NL1023309C2 - Altering the starch granule size in amyloplast-containing plant cells comprises providing the cells with an agent for modulating plastid division - Google Patents

Abstract

Altering the average starch granule size or size distribution in amyloplast-containing plant cells comprising providing the cells with an agent for modulating plastid division, is new. Independent claims are also included for: (1) amyloplast-containing plant cell comprising an agent for modulating plastid division; (2) transgenic plant whose genome contains a recombinant nucleic acid for modulating plastid division in the plant or an amyloplast-containing part thereof; (3) a starch-rich part of a plant as above.

Description

I *

Brief description: Method for modulating starch grain size in plants, transgenic plants with changed starch grain size and application of the starch grains.

Starch is a vegetable substance with a very wide field of application. Starch is the main component of the most important food crops. The type of starch has an important influence on the applicability of e.g. flour for preparing e.g. bread, cake and pasta.

In addition, starch in purified and modified form is used in dressings, sauces, desserts, binders and e.g. even in drilling fluids. Starch can also be converted by physical and / or chemical manipulations into a raw material for (biodegradable) packaging materials or alternative foods (e.g. snacks).

10

It has been found that the form in which starch is offered is important for the physical and / or chemical properties of the starch and the product in which the starch is processed. In the present invention, it has been found that plants can be induced to starch grains with a. Produce 15 changed size. This makes it possible to have desired sizes produced and applied. A non-limiting example is, for example, improving the mouthfeel of food products such as sauces and spreads. A larger starch grain, for example, gives a less blissful and more loose feeling in the mouth, which is experienced as more pleasant in certain applications.

Worldwide there is broad knowledge about the construction and storage of starch in plants such as the seed and tuber crops (such as grains and potatoes). Starch granules are formed in so-called amyloplasts. These are cell organelles that multiply by constricting. In one aspect, the present invention provides a method for changing the average starch grain size or the spread in the starch grain size in a set of amyloplast-containing plant cells, said plant cells being provided with a means for modulating a plastic-sharing process. It has surprisingly been found that the average size of the starch grains or the spread of the starch grain size in a plant cell can be modulated by interfering with the division of amyloplasts. It is preferable to interfere with the plastid division in such a specific way that processes other than plastid division related I in the cell are not, or hardly, affected. A suitable way for such a specific modulation is to manipulate the netting process with which a plastid divides into two separate plastids.

The process of plastid division is a precisely regulated process. Various genes are known to be involved in the division I of plastids in plants. Some of these genes are FtsZ genes and the Min I genes (see also Colleti 2000; Osteryoung 1998; WO 98/00436 and WO 01/81601).

FtsZ proteins form a contractile ring on the inside of the outer membrane of a plastid (Colleti (2000; Osteryoung 1998). With I constriction, the contractile ring becomes smaller until finally the truncation I takes place. For example, the Min genes appear, at least in bacteria involved in the placement of the ring during plastid division. It has been found in the present invention that it is possible to modulate the starch grain size distribution in plants by interference with the expression of proteins involved in plastid division in By interference with the Min protein expression, it is possible to achieve a larger or smaller spread in the average starch grain size, which is achieved by influencing the placement of the ring so that it is more centrally located (which leads to to a more homogeneous distribution of the grain size and therefore a lower spread) or, on the contrary, to a more extreme location, so that I a larger spread in the grain size is obtained. Preferably, the plastid sharing process is modulated by influencing the expression I 3 of FtsZ proteins and then preferably the expression of the FtsZ1 protein.

Means for modulating the plastid sharing process include means for manipulating the expression of nucleic acids encoding the endogenous proteins involved in the plastid sharing process. This can be done, for example, by making changes to regulator sequences in genes for these endogenous proteins, thereby obtaining a higher or lower expression of the endogenous protein. Such an intervention is possible by, for example, homologous recombination. In this embodiment, the agent is the nucleic acid with which the homologous recombination is catalyzed. In a preferred embodiment, the agent is a nucleic acid encoding an FtsZ gene or a functional part, derivative or analogue thereof. Preferably a nucleic acid from an FtsZ1 gene or a functional part, derivative or analogue thereof. Other means for modulating plastid division in a cell are peptides or antibodies that influence the function of a protein involved in the plastid introduction process or complex, for example an FtsZ protein or a Min protein. This influencing of the function need not necessarily affect the amounts of these components such as FtsZ in the cell.

By anchoring the entire coding region into the genome of the plant cell by means of an expression cassette, for example by using non-targeted integration into the genome via Agrobacterium tumefaciens mediated plant transformation, it has been possible to modulate the average starch grain size in plant cells. Lines with an average smaller starch grain and lines and with an average larger starch grain were found in the different clonal lines. The differences in the various lines are presumably the result of differences in expression of the nucleic acid by a different integration site or by * '. : The epigenetic mechanisms. An increase in the average grain size was also achieved by introducing so-called antisense molecules from an FtsZ gene. Without being bound by theory, it is believed that in both cases the expression of the FtsZ protein in the treated plant cell and its offspring is interfered with. This specific interference leads to an altered plastid division, probably via interference with the necking mechanism, and thus a deviation from the average size of the starch grain. It is assumed that a final decrease in FtsZ protein expression in the treated plant cell leads to an average larger starch grain while a final increase in

