EP0858513A2 - Modification of soluble solids using sucrose phosphate synthase encoding sequence - Google Patents

Abstract

This invention relates to methods for the utilization of sucrose phosphate synthase encoding sequences to modify the soluble solids in plant sink tissue and to modified plants, plant tissues and plant parts. The method finds use for example for changing the sweetness of plant parts such as fruits and tubers.

Description

MODIFICATION OF SOLUBLE SOLIDS USING SUCROSE

PHOSPHATE SYNTHASE ENCODING SEQUENCE

INTRODUCTION Technical Field

The present invention is directed to compositions and methods related to modification of the sweetness of selected plant tissues. The invention is exemplified by plants, plant parts, and plant cells transformed with one or more copies of a transgene comprising DNA encoding SPS and a transcriptional initiation region functional in plants.

Background

Sucrose is one of the primary end products of photosynthesis in higher plants. It is also the major carbohydrate transported to sucrose accumulating, or carbon sink, tissues for plant growth and development. Plant regions, such as leaf tissue, where sucrose is synthesized are commonly referred to as sucrose source tissue. Plant storage organs, such as roots or tubers, and fruits are examples of sink tissues. The sucrose translocates from the mature leaf (source) to any tissue requiring photoassimilate (sink), especially growing tissues including young leaves, seeds, and roots. Difficulties in the purification of sucrose phosphate synthase (SPS) from plants have interfered with efforts to characterize this enzyme. SPS catalyses the formation of sucrose phosphate, the sucrose precursor molecule, from fructose-6 phosphate and UDP-glucose in photosynthetically active plant cells. Sucrose phosphatase then acts on the sucrose phosphate moiety, in an irreversible reaction, to remove the phosphate and to release sucrose.

SPS is considered a rate limiting enzyme in the pathway providing sucrose to growύjg tissue, therefore the study of SPS and its activity is of special interest. In a recent publication, Walker and Huber, Plant Phys. (1989) 59:518-524, the purification and preliminary characterization of spinach (Spinachia oleracea) SPS was reported. However, monoclonal antibodies specific to the spinach SPS were found to be non-reactive with all other plants tested, "closely related" and "relatively unrelated species", including corn (Zea maize), soybean (Glycine max), barley (Hordeum vulgare), and sugar beet (Beta vulgaris). Thus, additional purified sources of SPS enzyme are needed for effective characterization of this factor. Especially of interest is the characterization of the corn SPS because of its very high export rates, as compared for example, to SPS levels of activity as found in the leaves of soybean. With the advent of biotechnology, the ability to modify various properties of plants, especially agronomically important crops, is of interest. In this regard, it would be useful to determine the coding sequence for an SPS gene to probe other crop sources, to use such coding sequences to prepare DNA expression constructs capable of directing the expression of the SPS gene in a plant cell and to express a DNA sequence encoding an SPS enzyme in a plant to measure the effects on crop yield due to the increased rate of sucrose translocation to growing tissues.

Relevant Literature The following references are related to expression of SPS in transgenic plants:

Somnewald, et al. (1994) Plant, Cell and Environment 77:649-658; Worrell, et al. (1991) The Plant Cell 5: 1121-1130; Micallef, et al. (1995) Planta 796:327-334; Foyer, et al.

(1994) Plant Physiol., 105(S), 23; Galtier et al. (1993) Plant Physiol. 707:535-543; and PCT Application No. WO 94/00563. The following references are related to isolation of DNA encoding SPS: Valdez-Alarcon et al. , (1996) Gene 170(2) :217-222; Sakamoto et al. ,

(1995) Plant Science (Shannon) 772(2):207-217; Heese et al, (1995) Mol. Gen. Genet., 247(A):515-520; Klein et al., (1993) Planta 190(4) :498-510; Salvucci et al., (1993) Plant Physiol., 102(2) :529-536; Sonnewald et al., (1993) 759(2): 174-181; and Herrera-Estrella et al. , (1991) J. Cell Biochem. Suppl. 0 (15 Part A) 148. PCT Application WO 94/00563 discloses antisense potato SPS placed behind a tuber promoter and used to alter the sucrose levels in potato. Acid invertase encoding sequences are described by Klann et al., (Plant Phys. (1992) 99:351-353).

SUMMARY OF THE INVENTION Methods for modifying the sweetness of plant sink tissues are provided in which sucrose phosphate synthase (SPS) activity and/or invertase activity in plant tissues are manipulated Also provided are nucleic acid constructs, vectors, plant cells, plant pans and plants containing at least one exogenously supplied copy of an SPS gene. The invention finds use in modifying carbohydrate partitioning in plant tissues and/or parts, which in turn can be used to alter plant growth, soluble solid content and/or sweetness, and/or to alter the sensitivity of plant growth to temperature and/or to levels of carbon dioxide and oxygen.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows an SDS-PAGE profile of corn SPS at various stages of SPS purification and the quality of the final preparation. Using an 8.5 % acrylamide gel. reducing conditions and staining with silver nitrate. The abbreviations used are as follows: M: Standard of molecular weight B-Galactosidase (116 kd), bovine Albumin (68 kd), Egg Albumin (45 kd), carbonic anhydrase (29 kd); H: Heparin fraction, 30 micrograms of proteins per well; FP: Final Preparation, 7.5 micrograms of proteins per well; FE: Final Extract, 7.5 micrograms of proteins per well; D: Fast-Flow DEAE fraction, 78.5 micrograms of proteins per well.

Figure 2 shows the results of a Western analysis of SPS using monoclonal antibodies. In Fig. 2A, membrane is incubated in the presence of the SPB3-2-19 antibody; in Fig. 2B, membrane is incubated in the presence of an antibody not directed against SPS (negative control anti-neomycin monoclonal antibody); in Fig. 2C, membrane is incubated in the presence of the SPB 13-2-2 antibody. The abbreviations used are as follows: M: standards of molecular weight radio-labeled by 1-125, (NEX-188 NEN) B-Galactosidase (116 kd), bovine albumin (68 kd), carbonic anhydrous (29 kd), trypsin inhibitor (20J kd), Alpha-Lactalbumin (14.4 kd), 150,000 cpm per lane; PA: proteins obtained after immunoaffinity chromatography (see below) with the SPB 13-2-2 monoclonal antibody, about 40 micrograms of proteins per lane; H: Heparin fraction, about 40 micrograms of protein per lane.

Figure 3 shows peptide sequences (SEQ ID NOS: 1-5) derived from SPS protein.

All peptides are typed N→C terminal. Figure 4 shows oligonucleotides used for the PCR reactions CD3 (SEQ ID NOS:

10-11) and CD4 (SEQ ID NOS: 12-13) in relation to the peptides (antisense sequences are presented upside down). Arrows point to the direction the oligonucleotides will prime the polymerase.

Figure 5 shows the characterization of CD3 and CD4 PCR reactions. Figure 5 A shows agarose gel electrophoresis of CD3 and CD4 PCR reactions. The sizes are given in kb. Figure 5B shows autoradiograph of Southern blot of CD3 and CD4 PCF reactions probed with oligonucleotides 4k5 (SEQ ID NO: 14).

Figure 6 shows schematic diagrams representing SPS cDNA and selected clones.

The upper bar represents the entire 3509 bp combined map. Translation stop and start paints are indicated.

Figure 7 shows the assembled SPS cDNA sequence (SEQ ID NO: 6). The sequences of clones SPS 90, SPS 61 and SPS 3 were fused at the points indicated in Fig. 2.

The SPS reading frame is translated (SEQ ID NOS: 6-7). All SPS protein derived peptide sequences are indicated. Figure 8 shows Western blots demonstrating the characteristics of rabbit SPS 90 and

SPS 30 antisera. The abbreviations used are: pAS** = preimmune serum, SPS 30 rabbit

_; AS** = immune serum anti-SPS 90. Molecular weight markers at left, where indicated. S = SPS 120 kd polypeptide ; S* = SPS 90 kd polypeptide ; S** = SPS 30 kd polypeptide.

Figure 9 shows analysis of protein from a 30-day old corn plant. Figure 9A shows a Comassie Blue-stained gel of total protein isolated from a 30 day old corn plant. M = size marker ; R = roots ; 1-8 = leaf numbers counting from the bottom of the plant. Leaf 5 has been cut into 5 segments from the leaf tip (5a) to the end of the sheatfi (5c). PEP = phosphoenolpyruvate carboxylase. Figure 9B shows the results of Western blot analysis using a mixture of antiSPS 30 and antiSPS 90 antisera against total plant protein isolated from a 30 day old corn plant. The signal corresponding to SPS appears at 120-140 kd. Figure 10 shows a schematic summary of a construction of plasmids pCGN627, pCGN639 and pCGN986. Figure 10A shows construction of pCGN627; Figure 10B shows construction of pCGN639; and Figure 10C shows construction of pCGN986.

Figure 11 shows partitioning between starch and sucrose as a function of temperature. The squares are data from control UC82B plants while triangles are data from transgenic tomatoes expressing SPS on a Rubisco small subunit promoter (pCGN3812).

Figure 12 shows maximum rates of photosynthesis for regenerated control (solid bars) and pCGN3812-24 transgenic (open bars) potatoes at three weeks after (panel A) and seven weeks after (panel B) planting. Figure 13 shows tuber dry mass for regenerated control (solid bars) and pCGN3812-24 transgenic (open bars) potatoes add 35 and 70 Pa carbon dioxide in highlight growth chambers (Figure 13 A) and open top chambers in the field (Figure 13B).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods for modifying the solids content of plant sink tissue which use a construct encoding SPS for example as a way of increasing the sweetness of fruit. The soluble solids include simple sugars, but also can include certain soluble polymers, and other soluble cell components. Total solids include more complex carbon compounds, such as starches and cellulose. The method provides for increasing the total solids in a plant sink tissue so that total solids are modified from a given ratio of total solids per unit weight of sink tissue, as measured in control plant cells, to a different ratio of total solids per unit weight of sink tissue. The amount of sucrose available to growing tissues in the plant is increased, and the increased sucrose results in increased total solids per unit weight in the sink tissues of the plant. The method generally comprises growing a plant having integrated into its genome a construct comprising as operably linked components in the 5' to 3' direction of transcription, a transcription initiation region functional in a plant cell and a DNA encoding SPS. The transcription initiation region may be constitutive or tissue specific. By tissue specific is intended that the region is preferentially expressed in cells of a particular plant tissue or part, for instance fruit or leaf as compared to other plant tissues. In one embodiment the method produces sink tissue having increased carbon as soluble solids, as an increased ratio of soluble solids per unit weight of sink tissue, as compared to that measured in control plant cells. This results from the increased levels of sucrose generating an increased rate of transportation of the available sucrose into the carbon sink tissue. In another embodiment, a method is provided to modify the soluble solids ratios in sink tissue, such as the ratio of sucrose to fructose, as compared to that measured in control plant cells or tissue. If the increased soluble solids in said sink tissue comprises fructose, a phenotype will result having an increased sweetness as opposed to the control tissue. A method is also disclosed, however, whereby a decreased ratio of fructose to sucrose, and whereby a reduced sweetness phenotype may be produced.

The use of constructs comprising encoding sequences to other sucrose metabolizing enzymes, such as acid invertase, or the utilization of such enzymes which are endogenous to the plant sink cells, can be advantageously used with this invention. For instance, acid invertase can be expressed in the cells or sink tissue from an expression construct, or, alternatively, the sink tissue can be prevented from converting sucrose to fructose and glucose by the use of an antisense acid invertase construct, whereby cells of the sink tissue will have a decreased acid invertase activity, and thereby a decreased ratio of fructose to sucrose as compared to cells in a control sink tissue. Fruit having increased total soluble solids and/or modified or increased fructose levels, as measured per unit weight are provided and include fruits such as tomato, strawberry and melon. The fruit has a modified sweetness phenotype, either from a total increase in sweetness by percentage of fruit weight, or from an increased ratio of fructose to sucrose in the soluble solids in the fruit.

Transgenic plants and plant parts are provided which have altered carbon partitioning and end-product synthesis through expression of a transgene required for sucrose synthesis. The transgenic plants, cells and plant parts such as leaf, fruit and root are characterized by modified levels of SPS activity compared to controls. By "modified SPS activity" is intended an increase or decrease in sucrose synthesis. Modification of SPS activity according to the subject invention alters the carbon partitioning between source tissue and sink tissue through an increase or decrease in sucrose synthesis. Altered carbon partitioning is manifested by one or more changes in development, growth and yield through modification of end-product synthesis and conversion in general. The protein and DNA encoding SPS of the subject invention is obtainable from any number of sources which contain an endogenous SPS. Among the preferred SPSs are those obtainable from corn or derived from corn SPS protein or nucleic acid using antibody and/or nucleic acid probes for SPS identification, amplification and isolation. The subject invention also provides a variety of SPS transgenes which have different promoter regions to regulate the transcription and level of SPS activity in plants or plant parts in a tissue-specific and growth-dependent manner. Among the preferred promoter regions are tiiose which provide for leaf, fruit and/or root specific expression of SPS. Preferably, the DNA encoding a SPS of interest in operably linked in a sense or antisense orientation to a selected transcription initiation region to provide for a sufficient level of expression of SPS in the desired tissue or tissues.

An advantage of increasing or decreasing SPS activity is the modification of sucrose synthesis, which is a key metabolic product that affects the interface between end-product synthesis and carbon partitioning for most plant systems. By "end-product" synthesis is intended the metabolic product interface between photosynthesis and plant growth and development. Information flow across the interface may occur by mass action or by signal transmission and transduction. Mass action effects occur when an increase in photosynthesis leads to faster growth resulting from an increase in the availability of photosynthate. Conversely, mass action feedback occurs when accumulation of end- products reduce the rates of photosynthetic reactions. Thus, an advantage of the subject invention is that modification of sucrose synthesis through SPS activity provides a central control point for modifying carbohydrate partitioning through end-product synthesis in a source tissue such as leaf and end-product conversion in a sink tissue such as growing leaf, fruit or root. For example, modulation of photosynthetic metabolism through expression of exogenous SPS is advantageously used to alter die synthesis of end-products such as starch, sucrose glucose, fructose, sugar alcohols, and glycine and serine from photorespiratory metabolism. SPS preferably is used to modulate end product synthesis of non- phosphorylated products of metabolism. Another advantage of the subject invention is that altering SPS activity provides a means for altering plant growth and yield of specific plant cells, plant tissues, plant parts and plants. In addition, by modulating the ability of a plant to synthesize sucrose, the growth response of a plant under a variety of different environmental conditions can be affected including carbon dioxide utilization, oxygen sensitivity, temperature-dependent growth responsiveness and expression of endogenous genes responsive to sugar content in general. Manipulation of growth conditions also permits the modulation of metabolism and the activity of the SPS transgene, for example, through light-mediated activation or deactivation of the SPS transgene and its product. Another advantage is the modification of overall soluble solids such as starch, sucrose, glucose and fructose in sink tissue such as fruit or root. SPS activity and sugar content also permit manipulation of endogenous gene expression and/or enzyme activity in the plant, such as the endogenous acid invertase found in ripening fruit to increase glucose and fructose levels as well as acid content. An additional advantage is that the onset of flowering, fruit number, mass, dimensions, and overall morphology can be modified by altering carbon partitioning. Thus, the subject invention permits the obtention of any transgenic plant or plant part which have any one of several readily selectable phenotypes related to SPS transgene expression and SPS protein activity.

In the subject invention, purification of com SPS protein is exemplified. By "protein" is intended any amino acid sequence, including a protein, polypeptide, or peptide fragment, whether obtained from plant or synthetic sources, which demonstrates the ability to catalyze the formation of sucrose phosphate. An SPS of this invention includes sequences which are modified, such as sequences which have been mutated, truncated, increased in size, contain codon substitutions as a result of the degeneracy of the DNA code, and the like as well as sequences which are partially or wholly artificially synthesized, so long as the synthetic sequences retain the characteristic SPS activity. SPS from sources in addition to com are obtainable by a variety of standard protocols employing protein properties, amino acid and nucleic acid information derived from com SPS. For example, antibody or nucleic acid probes derived from sequencing information permit isolation of a gene or parts of the gene including genomic DNA and cDNA encoding the target SPS of interest. For this purpose, degenerate and non-degenerate probes from hybridization studies with parts or all of the com SPS sequence can be used for identification, isolation and amplification of a gene or fragments encoding the SPS of interest. The SPS gene or fragments are assembled and evaluated by conventional recombinant DNA and biochemical techniques, and through nucleic acid and amino acid sequence database comparisons. As an example, SPSs derivable from com SPS sequences in this manner include potato, spinach, rice and sugar beet. In vitro and in vivo expression systems can be used to produce and test the SPS. The SPS activity can be evaluated by measuring formation of sucrose phosphate from fructose-6-phosphate and UDP-glucose substrates.

In order to obtain the nucleic acid sequences encoding the SPS, especially com SPS, substantially purified SPS was required. As demonstrated more fully in the examples, com SPS purified 500-fold was obtained in small quantities which were then ultimately used to obtain the peptide sequence which in turn led to the determination of the cDNA sequence.

Among the preferred proteins of the invention are the proteins having the above definition with a molecular weight from about 110 to about 130 kd, having the form of a monomer, a dimer or a tetramer and their derivatives, comprising at least one peptide having the following amino acid sequence: Thr-Trp-Ile-Lys (SEQ ID NO: 1) Tyr-Val-Val-Glu-Leu-Ala-Arg (SEQ ID NO: 2) Ser-Met-Pro-Pro-Ile-Trp-Ala-Glu-Val-Met-Arg (SEQ ID NO: 3) Leu-Arg-Pro-Asp-Gln-Asp-Tyr-Leu-Met-His-Ile-Ser-His-Arg (SEQ ID NO: 4) Trp-Ser-His-Asp-Gly-Ala-Arg (SEQ ID NO: 5) The invention also relates to a process to prepare proteins as above defined, having the following steps: (a) extracting SPS from parts containing SPS, which are preserved at low temperature, by grinding, centrifugation and filtration; (b) increasing the rate of SPS extraction from the extract so obtained by precipitation in an appropriate solvent, centrifugation and solubilization of the precipitate in a buffer solution; (c) purifying die protein so obtained by chromatography and, if desired, (d) preparing hybridomas, and monoclonal antibodies from an antigenic solution obtained at step (a), (b), or (c) above; (e) screening the hybridomas and raising monoclonal antibodies specifically directed against SPS; and (f) further purifying the SPS obtained at step (a), (b), or (c) with the monoclonal antibodies prepared. The invention more precisely relates to a process of preparation of com SPS having the following steps: (a) extracting SPS from parts of com plants by grinding, centrifugation, and filtration; (b) increasing the rate of SPS extraction from the extract so obtained by precipitation in polyethyleneglycol (PEG), centrifugation and solubilization of the precipitate obtained in a buffer solution; (c) purifying the protein so obtained by low pressure anion exchange chromatography and by chromatography on heparin sepharose, then by anion exchange high performance chromatography; (d) purifying the active pools by passage on two high performance chromatography columns, and if desired; (e) preparing hybridomas and monoclonal antibodies from an antigenic solution prepared from steps (a), (b), or (c); (0 screening the hybridomas and raising the monoclonal antibodies specifically directed against SPS; and (g) purifying the SPS preparation with the monoclonal antibodies so obtained.

