingly, in some embodiments, the mutant LLIP3.4 gene encodes a mutant LLIP3.4 protein, wherein said mutant LLIP3.4 is truncated. Typically, truncated LLIP3.4 comprises the N-terminal part of LLIP3.4, but lacks C-terminal parts. Truncated LLIP3.4 may for example be caused by a premature stop codon. It is also possible that mutant LLIP3.4 comprises the N-terminal part of LLIP3.4, lacks the C- terminal part, but comprise another C-terminus, which does not derive from LLIP3.4. This may for example be the case if the mutation is a mutation in a splice site, causing aberrant splicing, or if the mutation is a frame shift mutation.

[0157] In some embodiments wherein the the mutation in the LUP3.4 gene is a premature stop codon, said premature stop codon may be a stop codon positioned at any one of codons 1 to 171 of the LUP3.4 gene, for example of a LUP3.4 gene encoding SEQ ID NO: 23 and / or the LUP3.4 gene comprising the coding sequence as set forth in SEQ ID NO: 24, or a functional homologue of any one of the aforementioned having at least P7181 PC00

[0158] 70% sequence identity thereto. In some embodiments, said mutation is a premature stop codon positioned at codon 171 of the LUP3.4 gene, for example of a LUP3.4 gene encoding SEQ ID NO: 23 and / or the LUP3.4 gene comprising the coding sequence as set forth in SEQ ID NO: 24 and / or a LUP3.4 gene of SEQ ID NO: 73 but having a cytosine (C) in place of the thymine (T) at nucleotide 4950 of SEQ ID NO: 73, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. For example, the mutated LUP3.4 gene may be

[0159] LUP3.4_C511T(Q171stop) of SEQ ID NO: 73 or a functional homologue thereof having at least 70% sequence identity thereto, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. Thus, said mutant gene may encode a truncated version of LLIP3.4 consisting of amino acids 1 to 171 of SEQ ID NO: 23. In preferred embodiments, said mutant gene encodes a truncated version of LLIP3.4 consisting of amino acids 1 to 170 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto. For example, in some embodiments the QA- containing legume, such as the lupin plant, carries a cytosine (C) to thymine (T) mutation at a position corresponding to nucleotide 511 of SEQ ID NO: 24 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments of the present disclosure, the QA-containing legume is a NLL carrying a cytosine to thymine mutation at position 511 of SEQ ID NO: 24 or a functional homologue thereof having at least 70% sequence identity thereto.

[0160] In other embodiments the mutation in the LUP3.4 gene results in a gene encoding a mutated protein, such as mutant LLIP3.4, lacking at least the 192 most C-terminal amino acids of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto. Thus, in some embodiments mutant LLIP3.4 may comprise the N-terminal part of LLIP3.4 or a functional homologue thereof having at least 70% sequence identity thereto, but lack some or all of the C-terminal part. Preferably, said mutant LLIP3.4 lacks at least the 50 most C-terminal amino acids, such as at least the 100 most C terminal amino acids, such as at least the 150 most C-terminal amino acids, such as at least the 175 most C-terminal amino acids of wildtype LLIP3.4, such as LLIP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0161] In some embodiments wherein the the mutation in the LUP3.4 gene is a premature stop codon, said premature stop codon may be a stop codon positioned at any one of codons 1 to 330 of the LUP3.4 gene, for example of a LUP3.4 gene encoding SEQ ID NO: 23 and / or the LUP3.4 gene comprising the coding sequence as set forth in SEQ ID NO: 24, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. In some embodiments, said mutation is a premature stop codon positioned at codon 330 of the LUP3.4 gene, for example of a LUP3.4 gene encoding SEQ ID NO: 23 and / or the LUP3.4 gene comprising the coding sequence as set forth in SEQ ID NO: 24 and / or a LUP3.4 gene of SEQ ID NO: 74 but having a guanine (G) in place of the adenine (A) at nucleotide 6103 of SEQ ID NO: 74, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. For example, the mutated LUP3.4 gene may be LUP3.4_G990A(W330stop) of SEQ ID NO: 74 or a functional homologue thereof having at least 70% sequence identity thereto, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. Thus, said mutant gene may encode a truncated version of LLIP3.4 consisting of amino acids 1 to 330 of SEQ ID NO: 23. In preferred embodiments, said mutant gene encodes a truncated version of LLIP3.4 consisting of amino acids 1 to 329 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto. For example, in some embodiments the QA-containing legume, such as the lupin plant, carries a guanine (G) to adenine (A) mutation at a position corresponding to nucleotide 990 of SEQ ID NO: 24 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments of the present disclosure, the QA-containing legume is a NLL carrying a guanine to adenine mutation at position 990 of SEQ ID NO: 24 or a functional homologue thereof having at least 70% sequence identity thereto.

[0162] In other embodiments the mutation in the LUP3.4 gene results in a gene encoding a mutated protein, such as mutant LLIP3.4, lacking at least the 33 most C-terminal amino acids of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto. Thus, in some embodiments mutant LLIP3.4 may comprise the N-terminal part of LLIP3.4 or a functional homologue thereof having at least 70% sequence identity thereto, but lack some or all of the C-terminal part. Preferably, said mutant LLIP3.4 lacks at least the 10 most C-terminal amino acids, such as at least the 20 most C terminal amino acids, such as at least the 30 most C-terminal amino acids of P7181 PC00 wildtype LLIP3.4, such as LLIP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto.

[0163] In some embodiments of the present disclosure, the mutation in the LUP3.5 gene is a premature stop codon. Accordingly, in some embodiments, the mutant LLIP3.5 gene encodes a mutant LLIP3.5 protein, wherein said mutant LLIP3.5 is truncated. Typically, truncated LLIP3.5 comprises the N-terminal part of LLIP3.5, but lacks C-terminal parts. Truncated LLIP3.5 may for example be caused by a premature stop codon. It is also possible that mutant LLIP3.5 comprises the N-terminal part of LLIP3.5, lacks the C- terminal part, but comprise another C-terminus, which does not derive from LLIP3.5. This may for example be the case if the mutation is a mutation in a splice site, causing aberrant splicing, or if the mutation is a frame shift mutation.

[0164] In embodiments wherein the the mutation in the LUP3.5 gene is a premature stop codon, said premature stop codon may be a stop codon positioned at any one of codons 1 to 46 of the LUP3.5 gene, for example of a LUP3.5 gene encoding SEQ ID NO: 3 and / or the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. In some embodiments, said mutation is a premature stop codon positioned at codon 46 of the LUP3.5 gene, for example of a LUP3.5 gene encoding SEQ ID NO: 3 and / or the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4 and / or a LUP3.5 gene of SEQ ID NO: 75 but having a guanine (G) in place of the adenine (A) at nucleotide 700 of SEQ ID NO: 75, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. For example, the mutated LUP3.5 gene may be LUP3.5_G138A(Trp46Stop) of SEQ ID NO: 75 or a functional homologue thereof having at least 70% sequence identity thereto, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. Thus, said mutant gene may encode a truncated version of LLIP3.5 consisting of amino acids 1 to 46 of SEQ ID NO: 3. In preferred embodiments, said mutant gene encodes a truncated version of LLIP3.5 consisting of amino acids 1 to 45 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto. For example, in some embodiments the QA-containing legume, such as the lupin plant, carries a guanine (G) to adenine (A) mutation at a position corresponding to nucleotide 138 of SEQ ID NO: 4 or a functional P7181 PC00 homologue thereof having at least 70% sequence identity thereto. In some embodiments of the present disclosure, the QA-containing legume is a NLL carrying a guanine to adenine mutation at position 138 of SEQ ID NO: 4 or a functional homologue thereof having at least 70% sequence identity thereto.

[0165] In other embodiments the mutation in the LUP3.5 gene results in a gene encoding a mutated protein, such as mutant LLIP3.5, lacking at least the 321 most C-terminal amino acids of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto. Thus, in some embodiments mutant LLIP3.5 may comprise the N-terminal part of LLIP3.5 or a functional homologue thereof having at least 70% sequence identity thereto, but lack some or all of the C-terminal part. Preferably, said mutant LLIP3.5 lacks at least the 50 most C-terminal amino acids, such as at least the 100 most C terminal amino acids, such as at least the 150 most C-terminal amino acids, such as at least the 200 most C-terminal amino acids, such as at least the 250 most C-terminal amino acids, such as at least the 300 most C-terminal amino acids of wildtype LLIP3.5, such as LLIP3.5 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto.

[0166] In some embodiments of the present disclosure, the mutation in the HH130 gene is a premature stop codon. Accordingly, in some embodiments, the mutant HH130 gene encodes a mutant HH130 protein, wherein said mutant HH130 is truncated. Typically, truncated HH130 comprises the N-terminal part of HH130, but lacks C-terminal parts. Truncated HH130 may for example be caused by a premature stop codon. It is also possible that mutant HH130 comprises the N-terminal part of HH130, lacks the C- terminal part, but comprise another C-terminus, which does not derive from HH130. This may for example be the case if the mutation is a mutation in a splice site, causing aberrant splicing, or if the mutation is a frame shift mutation.

[0167] In embodiments wherein the the mutation in the HH130 gene is a premature stop codon, said premature stop codon may be a stop codon positioned at any one of codons 1 to 201 of the HH130 gene, for example of a HH130 gene encoding SEQ ID NO: 5 and / or the HH130 gene comprising the coding sequence as set forth in SEQ ID NO: 6 and / or a HH130 gene of SEQ ID NO: 76 but having a guanine (G) in place of the adenine (A) at nucleotide 1984 of SEQ ID NO: 76, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. In some P7181 PC00 embodiments, said mutation is a premature stop codon positioned at codon 201 of the HH130 gene, for example of a HH130 gene encoding SEQ ID NO: 5 and / or the HH130 gene comprising the coding sequence as set forth in SEQ ID NO: 6, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. For example, the mutated HH130 gene may be HH130_G 603 A(W201 stop) of SEQ ID NO: 76 or a functional homologue thereof having at least 70% sequence identity thereto, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. Thus, said mutant gene may encode a truncated version of HH130 consisting of amino acids 1 to 201 of SEQ ID NO: 5. In preferred embodiments, said mutant gene encodes a truncated version of HH130 consisting of amino acids 1 to 200 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto. For example, in some embodiments the QA-containing legume, such as the lupin plant, carries a guanine (G) to adenine (A) mutation at a position corresponding to nucleotide 603 of SEQ ID NO: 6 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments of the present disclosure, the legume is a NLL carrying a guanine to adenine mutation at position 603 of SEQ ID NO: 6 or a functional homologue thereof having at least 70% sequence identity thereto.

[0168] In other embodiments the mutation in the HH130 gene results in a gene encoding a mutated protein lacking at least the 178 most C-terminal amino acids of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto. Thus, in some embodiments mutant HH130 may comprise the N-terminal part of HH130 or a functional homologue thereof having at least 70% sequence identity thereto, but lack some or all of the C-terminal part. Preferably, said mutant HH130 lacks at least the 50 most C-terminal amino acids, such as at least the 100 most C terminal amino acids, such as at least the 150 most C-terminal amino acids of wildtype HH130, such as HH130 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto.

[0169] QA-containing legumes with reduced or decreased QA content

[0170] The present disclosure concerns QA-containing legumes, such as lupin plants, or parts thereof carrying at least one mutation in one or more QA transporter genes, preferably in one or more of the genes encoding LLIP3.1, LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, P7181 PC00 and / or HH130, as well as to methods using such QA-containing legumes and plant products thereof. The mutations may be any of the mutations described herein above in the section “Mutation in gene encoding QA transport protein”, and the QA-containing legumes may be any of the QA-containing legumes described herein, such as in the section “QA-containing legumes with transporter mutations” and / or “QA transporter mutation combinations”.

[0171] Interestingly, the seeds of the QA-containing legume, in particular of the lupin plant, preferably of the NLL, of the present disclosure, contains low (reduced) content of QAs, such as lupanine, 13-hydroxylupanine, and angustifoline. Thus, the reduced QA content may be a reduced QA content in the seeds, such as the mature seeds, of said QA-containing legume, for example a reduced content in the seed coat and / or seed embryo. In some embodiments, the QA content is increased in the pods, leaves, such as in the leaves, and / or stem, of said QA-containing legume.

[0172] In some embodiments, the seeds of the lupin plant(s) of the disclosure comprises at the most 1.5% QAs, such as at the most 1.4%, such as at the most 1.35%, such as at the most 1.33%, such as at the most 1.3% QAs, or less QAs, wherein the % is indicated as % of the dry seed weight. In some embodiments, the seeds, such as the mature seeds, of the lupin plant(s) of the disclosure comprises at the most 1.5% QAs, such as at the most 1.4%, such as at the most 1.35%, such as at the most 1.33%, such as at the most 1.3% QAs, or less QAs, wherein the % is indicated as % of the dry seed weight, wherein said QAs are lupanine, angustifoline, and 13-hydroxylupanine. Preferably, said lupin plant carries at least one mutation in at least the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof.

[0173] In some embodiments, the seeds of the lupin plant(s) of the disclosure comprises at the most 0.35% lupanine, such as at the most 0.3%, such as at the most 0.28%, such as at the most 0.26%, such as at the most 0.25% lupanine, or less lupanine, wherein the % is indicated as % of the dry seed weight. Preferably, said lupin plant carries at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof.

[0174] In some embodiments, the seeds of the lupin plant(s) of the disclosure comprises at the most 1% 13-hydroxylupanine, such as at the most 0.9%, such as at the most 0.8%, P7181 PC00 such as at the most 0.75%, such as at the most 0.7% 13-hydroxylupanine, or less 13- hydroxylupanine, wherein the % is indicated as % of the dry seed weight. Preferably, said lupin plant carries at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof.

[0175] In some embodiments, the seeds of the lupin plant(s) of the disclosure comprises at the most 0.5% angustifoline, such as at the most 0.4%, such as at the most 0.35%, such as at the most 0.32%, such as at the most 0.3% angustifoline, or less angustifoline, wherein the % is indicated as % of the dry seed weight. Preferably, said lupin plant carries at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof.

[0176] In some embodiments, the seeds of the lupin plant(s) of the disclosure comprises at the most 0.05% QAs, such as at the most 0.04%, such as at the most 0.03%, such as at the most 0.02%, or less QAs, wherein the % is indicated as % of the dry seed weight.

[0177] In some embodiments of the present disclosure, the seeds of the lupin plant(s) of the disclosure comprises at the most 0.05% of lupanine, 13-hydroxylupanine, and angustifoline, such as at the most 0.04%, such as at the most 0.03%, such as at the most 0.02%, or less of lupanine, 13-hydroxylupanine and / or esters thereof, and angustifoline, wherein the % is indicated as % of the dry seed weight.

[0178] In some embodiments, the lupanine content of mature seeds of a lupin plant carrying at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof, is reduced to at the most 50% of the content in a corresponding lupin plant not carrying at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof, such as at the most 40%, such as at the most 30%, such as at the most 25%, such as at the most 20% of the lupanine content of said corresponding lupin plant, and wherein the reduction is on a dry seed weight basis.

[0179] In other embodiments, the angustifoline content of mature seeds of a lupin plant carrying at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof, is reduced to at the most 70%, of the content P7181 PC00 in a corresponding lupin plant not carrying at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof, such as at the most 65%, such as at the most 60%, such as at the most 50%, such as at the most 40% of the angustifoline content of said corresponding lupin plant, and wherein the reduction is on a dry seed weight basis.

[0180] In some embodiments, the 13-hydroxylupanine content of mature seeds of a lupin plant carrying at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof, is reduced to at the most 75% of the content in a corresponding lupin plant not carrying at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene, or functional homologues thereof, such as at the most 70%, such as at the most 65%, such as at the most 60%, such as at the most 50%, such as at the most 40% of the 13-hydroxylupanine content of said corresponding lupin plant, and wherein the reduction is on a dry seed weight basis.

[0181] The lupin plant described in this section to carry at least one mutation in at least each of the LUP3. 1 gene and the LUP3.5 gene may, may for example carry a mutation resulting in a premature stop codon in each of the LUP3. 1 gene and LUP3.5 gene. The mutated LUP3. 1 gene may be LUP3.1_G360A(Trp120Stop) of SEQ ID NO: 72 and the mutated LUP3.5 gene may be LUP3.5_G138A(Trp46Stop) of SEQ ID NO: 75, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0182] In preferred embodiments of the present disclosure, the content (level or amount) of QAs is lower (less) in the seeds of a QA-containing legume carrying at least one mutation in at least one or more of the genes encoding LLIP3.1 , LLIP3.5, LLIP1.3, LLIP1.4, and / or HH130, compared to the content in seeds of a corresponding QA- containing legume not carrying said at least one mutation. For example, the content of QAs is lower in mature seeds of said QA-containing legume carrying at least one mutation in one or more of the genes encoding LLIP3.1 , LLIP3.5, LLIP1.3, LLIP1.4, and / or HH130, compared to the content in mature seeds of a corresponding QA- containing legume not carrying said at least one mutation. In some embodiments, the QA content is higher in pods, leaves, and / or stem of said QA-containing legume carrying at least one mutation in one or more of the genes encoding LLIP3.1 , LLIP3.5, LLIP1.3, LLIP1.4, and / or HH130, compared to the content in pods, leaves, and / or stem P7181 PC00 of a corresponding QA-containing legume not carrying said at least one mutation. In preferred embodiments, the QA content is lower (less) in seeds and higher in pods, leaves and / or stem of a QA-containing legume carrying at least one mutation in one or more of the genes encoding LLIP3.1 , LLIP3.5, LLIP1.3, LLIP1.4, and / or HH130, compared to the content in seed, pods, leaves and / or stem of a corresponding QA- containing legume not carrying said at least one mutation.

[0183] In other embodiments, the QA content in the seeds, preferably the mature seeds, of said QA-containing legume is reduced to at the most 70 % of the content in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein on a dry seed weight basis, such as at the most 65 %, such as at the most 60%, such as at the most 55%, such as at the most 50%, such as at the most 45%, such as at the most 40%, such as at the most 35%, such as at the most 30%, such as at the most 25 %, such as at the most 20%, or less, compared to the content in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein.