FtsZ protein expression leads to an on average smaller starch grain. Whatever the mechanism is, it appears that the average size of the starch grain can be influenced by introducing nucleic acid from an FtsZ gene or a functional part, derivative or analogue thereof, into the cell. A functional part of an FstZ gene is a part capable of reducing the expression of I endogenous FtsZ protein or increasing the amount of FtsZ protein (derived from I an endogenous gene or added gene), for example by an I antisense, a co - effect suppression effect, an RNAi, a selective restriction or an overexpression of the endogenous RNA and / or protein, or effect overexpression of a functional part of an FtsZ gene. Such strategies can be implemented by inserting expression cassettes for production of the nucleic acids into the cell or by introducing the nucleic acids. There are of course many more methods to lower or increase expression of endogenous FtsZ protein, and these are also part of the present invention. A functional part typically has a length of at least 19 base pairs and may involve a coding and / or a non-coding part of the gene. It is preferred to use a nucleic acid that is completely identical to the FtsZ gene or a functional part thereof. It should be understood, however, that minor changes that do not dramatically change the specific hybridization capabilities with IFtsZ nucleic acid in the plant cell are also tolerated. It is not possible to give the exact limits of the maximum permissible deviations, but a skilled person is capable of adequately assessing and designing them. Such nucleic acid derivatives of an FtsZ gene are therefore also part of the present invention. Nucleic acids can undergo various types of modifications and still functionally hybridize with their target sequence. Such analogues are of course also part of the invention. It is also possible today to make peptides that can hybridize to a target sequence in a nucleic acid-like manner.

Such peptides with PNA and morpholino-like peptides as examples are also suitable analogs of a nucleic acid sequence of a (functional part of) an FtsZ gene. It is also possible to use peptides or antibodies that influence the function of the FtsZ protein or plastid induction complex without changing the amounts of these components such as FtsZ.

An expression cassette as mentioned above is the state of the art for those skilled in the art. In addition to using expression cassettes with a constitutive promoter (e.g., 35S CaMV promoter) that are expressed throughout the plant, expression cassettes that express specific expression in a part of a plant are preferably used. A tuber or seed-specific promoter is preferably used. The advantage of such a promoter is the limitation of the influence on plastid division to those cells that are important for a large part of the starch production in a plant. Various tuber and seed-specific promoters are available and it is likely that more will follow.

The collection of plant cells containing amyloplast is preferably a transgenic plant. In this embodiment, the genome of the plant 30 is provided with a recombinant nucleic acid for modulating the process of plastid division in said plant or an amyloplast-containing part thereof. Preferably, the nucleic acid is introduced in the form of an expression cassette that expresses the nucleic acid. The transgenic plant or the genetically altered cell is of course part of the invention. Preferably, the plant belongs to the tuber, root or seed crops, or to the grains and, preferably, to the potato plants, cassava plants, wheat, rice, barley and maize. Such plants or plant cells may have undergone further genetic modifications. Preferred modifications include modifications that result in amylose-free starch-rich plant parts being produced. This is known, for example, from the potato industry.

The invention provides in particular the starch-rich part of a plant of the invention. From this, starch composition with the adjusted average grain size or spread therein can be recovered in a simple manner. Starchy parts include tubers, seeds and some roots. The invention also comprises a method for making such a starch composition, characterized in that a plant cell or a plant or amyloplast-containing part thereof according to the invention is used. A starch composition thus obtained is also provided. A starch composition is also provided which at least in part comprises such a starch composition. Starch compositions are often chemically or physically modified, these and other derivatives of starch composition are of course also part of the invention.

Starch or starch compositions are used in a wide variety of products. Such products are also covered by the present invention. Preferred products are food products, physically and / or chemically processed (bio) raw materials for the manufacture of a food product or utensil such as e.g. drilling fluids and packaging materials.

I 7 I It has been found that changes in the average size of starch granules have a clear effect on the mouthfeel of food products in which these compositions are incorporated. A larger average grain size results in a fuller taste sensation. While a smaller average grain size results in a more delicious taste sensation.

I This is particularly the case with sauces, spreads, custard and other thickened, more or less liquid food products intended for immediate use. The present invention thus also comprises the use of a starch composition or a product according to the invention for changing the mouthfeel of a food product, preferably a thickened more or less liquid food product intended for direct consumption and of which preferably the sauces, spreads and custard products. An example of a more or less liquid product is a custard or a pudding.

The invention also includes the use in grains. By means of the invention, the starch grain size distribution in flour products of cereals can be modulated which can be used to change, broaden or improve the applicability in flour products such as bread, cake or pasta. The same naturally applies to other crops whose starch-containing parts are used for the production of (baked) flour products. 1

In a further embodiment, the invention provides for the use of an agent for influencing the plastid division in a plant cell for influencing the size of starch granules in said plant cell. This effect applies in particular to plant cells containing amyloplasts with on average one starch core per amyloplast.

Examples H Example 1 Het The Fts-Z1 gene was isolated from potatoes. By means of a constitutive or tissue-specific promoter, constructs were made for overexpression or antisense suppression in the entire plant (35S promoter) or only in the starch producing organs (GBSS promoter).

H Potato plants were transformed by Agrobacterium transformation. A PCR analysis identified transgenic plants containing the relevant plant-foreign promoter FtsZ1 cassette. The effects on chloroplast sharing in the stomata were analyzed by fluorescence microscopy. Starch granules from H tubers of transgenic plants were analyzed using a coulter counter.