Preferably the corn is a com Pioneer corn hybrid strain 3184, the parts of plants are leaves which are kept at low temperature, for example between -50°C and -90°C, and purification in the polyethyleneglycol is realized first by precipitating at a final concentration in PEG about 6% , and then by precipitating at a final concentration of about

12% . The various chromatographies are performed in the following way: 1st chromatography, DEAE sepharose; 2nd chromatography, heparin sepharose (at this stage, the preparation obtained may be kept several days without loss of activity); 3rd chromatography, Mono Q chromatography; 4th chromatography, HPLC hydroxyapatite; and 5th chromatography, HPLC hydroxyapatite.

A variety of additional protein fractionation methods can be combined to generate a suitable purification scheme for SPS proteins and peptides from com and those in addition to com. If only very small amounts of denatured protein are needed, a high resolution technique may be used such as two-dimensional gel electrophoresis to obtain the protein in one step. When retention of activity is desired, a series of purification steps are designed to take advantage of different properties of the SPS of interest such as precipitation properties, charge, size, adsorptive properties and affinity properties as demonstrated for com SPS.

In general, purification follows the initial extraction and preparation of total protein, bulk precipitation followed by chromatographic procedures such as ion exchange, adsorption, gel filtration, affinity resins and non-denaturing electrophoresis methods so as to be substantially free from other proteins, particularly proteins of the source tissue. By "substantially free from other proteins" is meant that the protein has been partially purified away from proteins found in the source tissue or organism. Such a protein of this invention will demonstrate a specific enzymatic activity of at least greater than 0.05, more preferably at least greater than at least 0.30, wherein specific enzymatic activity (sA) is measured in units which correspond to 1 μmole (micromole) of sucrose formed per minute per mg of protein at 37°C. In a more preferred embodiment, the protein will demonstrate even more improved sA and increased purification factors (see, Table 5). The proteins can be further purified if desired, when retention of activity is less important, by electrophoretic procedures including native or denaturing poly aery lamide gel electrophoresis, isoelectric focusing and two dimensional gel electrophoresis. During the different steps of purification and thereafter, the SPS activity can be measured by two methods: (a) a method based on a colorimetric test or resorcinol test; and (b) a method based on the amount of one of the products formed during die transformation reactions where SPS is involved. Both methods are detailed in die experimental pan detailed hereunder. The exemplified invention relates to the enzyme comprising a corn SPS having a molecular weight from about 110 to 130 kilodalton (kd) and a specific activity of greater than 0.05 U. The invention relates more particularly to the enzyme comprising a com SPS having a specific activity of about 25 U. Antibodies to SPS are prepared as follows, or by other mediods known to those skilled in die art. Mice are immunized with several injections of enzymatic preparations. Different kinds of mice may be used, for example BALB/c. The antigen can be provided in complete Freunds adjuvant then in incomplete Freunds adjuvant. Several injections in mice are realized: good results have been obtained with diree injections of Mono Q, pools, (see above purification scheme) followed by three injections of final pools (days 0, 14, 27, 60, 90 and 105 for example). The first injections are administered sub-cutaneously, for example in the cushions, and die feet, die last injection is administered intravenously, in die tail for example. The preparation of spleen cellular suspensions from animals immunized as described above is made in a conventional way. The steps of fusion widi myeloma cells, of conservation of the hybridoma, of cloning, of antibodies production are made by conventional ways. To detect me hybridoma secreting the monoclonal antibodies raised against the antigen, two methods are used to select antibodies: a method of detection of antibodies as inhibitor of SPS activity; and a method of detection of antibodies precipitating SPS activities. In a preferred embodiment, these methods are the methods described in d e experimental section detailed hereunder.

Among the objects of the invention, are also provided lines of hybridoma cells, and in particular hybridoma cells described as: SPA 2-2-3 : 1-971; SPA 2-2-22 : 1-970; SPA 2-2-25 : 1-972; SPB 3-2-19 : 1-973; SPB 5-2-10 : 1-974; SPB 5-4-2 : 1-975; SPB 13-1-7 : 1-976; and SPB 13-2-2 : 1-977. Deposits of these hybridoma cells were made at the C.N.C.M. (Institut Pasteur Paris) on June 11, 1990. The invention relates also to monoclonal antibodies specifically directed against SPS.

The invention relates also to a process of preparation of proteins as defined above characterized in diat a preparation containing the so-called proteins is purified on a chromatography column having monoclonal antibodies as defined above specifically raised against d e proteins.

The invention relates also to cDNA coding for proteins as defined above, especially cDNA coding for co SPS. Among the preferred cDNA, most preferred is cDNA comprising a nucleotide sequence represented in Figure 7 (SEQ ID NO: 6). Thus, this invention relates to an extrachromosomal DNA sequence encoding a SPS as defined above. Any DNA sequence which is not incorporated into the genome of a plant is considered extrachromosomal, i.e., outside of die chromosome, for purposes of this invention. This includes, but is not limited to cDNA, genomic DNA, truncated sequences, single stranded and double stranded DNA. In a preferred embodiment, the DNA sequence is cDNA. In a different preferred embodiment, the DNA sequence is obtainable from com or is derived from the corn DNA sequence.

Among the preferred proteins and nucleic acid sequences of die invention is com SPS. The com SPS is represented in Figure 1 , which shows the presence of proteins at about 120. 95 and 30 kd. The proteins shown at 95 and 30 kd are considered to be breakdown products of the protein shown at 120 kd. The complete protein is believed to be a di- or tetrameric protein having as the basic sub-unit from about a 110 to about a 130 kd protein. The complete cDNA sequence of me com SPS is shown in Figure 7 (SEQ ID NO: 6).

The cDNA coding for sucrose phosphate synthase has been prepared in die following way: (1) sequencing of peptide fragments from purified SPS. Widi die purified preparations of SPS previously obtained, following separation on an acrylamide gel, a 120 kd minor band (corresponding to the total protein sequence) and two 90 kd and 30 kd major bands are obtained. Bodi major polypeptides are separated by electrophoresis and electroeluted. By trypsin digestion and sequencing of the fragments so obtained, the sequence of 5 peptides has been determined. This amino acid sequence makes it possible to determine the corresponding degenerate nucleotide sequence.

(2) Com leaf isolation. Total RNA is isolated according to Turpen and Griffith (1986, Biotechniques 4:11-15) for poly(A) RNA preparation, the standard oligo dT cellulose column is used.

(3) cDNA library construction. cDNA is synd esized using the protocol of a kit supplied by Promega except that M-MLV reverse transcriptase is used instead of AMV reverse transcriptase. The length of cDNA obtained is from 500 to several thousand base pairs. Eco l linkers are added to die blunt ended cDNA and diis material is cloned into a second generation lambda GT11 expression vector. Total library size is about 1.5xl0⁶ plaques.

(4) Utilization of PCR to synthesizing a nucleotide sequence specific for SPS. The oligonucleotides derived from peptides Bl l (SPS 30 kd) (SEQ ID NO: 3) and 4K (90 kd) (SEQ ID NO: 4) described in figure 3 are used as primers in a PCR reaction. It has been assumed that peptides derived from SPS 30 and SPS 90 are degradation products of protein SPS 120 kd, and diat d e peptides derived from SPS and SPS 90 are encoded by the same RNA.

With this hypothesis, by using in proper polarity pairs of oligonucleotides conesponding to the peptidic sequences in a PCR reaction, one may obtain the synthesis of the DNA, connecting the two location. Since it is a priori not know in which order die peptides are located relative to each odier, one has to do die two different possibilities (Fig. 4). Only the oligonucleotide couple CD3 syndiesizes a cDNA of defined lengdi (1200 bp) (Fig. 5). (5) cDNA library screening. When 250,000 lambda clones GT11 are screened using the 1200 bp long PCR cDNA, 16 positives are obtained. Sizes of die inserts ranged from 0.3 kb to 2.8 kb (see Fig. 6 for die two longest clones). The sequence is not complete in 5' . In a second round of library screening widi a 400 bp DNA fragment conesponding to the most 5' fragment of the clone SPS 3, a SPS 61 clone extending further 5' widiout having the 5' end of die reading frame is obtained (Fig. 6).

(6) Creation and screening of a second cDNA library in order to clone die 5' sequence of cDNA coding for SPS. A oligonucleotide complementary to the 5' sequence of clone SPS 61 is used as a primer for cDNA synthesis. After second strand reaction is completed, the cDNA is cloned into bacteriophage lambda GT11. The library includes about one million clones. The SPS 90 and SP 77 were obtained by screening this library with SPS 61 (Fig. 6).

(7) The assembled SPS reading frame. DNA sequences which encode die SPS may be employed as a gene of interest in a DNA construct or as probes in accordance with this invention. When provided in a host cell, the sequence can be expressed as a source of SPS. More preferred is d e SPS sequence in a vegetal cell under the regulatory control of a transcriptional and translational initiation region functional in plants. Vegetal cell means any plant cell being able to form undifferentiated tissues as callus or differentiated tissues as embryos, parts of plants, whole plants or seeds. Plants means for example plants producing grain seeds such as cereals, and includes wheat, barley, corn, and oat; leguminous plants such as soybean; oleaginous plants such as turnesol; tuberous plants such as potato; plants with roots such as beet; and fruit such as tomato. The sucrose phosphate syndiase is a key enzyme, in sucrose regulation mechanisms, but also in carbon partitioning regulation between starch and sucrose during photosynthesis (see J. Preiss, Tibs January 1984, page 24, or Stitt and Coll, (1987) Biochemistry of Plants, 70:3-27). Of particular interest are plants of the nightshade family Solanaceae, including the genetically similar but physiologically disparate plants potato (Solanium tuberosum) and tomato (Hycopersicon esculentum).

When provided in a DNA construct for integration into a plant genome, the sequence can encode a sense strand or an anti-sense strand. By increasing the amount of SPS available to me photosynthetically active plant cell by the expression of additional SPS, an increased flow of sucrose can be provided to growing tissues resulting, for example, in increased plant yields; by decreasing the amount of SPS available to the photosynthetically active plant cell, the rate of sucrose release from the plant cell may be hindered, resulting in less new plant growth. Controlling die rate of transport and the amount of sucrose available to growing tissues can be used to increase or decrease the total solids in a plant sink tissue from a given ratio of total solids per unit weight sink tissue. Total solids include soluble solids and insoluble solids such as sugars, starches and cellulose. Of particular interest are the soluble solids, which include the sugars sucrose, fructose, and glucose, soluble organics, polymers and other soluble components of cells. Increased total solids in a plant sink tissue may be in the form of an increase in glucose and/or fructose levels. Where the increase comprises fructose, for example, the resulting phenotype is increased sweetness. Where fructose levels are lowered a reduced sweetness phenotype is produced. Of particular interest is fruit having a modified sweetness phenotype. Increasing or decreasing die flow and/or amount of sucrose available to fruit tissue increases or decreases the conversion of sucrose to glucose and fructose by acid invertase, and thus die sweetness of fruit. In tomato fruit, for example, glucose and fructose are produced from sucrose by a vacuolar acid invertase that is active during fruit ripening. As fructose is twice as sweet on a molar basis as glucose, an increase in fructose levels or a fructose to glucose ratio can result in an increased sweetness of me fruit. Of particular interest is fruit of die plant family Solanaceae. Sink tissue solids can be modified widi SPS levels and/or activity in conjunction widi endogenous sucrose and starch metabolizing enzymes, such as acid invertase for sucrose and glycogen synthase for starch. Modification can be used to enhance or inhibit enzymatic activity, for example through sense or antisense expression. By increasing or decreasing SPS activity in plants, the interaction between photosynthesis and die synthesis of end products, such as sucrose and starch, can be modified. Of particular interest is the modification of the starch to sucrose ratio in a vegetal cell dirough the expression of a transgene encoding SPS. Modifying the starch to sucrose ratio in vegetal cell may transduce the affect through end-product syndiesis, signal transduction and/or translocation to odier vegetal cells, particularly the vegetal cells of leaf, fruit and root. In some plants, me change in carbohydrate partitioning can also affect the sensitivity of d e altered plant to carbon dioxide and oxygen. Increasing sucrose syndiesis can result in greater capacity for photosynthesis at elevated carbon dioxide, particularly in the potato. Conversely, decreasing sucrose synthesis (increasing starch synthesis) induces oxygen insensitivity. Such an effect can be obtained by expressing antisense SPS.

A sucrose metabolizing enzyme can also be modified dirough sense or antisense expression. Sequences to be transcribed are ligated to die 3' end die plant transcription initiation region. In the sense constructs, an mRNA strand is produced which encodes die desired sucrose metabolizing enzyme, while in antisense constructs, an RNA sequence complementary to an enzyme coding sequence is produced. The sense strand is desirable when one wishes to increase the production of a sucrose metabolizing enzyme in plant cells, whereas the antisense strand may be useful to inhibit production of a related plant sucrose metabolizing enzyme. The inhibition of acid invertase in tomato fruit, for instance, can lead to fruit having elevated levels of sucrose in d e tomato fruit. The sequence to acid invertase is known (Klann et al., (1992) Plant. Phys. (1992) 99:351-353). Expression of other sucrose metabolizing enzymes may result in alterations to other carbon components, for instance the expression of starch synthesizing enzymes to act in concert with the increase availability of sucrose may result in increased starch levels in die sink tissue. The transformation of plants using glycogen syndiesis enzymes (glgA, glgB and glgC) to modify starch compositions is described in U. S. Patent No. 5,349,123.

The presence of sucrose metabolizing enzyme sequences in the genome of a plant host cell may be confirmed, for example by a Southern analysis of DNA or a Northern analysis of RNA sequences or by PCR methods. In addition to sequences providing for transcriptional initiation in a plant cell, also of interest are sequences which provide for transcriptional and translational initiation of a desired sequence encoding a sucrose metabolizing enzyme. Translational initiation regions may be provided from the source of the transcriptional initiation region or from the gene of interest. In this matter, expression of the sucrose metabolizing enzyme in a plant cell is provided. The presence of the sucrose metabolizing enzyme in the plant host cell may be confirmed by a variety of memods including an immunological analysis of the protein (e.g. Western or ELIZA), as a result of phenotypic changes observed in die cell, such as altered soluble solids content or by assay for increased enzyme activity, and the like.

Other sequences may be included in die nucleic acid construct providing for expression of the sucrose metabolizing enzymes ("expression constructs") of mis invention, including endogenous plant transcription termination regions which will be located 3 ' to me desired sucrose metabolizing enzyme encoding sequence. For instance, transcription termination sequences derived from a patatin gene may be utilized when the sink tissue is potato tubers. Transcription termination regions may also be derived from genes other than those used to regulate the transcription in the nucleic acid constructs of this invention. Transcription termination regions may be derived from a variety of different gene sequences, including the Agrobacterium, viral and plant genes discussed above for their desirable 5' regulatory sequences. Further constructs are considered which provide for transcription and/or expression of more dian one sucrose metabolizing enzyme. For example, one may wish to provide enzymes to plant cells of d e sink tissue which provide for modification of the type of soluble solids to be produced therein, as well as for enhancing or otherwise modifying die increase or decrease in overall soluble solids production. An example of enzymes which may prove useful in modifying soluble solids ratios is the acid invertase enzyme.

In developing die nucleic acid constructs of this invention, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector, e.g. a plasmid, which is capable of replication in a bacterial host, e.g. E. coli. Numerous vectors exist that have been described in die literature, many of which are commercially available. After each cloning, the cloning vector with die desired insert may be isolated and subjected to further manipulation, such as restriction, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the components of the desired sequence. Once the construct has been completed, it may then be transfened to an appropriate vector for further manipulation in accordance widi the manner of transformation of the host cell.

The constructs of this invention providing for transcription and/or expression of sucrose metabolizing enzyme sequences of this invention may be utilized as vectors for plant cell transformation. The manner in which nucleic acid sequences are introduced into the plant host cell is not critical to diis invention. Direct DNA transfer techniques, such as electroporation, microinjection or DNA bombardment may be useful. To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers. The use of plant selectable markers is preferred in this invention as the amount of experimentation required to detect plant cells is greatly reduced when a selectable marker is expressed. Useful selectable markers include enzymes which provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, and the like. Similarly, enzymes providing for production of a compound identifiable by color change, such as GUS, or luminescence, such as luciferase are useful.

An alternative method of plant cell transformation employs plant vectors which contain additional sequences which provide for transfer of the desired sucrose metabolizing enzyme sequences to a plant host cell and stable integration of these sequences into the genome of the desired plant host. Selectable markers may also be useful in diese nucleic acid constructs to provide for differentiation of plant cells containing the desired sequences from those which have only the native genetic material. Sequences useful in providing for transfer of nucleic acid sequences to host plant cells may be derived from plant pathogenic bacteria, such as Agrobacterium or Rhizogenes, plant pathogenic viruses, or plant transposable elements.

A sucrose metabolizing enzyme considered in this invention includes any sequence of amino acids, such as protein, polypeptide, or peptide fragment, which demonstrates the ability to catalyze a reaction involved in the syndiesis or degradation of sucrose or a precursor of sucrose. These can be endogenous plant sequences, by which is meant any sequence which can be naturally found in a plant cell, including native (indigenous) plant sequences as well as sequences from plant viruses or plant padiogenic bacteria, such as Agrobacterium or Rhizobium species that are naturally found and functional in plant cells. It will be recognized by one of ordinary skill in the art that sucrose metabolizing enzyme sequences may also be modified using standard techniques of site specific mutation or PCR, or modification of the sequence may be accomplished in producing a synthetic nucleic acid sequence and will still be considered a sucrose biosynthesis enzyme nucleic acid sequence of d is invention. For example, wobble positions in codons may be changed such that the nucleic acid sequence encodes die same amino acid sequence, or alternatively, codons can be altered such diat conservative amino acid substitutions result. In either case, the peptide or protein maintains the desired enzymatic activity and is dius considered pan of die instant invention. A nucleic acid sequence to a sucrose metabolizing enzyme may be a DNA or RNA sequence, derived from genomic DNA, cDNA, mRNA, or may be synthesized in whole or in part. The structural gene sequences may be cloned, for example, by isolating genomic DNA from an appropriate source, and amplifying and cloning the sequence of interest using a polymerase chain reaction (PCR). Alternatively, the gene sequences may be synmesized, either completely or in pan, especially where it is desirable to provide plant-prefened sequences. Thus, all or a portion of the desired structural gene may be synthesized using codons prefened by a selected plant host. Plant- preferred codons may be determined, for example, from the codons used most frequently in the proteins expressed in a particular plant host species. Other modifications of die gene sequences may result in mutants having slightly altered activity. Once obtained, a sucrose metabolizing enzyme may be utilized widi die SPS sequence in a variety of ways.