[0184] In preferred embodiments, the QA-containing legume is a lupin plant, such as NLL, carrying: i. a mutation in the LUP3. 1 gene, for example of in a LUP3.1 gene encoding SEQ ID NO: 1 and / or in the LUP3.1 gene comprising the coding sequence as set forth in SEQ ID NO: 2, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, said mutation leading to reduced LLIP3.1 activity or loss-of-function of LLIP3.1, ii. a mutation in the LUP3.5 gene, for example of in a LUP3.5 gene encoding SEQ ID NO: 3 and / or in the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5, and iii. a mutation in the HH130 gene, for example of in a HH130 gene encoding SEQ ID NO: 5 and / or in the HH130 gene comprising the coding sequence as set forth in SEQ ID NO: 6, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, said mutation leading to a reduced HH130 activity or loss of function of HH130, P7181 PC00 and having a lower QA content in the seeds compared to corresponding lupin plant not carrying said mutation in the LUP3. 1 gene, LUP3.5 gene, and the HH130 gene.

[0185] In particular, the QA content in the seeds of said lupin plant carrying said mutation in the LUP3. 1 gene, LUP3.5 gene, and the HH130 gene, is reduced to at the most 70 % of the content in a corresponding lupin plant carrying said mutation in the LUP3. 1 gene, LUP3.5 gene, and the HH130 gene, on a dry seed weight basis, such as at the most 65%, such as at the most 60%, such as at the most 55%, such as at the most 50%, such as at the most 45%, such as at the most 40%, such as at the most 35%, such as at the most 30%, such as at the most 25 %, such as at the most 20%, or less, compared to the content in said corresponding lupin plant.

[0186] In some embodiments, the seeds, such as the mature seeds, of said lupin plant carrying said mutation in the LUP3. 1 gene, LUP3.5 gene, and the HH130 gene, comprises at the most 0.02 % QAs, such as lupin QAs, wherein the % is indicated as percentage (%) of dry seed weight, wherein the % is indicated as % of the dry seed weight.

[0187] In some embodiments, the seeds, such as the mature seeds, of said lupin plant comprises at the most a total of lupanine, 13-hydroxylupanine and / or esters thereof, and angustifoline of at the most 0.02%, wherein the % is indicated as % of the dry seed weight. Preferably, said lupin plant is carrying said mutation in the LUP3.1 gene, LUP3.5 gene, and the HH130 gene, said lupin plant is carrying a mutation in the LUP3. 1 gene and LUP3.5 gene, or said lupin plant is carrying a mutation in the LUP3.4 gene or HH130 gene, or functional homologues of any one of the aforementioned.

[0188] In some embodiments, the lupanine content in the seeds, preferably the mature seeds, of said QA-containing legume is reduced to at the most 50 % of the content of said lupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 35 % of the content of said lupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 30 %, such as at the most 25 %, such as at the most 20 %, or less, wherein the reduction is on a dry seed weight basis. P7181 PC00

[0189] In other embodiments, the 13-hydroxylupanine content in the seeds, preferably the mature seeds, of said QA-containing legume is at the most 75 % of the content of said 13-hydroxylupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 65 % of the content of said 13-hydroxylupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 60 %, such as at the most 55 %, such as at the most 50 %, or less, wherein the reduction is on a dry seed weight basis.

[0190] In other embodiments, the content in the seeds of 13-hydroxylupanine and esters thereof, preferably in the mature seeds, of said QA-containing legume is at the most 75 % of the content of said 13-hydroxylupanine and esters thereof in a corresponding QA- containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 65 % of the content of said 13-hydroxylupanine and esters thereof in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 60 %, such as at the most 55 %, such as at the most 50 %, or less, wherein the reduction is on a dry seed weight basis.

[0191] In some embodiments, the angustifoline content in the seeds, preferably the mature seeds, of said QA-containing legume at the most 70 % of the content of said angustifoline in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 65 % of the content of said angustifoline in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 60 %, such as at the most 55 %, such as at the most 50 %, such as at the most 45 %, such as at the most 40 %, wherein the reduction is on a dry seed weight basis.

[0192] Within the present disclosure, the esters of 13-hydroxylupanine may be one or more of 13-trans-cinnamoyloxylupanine, 13-hydroxydihydrocoumaroyloxylupanine, 13-cis- cinnamoyloxylupanine, 13-coumaroyloxylupanine, and 13-tigloyloxylupanine.

[0193] In preferred embodiments, the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 0.02 % lupanine, 13-hydroxylupanine, and angustifoline, wherein the % is indicated as % of the dry seed weight. In other words, in P7181 PC00 some embodiments, the sum of the content of lupanine, 13-hydroxylupanine, and angustifoline within the seeds, preferably the mature seeds, of said QA-containing legume amounts to at the most 0.02 %, wherein the % is indicated as % of the dry seed weight.

[0194] QA-containing legume specifics

[0195] The present disclosure concerns QA-containing legumes, such as lupin plants, or part thereof carrying a mutation in one or more of LLIP3.1, LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, and / or HH130, as well as to methods using such legumes and plant products thereof. The mutations may be any of the mutations described herein above in the section “Mutation in gene encoding QA transport protein”.

[0196] Said part of the QA-containing legume may be seeds, such as mature seeds and / or dry seed.

[0197] The QA-containing legume may be a legume of the Genisteae clade containing one or more QAs. In preferred embodiments of the present disclosure, the QA-containing legume is a lupin plant. The lupin plant may be any lupin plant, for example any Lupinus spp.. Preferably, the lupin is a lupin crop. Thus, the lupin plant may be selected from white lupin (L. albus), yellow lupin (L. luteus L.), Andean lupin (L mutabilis) and narrow-leafed lupin (NLL, L. angustifolius).

[0198] In some embodiments of the present disclosure, the lupin plant is of the species narrow-leafed lupin (L. angustifolius), which herein also is referred to as NLL.

[0199] The QA-containing legume, such as the lupin plant, carrying said mutation(s) may be produced by any useful means. In some embodiments, it is preferred that the QA- containing legume is produced by non-GMO methods.

[0200] In some embodiments of the present disclosure, the QA-containing legume is produced by a method comprising a step of random mutagenesis or is progeny of a QA- containing legume produced by a method comprising a step of random mutagenesis.

[0201] The “random mutagenesis” or “mutagenesis” may be performed by any useful method, for example by incubating QA-containing legumes, such as lupin plants, or seeds P7181 PC00 thereof with a mutagen. Non-limiting examples of useful mutagens include ethyl methanesulfonate (EMS), nitrous acid, mitomycin C, N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB), 1 , 2, 7, 8-diepoxyoctane (DEO), methyl methane sulfonate (MMS), N-methyl- N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4- NQO), 2-methyloxy-6-chloro-9(3-[ethyl-2-chloroethyl]-aminopropylamino)- acridinedihydrochloride (ICR-170), 2-amino purine (2AP), or hydroxylamine (HA). These chemicals can cause base-pair substitutions, frameshift mutations, deletions, transversion mutations, transition mutations, incorrect replication, and the like. In some embodiments, the mutagenesis can be carried out in vivo. Sometimes the mutagenic process involves the use of the legume’s DNA replication and repair mechanisms to incorporate and replicate the mutagenized base or bases.

[0202] In some embodiments of the present disclosure, the random mutagenesis is EMS mutagenesis. An example of an EMS mutant library of NLL cv. Oscar has been described in Mancinotti et al., 2023.

[0203] In particular, QA-containing legumes, such as lupin plants, carrying a particular mutation in the gene encoding LUP3.1 , LUP3.4, LUP3.5, LUP1.3, LUP1.4, or HH130 may be prepared and identified using the FIND-IT method, which for example is described by Knudsen et al., 2022 and in patent application WO 2018 / 001884. The FIND-IT method allows identification of any specific single-nucleotide substitution caused by a mutagen from a library generated by random mutagenesis. Thus, even though the library is prepared by random mutagenesis, the identification of a given specific mutation is reproducible as long as a sufficiently large library is created.

[0204] Thus, in some embodiments QA-containing legumes, such as lupin plants, carrying a particular mutation in the gene encoding LLIP3.1 , LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, or HH130 are prepared essentially as described in international patent application WO 2018 / 001884 or as described in Knudsen et al., 2022 using primers and probes designed to identify a particular mutation in the gene encoding LLIP3.1 , LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, or HH130. The primers are preferably designed so that they are capable of amplifying a fragment of the gene encoding LLIP3.1 , LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, or HH130 comprising the site of the desirable mutation, and the probes are preferably designed to distinguish between wildtype and mutant at the site of the desirable mutation. Suitable primers and probes for identification of a NLL P7181 PC00 carrying a guanine (G) to adenine (A) mutation at nucleotide 360 of SEQ ID NO: 2 or a guanine (G) to adenine (A) mutation at nucleotide 138 of SEQ ID NO: 4 are described herein below in Example 1. Suitable primers and probes for identification of a NLL carrying a cytosine (C) to thymine (T) mutation at nucleotide 511 of SEQ ID NO: 24 or a guanine (G) to adenine (A) mutation at nucleotide 990 of SEQ ID NO: 24 are described herein below in Example 1. The skilled person will be able design suitable primers and probes for identification of a NLL carrying a mutation in LUP1.3, LUP1.4, or HH130 based on the information of the sequences of SEQ ID NOs: 8, 10, 6, respectively, given herein. For example, for identification of a NLL carrying a mutation in HH130, suitable primers for the identification of a HH 130 knockout mutant are SEQ ID NO: 60 (a target specific forward primer for HH130) and 61 (a target specific reverse primer for HH130), and suitable probes are SEQ ID NO: 62 (mutant specific probe (FAM labelled)) and 63 (wildtype specific probe (HEX labelled)), for example as described in Example 4 herein. Thus, an NLL carrying a knockout mutation in HH130, i.e. being a HH130 knockout mutant, may be screened out and isolated from the EMS mutant library of NLL cv. Oskar (Mancinotti et aL, 2023) using the primers and probes described herein and in a similar manner as described herein below in Example 1 for LUP3.1 and LUP3.5 knockout mutant NLLs.

[0205] QA-containing legumes, such as lupin plants, carrying a particular mutation in the gene encoding LUP3.1 , LUP3.4, LUP3.5, LUP1.3, LUP1.4, or HH130 may also be prepared using various site directed mutagenesis methods, which for example can be designed based on the nucleic acid sequence coding for SEQ ID NOs: 1 , 23, 3, 7, 9, or 5, respectively. In some embodiments, the QA-containing legume, such as the lupin plant, is prepared using any one of CRISPR, a TALEN, a zinc finger, meganuclease, and a DNA-cutting antibiotic as described in WO 2017 / 138986. In some embodiments, the QA-containing legume, such as the lupin plant, is prepared using CRISPR / cas9 technique, e.g. using RNA-guided Cas9 nuclease. This may be done as described in Lawrenson et al., 2015, except that the single guide RNA sequence is designed based on the coding sequence or gene sequence of LUP3.1 , LUP3.4, LUP3.5, LUP1.3, LUP1.4, or HH130. In some embodiments, the QA-containing legume, such as the lupin plant, is prepared using a combination of both TALEN and CRISPR / cas9 techniques, e.g. using RNA-guided Cas9 nuclease. This may be done as described in Holme et al., 2017 except that the TALEN and single guide RNA (gRNA) sequence are designed based on the genes sequences provided herein. Several design tools for P7181 PC00 designing gRNAs are available, e.g. CHOPCHOP v3 (Labun et al., 2019) or CRISPR-P 2.0 (Lei et al., 2014). The skilled person will be able design suitable gRNAs targeting any one of the genes encoding LLIP1.3 and LLIP1.4, based on the information of the coding sequences of SEQ ID NOs: 8 and 10, respectively, given herein. For example, for targeting the LUP1.3 gene comprising the coding sequence set forth in SEQ ID NO: 8, suitable gRNAs may be any of the sequences set forth in SEQ ID NOs: 64 or 65. For targeting the LUP1.4 gene comprising the coding sequence set forth in SEQ ID NO: 10, suitable gRNAs may be any of the sequences set forth in SEQ ID NOs: 66 or 67. Examples of CRISPR / cas9 component delivery options into QA-containing legumes, such as lupin plants, are provided in Pigeaire et al., 1997, Ellison et al., 2020, and Mancinotti et al., 2021. Without being bound by theory, it may be possible to sequentially introduce a deleterious mutation, such as a mutation resulting in a stop codon, in the genes encoding LLIP1.3 and LLIP1.4, either simultaneously or sequentially, in order to obtain a lupin plant harbouring a mutation in the genes encoding each of LLIP1.3 and LLIP1.4.

[0206] In some embodiments, the QA-containing legume is prepared using homology directed repair, a combination of a DNA cutting nuclease and a donor DNA fragment. This may be done as described in Sun et al., 2016 except that the DNA cutting nuclease is designed based on the gene sequences provided herein and the donor DNA fragment is designed based on the coding sequence of the one of the mutated QA-containing legume variants provided herein.

[0207] It is understood that the disclosure also covers QA-containing legumes, such as lupin plants, which are progeny of QA-containing legumes produced as described above.

[0208] The QA-containing legumes, such as lupin plants, may be in any suitable form. For example, the QA-containing legume, such as lupin plant, according to the present disclosure may be a viable QA-containing legume, such as lupin plant, a dried legume, a homogenized legume, seeds of said QA-containing legume, such as lupin seeds, or flour of seeds of said QA-containing legume, such as flour of lupin seeds (lupin flour). The QA-containing legume may be a mature legume, an embryo, a seed, or the like.

[0209] Parts of QA-containing legumes, such as lupin plants, may be any suitable part of the legume, such as seeds, embryos, leaves, stems, roots, flowers, or fractions thereof. A P7181 PC00 fraction may, for example, be a section of a seed, embryo, leaf, stem, root, or flower. Parts of QA-containing legumes, such as lupin plants, may also be a fraction of a homogenate or a fraction of flour of seeds of said QA-containing legume, such as lupin flour.

[0210] In some embodiments of the present disclosure, parts of QA-containing legumes, such as lupin plants, may be cells of said legume, such as viable cells that may be propagated in vitro in tissue cultures. In other embodiments, however, the parts of QA- containing legumes, such as lupin plants, may be viable cells that are not capable of maturing into an entire plant / legume, i.e. cells that are not a reproductive material.

[0211] It is preferred that the QA-containing legume, such as the lupin plant, has not exclusively been obtained by means of an essentially biological process or is progeny of such a legume. For example, the QA-containing legume, such as the lupin plant, may comprise a mutation in one or more of the genes encoding LLIP3.1 , LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, or HH130, wherein said mutation has been induced by chemical and / or physical agents, such as sodium azide or EMS.

[0212] Thus, the QA-containing legumes may have been prepared by a method involving a step of induced mutagenesis or said lupin plant may be progeny of a QA-containing legume prepared by a method involving a step of induced mutagenesis. Said induced mutagenesis may for example be treatment with a mutagenizing chemical, such as sodium azide or EMS.

[0213] In some embodiments of the present disclosure, the QA-containing legume, described herein, has not exclusively been obtained by means of an essentially biological process.

[0214] In addition to the mutation in one or more of the genes encoding LLIP3.1, LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, or HH130, the QA-containing legume may comprise other mutations.

[0215] In some embodiments of the present disclosure, the QA-containing legume, or a part thereof, carrying a mutation in one or more of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene, and / or the LUP1.4 gene, has similar P7181 PC00 agricultural properties compared to a similar QA-containing legume not carrying said mutation in one or more of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene, and / or the LUP1.4 gene. In some embodiments, the QA-containing legume carrying a mutation in one or more of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene have suitable agronomic properties. Suitable agricultural or agronomic properties include, but are not limited to suitable growth rates, high yield, good viability or resistance against infections and / or pests. Preferably the QA-containing legumes, such as the lupin plants, have similar agronomic properties compared to a reference wildtype QA-containing legume. Said reference wildtype QA-containing legume may be any wildtype QA-containing legume not carrying said mutation in one or more of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene, but otherwise similar. For example, it may be a wildtype lupin plant of the same variety as the parent lupin plant. A reference wildtype QA-containing legume may also be referred to as “a corresponding QA-containing legume”.

[0216] For example, in some embodiments the QA-containing legume has approximately the same yield as a similar QA-containing legume not carrying said mutation in one or more of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene, and / or the LUP1.4 gene, when grown under the same conditions.

[0217] The “yield” refers to the production of seeds, and is preferably based upon the weight and / or the amount of the dry seeds produced by a QA-containing legume. If the QA- containing legumes are grown in the field, the yield may be based upon the weight and / or the amount of the dry seeds produced by the legumes in a given cultivated area at a favourable sowing density. Preferably, the QA-containing legume of the disclosure has a comparable yield (or even an improved yield) compared to a reference wildtype QA-containing legume.

[0218] With respect to lupins, there are additional factors which also may be considered in the art of generating a commercial lupin variety useful for consumption, addition to said mutation in one or more genes encoding LLIP3.1 , LLIP3.4, LLIP3.5, LLIP1.3, LLIP1.4, or HH130, for example seed yield and size. The skilled lupin breeder will be able to select and develop lupin plants, which - following crossings with other lupin plants - will result in superior cultivars and which maintain said mutation in one or more genes encoding P7181 PC00

[0219] LUP3.1 , LUP3.4, LUP3.5, LUP1.3, LUP1.4, or HH130. Alternatively, the breeder may utilize QA-containing legumes of the present disclosure for further mutagenesis to generate new cultivars carrying additional mutations in addition to the mutation in one or more genes encoding LUP3.1 , LUP3.4, LUP3.5, LUP1.3, LUP1.4, or HH130.

[0220] The disclosure also comprises QA-containing legumes, such as lupin plants, carrying a mutation in one or more of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene prepared from plant breeding methods, including methods of selfing, backcrossing, crossing to populations, and the like. Backcrossing methods can be used with the present disclosure to introduce into another cultivar the mutation in one or more genes encoding LLIP3.1 , LLIP3.4, LLIP3.5, LUP1.3, LUP1.4, or HH130.

