Abap1 Docking Protein

ABAPl DOCKING PROTEIN

TECHNICAL FIELD

The present invention relates generally to the manipulation of cellular processes involved in plant flowering and dormancy. More particularly, the present invention relates to a novel barley protein termed ABAPl , its docking protein DPI , and methods for using these proteins in the manipulation of hormone responses in plants.

BACKGROUND ART

Flowering, seed development, and seed dispersal are critical developmental steps in the life cycle of plants, and progression from one stage to the next is determined, in part, by the activation or repression of multiple regulatory genes in response to environmental cues. Methods to activate or repress such genes, and thereby manipulate the transition between stages of the plant life cycle, have been the subject of much research by commercial producers seeking to increase crop yield, hardiness, or product quality.

The transition to flowering occurs through highly coordinated processes and requires the integration of multiple regulatory pathways. For example, several plants utilize long days and cold temperature as environmental sensors of seasonal progression and gibberellic acid (hereinafter "GA") as a developmental indicator. These regulatory pathways are also involved in the control of the time of flowering through a coordinated interaction between the endogenous developmental factors and the surrounding environmental cues.

Following flowering, further regulatory pathways are activated or inhibited to permit seed ripening, dessication, and seed dispersal. In the production of certain crops, it is necessary that the seeds be fully ripe prior to harvesting in order to achieve optimal characteristics of any product that is produced from the seed. For example, in the production of canola oil, failure to complete seed ripening of the canola crop generally results in lower oil quality due to the presence of chlorophyll within the seed, even when the seed is treated with dessicants. Similarly, seed dormancy periods are highly regulated by pathways that respond to various environmental stress factors, for example drought or salt exposure. Dormant periods are characterized by cessation of growth or development and the suspension of metabolic processes.

In the field of stress responses, certain advances have been made in determining the plant proteins and regulatory pathways responsible for adaptation to stress conditions, and as a result, plants can now be genetically engineered to withstand a greater degree of environmental stresses, and to quickly recover and reinitiate the reproductive cycle following periods of stress.

It is generally held that the presence of seed dormancy ensures plant survival by distributing germination over time to account for potentially adverse conditions. In economically important species, selection pressure has frequently resulted in the modification of germination and dormancy. For example, germination of seeds prior to harvest, termed pre-harvest sprouting, can be a significant problem in cereal production, reducing grain quality and resulting in major economic losses.

Conversely, prolonged dormancy can be a problem in plant propagation of forestry and horticultural species.

Abscisic Acid

The plant hormone abscisic acid (hereinafter "ABA") regulates various physiological processes in plant development and is a key hormone in plant abiotic stress responses. These roles include agronomically important processes, such as its involvement in seed dormancy, synthesis of storage proteins, and lipid accumulation and its mediation of stress-induced processes.

Through a network of signaling intermediates, ABA regulates key events during growth and development, the most profound being those related to the promotion of seed dormancy and stress tolerance. In seeds, ABA acts via at least two routes, one inhibits seed germination, and the second maintains dormancy status of mature seeds. Following perception of ABA by plant cells, the cellular responses can be either very quick, such as ion channeling in guard cells, or slow and require changes in gene expression. In both situations, it is assumed that cellular response to ABA requires some kind of interaction between ABA molecules and receptors followed by protein phosphorylation, finally causing the transcription of genes involved in stress-induced processes.

Certain ABA mutants have been identified in the prior art, having different responses to ABA, and the molecular mechanism underlying ABA perception is therefore still poorly understood. For example, in high-mountain potatoes, exogenously applied ABA favors tuberization whereas GA favors flowering. In addition, the ABA-deficient mutants of Arabidopsis, in addition to a dwarf habit, flower early.

High-affinity binding sites for ABA have been reported in membrane fractions and guard cell plasmalemma of Viciafaba (S), microsomal fractions from

Arabidopsis thaliana (9), the cytosol of the developing flesh of apple fruits and more recently, an ABA-specific binding site was purified from the epidermis of broad bean leaves. The site of ABA perception has also been located at the extracellular side of the plasma membrane of barley aleurone tissue. However, due to difficulties in purifying ABA-binding proteins, most studies on ABA binding have been carried out by either using total protein extracts or histochemical probes. Furthermore, it has always been difficult to relate these proteins to any physiological role of ABA in plants. Despite numerous attempts to isolate membrane-bound hormone receptors in plants, little progress has been made in identifying ABA receptors owing to their low abundance relative to other proteins in plant cells.