Other endogenous plant sequences may be useful in nucleic acid constructs of this invention, for example to provide for transcription of the sucrose metabolizing enzyme sequences. Transcriptional regulatory regions are located immediately 5' to me DNA sequences of die gene of interest, and may be obtained from sequences available in die literature, or identified and characterized by isolating genes having a desirable transcription pattern in plants, and studying die 5' nucleic acid sequences. Numerous transcription initiation regions which provide for a variety of constitutive or regulatable, e.g. inducible, expression in a plant cell are known. Among sequences known to be useful in providing for constitutive gene expression are regulatory regions associated with Agrobacterium genes, such as for nopaline syndiase (Nos), mannopine syndiase (Mas), or octopine syndiase (Ocs), as well as regions coding for expression of viral genes, such as the 35S and 19S regions of cauliflower mosaic virus (CaMV). The term constitutive as used herein does not necessarily indicate diat a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in abundance is often detectable.

In providing for transcription and/or expression of the sucrose metabolizing enzyme sequences, for various reasons one may wish to limit the expression of diese enzymes to plant cells which function as carbon sinks. Towards d is end, one can identify useful transcriptional initiation regions that provide for expression preferentially in specific tissue types, such as roots, tubers, seeds or fruit. These sequences may be identified from cDNA libraries using differential screening techniques, for example, or may be derived from sequences known in the literature.

Many tissue specific promoter regions are known, such as the Rubisco small subunit promoter which preferentially is expressed in leaf tissue, the patatin promoter which is preferentially in potato mbers. Other transcriptional initiation regions which preferentially provide for transcription in certain tissues or under certain growth conditions, include those from napin, seed or leaf ACP, zein, and the like. Fruit specific promoters are also known, one such promoter is the E8 promoter, described in Deikman et al. (1988) EMBO J. 2.3315-3320; and DellaPenna et al. (1989) Plant Cell 7:53-63, the teachings of which are incorporated herein by reference. An E8-SPS construct (fruit-specific promoter) will express SPS in a fruit-specific manner, whereby the levels of sucrose produced in die fruit may be elevated. If coupled widi antisense acid invertase, the increase in sucrose would be maintained. This is a particular issue in tomatoes where acid invertase present in die fruit drives the production of glucose and fructose from sucrose..

The protein and DNA encoding SPS of the subject invention is obtainable from any source containing an endogenous SPS and can be wholly or partially synthetic. Among the prefened SPSs are those obtainable from co . By "obtainable from com" is meant that die sequence, whether an amino acid sequence or nucleic acid-sequence, is related to a corn SPS, including a SPS recovered through use of nucleic acid probes, antibody preparations, sequence comparisons or derivatives obtained through protein modeling or mutagenesis for example. Thus, one skilled in the art will readily recognize d at antibodies, nucleic acid probes (DNA and RNA) and die like can be prepared and used to screen odier plant sources for SPS and recover it. Typically, a homologously related nucleic acid sequence will show at least about 60% homology, and more preferably at least about 70% homology between the com SPS and die given plant SPS of interest, excluding any deletions which may be present. Homology is found when diere is an identity of base pairs and can be determined upon comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions conducted under relatively stringent conditions, e.g. , under conditions where diere is a fairly low percentage of non-specific binding widi com SPS probes.

Probes can be considerably shorter than the entire sequence, but should be at least about 10, preferably at least about 15, more preferably at least 20 nucleotides in length. Longer oligonucleotides are also useful, up to full length of the gene encoding die polypeptide of interest. Bodi DNA and RNA probes can be used. A genomic library prepared from me plant source of interest can be probed with conserved sequences from com SPS to identify homologously related sequences. Use of the entire com SPS cDNA may be employed if shorter probe sequences are not identified. Positive clones are dien analyzed by restriction enzyme digestion and/or sequencing. In this general manner, one or more sequences can be identified providing bodi die coding region, and die transcriptional regulatory elements of the SPS gene from such plant source. As an example, probes derived from com SPS are used for isolating SPS from corn and sources in addition to com. A probe or a battery of probes representing all or segments of die SPS coding region of com SPS are preferably used. The corn SPS sequences can be compared by conventional gene bank searches and die conserved and nonconserved regions used in the design of additional probes if needed. In addition, die conserved and nonconserved regions for probe design are identifiable dirough standard hybridization techniques or, for example, by comparing amino acid and/or nucleic acid sequences of co SPS to SPS sequences from diverse sources including rice, potato, sugar beet, spinach ,or Arabidopsis thaliana. which is a flowering plant member of the mustard family Brasicaceae. In use. probes are typically labeled in a detectable manner (for example with ³²P- labelled or biotinylated nucleotides) and are incubated widi single-stranded DNA or RNA from the plant source in which the gene is sought, aldiough unlabeled oligonucleotides are also useful. Hybridization is detected by means of die label after single-stranded and double-stranded (hybridized) DNA or DNA/RNA have been separated, typically using nitrocellulose paper or nylon membranes. Hybridization techniques suitable for use with oligonucleotides are well known to those skilled in the art.

From the cDNA sequences, one skilled in the art can obtain the corresponding genomic DNA sequences related d ereto to obtain the coding region of the SPS, including intron sequences, transcription, translation initiation regions and/or transcript termination regions of the respective SPS gene. The regulatory regions can be used widi or widiout the SPS gene in various probes and/or constructs. The complete SPS reading frame can be assembled using restriction enzyme fragments of SPS 90, SPS 61 and SPS 3, see Fig. 6.

When expressed in E. coli, d e SPS cDNA produces a protein which is recognized by anti-SPS antisera and has die same electrophoretic mobility as SPS extracted from com leaves. We show that diis E. coli SPS is as active as plant SPS, i.e. for complete enzymatic activity in E. coli no other plant factor is needed but die SPS cDNA.

Plants obtained by the method of transformation and containing fusions of SPS cDNA to tissue specific promoters in order to modify or alter the composition of certain plant organs are also included.

A DNA construct of diis invention can include transcriptional and translational initiation regulatory regions homologous or heterologous to the plant host. Of particular interest are transcriptional initiation regions from genes which are present in the plant host species, for example, the tobacco ribulose biphosphate carboxylase small subunit (SSU) transcriptional initiation region; die cauliflower mosaic virus (CaMV) 35S transcriptional initiation region, including a "double" 35S CaMV promoter, die tomato fruit-specific E8 (E8) transcriptional initiation region, and those associated widi T-DNA, such as the opine synthase transcriptional initiation region, e.g., octopine, mannopine, agropine, and the like. Any one of number of regulatory sequences may be preferred in a particular situation, depending upon whedier constitutive or tissue and/or timing induced transcription is desired, the efficiency of a particular promoter in conjunction with die heterologous SPS, the ability to join a strong promoter wid a control region from a different promoter to provide for inducible transcription, ease of construction and the like. For example, tissue specific promoters can be employed to selectively modify or alter the composition of certain plant organs. Promoters which function in, or are specific by fmit, root and/or leaf are examples. These regulatory regions find ample precedence in the literature. The termination region may be derived from the 3 '-region of die gene from which the initiation region was obtained, from die SPS gene, or from a different gene. Preferably the termination region will be derived from a plant gene, particularly, the tobacco ribulose biphosphate carboxylase small subunit termination region ; a gene associated widi the Ti- plasmid such as die octopine syn iase termination region or the tml termination region. In developing the expression cassette, the various fragments comprising the regulatory regions and open reading frame may be subjected to different processing conditions, such a ligation, restriction, resection, in vitro mutagenesis, primer repair, use of linkers and adapters, and the like. Thus, nucleotide transitions, transversions, insertions, deletions, or the like, nay be performed on die DNA which is employed in the regulatory regions and/or open reading frame.

During die construction of the expression cassette, the various fragments of die DNA will usually be cloned in an appropriate cloning vector, which allows for amplification of the DNA, modification of d e DNA or manipulation by joining or removing of the sequences, linkers, or the like. Normally, the vectors will be capable of replication in at least a relatively high copy number in E. coli. A number of vectors are readily available for cloning, including such vectors as pBR322, pUC series, M13 series, etc. The cloning vector will have one or more markers which provide for selection or transformants . The markers will normally provide for resistance to cytotoxic agents such as antibiotics, heavy metals, toxins, or the like. By appropriate restriction of the vector and cassette, and as appropriate, modification of the ends, by chewing back or filling in overhangs, to provide for blunt ends, by addition of linkers, by tailing, complementary ends can be provided for ligation and joining of die vector to the expression cassette or component thereof. After each manipulation of the DNA in the development of die cassette, die plasmid will be cloned and isolated and, as required, the particular cassette component analyzed as to its sequence to ensure that the proper sequence has been obtained. Depending upon the nature of the manipulation, the desired sequence may be excised from the plasmid and introduced into a different vector or die plasmid may be restricted and die expression cassette component manipulated, as appropriate.

The manner of transformation of E. coli with the various DNA constructs (plasmids and viruses) for cloning is not critical to diis invention. Conjugation, transduction, transfection or transformation, for example, calcium phosphate mediated transformation, may be employed. In addition to the expression cassette, depending upon the manner of introduction of the expression cassette into the plant cell, odier DNA sequences may be required. For example when using the Ti- or Ri-plasmid for transformation of plant cells, as described below, at least the right border and frequently bodi the right and left borders of the T-DNA of the Ti- or Ri-plasmids will be joined as flanking regions to the expression cassette. The use of T-DNA for transformation of plant cells has received extensive study and is amply described in Genetic Engineering, Principles and Methods (1984) Vol 6 (Eds. Setlow and Hollaender) pp. 253-278 (Plenum, NY) ; A. Hoekema, in: The Binary Plant Vector System (1985) Offsetdrukkerij Ranters, 8.V. Alblasserdam.

Alternatively, to enhance integration into the plant genome, terminal repeats of transposons may be used as borders in conjunction widi a transposase. In this situation, expression of the transposase should be inducible, so that once die expression cassette is integrated into the genome, it should be relatively stably integrated and avoid hopping. The expression cassette will normally be joined to a marker for selection in plant cells. Conveniently, the marker may be resistance to a biocide, particularly an antibiotic, such as Kanamycin, G418, Bleomycin, Hygromycin, Chloramphenicol, or the like. The particular marker employed will be one which will allow for selection of transformed plant cells as compared to plant cells lacking die DNA which has been introduced.

A variety of techniques are available for die introduction of DNA into a plant cell host. These techniques include transformation with Ti-DNA employing A. tumefaciens or A. rhizogenes as the transforming agent, protoplast fusion, injection, electroporation, DNA particle bombardment, and die like. For transformation with Agrobacterium, plasmids can be prepared in E. coli which plasmids contain DNA homologous widi the Ti-plasmid, particularly T-DNA. The plasmid may be capable of replication in Agrobacterium, by inclusion of a broad spectrum prokaryotic replication system, for example RK290, if it is desired to retain die expression cassette on a independent plasmid rather than having it integrated into the Ti-plasmid. By means of a helper plasmid, the expression cassette may be transferred to d e A. tumefaciens and die resulting transformed organism used for transforming plant cells. Conveniently, explants may be cultivated with die A. tumefaciens or A. rhizogenes to allow for transfer of the expression cassette to the plant cells, and die plant cells dispersed in an appropriate selection medium. The Agrobacterium host will contain a plasmid having die vir genes necessary for transfer. After transformation, the cell tissue (for example protoplasts, explants or cotyledons) is transfened to a regeneration medium, such as Murashige-Skoog (MS) medium for plant tissue and cell culture, for formation of a callus. Cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. , Plant Cell Reports (1986) 5:81-84. The transformed plants may then be analyzed to determine whe ier the desired gene product is still being produced in all or a portion of the plant cells. After expression of the desired product has been demonstrated in the plant, the plant can be grown, and either pollinated with die same transformed strain or different strains and the resulting hybrid having die desired phenotypic characteristic identified. Two or more generations may be grown to ensure that die subject phenotypic characteristic is stably maintained and inherited. To identify die desired phenotypic characteristic, transgenic plants which contain and express a given SPS transgene are compared to control plants. Preferably, transgenic plants are selected by measurement of SPS activity in leaf, fruit and/or root. The SPS activity may be periodically measured from various stages of growth through senescence and compared to diat of control plants. Plants or plant pans having increased or decreased SPS activity compared to controls at one or more periods are selected. Transgenic plants exhibiting SPS activity from about 1 to 12 fold d at of control plants are prefened, with about 1 to 5 fold being more preferred, depending on a desired secondary trait. The activity can be compared to one or more odier traits including SPS type, transcription initiation type, translation initiation type, termination region type, transgene copy number, transgene insertion and placement.

When evaluating a phenotypic characteristic associated widi SPS activity, the transgenic plants and control plants are preferably grown under growdi chamber, greenhouse, open top chamber, and/or field conditions. Identification of a particular phenotypic trait and comparison to controls is based on routine statistical analysis and scoring. Statistical differences between plants lines can be assessed by comparing SPS activity between plant lines within each tissue type expressing SPS. Expression and activity are compared to growth, development and yield parameters which include plant part morphology, color, number, size, dimensions, dry and wet weight, ripening, above- and below-ground biomass ratios, and timing, rates and duration of various stages of growth through senescence, including vegetative growth, fmiting, flowering, and soluble solid content including sucrose, glucose, fructose and starch levels. To identify transgenic plants having other traits, the plants can be tested for photosyndietic and metabolic activity, as well as end-product syndiesis. For example, material isolated from transgenic plant cells and plant parts such as leaf, fruit and root are measured for end-products such as starch, sucrose, glucose, fructose, sugar alcohols, and glycine and serine from photorespiratory metabolism following standard protocols. Sweetness based on sugar content, particularly fructose, can be tested as well. For some plants, it may be necessary to modify growth conditions to observe the phenotypic effect. As an example, oxygen, carbon dioxide and light can be controlled and measured in an open gas chamber system, and carbon partitioning measured by C labeling of carbon dioxide or other metabolic substrates. Carbon partitioning also can be determined in extracts from fruit, leaf and/or root by chromatographic techniques or by Brix using a sugar refractometer. These characteristic also can be compared against or induced by growth conditions which vary gas exchange parameters, light quality and quantify, temperature, substrate and moisture content between lines within each type of growing condition.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1

Purification of Sucrose Phosphate Svnthase of Com 1 J - Method of determination of enzvmatic activity (SPS) During purification SPS activity is followed in 2 ways: a) eidier by means of a colorimetric test (Kerr c?t al. , Planta. , 1987, 770:515-519) called resorcinol test described below.

Sucrose Phosphate Synthase catalyzes the reaction:

UDPG + Fructose 6-P < = > Sucrose 6-P + UDP UDPG : Uridine Di-Phospho Glucose

Fructose 6-P or F6P : Fructose 6-Phosphate Sucrose 6-P : Sucrose 6-Phosphate

The sucrose 6-P formed reacts widi die resorcinol to give a red-colored compound quantifiable by spectrophotometry at 520 nm (nanometer) (Optical Density (O.DJ = 520 nm). In practice, to 45 μl (microliter) of enzymatic preparation 25 μl of a buffered solution containing die two substrates is added (UDPG 70 mM, F6P 28 mM, MgCl, 15 mM, HEPES 25 mM pH 7.5). After incubation at 37°C, the reaction is stopped by adding

70 μl of NaOH in solution and heating at 95°C during 10 min. After cooling, 0.25 ml of a solution 0.1 % resorcinol in ethanol 95% is added; then 0.75 ml of HCl 30% is added. The OD at 520 mm is read after incubation for 8 min at 80°C, and cooling, b) or by means of a coupled enzymatic system (Harbron et al. , Anal. Biochem. 1980, 707:56-59) being composed in d e following way: UDPG + F6P < = > Sucrose 6-P + UDP

SPS UDP + ATP < = > ADP + UTP

Nucleoside Diphosphokinase NP₂K ADP + PEP < = > Pyruvate + ATP

Pyruvate kinase PK Pyruvate + NADH < = > NAD -I- lactate

Lactate dehydrogenase LDH

The disappearance of die NADH absorption at 340 nm is monitored: 1 mole of

NAD formed or 1 mole of NADH consumed corresponds to 1 mole of sucrose 6 P formed.

In practice, in a quartz spectrophotometric tun diermostated at 37°C, the following solution are added.

- 540 μl of HEPES buffered 50 mM, MgCl₂ 10 mM, KC1 20 mM pH = 7.5, - 250 μl of a mixture of substrates PEP (1.6 mM NADH 0.6 mM, ATP 4 mM UDPG 112 mM),

- 60 μl of an enzyme mixture (LDH 166.7 U/ml PK 333.3 U/ml, NPzK 66.7 U/ml),

- 100 μl of F6P 112 mM.

After homogenization, 50 μl of the preparation containing SPS is added, d e diminution of optical density at 340 nm is added widi a spectrophotometer (UVIKON 860, KONTRON instruments). The measure is done widi the kinetic of the machine.

1.2 Purification of the SPS (preparation of the immunogenl 1.2J Extraction The starting material for the purification are nature leaves of young corn plants (Zea mays L. cv Pioneer 3184), which have been harvested in late morning, cut up, deveined, frozen in liquid nitrogen and stored at -70°C.

250 g of leaves are suspended in 1 liter of 50 mM HEPES 10 mM MgCl₂, 1 mM EDTA, 5 mM DTT, pH = 7.5 buffer (extraction buffer) which has observed to it 11 g of Polyvinyl-pyrrolidone, nitrogen is bubbled dirough and the suspension is cooled to 0°C. The leaves are ground, until a homogeneous liquid is obtained. This ground product is filtered, and then centrifuged at 14,000 xg for 20 minutes at 4°C. While the bubbling through of nitrogen is maintained, a solution of 50% polyediylene glycol (PEG 8000 "Breox" at 50% w/v of extraction buffer) is added to the supernatant until a final concentration of PEG of 6% is reached. Then the suspension is cooled at 0°C. After centrifuging at 14,000 g for 20 minutes the supernatant has added to it 50% PEG until a final concentration of PEG of 12% is reached. After a repeated centrifugation, the supernatant is discarded and die residue is solubilized with 60 ml of 50 mM HEPES, 10 mM MgCl₂. 1 mM EDTA, 5 mM DTT, 10% ethylene glycol (EG), 0.08 M KC1, pH 7.5 buffer (recovery buffer). This solution is clarified by centrifuging at 40,000 g for 10 minutes. The supernatant constitutes the final extract.

1.2.2 Low pressure anion-exchange chromatography: fast-flow DEAE Sepharose exchanger The final extract is chromatographed on a column 25 mm x 162 mm of 80 ml of

Fast-Flow DEAE Sepharose (Pharmacia) equilibrated with recovery buffer. After washing the column with die same buffer, the proteins adsorbed on die support are eluted by means of a linear gradient with increasing ionic strength between 0.08 M KC1 and 0.35 M KC1 in the 50 mM HEPES, 10 mM MgCl₂, 1 mM EDTA, 5 mM DTT, 10% EG, pH 7.5 buffer (buffer A). The flow rate applied during this experiment is 180 ml/h and chromatography is executed at 4°C.