I Material and methods

I The cloning of potato FtsZ

Potato FtsZ-1 cDNA was cloned using the Clontech SMART1® RACE cDNA amplification kit according to the recommendations of the I producers. A potato EST sequence (dbEST 1d 53498s38) was used to construct the δ 'primer (SP 107, Table 1). In order to

To obtain fragments that are visible on gel, the touchdown PCR was used

I carried out with 30 cycles. The PCR products were cloned in pGEM®-T easy I (Promega). The clones were tested for the presence of FtsZ using nested PCR using the primers SP102 and SP 103. The complete sequence of one clone I was determined (BaseClear). 2 2 30 I 1 I 9 I Vector Construction I In order to clone the FtsZ coding region into expression vectors I, the pGEM®-T easy plasmid containing the FtsZ-PCR fragment was digested with the restriction enzyme EcoRI. The overhanging ends of the FtsZ fragment were removed with Klenov DNA polymerase resulting in a blunt EcoRI-FtsZ fragment. The blunt EcoRI-FteZ-I fragment was cloned in two orientations between the blunt ends of an Narrow-opened pBluescript SK + plasmid enclosing the FtsZ fragment I by unique polycloning sites that allow the FtsZ fragment to be easily transferred to other cloning plasmids.

To generate the 35S FtsZ sense construct, the GFP gene in pMP2164 I (containing a 35S promoter) was replaced by FtsZ. with the help of Ncol and I Pstl sites. For construction of the antisense orientation (35S antisense I FtsZ), the GFP gene in pMP2164 was removed by cutting with Ncol and I Notl and blunting with Klenov. After ligation of the blunt I EcoRI-FtsZ fragment described above instead of the GFP gene, the anti-sense FtsZ-I orientation was checked via restriction enzyme analysis. The EcoRI fragments I of the resulting plasmids with the expression cassettes for 35S-FtsZ and 35S-I antisense-FtsZ were cloned into the plant vector pMOG402.

For the construction of tissue-specific promoter FtsZ fusions, the plant vector pPGB121S [Kuipers et al., 1995], which contains the GBSS promoter, was used. FtsZ was cloned as BamH1-Sal I fragments in both directions.

All plant vectors were introduced into Agrobacterium tumefaciens I MOG101 [Hood et al., 1993] through 3-point crossings.

I 25 I Potato transformation I In vitro potato plants (cultivar Kardal, supplied by Avebe) were used to obtain transgenic plants via the stem transformation method from Visser (1991) using the above-mentioned transgenic Agrobacterium tumefaciens MOG10 strains with verschillende Η the 4 different FtsZ gene constructs (35S FtsZ sense, 35S FtsZ antisense, H GBSS FtsZ sense and GBSS FtsZ antisense). Plants growing on H selection medium containing kanamycin were tested for the presence of the FtsZ construct by PCR analysis. 1-2 cm 2 leaf was ground H 5 with a potter in 200 μΐ CTAB extraction buffer (2% CTAB (N-cetyl-NNN-)

trimethylammonium bromide), 1.4 M NaCl, 20 mM EDTA, 100 mM Tris, pH

8.0) at room temperature. After washing the potter with 300 μΐ CTAB and combining the sample and the wash solution, the sample was incubated for 10 min at 37 ° C with 10 μΐ RNAse (10 mg / ml).

The sample was then incubated with 100 μΐ chloroform at 65 ° C and extracted twice with phenol / chloroform / isoamyl alcohol (24/24/1) and once with chloroform at room temperature. The DNA was precipitated with 10 μΐ NaAC (pH 4.8) and 1 ml ethanol. The DNA was washed, dried and dissolved in 50-200 μΐ 10 mM Tris, 1 mM EDTA (pH 8.0). One μΐ I 15 was used for PCR reactions in a final volume of 25 μΐ. The forward PCR primer was selected in the promoter region: primer as-1b for 35S-I promoter constructs and primer GBSS1 for GBSS promoter constructs. For I sense constructs, SP103 was used as the reverse PCR primer and for I antisense constructs, SP108 was used as the reverse PCR primer.

In order to be able to pot them in soil, in vitro plants were grown in a medium without kanamycin. The plants were grown for a number of weeks in pots of sufficient size in a greenhouse (Karna).

Chloroplast visualization I Chloroplasts in stomata of the lower leaves epidermis I were examined by fluorescence microscopy (Biorad I MRC1024ES; excitation 488 nm; emission 680 DF 32 nm).

11

Results

The cloning of potato FtsZ-1

Potato FtsZ-1 was cloned using the Clontech SMART ™ RACE cDNA amplification kit. The sequence present in the database was used as the 5 "primer (SP107, Table 1) and the 3 'RACE primer as the 3" primer. The PCR product was cloned into pGEM®-T easy (Promega). Twenty-four clones were tested with a second PCR reaction with nested primers (SP102 and SP103, Table 1). Two inserts of about 1500 bp in length were positive. The sequence of one clone was determined (pFtsZ-1). The nucleotide sequence and the derived protein sequence can be found in Figure 1. The protein sequence was 79% identical (86% identity) to FtsZ from Arabidopsis (Figure 2).

