NL9300078A - Protein with anti-nutritional properties. - Google Patents

Description

PROTEIN WITH ANTI-NUTRITIONAL PROPERTIES

The invention relates to proteins with anti-nutritional properties. In the animal feed industry it is known that certain feed components, such as legume seeds, for example pea (Pisum sativum) and bean (Phaseolus vulgaris), cause problems. These ingredients contain proteins with anti-nutritional properties, also called anti-nutritional factors (ANF), such as lectins, trypsin inhibitors, hemagglutinins and tannins. These ANF can bind to intestinal epithelial cells of animals such as pigs fed with flour of the above uncooked feed ingredients, thereby damaging the intestines (Hendriks. Et al., 1987; Huisman et al., 1990 (a); Huisman et al .; 1990 (b); Pusztai, 1989). In order to counteract this adverse effect of ANF, legume seeds must be heat-treated for a significant period of time. Apparently, the ANF are toxic in the native state and lose their toxicity upon irreversible denaturing. A standard heat treatment is toasting, which is heating in the presence of steam for 40 minutes at 104 ° C and 19% moisture (Huisman et al., 1990 (b)). Toasting costs energy and reduces the nutritional value of the seed and seed meal, and therefore makes the use of legume from leguminous plants as feed for pigs economically advantageous.

The object of the invention is now to provide ANF which have reduced or completely nullified anti-nutritional activity in warm-blooded animals and humans. Such ANF need to be subjected to a less extensive heat treatment or even not at all in order to serve as feed.

It has now been found that ANF can be provided with a reduced anti-nutritional effect, i.e. they retain their activity in the plant, at ambient temperature, but lose their activity at the elevated temperature in the intestines of livestock, such as pigs. Such ANF maintain their desired activity in the plant, but have a reduced or completely nullified anti-nutritional activity in humans and pets. Seeds containing such an ANF can be fed to livestock without prior heat treatment or with less heat treatment than the current method.

Accordingly, the invention provides a protein with anti-nutritional properties that is mutated by replacing at least one amino acid of the natural protein sequence with another amino acid in at least one surface loop of the protein, said surface loop containing one or more amino acids containing of the ligand binding site, whereby the mutated protein has a reduced anti-nutritional effect.

In the following, the invention will be further explained and described in more detail with reference to leguminous lectins as examples of proteins with anti-nutritional properties.

Lectins from leguminous plants are carbohydrate-binding proteins that are present in large amounts in seeds and in small amounts in carrots (Diaz et al., 1990). Lectins in roots are involved in vivo in the host-specific symbiosis with Rhi zobium bacteria (Diaz et al., 1989). The function of lectin in the seeds is not known, but it has been hypothesized to protect seeds and plants from herbivorous animals, including insects (Boulter et al., 1990; Bourne et al., 1990; Chrispeels et al., 1991 ; Pusztai, 1989).

Lectins from leguminous plants are among the most studied carbohydrate-binding proteins (Sharon et al., 1989; Sharon et al., 1990). The nucleotide sequence and structure of a large number of leguminous lectins is known (Becker et al., 1975; Einspahr et al., 1988; Ein-spahr et al., 1986; Reeke et al., 1986; Shaanan et al., 1991; Sharon et al., 1990). For example, the pea lectin, Pisum sativum L., (PSL) is a so-called 'B-barrel' protein (Richardson et al., 1987) organized as a dimer, as are the other lectins of the Vicieae group of the Leguminosae family (Sharon et al., 1990). Both monomers have been converted into a small and a larger B chain in a post-translation process. The mature protein therefore has an a2B2 configuration (Higgins et al., 1983). The overall three-dimensional structure of monomers of other butterfly-flower lectins is very similar to that of the PSL monomer, despite differences in post-translation processes and multimerization. The polypeptide chain of the native lectins winds into anti-parallel 'B-sheet' structures that form the framework of the molecule. These B-sheets are connected via larger and smaller surface loops. The metal and carbohydrate bonding sites are located on these surface loops. (Einspahr et al., 1986; Einspahr et al., 1988).

For example, Einspahr et al., 1986, denote the structure of PSL in which the following surface loops are present, designated by the amino acid numbers: (B) 9-17, 28-39, 47-62, 77-81, 87-115, 122- 136, 142-147, 151-160, 167-172; (tt) 195-206, 215-222. The structure and, therefore, the locations of the surface loops of other leguminous lectins are also known.