The SPS activity is eluted at about 0J7 M KC1.

1.2.3 Chromatography on heparin Sepharose The fractions containing the SPS activity are collected and diluted to one fifth in buffer A, then added to 12 ml of heparin Sepharose previously equilibrated with buffer A.

After one hour of incubation wi i gentle agitation at 4°C, the gel is washed with about 10 volumes of buffer A + 0.05 M KC1, then repacked in a chromatography column.

The proteins adsorbed are eluted in an isocratic way by means of a 10 mM CAPS, 10 mM MgCl₂, 1 mM EDTA, 5 mM DTT, 10% EG, 0.01 % Tween 80, 1 mg/ml heparin. 1 % Fructose, 0.25 M KC1, pH 10 buffer, delivered at 60 ml/h. Chromatography is executed at 4°C. The fractions containing the SPS activity are collected (heparin fraction) and preserved on ice until the following purification stage. The enzyme at this stage is stable for a least one week. The following purification steps are carried out using a system of High Performance

Liquid Chromatography (HPLC) ; he purification is followed by means of a detector fitted widi a filter enabling absorbency in the ultra-violet at 280 nm (A280) to be measured. The buffers and the fractions recovered are kept at low temperature.

1.2.4 High performance anion-exchange chromatography: Mono O

The heparin fraction is diluted by adding one diird volume of 20 mM Triethanolamine, 10 mM MgCl₂, 1 mM EDTA, 10 mM DTT, 3 % EG, 0.3% Tween 80. pH 7.5 buffer (buffer A) and loaded on an FPLC Mono Q HRlO/10 column, (10 x 100 mm Pharmacia) previously equilibrated widi d e same buffer which has added to it NaCl (final concentration 0J8 M). After the A280 has returned to 0, die proteins adsorbed on die chromatography support are eluted by means of a salt-complex gradient with buffer A (see above) and buffer B (buffer A + NaCl, 1 M) on a Mono Q column as shown below in Table 1.

ϊahk L Salt Gradient for Mono O Column time (minutes) J B o 18

0J 24

15 24 19 26

23 26

33 31

38 31

41 100 43 18

The flow rate applied to die Mono Q column is 180 ml/h. The SPS activity is eluted between 0.26 and 0.31 M NaCl. The active fractions are collected togedier ("Mono Q fraction").

1.2.5 HPLC on Hydroxyapatite

The Mono Q fraction is loaded on an HPLC column of hydroxyapatite 4 mm x 75 mm neutralized with 20 mM KH₂PO₄/K₂HPO₄, 3% EG, 0.3% Tween 80, 5 mM DTT, pH

7.5 buffer. After the A280 absorbance has returned to 0, die proteins adsorbed to d e column are eluted by means of the following phosphate gradient using buffer A (see above) and buffer B (the same as buffer A additionally containing but 500 mM Phosphate of K) as shown below in Table 2. I_U____Z

Phosphate Gradient for Hvdroxvapatite Column time (minutes ___H

0 2 5 11

9 13

14 13

29 40

31 100 32 100

35 2

The flow rate applied to die column is 60 ml/h. At this stage, the phosphate will partially inhibit SPS activity and therefore it is difficult to calculate a specific activity and also a purification factor (see Table 1) at this stage. The SPS activity is eluted under diese conditions with about 60 mM phosphate. The active fractions are collected togedier and constitute the HAC fraction.

1.2.6 HPLC on DEAE 5PW The HAC fraction is loaded on an anion-exchange HPLC column of Di Ethyl

Amino Ediyl type (DEAE-5PW) previously neutralized with a buffer of 20 mM

Triethanolamine, 10 mM MgCl₂, 1 mM EDTA, 3% EG, 2.5 mM DTT, 2% betaine, pH

7.5 buffer (buffer A) + 0.15 M NaCl.

After the A280 absorbance has returned to 0, d e proteins adsorbed to die column are eluted by means of the following NaCl gradient using buffer A (see above) and buffer

B (the same as buffer A but additionally containing 1 M NaCl) as shown below in Table 3.

Tahle 3

Sail Gradient for DEAE Column time (minutes) ___

0 15 OJ 20

5 20

22 35

27 35

30 100 31 15

The flow rate applied to the column is 60 ml/h. The SPS activity is eluted with about 0.3M NaCl.

1.2.7 Preparation of the final preparation: concentration

The final preparation is concentrated by HPLC chromatography on a Mono Q HR5/5 exchanger (5 X 50 mm, Pharmacia) and rapid elution. The DEAE 5PW fraction (or the G200 fraction) is diluted to two diirds widi buffer A (see 1.2.6) and loaded on die column which previously has been neutralized with buffer A + 0J8 M NaCl. The following gradient is then applied on die column using buffer A and B (see 1.2.6) as shown below in Table 4.

ϊahte

Gradient for Concentration time (minutes) i B

0 18

10 40

12 100

13 18

The flow rate applied to die column is 60 ml/h. The SPS activity is eluted with about 0.3 M NaCl. The final preparation is stored at -20°C until used.

The results obtained at die various purification stages in terms of quantities of proteins recovered and of SPS activity are summarized in Table 5 below. Table 5

Purification of Com SPS

'sA = Specific enzymatic activity: 1 U conesponds to 1 μmole of sucrose formed per minute per mg of protein at 37°C. The measurement of die quantity of proteins is carried out using die Bradford mediod. As Tween interferes enormously with this method, it is not possible to determine die proteins and then to calculate an sA at die level of die stages containing one. Furthermore, as phosphate is an inhibitor of SPS activity, the determination during the HAC stage gives an underestimated result. ²pF= Purification factor

³Y= Yield. The increasing yield during die initial stages of purification can be explained by the elimination, during purification, of certain inhibitors of SPS activity. ⁴( )= approximate value ⁵-= not determined

An SDS-PAGE profile at various stages of the purification process and die quality of die final preparation is given in Figure 1. The 120, 95 and 35 kd proteins are conelated to d e SPS activity. The 35 and 95 kd proteins are very likely breakdown products of the 120 kd protein as it can be shown by die nucleotide sequence coding for the SPS protein. Furthermore, the antibodies directed against the 35 and 95 kd proteins also recognize die protein 120 kd in immunodetection after membrane transfer, which demonstrates an antigenic identity between these three proteins (see below). It must be pointed out, however, that die addition of protease inhibitors in the buffers during purification has not enabled us to obtain a single 120 kd protein. Gel permeation chromatographies were carried out in order to determine the apparent molecular weight of the native SPS protein. Briefly, the HAC fraction was concentrated by HPLC chromatography on a Mono Q HR 5/5 inchanger (see 1.2.7). The active fractions were collected togedier (about 2 ml) and loaded on an G 200 column previously washed with a buffer containing 20 mM triethanolamine, 10 mM MgCl₂, 1 mM EDTA, 3 % E.G., 2.5 mM DTT, 2% betain, 0.3 M NaCl pH 7.5. The SPS activity was eluted with a major protein peak corresponding to an apparent mass of 270-280 kda which is in agreement with the results obtained by Harbron et al. (Arch. Biochem. Biophys. , 1981, 272:237-246) with the spinach SPS. It can be noted diat the chromatography on a TS lambda 60000 permeation column lead to die elution of die SPS activity at a retention time corresponding to an apparent mass of 440 kda which is close to die value obtained by Doehlert and Huber (Plant Physiol , 1983, 75:989-994) with the spinach SPS, using an AcA34 permeation column.

The SPS protein seems therefore to be a di or tetrameric protein having as the basic sub-unit a 120 kda protein (homodimeric or homo-tetrameric). The results of SDS page analysis at various stages of purification are shown in Figure 1. The bands of proteins visible at about 120 kd (1), 95 kd (2) and 35 kd (3) are conelated, during the chromatography stages, with the appearance of SPS activity in the respective fractions.

Example 2

Process for the Preparation of Monoclonal Antibodies Directed Against SPS

2.1 Immunizations BALB/c mice were immunized by subcutaneous injection (pads and paws) according to the following methodology: Day 0 injection of about 5 micrograms of proteins (or about 0.3 U SPS per mouse): Mono Q pool emulsified volume for volume widi Freund's Complete Adjuvant (FCA).

Day 14 injection of about 5 micrograms of proteins (or about 0.3 U SPS per mouse): Mono Q pool emulsified volume for volume with Freund's Incomplete Adjuvant

(FIA).

Day 27 Idem D14

Day 0 + 60 injection of about 20 micrograms of proteins: final pool in FIA Day 0 + 90 injection of about 12 micrograms of proteins: final pool in FIA Day 0 + 135 injection by intravenous route (IV) in die tail of about 20 micrograms of proteins : final pool .

Fusion is achieved 3 days after the IV immunization. The sera were removed at D34, D61, D98 and D 159 in order to measure the immune response (see screening).

2J J Screening method

Two memods were used to detect antibodies specific to the SPS used for immunizations:

- detection method of antibodies inhibiting die SPS activity

- detection method of antibodies directed against the SPS (inhibiting or not).

a) Detection mediod of antibodies inhibiting die SPS activity

This mediod of screening allows the detection of antibodies which interfere with the active site of the SPS or on a site close to die latter, and therefore prevent the access of substrates. In practice, 70 μl of serum or of supernatant of hybridoma culture diluted in a suitable way was mixed widi 70 μl of SPS preparation (Heparin fraction). After one hour of incubation at ambient temperature, the residual SPS activity was determined by coupled enzymatic determination (see 1.1). The results are expressed as a percentage of inhibition as compared to die same SPS preparation treated in die same way but without antibodies.

b) Detection mediod of antibodies directed against SPS (inhibiting or not)

This method is based on die precipitation of die antibody-SPS complex by goat anti-mouse IgG coupled to sepharose beads (GAM sepharose). In practice, 60 μl of serum or supernatant of hybridoma culture diluted in any suitable manner were added to 60 μl of SPS preparation (Heparin fraction). After 2 hours of incubation at ambient temperature, the mixture was added to 50 μl of 25% GAM-Sepharose previously washed diree times with a buffer of 50 mM HEPES, 10 mM MgCl₂, 1 mM EDTA, 10% EG, 5 mM DTT, pH 7.5. The mixture was incubated overnight at 4°C wid strong agitation. After centrifuging the mixture for 5 minutes at 3000 rpm, the residual SPS activity in the supernatant was determined by coupled enzymatic determination (see 1.1). The results are expressed as a percentage of precipitation (% prec.) as compared to the same SPS preparation treated in the same way without antibodies.

2.1.2 Results

10 mice were immunized according to the protocol described previously. The following table gives the results of the precipitation determinations carried out with the heteroantisera of the 10 mice on D159. The sera are diluted to one two-hundreddi. ϊahk ό

Percentage Precipitation of Antihodv-SPS Complex Mouse 1 2 3 4 5 6 7 8 9 10

% Prec. 45 22 32 64 36 30 22 16 39 37

Additional dilutions of the serum of mouse 4 give the following results:

lahkl

The spleens of mice 1 and 4 were used for the fusion with myeloma cells.

2.2 Cellular fusion

The splenocytes of the mice were fused with myeloma cells of SP2/0-Agl4 mice according to a ratio of 2: 1 in the presence of 45% polyethylene glycol 1500. The selection of the hybridomas was effected by adding hypoxandiine and azaserine to the culture medium 24 and 48 hours after fusion.

The hybridomas were cloned and sub-cloned by the mediod of limited dilution.

2.2J Results of the screening of hybrids and clones

Results from screening of hybrids, clones and sub-clones are shown below in Table 8. J_ύ_____

Hybrid. Clone and Sub-clone Screening

Hybrids

Mouse 4 (SPA fusion) 2 positive hybrids out of 45 SPA2 : 38 % prec. SPA 19: 7 % prec.

Clones

SPA fusion SPB fusion

2 clones retained out of 36 7 clones retained out of 46 SPA2-2 : 85 % prec. SPB3-2 : 19 % prec. SPA 19-7 : 8 % prec. SPB5-1 : 76 % prec. SPB5-2 : 71 % prec. SPB5-3 : 45 % prec. SPB5-4 : 24 % prec. SPB13-1 : 79 % prec. SPB 13-2 : 53 % prec.

Sub-Clones

SPA fusion SPB fusion sub-clones retained out of 48 sub-clones retained out of 72 SPA2-3 : 60 % prec. SPB3-2-19 : 21 % prec. SPA2-2-33 : 33 % prec. SPB5-2-10 : 86 % prec. SPA2-2-25 : 92 % prec. SPB5-4-2 : 46 % prec. SPB13-1-7 : 87 % prec. SPB13-2-2 : 93 % prec.

2.2.2 Production of anti-SPS monoclonal antibodies

The hydridomas were injected by die intra-peritoneal route into female BALB/c mice previously treated wid pristane. The monoclonal antibodies were partially purified from ascites fluids precipitated widi 18% sodium sulphate. The proteins so precipitated were dissolved dien dialyzed against PBS (F18).

2.2.3 Characterization of anti-SPS monoclonal antibodies a) Typing

The typing was done using an ELISA test. Anti-IgG rabbit and anti-IgM mouse antibodies (Zymed) were fixed at the bottom of the wells of a 96-well plate. After one night at ambient temperature me unoccupied sites were saturated widi a solution of 3 % bovine serum albumin in PBS. After one hour of incubation at 37°C and several washes, the various F18's were deposited in d e wells. After incubation and several washes, goat or rabbit antibodies, anti-class and anti-sub class mouse immunoglobulins linked with peroxidase. were added. After one hour at 37°C, the antibody type was identified using an H₂0₂/ABTS system. All the anti-SPS monoclonal antibodies were found to be of IgG, type, b) Inhibition of SPS activity

The determination of the capacity of the antibodies to inhibit d e SPS activity was canied out by die technique mentioned previously (see 2.1.1 a) using F18's. The results are shown below in Table 9.

c) Immuno-precipitation of the SPS activity

The determination of the ability of the antibodies to immunoprecipitate the SPS activity was carried out by die technique mentioned previously (see 2.1,1 b) using F18's. The results are shown below in Table 10. Example 3

Use of the Monoclonal Antibodies for the Characterization and Purification of SPS

3J Characterization of Co SPS

This characterization was canied out widi SPB3-2-19 and SPB13-2-2 antibodies by the technique of immuno-detection after transfer of the proteins from an electrophoresis gel under denaturing conditions (SDS-PAGE) on nitrocellulose membrane (Western). After electrophoretic separation in a 12.5% acrylamide gel (Nature 277 (1970) 680-685), d e proteins were transferred onto a 0.22 μm nitrocellulose membrane (Schleicher and Schuell). The buffer was a standard electrophoresis buffer (3.03 g/1. TRIS base, 14.4 g/1. Glycine, 0.1 % SDS, pH 8.3, 20% methanol).

After transfer, the membrane was put in a blocking bath (0.5% Casein in PBS). After one hour at 37°C under gentle agitation, the membrane was washed 3 to 4 times in a washing buffer (0.1 % Casein, 0.5% Tween 20, in PBS) men incubated widi a solution of 10 micrograms/ml of the monoclonal antibody to be tested. A part of the membrane was incubated in parallel widi a non-immune antibody (negative control). After one hour of incubation at ambient temperature followed by 9 or 10 washes, die membrane was incubated in the presence of an anti-mouse antibody labeled wid ¹²⁵I diluted in a washing buffer (50,000 cpm per cm² of membrane). After one hour of incubation at ambient temperature followed by 9 or 10 washes, the membrane was dried, d en autoradiographed (X-OMAT AR Kodak film and Crone XTRA Life Dupont amplifying screen). The results of the autoradiography are shown in Figure 2. In the autoradiograph, a strong signal is observed at the protein bands 120 kd, 95 kd and 35 kd which correlates widi the previous results (see first part).

3.2 Purification of Sucrose Phosphate Svnthase bv Immunoaffinity Chromatography A methodology for the purification of com Sucrose Phosphate Syndiase on an immunoaffinity support has been perfected in order to increase the quantity of protein recovered while reducing the number of purification stages and to obtain quantities sufficient for protein sequencing.

3.2J Preparation of the immuno-adsorhent The F18 (see 2.2.2) conesponding to the SPB 13- 1-7 antibody or to the SPB 13-2-2 antibody were mixed with activated CH-Sepharose, (1 mg of antibody per ml of gel). After incubation for 2 hours at ambient temperature, the sites not occupied by die antibodies were saturated with 1M edianolamine, pH 9. The support was then washed alternately with OJM acetate, 0.5 M NaCl, pH 4 buffer and OJ M TRIS. 0.5 M NaCl. pH 8 buffer. The immunoaffinity support dius prepared was preserved at 4°C in a 50 mM HEPES. 10 mM MgCl₂, 1 mM EDTA, 1 mM PMSF, zero 0.01 % sodium nitride (azide), pH 7.5 buffer.

3.2.2 Immunoaffinity Chromatography

50% PEG was added to the Heparin fraction of SPS (see 1.2.3.) to give a final concentration of PEG of 20%. After incubation for 30 minutes at 4°C with gentle agitation, the mixture was centrifiiged at 1600 g for 30 minutes. The protein deposit was taken up in half of the initial volume with the 50 mM HEPES, 10 mM MgCl₂, 1 mM

EDTA, 10% ethylene glycol, pH 7.5 buffer. This stage allows die previous buffer, which is incompatible with the immunoaffinity chromatography, step to be eliminated, and die proteins to be concentrated. The yield of SPS activity was from 80 to 90% .