15 Agrobacterium strains

The FtsZ-1 insert (ifcoRI blunt fragment) was transferred to pBluescript SK + (Smal) in both directions. Subsequently, the FtsZ coding regions were cloned into the pPGB121S plant vector, creating sense and anti-sense constructs driven by the tuber-specific GBSS-20 promoter.

For the constructs containing the constitutive CaMV 35S promoter, the FtsZ coding region was cloned in pMP2164 between the 35S promoter and the NOS terminator in both directions. The resulting constructs were cloned as an EcoRI fragment in the plant vector pMOG402.

The binary vectors were introduced into the Agrobacterium tumefaxiiens strain MOG101 by 3-point crossings.

Plant transformation

Sterile potato plants (Kardal cultivar) were genetically modified by the stem transformation method [Visser, 1991] using 1 n 9 33 after the Agrobacterium strains described above. Independently I transformed shoots were grown on selection medium and for each I of the 4 constructs 25 to 30 plants were grown in trays 10 cm high. DNA from each transformant was isolated from the leaf and used for PCR reactions. The forward PCR primers were located in the 35S (As-1b) or GBSS (GBSS1) promoter and the reverse PCR primers in the I FtsZ coding region (SP103 or SP108) (Table 1). The results can be found in Table 2. Most transformants contain the correct PCR fragment indicative of the presence of the desired expression cassette in the plant genome.

I Chloroplasts I The plants with the FtsZ (sense of antisense) gene driven by the 35S CaMV promoter were analyzed by fluorescence microscopy to examine the number and size of the chloroplasts in stomata, as it is expected that a change in FtsZ affects plastid sharing. In Figure 3, representative areas of the epidermis of the lower leaves are depicted. Some transformants I have fewer and larger chloroplasts in their stomata (S4, S10, S11, A5, A6, A8, All) and one plant has clearly smaller chloroplasts (S1).

I Tuber induction and starch analysis I All plants with the correct PCR fragment were further grown in vitro I and one plant from each transformant was planted in soil at Avebe (Karna).

I 25 I Partial conclusion

Potato plants were produced which in the genome contained the potato I FtsZ coding region in sense or antisense direction under control of the constitutive 35S promoter or the tuber-specific GBSS promoter. The 13 chloroplasts in the stomata of the 35S transformants were analyzed by fluorescence. In some plants, the number or size of the plastids differs, indicating that the expression of FtsZ interfered with plastid division.

5

Example 2

In the first example, only the plastid division in the stomata of the transgenic potato plants with 35S-10 promoter constructs was studied. In example 2, the starch grain size distribution of the starch in the transgenic potato plants was also studied, which contained the 4 different FtsZ constructs.

The first generation of Kardal plants with overexpression (GS) or suppression (35S-AS) of the expression of the FtsZ1 gene contains a number of plants with starch grain sizes between 40 and 54 µm, while the starch grain size of the control plants is around 30 µm (Caspers et al). A selection of these plants was grown in greenhouses on larger plots.

Starch was isolated from these plants and the particle size distributions of the starch from these plants were determined with a coulter counter.

20

Material and method

Five plants from each selected control clones (4 pieces), GBSS-FtsZ (GS) clones (4 pieces) and 35S-asFtsZ (35S-AS) clones (2 pieces) were grown under greenhouse conditions in pots at a growing station .

Starch was isolated from the largest harvested tubers (approx. 20-30 g / tuber). For starch isolation, approximately 100 grams of tuber (the five largest tubers) were ground in a Wearing blender. An amount of demi water was added, with a ratio between the weight of the tubers 30 and the weight of the demi water from 2 to 1. To prevent brown discoloration, 1000 ppm of sodium hydrogen sulfite (sodium bisulfite) was added and the tubers were kept for 1 milled minute. The slurry was then filtered through a cheese cloth and the starch precipitated. The supernatant was decanted and replaced with 150 ml of demi water. After settling of the starch, the supernatant liquid was poured off and the starch was transferred to an aluminum dish. The starch was dried overnight in an incubator at 37 ° C.

For the determination of the particle size distributions, the

Sympatec laser diffraction method used with the following specifications: I Sympatec HELOS (H1140), measurement conditions: 10 s (QUIXEL) no focus ref.

I opt. <L%, measuring range: R4: 0.5 /1.8...350 μm, measuring duration: 10 s, QUIXEL (HD23xx model): Quix sp20%, noclean lowl. 60s ultrasonic. The results were analyzed as volume size distribution.

Results The particle size distribution was measured in 4 selected clones I of the non-transformed Kardal control group, 4 clones of GS with I FtsZ1 overexpression and 2 clones of 35 S-AS with repression of FtsZ1 expression. Volume particle size distributions for each potato line can be found in Table 3. The 4 Kardal clones from the control group have an average volume median diameter of 42.0 µm with values ranging between 38.2 µm and 43.8 µm. The volume median diameter of the 35S-AS clones is generally equal to or slightly larger (up to 23% increase) than the

Kardal control group, with values of 45.3 pm and 51.8 pm, respectively.

I The volume median diameter of the GS clones is much higher than the diameter I found for the Kardal plants from the control group, with values 15 to 73.7 μιη. The significance of the measured diameter increases in the 35S-AS and GS clones is underlined by the slight variation in diameter measurements at the 4 independent control plant groups.