[0221] A way to accelerate the process of plant breeding comprises the initial multiplication of generated mutants by application of tissue culture and regeneration techniques. Thus, another aspect of the present disclosure is to provide cells, which upon growth and differentiation produce QA-containing legumes carrying the mutation in one or more of the genes encoding LUP3.1 , LUP3.4, LUP3.5, LUP1.3, LUP1.4, or HH130. QA- containing legumes, such as lupin plants, of the present disclosure may be propagated through any conventional methods, such as growing legumes in the field.

[0222] Methods of reducing QA content in seeds of a QA-containing legume

[0223] The present disclosure also relates to methods of decreasing the content of one or more QAs in a QA-containing legume or parts thereof.

[0224] Thus, provided is a method of reducing or decreasing the QA content in QA-containing legume or part thereof, comprising modifying the activity of at least one QA transport protein in cells of said QA-containing legume. Said QA transport protein may be LUP3.1 (SEQ ID NO: 1), LUP3.4 (SEQ ID NO: 23), LUP3.5 (SEQ ID NO: 3), HH130 (SEQ ID NO: 5), LUP1 .3 (SEQ ID NO: 7), or LUP1 .4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0225] In some embodiments, the method concerns decreasing the QA content in seeds, such as mature seeds, of said QA-containing legume. Accordingly, the method may be a P7181 PC00 method for reducing or decreasing the QA content in seeds, such as mature seeds, of said QA-containing legume.

[0226] In other embodiments, the method concerns increasing the QA content in pods, leaves, and / or stem, of said QA-containing legume. Accordingly, the method may be a method for increasing the QA content in pods, leaves, and / or stem, of said QA-containing legume.

[0227] In some embodiments, the method comprises modifying, such as reducing, the activity of at least two of LUP3.1 (SEQ ID NO: 1), LUP3.4 (SEQ ID NO: 23), LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, such as modifying the activity of: i. LUP3.1 (SEQ ID NO: 1) and LUP3.5 (SEQ ID NO: 3); ii. LUP3.1 (SEQ ID NO: 1) and HH130 (SEQ ID NO: 5); iii. LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5); iv. LUP3.4 (SEQ ID NO: 23) and HH130 (SEQ ID NO: 5); or v. LUP3.1 (SEQ ID NO: 1), LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0228] In other embodiments, the method comprises modifying, such as reducing, the activity of LUP1.3 (SEQ ID NO: 7) and / or LUP1.4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, such as modifying the activity of: i. LUP1.3 (SEQ ID NO: 7) and LUP1.4 (SEQ ID NO: 9); or ii. LUP3.5 (SEQ ID NO: 3) and LUP1.4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0229] In some embodiments, the method is a method of reducing the activity of at least two of LUP3.1 (SEQ ID NO: 1), LUP3.4 (SEQ ID NO: 23), LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. In other embodiments, the method is a method of reducing the activity of LUP1.3 (SEQ ID NO: 7) and LUP1.4 (SEQ ID NO: 9), P7181 PC00 or of LLIP3.5 (SEQ ID NO: 3) and LLIP1.4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto. Thus, the method may comprise reducing the activity of at least one QA transport protein in cells of said QA-containing legume or part thereof.

[0230] The reduced activity of any one of LUP3.1 (SEQ ID NO: 1), LUP3.4 (SEQ ID NO: 23), LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5), LUP1.3 (SEQ ID NO: 7), LUP1.4 (SEQ ID NO: 9), or functional homologues of any one of the aforementioned having at least 70% sequence identity thereto, may be a consequence of a mutation in the gene encoding said polypeptide or a mutation in said polypeptide. Mutations resulting in reduced activity compared to a wildtype polypeptide are described herein above, for example in the section “Mutation in gene encoding QA transport protein”.

[0231] Thus, it is to be understood, that activity of a certain protein (polypeptide) for the present disclosure is a comparable activity and “reduced activity” herein refers to reduced activity of a mutant protein compared to the activity of a corresponding wildtype protein. Thus, with respect to activity in relation to a protein of the present disclosure it is in general made to SEQ ID NO: 1 as reference sequence for LLIP3.1 , to SEQ ID NO: 23 as a reference sequence for LLIP3.4, to SEQ ID NO: 3 as reference sequence for LLIP3.5, to SEQ ID NO: 5 as reference sequence for HH130, to SEQ ID NO: 7 as reference sequence for LLIP1.3, and to SEQ ID NO: 9 as reference sequence for LLIP1.4, or functional homologues of any one of the aforementioned having at least 70% sequence identity thereto. Therefore, it is understood that the sequence of wildtype LUP3.1 , LUP3.4, LUP3.5, HH130, LUP1.3, and LUP1.4 from other lupin species or varieties may differ to some extent from the polypeptide sequences of SEQ ID NO: 1 , SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively.

[0232] The QA-containing legume may be any of the legumes described herein.

[0233] Plant product and methods of producing such

[0234] The present disclosure also provides methods of producing a plant product containing the QA-containing legume or part thereof described herein. Thus, provided herein is also a plant product comprising the QA-containing legume or part thereof, as described herein. P7181 PC00

[0235] In preferred embodiments, said QA-containing legume is a lupin plant, such as a lupin plant of the species narrow-leafed lupin (NLL, L. angustifolius). QAs of lupins may also sometimes be referred to as lupin QAs or lupin-type QAs. The primary QAs of lupin seeds are lupanine, angustifoline, and 13-hydroxylupanine.

[0236] The plant product may be selected from the group consisting of: i. an extract prepared from seeds of said QA-containing legume; ii. flour prepared from the seeds of said QA-containing legume and / or from the extract, such as lupin flour prepared from the seeds of lupin plants; iii. kernel flour prepared from the endosperm of the seeds of said QA-containing said legume, such as lupin kernel flour prepared from the endosperm of seeds of said lupin plants; iv. hulls prepared from the seeds of said QA-containing legume or part thereof, such as prepared from the seeds and / or dried seeds of said QA-containing legume, for example from said lupin plants; v. meal prepared from the seeds of said QA-containing legume or part thereof, such as prepared from seeds or dried seeds of said QA-containing legume; vi. plant flakes, such legume flakes of said QA-containing legume, prepared from the seeds of said QA-containing legume; and vii. dry seeds prepared from the seeds of said QA-containing legume.

[0237] Flour is usually prepared by grinding or milling dry seeds of the QA-containing legume, such as the lupin plant. The flour can be prepared by either wet or dry milling, preferably by dry milling. Similarly, meal is usually prepared by more coarsely grinding or more coarsely milling dry seeds of the QA-containing legume, such as the lupin plant.

[0238] Provided herein is also a plant cell of the QA-containing legume or part thereof described herein.

[0239] Also provided is one or more seeds from a QA-containing legume described herein. Said seeds may be processed into a flour or meal by techniques known in the field. Thus, provided herein is seed flour and / or seed meal from said seed, for example lupin P7181 PC00 flour and / or lupin meal. Various other food products may be derived therefrom, such as pasta e.g. lupin pasta, or yoghourt, such as lupin yoghurt.

[0240] In some embodiments of the present disclosure, the methods concerns producing a plant product, said method comprising the steps of: i. providing seeds of a QA-containing legume described herein; and ii. processing said seeds into a plant product, wherein the processing preferably comprises or consists of drying said seeds into dry seeds.

[0241] In preferred embodiments, said seeds are mature seeds and / or dry seeds.

[0242] In some embodiments, said method of producing a plant product concerns producing a meal and step ii. comprises milling and / or grinding dry seeds of said QA-containing legume.

[0243] In other embodiments, said method of producing a plant product concerns producing a flour and step ii. comprises milling and / or grinding dry seeds of said QA-containing legume.

[0244] Said method of producing a plant product may be a method of producing hulls, and step ii. comprises dehulling the seeds. For example, step ii. may comprise drying said seeds into dry seeds and dehulling said dry seeds to obtain hulls.

[0245] In some further embodiments, said method of producing a plant product concerns producing a kernel flour and step ii. comprises dehulling the seeds to obtain endosperms of said seeds of said QA-containing legume, milling and / or grinding said endosperms. Preferably, said seeds are dry seeds, and step ii. optionally also comprises drying said seeds into dry seeds.

[0246] In other embodiments, said method of producing a plant product concerns producing a meal and step ii. comprises milling and / or grinding dry seeds of said QA-containing legume.

[0247] In other embodiments, said method of producing a plant product concerns producing debittered seeds of said QA-containing legume, and step ii. comprises soaking and / or P7181 PC00 boiling the dry seeds of said QA-containing legume. Step ii. may further comprise rinsing the dry seeds. By being soaked in water or another suitable liquid, at least some of the QAs of the dry seeds of said QA-containing legume leach out. The part of the method related to soaking may take up to several days. Boiling said dry seeds, optionally after soaking, also serves to reduce, such as further reduce the content of one or more QAs in the seeds. To remove or attempt removing any remaining QAs, the soaked and / or boiled (dry) seeds may be rinsed with water or another suitable liquid.

[0248] In other embodiments, the plant product is leaching water and step ii. comprises boiling the dry seeds of said QA-containing legume.

[0249] In further some embodiments, the method of producing a plant product concerns producing plant flakes of said QA-containing legume or part thereof, and step ii. comprises chopping the dry seeds of said QA-containing legume.

[0250] Alternatively or in addition, the method of producing a plant product concerns producing an extract and step ii. comprises preparing an extract of said seeds.

[0251] The method of producing a plant product may concern producing a protein isolate and step ii. comprises isolating proteins from said seeds, for example said seeds are seeds of a lupin plant described herein.

[0252] For example, the method may concern producing a protein isolate and / or an extract, comprising: a. providing seeds of a QA-containing legume, described herein, or part thereof and / or an extract of the QA-containing legume and / or flour of seeds of the QA- containing legume; b. preparing a protein isolate and / or an extract of said seeds, extract and / or flour.

[0253] The “extract" may be any purified or substantially purified fraction, compound or molecule of the QA-containing legume. The extract may be prepared by extraction with a solvent, i.e. any liquid that can dissolve or substantially disperse another substance.

[0254] Protein isolates may be prepared by any useful method, for example methods comprising one or more of the following: P7181 PC00

[0255] • Solubilisation of proteins by dispersing QA-containing legume flours, such as lupin flours, in strong alkaline and acid aqueous solution

[0256] • Solubilisation of proteins by dispersing QA-containing legume flours, such as lupin flours, in aqueous solutions with high salt concentration

[0257] • Precipitation / formation of protein rich aggregates by adjusting pH close to isoelectric point

[0258] • Precipitation / formation of protein rich aggregates by decreasing ionic strength of solution

[0259] • Concentration of proteins in the QA-containing legume flours, such as lupin flours, by a physical process based on particle size and density called air classification

[0260] • Air classification e.g. essentially as described in Silventoninen et al., 2018 or in Fenn et al., 2022.

[0261] Uses

[0262] The present disclosure also provides uses of the QA-containing legume or part thereof described herein.

[0263] In some embodiments, said use of the QA-containing legume or part thereof, described herein, is use in a food product. For example, said use may be use of seeds of the QA- containing legume, such as the lupin plant, described herein, in a food product. The food product may be one of the plant products described herein in the section “Plant product and methods of producing such”.

[0264] In other embodiments, said use of the QA-containing legume or part thereof, described herein, as animal feed.

[0265] Examples

[0266] Example 1: Quinolizidine alkaloid transporters and their applications

[0267] Aim

[0268] To identify and characterise transport proteins involved in the long-distance transport of seed-bound QAs in bitter narrow-leafed lupin (NLL). P7181 PC00

[0269] Materials and Methods

[0270] Candidate selection

[0271] Several RNAseq datasets of bitter and sweet NLL (Yang et al., 2017, Kamphuis et al., 2015) were used to select QA transporter candidate genes based on one or more of the following criteria: transmembrane domain prediction (TMHMM-2.0; Krogh et al., 2001), overall expression pattern, expression levels in key organs such as pods and seeds, and predicted sub-cellular localization.

[0272] High-throughput screen for QA transporters Preparation of NLL extract. To prepare an extract containing all QAs accumulating in NLL, we extracted a mix of seed, pod and leaf tissues from field-grown plants at several developmental stages. The frozen tissue mix was ground to powder using a mortar and pestle in the presence of liquid nitrogen. 50 mg of the homogenized powder was resuspended in 1 mL 80 % methanol and 0.06% formic acid and vortexed.

[0273] Samples were incubated for 30 min at 4°C, vortexed again and centrifuged at 20,000 x g for 15 min. The supernatant was run through a C18 column (Phenomenex, Strata® C18-E, 200 mg / 3 mL 8B-S001-FBJ). The flow-through was evaporated to dryness, and the pellet was stored at -20°C.

[0274] Cloning of CDSs into pNB1u. Of the 141 selected CDSs, we were able to clone 121 from cDNA of NLL cv. Oskar (bitter) as follows. cDNA was synthesized from pod and seed RNA extracted with the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich) and also from RNA stored from the previous RNAseq experiment described by Yang et al (Yang et al., 2017). For cDNA synthesis we used either the SuperscriptTM III First- Strand Synthesis System (Thermo Fisher Scientific) with both (dT)2o and random hexamer primers, or the iScript cDNA Synthesis Kit (Bio-Rad). The CDSs were amplified by PCR and cloned via USER cloning (Nour-Eldin et al., 2010) into the expression vector pNB1u (Nour-Edlin et al., 2006) containing the 5’- and 3’-UTRs of the Xenopus b-globin gene. Cloned sequences were verified by Sanger sequencing.

[0275] Synthesis ofcRNA. For expression in Xenopus oocytes, cRNA was synthesized as described in Jorgensen et al. (2016). In brief, transcriptionally active units including the T7 promoter and both 5’- and 3’-UTRs were amplified by PCR using the primer pair of SEQ ID NO: 68 (forward primer) and SEQ ID NO: 69 (reverse primer). In vitro P7181 PC00 transcription was carried out on the purified PCR products using the mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific) to yield cRNA. The quality of the cRNA was verified by visualisation on agarose gels as well as via NanoDrop (Thermo Fisher Scientific) measurements. The individual cRNAs were diluted to 500 ng / pl and stored at -80°C.

[0276] Functional screen. Prior to injection, five candidate cRNAs were pooled to give a final concentration of 100 ng / pl per cRNA. Xenopus oocytes (Ecocyte Bioscience, http: / / ecocyte-us.com) were injected with 50 nL pooled cRNA or nuclease-free water using the Drummond NANOJECT II (Drummond Scientific) and incubated for three days at 16°C in Kulori buffer pH 7.4 (90 mM NaCI, 1 mM KCI, 1 mM MgCh, 1 mM CaCh, 10 mM HEPES). After this, assays were carried out as described in Jorgensen et al. (2017). Oocytes were first pre-incubated for 5 min in Kulori buffer pH 5.5 and then incubated for one hour in NLL plant extract dissolved in Kulori buffer pH 5.5 (250 mg plant extract in 5 ml Kulori). Oocytes were then washed three times in cold Kulori pH 7.4, and five oocytes per pool were placed in an Eppendorf tube representing one replicate. Each cRNA pool was tested in triplicate. The remaining Kulori buffer was pipetted off and the oocytes were homogenised in 60 ml extraction buffer [65 % MeOH, 0.06% formic acid, 22 pM caffeine (internal standard)]. Homogenised oocytes were kept at -20°C for 2 hours. After this, the mixture was spun down, and a fraction of the supernatant was diluted 4X with water. The dilutions were filtered through a 0.22-pM PVDF membrane and stored at -20°C. QA analysis was performed as described below.

[0277] QA analysis

[0278] The analysis of QAs was performed via LC-MS as described by Otterbach et al. (2019). Briefly, separation was carried out via LIPLC using a C18 column with increasing acetonitrile concentrations. The LIPLC was coupled to a quadrupole-time-of-flight mass spectrometer operated in positive mode including electrospray ionization. The identification of lupanine was made by comparison to a commercial standard (Innosil, Poland). For all other QAs (13-hydroxylupanine, angustifoline, etc), the identification relied on exact masses, fragmentation patterns, UV spectra, and relative retention times as detailed by Otterbach et al. (2019). Peak areas of individual QAs were quantified using extracted ion chromatograms and normalised according to the internal standard caffeine. P7181 PC00

[0279] Probing the NLL PUP family for QA transport activity

[0280] We selected 14 NLL PLIPs with expression in leaves, stems, and / or pods (biosynthetic organs). This included the four that were originally identified as QA transporters in the high-throughput screen. The CDSs that had not yet been tested were cloned into pNB1u and tested for QA uptake in Xenopus oocytes as described above for the high- throughput screen. The oligonucleotides (primers) used for the cloning of LUP3.1, LUP3.4, and LUP3.5 that led to QA transport upon expression in Xenopus oocytes are shown in Table 1.

[0281] Table 1. SEQ ID NOs of oligonucleotides used for the cloning of LUP3.1 , LUP3.4, and LUP3.5 from NLL cDNA into pNB1u. The sequences are shown in the 5’->3’ direction. Nucleotides in lowercase represent the USER-tails added for cloning purposes.

[0282] Sub-cellular localisation of selected LUPs in Nicotiana benthamiana

[0283] The CDSs of LUP2.1, LUP3.1 , and LUP3.5 were USER-cloned (Nour-Eldin et al., 2010) in frame into pPS48YFPu or pPS48u (Nour-Eldin et al., 2006) encoding YFP either upstream or downstream of the insertion site. As controls, we used plasmids encoding a plasma membrane CFP marker (AtPIP2A in plasmid pm-ck / CD3-1001) and tonoplast mCherry marker (g-TIP in plasmid vac-rk / CD3-975) (Nelson et al., 2007). After transformation into Agrobacterium Agl-1 , cells were cultured at 28°C with shaking. Overnight cultures were diluted to an QD600 of 0.025 and cultured for an additional 20 h. Pellets were collected by centrifugation and resuspended in infiltration medium (10 mM MES, 10 mM MgCh, 100 pM acetosyringone, pH 5.6) to an QD600 of 0.05. Suspensions were kept at room temperature for 3 hrs. Suspensions of individual constructs were infiltrated into the abaxial side of leaves of 4-week-old Nicotiana benthamiana using 1-ml needleless syringes. At 3 d post-infiltration, leaf discs were excised and mounted with water for observation by an inverted confocal laser scanning microscope (Leica Stellaris 8). Excitation / emission wavelengths were 514 / 519-616 for YFP, 440 / 446-563 for CFP, 568 / 593-632 for mCherry and 651 / 656-750 for visualising chloroplasts. Images were acquired and processed using the microscope imaging software LAS X (Leica). P7181 PC00

[0284] Electrophysiological studies of selected LUPs

[0285] Oocyte and electrode preparation. cRNA of LLIP3.1 , LLIP3.5 and LLIP2.1 was injected separately into oocytes at a concentration of 500 ng / pl. Injected oocytes were incubated in Kulori buffer pH 7.4 (90 mM NaCI, 1 mM KCI, 1 mM MgCh, 1 mM CaCh, 10 mM HEPES) for 2-3 days at 16°C before recording currents. Electrode preparation was done by immersing the silver wires in 3M KCI and 1.5M acetate, leaving only a short end (less than 1 cm) of each wire in the air. Intracellular glass microelectrodes had tip resistances between 300 and 1500 kQ.