The solution obtained was applied with a flow rate of 0.1 ml/min over 1 ml of immunoaffinity support packed in a column and on which had been fixed an antibody not directed against the SPS (activated CNBr-Sepharose, on which an antineomycin antibody is fixed). This first stage allows the elimination of certain contaminants which are fixed nonspecifically on the chromatography support. The effluent of die non-specific column was in turn applied to die anti-SPS immunoaffinity support (2 ml in an 11 x 20 mm column) with a flow rate of 0J ml/min. These two stages were carried out at laboratory temperature. The column was washed widi 10 ml of load buffer and dien with a washing buffer (load buffer with the addition of 0.25 M NaCl and 0.3% Tween 20) until absorbency in ultra-violet at 280 nm was close to base level. The proteins adsorbed on die support were eluted with a solution of 50 mM triethylamine, pH 11. This elution was carried out at 4°C and the immunoaffinity column was reversed to obtain an optimum yield. The

SDS-PAGE profile of the final preparation obtained corresponds to that obtained using the standard protocol (see 1). It must be noted diat die elution mediod of the proteins adsorbed on die immunoaffinity support irreversibly destroys die SPS activity but the recovery yield of the eluted SPS proteins is optimal compared to tests carried out in native elution conditions. The eluate of the immunoaffinity column was desalted using a Sephadex G25 column, against a 0.14% Glycerol, 0.07% 2-mercapto-ethanol, 0.04% SDS, O.9 mM TRIS pH 6.8 buffer (electrophoresis buffer in reducing conditions diluted 70 times). After desalination, die protein preparation was concentrated 70 times widi a concentrator under vacuum and the SPS proteins were purified by SDS-PAGE (see below). Example 4 Partial Sequencing of SPS Polypeptides 4J Purification of SPS Polypeptides for Sequencing

Samples of a purified protein preparation obtained as described in Example 3.2.2. were subjected to preparative SDS-PAGE. After electrophoresis, the protein bands were visualized with KC1 treatment as described by Bergman and Joernvall (Eur. Biochem. (1978) 769:9-12) and die bands observed at 90kd and 30kd were excised. The proteins from diese gel fragments were electroeluted using an Electrophoretic Concentrator according to manufacturer's instructions (ISCO ; Lincoln, NE) in 4 mM sodium acetate, pH8. After electroelution, protein yields were quantitated by comparison to a bovine serum albumin (BSA) standard on a Comassie Blue-stained gel. Approximately 30 mg of the 30 kd protein and 75 μg of the 90 kd protein were obtained.

4.2 Trvptic Digestion and Protein Sequencing of SPS polypeptides The proteins were concentrated by acetone precipitation, and resuspended in 50 mM ammonium carbonate buffer, pH 8. Tryptic digestion and HPLC purification were performed as described by Sturm and Chrispeels (Biol. Chem. (1987) 262: 13392-13403). Briefly, digestion was performed by addition of trypsin (5% of SPS protein), and incubation for two hours at 37°C. The digestion was then repeated. The proteins were concentrated by lyophilization and resuspended in 50mM sodium phosphate buffer, pH 2.2. This mixture was subjected to reverse phase HPLC separation by application to a C18 column in phosphate buffer. Elution was performed using an increasing gradient of acetonitrile. Eluted material from the phosphate buffer/acetonitrile gradient was monitored at 214 nm. The fractions corresponding to peaks of absorbance at 214 nm were collected, lyophilized, resuspended in 0J % trifluoroacetic acid, reapplied to the C18 column

(equilibrated widi 0J % trifluoroacetic acid), and eluted using an acetonitrile gradient. Eluted material from the trifluoroacetic acid/acetonitrile gradient was monitored at 214 nm. The fractions corresponding to peaks of absorbance at 214 nm were collected, lyophilized, and subjected to standard Edman degradation protein sequencing on an automated protein sequencer (Applied Biosystems ; Foster City, CA). Sequences of five peptides were obtained. See Fig. 3 (SEQ ID NOS: 1-5). Example Isolation and Assembly of a Full-length cDNA for SPS 5J RNA Isolation from Co Leaf

Total RNA was isolated from com leaves (see 1.2.1.) according to the method of Turpen and Griffith (Biotechniques (1986) 4: 11-15). Briefly, 250 gm of material was homogenized in 4M guanidine thiocyanate and 2% sarcosyl. The mixture was then centrifiiged and die cleared supernatant was layered into a 5.7 M CsCl cushion and centrifiiged for 5.5 hours at 50,000 rpm. The RNA pellet was dissolved in water, extracted with phenol and chloroform, and precipitated widi edianol. The resulting pellet was resuspended in water. The final yield from the RNA isolation step was quantitated by UV spectrophotometry .

5.2 Polv(A) RNA Isolation

A saturated suspension of cellulose powder/water was added to die RNA/water mixture obtained in 5J, at 10% of the total volume, to remove residual polysaccharides. After centrifugation, the supernatant, containing the RNA, was applied to an oligo(dT)- cellulose column as described by Maniatis et al. (Molecular Cloning: A Laboratory Manual, (1982) Cold Spring Harbor, New York). The fraction containing the poly(A) + RNA was then reapplied to die column. The eluted fraction containing the poly(A)+ RNA was extracted widi phenol, and die RNA was precipitated widi edianol. Analysis by gel electrophoresis showed complete absence of ribosomal RNA.

5.3 Construction of Total Corn Leaf Library cDNA synthesis was performed according to die manufacturer's instructions (RiboClone cDNA Synthesis System by Promega, Madison, WI), using five μg of poly(A)+ RNA as template, except that M-MLV reverse transcriptase (BRL ; Bethesda, MD) was substituted for AMV reverse transcriptase. EcoRI linkers were added to the blunt-ended cDNA, and die resulting fragments were cloned into an expression vector (LambdaZAP, Stratagene ; La Jolla, CA) according to die manufacturer's instructions. The resulting library contained approximately 1.5 x 10⁶ transformants.

5.4 PCR Generation of a Partial SPS cDNA Probe

Using the sequence information from the peptides of Example 4 (SEQ ID NOS: 8-9) and the polymerase chain reaction (PCR), a 1200 bp SPS cDNA fragment was generated. Total corn leaf cDNA (5. J was used as a template, and degenerate oligonucleotides (SEQ ID NOS: 10-13), designed from two peptide sequences of die 30kd and 90kd SPS polypeptides, were used as primers. These primer sets were designated as CD3 (SEQ ID NOS: 10-11) and CD4 (SEQ ID NOS: 12-13). See Fig. 4. PCR was carried out, according to die manufacturer's instructions (GeneAmp DNA Amplification Reagent Kit and DNA Thermal Cycler of Perkin Elmer Cetus ; Norwalk, CT) except that the reaction was carried out for 30 cycles, and die annealing steps were programmed to be at 50°C for 1 minute. The PCR reactions were analyzed by agarose gel electrophoresis. Use of the conect set of primers, CD3, resulted in a 1200 bp band being generated by the PCR reaction. PCR using the other set of primers, CD4, gave no specific signals. See Fig. 5. Southern analysis (see Fig. 5) confirmed that the PCR band was not an artifact. The probe 4K5 (SEQ ID NO: 14) was used because die corresponding sequence of die probe was predicted to be wid in the 1200bp fragment if the fragment corresponded to the SPS sequence. The probe hybridized to die 1200 bp band generated by PCR using die primer set CD3 but not to PCR products generated by the primer set CD4. See Fig. 5.

5.5 Isolation of SPS Bacteriophage La hda cDNA Clones

The 1200 bp PCR-generated fragment was labeled with ³²P (as per the Random Primed DNA Labeling Kit, Boehringer Mannheim, Indianapolis, IN) and used as a probe to screen approximately 250,000 plaques of the cDNA library (5.3 J. The inserts of the positive clones were analyzed by restriction analysis widi EcoRI, and the clones with die longest inserts, SPS#3 and SPS#18, were selected for further analysis. See Fig. 6. A 0.4 kb H dIII/EcoRI fragment from the 5' end of SPS#3 was isolated, dien labeled widi ³²P by random priming (Random Primed DNA Labeling Kit) and used as a probe to re-screen the library. Another clone, designated SPS#61, which extends further upstream than SPS#3, was isolated. See Fig. 6. DNA sequencing indicated diat die 5' end of the SPS reading frame was not reached.

To isolate cDNA clones diat included more of the 5' region than SPS#3 or SPS#61 , a new cDNA library was prepared, as per Example 5.3., (RiboClone cDNA Syndiesis System by Promega ; Madison, WI) using M-MLV reverse transcriptase instead of AMV reverse transcriptase. However, instead of using oligo (dT) as a primer, a synthetic 17 bp primer, 23B, derived from the 5' sequence of the SPS#61 clone, was used (see Fig. 6). This resulted in cDNAs that contain only regions upstream of the SPS#61 5' region. The library was screened widi die ³²P-labeled EcoRI insert from SPS#61 , and 16 positive clones were obtained. The clones with die longest inserts, SPS#77 and SPS#90, were selected for further analysis. DNA sequencing of SPS#77 and SPS#90 showed d at the region of overlap (greater than 100 bp) widi SPS#61 was identical in all clones, and that both extended further upstream into die 5' region. See Fig. 6. PCR was carried out using single-stranded cDNA (from a reverse transcriptase reaction corn leaf RNA (5.2J primed wid oligo (dT) as described above) as template and primers selected from the SPS#90 and SPS#3 sequences, confirmed that SPS#90 and SPS#3 originate from the same mRNA transcript. The fragment resulting from this PCR reaction was 750 bp in length, consistent widi die size predicted from the DNA sequence. The 750 bp fragment was subcloned into a Bluescript-derived vector as a Sall/HindlU fragment. Four of the resulting subclones were partially sequenced, and the sequence obtained matched d e existing DNA sequence.

5.6 Assembly of the SPS Reading Frame

Both DNA strands of SPS#90, SPS#61, and SPS#3 were sequenced, using the method of Sanger et al. (PNAS (1977) 74:5463-5467). All three sequences can be combined to form one contiguous sequence of 3509 bp. See Fig. 7 (SEQ ID NO: 6). Primer extension experiments using com leaf poly(A) RNA and an antisense primer showed diat die 5 ' end of our DNA sequence represents sequences form the actual 5 ' end of the SPS in RNA. In the SPS reading frame, as defined by die five peptide sequences (SEQ ID. NOS. : 1-5 respectively) (see Fig. 3), the first mediionine codons are located at bp 112 and bp 250. See Fig. 7 (SEQ ID NO: 6). The codon at bp 112 is similar to the consensus eukaryotic translational start site (Kozak, Cell (1986) 44:283-292) and is located 54 bp downstream of a TAG stop codon (bp 58). It is proposed diat this codon represents the translational start of the SPS polypeptide in vivo. After a 1068 codon reading frame, translation is stopped by TGA. The following 193 bp contain the 3' untranslated region including a poly(A) addition signal, AAATAAA.

The full-lengdi SPS coding region can be assembled by combining die 529 bp BαmHI/H.Λdlll fragment of SPS#90, the 705 bp Hindlll fragment of SPS#61 and the 2162 bp Hindlll/ EcoRI fragment from SPS#3 (see Fig. 6).

Example 6 Detection of SPS Polypeptides by Specific Antisera 6J Preparation of Antibodies to SPS

Samples of purified protein preparations obtained by the mediod described in 3.2.2. were subjected to SDS-PAGE electrophoresis. The proteins in the gel were fixed and stained. The bands corresponding to die 90kd and 30kd polypeptides were excised. Using this material, polyclonal antisera were raised in rabbits by conventional procedures. Western analysis (as described by Oberfelder, Focus (1989) 77 (1): 1-5) showed diat the antibodies isolated from the rabbit immunized with SPS 30 recognized die bands corresponding to the SPS#30 and SPS#120 peptides on a SDS PAGE gel, and that the antibodies isolated from the rabbit immunized with SPS#90 recognized the bands corresponding to die SPS#90 and SPS#120 polypeptides (see Fig.8).

6.2 Immunological localization of SPS in the Com Plant

Total proteins were extracted from leaves of a 30 day-old com plant, harvested at 11 :00 am, by boiling in SDS buffer. The protein extracts were loaded on duplicate

SDS-PAGE gels. One gel was stained widi Comassie Blue, while die odier was subjected to Western analysis, using a mixture of SPS#30 and SPS#90 antisera as probe. See Fig. 9. The prominent bands appearing on the Comassie Blue-stained gel were identified as phosphoenolpyruvate carboxylase (PEPcase), an enzyme involved in C4 photosynthesis. The Western blot showed die presence of the SPS band. The SPS protein pattem was very similar to the PEPcase protein pattem: not present in roots, nor present in die section of leaf closest to die stem, nor present in very young leaves. This pattem corresponds with expression associated widi photosyndiesis, and is the pattem expected for SPS.

Example 7

Construction of Expression Construct Plasmids 7.1 Construction of the full-length SPS reading frame

Clone SPS#90 was digested with Hindlll and ligated with die 705 bp H dIII fragment from clone SPS#61 to create a plasmid containing the 5' end of die SPS coding region. The resulting plasmid was digested with ItømHI and partially digested wid

Hindlll, resulting in a 1340 bp BamHl/ Hindlll fragment containing the 5' end of the coding region. The 3 ' end of the SPS coding region was obtained by digestion of SPS#3 wid EcoRI and partial digestion widi Hindlll, resulting in a 2162 bp H dIII/EcoRI fragment. This 2162 bp H dIII/EcoRI fragment, carrying the 3' end, was ligated wid the 1340 BamΑl/EcόRl fragment carrying the 5' end into a ifø/nΗI/EcσRI-digested pUC-derivative plasmid Bluescript, to create a plasmid carrying the entire 3403 bp SPS coding region and 3' untranslated transcription termination region. 7.2 Expression of SPS in E. coli

When cloning the 3403 bp BamtlllEcdKl SPS fragment into the plasmid Bluescript SK (Stratagene, La Jolla, CA), a translational fusion between the plasmid coded lacZ sequence and the SPS reading frame was created. The resulting fusion protein contains 30 N-terminal amino acids from the β-galactosidase and d e complete SPS polypeptide. The fusion protein was expressed in E. coli under the Bluescribe plasmid lacZ promoter. Preparation of total protein followed by Western analysis using anti-SPS antisera (see 6.1.) shows a band comigrating widi native plant SPS. For the SPS activity test, die E. coli cells containing the SPS expression construct as described were opened with lysozyme and sonication. Soluble protein was desalted by a Sephadex G-25 column. This protein extract was assayed for SPS activity analogous to the method described in 1.1. a., except that die reagent andirone was used instead of resorcinol (Handel, Analytical Biochemistry, (1968) 22:280-283). This test showed that the SPS protein, expressed from the cDNA in E. coli does have SPS enzyme activity. By comparison to native plant enzyme it seems to have die same specific activity.

7.3. Construction of the Tobacco Small Suhunit (SSU) Promoter-Transcriptional Fusions The SPS coding region can be conveniently cloned as a BamHl/ EcoR (bp 106 - bp 3506) fragment 3' of a tobacco small subunit promoter. A SSU promoter for expression of the SPS coding region, was prepared as follows. The SSU promoter region from pCGN627 (described below) was opened by Kpnl and die 3' overhang removed. After £α>RJ digestion, the 3403 bp BamHl (filled in) EcoRI SPS cDNA fragment (see, Example 7.1.) was inserted. After the SPS coding region was ligated into d e SSU promoter, d e SSU/SPS region was ligated into a binary vector and integrated into a plant genome via Agrobacterium tumefaciens-mediated transformation. (The SPS region carries its own transcription termination region in the cDNA sequence). Insertion of the SSU/SPS construct into die binary vector pCGN1557 resulted in pCGN3812.

PCGN627 The 3.4 kb EcøRI fragment of TSSU3-8 (O'Neal et al. , Nucleic Acids Res. (1987)

75:9661-8677), containing the small subunit promoter region, was cloned into the EcoRI site of M13mpl8 (Yanisch-Perron et al, Gene (1985) 55:103-119) to yield an M13 clone 8B. Single-stranded DNA was used as a template to extend die oligonucleotide primer "Probe 1 " (O'Neal et al. , Nucleic Acids Research (1987) 75:8661-8677) using the Klenow fragment of DNA polymerase I. Extension products were treated widi mung bean nuclease and dien digested widi Hindlll to yield a 1450 bp fragment containing die SSU promoter region. The fragment was cloned into HinaHl-Smal-digested pUC13 (Yanisch-Perron et al.. Gene (1985) 53: 103-119) to yield pCGN625. pCG2J625 was digested with Hindlll, the ends blunted with Klenow, and the digested plasmid re-digested widi EcόRI. The EcøRI/blunted-Hmdiπ fragment containing the SSU promoter region was ligated widi Smal/EcøRI-digested pUC18 to yield pCGN627.

7.4. Construction of a CaMV Promoter-SPS Transcriptional Fusion

The 35ESS promoter-DNA fragment from cauliflower mosaic virus was fused to die SPS DNA as follows. The plasmid pCGN639 was opened by BamHl and EcόR and the 3403 bp BamHl-EcoKl SPS cDNA fragment (described in Example 7J) was cloned into diis plasmid. The hybrid gene was removed from diis plasmid as a 4.35 kb Xbal-Ecό l fragment and ligated into a binary vector (McBride and Summerfelt, Plant Mol. Bio. (1990) 74:269-276) and integrated into a plant genome via Agrobacterium tumefaciens mediated transformation. Insertion of the CaMV/SPS construct into the binary vector pCGN1557 (McBride and Summerfelt supra) results in pCGN3815.

7.4J . Construction of pCGN639 pCGN164 was digested widi EcoRW and BamHl to release a EcoRV -BamHl fragment which contained a portion of die 35S promoter (bp 7340-7433). pCG8638 was digested with Hmdlll and EcoRV to release a ///ndlll-EcøRV fragment containing a different portion of the 35S promoter (bp 6493-7340). These two fragments were ligated into pCGN986 which had been digested widi Hindlll and BamHl to remove the

H//ιdIII-5α7iHI fragment containing die 35S-promoter ; this ligation produced pCGN639, which contains the backbone and tml-3' region from pCGN986 and die two 35S promoter fragments from pCGN164 and pCGN638.

7.4.2. Construction of pCGN 164

The Alul fragment of CaMV (bp 7144-7735) (Gardner et al. , Nucl. Acids Res. (1981) 9:2871-2888) was obtained by digestion wid Alul and cloned into the Hindi site of M13mp7 (Vieira and Messing, Gene (1982) 79:259-268) to create C614. An EcoRl digest of C614 produced die EcoRI fragment from C614 containing the 35S promoter which was cloned into d e EcoRI site of pUC8 (Vieira and Messing, supra) to produce pCGN146. To trim the promoter region, the BgUl site (bp 7670) was treated with BgUl and BalSl and subsequently a BgUl linker was attached to d e fto/31 treated DNA to produce pCGN147. pCGN147 was digested widi EcoRI/HpΛI and me resulting EcoRI-Hphl fragment containing the 35S promoter was ligated into EcoRl-Smal digested M13mp8 (Vieira and Messing, supra) to create pCGN164.

7.4.3. Construction of pCGN638 Digestion of CaMVIO (Gardner, et al. , Nucl Acids Res. (1981) 9:2871-2888) with

BgUl produced a BgUl fragment containing a 35S promoter region (bp 6493-7670) which was ligated into the BamHl site of pUC19 (Norrander et al , Gene (1983) 26: 101-106) to create pCGN638.

7.4.4. Construction of pCGN986 pCGN986 contains a cauliflower mosaic virus 35S (CaMV35) promoter and a T-DNA tml-3' region widi multiple restriction sites between them. pCGN986 is derived from another cassette, pCGN206, containing a CaMV35S promoter and a different 3' region, the CaMV region VI 3'-end and pCGN971Ε, a tml 3' region. pCGN148a containing a promoter region, selectable marker (kanamycin with 2 ATG's) and 3' region, was prepared by digesting pCGN528 widi BgUl and inserting the BamHl-BgUl promoter fragment from pCGN147 (see 7.4.2. above). This fragment was cloned into die BgUl site of pCGN528 so d at the BgUl site was proximal to the kanamycin gene of pCGN528.