Two GS clones (GS04 and GS25) in particular have a considerably higher median median diameter than the control group. Compared to the average of the Kardal control group, the increase in volume median diameter of clone GS04 is 75% and that of clone GS25 is 45%. The differential particle size distributions of each potato clone are shown in Figure 4 and Table 4.

10

Partial conclusions

Kardal genetically modified with a 35S antisense FtsZ gene construct that suppresses FtsZ expression generally has a small effect on the volume median diameter (up to 23% increase) compared to the Kardal control group.

Kardal that is genetically modified with a GBSS-FtsZ gene construct that causes over-expression of FtsZ leads to an increase in the volume median diameter of up to 75%.

^ λ o o o η n I Literature I 1. Colleti KS, Tattersall EA, Pyke KA, Froelich JE, Stokes KD, Osteryoung 5 KW (2000). A homologue of the bacterial cell division site-determining factor MinD mediation placement of the chloroplast division apparatus.

I Current Biol 10, 507-516.

2. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993). New Agrobacterium helper plasmids for gene transfer to plants. Transgene Res 2, 208-218.

I 10 3. Kuipers AGJ, Soppe WJJ, Jacobseb E Visser RGF (1995). Factors affecting the inhibition by antisense RNA or grain-bound starch synthase gene I expression in potato. Mol Gen Genet 246, 745-755.

4. Osteryoung KW (2000). Organelle fission. Crossing the evolutionary divide.

I Plant Phys 123, 1213-1216.

5. Osteryoung KW, Stokes KD, Rutherford SM, Percival AL Lee WY (1998).

I Chloroplast division in higher plants requires members of two functional I divergent families with homology to bacterial ftsZ. Plant Cell 10, 1991-2004.

I 6. Visser RGF (1991). Regeneration and transformation of potato by 20 Agrobacterium tumefiens. In: Lindsay K (ed) Plant Tissue Culture

Manual, Kluwer Academic Publishers, Dordrecht. Vol B5, pp 1-9.

7. Caspers M.P.M., Brunt K. (2001), TNO report starch grain analysis transgenic potato lines, TNO Nutrition and Food Research.

25 17

Table 1. Primers used for PCR reactions.

Name_Sequence_

SP 102__GCGCCTCGAGCTTCTATGCTATAAACACGG

SP103__GGCCGAGCTCCGTCCTTCAAAGCTGAAAGG

SP 107__TTCACTAGTGATTCCATGGCGATTTTAGGG

SP108__GGAGCAAAGCTACTTGAGAAGGGC _

As-lb__CCACTGACGTAAGGGATGAC_

GBSSl GAGGGAGITGGTTTAGTTTTTAGA I

1 n O O o η λ I 18 I Table 2. Transgenic potato plants.

Table 2A. 35S sense plants ____ I DNA-PCR 35S-ftsz Micro-Large Small isolation analyzes present scopic plastids plastids observing _____g___ I ι + + + + __ + _ I 2 + + + + __ I _3 __ + _ + ~ + + ___ I _4__ + __ + __ + __ + __ + ___ I _5 __ + ___ + __ + __ + ___ I _6 __ + __ + __ + __ + ___ _7 __ + __ + __ + ___ + ___ _8 __ + __ + __ + __ + ___ I _9__ + __ + __ + __ + ___ JO __ + __ + __ + __ + __ + __ I _n __ + __ + __ + __ + __ + __ I _12 __ + __ + __ + __ + ___ I _13 __ + __ + __: __; ___ I 14 + __ + __-____ _15 __ + __ + __ + __ + ___ + / many _16 __ + __ + __ + __ + ___ I 17 + __ + __-____ I 18 + __ + __-____ I _19 __ + __ +, ____ I _20 __ + __ + __-_____ I 21 __ + __ + __-____ I 22 + __ + __-____ I _23 __ + __ + __ + __ + __ + __ I _24 __ + __ + __ + __ + ___ I _25 __ + __ + __ + __ + ___ I _26 __ + __ + __ + __ + ___ I _27 __ + __ + __-____ I _28 ___ + __ + __-____ I 29 __ + __ + __ + __ + ___ i3o 1+ 1+ 1+ 1+ /.--5. .Λ Ztt _ 19