DISCLOSURE OF THE INVENTION

Exemplary embodiments of the present invention are directed to a nucleotide coding sequence for controllable expression of a protein designated as DPI and configured for binding to the ABAPl protein, nucleotide molecules and constructs comprising the DPI nucleotide coding sequence, the amino acid sequence characterizing the DP 1 protein, methods for modulating the effects on plants resulting from the binding of ABA to the ABAPl protein whereby nucleotide molecules or constructs comprising the DPI nucleotide sequence are stably introduced into the genomes of seed-producing recipient plants, methods for affecting the germination, metabolism, physiological performance and productivity of the recipient plants, and recipient seeds comprising the nucleotide molecules. One exemplary embodiment of the present invention is directed to a nucleotide coding sequence identified as SEQ ID NO: 4 for expression of an ABAPl- binding protein comprising an amino acid sequence identified as SEQ ID NO: 3.

According to one aspect, there is provided an ABAPl -binding protein comprising the amino acid sequence identified as SEQ ID NO: 3, and is referred to herein as the DPI protein. The DPI protein is associated with the plasma membrane of plant cells.

Another exemplary embodiment of the present invention is directed to methods modulating one or more of seed development, seed maturation, seed quality, germination vigor, vegetative growth and flowering by affecting the binding of ABAPl proteins to DPI proteins associated with plant cell membranes. The methods generally comprise; first, constructing a nucleotide molecule by fusing a nucleotide coding sequence set forth in SEQ ID NO: 4 with a regulatory nucleotide sequence selected for over-expression or for under-expression of the coding sequence. Second, stably introducing said nucleotide molecule into the genome of a recipient cell from a seed-producing parent plant. Third, culturing the recipient cell into a recipient whole plant and then further culturing the plant to produce mature recipient seeds, and fourth, harvesting the mature recipient seeds. The recipient cell, recipient vegetative tissues comprising the recipient whole plant produced from the recipient cells, and recipient seeds produced therefrom, and plants and seeds subsequently produced from the recipient seeds, will express, relative to the parent seed-producing plant, modified amounts of the AB AP 1 -binding protein referred to herein as DPI, said DPI protein comprising an amino acid sequence identified as SEQ ID: 3.

According to one aspect, there is provided a method for increasing the binding of ABAPl proteins to plant cell membranes by over-expressing therein nucleotide coding sequence SEQ ID NO: 4 thereby increasing DPI proteins in the plant cell membranes. The consequence will be decreased binding of ABA by the ABAPl proteins resulting in greater ABA-mediated effects on seed development, maturation, quality and germination, plant growth, development and flowering.

According to another aspect, there is provided a method for decreasing the binding of ABAPl proteins to plant cell membranes by under-expressing therein nucleotide coding sequence SEQ ID NO: 4 thereby decreasing DPI proteins in the plant cell membranes. The consequence will be increased binding of ABA by the ABAPl proteins resulting in reduced ABA-mediated effects on seed development, maturation, quality and germination, plant growth, development and flowering.

According to a further aspect, the nucleotide molecule may be introduced into the genome of a recipient cell by one of ballistic transformation, Agrobacterium- transformation, or electropolation.

According to another aspect, the recipient cell may be selected from a monocot seed-producing plant, or alternatively, from a dicot seed producing plant.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures, wherein:

Fig. Ia is a nucleic acid sequence SEQ ID NO: 4 of the gene encoding the DPI protein;

Fig Ib is an amino acid sequence SEQ ID NO: 3 comprising the DPI protein;

Fig. 2 is a graph depicting the dose-dependent effect of ABA on interaction between ABAPl and DPI in GST pull-down assays;

Fig. 3 is a micrograph identifying the cellular localization in onion epidermal cells of: (a) ABAPl, (b) DPI, and (a) both ABAPl and DPI in the presence of ABA

Fig. 4 shows the effects of ABA and GA on the interactions between the

ABAPl and BP80 proteins wherein: (a) is a SDS-PAGE gel showing the effects of increasing ABA concentrations on co-immunoprecipitation of ABAPl and BP-80, and (b) is an SDS-PAGE gel showing the effects of increasing concentrations of GA on reversing the effects of ABA; and

Fig. 5 is a western blot of a SDS-PAGE gel showing precipitation of bound

ABAPl -BP-80 proteins in the absence of ABA. DETAILED DESCRIPTION OF THE INVENTION