[0286] Electrophysiological measurements. Lupanine-induced currents were recorded using the TEVC on the automated Roboocyte2 system (Multi-channel Systems). To test electrogenicity, oocytes expressing the LUP genes were clamped and continuously perfused with MES-based eKulori buffer (2 mM LaCI3, 90 mM NaCI, 1 mM KCI, 1 mM MgCh, 1 mM CaCh and 10 mM MES pH 5.5). Only oocytes with more than -11mV membrane potential after impalement were chosen for the measurements. Individual oocytes were voltage-clamped to the membrane potential of -60 mV, and the current was continuously recorded under the following conditions: 1) Baseline signal for 20 s, 2) Perfusion with 3 mM lupanine tartrate in eKulori buffer for 35 s, 3) Wash-off in eKulori buffer for 40 s.

[0287] KM determination. For the kinetics experiments, l-V curve recordings were done while perfusing with different lupanine tartrate concentrations in an eKulori buffer at pH 5.5. Individual oocytes were clamped at - 60 mV, perfused with the test solution until the current stabilised, and then subjected to 100-ms stepwise changes of membrane potential from - 140 mV to 20 mV. For each measurement, the current was recorded first with eKulori buffer alone, then with the same eKulori buffer containing lupanine tartrate, and finally with eKulori alone as a wash-off step.

[0288] Apparent affinity constants were estimated from concentration-response curves. Induced currents were obtained by subtracting the steady-state currents on eKulori buffer alone from the currents in the presence of lupanine tartrate in eKulori buffer at each of the tested voltages. The normalised currents at different membrane potentials were plotted against the applied substrate concentrations and fitted to the Michaelis- P7181 PC00

[0289] Menten equation using the R package “propogate”. The R package “ggplot2” was used for data visualisation.

[0290] Isolation and phenotyping of individual LUP3. 1 and LUP3.5 knockouts

[0291] The EMS mutant library of NLL cv. Oskar described in Mancinotti et al. (2023) was screened for early stop codon mutations in LLIP3.1 and LLIP3.5 as specified in Knudsen et aL, 2022. For each gene, we used a TaqMan assay with target-specific primers as well as two differently labelled probes: a mutant-specific one and a wildtypespecific one. The SEQ ID NOs of the primers and probes (oligonucleotides) are given in Table 2. One individual heterozygous M2 mutant seed was identified for each of the mentioned targets. The identified mutants corresponded to G360A (Trp120Stop) and G138A (Trp46Stop), respectively.

[0292] Table 2. SEQ ID NOs of oligonucleotides used for primers and the TaqMan probes that led to the isolation of the LLIP3.1 and the LLIP3.5 knockout mutants from a previously constructed EMS mutant library of NLL cv. Oskar.

[0293] The M2 heterozygous mutant seeds were sown in 16 cm-wide, 20-cm deep pots filled with commercial peat-based potting soil and grown in a growth chamber under conditions to decrease time to flowering and accelerate generation turnover, as described in (Croser et al., 2016). Specifically, plants were grown in a growth chamber under 310 uE of far-red enriched lighting (LEDs of model BX120c2 with spectrum AP67; Valoya, Finland), 24 / 20°C day / night, 60 % relative humidity, and a 20 h photoperiod. Approximately 2 to 3 weeks after flowering, plants were moved to regular growth chambers with 20 / 18°C day / night, 220 uE of light, 60 % relative humidity, and a 16 h photoperiod. After the plants had developed mature seeds, the M3 seed was collected and M3 plants were grown also as described above. Approximately two weeks after germination, leaf material from each plant was collected for DNA extraction and genotyping by Sanger sequencing to identify wildtype, heterozygous, and homozygous mutant plants, using the primers (oligonucleotides) with SEQ ID NOs indicated in Table 3. P7181 PC00

[0294] Table 3. SEQ ID NOs of oligonucleotides used to confirm the inactivating mutations in the LLIP3.1 and LLIP3.5 knockout mutants post-isolation by PCR amplification and Sanger sequencing. The same method was used for genotyping of the progeny as well as the derived crosses.

[0295] The mutated gene of LLIP3.1 (Trp120Stop) is as set forth in SEQ ID NO: 72 (LUP3. 1_G360A(Trp120Stop)). The mutated gene of LLIP3.5 (Trp46Stop) is as set forth in SEQ ID NO: 75 LUP3.5_G138A(Trp46Stop)).

[0296] At 30 days after anthesis (DAA), immature seeds and pods, as well as leaf tissue was harvested from wildtype and homozygous individuals. Mature seeds from these plants was also collected. Tissues were stored at -70°C. To analyse QA content, each sample was crushed to a fine powder using a mortar and pestle with liquid nitrogen.

[0297] Approximately 30 mg of each powder was weighed, and metabolites were extracted using 1 ml of extraction solvent (60 % methanol in water, 0.06 % formic acid, and either 5 ppm [for pod and leaf tissue] or 50 ppm [for seed tissue] caffeine as an internal standard). The mixtures were shaken vigorously for 90 min at room temperature after which they were centrifuged at maximum speed for 1 minute. The supernatant was collected and diluted 1:5 with water. Seed samples were further diluted 1 :10 with 12 % methanol in water and 0.012 % formic acid. Each diluted extract was passed through a 0.22 pm filter and transferred to a glass vial for QA analysis, as described above.

[0298] Crossing and first chemotyping of the double LUP3.1 x LUP3.5 knockout mutant The individual LUP mutants were crossed to give combinations of wildtype, heterozygous, and homozygous genotypes at both loci. For each cross, forceps were used to carefully emasculate flowers on the main stem at their immature stage (before the anthers had extended past the pistil). Two days later, the emasculated flowers were fertilised with ripe pollen from the paternal plants. The genotypes of the crossed plants were assessed as described above for the individual LUP mutants. The chemotyping of the double mutant compared to a control genotype (wildtype (WT) for LUP3.1 and P7181 PC00 heterozygous for LLIP3.5) was carried out as described above for the single mutants, except that seeds were only analysed at maturity.

[0299] Simultaneous chemotyping of single and double mutants

[0300] Seeds of single mutants (LLIP3.1 and LLIP3.5), double mutant (LUP3.1 / LUP3.5), and a wildtype control were sown in pots as described above. Plants were grown in a walk-in growth chamber at 21 / 19°C day / night, 300 uE of light, 60 / 65% day / night relative humidity, and a 16 h photoperiod. Tissues were harvested and analyzed as indicated above.

[0301] Results

[0302] Selection of and screening for QA transporters in Xenopus oocytes

[0303] We conducted a broad in silico search to identify candidate QA transporters of the bitter NLL cultivar Oskar potentially having different functions: 1) exporters from biosynthetic cells, 2) phloem loaders in above-ground tissues, and 3) transporters within the seed. In total, we identified 141 candidates, of which we successfully cloned 121 from cDNA.

[0304] We tested these 121 candidates in a high-throughput transport screen in Xenopus laevis oocytes. Genes were expressed in pools of five, and oocytes were incubated in an extract of NLL leaves, stems, and pods representing all QAs found in NLL. In four of these pools, we could observe an over-accumulation of QAs in oocytes compared to the water- injected controls. The resulting QA profiles differed, with one pool overaccumulating only lupanine, another pool over-accumulating the three “core QAs” (lupanine, 13-hydroxylupanine, and angustifoline), and the last two pools overaccumulating all QAs (including the various esters of 13-hydroxylupanine). We then tested the genes found in these four pools separately and found that we could unambiguously assign the QA transport activity to a single gene in each pool.

[0305] A specific clade of purine uptake permeases houses at least 11 QA transporters The four QA transporters identified in our high-throughput oocyte screen all belonged to the same family of purine uptake permeases (PUPs). We conducted an overall search for PUPs in the NLL genome (Hane et al., 2017) and found 35 unique CDSs excluding duplicates and a few likely pseudo-genes. Out of these we selected 14 P7181 PC00 further QA transporter candidates. Using the Xenopus oocyte system in conjunction with NLL extracts, we identified a total of 11 QA transporters (including the four hits of the original screen). We designated these as LUPs (Lupin alkaloid Uptake Permeases) (Table 4). The LUPs displayed one of four distinct substrate specificities: they could either transport all QAs (core and esterified), core QAs only, lupanine only, or angustifoline only (Fig. 1).

[0306] Table 4. List of 11 NLL genes encoding membrane proteins able to mediate QA uptake into Xenopus oocytes as shown in Fig. 1. The genes belong to the larger family of purine uptake permeases (PUPs). We renamed these specific genes “LUPs”, which is short for Lupin alkaloid Uptake Permeases. AA: amino acid

[0307] Identification of target LUPs fortransport engineering

[0308] We first focused our attention on the likely transport of QAs from pods to seeds, and, in particular, on the loading of QAs into the pod vasculature. The latter was enabled by a recent tissue-specific RNAseq dataset obtained using laser capture microdissection (Frick et al., 2023). By examining the expression patterns of all LUP genes in the available RNAseq datasets, we found that LUP3.1 , LUP3.5, and LUP2.1 displayed high expression in the pod vasculature (Fig. 2).

[0309] Subcellular localization of selected LUPs P7181 PC00

[0310] Phloem loaders typically display a plasma membrane localization. We tested the subcellular localisation of LLIP3.1 , LLIP3.5, and LLIP2.1 by expressing fluorescent protein fusions in N. benthamiana. For LLIP3.1 and LLIP2.1 , we first confirmed the QA importing activity of the N- and C-terminal YFP fusions were active in Xenopus oocytes. We then imaged leaves of N. benthamiana expressing the only the N-terminal fusions, and we observed a plasma membrane localization in both cases (Fig. 3). As we had not tested the functionality of N- and C-terminal fusions for LLIP3.5 in Xenopus oocytes, we imaged leaves of N. benthamiana expressing both types of fusion, and these also localized to the plasma membrane (Fig. 3).

[0311] Electrogenicity and kinetics of the selected LUPs towards lupanine

[0312] To carry out electrophysiological studies with a pure QA as transport substrate, we purified lupanine from seeds of NLL cv. Oskar using flash chromatography. We then tested the electrogenicity of lupanine transport by LLIP3.1 , LLIP3.5, and LLIP2.1 using the voltage-clamp method in Xenopus oocytes. With the clamp set at -60 mV, the application of 3 mM lupanine resulted in negative currents for oocytes expressing each of the three transporters. This suggests a positive charge influx into the oocytes. By contrast, water- injected oocytes responded minimally to the application of lupanine. In these experiments, LLIP3.1 gave the most negative current, LLIP3.5 gave an intermediate one, and LLIP2.1 gave the least negative one (Fig. 4A). These magnitudes may or may not reflect the intrinsic speeds of transport, as the protein levels upon expression in oocytes were not necessarily the same.

[0313] To compare their affinities towards lupanine, we constructed saturation curves for LLIP3.1 , LLIP3.5, and LLIP2.1 at -60 mV. The curves were fitted to Michaelis-Menten kinetics, which gave KM values of 3.3 mM, 2.1 mM, and 10.6 mM, respectively (Fig. 4B and Fig. 8). Accordingly, these LUPs can be considered low-affinity transporters, and the affinities of LUP3.1 and LUP3.5 towards lupanine appear higher than that of LUP2.1.

[0314] Isolation and phenotyping of LU P3.1 and LUP3.5 knockout mutants

[0315] We have recently generated an EMS mutant population of NLL cv. Oskar composed of around 100.000 individuals obtained using low amounts of mutagen (Mancinotti et al 2023). We screened the population and identified one heterozygous individual with a pre-mature stop codon mutation for LUP3.1 , and one for LUP3.5. We selfed the heterozygous individuals to obtain mixed progeny including wildtype and homozygous P7181 PC00 siblings. These were chemotyped using the corresponding wildtype siblings as controls. For the phenotyping, we analysed the QA content of leaves, pods, and seeds at 30 DAA (the midpoint in seed development) as well as the QA content of mature seeds.

[0316] For LLIP3.1 , we observed a reduction to around 50% in lupanine and angustifoline content in seeds at 30 DAA. However, this reduction could not be observed in the mature seeds (Fig. 5A). For LLIP3.5, we did not observe any differences in seed QA content at 30 DAA but observed a reduction to around 60% of both lupanine and angustifoline in the mature seeds. This reduction was accompanied by a 2.5-3-fold increase in lupanine, angustifoline, and 13-hydroxyupanine in the pods at 30 DAA, lending further to the hypothesis that pods are an important source of QAs for the seeds (Fig. 5B).

[0317] Crossing and chemotyping of the double LUP3.1 x LUP3.5 knockout mutant Given the mild chemotypes observed for the individual LUP mutants, we hypothesised that the genes were acting redundantly and proceeded to generate a double mutant by crossing. We first chemotyped the double mutant in a small growth chamber in comparison to a control that was wildtype for LLIP3.1 and heterozygous for LLIP3.5. We analysed QA content in leaves and pods at 30 DAA as well as in the mature seeds (Fig. 6). Compared to the control, the leaves at 30 DAA seemed unaffected, while the pods at 30 days after anthesis (DAA) accumulated ~2.5 times more lupanine as well as -2 times more 13-hydroxylupanine. Gratifyingly, the double mutant gave mature seeds where lupanine content was reduced to -20%, angustifoline to -40%, and 13- hydroxylupanine to -50%.

[0318] We then carried out another chemotyping round in a larger growth chamber where we used fully wildtype plants as controls and included the single mutants as well (Fig. 7). In this round, the double mutant also accumulated lower QA levels in mature seeds, with lupanine reduced to -30%, angustifoline to -60%, and 13-hydroxylupanine to -60% compared to the wildtype control. Concomitantly, QA levels in developing pods of the double mutant were increased between 3 times (angustifoline and 13- hydroxylupanine) and 4 times (lupanine). As seen before, the single LLIP3.1 knockout did not show any marked differences in QA content, while the LLIP3.5 displayed a mild reduction in the mature seeds, this time only angustifoline being reduced to -60%. P7181 PC00

[0319] Estimation of QAs by mass in the LUP3. 1 / LUP3.5 double mutant

[0320] The LUP3.1 / LUP3.5 double mutant gave mature seeds where lupanine content was reduced to -20%, angustifoline to -40%, and 13-hydroxylupanine to -50% compared to a bitter control plant. The QA content in seeds of bitter NLL was quantified previously in Mancinotti et al., 2023 and listed in Table 5.

[0321] Table 5. Quantification of QA content in the seeds of bitter NLL cv. Oskar, data from Mancinotti et al., 2023.

[0322] Therefore, the total QA amount by mass in the LUP3.1 / LUP3.5 double mutant was estimated at around 1 .33 % of seed dry weight (Table 6), implying a total QA reduction down to 36%.

[0323] Table 6. Estimation of total alkaloid content in the seeds of narrow-leafed lupin plants

[0324] (NLLs)

[0325] Conclusion

[0326] In this example we identified transport proteins involved in the long-distance transport of seed-bound QAs in NLL. We started by conducting a high-throughput functional screen of gene candidates in Xenopus oocytes, and examining gene expression and protein subcellular localization in planta. We identified two immediate gene targets, i.e. LUP3.1 and LUP3.5, likely involved in the transport of seed-bound QAs from pods to seeds. We P7181 PC00 obtained NLL mutant lines where the respective genes had been knocked-out. Crossing these lines to create a double-knockout plant resulted in the reduction of seed QA content down to 36%, with no reduction of alkaloid levels in other plant organs.

[0327] Example 2: Identification of quinolizidine alkaloid exporters in narrow-leafed lupin Aim

[0328] To identify QA exporters of bitter narrow-leafed lupin (NLL).

[0329] Materials and Methods

[0330] Selection of candidate genes

[0331] QA exporter candidates were selected using tissue-specific transcriptome of bitter narrow-leafed lupin (cv. Oskar) (Frick et al., 2023) based on transmembrane domain predictions (TMHMM-2.0; Krogh et al., 2001) and expression patterns..

[0332] Candidate cloning and cRNA preparation

[0333] The cloning of candidate CDSs was performed as described in Example 1 , using primers designed to anneal to the beginning and the end of each CDS. cRNA preparation was also performed as in Example 1 with the following modifications: After the in vitro transcription, cRNA was purified using AcroPrep™ Filter Plates (Cytiva) with centrifugation at 5000 rpm for 10min. For each candidate, the concentration of the purified cRNA was normalized to 500 ng / pl and aliquoted prior to storing at -20°C.

[0334] Exporter screen and functional characterization in Xenopus oocytes

[0335] The preparation of plant extracts was performed as described in Example 1 with some modifications:

[0336] In brief, 6 g homogenized plant tissue was resuspended in 150 mL of 80 % methanol with 0.06 % formic acid. The flow-through was distributed in 6.5-ml portions in 15-ml centrifuge tubes before evaporation. The evaporation was performed using a SpeedVac at 1000 rpm for 8 h. The dried extract was dissolved in 8 ml Kulori buffer (90 mM NaCI, 1 mM KCI, 1 mM MgCI2, 1 mM CaCI2, 10 mM HEPES).