Table 2B. 35S antisense plants____ DNA-PCR- Ftsz-35S Micro-Large Small isolation analyzes present scopic plastids plastids _____observation___ _1 __ + __ + __-__ + ___ _2 __ + __ + __ + ___ _3 __ + __ + __-__ + ___ _4 __ + __ + __ + __ + __ + __ + ___ _5 __ + __ + __ + __ + __ + / strange__ _6 __ + __ + __ + __ + __ + / strange__ _7 __ + __ + __ + __ + ___ 8 + + + 4- + _9 __ + __ + __ + __ + ___ 10 __ + __ + __ + __ + ___ 11 __ + __ + __ + __ + __ + __ 12 __ + __ + __ + __ + ___ 13 __ + __ + __ + __ + __ + __ 14 __ + __ + __ + __ + __ + __ 15 _ + + + + _ + __ 16 __ + _ + ~ + + ___ 17 __ + __ + __ + __ + ___ 18 __ + __ + _ + _ + ___ in iii are missing by some ________________________ skin mouths _ 20 __ + __ + __ + __ + _ 21 __ + __ + __ + __ + ___. _22 __ + __ + __-_____ 23 __ + __ + __ + __ + ___ 24 __ + __ + __ + ___ + ___ 25 __ + __ + _ + + _ + __ 26 __ + _ + + ~ + + 27 __ + __ + __ + __ + ___ 28 __ + __ + __ + __ + __ + __ 29 __ + __ + __ + __ + __ + / many__ 1023309 I 20 I Table 2C. GBSS sense plants_ I DNA-PCR-GBSS-ftsz I __ isolation__analysis present I _1 __ + __ + __ + _ I _2 __ + __ + __ + _ I _3 __ + __ + __ + _ I _4 __ + __ + __ + _ I _5 __ + __ + __ + _ _6 __ + __ + __ + I _7 __ + __ + __ + _ I _8 __ + __ + __ + _ I _9 __ + __ + __ + _ I JO __ + __ + __ + _ I 11 + __ + __ + _ I 12 + __ + __ + _ I 13 + __ + __ + _ I J4 __ + __ + __ + _ I J5 __ + __ + __ + _ I J6 __ + __ + __ + _ I J7 __ + __ + __ + _ I J8 __ + __ + __ + _ I 19 + __ + __ + _ _20 __ + __ + __ + _ I 21 + __ + __ + _ I _22 __ + __ + __ + _ I 23 + + _ + __ _24 __ + __ + __ + _ I _25 __ + __ + __ + _ 21

Table 2D. GBSS antisense plants

DNA-PCR-Ftsz-GBSS

__ insulation__analysis present _1 __ + __ + __ + _ _2 __ + __ + __ + _ _3 __ + __ + __ + _ 4 + + + _5 ___ + __ + __ + _ 6 + + + 7 ~ + + _8 __ + __ + __ + _ _9__ + __ + __ + _ 10 __ + __ + __ + _ 11 __ + __ + __ + _ 12 __ + __ + __ + _ 13 __ + __ + __ + _ 14 __ + __ + __ + _ 15 __ + __ + __ + _ 16 __ + __ + __ + _ 17 __ + __ + __ + _ '18 __ + __ + __ + _ _19 __ + __ + __: _ 20 __ + __ + __ ± _ 21 __ + __ + __ + _ 22 + __ + __ + / - 23 __ + __ + __ + _ 24 __ + __ + __ + _ 25 __ + __ + __ + __ 26 __ + __ + __ + _ 1 27 + + 1 + 1 0233 no 22

Table 3. Volume particle size distribution in starch samples as measured by laser diffraction 5 ______ ^^ Sample ^^^ dlMuiO ^^

Check 1__38.2__13.4__36.1__59.1

Check 2__42; 6__14.0__37.1__64.2

Check 3__43.5__10.1__31.0__78.4

Check 4 43.8__12.7 34.9__67.4

Average 42.0 12.6 34.8 67.3 from control group GS04__73 / 7__19.8__56Λ__166.2 GS12__56 / 7__15.7__401__96.0 GS14 47.3__14 / 3__4L0__75.6 GS25__609__16 / 7__44J) __ 103.5 35S-AS15__5L8__102__44J3__28.3 1.4 -3 AS28.8 45.3 13.4-vol. -median diameter (μηα) 210% of the starch particle diameter is smaller than the value in μηα (ie 90% of the starch particle diameter is larger than the value) 10 350% of the starch particle diameter is smaller than the value in μηα (ie 50% of the starch particle diameter starch particle diameter is greater than the value) 490% of the starch particle diameter is smaller than the value in μηα (ie 10% of the starch particle diameter is greater than the value) λ oooQfin 23

Table 4

Particle _ Differential frequency (%) __ diameter Control G- G- G- G- 35S- 35S- (μιη) Average S04 S12 S14 S25 AS15 AS28 1.8__0.06 0.04 0.05 0.06 0.05 0.05 0.07 2.2 __0.08 0.05 0.06 0.07 0.07 0.07 0.08 2.6 __0.08 0.05 0.06 0.06 0.06 0.06 0.07 3.0 __0.07 0.04 0.05 0.05 0.05 0.05 0.06 3.6 __ 0.06 0.04 0.04 0.04 0.04 0.05 4.4 __0.05 0.03 0.04 0.03 0.03 0.03 0.03 5.2 __0.04 0.02 0.03 0.03 0.02 0.02 0.03 6.2 __0.04 0.02 0.03 0.02 0.02 0.02 0.02 7.4 __0.04 0.02 0.03 0.03 0.02 0.02 0.02 8.6 0.06 0.03 0.05 0.04 0.03 0.03 0.04 10.0 __0.10 0.05 0.08 0.07 0.05 0.05 0.07 12.0 __0.17 0.08 0.13 0.12 0.10 0.10 0.15 15.0 __0.32 0.14 0.22 0.23 0.19 0.30 18.0 __0.50 0.22 0.32 0.38 0.31 0.32 0.51 21.0 __0.71 0.30 0.42 0.53 0.44 0.45 0.73 25.0 __0.97 0.41 0.55 0.72 0.62 0.61 1.00 30.0 __1.32 0.57 0.72 1.00 0.88 0.85 1.34 36.0 __1.68 0.78 0.95 1.35 1.20 1.17 1.67 42.0 1.86 1.05 1.20 1.49 1.53 1.80 50.0 1.75 1.36 1.45 1.81 1.70 1.83 1.65 60.0 __1.25 1.66 1.65 1.70 1.68 1.90 1.16 72.0 __0.61 1.72 1.62 1.23 1.31 1.49 0.58 86.0 __0.20 1.34 1.25 0.64 0.74 0.78 0.23 102.0 __0.08 0.73 0.71 0.24 0.31 0.28 0.10 122.0 __0.06 0.33 0.31 0.09 0.13 0.09 0.09 146.0 __0.08 0.20 0.15 0.06 0.10 0.07 0.12 174.0 __0.12 0.24 0.13 0.09 0.13 0.10 0.18 206.0 __0.14 0.31 0.15 0.13 0.19 0.15 0.21 246.0 __0.13 0.34 0.17 0.13 0.23 0.18 0.19 294.0 __0.11 0.33 0.14 0.09 0.26 0.15 0.13 1 350.0 0.00 0.27 0.00 0.00 0.27 0.00 0.00 a η ooo η n