We have previously disclosed a plant gene (GenBank Accession No. AF127388) characterized by the nucleotide sequence set forth in SEQ ID NO: 1, encoding a protein designated ABAPl, that has a unique ABA-binding site. The ABAPl protein is characterized by the amino acid sequence set forth in SEQ ID NO: 2. We also disclosed methods to isolate ABAPl, and to manipulate ABAPl expression and concentrations in plants, to modulate the effects of ABA in those plants. Furthermore, we have identified the Arabidopsis RNA-binding protein FCA (i.e., flowering control protein) as an ABA receptor, useful for modulating the transition from the vegetative stage to the reproductive stage in plants. FCA shares sequence homology at the C-terminus with ABAPl, although FCA is not common in seeds and its loss in fca-l mutants does not alter ABA inhibition of stomatal opening or seed germination. ABAPl specifically binds ABA and GA, and up-regulates genes that promote dormancy. We have previously shown that ABAPl possesses a WW domain capable of facilitating a protein:protein interaction, and that ABAPl is an integral component of the ABA signaling that regulates seed germination. Therefore, it is desirable to provide molecules that specifically bind to ABAPl or inhibit binding of ABAPl to other proteins. Such molecules may be useful in the manipulation of ABA signaling pathways. Moreover, it is desirable to determine whether ABA signaling pathways may be manipulated to affect the plant life cycle and whether any role of ABA or its binding proteins may be commercially exploited.

We have surprisingly found that the ABAPl protein binds to a vacuolar sorting receptor protein named BP-80 (i.e., a binding protein of 80 kDa), and that ABA disrupts the binding interactions between ABAPl and BP-80, i.e., the extent of ABAPl binding to BP-80 is directly proportional to the concentration of ABA. As ABA concentrations increase, the extent of ABAPl -BP-80 binding decreases, whereas when ABA concentrations decrease, the extent of ABAPl -BP-80 binding increases. However, the inhibitory effects of ABA on binding of the ABAPl and BP- 80 proteins are not reversed by increasing concentrations of GA. BP-80 is an integral vacuolar membrane sorting protein that functions to direct lytic proteins to lytic vacuoles contained within cells comprising plant tissues. In seeds, lytic vacuoles store carbohydrases, proteases and lipases which are essential for seed germination. Furthermore, we have now discovered that a 35-kDa fragment of the BP-80 protein comprising the amino acid sequence identified herein as SEG ID NO: 3 and referred to as "DPI", specifically represents the ABA and ABAPl binding domain of the BP- 80 protein. The DPI protein is encoded by the nucleotide sequence identified herein as SEQ ID NO: 4.

Accordingly, those skilled in these arts will understand that by controllably manipulating and expressing the ABAPl, the BP-80, and the DPI proteins, it is now possible to manipulate the genome of recipient plant cells to affect the delivery of lytic proteins to the lytic vacoules via the BP-80 sorting proteins, thereby making it possible to control seed development, maturation, quality and germination vigor.

Accordingly, manipulation of the expression of the ABAPl protein and/or the BP-80 protein and/or the DPI protein will enable controllable modulation of accumulation and storage of proteins within the lytic vacuole thereby inhibiting the transport of lytic proteins to the lytic vacuoles, thereby affecting seed quality properties and germination characteristics. Furthermore, it is now also possible to manipulate the expression of the ABAPl and DPI proteins in recipient plant cells for manipulably controlling plant vegetative growth and flowering.

ABAPl

Plants were genetically modified by transformation with a vector containing a barley ABAPl cDNA sequence SEQ ID NO: 1 in either, sense or antisense orientation. Full length ABAPl sequences may be inserted into the vector, or an appropriate portion of the ABAPl may be selected and used in accordance with the invention. In the following examples, barley was used as a representative monocot species, and Arabidopsis was used as a representative dicot species.

Generally; direct transformations may be conducted by any one of a number of methods known to those of skill in the art, such as electroporation, PEG precipitation, microprojectile bombardment or infection with Agrobacterium.

Arabidopsis plants overexpressing the barley ABAPl protein (SEQ ID NO: 2) produced 20-50% more seeds per plant than unmodified Arabidopsis plants. In addition, the modified plants produced larger siliques or seed pods than unmodified plants. Still further, modified plants were generally taller, appeared more robust, and had generally greater biomass than unmodified plants. By contrast, Arabidopsis ABAPl knockdown plants showed decreases in biomass.