As noted above, according to the invention, the lectin must be mutated in at least one surface loop, this surface loop containing one or more amino acids that are part of the monosaccharide binding site. For many lectins, details of the monosaccharide binding site are known, especially which amino acids are involved in the binding of monosaccharides. It turns out that the monosaccharides bind to all lectins in similar places and in much the same way (Einspahr et al., 1986; Bourne et al., 1990 (a) and (b)).

Preferably, in the mutated lectin, an amino acid has been replaced in a portion of the surface loop containing a conserved box of hydrophobic amino acids.

As mentioned earlier, the three-dimensional structures of the lectins of different leguminous plants are almost identical. The degree of preservation is therefore high. This is apparent, for example, from Sharon et al., 1990.

In a still further preference, in the mutated lectin, an amino acid with a large hydrophobic side chain has been replaced by another amino acid.

Amino acids with a large hydrophobic side chain are valine, leucine, isoleucine, phenylalanine, tryptophan and methionine. The replacement of such amino acids with smaller side chain amino acids has a major impact on the stability of the lectin molecule. A particularly suitable replacement amino acid with a small side chain is alanine.

According to a specific embodiment, an amino acid is replaced in the surface loop consisting of amino acids 87-115 of the p-chain lectin B chain or the corresponding amino acids of lectins of other leguminous plants.

This surface loop is described for pea lectin in Einspahr et al., 1988. This loop is shown in Figure 1A and Table 1. The -NH group of glycine-99 is part of the monosaccharide binding site (Bourne et al., 1990 (a)). The loop is rotated and the amino acids at the site of rotation are conserved in all leguminous lectins (amino acids 101-104 in PSL; "conserved box"). In Table 1, the primary sequences of this surface loop of 14 different legume lectins were compared (Sharon et al., 1990). The numbering is that of the PSL sequence. Considering the great similarity in structure between the different lectins, also with regard to this surface loop, and the great degree of

Table 1 87 100 115

PSL IAPVDTK PQT 6GGY LGVP NSAEYDKTTQT

LOL IAPVDTK PQT GGGY LGVP NSKDYDKTSQT

LCL IAPVDTK PQT GGGY LGVPYNGKEYDKTSQT

VFL IAPVDTK PQT GGGY LGVP NGKDYDKTAQT

SBA LAPIDTK PQTH AGY LGLP NENE SGDQV

DBA LVPVGSE PRRN GY LGVPDSDVYNNSAGQT

LTA LAPVGTEIPDDSTGGF LGIPDGS NGFNQF

PHA-L LVPVGSQ PKDK GGF L G L P D G S NSNFHT

PHA-E LLPVGSQ PKDK GGL LGLPNNYK YDSNAHT

SL LAPTDTQ PKS GGGY LGIP KDAESNET V

LBL LVPVDSQ PKKK GRLLGLPNKS ENDINALT

DL IANTDTSIPSGS GGRLLGLFP DANAD T I

CONA ISNIDSSIPSGST GRLLGLFP DANAD T I

ECORL MGPTKSK PAQ GYGY LGIPFNSKQ DNSYQT

Abbreviations: PSL = Pisum sativum lectin SL = Onobrychis vicifolia (sanfoin) lectin LOL = Lathyrus ochrus lectin LBL = Phaseolus limensis (lima bean) lectin LCL = Lens culinaris lectin DL = Dioclea grandiflora lectin VFL = Vicia faba lectine (favine) CONA ensiformis lectin (Concanavalin A) SBA = Glycine max (soybean) lectin ECORL = Erythrina corallodendron lectin DBA = Dolichos biflorus lectin LTA = Lotus tetragonolobus lectin PHA = Phaseolus vulgaris lectin (Phytohaemagglutinin) preservation of amino acids, can be obtained without preservation of amino acids, L extrapolated to the other leguminous lectins.

The aforementioned surface loop is present in all finer-flowered lectins, including lectin from the jackbean, Canavalia ensiformis. (Concanavalin A), but is interrupted there by a circular permutation (Fig. 1B). Nevertheless, the overall configuration of the loop including the preserved box is the same as in PSL.

Particularly preferably, in the above surface loop, an amino acid is replaced from the conserved box, amino acids 101-104 of the β chain of PSL or the corresponding amino acids of lectins from other leguminous plants.