[0337] The expression of candidates in Xenopus oocytes was achieved by microinjection of cRNA as described in Example 1. The diffusion-based export assays were then carried out in two steps (Fig. 10). In step 1 , QAs were introduced into the cytosol of Xenopus oocytes by incubating the oocytes in a lupin plant extract in Kulori buffer at pH 8.5. P7181 PC00

[0338] After one hour, the oocytes were washed three times in Kulori buffer pH 8.5, and some of the oocytes were harvested as controls (reference; “Oocytes #1” in Fig. 10). In step 2, we allowed for QA export by incubating the remaining oocytes in Kulori buffer at pH 5.5, mimicking the plant cell apoplast. For step 2, we used 96-well Il-bottom microtiter plates (Greiner Bio-One), with 150 mL of buffer and 3 oocytes per well. The incubated oocytes and a sample of the external medium were collected after 1 hour (“Oocytes #2” and “Medium #2” in Fig. 10, respectively).

[0339] Analysis of QAs using LC-MS

[0340] The preparation of oocyte samples was performed as described in Example 1 but using an extractant consisting of 65% MeOH, 0.06% formic acid, and 8 pM caffeine (internal standard). For the preparation of media samples, 10 pL of medium were diluted in 90 pL of extractant consisting of 16.25% MeOH, 0.015% formic acid, 2 pM caffeine). All diluted samples were filtered and QAs were analysed via LC-MS as described in Example 1.

[0341] Results

[0342] Selection of QA exporter candidates

[0343] To select candidate genes potentially involved in QA export from biosynthetic cells, we used a tissue-specific transcriptome of NLL (cv. Oskar) generated by laser-capture microdissection (Frick et al., 2023). In total, 16 candidate transporter genes were selected. The expression pattern of these candidates are visualized in Fig. 9. All candidates followed a similar expression pattern as the QA biosynthesis genes LDC (Bunsupa et al., 2012) and CAO (Yang et al., 2017), with relatively high expression in epidermal tissues and relatively low expression in other tissues. The selected genes coded for transporters of known families such as PUP, NPF, amino acid, and sugar transporters, as well as a few proteins of unknown function.

[0344] Diffusion-based QA export screen

[0345] After testing the diffusion of QAs from lupin extracts into Xenopus oocytes at different basic pHs (not shown), we designed a diffusion-based export assay with two incubation steps (Fig. 10). In step 1, we introduced QAs into the cytosol of oocytes by incubating cRNA-injected oocytes in a lupin extracts at pH 8.5. After one hour, some oocytes were harvested as a baseline reference for internal QA content (Oocytes #1 in Fig. 10). In step 2, we incubated the remaining oocytes in a clean Kulori buffer at pH 5.5 to mimic P7181 PC00 the plant cell apoplast. After one hour, these oocytes (Oocytes #2 in Fig. 10) were harvested and compared to Oocytes #1 with respect to QA content. We also took a sample of the buffer after incubation (Medium #2) for comparison to water-injected oocytes.

[0346] We grouped our 16 candidates into 4 pools and carried out a diffusion-based QA export screen (Fig. 11). Based on diffusibility under step 1 of our assay (QA-loading step), we only quantified lupanine, angustifoline, 13-coumaroyloxylupanine, 13-trans- cinnamoyloxylupanine, and 13-cis-cinnamoyloxylupanine. The results showed that only lupanine and angustifoline were retained inside the water-injected oocytes after the incubation clean buffer (step 2); therefore, we focused on these two QAs. Among the candidate pools, pool 2.9 showed a significant decrease in lupanine and angustifoline levels in oocytes at this incubation step (compare Oocytes #1 with Oocytes #2 in Fig. 11 A). The same pool also showed an increase in lupanine and angustifoline levels in the clean buffer (Medium #2) when comparing to water-injected oocytes (Fig. 11 B). We set out to identify the potential QA exporter in pool 2.9. The assays were carried out exactly as in the preceding screen, but with the individual candidate genes (HH001L, HH001S, and HH003). The results identified HH001L (gene Lup028431.1 in the nomenclature by Hane et al. 2017; here renamed as LLIP1.3, amino acid sequence of LLIP1.3 is set forth in SEQ ID NO: 7) as the exporter of lupanine and angustifoline, as evidenced by a decrease in QA content in the loaded oocytes upon incubation in clean buffer (Fig. 11 C) and an increase in QA content in said buffer (Fig. 11 D). A replicate of these results is presented in Fig. 14.

[0347] The PUP family may play a role in QA export Protein annotation revealed that HH001 L is a member of the PUP family. In NLL, 11 members of this family were shown in Example 1 to enable QA import in Xenopus oocytes. These PUPs were dubbed as LUPs (lupin alkaloid permeases) in Example 1. In addition, LUP3.1 and LUP3.5 were shown to play a role in loading QAs into the pod vasculature for subsequent transport to the seeds (Example 1). Surprisingly, HH001 L belongs to the same clade, yet previous uptake assays in Xenopus ooctyes had not detected any QA import activity. Following the naming scheme proposed for the LUPs, we renamed HH001 L as LUP1.3. P7181 PC00

[0348] The export activity of LLIP1 .3 suggests that other members of the same clade might also function as QA exporters. Using the same QA export assay, we tested five members of the mentioned PUP clade as well as a PUP transporter just outside this clade (Lup002816) (Fig. 12). First, the closest homolog of LUP1.3 (85.9% identity) was also able to export QAs, and we renamed this transporter LUP1.4 (gene Lup028432.1 in the nomenclature by Hane et al., 2017, amino acid sequence of LUP1 .4 is set forth in SEQ ID NO: 9). Just like LUP1.3, LUP1.4 had previously tested negative for QA import (Example 1). Interestingly, the two LUPs previously implicated in QA import into the pod vasculature, LUP3.1 and LUP3.5, could also act as QA exporters in our export assay. The same was the case for a third LUP highly expressed in the pod vasculature, LUP2.1. This indicates bidirectional transport abilities for LUP3.1 , LUP3.5 and LUP2.1. However, not all the tested LUPs showed QA export activity in our assay. For example, the seed-specific QA importer LUP3.4 tested negative for QA export ability. Finally, the PUP transporter Lup002816 did not show QA export ability and had previously also tested negative for QA import (Example 1).

[0349] Expression patterns of LUP1.3 and LUP1.4

[0350] Analysis of an existing RNAseq dataset revealed that both LUP1 .3 and LUP1 .4 are highly expressed in the epidermis of pod, leaf, and stem tissues, and thus displaying similarity with lupin QA biosynthetic genes such as LDC (Fig. 13). In addition, LUP1.4 is expressed to high levels in the pod mesocarp and to medium levels in the leaf mesophyl.

[0351] Conclusion

[0352] In this experiment, we developed a diffusion-based QA export assay and used it to identity two unidirectional QA exporters, LUP1.3 and LUP1.4, located at the biosynthesis sites.

[0353] The activities and expressions patterns of LUP1 .3 and LUP1 .4 suggest functional redundancy in releasing QAs to the apoplast for long-distance transport. Interestingly, LUP1.4 may also facilitate long-distance transport by preventing the overaccumulation of QAs in the pod mesocarp and in the leaf mesophyll.

[0354] Surprisingly, both LUP1.3 and LUP1.4 belong to the PUP transporter family (data of phylogenetic analysis not shown), which also includes the two transporters identified in P7181 PC00

[0355] Example 1 herein and shown to import QAs into the pod vasculature, LLIP3.1 and LLIP3.5. Using the oocytes diffusion-based assay described herein, we demonstrated that LUP3.1 and LUP3.5 also possess QA export capabilities, thus realising that they have bidirectional transporter capability.

[0356] The results of experiment also indicate that the LUP3.4 transporter identified in Example 1 does not have QA export capabilities, suggesting that it is an exclusive QA importer.

[0357] Example 3: An expanded screen for quinolizidine alkaloid transporters identified a novel importer from the UMAMIT family

[0358] Aim

[0359] To identify QA transporters involved in post-phloem transportation in seeds of bitter narrow-leafed lupin (NLL), i.e. seed transporters.

[0360] Materials and Methods

[0361] RNAseq dataset of seed coat and embryo tissues

[0362] NLL cv. Oskar plants were grown in a growth chamber on commercial peat-based soil. Pots were 20-cm deep with a diameter of 16 cm and housed 3-6 plants each. Growing conditions included 20 hours of light (310 mE), a day / night temperature regime of 20°C / 18°C, and a relative humidity of 60%. Seeds were harvested at approximately 30 days after anthesis, dissected into seed coats and embryos, and stored at -70°C. The Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) was used to extract RNA, which was quality-checked on agarose gels and on a BioAnalyzer (Agilent Technologies). Seed coats and embryos were extracted in triplicate. The RNA was sequenced by Macrogen (South Korea) on a HiSeq2500 Illumina upon construction of paired-end TruSeq libraries. Raw Illumina reads were pre-processed as described by Frick et al., 2023.

[0363] Transcript expression of processed RNAseq data was quantified as described by Frick et al., 2023.

[0364] Isolation and phenotyping of two allelic LUP3.4 knockout mutants

[0365] The mutant library of NLL cv. Oskar was previously constructed as described by Mancinotti et al., 2023. Two allelic knockout mutants in LUP3.4 were identified essentially as described in Example 1 (Q171stop corresponding to C511T, and W330stop corresponding to G990A). The primers and probes used for the respective P7181 PC00

[0366] TaqMan assays are given in Table 7. Heterozygous individuals were taken to homozygosity as described in Example 1. Genotyping was carried out by Sanger sequencing using the primers listed in Table 7. Mature seeds were collected at dissected into seed coats and embryos, and QA analysis was performed as described in Example 1.

[0367] Table 7. SEQ ID NOs of primers and probes (oligonucleotides) used for the isolation of two allelic LLIP3.4 knockout mutants and their genotyping via Sanger sequencing.

[0368] The mutated gene of LLIP3.4 (Q171stop) is as set forth in SEQ ID NO: 73 (LUP3.4_C511T(Q171stop)). The mutated gene of LLIP3.4 (W330stop) is as set forth in SEQ ID NO: 74 {LUP3.4_G990A(W330stop)).

[0369] Transporter candidate selection

[0370] We used the RNAseq dataset of seed coat and embryo described in a previous section to obtain a broad list of candidate seed NLL transporters. We first selected transcripts that were highly expressed in either seed coat or embryo (TPM>20). We then filtered for transcripts encoding proteins with more than 5 transmembrane domains as predicted by the TMHMM server v.2.0 (Krogh et al., 2001) (http: / / www.cbs.dtu.dk / services / TMHMM / ). This led to 180 remaining transcripts. We used BLAST searches to retrieve the respective full-length CDSs from the annotated NLL genome (Hane et al., 2017).

[0371] Expression in Xenopus oocytes and QA uptake assays P7181 PC00

[0372] Transporter candidates were expressed in Xenopus oocytes and screened for QA uptake as described in Example 1.

[0373] Results

[0374] Constructing an RNAseq dataset of seed coat and embryo tissues

[0375] To assess gene expression in seeds, we constructed a tissue-specific RNAseq dataset of seed coat and seed embryo tissues at approximately 30 days after anthesis (DAA). The data was mapped onto the de novo transcriptome by Yang et al., 2017, to give gene expression values in transcripts per million (TPM). This dataset showed that, apart from the expression patterns described in Examples 1 and 2, LLIP3.5 and LLIP1.4 are also expressed in the seed coat (Fig. 19).

[0376] LUP3.4 contributes marginally to QA transport into embryos

[0377] Among the 13 PUP transporters from NLL able to mediate QA transport in Xenopus oocytes (LUPs) (Examples 1 and 2), we chose LUP3.4 for further analysis because it was the only one expressed in seeds (Example 1). Our RNAseq dataset of seeds tissues revealed that LUP3.4 was exclusively expressed in the embryo. Previous transport assays had shown the ability of LUP3.4 to mediate QA import into oocytes (Example 1). In contrast to the LUPs involved in phloem loading (LUP3.1 and LUP3.5), LUP3.4 had tested negative for QA export from oocytes (Example 2). This implied a unidirectional transport mechanism and suggested a role in QA import into the embryo.

[0378] We isolated an early stop codon mutant of LUP3.4 (Q171 stop) from the previously constructed mutant NLL library obtained using low amounts of EMS mutagen. Compared to wildtype (WT) siblings, QA contents were marginally lower in the mature embryo, significantly so for 13-hydroxylupanine and angustifoline. At the same time, the trace amounts of QAs normally left in the mature seed coat were increased multiple fold for all three core QAs. The QA content in whole seeds (measured independently) did not change (Fig. 16).

[0379] We isolated a second early stop codon mutant of LUP3.4 (W330Stop). This mutant showed a similar chemotype to the allelic mutant described above. However, the QA reduction in the embryo was only significant for 13-hydroxylupanine, and the QA increase in the seed coat was only significant for angustifoline and 13-hydroxylupanine (Fig. 17). The subtle phenotype of both LUP3.4 knockout mutants indicates a role in P7181 PC00 import of QAs into embryos and suggest the additional involvement of other transporters.

[0380] Identification of a novel QA transporter expressed in seeds of NLL

[0381] To find QA transporters in NLL seeds (Fig. 15), we selected and cloned 180 genes that were highly expressed in either embryos or seed coats (TPM>20) and coded for proteins with more than 5 predicted transmembrane domains. We grouped the genes into 33 pools and tested their QA import ability in Xenopus oocytes using an NLL plant extract. Among these pools, only one (Pool 4.3) showed higher QA intake compared to the water-injected control (Fig. 18A). Interestingly, none of the candidates in this pool belonged to the PUP transporter family.

[0382] To identify the seed QA transporter in pool 4.3, we tested the candidate genes individually in Xenopus oocytes. Among them, only HH130 (corresponding to Lup017053.1.1, SEQ ID NO: 5 encoded by SEQ ID NO: 6) mediated significant uptake for all QAs (Fig. 18B). Like LUP3.4, HH130 is expressed highly in the embryo and is not expressed in the seed coat (Fig. 19). However, unlike LUP3.4 (which is exclusively expressed in the seed), HH130 is also expressed in other organs, with consistently high expression in the vasculature (Fig. 19). Combined with its QA uptake ability, the expression patterns of HH130 indicates a functional redundancy compared not only to the embryo importer LUP3.4 but also to the phloem loaders LUP3.1 and LUP3.5.

[0383] Conclusion

[0384] In this experiment, we show that LUP3.4 contributes to the import of QAs into the seed embryo; however, given the subtle phenotype of the knockout mutant, other transporters must also be involved.

[0385] We also used the QA uptake assay in Xenopus oocytes as described in Example 1 to identify the QA transporter HH130. The gene encoding HH130 corresponds to gene Lup017053.1.1 (SEQ ID NO: 6) in the NLL genome draft (Hane et al., 2017), where it is annotated as a WALLS ARE THIN 1 (WATI)-like protein and is thus not a PUP in contrast to the QA transporters of Examples 1 and 2.

[0386] Given the expression of HH 130 in both the vasculature and the seed embryo of NLL and its proven QA import capability, the experiments presented in Example 3 suggests P7181 PC00 that HH130 has redundant activity with each of LLIP3.1 and LLIP3.5 in the vasculature, and with LLIP3.4 in the seed embryo.

[0387] Finally, the observed expression of LLIP1.4 and LLIP3.5 in the seed coat suggests a combined involvement in QA export from this tissue.

[0388] Example 4: Generation of mutants

[0389] Aim

[0390] To generate NNLs having a mutation in HH130 or both HH130 and LUP3.4.

[0391] Generation of the HH130 mutant

[0392] The previously constructed narrow-leafed lupin (NLL, Lupinus angustifolius) cv. Oskar mutant library (Mancinotti et al., 2023) was screened using the FIND-IT technology as described in Knudsen et al., 2022 to identify a knockout mutation in the HH130 gene.

[0393] A homozygous seed of mutant HH130 (W201stop) was identified by using TaqMan assays with the following primers: HH130 target specific forward primer of SEQ ID NO: 60, HH130 target specific reverse primer of SEQ ID NO: 61 , HH130 mutant specific probe (FAM labelled) of SEQ ID NO: 62, and HH130 wildtype specific probe (HEX labelled) of SEQ ID NO: 63.

[0394] The seed was sown in 16 cm-wide, 20 cm-deep pots in commercial potting mix (Pindstrup Faerdigblanding 2), and grown in a growth chamber at 21 °C / 19°C day / night temperature, 16h photoperiod, and 300 pmol m-2 s-1 light intensity. The plant was genotyped by Sanger sequencing of the mutated region using the HH130 target specific forward primer (SEQ ID NO: 60) and the HH130 target specific reverse primer (SEQ ID NO: 61), and the genotype was confirmed.

[0395] Generation of the double HH130 and LUP3.4 mutant

[0396] To generate a LLIP3.4 (W330stop) / HH130 (W201stop) double mutant of NLL cv. Oskar, the homozygous single mutants (generated as described in Examples 3 and 4, respectively) were crossed by first emasculating a few flowers at their immature stage (before another extension past the pistil) and then a day later fertilizing the emasculated flowers with mature pollen from the paternal plant. The LUP3.4 (W330stop) and HH130 P7181 PC00

[0397] (W201stop) mutants were crossed both ways by having both genotypes act as maternal and paternal plant in different crosses.

[0398] The mutated gene of LLIP3.4 (W330stop) is as set forth in SEQ ID NO: 74 (UJP3.4_G990A(W330stop)). The mutated gene of HH130 (W201stop) is as set forth in SEQ ID NO: 76 {HH130_G603A(W201stop)).

[0399] Seeds from this crossing are tested for QA content (chemotyping) essentially as described in Example 1 and are expected to have a reduced QA contents.

[0400] Conclusion

[0401] In this experiment a HH130 (W201stop) single mutant of NLL cv. Oskar as well as a LLIP3.4 (W330stop) / HH130 (W201stop) double mutant of NLL cv. Oskar was successfully obtained.

[0402] Example 5: Chemotyping of LU P3. 1 and LUP3.5 single and double mutants

[0403] Aim

[0404] To chemotype LUP3.1 and LUP3.5 single and double mutants in comparison to a wildtype plant of narrow-leafed lupin (NLL, Lupinus angustifolius) cv. Oskar.