Barley ABAPl antisense knockdown plants yielded fewer seeds, were slow to flower, appeared to be unable to tiller, and generally did not thrive. Moreover, although the ABAPl deficient plants produced an appropriate number of reproductive spikelets, the spikelets were infertile, producing no seeds.

It was found that ABAP 1 inhibits seed germination in barley plants and retards radical and plumule growth in barley embryos. Overexpression of ABAPl in plants also enhances silique size, seed number, seed oil content, and plant biomass in both monocot and dicot plant species. The increases in seed number may be due to either an ABAPl dependent increase in the number of female gametes produced by these plants, or ABAP I overexpression may improve embryo viability. By contrast, reducing expression of ABAPl in plants favors vegetative growth and results in plant infertility.

The plants produced in accordance with the invention may be commercially exploited to enhance plant tissue development. For example, plants overexpressing ABAPl produce more seeds, and also have other enhanced properties, such as seed oil content and increased biomass. Similarly, plants containing the ABAPl antisense vector under control of an inducible promoter may be triggered to induce vegetative growth at an appropriate time in the plant life cycle or if environmental conditions are not appropriate for supporting seed development.

The methods of the present invention are described above with reference to Barley and Arabidopsis as representative of monocot and dicot plants, respectively. As the effects on both monocot and dicot plants were similar, it is reasonable that similar studies in other plant species would confirm that the methods of the present invention may be universally applicable to commercially valuable plants. Moreover. DPI and variants may similarly be introduced into plant cells using similar methods.

Example 1 - Methods for Preparing Constructs

In the presently described embodiments, the ABAPl cDNA sequence identified as SEQ ID NO: 1 (open reading frame of GenBank Accession AF127388) was cloned in sense or antisense orientation into PUTV 45 vector digested with BaniHl and Sacl under the control of a ubiquitin promoter. The vector included a Basta resistance gene to simplify later identification of successfully transformed plants. Successfully transformed plants were identified by spraying of plants with Basta (20OuM) after 7 days of growth. Seeds were harvested from healthy plants, and presence of the transgene was verified by PCR assay.

Although the open reading frame of ABAPl was used in the present embodiment, other effective ABAPl sequences may be selected by methods known to those skilled in the art. For example, in such applications, it is generally preferable that the DNA sequence contains at least about 15 nucleotides, but may contain as many as 2,000 nucleotides. Preferably the sequence is selected from the coding or noncoding region of the ABAPl cDNA SEQ ID NO: 1.

The selected DNA sequence was then subcloned in either sense (5' - 3') or antisense (3' - 5 ' ) orientation downstream of a promoter by known methods to form an expression cassette. The promoter is preferably one that is functional in plants and/or seeds, and is more preferably a promoter functional during plant seed development. The promoter may be an inducible promoter to provide appropriate control over the expression of the selected DNA sequence.

Other methods to overexpress proteins in plant species are known, such as targeted insertion into the plant genome, and may similarly be used to overexpress ABAPl in monocot or dicot plants in accordance with the invention.

Example 2 - Monocot Transformation

Transformation of monocots is preferably accomplished by microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall.

Monocot transformation in the present study was carried out by embryo bombardment. Mature embryos of McLeod and Harrington barley cultivars were hand-isolated from imbibed de-husked seeds. Gold powder particles (1 mg) were mixed with different plasmid concentrations in a ratio of I.T:I of reporter:effector:insert, precipitated for 2 h at -20 0C before being re-suspended in 95% ethanol. Bombardment included 500 ng DNA/shot and was otherwise carried out as described by Isabel-LaMondeda et al, 2003, Plant J, vol. 33, p. 329). After bombardment, embryos were subjected to different ABA treatments and incubated in the dark for different time intervals and germination percentage was recorded and tissue collected.

Example 3 - Dicot Transformation

The generally preferred method for dicot transformation is infection of plant cells with Agrobacterium tumefaciens by leaf-disk protocol (Horsch et al., 1985, Science 227: 1229-1231).

In the present dicot study, the assembled vector was transformed into the

LBA4404 Agrobacterium strain. Transformation into Arabidopsis was accomplished by dipping Arabidopsis inflorescence shoots for 3 seconds into 200nil of LB4404 infiltration medium (112 x Murshiage and Skoog salts, 1 x Gamborgs B5 vitamins, 5% (w/v) sucrose and 0.044 benzylamino purine) containing the Agrobacterium transformed with ABAP 1 sense or antisense vector. Plants were then transferred to a growth room and exposed to 16 hours of light per day at approximately 22 degrees C until seed harvest. Following transformation, harvested dried seeds were sown, stratified for 3 days at 4 degrees C, and then placed in the growth room.