In particular, when valine-103 of PSL is replaced by alanine, a mutated lectin V103A is obtained, which has the following properties: - In hemagglutination tests at room temperature, there was no difference in properties between native (wild-type, wt) PSL and PSL V103A are being observed. It can be deduced from this that the in vivo properties of PSL V103A have not changed.

The agglutination activity was determined after incubation at 70 ° C. Wt-PSL lost activity after incubation for 20-25 minutes, while PSL V103A was inactivated after 5 minutes incubation. Incubation at 70eC irreversibly inactivated both lectins.

- Another test showed that PSL V103A was irreversibly inactivated at 70eC, while wt-PSL lost its activity at 80 ° C.

-The main result was found that PSL V103A is still active at 28 ° C, but inactive at 37 ° C. Wt-PSL, on the other hand, is still fully active at 45 ° C, and only loses its activity at 55 ° C. Inactivation at these temperatures is reversible; the activity can be restored by cooling.

Given the high degree of conservation, replacement of the amino acid of lectins from other leguminous plants with valine-103 of PSL can be expected to give the same results.

The invention further provides a method for the preparation of the above-described mutated proteins with a reduced anti-nutritional effect, wherein in a protein-encoding expression vector the DNA sequence is altered by site-directed mutagenesis such that upon expression in a suitable host cell the mutated protein is formed. This method will be described in detail in the Materials and Methods section. Site-directed mutagenesis is a method known per se for specifically mutating proteins.

The invention also provides a DNA sequence comprising a sequence encoding a mutated protein as described above.

In the specific case of PSL V103A, the coding sequence is the same as the wt-PSL coding sequence, except that the codon for valine-103, GTT, is changed to GCT (which encodes alanine).

The invention also provides an expression vector comprising the mutant protein coding sequence and a suitable promoter sequence. Examples of promoter and expression vector are given in the Materials and Methods section.

The invention also provides a plant cell containing the above DNA sequence containing the promoter sequence and the mutant protein coding sequence, which sequence is expressed in the plant cell to form the mutated protein with a reduced anti-nutritional effect.

The plant cell is in particular a butterfly flower cell. The gene for the mutated protein is built into this cell and it is ensured that the gene originally present for the wt protein is not expressed, for example by the anti-sense approach, see for example van der Krol et al., 1988. The plant cell can also be a cell of a plant in which the protein in question does not naturally occur. EP-A-0.351.924 discloses transgenic plants, such as tobacco or potato, which contain a PSL gene and in which the lectin is also expressed. Instead of the gene for wt-PSL, the gene of the present invention could also be used here.

Finally, the invention also relates to plants regenerated from the above-mentioned plant cell, as well as to plant parts, in particular the seeds thereof. For the regeneration of pea plants, reference can be made to Schroeder et al.

The invention is now illustrated by the following example and with reference to the drawings, in which:

Fig. 1 represents the three-dimensional structures of the monomers of PSL (A) and CON A (B). The surface loop of both molecules is shown in bold lines. Arrows indicate the sites involved in the circular permutation of CON A. An asterisk indicates the site of the sugar binding site in both molecules (Becker et al., 1975; Einspahr et al., 1986);

Fig. 2 shows the position of Val-103 and Phe-104 in detail. The C-a trace of the surface loop (A) and the top β-sheet (B) is indicated. The position of the sugar binding site is indicated by an asterisk. Relevant amino acids are numbered and their side chains are shown in bold lines;

Fig. 3 represents an immunoblot of crude E. coli extracts containing PSL.

Lane 1: Marker (lectin from pea seed); lane 2: wt-PSL; lane 3: PSL V103A; lane 4: PSL F104A; lane 5: PSL V103A / -F104A. The polyclonal antiserum used does not react with the 6 kD α subunit present in lane 1;

Fig. 4 represents the hemagglutination assay with different concentrations of wt and mutated PSL;

Fig. 5 depicts the haemagglutination inhibition assay with wt and mutated PSL. The PSL concentration is 250 Mg.ml ”1. The concentrations of glucose (GLC), mannose (MAN) and galactose (GAL) are indicated;

Fig. 6 depicts the irreversible inactivation of wt and mutated PSL. The measurements were performed in duplicate;

Fig. Shows 7 temperature ranges. The PSL concentration was 250 Mg.ml-1. The temperature was increased by 1 ° C min-1, and samples were taken at the indicated temperatures;

Fig. 8 represents the hemagglutination assay at elevated temperatures. The PSL concentration is 250 Mg.ml ”1; no PSL has been added in the last row;

Fig. 9 presents DSC patterns of wt and mutated PSL. The peaks indicate the denaturing temperature (Tm), the area under the curves represents the total denaturing energy (ΔΗ). See also Table 2.