[0405] Materials and Methods

[0406] We carried out an additional round of chemotyping for the single and double mutants of LUP3.1 and LUP3.5 (generated as described in Example 1) in comparison to a wildtype plant, where we analyzed QA content in mature seeds and developing pods.

[0407] The experiment was performed under the same conditions described in Example 1 , except that a different growth chamber was used. Only fully filled, white seeds, were harversted for chemotyping.

[0408] Results

[0409] In this round of chemotyping, the double mutant LUP3.1 / LUP3.5 again accumulated lower QA levels in mature seeds compared to the wildtype plant. QA levels in developing pods of the double mutant were again increased compared to the wildtype plant. P7181 PC00

[0410] Conclusion

[0411] A reduction in QA content was observed for the double mutant LUP3.1 / LUP3.5 compared to the wildtype plant. Sequence overview P7181PC00 P7181PC00 P7181PC00 P7181PC00 P7181PC00 P7181PC00 P7181PC00 P7181PC00 P7181PC00 P7181 PC00

[0412] SEQ ID NO: 72 - LUP3.1_G360A(Trp120Stop), synthetic construct, nucleic acid

[0413] SEQ ID NO: 73 - LUP3.4_C511T(Q171stop), synthetic construct, nucleic acid

[0414] SEQ ID NO: 74 - LUP3.4_G990A(W330stop), synthetic construct, nucleic acid

[0415] SEQ ID NO: 75 - LUP3.5_G138A(Trp46Stop), synthetic construct, nucleic acid

[0416] SEQ ID NO: 76 - HH130_G603A(W201 stop) , synthetic construct, nucleic acid P7181 PC00

[0417] References

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[0422] Hane et al., 2017. A comprehensive draft genome sequence for lupin (Lupinus angustifolius), an emerging health food: insights into PhD Thesis - Transport engineering in narrow-leafed lupin 23 plant-microbe interactions and legume evolution. Plant Biotechnology Journal, 15(3). https: / / doi.org / 10.1111 / pbi.12615

[0423] Holme et al., 2017. Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR / Cas9 and TALENs. Plant Mol Biol 95, 111-121. DOI 10.1007 / sl 1103-017-0640-6

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[0432] Mancinotti et al., 2021. Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin. Plant Methods, 17(1). https : / / do i . org / 10.1186 / s 13007-021 -00832-4

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[0437] Otterbach et al., 2019. Quinolizidine alkaloids are transported to seeds of bitter narrow- leafed lupin. Journal of Experimental Botany, 70(20), 5799-5808. https: / / doi.Org / 10.1093 / jxb / erz334

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[0440] Sun et al., 2016. Precise Genome Modification via Sequence-Specific Nucleases- Mediated Gene Targeting for Crop Improvement. Front Plant Sci. 2016 Dec 20;7:1928. doi: 10.3389 / fpls.2016.01928.

[0441] Yang et al., 2017. Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes. Journal of Experimental Botany, 68(20). https: / / d0i.0rg / l 0.1093 / jxb / erx362

[0442] Zhang et al., 2007. Review: Nutrient loading of developing seeds. Functional Plant Biology, 34(4). https: / / doi.org / 10.1071 / FP06271 P7181 PC00

[0443] Fenn D et al., 2022, Physicochemical, anti- nutritional, and functional properties of airclassified protein concentrates from commercially grown Canadian yellow pea (Pisum sativum) varieties with variable protein levels, Cereal Chemistry. 2022;99:157-168, DOI: 10.1002 / cche.10506

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[0445] Lawrenson et al., Genome Biology (2015) 16:258; DOI 10.1186 / s13059-015-0826-7

[0446] Items

[0447] 1. A quinolizidine alkaloid (QA)-containing legume or part thereof, wherein said QA- containing legume carries a mutation in at least one gene encoding a QA transport protein, preferably wherein said QA-containing legume has reduced content of one or more QAs in the seeds, compared to a corresponding QA-containing legume not comprising said mutation.

[0448] 2. The QA-containing legume or part thereof according to item 1 , wherein said QA transport protein is LUP3.1 , LUP3.5, HH130, LUP1.3, or LUP1.4, and wherein said mutation is one or more of: i. a mutation in the LUP3. 1 gene leading to reduced LUP3.1 activity or loss-of- function of LUP3.1 ; ii. a mutation in the LUP3.5 gene leading to reduced LUP3.5 activity or loss-of- function of LUP3.5; and / or iii. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; or wherein said mutation is one or more of: iv. a mutation in the LUP1.3 gene leading to a reduced LUP1.3 activity or loss of function of LUP1.3; and / or v. a mutation in the LUP1.4 gene leading to reduced LUP1.4 activity or loss of function of LUP1.4.

[0449] 3. The QA-containing legume or part thereof according to any one of the preceding items, wherein said QA transport protein is LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , or LUP5.1 , and wherein said mutation is one or more of: P7181 PC00 i. a mutation in the LUP3. 1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1; ii. a mutation in the LUP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5; iii. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; iv. a mutation in the LUP1.3 gene leading to a reduced LLIP1.3 activity or loss of function of LLIP1.3; v. a mutation in the LUP1.4 gene leading to reduced LLIP1.4 activity or loss of function of LLIP1.4; vi. a mutation in the LLIP1.1 gene leading to reduced LLIP1.1 activity or loss-of- function of LU P 1.1 ; vii. a mutation in the LUP1.2 gene leading to reduced LUP1.2 activity or loss-of- function of LUP1.2; viii. a mutation in the LUP2.1 gene leading to reduced LUP2.1 activity or loss-of- function of LUP2.1 ; ix. a mutation in the LUP2.2 gene leading to reduced LUP2.2 activity or loss-of- function of LUP2.2; x. a mutation in the LUP3.2 gene leading to reduced LUP3.2 activity or loss-of- function of LUP3.2; xi. a mutation in the LUP3.3 gene leading to reduced LUP3.3 activity or loss-of- function of LUP3.3; xii. a mutation in the LUP3.4 gene leading to reduced LUP3.4 activity or loss-of- function of LUP3.4; xiii. a mutation in the LUP4.1 gene leading to reduced LUP4.1 activity or loss-of- function of LUP4.1 ; and / or xiv. a mutation in the LUP5.1 gene leading to reduced LUP5.1 activity or loss-of- function of LUP5.1.

[0450] 4. A quinolizidine alkaloid (QA)-containing legume or part thereof, wherein said QA- containing legume carries one or more of: i. a mutation in the LUP3. 1 gene leading to reduced LUP3.1 activity or loss-of- function of LUP3.1; ii. a mutation in the LUP3.5 gene leading to reduced LUP3.5 activity or loss-of- function of LUP3.5; and / or P7181 PC00 iii. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; or wherein said QA-containing legume carries one or more of: iv. a mutation in the LUP1.3 gene leading to a reduced LLIP1.3 activity or loss of function of LLIP1.3; and / or v. a mutation in the LUP1.4 gene leading to reduced LLIP1.4 activity or loss of function of LLIP1.4.

[0451] 5. A quinolizidine alkaloid (QA)-containing legume or part thereof, wherein said QA- containing legume carries one or more of: i. a mutation in the LUP3. 1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1; ii. a mutation in the LUP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5; iii. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; iv. a mutation in the LUP1.3 gene leading to a reduced LLIP1.3 activity or loss of function of LLIP1.3; v. a mutation in the LUP1.4 gene leading to reduced LLIP1.4 activity or loss of function of LLIP1.4; vi. a mutation in the LUP1. 1 gene leading to reduced LLIP1.1 activity or loss of function of LU P 1.1 ; vii. a mutation in the LUP1.2 gene leading to reduced LUP1.2 activity or loss of function of LUP1.2; viii. a mutation in the LUP2. 1 gene leading to reduced LUP2.1 activity or loss of function of LUP2.1 ; ix. a mutation in the LUP2.2 gene leading to reduced LUP2.2 activity or loss of function of LUP2.2; x. a mutation in the LUP3.2 gene leading to reduced LUP3.2 activity or loss of function of LUP3.2; xi. a mutation in the LUP3.3 gene leading to reduced LUP3.3 activity or loss of function of LUP3.3; xii. a mutation in the LUP3.4 gene leading to reduced LUP3.4 activity or loss of function of LUP3.4; P7181 PC00 xiii. a mutation in the LUP4. 1 gene leading to reduced LLIP4.1 activity or loss of function of LLIP4.1 ; and / or xiv. a mutation in the LUP5. 1 gene leading to reduced LLIP5.1 activity or loss of function of LLIP5.1.

[0452] 6. The QA-containing legume according to any one of the preceding items, wherein each of LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1, LUP1.2, LUP2.1, LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1, and LUP5.1 is a QA transport protein.

[0453] 7. The QA-containing legume or part thereof according to any one of the preceding items, wherein said QA transport protein is LLIP3.1 as set forth in SEQ ID NO: 1, LUP3.5 as set forth in SEQ ID NO: 3, HH130 as set forth in SEQ ID NO: 5, LUP1.3 as set forth in SEQ ID NO: 7, LLIP1.4 as set forth in SEQ ID NO: 9, LLIP1.1 as set forth in SEQ ID NO: 13, LUP1.2 as set forth in SEQ ID NO: 15, LUP2.1 as set forth in SEQ ID NO: 21 , LUP2.2 as set forth in SEQ ID NO: 27, LUP3.2 as set forth in SEQ ID NO: 11, LUP3.3 as set forth in SEQ ID NO: 25, LUP3.4 as set forth in SEQ ID NO: 23, LUP4.1 as set forth in SEQ ID NO: 19, or LUP5.1 as set forth in SEQ ID NO: 17, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, such as at least 71% sequence identity, such as at least 72% sequence identity, such as at least 73% sequence identity, such as at least 74% sequence identity, such as at least 75% sequence identity, such as at least 76% sequence identity, such as at least 78% sequence identity, such as at least 79% sequence identity, such as at least 80% sequence identity, such as at least 81% sequence identity, such as at least 82% sequence identity, such as at least 83% sequence identity, such as at least 84% sequence identity, such as at least 85% sequence identity, such as at least 86% sequence identity, such as at least 87% sequence identity, such as at least 89% sequence identity, such as at least 90% sequence identity, such as at least 91% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 94% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto. P7181 PC00

[0454] 8. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LUP3. 1 gene is a gene encoding LLIP3.1 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto.

[0455] 9. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LUP3. 1 gene comprises the coding sequence as set forth in SEQ ID NO: 2 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 2, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0456] 10. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LUP3.5 gene is a gene encoding LLIP3.5 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto.

[0457] 11. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LUP3.5 gene comprises the coding sequence as set forth in SEQ ID NO: 4 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 4, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0458] 12. The QA-containing legume or part thereof according to any one of the preceding items, wherein the HH130 gene is a gene encoding HH130 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0459] 13. The QA-containing legume or part thereof according to any one of the preceding items, wherein the HH130 gene comprises the coding sequence as set forth in SEQ ID NO: 6 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 6, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0460] 14. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.1 activity and / or the LLIP3.5 activity is QA import protein activity, preferably wherein said QA import protein activity comprises import of said one or more QAs into the vasculature of said QA-containing legume, such as of said lupin plant.

[0461] 15. The QA-containing legume or part thereof according to any one of the preceding items, wherein the HH130 activity and / or the LLIP3.4 activity is QA import protein activity, preferably wherein said QA import protein activity comprises import of said one or more QAs into the seeds, such as into the seed embryos, of said QA- containing legume, and / or wherein the HH130 activity comprises import of said one or more QAs into the pod vasculature, preferably wherein said QA-containing legume is a lupin plant.

[0462] 16. The QA-containing legume or part thereof according to any one of the preceding items, wherein the QA import protein activity is reduced.

[0463] 17. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.1 activity and / or the LLIP3.5 activity is QA export protein activity, optionally wherein said QA export protein activity of LLIP3.5 comprises export of said one or more QAs from the seeds, such as the seed coats, of said QA- containing legume, such as of sad lupin plant. P7181 PC00

[0464] 18. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.1 activity and / or the LLIP3.5 activity is QA export protein activity and wherein said QA is at least one of lupanine, 13-hydroxylupanine and esters thereof, and angustifoline.

[0465] 19. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.1 activity, the LLIP3.5 activity, and / or the HH130 activity is QA import protein activity and wherein said QA is at least one of lupanine, 13-hydroxylupanine and esters thereof, and angustifoline.

[0466] 20. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.3 gene is a gene encoding LLIP1.3 of SEQ ID NO: 7 or a functional homologue thereof having at least 70% sequence identity thereto.

[0467] 21 . The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.3 gene comprises the coding sequence as set forth in SEQ ID NO: 8 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 8, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0468] 22. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.4 gene is a gene encoding LLIP1.4 of SEQ ID NO: 9 or a functional homologue thereof having at least 70% sequence identity thereto.

[0469] 23. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.4 gene comprises the coding sequence as set forth in SEQ ID NO: 10 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 10, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at P7181 PC00 least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0470] 24. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.3 activity and / or the LLIP1.4 activity is QA export protein activity, wherein said QA export protein activity comprises export of said QA from QA source tissue, such as biosynthetic tissue of said QAs, and / or from the seed coat of seeds, of said QA-containing legume, optionally wherein said biosynthetic tissue is the epidermis of one or more of pods, leaves, and / or stems.

[0471] 25. The QA-containing legume or part thereof according to any one of the preceding items, wherein the QA export protein activity is reduced.

[0472] 26. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.3 activity and / or the LLIP1.4 activity is QA export protein activity and wherein said QA is at least one of lupanine, 13-hydroxylupanine and esters thereof, and angustifoline.

[0473] 27. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.1 gene is a gene encoding LLIP1.1 of SEQ ID NO: 13 or a functional homologue thereof having at least 70% sequence identity thereto.

[0474] 28. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.1 gene comprises the coding sequence as set forth in SEQ ID NO: 14 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 14, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto. P7181 PC00

[0475] 29. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.2 gene is a gene encoding LLIP1.2 of SEQ ID NO: 15 or a functional homologue thereof having at least 70% sequence identity thereto.

[0476] 30. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP1.2 gene comprises the coding sequence as set forth in SEQ ID NO: 16 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 16, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0477] 31. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP2.1 gene is a gene encoding LLIP2.1 of SEQ ID NO: 21 or a functional homologue thereof having at least 70% sequence identity thereto.

[0478] 32. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP2.1 gene comprises the coding sequence as set forth in SEQ ID NO: 22 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 22, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0479] 33. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP2.2 gene is a gene encoding LLIP2.2 of SEQ ID NO: 27 or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0480] 34. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP2.2 gene comprises the coding sequence as set forth in SEQ ID NO: 28 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 28, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0481] 35. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.2 gene is a gene encoding LLIP3.2 of SEQ ID NO: 11 or a functional homologue thereof having at least 70% sequence identity thereto.

[0482] 36. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.2 gene comprises the coding sequence as set forth in SEQ ID NO: 12 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 12, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0483] 37. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.3 gene is a gene encoding LLIP3.3 of SEQ ID NO: 25 or a functional homologue thereof having at least 70% sequence identity thereto.

[0484] 38. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.3 gene comprises the coding sequence as set forth in SEQ ID NO: 26 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 26, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at P7181 PC00 least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0485] 39. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.4 gene is a gene encoding LLIP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto.

[0486] 40. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP3.4 gene comprises the coding sequence as set forth in SEQ ID NO: 24 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 24, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0487] 41. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP5.1 gene is a gene encoding LLIP5.1 of SEQ ID NO: 17 or a functional homologue thereof having at least 70% sequence identity thereto.

[0488] 42. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP5.1 gene comprises the coding sequence as set forth in SEQ ID NO: 18 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 18.

[0489] 43. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP4.1 gene is a gene encoding LLIP4.1 of SEQ ID NO: 19 or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0490] 44. The QA-containing legume or part thereof according to any one of the preceding items, wherein the LLIP4.1 gene comprises the coding sequence as set forth in SEQ ID NO: 20 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 20, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

[0491] 45. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3. 1 gene, said mutation leading to reduced LLIP3.1 activity or loss-of-function of LLIP3.1, and a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5.

[0492] 46. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3. 1 gene comprising the coding sequence as set forth in SEQ ID NO: 2 and a mutation in the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0493] 47. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3. 1 gene, said mutation leading to reduced LLIP3.1 activity or loss-of-function of LLIP3.1, a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5, and a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130. P7181 PC00

[0494] 48. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3. 1 gene comprising the coding sequence as set forth in SEQ ID NO: 2, a mutation in the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4, and a mutation in the HH130 gene comprising the coding sequence as set forth in SEQ

[0495] ID NO: 6, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0496] 49. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP1.3 gene, said mutation leading to reduced LLIP1.3 activity or loss-of-function of LLIP1.3, and a mutation in the LUP1.4 gene, said mutation leading to reduced LLIP1.4 activity or loss-of-function of LLIP1.4.

[0497] 50. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP1.3 gene comprising the coding sequence as set forth in SEQ ID NO: 8 and a mutation in the LUP1.4 gene comprising the coding sequence as set forth in SEQ

[0498] ID NO: 10, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0499] 51. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5, and a mutation in the LUP1.4 gene, said mutation leading to reduced LLIP1.4 activity or loss-of-function of LLIP1.4. P7181 PC00

[0500] 52. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3.5 gene comprising the coding sequence as set forth in SEQ ID NO: 4 and a mutation in the LUP1.4 gene comprising the coding sequence as set forth in SEQ ID NO: 10, or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0501] 53. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3.4 gene, said mutation leading to reduced LLIP3.4 activity or loss-of-function of LLIP3.4, and a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130.

[0502] 54. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the LUP3.4 gene comprising the coding sequence as set forth in SEQ ID NO: 24, and a mutation in the HH130 gene comprising the coding sequence as set forth in SEQ ID NO: 6, or a functional homologue of any one of the aforementioned at least 70% sequence identity thereto.

[0503] 55. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130.