The shuttle vector used for this construct pCGN528, is made as follows: pCGN525 was made by digesting a plasmid containing Tn5, which harbors a kanamycin gene

(Jorgensen et al , Mol. Gen. Genet. (1979) 177:65), with Hi^' idlll-BαmHI and inserting die Hindϊϊ-BamHl fragment containing the kanamycin resistance gene into the HmdlH-SαmHI sites in the tetracycline gene of pACYC184 (Chang and Cohen, J. Bacteriol (1978) 134: 1141-1156). pCGN526 was made by inserting die BamHl fragment 19 of pTiA6 (Thomashow et al. , Cell (1980) 19:729-739) modified with XΛoI linkers inserted into die Smal site, into die BamHl site of pCGN525. pCGN528 was obtained by deleting die small Xhόl and religating. pCGN149a was made by cloning the ftamHI kanamycin gene fragment from pMB9KanXXI into the BamHl site of ρCGN148a. pMB9KanXXI is a ρUC4K variant (Vieira and Messing, Gene (1982) 79:259-268) which has die Xhόl site missing but contains a functional kanamycin gene from Tn903 to allow for efficient selection in Agrobacterium. pCGN149a was digested widi Hmdlll and BamHl and ligated which pUC8 (Vieira and Messing, supra) digested widi H diπ and BamHl to produce pCGE169. This removes the Tn9O3 kanamycin marker. pCGN565 and pCGN169 were both digested widi H dIII and Pstl and ligated to form pCGN203, a plasmid containing die CaMV 35S promoter and part of the 5 '-end of the Tn5 kanamycin gene (up to die Pstl site, (Jorgensen et al. , Mol. Gen. Genet. (1979) 777:65). pCGN565 is a cloning vector based on pUC8-Cm (K. Buckley, Ph.D. Thesis, UC San Diego 1985), but containing the polylinker from pUC18 (Yanisch-Perron et al , Gene (1985) 55:103-119). A 3' regulatory region was added to pCGN203 from pCGN204 (an EcoRI fragment of CaMV (bp 408-6105) containing die region VI 3' cloned into pUC18 (Gardner <?t al. , Nucl. Acids Res. (1981) 9:2871-2888) by digestion widi HindEl and Pstl and ligation. The resulting cassette, pCGN206, is die basis for die construction of pCGN986.

The pTiA6 T-DNA tml 3 '-sequences were subcloned from die Bam\9 T-DNA fragment (Thomashow et al , Cell (1980) 79:729-739) as a flαmHI-EcoRI fragment (nucleotides 9062 to 12,823, numbering as in Barker et al , Plant Mol Biol (1983) 2:335-350) and combined with the pACYC184 (Chang and Cohen, /. Bacteriol (1978) 134: 1141-1156) origin of replication as an EcoRI-Hwdll fragment and a gentamycin resistance marker (from plasmid pLB41), (D. Figurski) as a BamHl-HindU fragment to produce pCGN417. The unique Smal site of pCGN417 (nucleotide 11 ,207 of the Bam\9 fragment) was changed to a Sacl site using linkers and die BamHl-Sacl fragment was subcloned into pCGN565 to give pCGN971. The BamHl site of pCGN971 was changed to an EcoRI site using linkers to yield pCGN971Ε. The resulting EcoRI-SacI fragment of pCGN971Ε, containing the tml 3' regulatory sequence is joined to pCGN206 by digestion with EcoRI and Sacl to give pCGN975. The small part of the Tn5 kanamycin resistance gene was deleted from the 3 '-end of die CaMV 35S promoter by digestion widi SaU and

BgUl, blunting the ends and ligating with SaU linkers. The final expression cassette, pCGN986, contains the CaMV 35S promoter followed by two SaU sites, an Xbal site, BamHl. Smal, Kpn sites and the tml 3' region (nucleotides 11207-9023 of the T-DNA). A schematic summary of the construction of the various plasmids is shown in Figures 10A through IOC.

Example 8 Transgenic SPS Tomato Plants

8J Production of Tissue-Specific SPS "Sense" Transgenic Tomato Plants

Tomato plants were transformed widi expression cassettes containing SPS encoding sequences (pCGN3812, pCGN3815, pCGN3342, and pCGN3343) via Agrobacterium tumefaciens mediated transformation (Fillatti, et al , Bio/Technology (1987) 5:726-730) and regenerated. Preparation of pCGN3812, a tobacco SSU/SPS construct, and pCGN3815, a CaMV 35S/SPS construct are described in Examples 7.3 and 7.4, respectively. The fruit- specific E8/SPS constructs pCGN3342 and pCGN3343 were prepared as described for pCGN3812 with die following modifications. Approximately 2J kb of the 5' region conesponding to die tomato derived E8 fruit-specific promoter replace the SSU promoter region in pCGN3812. The E8 promoter is described in Deikmann et al. (1988) EMBOJ, 2:3315-3320; and Delia Penna et al (1989) Plant Cell, 7:53-63. The pCGN3342 and pCGN3343 constructs also contain a SPS cDNA sequence truncated at the Apό site just 3' of the SPS coding region (at nucleotide 3318), and fused to a 1.2 kb region of the A. tumefaciens tml 3' terminator region from pTiA6 (Barker et al. , (1983) Plant Mol. Biol, 2:335-350; sequence 11208-10069 of the T-DNA region from A. tumefaciens Ti plasmid pTi 15955). Constructs pCGN3342 and pCGN3343 represent opposite orientations of the E8-corn SPS-tml insert in the binary vector pCGN1557, which contains die kanamycin nptll marker gene under the control of die CaMV 35S promoter region and the tml 3' terminator region described above for pCGN3318 (McBride and Sumerfelt, Plant Mol. Biol. (1990) 74:269-276). Tomato plant lines are designated widi a number corresponding to the constmct used for transformation. Tomato lines arising from separate transformation events are signified by a hyphen and a number following the construct/plant designation.

8.2 Immunohlot Results Leaves from transformed tomato plants (pCGN3812 and pCGN3815) and control tomato and com leaves were tested as described in Example 6.2 for SPS activity using the SPS #30 and SPS #90 peptide polyclonal antisera of Example 6. No cross reactivity between the antisera and the control (endogenous) tomato leaves was seen. This indicates that the com and tomato SPS are not highly related. As to the transgenic tomato plants, leaf extracts from tomato plants containing the pCGN3815 or pCGN3818 constructs showed signals up to levels several times those observed in the untransformed corn leaf extracts.

8.3 SPS Activity

Leaf extracts also were tested for SPS activity according to die resorcinol protocol described in Example 1.1. a. In comparison to leaf extracts from control plants, leaves from transformed tomato plants containing d e SPS gene showed up to 12-fold increases in SPS activity. Higher SPS activity also was observed in some leaf extracts from transgenic tomato plants containing the corn SPS gene as compared to control com leaf extracts.

8.4 Starch and Sucrose Levels

Leaf tissue was analyzed for starch and sucrose levels according to die mediod of Haissig, et al , Physiol. Plan (1979) 47: 151-157. Two controls were used, leaves from an untransformed plant and leaves from a transformant which did not show any com SPS immunoblot signal. The starch and sucrose levels of these two plants were essentially the same, and had an almost equal percentage of starch (mg/lOOmg dry weight) and sucrose (mg/lOmg dry weight). High-expressing plants containing pCGN3812 (pCGN3812-9 and pCGN3812-l 1) showed bodi a reduction in leaf starch by 50% and an increase in sucrose levels by a factor of two. Thus, the extra sucrose synthesis provided by die exogenous SPS activity had a profound affect on carbohydrate partitioning. These data indicate diat die presence of high levels of corn SPS activity resulting from a sufficient level of transgenic expression of a SPS transgene functional in tomato leaves cause a modification of carbohydrate partitioning in this tissue.

8.5 Oxygen Sensitivity

The interaction between photosynthesis and die syndiesis of end products in tomatoes expressing com SPS was evaluated by gas exchange analysis. Oxygen sensitivity of plants was induced by lowering growth temperature and then O₂ sensitivity measured as the rate of photosynthesis in low O₂ (Sage and Sharkey (1987) Plant Physiol. 84:658-664). Photosynthesis of tomato plants expressing corn SPS became oxygen insensitive at 14.2°C (measured in 35 Pa CO₂ ), whereas untransformed controls became insensitive at 17.3°C. Change in the growth temperature from 22°C to 30°C during die day did not affect this pattem. Furthermore, the transformed plants did not acclimate following growth at high CO, (Wonell et al (1991). The Plant Cell 5:1121-1131). These data show that the SPS expressing plants have a reduced ceiling imposed on photosyndiesis by end product syndiesis at lower temperatures. The data also show that die temperature at which photosynthesis becomes oxygen insensitive can be modulated by SPS activity through its effect on chloroplasts, photosynthetic capacity and end product synthesis and sink transport/conversion.

8.6 Temperature Effect on Partitioning The effect of temperature on starch and sucrose partitioning was evaluated in tomato plants transformed widi pCGN3812 (see 7.3). The transformed tomato plants were compared to control UC82B plants. The rate of starch plus sucrose synthesis as a function of temperature was assayed by feeding a pulse of ^l4 CO₂ to leaves at a normal partial pressure then chasing with unlabeled CO₂ for a long enough period of time to permit incorporation of the labeled carbon into starch, sucrose, fructose, glucose or another end product but for a short enough period of time so mat very little of die carbon was exported from the leaf source tissue. Analysis of end product syndiesis showed diat sucrose synthesis appeared more sensitive to temperature than did starch syndiesis. For example, plants expressing about 5-fold more SPS activity compared to controls did not partition more carbon to sucrose at the lowest temperature. This indicates d at the control coefficient for SPS approaches zero as metabolic activity of the plant is reduced widi temperature under diese conditions. The additional SPS activity also changed the oxygen sensitivity in diis same temperature range. The above results show that partitioning between starch and sucrose, end-product syndiesis/sink transport and conversion can be modulated as a function of temperature. (See Fig. 11).

8.7 Xield

Manipulation of yield by modification of end-product synthesis is related to growth conditions and reproductive/vegetative sink. The effect of growth conditions on tomato yield was evaluated in homozygous SSU/SPS (Rubisco small subunit promoter-SPS), 35/SPS (CaMV 355 promoter-SPS) and E8/SPS (E8 fruit-specific promoter-SPS) tomato plant lines grown under growth chamber, open-top chamber and field conditions following standard mediods in the art.

When compared to untransformed tomato plants, variation in yield increase was observed in the growth chamber, open-top chamber and field trials. Differences observed in fmit yield may be due to earlier flowering and die number of fruits set and filled for plants grown in growth chambers and pots compared to those grown in the field. Also, tomatoes expressing SPS behind die CaMV 35 S promoter grew better than tomatoes expressing the gene behind a Rubisco small subunit promoter under growth chamber conditions. These data indicate a promoter effect. Additionally, studies in temperature controlled growth rooms show that diere was more yield penalty in the SPS tomatoes at low temperatures than at high temperature. These data are in accordance widi the partitioning data showing a reduction in modulation of sucrose levels at low temperature in tomato plants.

8.7J Soluhle Solids In T2 SSU/SPS Tomato Plants Grown Under Growth Chamber and Greenhouse Conditions Leaf-specific SSU/SPS tomato lines 3812-9 and 3812-11 were evaluated for soluble solid content. Extracts of fruit from these tomato lines and controls were grown and harvested in a Biotron growth chamber or under standard greenhouse conditions and served as the tissue source. T2 plants from the 3812-9 and 3812-11 lines were segregating as the original lines were shown to contain at least two SSU-SPS insertions. For growth chamber conditions, T2 plants were illuminated by metal halide lamps at peak level of 500 μmol photons/m/s (pot level), at a temperature of 26°C for die 16h day and 18°C at night, and a relative humidity of 60% . Plants were watered daily widi half-strength Hoagland's solution (Hoagland and Amon, Calif. Argicult. Exp. Sta. Cir. (1938) 557: 1-39). Soluble solids were evaluated as Brix units per unit weight fruit tissue measured for the average of three fruits per plant. Transgenic SSU/SPS plants grown under growdi chamber conditions exhibited substantial increases in soluble solids compared to controls. The soluble solids measured in a segregating T2 population of 3812-11 plants grown under greenhouse conditions showed the same effect, but overall increases were reduced compared to

SSU/SPS plants in growth chamber tests.

8.7.2 Soluhle Solids In T4 SSU/SPS and 35S/SPS Tomato Plants Grown Under Greenhouse Conditions

Homozygous SSU/SPS tomato lines were generated from original SSU/SPS 3812-9 transformants in UC82-B tomatoes following standard products. Two homozygous lines designated A and B were grown under greenhouse conditions and fmit evaluated for soluble solid content using Brix analysis measured per unit weight fmit tissue. Soluble solids were measured as an average of diree plants per line and diree fmit per plant. The average soluble solid content for die SSU/SPS 3812-9 lines was increased significantly compared to die UC82-B controls. The data was shown to be significant at a 0.01 % level (99%), according to least significant difference (LSD) statistical analysis.

Homozygous lines of tomato plants transformed widi d e 35S/SPS constmct of pCGN3815 were generated to compare die homozygous leaf-specific SPS constmct results to homozygous constitutive expression constmct. In one line, designated 3815-13-2, a substantial increase in fmit yield was observed, as measured for bodi fmit size and fmit number, compared to non-transformed controls and, surprisingly, compared against die SSU/SPS leaf-specific homozygous line controls. The 3815-13-2 plants also produced a second flush of fruit.

8.7.3 Soluhle Solids In Field Grown T4 SSU/SPS Tomato Plants

Tomato plants homozygous for die SSU/SPS constmct were generated from T4 crosses of original 3812-9 transformants as described in Example 8J . Tomato lines designated A and B, which arose from separate crossing events, were grown under field conditions following standard field trial protocols. Soluble solids were obtained from fmit extracts of replicate plants as described for growth chamber and greenhouse smdies. The soluble solids were evaluated by determining the average refractive index (RI) and specific sugar content per unit weight fmit tissue using high pressure liquid chromatography (HPLC). The RI measurements permitted analysis of overall sugar and acid content and die HPLC analysis for contributions by individual sugars. Bodi mediods of analysis were conducted following standard protocols. The results are reported in Table 11 below.

The transgenic tomato lines A and B consistently showed higher sugar and acid content compared to die controls. Sucrose, glucose and fructose levels were increased substantially in tomato fruit of die A and B lines, compared to the controls. Surprisingly, the contribution of glucose and fructose to die overall increase in soluble solids was pronounced compared to sucrose, indicating a net partitioning and conversion of photoassimilate to die fmit sink tissue.

8.7.4 Soluble Solids In Fruit-Specific E8/SPS Tomato Plants Grown Under Greenhouse

Conditions

The soluble solids in fruit from tomato plant lines 3342 and 3343 expressing die fruit-specific E8-SPS constructs were evaluated as follows. Tomato plant lines arising from separate transformation events widi pCGN3342 and pCGN3343 were grown under standard Greenhouse conditions. Soluble solids from replicate lines and trials were measured using RI, SPS specific activity and HLPC analyses. As a control, untransformed tomato plants and leaf-specific SSU/SPS tomato line were examined in parallel for each trial. Representative data for soluble solid content and distribution are reported in Tables 12-14 below.

Soluble solids measured as refractive index (RI) per unit weight fmit tissue.

Xahl£j3

Soluhle Solids and Sugar Content In Fruit-Specific E8/SPS Tomato Plants

Dale RI Sugar Concentration (%)

Sucrose

A 4.4 0.00 B 7.9 0.00 C 6.1 0.00 D 7.5 0.00 E 7.2 0.00 F 8.4 0.00

( l G 8.5 0.52 H 4.0 0.00 I 8.5 0.00

oo ∞ m t~- co r~ co -^ ^■— 00 —< o co r- co a o "-■ CN 00

a CO Tf l- co T «n T co CN CO TJ-

u| o Q Q Q QQQ^Q Q Q Q

3 z Z Z Z o Z Z Z Z O z z

Tomato plant lines expressing the fmit-specific E8/SPS constructs consistently showed an increase in soluble solids reflected by overall sugar content, acid content and distribution. To assess die correlation between SPS activity and altered soluble solid content, SPS activity was measured in fmit from control tomato plants and compared to diat in fruit from E8/SPS tomato lines 3343-6 and 3342-11. Control fmit from tomato line FL7060 was assayed with a SPS activity rate of 17.8 μmols sucrose/gram fresh weight/hour. Activity was much higher in the transgenic lines, with die 3343-6 event having a rate of 67.5 μmols sucrose/grown fresh weight/hour and die 3342-11 event measured at 36.6 μmols sucrose/gram fresh weight/hour. These results show diat die increase of fmit-specific activity of the SPS conelates to die increase in sugar content of fruit.

Example 9 Transgenic SPS Potato Plants 9.1 Production of SPS Potato Plants Potato plants were transformed widi expression cassettes containing SPS coding sequences (pCGN3812) via Agrobacterium tumefaciens mediated transformation (Fillatti et al, supra) and regenerated. Preparation of pCGN3812, a tobacco SSU/SPS constmct, is described in Example 4.3.

9.2 Oxygen Sensitivity

Potato is adapted to cool weadier and has a large vegetative sink, whereas the genetically similar tomato has a large reproductive sink. To evaluate whether potato has a relatively higher capacity for starch plus sucrose synthesis, allowing it to avoid oxygen insensitivity in the range of 12°C to 20°C, oxygen sensitivity was examined in potatoes expressing the com SPS gene. Potatoes expressing the co SPS exhibited a higher capacity for photosynthesis in elevated CO₂ when die plants were three weeks old compared to controls. When die potato com SPS expressing plants were six to seven weeks old wid developing tubers, they showed die acclimation to elevated CO₂ found in many plants and the controls (Fig. 12). These data show that responsiveness of plant growth to elevated CO₂ in plants having diverse physiological systems can be modulated by manipulating sucrose synthesis through an SPS which functions in plants. 9.3 Tuber Yield

Transformed potatoes expressing co SPS exhibited greater tuber yield when grown in bod large chambers and in open top chambers out-of-doors (Fig. 13). Because yield in potato is tuber mass and not fruit, the effect in potato appear different from the effect seen in tomato. Collectively, the tomato and potato yield data indicate that modification of SPS activity through expression of an exogenous transgene encoding SPS directly effects net sucrose synthesis and mass action in a similar manner in diverse plant systems, even though sucrose metabolism and its systemic effects may differ, which can be used to manipulate yield.