Claims (18)

A method for changing the average starch grain size or spreading the starch grain size in a collection of plant cells containing amyloplast wherein said plant cells are provided with a means for modulating a process of plastid division I. 2.. The method of claim 1, wherein the agent is a nucleic acid I of an FtsZ gene or a functional part, derivative or analogue thereof. The method of claim 2, wherein the agent is a nucleic acid I of an FtsZ1 gene or a functional part, derivative or analogue thereof. I 10 A method according to any one of claims 1-3, wherein the plant cell set is a plant made transgenic by providing the genome with a recombinant nucleic acid to modulate the process I of plastid division in said plant or an amyloplast containing part I thereof. An amyloplast-containing plant cell comprising an agent for altering the plastid division process in said cell. 6. A transgenic plant containing a recombinant nucleic acid in the genome for modulating a plastid division process in said plant or an amyloplast-containing part thereof A transgenic plant according to claim 6, wherein the plant belongs to the tuber or seed crops, or to the grains. A potato plant, a grain plant or a cassava plant according to claim 7. A starch-rich part of a plant according to any one of claims 6-8. I 25 A starchy part of a plant according to claim 9, wherein the I part comprises a tuber, a root or a seed. m A method for making a starch composition, characterized in that a plant cell according to claim 5, or a plant or amyloplast-containing part thereof according to any of claims 6-10, is used. A starch composition, characterized in that at least a part thereof is obtained according to claim 11, or a derivative of such a starch composition. A product comprising a starch composition or a derivative thereof according to claim 12. 14. A product according to claim 13, wherein the product is a food product. A product according to claim 13, wherein the product is a physically and / or chemically processed (bio) raw material for the manufacture of a food product or utensil. The use of a starch composition according to claim 12 or a product according to claim 13, for changing the mouthfeel of a food product. 17. In the nucleic acid of Figure 1 or a functional part thereof. A protein molecule according to Figure 1 or a functional part thereof. 1 Π 9 QQ η n (I Figure 1. DNA sequence and deduced protein sequence of potato-FtsZl. ATGGCGATTTTAGGGCTCTCAAATCCAGCAGAATTAGCTTCTTCTCCTTCTTCTTCCTTA MAÏLGLSNPAELASSPSSSL ACTTTTTCCCACAGGCTACATACTTCCTTCATTCCTAAACAATGCTTCTTCACCGGAGTT TFSHRLHTSFIPKQCFFTGV CGCCGGAAAAGTTTTTGCCGGCCTCAACGTTTCAGCATTTCAAGTTCATTTACTCCGATG RRKSFCRPQRFSISSSFTPM GATTCTGCTAAGATTAAGGTCGTCGGCGTCGGTGGAGGTGGAAACAATGCCGTTAACCGT DSAKI KVVGVGGGGNNAVNR ATGATTGGTAGTGGCTTACAGGGTGTTGACTTCTATGCTATAAACACGGATGCTCAAGCA MIGSGLQGVDFYAI NTD AQA CTGGTACAGTCTGCTGCCGAGAACCCACTTCAAATTGGAGAACTTCTGACTCGTGGGCTT LVQSAAENPLQIGELLTRGL GGTACTGGTGGCAATCCTCTTTTAGGGGAACAGGCAGCGGAGGAGTCAAAGGAAGCTATT LVQSAAENPLQIGELLTRGL