DPI -An ABAPl Docking Protein A 35 kDa protein referred to herein as DPI (previously termed ABA45) has been cloned from barley aleurones. DPI comprises the amino acid sequence listed in SEQ ID NO: 3 (Fig. Ib) and is encoded by the nucleotide sequence listed in SEQ ID NO: 4 (Fig Ia), is a GRAM-domain containing protein, and possesses consensus motifs (PPY and TPSP) that interact with WW domains. DPI includes a long transmembrane domain, suggesting association with aleurone plasma membranes. DPI also includes domains for SH3 interaction, and for binding kinases and phosphatases, suggesting a role in signaling.

DPI contains Epidermal Growth Factor (EGF)-like domains and a protease- associated domain, suggesting a role in vacuolar sorting and Ca++ signaling. Ca++ is critical in most ABA and GA responses and DPI may therefore be a critical factor in regulating hormonal responses involving ABA and GA. Each of the above DPI features may be modified, inactivated, removed, or manipulated to achieve various intracellular effects, including increased or decreased seed germination or dormancy, improved response to stress, or altered calcium signaling within the cell or plant.

Example 4 -Isolation and Characterization of DPI

DPI was initially isolated along with ABAPl during screening of an expression library with anti-idiotypic ABA antibodies. DPI was identified as an ABA-responsive protein, and it was found that DP 1 could bind ABAPl in vitro. As shown in Fig. 2, (+)-ABA inhibits ABAPl-DPl interaction in GST pull-down assays in a dose-dependent manner. GST pull-down assays also demonstrated that the in vitro interaction between ABAPl and DPI is affected by the presence of ABA in a similar fashion as FCA-FY, which is indicative of conformational change in the ABAPl WW domain following ABA binding. The interaction was sensitive to ABA at concentrations approaching the low end of the physiological range.

Example 5

The cellular localization of ABAPl-DPl interaction in vivo was determined using fluorescence tagged ABAPl and DPI expressed in onion epidermal cells. As shown in Figs. 3 (a) and 3(b), ABAPl and DPI are attached to the plasma membrane but can also be associated with the nucleus.

As shown in Fig. 3(c), in the presence of ABA, ABAPl in onion epidermal cells became less associated with the plasma membrane, with more ABAPl being found in the nucleus.

Example 6

A pulldown experiment was conducted to confirm the effects of ABA on the binding between the ABAPl and BP-80 proteins. One of the proteins was synthesized with a portion of the glutathione sulfo-transferase (GST) enzyme attached to the N- terminal. The other protein was in vzYrotranslated in the presence of 35S-methionine. Separate batches of the two proteins were mixed together and incubated in solutions that contained increasing concentrations of ABA. Bound ABAPl -BP-80 protein complexes were "pulled down" with affinity beads coated with GST antibodies. The ABAP1-BP-80 complexes were then eluted from the beads and run through SDS- PAGE gels which were then assessed for the presence of the 35S-methionine which confirmed the presence of the bound ABAP1-BP-80 protein complex. Fig. 4(a) shows that ABAP1-BP-50 bound together in the absence of ABA or in a very low concentration of ABA, i.e., 0.01 μM. However, increasing the ABA concentration to 0.05 μM and greater, effectively prevented the binding of ABAPl to BP-80. Fig. 4(b) shows that the inhibition of ABAPl binding to BP-80 by 0.05 μM ABA was not alleviated by increasing GA concentrations. Fig. 5 shows the results of a pull-down assay conducted with ABAPl and BP-80 with pea extract in the absence of ABA. The PB-80 protein bound to ABAPl was visualized with a BP-80 antibody.

It appears that ABAPl is attached to the plasma membrane through its interactions with DPI, and that ABA binding disturbs this interaction resulting in dissociation of ABAPl from the plasma membrane, thereby increasing the appearance of ABAPl in the cytosol, and causing accumulation of ABAPl in the nucleus. This is supported by previous studies in seeds using tethered ABA or microinjected ABA, which have shown that ABA is likely perceived by two different sites: the first located at the plasma membrane mediating ABA inhibition of GA; and the second in the cytosol activating the Enz promoter.