The above results demonstrate that transgenic plants can be constructed which have altered carbon partitioning through expression of a gene required for sucrose synthesis. Plants transformed wid a DNA expression constmct capable of controlling the expression of an SPS gene exhibited modification of starch and sucrose levels, CO₂ and/or O₂ sensitivity, temperature dependent growdi responsiveness, and overall modification of carbon partitioning between source tissue such as leaf and sink tissue such as fmit or root. The data also show diat the plant growth and yield were affected by altered carbon partitioning, as illustrated in two different plants of the nightshade family Solanaceae, potato and tomato. The data also show diat control of carbohydrate partitioning through modification of end-product synthesis, for example, sucrose synthesis and conversion to other sugars in sink tissue, such as glucose and fructose provide means for altering plant growth and yield of specific plant tissues, plant parts and/or whole plant systems. In particular, increased SPS activity and tissue-specific SPS activity was demonstrated to produce a net increase in overall soluble solids in sink tissue such as fmit. Increases in the sugars sucrose, glucose and fructose represented soluble sugars analyzed in the soluble solids, widi contributions by glucose and fructose being higher than sucrose. The SPS activity and sugar content data indicate diat die endogenous acid invertase found in ripening tomato fruit contributed to die observed increases in glucose and fructose.

Acid levels in d e fruit-specific E8/SPS constructs also were observed, correlating acid content to an increase in sugar content. These data collectively show diat SPS can be used to alter the overall content and ratio of soluble solids in a plant sink tissue, resulting in a demonstrable phenotype in plants, such as fmit having modified sweetness. Also, tomatoes expressing SPS behind die CaMV 35S promoter grew better than tomatoes expressing die gene behind a

Rubisco small subunit promoter under growth chamber conditions. These data indicate a promoter effect which can be manipulated to control SPS activity in particular plant cells, plant parts and diroughout the plant. In general, the results show that plant growth and yield can be enhanced dirough transgenic expression of SPS, even though its effect on photosynthesis may be small.

Example 10

Soluble Solids in T2 SSU-SPS Plants Investigation of the soluble solids in the f its of die SSU-SPS lines was initially done on extracts from fmit of 3812-9 and 3812-11 lines grown in a Biotron incubator. T2 plants were illuminated by metal halide lamps at a peak level of 500 μmol photons/m s (pot level), 26 C for the 16 h day and 18 C at night, and a relative humidity of 60% . Plants were watered daily with half-strength Hoagland's solution (Hoagland and A on, Calif. Agricult. Exp. Sta. G^'r.(1938) 557: 1-39). These lines were segregating as the original lines contained at least 2 insertions.

Brix analysis (soluble solids) on extracts from diese plants revealed lines widi Brix readings as much as 40% higher than tiie controls. The extracts measured were die average of 3 fmit from one plant.

Measurements were also taken for fruit from a segregating T2 population of 3812-11 plants in the greenhouse. The controls averaged a Brix reading of 3.5 while the transgenics averaged 4.0, an increase of 14%.

Example 1 1 Homozygous Plants T4 homozygous lines were generated from original 3812-9 transformants in UC82-B tomatoes. The original line segregated 15: 1 for Kan resistance, indicating that it had two insertion sites. Two homozygous lines were generated and verified to be different by Southern border analysis. These lines were designated A and B.

Individual homozygote (T4) lines were grown in the greenhouse, with three fmit taken from each plant and 3 plants analyzed from each line. The Brix of the UC82B controls was 3.35 while die Brix on the 3812-9 lines ranged from 3.7 to 4J. This is an increase from 12% to 24% . Statistics (LSD) on all the lines in which fruit from 3 plants were analyzed showed these results to be significant at a .01 % level (99%). Measurements were also made on homozygous lines of tomato plants transformed with die 35S CaMV promoter-SPS constmct pCGN3815. In one line, 3815-13-2 mere was a substantial increase in yield of tomatoes, in terms of an increase in both fruit size and in fmit number, as measured against non-transformed control plants and as against SSU-SPS homozygous line controls. The 3815-13-2 plants also produced a second flush of fmit. A second transgenic line containing the pCGN3815 constmct did not produce diese dramatic yield increases.

Example 12 Brix Analysis of Field Trial SSU-SPS Fmit Field trial results of RI measurements are provided in Table 15. The R/I (refractive index) was measured several times on the fruit of these plants (Table 15). R/I is a measure of soluble solids and is indicative of sugars and acids. The transgenic A and B lines consistently had a higher R/I than the control UC82-B.

ϊahl£j5

Summary of Refractive Index Measurements

Example 13 HPLC Analysis on SSU-SPS Fmit Sugars Fmit from the SPS plants described in Example 12 were further analyzed by HPLC to determine contributions of individual sugars to the increased soluble solids content. As seen in Table 16, sucrose did not increase as much as might be expected based on die fact d at sucrose is the sugar transported by die plant into the fmit. Glucose was not increased as much as fmctose. which increased nearly 50% .

It is evident from die above results, that plant cells and plants can be produced which have improved properties or may produce a desired phenotype. In accordance with die subject invention, it is now seen that SPS sequences may be introduced into a plant host cell and be used to express die enzyme to increase soluble solids content in fmit. Moreover, it is seen that the SPS may be used to alter the overall content and ratio of soluble solids in plant sink tissue, resulting in a demonstrable phenotype in planta, such as altered fruit sweetness. In this manner, fruits, such as tomato fruit, having modified sweetness may be obtained.

Example 14 Fmit Specific Expression of SPS E8-SPS constructs designated as pCGN3342 and pCGN3343 contain the tomato E8 promoter comprising the approximately 2J kb 5' region of the E8 promoter. A description of this promoter region can be found in Deikman et al, supra, and in Deikman et al (Plant Physiol (1992) 700:2013-2017).

This E8 promoter is fused to die same SPS encoding sequence used for pCGN3812 and pCGN3815, only die SPS sequence used in these constructs has been tmncated at die Apol site just 3' of die SPS encoding sequence (at nucleotide 3318), and fused to a 1.2 kb region of die tml 3' region from pTiA6 (Barker et a , (1983) Plant Mol Biol. 2:335-350; sequence 11207- 10069 of die T-DNA region from die Agrobacterium tumefaciens Ti plasmid pTil5955). Constructs pCGN3342 and pCGN3343 are the opposite orientations of diis E8-SPS-tm7 constmct in the 35S kan binary, pCGN1557 (McBride and Summerfelt, supra). Tomato lines arising from separate transformation events using pCGN3342 and pCGN3343 are signified by the constmct number followed by a hyphen and an event number.

Table 17 provides data from RI measurements of soluble solids in tomatoes from greenhouse smdies of TI plants. The RI was measured several times on d e fmit of diese plants.

Assays were made for die SPS activity in control and transgenic fmit from die 3343-6 and 3342-11 events. The control 7060 fmit was assayed with a SPS activity rate of 17.8 μmols sucrose/g/hr. This demonstrates that the increase sugar concentration of fmit in transgenic tomatoes over the control conelates to an increase of SPS activity in the fmit.

Tables 18 and 19 provide an analysis of individual sugars as measured by HPLC from two separate trials, to determine contributions of each sugar to the increased soluble solids content observed in transgenic E8-SPS fruit. The data of Table 18 and 19 demonstrate that increased SPS activity from transgenic expression in fruit by a fruit specific promoter can produce an overall net increase in sugars in the fmit. Due to the endogenous acid invertase found in ripening tomato fmit, increases in sugar are found in glucose and fmctose.

It also appears that there is a conelating increase in acid levels with an increase in sugar content in fruit transformed widi E8-SPS.

ϊable_Kt

Sugars of Tomatoes of Plants Transformed with SPS Gene

en

Xahlf LZ

σ. t

CJ t B R

3 to o to to ee

∞ι o ε

— H co , fr¬ O

E2 m C P No P P B B β efl

Oil 3 O

s

Iah J2

Sugars and Acids of SPS Tomato Lines

All publications and patent applications mentioned in die specification are indicative of the level of skill of those skilled in d e art to which diis invention pertains. All referenced publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now been fully described, it would be apparent to one of ordinary skill in die art that many changes and modifications can be made thereto without departing from die spirit or scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(1) APPLICANT: Van Assche, C. Lando, D. Bruneau, J. M. Voelker, T. Gervais, M.

(ll) TITLE OF INVENTION: MODIFICATION OF SUCROSE PHOSPHATE

SYNTHASE IN PLANTS

(ill) NUMBER OF SEQUENCES: 14

(lv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Law Offices of Barbara Rae-Venter

(B) STREET: 260 Sheridan Avenue, Suite 440

(C) CITY: Palo Alto

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94306

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: NOT YET ASSIGNED

(B) FILING DATE: NOT YET ASSIGNED

(C) CLASSIFICATION: NOT YET ASSIGNED

(vn) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/175,471

(B) FILING DATE: 27-DEC-1993

(vin) ATTORNEY/AGENT INFORMATION:

(A) NAME: Barbara Rae-Venter

(B) REGISTRATION NUMBER: 32,750

(C) REFERENCE/DOCKET NUMBER: CGNE.072.02US

(lx) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415)328-4400

(B) TELEFAX: (415)328-4477 (2) INFORMATION FOR SEQ ID NO:1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS :

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

Thr Trp lie Lys

1

(2) INFORMATION FOR SEQ ID NO:2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

Tyr Val Val Glu Leu Ala Arg 1 5

(2) INFORMATION FOR SEQ ID NO:3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3.

Ser Met Pro Pro lie Trp Ala Glu Val Met Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO: 4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

Leu Arg Pro Asp Gin Asp Tyr Leu Met His lie Ser His Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Trp Ser His Asp Gly Ala Arg

1 5 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3509 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 112..3315

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:6:

GAATTCCGGC GTGGGCGCTG GGCTAGTGCT CCCGCAGCGA GCGATCTGAG AGAACGGTAG 60

AGTTCCGGCC GGGCGCGCGG GAGAGGAGGA GGGTCGGGCG GGGAGGATCC G ATG GCC 117

Met Ala

1

GGG AAC GAG TGG ATC AAT GGG TAC CTG GAG GCG ATC CTC GAC AGC CAC 165 Gly Asn Glu Trp lie Asn Gly Tyr Leu Glu Ala lie Leu Asp Ser His 5 10 15

ACC TCG TCG CGG GGT GCC GGC GGC GGC GGC GGC GGG GGG GAC CCC AGG 213 Thr Ser Ser Arg Gly Ala Gly Gly Gly Gly Gly Gly Gly Asp Pro Arg 20 25 30

TCG CCG ACG AAG GCG GCG AGC CCC CGC GGC GCG CAC ATG AAC TTC AAC 261 Ser Pro Thr Lys Ala Ala Ser Pro Arg Gly Ala His Met Asn Phe Asn 35 40 45 50

CCC TCG CAC TAC TTC GTC GAG GAG GTG GTC AAG GGC GTC GAC GAG AGC 309 Pro Ser His Tyr Phe Val Glu Glu Val Val Lys Gly Val Asp Glu Ser 55 60 65

GAC CTC CAC CGG ACG TGG ATC AAG GTC GTC GCC ACC CGC AAC GCC CGC 357 Asp Leu His Arg Thr Trp lie Lys Val Val Ala Thr Arg Asn Ala Arg 70 75 80 GAG CGC AGC ACC AGG CTC GAG AAC ATG TGC TGG CGG ATC TGG CAC CTC 405 Glu Arg Ser Thr Arg Leu Glu Asn Met Cys Trp Arg lie Trp His Leu 85 90 95

GCG CGC AAG AAG AAG CAG CTG GAG CTG GAG GGC ATC CAG AGA ATC TCG 453 Ala Arg Lys Lys Lys Gin Leu Glu Leu Glu Gly lie Gin Arg lie Ser 100 105 110

GCA AGA AGG AAG GAA CAG GAG CAG GTG CGT CGT GAG GCG ACG GAG GAC 501 Ala Arg Arg Lys Glu Gin Glu Gin Val Arg Arg Glu Ala Thr Glu Asp 115 120 125 130

CTG GCC GAG GAT CTG TCA GAA GGC GAG AAG GGA GAC ACC ATC GGC GAG 549 Leu Ala Glu Asp Leu Ser Glu Gly Glu Lys Gly Asp Thr lie Gly Glu 135 140 145

CTT GCG CCG GTT GAG ACG ACC AAG AAG AAG TTC CAG AGG AAC TTC TCT 597 Leu Ala Pro Val Glu Thr Thr Lys Lys Lys Phe Gin Arg Asn Phe Ser 150 155 160

GAC CTT ACC GTC TGG TCT GAC GAC AAT AAG GAG AAG AAG CTT TAC ATT 645 Asp Leu Thr Val Trp Ser Asp Asp Asn Lys Glu Lys Lys Leu Tyr lie 165 170 175

GTG CTC ATC AGC GTG CAT GGT CTT GTT CGT GGA GAA AAC ATG GAA CTA 693 Val Leu lie Ser Val His Gly Leu Val Arg Gly Glu Asn Met Glu Leu 180 185 190

GGT CGT GAT TCT GAT ACA GGT GGC CAG GTG AAA TAT GTG GTC GAA CTT 741 Gly Arg Asp Ser Asp Thr Gly Gly Gin Val Lys Tyr Val Val Glu Leu 195 200 205 210

GCA AGA GCG ATG TCA ATG ATG CCT GGA GTG TAC AGG GTG GAC CTC TTC 789 Ala Arg Ala Met Ser Met Met Pro Gly Val Tyr Arg Val Asp Leu Phe 215 220 225

ACT CGT CAA GTG TCA TCT CCT GAC GTG GAC TGG AGC TAC GGT GAG CCA 837 Thr Arg Gin Val Ser Ser Pro Asp Val Asp Trp Ser Tyr Gly Glu Pro 230 235 240

ACC GAG ATG TTA TGC GCC GGT TCC AAT GAT GGA GAG GGG ATG GGT GAG 885 Thr Glu Met Leu Cys Ala Gly Ser Asn Asp Gly Glu Gly Met Gly Glu 245 250 255 AGT GGC GGA GCC TAC ATT GTG CGC ATA CCG TGT GGG CCG CGG GAT AAA 933 Ser Gly Gly Ala Tyr lie Val Arg lie Pro Cys Gly Pro Arg Asp Lys 260 265 270

TAC CTC AAG AAG GAA GCG TTG TGG CCT TAC CTC CAA GAG TTT GTC GAT 981 Tyr Leu Lys Lys Glu Ala Leu Trp Pro Tyr Leu Gin Glu Phe Val Asp 275 280 285 290

GGA GCC CTT GCG CAT ATC CTG AAC ATG TCC AAG GCT CTG GGA GAG CAG 1029 Gly Ala Leu Ala His lie Leu Asn Met Ser Lys Ala Leu Gly Glu Gin 295 300 305

GTT GGA AAT GGG AGG CCA GTA CTG CCT TAC GTG ATA CAT GGG CAC TAT 1077 Val Gly Asn Gly Arg Pro Val Leu Pro Tyr Val lie His Gly His Tyr 310 315 320

GCC GAT GCT GGA GAT GTT GCT GCT CTC CTT TCT GGT GCG CTG AAT GTG 1125 Ala Asp Ala Gly Asp Val Ala Ala Leu Leu Ser Gly Ala Leu Asn Val 325 330 335

CCA ATG GTG CTC ACT GGC CAC TCA CTT GGG AGG AAC AAG CTG GAA CAA 1173 Pro Met Val Leu Thr Gly His Ser Leu Gly Arg Asn Lys Leu Glu Gin 340 345 350

CTG CTG AAG CAA GGG CGC ATG TCC AAG GAG GAG ATC GAT TCG ACA TAC 1221 Leu Leu Lys Gin Gly Arg Met Ser Lys Glu Glu lie Asp Ser Thr Tyr 355 360 365 370

AAG ATC ATG AGG CGT ATC GAG GGT GAG GAG CTG GCC CTG GAT GCG TCA 1269 Lys lie Met Arg Arg lie Glu Gly Glu Glu Leu Ala Leu Asp Ala Ser 375 380 385

GAG CTT GTA ATC ACG AGC ACA AGG CAG GAG ATT GAT GAG CAG TGG GGA 1317 Glu Leu Val lie Thr Ser Thr Arg Gin Glu lie Asp Glu Gin Trp Gly 390 395 400

TTG TAC GAT GGA TTT GAT GTC AAG CTT GAG AAA GTG CTG AGG GCA CGG 1365 Leu Tyr Asp Gly Phe Asp Val Lys Leu Glu Lys Val Leu Arg Ala Arg 405 410 415

GCG AGG CGC GGG GTT AGC TGC CAT GGT CGT TAC ATG CCT AGG ATG GTG 1413 Ala Arg Arg Gly Val Ser Cys His Gly Arg Tyr Met Pro Arg Met Val 420 425 430 GTG ATT CCT CCG GGA ATG GAT TTC AGC AAT GTT GTA GTT CAT GAA GAC 1461 Val lie Pro Pro Gly Met Asp Phe Ser Asn Val Val Val His Glu Asp 435 440 445 450

ATT GAT GGG GAT GGT GAC GTC AAA GAT GAT ATC GTT GGT TTG GAG GGT 1509 lie Asp Gly Asp Gly Asp Val Lys Asp Asp lie Val Gly Leu Glu Gly 455 460 465

GCC TCA CCC AAG TCA ATG CCC CCA ATT TGG GCC GAA GTG ATG CGG TTC 1557 Ala Ser Pro Lys Ser Met Pro Pro lie Trp Ala Glu Val Met Arg Phe 470 475 480

CTG ACC AAC CCT CAC AAG CCG ATG ATC CTG GCG TTA TCA AGA CCA GAC 1605 Leu Thr Asn Pro His Lys Pro Met lie Leu Ala Leu Ser Arg Pro Asp 485 490 495

CCG AAG AAG AAC ATC ACT ACC CTC GTC AAA GCC TTT GGA GAG TGT CGT 1653 Pro Lys Lys Asn lie Thr Thr Leu Val Lys Ala Phe Gly Glu Cys Arg 500 505 510

CCA CTC AGG GAA CTT GCA AAC CTT ACT CTG ATC ATG GGT AAC AGA GAT 1701 Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu lie Met Gly Asn Arg Asp 515 520 525 530

GAC ATC GAC GAC ATG TCT GCT GGC AAT GCC AGT GTC CTC ACC ACA GTT 1749 Asp lie Asp Asp Met Ser Ala Gly Asn Ala Ser Val Leu Thr Thr Val 535 540 545

CTG AAG CTG ATT GAC AAG TAT GAT CTG TAC GGA AGC GTG GCG TTC CCT 1797 Leu Lys Leu lie Asp Lys Tyr Asp Leu Tyr Gly Ser Val Ala Phe Pro 550 555 560

AAG CAT CAC AAT CAG GCT GAC GTC CCG GAG ATC TAT CGC CTC GCG GCC 1845 Lys His His Asn Gin Ala Asp Val Pro Glu lie Tyr Arg Leu Ala Ala 565 570 575

AAA ATG AAG GGC GTC TTC ATC AAC CCT GCT CTC GTT GAG CCG TTT GGT 1893 Lys Met Lys Gly Val Phe lie Asn Pro Ala Leu Val Glu Pro Phe Gly 580 585 590