[0504] 56. The QA-containing legume or part thereof according to any one of the preceding items, wherein said plant carries a mutation in the HH130 gene comprising the coding sequence as set forth in SEQ ID NO: 6, or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0505] 57. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene is one of the following mutations: i. a mutation leading to a premature stop codon; ii. a mutation in a splice site; iii. a frame-shift mutation; iv. deletion of the entire LUP3. 1 gene, the LUP3.4 gene, LUP3.5 gene, HH130 gene, LUP1.3 gene, and / or LUP1.4 gene or a part thereof; v. a mutation in the active site of LLIP3.1, LLIP3.4, LLIP3.5, HH130, LLIP1.3 and / or LUP1.4 leading to reduced or abolished LUP3.1, LUP3.4, LUP3.5, HH130, LLIP1.3 and / or LLIP1.4 activity or; vi. a mutation in the promoter region regulating transcription of the LUP3. 1 gene, the LUP3.4 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene leading to reduced or abolished transcription or translation.

[0506] 58. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3. 1 gene is a premature stop codon.

[0507] 59. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at any one of codons 1 to 120 of the LUP3.1 gene.

[0508] 60. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at codon 120 of the LUP3.1 gene.

[0509] 61. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.1 gene results in a gene encoding a mutated protein lacking at least the 237 most C-terminal amino acids of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0510] 62. The QA-containing legume or part thereof according to any one of the preceding items, wherein the QA-containing legume carries a guanine to adenine mutation at a position corresponding to nucleotide 360 of SEQ ID NO: 2 or a functional homologue thereof having at least 70% sequence identity thereto.

[0511] 63. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3. 1 gene introduces a premature stop codon positioned at codon 120 and / or wherein the mutated LUP3. 7_gene is LUP3. 1 G360A(Trp120Stop) of SEQ ID NO: 72 or a functional homologue thereof having at least 70% sequence identity thereto.

[0512] 64. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.4 gene is a premature stop codon.

[0513] 65. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at any one of codons 1 to 171 or at any one of codons 1 to 330 of the LUP3.4 gene.

[0514] 66. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at codon 171 of the LUP3.4 gene or at codon 330 of the LUP3.4 gene.

[0515] 67. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.4 gene results in a gene encoding a mutated protein lacking at least the 192 most C-terminal amino acids of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto or at least the 33 most C-terminal amino acids of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto.

[0516] 68. The QA-containing legume or part thereof according to any one of the preceding items, wherein the QA-containing legume carries a guanine to adenine mutation at a position corresponding to nucleotide 990 of SEQ P7181 PC00

[0517] ID NO: 24 or a functional homologue thereof having at least 70% sequence identity thereto, or a cytosine to thymine mutation at position 511 of SEQ ID NO: 24 or a functional homologue thereof having at least 70% sequence identity thereto.

[0518] 69. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.4 gene introduces a premature stop codon positioned at codon 990 and / or wherein the mutated LUP3.4 gene is LUP3.4_G990A(W330stop) of SEQ ID NO: 74 or a functional homologue thereof having at least 70% sequence identity thereto.

[0519] 70. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.4 gene introduces a premature stop codon positioned at codon 171 and / or wherein the mutated LUP3.4 gene is LUP3.4_C511T(Q171stop) of SEQ ID NO: 73 or a functional homologue thereof having at least 70% sequence identity thereto.

[0520] 71. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.5 gene is a premature stop codon.

[0521] 72. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at any one of codons 1 to 46 of the LUP3.5 gene.

[0522] 73. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at codon 46 of the LUP3.5 gene.

[0523] 74. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.5 gene results in a gene encoding a mutated protein lacking at least the 321 most C-terminal amino acids of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0524] 75. The QA-containing legume or part thereof according to any one of the preceding items, wherein the QA-containing legume carries a guanine to adenine mutation at a position corresponding to nucleotide 138 of SEQ ID NO: 4 or a functional homologue thereof having at least 70% sequence identity thereto.

[0525] 76. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the LUP3.5 gene introduces a premature stop codon positioned at codon 46 and / or wherein the mutated LUP3.5 gene is LUP3.5_G138A(Trp46Stop) of SEQ ID NO: 75 or a functional homologue thereof having at least 70% sequence identity thereto.

[0526] 77. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the HH130 gene is a premature stop codon.

[0527] 78. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at any one of codons 1 to 201 of the HH130 gene.

[0528] 79. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation is a premature stop codon positioned at codon 201 of the HH130 gene.

[0529] 80. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the HH130 gene results in a gene encoding a mutated protein lacking at least the 178 most C-terminal amino acids of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto.

[0530] 81. The QA-containing legume or part thereof according to any one of the preceding items, wherein the QA-containing legume carries a guanine to adenine mutation at a position corresponding to nucleotide 603 of SEQ ID NO: 6 or a functional homologue thereof having at least 70% sequence identity thereto. P7181 PC00

[0531] 82. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutation in the HH130 gene introduces a premature stop codon positioned at codon 201 and / or wherein the mutated HH130 gene is HH130_G603A(W201stop) of SEQ ID NO: 76 or a functional homologue thereof having at least 70% sequence identity thereto.

[0532] 83. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutated LUP3. 1 gene encodes a protein consisting of amino acids 1 to 119 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto.

[0533] 84. The QA-containing legume or part thereof according to any one of the preceding items, wherein mutated LUP3.4 gene encodes a protein consisting of amino acids 1 to 170 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto.

[0534] 85. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutated LUP3.4 gene encodes a protein consisting of amino acids 1 to 329 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto.

[0535] 86. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutated LUP3.5 gene encodes a protein consisting of amino acids 1 to 45 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto.

[0536] 87. The QA-containing legume or part thereof according to any one of the preceding items, wherein the mutated HH130 gene encodes a protein consisting of amino acids 1 to 200 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto.

[0537] 88. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the QA content in the seeds, preferably the mature seeds, of said QA-containing legume is reduced to at the most 75% of the content in a corresponding QA-containing legume not comprising said mutation in at least one P7181 PC00 gene encoding a QA transport protein on a dry seed weight basis, such as at the most 70 % of the content in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein on a dry seed weight basis, such as at the most 65 %, such as at the most 60%, such as at the most 55%, such as at the most 50%, such as at the most 45%, such as at the most 40%, such as at the most 35%, such as at the most 30%, such as at the most 25 %, such as at the most 20%, or less, compared to the content in a corresponding QA- containing legume not comprising said mutation in at least one gene encoding a QA transport protein.

[0538] 89. The QA-containing legume or a part thereof according any one of the preceding items, wherein the lupanine content in the seeds, preferably the mature seeds, of said QA-containing legume is reduced to at the most 50 % of the content of said lupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 40 %, such as at the most 35 % of the content of said lupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 30 %, such as at the most 25 %, such as at the most 20 %, or less, wherein the reduction is on a dry seed weight basis, preferably wherein the QA-containing legume is as defined in any one of items 45 to 56, such as in items 45 to 46 or 47 to 48.

[0539] 90. The QA-containing legume or a part thereof according to any one of items 45 to 48, wherein the 13-hydroxylupanine content in the seeds, preferably the mature seeds, of said QA-containing legume at the most 75% of the content of said 13-hydroxylupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 70%, such as at the most 65 % of the content of said 13-hydroxylupanine in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 60 %, such as at the most 55 %, such as at the most 50 %, such as at the most 40%, or less, wherein the reduction is on a dry seed weight basis, preferably wherein the QA-containing legume is as defined in any one of items 45 to 56, such as in items 45 to 46 or 47 to 48. P7181 PC00

[0540] 91 . The QA-containing legume or a part thereof according to any one of items 45 to 48, wherein the angustifoline content in the seeds, preferably the mature seeds, of said QA-containing legume at the most 70 % of the content of said angustifoline in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 65 % of the content of said angustifoline in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein, such as at the most 60 %, such as at the most 55 %, such as at the most 50 %, such as at the most 45 %, such as at the most 40 %, wherein the reduction is on a dry seed weight basis, preferably wherein the QA-containing legume is as defined in any one of items 45 to 56, such as in items 45 to 46 or 47 to 48.

[0541] 92. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 0.02 % QAs, such as lupin QAs, wherein the % is indicated as percentage (%) of dry seed weight, wherein the % is indicated as % of the dry seed weight, preferably wherein the QA-containing legume is as defined in any one of items 45 to 56, for example in items 47 to 50, such as in items 45 to 46 or 47 to 48.

[0542] 93. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 0.02 % lupanine, 13-hydroxylupanine and / or esters of 13-hydroxylupanine, and angustifoline, wherein the % is indicated as % of the dry seed weight, preferably wherein the QA-containing legume is as defined in any one of items 45 to 56, for example in items 47 to 50, such as in items 45 to 46 or 47 to 48.

[0543] 94. The QA-containing legume or a part thereof according to item 93, wherein the esters of 13-hydroxylupanine are one or more of 13-trans- cinnamoyloxylupanine, 13-hydroxydihydrocoumaroyloxylupanine, 13-cis- cinnamoyloxylupanine, 13-coumaroyloxylupanine, and 13-tigloyloxylupanine. P7181 PC00

[0544] 95. The QA-containing legume or part thereof according to any one of the preceding items, wherein the reduced QA content is a reduced QA content in the mature seeds.

[0545] 96. The QA-containing legume or part thereof according to any one of the preceding items, wherein the reduced QA content in the seeds is a reduced QA content in the mature seeds.

[0546] 97. The QA-containing legume or part thereof according to any one of the preceding items, wherein the QA content is increased in the pods, leaves, such as in the leaves, and / or stem, of said QA-containing legume.

[0547] 98. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 1.5% QAs, such as at the most 1.4%, such as at the most 1 .35%, such as at the most 1.33%, such as at the most 1.3% QAs, or less QAs, wherein the % is indicated as % of the dry seed weight.

[0548] 99. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 1.5% QAs, such as at the most 1.4%, such as at the most 1 .35%, such as at the most 1.33%, such as at the most 1.3% QAs, or less QAs, wherein the % is indicated as % of the dry seed weight, wherein said QAs are lupanine, angustifoline, and 13-hydroxylupanine.

[0549] 100. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 0.35% lupanine, such as at the most 0.3%, such as at the most 0.28%, such as at the most 0.26%, such as at the most 0.25% lupanine, or less lupanine, wherein the % is indicated as % of the dry seed weight.

[0550] 101 . The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 1% 13-hydroxylupanine, such as at the P7181 PC00 most 0.9%, such as at the most 0.8%, such as at the most 0.75%, such as at the most 0.7% 13-hydroxylupanine, or less 13-hydroxylupanine, wherein the % is indicated as % of the dry seed weight.

[0551] 102. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, such as the mature seeds, of said lupin plant comprises at the most a total of lupanine, 13-hydroxylupanine, and angustifoline of at the most 0.02%, wherein the % is indicated as % of the dry seed weight, preferably said QA-containing legume is a lupin plant, preferably carrying said mutation in the LUP3. 1 gene, LUP3.5 gene, and the HH130 gene.

[0552] 103. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, such as the mature seeds, of said lupin plant comprises at the most a total of lupanine, 13-hydroxylupanine, and angustifoline of at the most 0.02%, wherein the % is indicated as % of the dry seed weight, preferably said QA-containing legume is a lupin plant, preferably carrying said mutation in the LUP3. 1 gene and the LUP3.5 gene.

[0553] 104. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, such as the mature seeds, of said lupin plant comprises at the most a total of lupanine, 13-hydroxylupanine, and angustifoline of at the most 0.02%, wherein the % is indicated as % of the dry seed weight, preferably said QA-containing legume is a lupin plant, preferably carrying said mutation in the LUP3.4 gene and the HH130 gene.

[0554] 105. The QA-containing legume or a part thereof according to any one of the preceding items, wherein the seeds, preferably the mature seeds, of said QA- containing legume comprises at the most 0.5% angustifoline, such as at the most 0.4%, such as at the most 0.35%, such as at the most 0.32%, such as at the most 0.3% angustifoline, or less angustifoline, wherein the % is indicated as % of the dry seed weight.

[0555] 106. The QA-containing legume, or a part thereof, according to any one of the preceding items, wherein the QA-containing legume has similar agricultural properties compared to a similar QA-containing legume not carrying said mutation in P7181 PC00 one or more of the LUP3. 1 gene, the LUP3.5 gene, the LUP3.4 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene.

[0556] 107. The QA-containing legume, or a part thereof, according to any one of the preceding items, wherein the QA-containing legume has approximately the same yield as a similar QA-containing legume not carrying said mutation in one or more of the LUP3. 1 gene, the LUP3.5 gene, the LUP3.4 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene, when grown under the same conditions.

[0557] 108. The QA-containing legume, or a part thereof, according to any one of the preceding items, wherein the QA-containing legume has not exclusively been obtained by means of an essentially biological process.

[0558] 109. The QA-containing legume or method according to any one of the preceding items, wherein said part of the QA-containing legume is seeds, such as mature seeds.

[0559] 110. A method of reducing or decreasing the QA content in QA-containing legume or part thereof, comprising modifying the activity of at least one QA transport protein in cells of said QA-containing legume.

[0560] 111. The method according to item 110, wherein said QA transport protein is LUP3.1 (SEQ ID NO: 1), LUP3.4 (SEQ ID NO: 23), LUP3.5 (SEQ ID NO: 3), HH130 (SEQ ID NO: 5), LUP1.3 (SEQ ID NO: 7), or LUP1.4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0561] 112. The method according to any one of items 110 to 111 , comprising modifying the activity of at least two of LLIP3.1 (SEQ ID NO: 1), LLIP3.4 (SEQ ID NO: 23), LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, such as modifying the activity of: i. LUP3.1 (SEQ ID NO: 1) and LUP3.5 (SEQ ID NO: 3); ii. LUP3.1 (SEQ ID NO: 1) and HH130 (SEQ ID NO: 5); iii. LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5); P7181 PC00 iv. LLIP3.4 (SEQ ID NO: 23) and HH130 (SEQ ID NO: 5); or v. LUP3.1 (SEQ ID NO: 1), LUP3.5 (SEQ ID NO: 3), and HH130 (SEQ ID NO: 5), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0562] 113. The method according to any one of items 110 to 112, comprising modifying the activity of LUP1.3 (SEQ ID NO: 7) and / or LUP1.4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto, such as modifying the activity of: i. LUP1.3 (SEQ ID NO: 7) and LUP1.4 (SEQ ID NO: 9); or ii. LUP3.5 (SEQ ID NO: 3) and LUP1.4 (SEQ ID NO: 9), or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.

[0563] 114. The method according to any one of items 110 to 113, comprising reducing the activity of at least one QA transport protein in cells of said QA- containing legume or part thereof.

[0564] 115. The methods according to any one of items 110 to 114, wherein the QA- containing legume is as defined in any one of items 1 to 109 or 136.

[0565] 116. The methods according to any one of items 110 to 115, wherein the QA content is decreased in seeds, such as mature seeds, of said QA-containing legume.

[0566] 117. The methods according to any one of items 110 to 116, wherein the QA content is increased in pods, leaves, and / or stem, of said QA-containing legume.

[0567] 118. The method according to any one of items 110 to 117, wherein the method is a method for reducing or decreasing the QA content in seeds, such as mature seeds, of said QA-containing legume, or a method for increasing the QA content in pods, leaves, and / or stem, of said QA- containing legume. P7181 PC00

[0568] 119. A plant product comprising the QA-containing legume or part thereof, as defined in any one of items 1 to 109 or 136.

[0569] 120. The plant product according to item 119, wherein the plant product is selected from the group consisting of: i. an extract prepared from seeds of said QA-containing legume; ii. flour prepared from the seeds of said QA-containing legume and / or from the extract, such as lupin flour prepared from the seeds of said lupin plants; iii. kernel flour prepared from the endosperm of the seeds of said QA-containing said legume, such as lupin kernel flour prepared from the endosperm of seeds of said lupin plants; iv. hulls prepared from the seeds of said QA-containing legume or part thereof, such as prepared from the seeds and / or dried seeds of said QA-containing legume, for example from said lupin plants; v. meal prepared from the seeds of said QA-containing legume or part thereof, such as prepared from seeds or dried seeds of said QA-containing legume; vi. plant flakes, such legume flakes of said QA-containing legume, prepared from the seeds of said QA-containing legume; and vii. dry seeds prepared from the seeds of said QA-containing legume.

[0570] 121. A plant cell of the QA-containing legume or part thereof as defined in any one of items 1 to 109 or 136.

[0571] 122. Seeds from the QA-containing legume as defined in any one of items 1 to 109 or 136.

[0572] 123. Seed flour from the seeds of item 122.

[0573] 124. Use of the QA-containing legume or part thereof, such as the seed, as defined in any one of items 1 to 109 or 136 in a food product.

[0574] 125. Use of the QA-containing legume or part thereof, according to any one of items 1 to 109 or 136, as animal feed. P7181 PC00

[0575] 126. A method of producing a plant product, said method comprising the steps of: i. providing seeds of a QA-containing legume as defined in any one of items 1 to 109 or 136, preferably wherein said seeds are mature seeds; and ii. processing said seeds into a plant product, wherein the processing preferably comprises or consists of drying said seeds into dry seeds, preferably wherein the plant product is as defined in any one of items 119 to 120.

[0576] 127. The method according to item 126, wherein the plant product is a flour and step ii. comprises milling and / or grinding dry seeds of said QA-containing legume.

[0577] 128. The method according to item 126, wherein the plant product is hulls and step ii. comprises dehulling the seeds, optionally wherein said seeds are dry seeds.

[0578] 129. The method according to item 126, wherein the plant product is a kernel flour and step ii. comprises dehulling the seeds, optionally wherein said seeds are dry seeds, to obtain endosperms of said seeds of said QA-containing legume, milling and / or grinding said endosperms.

[0579] 130. The method according to item 126, wherein the plant product is a meal and step ii. comprises milling and / or grinding dry seeds of said QA-containing legume.

[0580] 131. The method according to item 126, wherein the plant product is debittered seeds, and step ii. comprises soaking and / or boiling the dry seeds of said QA- containing legume.

[0581] 132. The method according to item 126, wherein the plant product is flakes of said QA-containing legume, and step ii. comprises chopping the dry seeds of said QA-containing legume.