EXAMPLE

Materials and Methods Bacterial Strain E. coli strain DH5aF + (supE44 hsdR17 recAl endAl avrA96 thi-1 jrelAl) was used for expression of wild type and mutant lectin genes and for the production of PSL. The bacteria were grown in Luria Complete medium (Maniatis et al., 1982) (LC) at 37 ° C.

Clonerina and Place-oriented mutaoenesis of PSL

psl cDNA (Stubbs et al., 1986) was modified as previously described (van Eijsden et al., 1992) by introducing an additional EcoRI restriction site, resulting in almost complete removal of the signal sequence, and donated in pUC 18 (Yannisch-Perron et al., 1985), to obtain the expression vector pMP 2809. The N-terminus of the lectin formed was determined by protein sequencing by automated Edman degradation with an Applied Biosystems 477A Protein Sequencer.

Mutations were introduced using the polymerase chain reaction (per) as previously described (van Eijsden et al., 1992). For each mutation, an 89 bp EcoRV / BamH1 frame from pMP 2809 was multiplied, using one mutagen and one non-mutagenic primer. The codon for valine-103, GTT, was changed to GCT (encoding alanine) to form V103A. The codon for phenylalanine-104, TTC, was changed to GCC (which also encodes alanine) to form F104A. The double mutant was prepared by combining both mutations in a single mutagenic primer. The entire multiplied DNA was sequenced by the method of Sanger et al. (Sanger et al., 1977), using sequenase 2.0® (USB, Cleveland, Ohio, USA). After checking the 89 bp fragment by DNA sequence analysis, the original EcoRV / -BamHl fragment from pMP 2809 was replaced by the fragments containing the mutations, yielding pMP 3203, pMP 3204 and pMP 3211, respectively.

Isolation of PSL from E.coli

The isolation of PSL from E.coli was performed as previously described (Prasthofer et al., 1988; Van Eijsden et al., 1992), with some modifications: E.coli DH5aF + cells, containing pMP 2809 or one of its derivatives were grown in 2 liters LC containing 100 μg / ml carbenicillin at 37 ° C, and were induced in the mid-exponential phase by addition of IPTG (isopropyl-BD-thiogalactopyranoside; Boehringer, Mannheim) to the medium to a final concentration of 0.5 mM. After induction, the cells were grown for an additional 16 hours at 37 ° C, harvested and washed in TBS (10 mM Tris-HCl, pH 6.8 containing 150 mM NaCl). All further steps were performed at 4 ° C unless otherwise noted. The cells were resuspended in 25 ml TBS containing 0.5 mM PMSF (phenylmethylsulfonyl fluoride) and lysed in a "French pressure" cell (American Instrument company, Silver Spring, MA, USA) at a pressure of 1500 psi. Inclusion bodies containing PSL were collected by centrifugation at 15000 rpm for 30 minutes. The protein was denatured overnight in TBSm (TBS containing 1 mM MnCl 2 and 1 mM CaCl 2) in the presence of 7 M guanidine HCl. Membranes and residual aggregates were removed by ultracentrifugation for one hour at 175000 xG. The supernatant was quickly diluted 25-fold in TBSm containing 1.5 M urea at 0 ° C. The proteins were allowed to refold for at least 24 hours, after which the solution was dialyzed extensively against deionized water and then lyophilized. Finally, the lyophilized proteins were redissolved in TBSm and purified by affinity chromatography at room temperature on Sephadex G-75 (Pharmacia, Uppsala, Sweden) in TBSm (Diaz et al., 1990).

SDS-PAGE of PSL fractions was performed with a 15% running gel according to Lugtenberg et al. (1975). The gels were then blotted onto PVDF membrane (Millipore, Bedford, MA, USA). Immunochemical staining using anti-PSL polyclonal antibodies was then performed according to Diaz et al. (1990).