Under normal conditions (when the level of ABA is below Kd 28nM), the physiological consequence of ABAPl-DPl dissociation in the presence of ABA is related to an inhibition of germination and ABAPl accumulation in the nucleus, where it likely mediates RNA processing. Germination therefore requires protein:protein interaction of ABAPl-DPl such that ABAPl is tethered to the plasma membrane, and/or germination inhibition involves interaction of ABA with ABAPl, releasing ABAPl from its association with the plasma membrane.

The ultimate effect of DPI interaction with ABAPl is to regulate signal transduction in the presence or absence of ABA (ie, if ABA is not present or is bound to FCA or ABAPl) and control time to flowering or seed dormancy or ripening. This is likely accomplished through altered calcium handling/signaling by the cell.

Although ABAPl does not possess the common RNA recognition motif

(RRM) to bind RNA, it does possess a WW interaction domain harboring residues homologous to the Prp40 splicing factor. Several WW-containing proteins are Prp40 orthologs such as FBPI I. There is substantial evidence to link nuclear WW- containing proteins to transcription and splicing. For example, YAP, a WW containing protein, interacts with PEBP2 transcription factor and Nedd4, another WW containing protein, interacts with RNA polymerase 11 , interactions that affect the machinery of RNA processing. Hormone receptor mobility, therefore, is likely a factor in ABA signal transduction since this phenomenon had been also observed in mammalian glucocorticoid receptor following ligand binding.

Other suitable nucleotide and amino acid sequences homologous to SEQ ID NO: 4 and SEQ ID NO: 3, respectively, may be generated and tested for use in accordance with the invention. For example, a homologous protein that retains the ability to bind ABAPl but has a defective transmembrane region may be created and used to manipulate a plant life cycle. Such a protein may be supplied to a plant cell in excess to bind available ABAPl in the nucleus or cytosol. With a defective transmembrane portion, the DPI protein would not anchor ABAPl to the membrane, resulting in inappropriate cell signaling. Further, proteins may be created that have varying affinities for ABAP 1 , which would also have varied effects on the plant life cycle. Similarly, modifications may be made to ABAPl, for example, to increase or decrease affinity for ABA and/or DPI , altering hormone response in the plant.

The above-described embodiments have been provided as examples, for clarity in understanding the invention. A person of skill in the art will recognize that alterations, modifications and variations may be effected to the embodiments described above while remaining within the scope of the invention as defined by the claims appended hereto.

CLAIMS

1. An isolated nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 4, said nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO: 3.

2. A nucleotide construct comprising the isolated nucleic acid molecule of claim 1 fused with a regulatory nucleotide sequence selected for over-expression or under- expression of said nucleotide sequence set forth in SEQ ID NO: 4.

3. A method for controllably modulating delivery of lytic proteins to lytic vacoules contained within plant cells thereby controllably affecting at least one of seed development, seed maturation, seed quality, germination vigor, vegetative growth, and flowering, said method comprising the steps of:: constructing a nucleotide molecule by fusing a nucleotide coding sequence set forth in SEQ ID NO: 4 with a regulatory nucleotide sequence selected for over- expression or for under-expression of said coding sequence in said plant cells; stably introducing said nucleotide molecule into the genome of a recipient cell from a seed-producing plant; culturing said recipient cell into a whole recipient plant comprising recipient vegetative tissues; further culturing said whole recipient plant to produce and mature recipient seeds therein, and harvesting said mature recipient seeds; and germinating said mature recipient seeds and culturing plants therefrom.

4. A method according to claim 3, wherein the recipient cell is selected from the group consisting of monocot seed-producing plants.

5. A method according to claim 3, wherein the recipient cell is selected from the group consisting of dicot seed-producing plants.

6. A method according to claim 3, wherein said nucleotide molecule is introduced into said recipient cell by a transformation method selected from the group consisting of ballistic transformation, Agrobacterium-mediated transformation, and electropolation.

7. A plant cell having stably incorporated into its genome at least one nucleotide construct according to claim 2.

8. A transformed plant having stably incorporated into its genome at least one nucleotide construct according to claim 2.

9. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:

3.

10. A composition configured for controllably affecting at least one of seed development, seed maturation, seed quality, germination vigor, vegetative growth, and flowering, the composition comprising the isolated protein of claim 9.

11. A method for controllably modulating delivery of lytic proteins to lytic vacoules contained within plant cells thereby controllably affecting at least one of seed development, seed maturation, seed quality, germination vigor, vegetative growth, and flowering, said method comprising the steps of: culturing a plant, and controllably applying the composition of claim 10 to said plant.

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