[0582] 133. The method according to item 126, wherein the plant product is a protein isolate and step ii. comprises isolating proteins from said seeds. P7181 PC00

[0583] 134. The method according to item 126, wherein the plant product is an extract and step ii. comprises preparing an extract of said seeds.

[0584] 135. The method according to item 126, wherein the plant product is leaching water and step ii. comprises boiling the dry seeds of said QA-containing legume.

[0585] 136. The QA-containing legume, the method, the plant cell, the seeds, the seed flour, the use, and / or the plant product according to any one of the preceding items, wherein the QA-containing legume is a lupin plant, such as a lupin plant of the species narrow-leafed lupin (NLL, Lupinus angustifolius), white lupin (Lupinus albus), yellow lupin (Lupinus luteus L.), or Andean lupin (Lupinus mutabilis), preferably said lupin plant is a NLL.

Claims

P7181 PC00Claims1. A quinolizidine alkaloid (QA)-containing legume or part thereof, wherein said QA- containing legume carries a mutation in at least one gene encoding a QA transport protein, preferably wherein said QA-containing legume has reduced content of one or more QAs in the seeds, compared to a corresponding QA-containing legume not comprising said mutation.

2. The QA-containing legume or part thereof according to claim 1 , wherein said QA transport protein is LUP3.1 , LUP3.5, HH130, LUP1.3, LUP1.4, LUP1.1 , LUP1.2, LUP2.1 , LUP2.2, LUP3.2, LUP3.3, LUP3.4, LUP4.1 , or LUP5.1 , and wherein said mutation is one or more of: i. a mutation in the LLIP3.1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1 , preferably wherein the wildtype LLIP3.1 gene is a gene encoding LLIP3.1 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP3.1 gene comprises the coding sequence as set forth in SEQ ID NO: 2 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 2; ii. a mutation in the LLIP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5, preferably wherein the wildtype LLIP3.5 gene is a gene encoding LLIP3.5 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP3.5 gene comprises the coding sequence as set forth in SEQ ID NO: 4 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 4; iii. a mutation in the HH130 gene leading to reduced HH130 activity or loss-of- function of HH130, preferably wherein the wildtype HH130 gene is a gene encoding HH130 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype HH130 gene comprises the coding sequence as set forth in SEQ ID NO: 6 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 6; iv. a mutation in the LLIP1 .3 gene leading to reduced LLIP1.3 activity or loss-of- function of LLIP1.3, preferably wherein the wildtype LLIP1.3 gene is a gene encoding LLIP1.3 of SEQ ID NO: 7 or a functional homologue thereof having atP7181 PC00 least 70% sequence identity thereto, further optionally wherein the wildtype LLIP1 .3 gene comprises the coding sequence as set forth in SEQ ID NO: 8 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 8; v. a mutation in the LLIP1 .4 gene leading to reduced LLIP1.4 activity or loss-of- function of LLIP1.4, preferably wherein the wildtype LLIP1.4 gene is a gene encoding LLIP1.4 of SEQ ID NO: 9 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP1 .4 gene comprises the coding sequence as set forth in as set forth in SEQ ID NO: 10 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 10; vi. a mutation in the LLIP3.4 gene leading to reduced LLIP3.4 activity or loss-of- function of LLIP3.4, preferably wherein the wildtype LLIP3.4 gene is a gene encoding LLIP3.4 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP3.4 gene comprises the coding sequence as set forth in SEQ ID NO: 24 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 24; vii. a mutation in the LLIP1.1 gene leading to reduced LLIP1.1 activity or loss-of- function of LLIP1.1 , preferably wherein the wildtype LLIP1.1 gene is a gene encoding LLIP1.1 of SEQ ID NO: 13 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP1.1 gene comprises the coding sequence as set forth in SEQ ID NO: 14 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 14; viii. a mutation in the LLIP1.2 gene leading to reduced LLIP1.2 activity or loss-of- function of LLIP1 .2, preferably wherein the wildtype LLIP1.2 gene is a gene encoding LLIP1.2 of SEQ ID NO: 15 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP1 .2 gene comprises the coding sequence as set forth in SEQ ID NO: 16 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 16; ix. a mutation in the LLIP2.1 gene leading to reduced LLIP2.1 activity or loss-of- function of LLIP2.1 , preferably wherein the wildtype LLIP2.1 gene is a gene encoding LLIP2.1 of SEQ ID NO: 21 or a functional homologue thereof havingP7181 PC00 at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP2.1 gene comprises the coding sequence as set forth in SEQ ID NO: 22 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 22; x. a mutation in the LLIP2.2 gene leading to reduced LLIP2.2 activity or loss-of- function of LLIP2.2, preferably wherein the wildtype LLIP2.2 gene is a gene encoding LLIP2.2 of SEQ ID NO: 27 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP2.2 gene comprises the coding sequence as set forth in SEQ ID NO: 28 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 28; xi. a mutation in the LLIP3.2 gene leading to reduced LLIP3.2 activity or loss-of- function of LLIP3.2, preferably wherein the wildtype LLIP3.2 gene is a gene encoding LLIP3.2 of SEQ ID NO: 11 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP3.2 gene comprises the coding sequence as set forth in SEQ ID NO: 12 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 12; xii. a mutation in the LLIP3.3 gene leading to reduced LLIP3.3 activity or loss-of- function of LLIP3.3, preferably wherein the wildtype LLIP3.3 gene is a gene encoding LLIP3.3 of SEQ ID NO: 25 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP3.3 gene comprises the coding sequence as set forth in SEQ ID NO: 26 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 26; xiii. a mutation in the LLIP5.1 gene leading to reduced LLIP5.1 activity or loss-of- function of LLIP5.1 , preferably wherein the wildtype LLIP5.1 gene is a gene encoding LLIP5.1 of SEQ ID NO: 17 or a functional homologue thereof having at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP5.1 gene comprises the coding sequence as set forth in SEQ ID NO: 18 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 18; and / or xiv. a mutation in the LLIP4.1 gene leading to reduced LLIP4.1 activity or loss-of- function of LLIP4.1 , preferably wherein the wildtype LLIP4.1 gene is a gene encoding LLIP4.1 of SEQ ID NO: 19 or a functional homologue thereof having126P7181 PC00 at least 70% sequence identity thereto, further optionally wherein the wildtype LLIP4.1 gene comprises the coding sequence as set forth in SEQ ID NO: 20 or a functional homologue thereof encoding a protein having at least 70% sequence identity to the protein encoded by SEQ ID NO: 20.

3. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said QA transport protein is LLIP3.1 as set forth in SEQ ID NO: 1 , LUP3.5 as set forth in SEQ ID NO: 3, HH130 as set forth in SEQ ID NO: 5, LUP1.3 as set forth in SEQ ID NO: 7, LLIP1.4 as set forth in SEQ ID NO: 9, LLIP1.1 as set forth in SEQ ID NO: 13, LUP1.2 as set forth in SEQ ID NO: 15, LUP2.1 as set forth in SEQ ID NO: 21 , LUP2.2 as set forth in SEQ ID NO: 27, LUP3.2 as set forth in SEQ ID NO: 11 , LUP3.3 as set forth in SEQ ID NO: 25, LUP3.4 as set forth in SEQ ID NO: 23, LUP4.1 as set forth in SEQ ID NO: 19, or LUP5.1 as set forth in SEQ ID NO: 17, or functional homologues of any one of the aforementioned having at least 70% sequence identity thereto.

4. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said QA transport protein is LLIP3.1 , LLIP3.5, HH130, LLIP1.3, or LLIP1.4, and wherein said mutation is one or more of: i. a mutation in the LUP3. 1 gene leading to reduced LLIP3.1 activity or loss-of- function of LLIP3.1 ; ii. a mutation in the LUP3.5 gene leading to reduced LLIP3.5 activity or loss-of- function of LLIP3.5; and / or iii. a mutation in the HH130 gene leading to a reduced HH130 activity or loss of function of HH130; and / or wherein said mutation is one or more of: iv. a mutation in the LUP1.3 gene leading to a reduced LLIP1.3 activity or loss of function of LLIP1.3; and / or v. a mutation in the LUP1.4 gene leading to reduced LLIP1.4 activity or loss of function of LLIP1.4.

5. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said plant carries a mutation in the LUP3. 1 gene, said mutation leading to reduced LLIP3.1 activity or loss-of-function of LLIP3.1 , and127P7181 PC00 a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5.

6. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said plant carries a mutation in the LUP3. 1 gene, said mutation leading to reduced LLIP3.1 activity or loss-of-function of LLIP3.1, and a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5, wherein the wildtype LUP3. 1 gene is a gene encoding LLIP3.1 as set forth in SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto, and the wildtype LUP3.5 gene is a gene encoding LLIP3.5 as set forth in SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto.

7. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said plant carries a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130.

8. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said plant carries a mutation in the LUP3. 1 gene, said mutation leading to reduced LLIP3.1 activity or loss-of-function of LLIP3.1, a mutation in the LUP3.5 gene, said mutation leading to reduced LLIP3.5 activity or loss-of-function of LLIP3.5, and a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130, wherein the wildtype LUP3. 1 gene is a gene encoding LLIP3.1 as set forth in SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto, the wildtype LUP3.5 gene is a gene encoding LLIP3.5 as set forth in SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto, and the wildtype HH130 gene is a gene encoding HH130 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto.128P7181 PC009. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said plant carries a mutation in the LUP3.4 gene, said mutation leading to reduced LLIP3.4 activity or loss-of-function of LLIP3.4, and a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130.

10. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said plant carries a mutation in the LUP3.4 gene, said mutation leading to reduced LLIP3.4 activity or loss-of-function of LLIP3.4, and a mutation in the HH130 gene, said mutation leading to a reduced HH130 activity or loss of function of HH130, wherein the wildtype LUP3.4 gene is a gene encoding LLIP3.4 as set forth in SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto, and the wildtype HH130 gene is a gene encoding HH130 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto.

11. The QA-containing legume or part thereof according to any one of the preceding claims, wherein said plant carries a mutation in the LUP1.3 gene, said mutation leading to reduced LLIP1.3 activity or loss-of-function of LLIP1.3, and a mutation in the LUP1.4 gene, said mutation leading to reduced LLIP1.4 activity or loss-of-function of LLIP1.4.

12. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutation in the LUP3. 1 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene is one of the following mutations: i. a mutation leading to a premature stop codon; ii. a mutation in a splice site; iii. a frame-shift mutation; iv. deletion of the entire LUP3. 1 gene, LUP3.5 gene, HH130 gene, LUP1.3 gene, and / or LUP1.4 gene or a part thereof;129P7181 PC00 v. a mutation in the active site of LLIP3.1, LLIP3.5, HH130, LLIP1.3 and / or LLIP1.4 leading to reduced or abolished LLIP3.1 , LLIP3.5, HH130, LLIP1.3 and / or LLIP1.4 activity or; vi. a mutation in the promoter region regulating transcription of the LUP3. 1 gene, the LUP3.5 gene, the HH130 gene, the LUP1.3 gene and / or the LUP1.4 gene leading to reduced or abolished transcription or translation.

13. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutation in the LUP3. 1 gene is a premature stop codon, preferably a premature stop codon positioned at any one of codons 1 to 120 of the LUP3.1 gene, such as a premature stop codon positioned at codon 120 of the LUP3. 1 gene, and / or wherein the mutation in the LUP3. 1 gene results in a gene encoding a mutated protein lacking at least the 237 most C-terminal amino acids of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto, and / or wherein the mutation in the LUP3. 1 gene introduces a premature stop codon positioned at codon 120 and / or wherein the mutated LUP3.7_gene is LUP3.1 G360A(Trp120Stop) of SEQ ID NO: 72 or a functional homologue thereof having at least 70% sequence identity thereto.

14. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutation in the LUP3.5 gene is a premature stop codon, preferably a premature stop codon positioned at any one of codons 1 to 46 of the LUP3.5 gene, such as a premature stop codon positioned at codon 46 of the LU P3.5 gene, and / or wherein the mutation in the LUP3.5 gene results in a gene encoding a mutated protein lacking at least the 321 most C-terminal amino acids of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto, and / or wherein the mutation in the LUP3.5 gene introduces a premature stop codon positioned at codon 46 and / or wherein the mutated LUP3.5 gene is LUP3.5_G138A(Trp46Stop) of SEQ ID NO: 75 or a functional homologue thereof having at least 70% sequence identity thereto.

15. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutation in the LUP3.4 gene is a premature stop130P7181 PC00 codon, preferably a premature stop codon positioned at any one of codons 1 to 171 of the LUP3.4 gene, such as a premature stop codon positioned at codon 171 of the LUP3.4 gene, and / or wherein the mutation in the LUP3.4 gene results in a gene encoding a mutated protein lacking at least the 192 most C-terminal amino acids of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto, and / or wherein the mutation in the LUP3.4 gene introduces a premature stop codon positioned at codon 171 and / or wherein the mutated LUP3.4 gene is LUP3.4_C511T(Q171stop) of SEQ ID NO: 73 or a functional homologue thereof having at least 70% sequence identity thereto.

16. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutation in the LUP3.4 gene is a premature stop codon, preferably a premature stop codon positioned at any one of codons 1 to 330 of the LUP3.4 gene, such as a premature stop codon positioned at codon 330 of the LUP3.4 gene, and / or wherein the mutation in the LUP3.4 gene results in a gene encoding a mutated protein lacking at least the 33 most C-terminal amino acids of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto, and / or wherein the mutation in the LUP3.4 gene introduces a premature stop codon positioned at codon 330 and / or wherein the mutated LUP3.4 gene is LUP3.4_G990A(W330stop) of SEQ ID NO: 74 or a functional homologue thereof having at least 70% sequence identity thereto.

17. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutation in the HH130 gene is a premature stop codon, preferably a premature stop codon positioned at any one of codons 1 to 201 of the HH130 gene, such as a premature stop codon positioned at codon 201 of the HH130 gene, and / or wherein the mutation in the HH130 gene results in a gene encoding a mutated protein lacking at least the 178 most C-terminal amino acids of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto, and / or wherein the mutation in the HH130 gene introduces a premature stop codon positioned at codon 201, and / or131P7181 PC00 wherein the mutated HH130 gene is HH130_G603A(W201stop) of SEQ ID NO: 76 or a functional homologue thereof having at least 70% sequence identity thereto.

18. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutated LUP3. 1 gene encodes a protein consisting of amino acids 1 to 119 of SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto.

19. The QA-containing legume or part thereof according to any one of the preceding claims, wherein mutated LUP3.4 gene encodes a protein consisting of amino acids 1 to 170 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto.

20. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutated LUP3.4 gene encodes a protein consisting of amino acids 1 to 329 of SEQ ID NO: 23 or a functional homologue thereof having at least 70% sequence identity thereto.

21. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutated LUP3.5 gene encodes a protein consisting of amino acids 1 to 45 of SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto.

22. The QA-containing legume or part thereof according to any one of the preceding claims, wherein the mutated HH130 gene encodes a protein consisting of amino acids 1 to 200 of SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto.

23. The QA-containing legume or a part thereof according to any one of the preceding claims, wherein the QA content in the seeds, preferably the mature seeds, of said QA-containing legume is reduced to at the most 75% of the content in a corresponding QA-containing legume not comprising said mutation in at least one gene encoding a QA transport protein on a dry seed weight basis, such as at the most 70 %, such as at the most 65 %, such as at the most 60%, such as at the most 55%, such as at the most 50%, such as at the most 45%, such as at the most 40%,132P7181 PC00 such as at the most 35%, such as at the most 30%, such as at the most 25 %, such as at the most 20%, or less, compared to the content in a corresponding QA- containing legume not comprising said mutation in at least one gene encoding a QA transport protein.

24. A plant product comprising the QA-containing legume or part thereof, as defined in any one of claims 1 to 23 or 31 to 32, preferably wherein the plant product is selected from the group consisting of: i. an extract prepared from seeds of said QA-containing legume; ii. flour prepared from the seeds of said QA-containing legume and / or from the extract, such as lupin flour prepared from the seeds of said lupin plants; iii. kernel flour prepared from the endosperm of the seeds of said QA-containing said legume, such as lupin kernel flour prepared from the endosperm of seeds of said lupin plants; iv. hulls prepared from the seeds of said QA-containing legume or part thereof, such as prepared from the seeds and / or dried seeds of said QA-containing legume, for example from said lupin plants; v. meal prepared from the seeds of said QA-containing legume or part thereof, such as prepared from seeds or dried seeds of said QA-containing legume; vi. plant flakes, such legume flakes of said QA-containing legume, prepared from the seeds of said QA-containing legume; and vii. dry seeds prepared from the seeds of said QA-containing legume.

25. A method of producing a plant product, said method comprising the steps of: i. providing seeds of a QA-containing legume as defined in any one of claims 1 to 23 or 31 to 32, preferably wherein said seeds are mature seeds; and ii. processing said seeds into a plant product, wherein the processing preferably comprises or consists of drying said seeds into dry seeds, preferably wherein the plant product is as defined in claim 24.

26. A plant cell of the QA-containing legume or part thereof as defined in any one of claims 1 to 23 or 31 to 32.

27. Seeds from the QA-containing legume as defined in any one of claims 1 to 23 or 31 to 32.133P7181 PC0028. Seed flour from the seeds of claim 27.

29. Use of the QA-containing legume or part thereof, such as the seed, as defined in any one of claims 1 to 23 or 31 to 32 in a food product.

30. Use of the QA-containing legume or part thereof, as defined in any one of claims 1 to 23 or 31 to 32, as animal feed.

31. The QA-containing legume, the method, the plant cell, the seeds, the seed flour, the use, or the plant product according to any one of the preceding claims, wherein the QA-containing legume is a lupin plant, such as a lupin plant of the species narrow-leafed lupin (NLL, Lupinus angustifolius), white lupin (Lupinus albus), yellow lupin (Lupinus luteus L), or Andean lupin (Lupinus mutabilis), preferably said lupin plant is a NLL.

32. The QA-containing legume, the method, the plant cell, the seeds, the seed flour, the use, or the plant product according to any one of the preceding claims, wherein said QA is at least one of lupanine, 13-hydroxylupanine and / or esters thereof, and angustifoline.

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