Haemaqllutination determinations

The haemagglutination assay used to investigate the sugar binding capacity and sugar binding specificity of PSL has been previously described (Kijne et al., 1980). In order to investigate stability, a PSL solution of 250 μg.Ίs ^ 1-1 in TBSm was incubated at 70 ° C. Samples were taken at 5 minute intervals and placed on ice. These samples were then examined in the haemagglutination assay for residual activity.

In another test, a PSL solution of 250 μg.ml1 in TBSm was subjected to a temperature range using a Biozym thermal cycler (Wessex,

Andover Hampshire, England). The temperature was raised with 10C.min-1 from 20 ° C to 80 ° C. Samples were taken after each 10 ° C increase, placed on ice and examined in the hemagglutination assay.

Finally, haemagglutination assays were performed at different temperatures (28, 37, 45 and 55 ° C) instead of 20 ° C to attempt to differentiate between reversible and irreversible inactivation.

Differential Scanning Calorimetry (DSC)

In order to determine the denaturing temperature (Tm) and the denaturing energy (ΔΗ) of PSL, a Mettler TA-300 DSC device coupled to a TC-10 detector was used. The temperature ranges examined were between 5 and 100 ° C and the heating rate was 10 ° C. min-1. The samples contained 80% by weight of water.

Results

Production of wild-tvpe and mutated PSL in E.coli

The plasmid pMP 2809, containing psl cDNA, was expressed in E. coli. This led to the formation of unprocessed PSL containing five additional amino acids at the N-terminus, namely three amino acids of the pUC 18 sequence and two residues of the original signal peptide. This molecule was designated wt-PSL (Van Eijsden et al. 1992). Typically, 10-20 mg affinity purified wt-PSL could be isolated from a two liter E. coli culture by the inventors. Introduction of the mutations V103A, F104A and V103A / F104A in pMP 2809 gave the plasmids pMP 3203, pMP 3204 and pMP 3211, respectively. The yield of PSL F104A was approximately equal to that of Wt-PSL. PSL V103A, however, was much more difficult to obtain, as only about 2 mg could be isolated from a two liter culture. Wt and mutated PSL monomers all have an apparent molecular weight of about 28 kD as can be deduced from SDS-PAGE (Fig. 3), which is similar to that of unprocessed pea seeds from pea seeds. The double mutant, PSL V103A / F104A, was also generated in E. coli, but could not be isolated in an active form by affinity chromatography. The molecular weight of native wt and mutated PSL, as can be seen from gel filtration experiments, was found to be about 55 kD. It can be concluded from this result that the introduced mutations did not affect PSL dimerization.

Haemaqlutlnatie determinations

Wt-PSL, PSL V103A and PSL F104A all agglutinated a 2% suspension of human A + erythrocytes to a minimum concentration of about 16 Mg / ml (Fig. 4). No significant differences in the sugar-binding properties of the mutants could be observed: mannose inhibits hemagglutination at a minimum concentration of 3 mM, glucose inhibits hemagglutination at a minimum concentration of 12.5 mM, and galactose does not inhibit hemagglutination at a concentration of no less than 3 mM. 100 mM (Fig. 5).

After incubating PSL for various periods at 70 ° C, the residual agglutination activity was determined. Wt-PSL and PSL F104A lost their activity after incubation for 20-25 minutes at 70 ° C, while PSL V103A was inactivated after incubation for 5 minutes at 70 ° C. In all cases, the inactivation by incubation at 70 ° C was found to be irreversible, since the activity was not restored by incubation on ice for 30 minutes (Fig. 6).

Wt-PSL and PSL V103A were subjected to a temperature traetet ranging from 20 to 80 ° C in a "thermal cycler". PSL V103A irreversibly lost its activity at 70 ° C. Wt-PSL lost its activity at 80 ° C in the same assay (Fig. 7).

Incubation of the hemagglutination assays at elevated temperatures demonstrated that PSL V103A remains active at 28 ° C, but that the activity of this mutant is lost at 37 ° C (Fig. 8). PSL F104A was still active at 37 eC, but activity decreased at 45 ° C. Wt-PSL was fully active at 45 ° C, but lost activity at 55 ° C. Inactivation at these temperatures is reversible on cooling, as evidenced by the combined results of the tests shown in Figures 7 and 8. Incubation of the haemag glutination assays at these temperatures had no visible effect on the erythrocytes used.