GCAAATTCTCTAAAAGGTTCAGATATGGTTTTCATAACAGCAGGAATGGGTGGAGGTACA ANSLKGSDMVFITAGMGGGT GGATCTGGTGCGGCTCCTGTTGTGGCTCAAATAGCAAAAGAAGCAGGTTATTTGACTGTT GSGAAPVVAQIAKEAGYLTV GGTGTTGTTACATATCCTTTCAGCTTTGAAGGACGTAAAAGATCTGTGCAGGCTCTGGAA GVVTYPFSFEGRKRSVQALE GCAATTGAAAAACTTCAGAGAAATGTTGACACTCTTATAGTAATTCCCAATGATCGTCTA AIEKLQRNVDTLIVIPNDRL CTAGATATTGCCGATGAGCAGACACCACTTCAAGATGCTTTCCTTCTTGCAGATGATGTA LDIADEQTPLQDAFLLADDV TTACGTCAAGGTGTCCAAGGAATATCTGATATAATCACTATTCCTGGGCTTGTGAATGTG LRQGVQGISDIITIPGLVNV GATTTTGCCGATGTAAAGGCAGTGATGAAAGACTCTGGAACTGCTATGCTCGGAGTGGGG DFADVKAVMKDSGTAMLGVG GTTTCATCAAGCAAGGACCGAGCTGAAGAAGCAGCCGAACAAGCAACTCTGGCCCCTCTA VSSSKDRAEEAAEQATLAPL ATTGGGTCGTCAATTCAATCTGCAACTGGGGTAGTTTATAACATTACAGGAGGAAAAGAC IGSSIQSATGVVYNITGGKD ATAACTTTGCAAGAAGTGAATAGGGTGTCCCAGGTTGTTACCAGTCTGGCTGATCCCTCT ITLQEVNRVSQVVTSLADPS GCTAACATCATATTTGGTGCTGTTGTTGATGAGCGTTACAATGGTGAAATACACGTGACA ANIIFGAVVDERYNGEIHVT ATAATTGCAACTGGTTTCACCCAGTCGTTTCAGAAGACACTTCTATCTGACCCACGAGGA IIATGFTQSFQKTLLSDPRG GCAAAGCTACTTGAGAAGGGCTCTGGAATCAAAGAAAGCATGGCATCACCTGTTACCCTG AKLLEKGSGI KESMASPVTL AGATCATCAAACTCACCTTCAACAACCTCACGGACACCTACTCGAAGGCTGTTCTTTTAG RSSNSPSTTSRTPTRRLFF * CTCCTTCATATTGTCTTTTTTACAGCTTCATTACTCTCCTTTTTAGTTTCTTCCTTTGTA TTTAACATGTTTTGCTGAAGGGAACTGATTGGTGTTTGCATGTGGCTGTAGAGATAGTGA TTATTCTTTATCAG ATGCATACTCAGCTGTGCTCTCTTGTGACAGCACGTTTTTACGCAT GTAAAATATTGATACTTGTTTATTAGA31 λ λ o o o η λ Figure 2. Sequence comparison of FtsZ from Arabidopsis (AtFtsZ-1) and potato (StFtsZ-1). AtFtsZ-1: 1 MAIIPLAQLNELTISSSSSSFLTKSISSHSLHSSCICASSRISQFRGGFSKRRSDSTRSK MAI + L + ++ N SS SS LH LT SH + SI + FG R + RR StFtsZ-1: 1-MAILGLS NPAELASSPSSSLT FSHRLHTSFI PKQCFFTG --- --- VRRKSFCRPQ AtFtsZ-1: 61 + SMRLRCSFSPMESARIKVIGVGGGGNNAVNRMISSGLQSVDFYAINTDSQALLQFSAENP SF + PM + S A + IKV + GVGGGGNNAVNRMI SGLQ VDFYAINTD + QAL + Q + AenP StFtsZ-1: 50 RFSISSSFTPMDSAKIKWGVGGGGNNAVNRMIGSGLQGVDFYAINTDAQALVQSAAENP AtFtsZ-1: 121 LQIGELLTRGLGTGGNPLLGEQAAEESKDAIANALKGSDLVFITAGMGGGTGSGAAPWA lqigelltrglgtggnpllgeqaaeesk + aian + lkgsd + vfitagmgggtgsgaapwa StFtsZ-1: 110 lqigelltrglgtggnpllgeqaaeeskeaianslkgsdmvfitagmgggtgsgaapwa AtFtsZ-1: 181 QISKDAGYLTVGWTYPFSFEGRKRSLQALEAXEKLQKNVDTLIVIPNDRLliDIADEQTP QI + K + AGYLTVGVVTYPFSFEGRKRS + + QALEAIEKLQ NVDTLIVIPNDRLLDIAOEQTP StFtsZ-1: 170 QIAKEAGYLTVGWTYPFSFEGRKRSVQALEAIEKLQRNVDTLIVIPNDRLLDXADEQTP AtFtsZ-1: 241 LQDAFLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTAMLGVGVSSSKNRAE LQDAFLLADDVLRQGVQGXSDIITIPGLVNVDFADVKAVMKDSGTAMLGVGVSSSK + StFtsZ RAE-1: 230 LQDAFLLADDVLRQGVQGISDlITIPGLVNV DFADVKAVMKDSGTAMLGVGVSSSKDRAE AtFtsZ-1: 301 EAAEQATLAPLIGSSIQSATGWYNITGGKDITLQEVNRVSQWTSLADPSANIXFGAW EAAEQATLAPLIGSSIQSATGVVYNITGGKDITI.QEVNRVSQWTSLADPSANIIFGAW StFtsZ-1: 290 EAAEQATLAPLIGSSIQSATGWYNITGGKDITLQEVMRVSQWTSLADPSANIIFGAW AtFtsZ-1: 361 DDRYTGEIHVTIIATGFSQSFQKTLI / rDPRAAKLLDKMGSSGQQENKGMSIiPHQKQSPST D RY GEIHVTIIATGF + + + QSFQKTLL DPR akll SG + K + E + M + P + S ++ StFtsZ-1: 350 DERYNGEIHVTIIATGFTQSFQKTLLSDPRGAKLLEK —GSGIKES — MASPVTLRSSNS AtFtsZ-1: 421 ISTKSSSP-RRLFF 433 ST S + P RRLFF StFtsZ-1: 406 PSTTSRTPTRRLFF 419 in? 33no