CTC ACC CTG ATC GAG GCT GCG GCA CAC GGA CTC CCG ATA GTC GCT ACC 1941 Leu Thr Leu lie Glu Ala Ala Ala His Gly Leu Pro He Val Ala Thr 595 600 605 610 AAG AAT GGT GGT CCG GTC GAC ATT ACA AAT GCA TTA AAC AAC GGA CTG 1989 Lys Asn Gly Gly Pro Val Asp He Thr Asn Ala Leu Asn Asn Gly Leu 615 620 625

CTC GTT GAC CCA CAC GAC CAG AAC GCC ATC GCT GAT GCA CTG CTG AAG 2037 Leu Val Asp Pro His Asp Gin Asn Ala He Ala Asp Ala Leu Leu Lys 630 635 640

CTT GTG GCA GAC AAG AAC CTG TGG CAG GAA TGC CGG AGA AAC GGG CTG 2085 Leu Val Ala Asp Lys Asn Leu Trp Gin Glu Cys Arg Arg Asn Gly Leu 645 650 655

CGC AAC ATC CAC CTC TAC TCA TGG CCG GAG CAC TGC CGC ACT TAC CTC 2133 Arg Asn He His Leu Tyr Ser Trp Pro Glu His Cys Arg Thr Tyr Leu 660 665 670

ACC AGG GTG GCC GGG TGC CGG TTA AGG AAC CCG AGG TGG CTG AAG GAC 2181 Thr Arg Val Ala Gly Cys Arg Leu Arg Asn Pro Arg Trp Leu Lys Asp 675 680 685 690

ACA CCA GCA GAT GCC GGA GCC GAT GAG GAG GAG TTC CTG GAG GAT TCC 2229 Thr Pro Ala Asp Ala Gly Ala Asp Glu Glu Glu Phe Leu Glu Asp Ser 695 700 705

ATG GAC GCT CAG GAC CTG TCA CTC CGT CTG TCC ATC GAC GGT GAG AAG 2277 Met Asp Ala Gin Asp Leu Ser Leu Arg Leu Ser He Asp Gly Glu Lys 710 715 720

AGC TCG CTG AAC ACT AAC GAT CCA CTG TGG TTC GAC CCC CAG GAT CAA 2325 Ser Ser Leu Asn Thr Asn Asp Pro Leu Trp Phe Asp Pro Gin Asp Gin 725 730 735

GTG CAG AAG ATC ATG AAC AAC ATC AAG CAG TCG TCA GCG CTT CCT CCG 2373 Val Gin Lys He Met Asn Asn He Lys Gin Ser Ser Ala Leu Pro Pro 740 745 750

TCC ATG TCC TCA GTC GCA GCC GAG GGC ACA GGC AGC ACC ATG AAC AAA 2421 Ser Met Ser Ser Val Ala Ala Glu Gly Thr Gly Ser Thr Met Asn Lys 755 760 765 770

TAC CCA CTC CTG CGC CGG CGC CGG CGC TTG TTC GTC ATA GCT GTG GAC 2469 Tyr Pro Leu Leu Arg Arg Arg Arg Arg Leu Phe Val He Ala Val Asp 775 780 785 TGC TAC CAG GAC GAT GGC CGT GCT AGC AAG AAG ATG CTG CAG GTG ATC 2517 Cys Tyr Gin Asp Asp Gly Arg Ala Ser Lys Lys Met Leu Gin Val He 790 795 800

CAG GAA GTT TTC AGA GCA GTC CGA TCG GAC TCC CAG ATG TTC AAG ATC 2565 Gin Glu Val Phe Arg Ala Val Arg Ser Asp Ser Gin Met Phe Lys He 805 810 815

TCA GGG TTC ACG CTG TCG ACT GCC ATG CCG TTG TCC GAG ACA CTC CAG 2613 Ser Gly Phe Thr Leu Ser Thr Ala Met Pro Leu Ser Glu Thr Leu Gin 820 825 830

CTT CTG CAG CTC GGC AAG ATC CCA GCG ACC GAC TTC GAC GCC CTC ATC 2661 Leu Leu Gin Leu Gly Lys He Pro Ala Thr Asp Phe Asp Ala Leu He 835 840 845 850

TGT GGC AGC GGC AGC GAG GTG TAC TAT CCT GGC ACG GCG AAC TGC ATG 2709 Cys Gly Ser Gly Ser Glu Val Tyr Tyr Pro Gly Thr Ala Asn Cys Met 855 860 865

GAC GCT GAA GGA AAG CTG CGC CCA GAT CAG GAC TAT CTG ATG CAC ATC 2757 Asp Ala Glu Gly Lys Leu Arg Pro Asp Gin Asp Tyr Leu Met His He 870 875 880

AGC CAC CGC TGG TCC CAT GAC GGC GCG AGG CAG ACC ATA GCG AAG CTC 2805 Ser His Arg Trp Ser His Asp Gly Ala Arg Gin Thr He Ala Lys Leu 885 890 895

ATG GGC GCT CAG GAC GGT TCA GGC GAC GCT GTC GAG CAG GAC GTG GCG 2853 Met Gly Ala Gin Asp Gly Ser Gly Asp Ala Val Glu Gin Asp Val Ala 900 905 910

TCC AGT AAT GCA CAC TGT GTC GCG TTC CTC ATC AAA GAC CCC CAA AAG 2901 Ser Ser Asn Ala His Cys Val Ala Phe Leu He Lys Asp Pro Gin Lys 915 920 925 930

GTG AAA ACG GTC GAT GAG ATG AGG GAG CGG CTG AGG ATG CGT GGT CTC 2949 Val Lys Thr Val Asp Glu Met Arg Glu Arg Leu Arg Met Arg Gly Leu 935 940 945

CGC TGC CAC ATC ATG TAC TGC AGG AAC TCG ACA AGG CTT CAG GTT GTC 2997 Arg Cys His He Met Tyr Cys Arg Asn Ser Thr Arg Leu Gin Val Val 950 955 960 CCT CTG CTA GCA TCA AGG TCA CAG GCA CTC AGG TAT CTT TCC GTG CGC 3045 Pro Leu Leu Ala Ser Arg Ser Gin Ala Leu Arg Tyr Leu Ser Val Arg 965 970 975

TGG GGC GTA TCT GTG GGG AAC ATG TAT CTG ATC ACC GGG GAA CAT GGC 3093 Trp Gly Val Ser Val Gly Asn Met Tyr Leu He Thr Gly Glu His Gly 980 985 990

GAC ACC GAT CTA GAG GAG ATG CTA TCC GGG CTA CAC AAG ACC GTG ATC 3141 Asp Thr Asp Leu Glu Glu Met Leu Ser Gly Leu His Lys Thr Val He 995 1000 1005 1010

GTC CGT GGC GTC ACC GAG AAG GGT TCG GAA GCA CTG GTG AGG AGC CCA 3189 Val Arg Gly Val Thr Glu Lys Gly Ser Glu Ala Leu Val Arg Ser Pro 1015 1020 1025

GGA AGC TAC AAG AGG GAC GAT GTC GTC CCG TCT GAG ACC CCC TTG GCT 3237 Gly Ser Tyr Lys Arg Asp Asp Val Val Pro Ser Glu Thr Pro Leu Ala 1030 1035 1040

GCG TAC ACG ACT GGT GAG CTG AAG GCC GAC GAG ATC ATG CGG GCT CTG 3285 Ala Tyr Thr Thr Gly Glu Leu Lys Ala Asp Glu He Met Arg Ala Leu 1045 1050 1055

AAG CAA GTC TCC AAG ACT TCC AGC GGC ATG TGAATTTGAT GCTTCTTTTA 3335 Lys Gin Val Ser Lys Thr Ser Ser Gly Met 1060 1065

CATTTTGTCC TTTTCTTCAC TGCTATATAA AATAAGTTGT GAACAGTACC GCGGGTGTGT 3395

ATATATATAT TGCAGTGACA AATAAAACAG GACACTGCTA ACTATACTGG TGAATATACG 3455

ACTGTCAAGA TTGTATGCTA AGTACTCCAT TTCTCAATGT ATCAATCGGA ATTC 3509

(2) INFORMATION FOR SEQ ID NO:7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1068 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Ala Gly Asn Glu Trp He Asn Gly Tyr Leu Glu Ala He Leu Asp 1 5 10 15

Ser His Thr Ser Ser Arg Gly Ala Gly Gly Gly Gly Gly Gly Gly Asp 20 25 30

Pro Arg Ser Pro Thr Lys Ala Ala Ser Pro Arg Gly Ala His Met Asn 35 40 45

Phe Asn Pro Ser His Tyr Phe Val Glu Glu Val Val Lys Gly Val Asp 50 55 60

Glu Ser Asp Leu His Arg Thr Trp He Lys Val Val Ala Thr Arg Asn 65 70 75 80

Ala Arg Glu Arg Ser Thr Arg Leu Glu Asn Met Cys Trp Arg He Trp 85 90 95

His Leu Ala Arg Lys Lys Lys Gin Leu Glu Leu Glu Gly He Gin Arg 100 105 110

He Ser Ala Arg Arg Lys Glu Gin Glu Gin Val Arg Arg Glu Ala Thr 115 120 125

Glu Asp Leu Ala Glu Asp Leu Ser Glu Gly Glu Lys Gly Asp Thr He 130 135 140

Gly Glu Leu Ala Pro Val Glu Thr Thr Lys Lys Lys Phe Gin Arg Asn 145 150 155 160

Phe Ser Asp Leu Thr Val Trp Ser Asp Asp Asn Lys Glu Lys Lys Leu 165 170 175

Tyr He Val Leu He Ser Val His Gly Leu Val Arg Gly Glu Asn Met 180 185 190

Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly Gin Val Lys Tyr Val Val 195 200 205

Glu Leu Ala Arg Ala Met Ser Met Met Pro Gly Val Tyr Arg Val Asp 210 215 220 Leu Phe Thr Arg Gin Val Ser Ser Pro Asp Val Asp Trp Ser Tyr Gly 225 230 235 240

Glu Pro Thr Glu Met Leu Cys Ala Gly Ser Asn Asp Gly Glu Gly Met 245 250 255

Gly Glu Ser Gly Gly Ala Tyr He Val Arg He Pro Cys Gly Pro Arg 260 265 270

Asp Lys Tyr Leu Lys Lys Glu Ala Leu Trp Pro Tyr Leu Gin Glu Phe 275 280 285

Val Asp Gly Ala Leu Ala His He Leu Asn Met Ser Lys Ala Leu Gly 290 295 300

Glu Gin Val Gly Asn Gly Arg Pro Val Leu Pro Tyr Val He His Gly 305 310 315 320

His Tyr Ala Asp Ala Gly Asp Val Ala Ala Leu Leu Ser Gly Ala Leu 325 330 335

Asn Val Pro Met Val Leu Thr Gly His Ser Leu Gly Arg Asn Lys Leu 340 345 350

Glu Gin Leu Leu Lys Gin Gly Arg Met Ser Lys Glu Glu He Asp Ser 355 360 365

Thr Tyr Lys He Met Arg Arg He Glu Gly Glu Glu Leu Ala Leu Asp 370 375 380

Ala Ser Glu Leu Val He Thr Ser Thr Arg Gin Glu He Asp Glu Gin 385 390 395 400

Trp Gly Leu Tyr Asp Gly Phe Asp Val Lys Leu Glu Lys Val Leu Arg 405 410 415

Ala Arg Ala Arg Arg Gly Val Ser Cys His Gly Arg Tyr Met Pro Arg 420 425 430

Met Val Val He Pro Pro Gly Met Asp Phe Ser Asn Val Val Val His 435 440 445

Glu Asp He Asp Gly Asp Gly Asp Val Lys Asp Asp He Val Gly Leu 450 455 460 Glu Gly Ala Ser Pro Lys Ser Met Pro Pro He Trp Ala Glu Val Met 465 470 475 480

Arg Phe Leu Thr Asn Pro His Lys Pro Met He Leu Ala Leu Ser Arg 485 490 495

Pro Asp Pro Lys Lys Asn He Thr Thr Leu Val Lys Ala Phe Gly Glu 500 505 510

Cys Arg Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu He Met Gly Asn 515 520 525

Arg Asp Asp He Asp Asp Met Ser Ala Gly Asn Ala Ser Val Leu Thr 530 535 540

Thr Val Leu Lys Leu He Asp Lys Tyr Asp Leu Tyr Gly Ser Val Ala 545 550 555 560

Phe Pro Lys His His Asn Gin Ala Asp Val Pro Glu He Tyr Arg Leu 565 570 575

Ala Ala Lys Met Lys Gly Val Phe He Asn Pro Ala Leu Val Glu Pro 580 585 590

Phe Gly Leu Thr Leu He Glu Ala Ala Ala His Gly Leu Pro He Val 595 600 605

Ala Thr Lys Asn Gly Gly Pro Val Asp He Thr Asn Ala Leu Asn Asn 610 615 620

Gly Leu Leu Val Asp Pro His Asp Gin Asn Ala He Ala Asp Ala Leu 625 630 635 640

Leu Lys Leu Val Ala Asp Lys Asn Leu Trp Gin Glu Cys Arg Arg Asn 645 650 655

Gly Leu Arg Asn He His Leu Tyr Ser Trp Pro Glu His Cys Arg Thr 660 665 670

Tyr Leu Thr Arg Val Ala Gly Cys Arg Leu Arg Asn Pro Arg Trp Leu 675 680 685

Lys Asp Thr Pro Ala Asp Ala Gly Ala Asp Glu Glu Glu Phe Leu Glu 690 695 700 Asp Ser Met Asp Ala Gin Asp Leu Ser Leu Arg Leu Ser He Asp Gly 705 710 715 720

Glu Lys Ser Ser Leu Asn Thr Asn Asp Pro Leu Trp Phe Asp Pro Gin 725 730 735

Asp Gin Val Gin Lys He Met Asn Asn He Lys Gin Ser Ser Ala Leu 740 745 750

Pro Pro Ser Met Ser Ser Val Ala Ala Glu Gly Thr Gly Ser Thr Met 755 760 765

Asn Lys Tyr Pro Leu Leu Arg Arg Arg Arg Arg Leu Phe Val He Ala 770 775 780

Val Asp Cys Tyr Gin Asp Asp Gly Arg Ala Ser Lys Lys Met Leu Gin 785 790 795 800

Val He Gin Glu Val Phe Arg Ala Val Arg Ser Asp Ser Gin Met Phe 805 810 815

Lys He Ser Gly Phe Thr Leu Ser Thr Ala Met Pro Leu Ser Glu Thr 820 825 830

Leu Gin Leu Leu Gin Leu Gly Lys He Pro Ala Thr Asp Phe Asp Ala 835 840 845

Leu He Cys Gly Ser Gly Ser Glu Val Tyr Tyr Pro Gly Thr Ala Asn 850 855 860

Cys Met Asp Ala Glu Gly Lys Leu Arg Pro Asp Gin Asp Tyr Leu Met 865 870 875 880

His He Ser His Arg Trp Ser His Asp Gly Ala Arg Gin Thr He Ala 885 890 895

Lys Leu Met Gly Ala Gin Asp Gly Ser Gly Asp Ala Val Glu Gin Asp 900 905 910

Val Ala Ser Ser Asn Ala His Cys Val Ala Phe Leu He Lys Asp Pro 915 920 925

Gin Lys Val Lys Thr Val Asp Glu Met Arg Glu Arg Leu Arg Met Arg 930 935 940 Gly Leu Arg Cys His He Met Tyr Cys Arg Asn Ser Thr Arg Leu Gin 945 950 955 960

Val Val Pro Leu Leu Ala Ser Arg Ser Gin Ala Leu Arg Tyr Leu Ser 965 970 975

Val Arg Trp Gly Val Ser Val Gly Asn Met Tyr Leu He Thr Gly Glu 980 985 990

His Gly Asp Thr Asp Leu Glu Glu Met Leu Ser Gly Leu His Lys Thr 995 1000 1005

Val He Val Arg Gly Val Thr Glu Lys Gly Ser Glu Ala Leu Val Arg 1010 1015 1020

Ser Pro Gly Ser Tyr Lys Arg Asp Asp Val Val Pro Ser Glu Thr Pro 1025 1030 1035 1040

Leu Ala Ala Tyr Thr Thr Gly Glu Leu Lys Ala Asp Glu He Met Arg 1045 1050 1055

Ala Leu Lys Gin Val Ser Lys Thr Ser Ser Gly Met 1060 1065

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Possible peptide encoding sequences" (iii) HYPOTHETICAL: YES (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 :

WSNATGCCNC CNATHTGGGC NGARGTNATG MGN 33 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Possible peptide encoding sequences"

(iii) HYPOTHETICAL: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

YTNMGNCCNG AYCARGAYTA YYTNATGCAY ATHWSNCAYM GN 42

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic oligonucleotide mixture"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ATGCCNCCNA THTGGGCNGA 20

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic oligonucleotide mixture"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

TGCATNAGRT ARTCYTGRTC 20

(2) INFORMATION FOR SEQ ID NO:12:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic oligonucleotide mixture"

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:12:

TCNGCCCADA TNGGNGGCAT 20

(2) INFORMATION FOR SEQ ID NO:13:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic oligonucleotide mixture" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

GAYCARGAYT AYCTNATGCA 20

(2) INFORMATION FOR SEQ ID NO:14 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Synthetic oligonucleotide mixture"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

TGRTCNGGNC KNAR 14

Claims

What is claimed is:

1. A method of modifying sweetness of a plant part, said method comprising: growing a plant having integrated into its genome at least one copy of an exogenous

DNA sequence encoding a polypeptide having sucrose phosphate synthase activity under conditions whereby said DNA sequence is expressed and sweetness of said plant part is modified.

2. The method of Claim 1, wherein said plant part has an increased level of sucrose as compared to that of a control plant part.

3. The method of Claim 1, wherein said plant part has an increased level of fructose as compared to that of a control plant part.

4. The method of Claim 1, wherein said plant part has an increased level of glucose as compared to that of a control plant part.

5. The method of Claim 1, wherein expression of said DNA sequence is controlled by a tissue specific transcription initiation region.

6. The method of Claim 5, wherein said tissue specific transcription initiation region comprises a fruit specific promoter.

7. The method of Claim 1, wherein said plant is of the family Solenciae.

8. A method of modifying the ratio of soluble solids in a plant sink tissue, said method comprising:

growing a transgenic plant having acid invertase in cells of said sink tissue, said transgenic plant having integrated into its genome at least one copy of an exogeneous DNA sequence encoding a polypeptide having sucrose phosphate synthase activity under conditions whereby said DNA sequence is expressed and the ratio of soluble solids of said plant sink tissue is modified as compared to a control plant sink tissue.

9. The method according to Claim 8, wherein said invertase is endogenous to said cells.

10. A plant sink tissue comprising:

a modified ratio of soluble solids as compared to a control sink tissue, wherein said ratio is modified according to the method of Claim 8.