Differential Scanning Calorimetry (DSC)

The denaturing temperature (Tm) and denaturing energy (ΔΗ) of wt-PSL and the two mutants were determined by DSC. The results are shown in Table 2. The DSC curves are shown in Fig. 9. Tm and ΔΗ of PSL isolated from pea seeds are not significantly different from the Tm and ΔΗ of wt-PSL isolated from E.coli (data not reported). The Tm of PSL V103A is about 10eC lower than that of wt-PSL, while ΔΗ is only about 55% of the ΔΗ of wt-PSL. PSL F104A shows no significant difference in Tm or ΔΗ compared to wt-PSL. These results confirm the findings of the hemagglutination tests.

Table 2 PSL_Tm f ° a_ΔΗ fkCal / moli

Wt 82.3 (± 0.7) 164 (± 6) V103A 71.4 (± 1.0) 88 (± 4) F104A 81.8 (± 0.5) 145 (± 15)

The one shown in FIG. DSC curves shown in 9 show single denaturing peaks in wt-lectin and in the mutated lectins. However, the optimum of the curve of V103A has shifted about 11 ° C and is at 71 ° C. This means that the mutation in PSL V103A facilitates total denaturing of the protein. A local effect on the surface loop conformation could not be demonstrated in this test.

Using the various hemagglutination tests described above, a distinction can be made between reversible and irreversible inactivation of PSL. Reversible inactivation of a PSL solution in TBSm occurs at temperatures between 28 and 55eC, while irreversible inactivation occurs between 60 and 80eC. PSL V103A is less stable than wt-PSL or PSL F104A. PSL F104A is somewhat less stable than wt-PSL at 45eC, but this difference is not found at higher temperatures. Irreversible inactivation is likely to coincide with complete denaturation of the protein, while reversible inactivation is likely attributable to local conformational changes in the surface loop, which cause local denaturing of this loop or an overall decrease in sugar binding affinity.

Refolding of denatured PSL appears to require very specific conditions. This phenomenon is particularly common in renaturation of PSL from E.coli inclusion bodies, which would also explain why PSL V103A is more difficult to isolate than wild-type lectin and why the double mutant, V103A / F104A, cannot become in an active form. isolated.

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Claims (14)

Protein with anti-nutritional properties mutated by replacing at least one amino acid of the natural protein sequence with another amino acid in at least one surface loop of the protein, said surface loop containing one or more amino acids that are part of the ligand binding site, causing the mutated protein to have a reduced anti-nutritional effect. Protein according to claim 1, characterized in that at least one amino acid is replaced in a portion of the surface loop containing a conserved box of hydrophobic amino acids. Protein according to claim 1 or 2, characterized in that at least one amino acid with a large hydrophobic side chain has been replaced by another amino acid. Protein according to claim 3, characterized in that the replacement amino acid is alanine. Protein according to any one of claims 1 to 4, characterized in that it is a leguminous lectin. Butterfly-flower lectin according to claim 5, characterized in that the surface loop in which at least one amino acid has been replaced consists of amino acids 87-115 of the β-chain of pea lectin or the corresponding amino acids of lectins of other leguminous plants. Butterfly-flower lectin according to claim 6, characterized in that at least one of the amino acids 101-104 of the pea-lectin β-chain or the corresponding amino acids of lectins of other leguminous plants has been replaced by another amino acid. Butterfly-flower lectin according to claim 7, characterized in that valine-103 of the β-chain of pea lectin or the corresponding amino acid of lectins of other leguminous plants has been replaced by alanine. Method for the preparation of a protein with anti-nutritional properties according to any one of claims 1-8, characterized in that the DNA sequence in a protein-encoding expression vector is altered by site-directed mutagenesis such that upon expression in a appropriate host cell the mutated protein is generated. DNA sequence comprising a sequence encoding a protein according to any one of claims 1-8. Expression vector comprising a DNA sequence as defined in claim 10 and a suitable promoter sequence. Plant cell comprising a DNA sequence according to claim 10, which is expressed in the plant cell to form the mutated protein with a reduced anti-nutritional effect. Butterfly-flowered cell according to claim 12. Plant regenerated from a plant cell as defined in claim 12 or 13, as well as plant parts, in particular seeds.