EP0858513A2 - Modification de solides solubles a l'aide d'une sequence codant la phosphate synthase de saccharose - Google Patents

Modification de solides solubles a l'aide d'une sequence codant la phosphate synthase de saccharose

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Publication number
EP0858513A2
EP0858513A2 EP96940757A EP96940757A EP0858513A2 EP 0858513 A2 EP0858513 A2 EP 0858513A2 EP 96940757 A EP96940757 A EP 96940757A EP 96940757 A EP96940757 A EP 96940757A EP 0858513 A2 EP0858513 A2 EP 0858513A2
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European Patent Office
Prior art keywords
sps
die
plant
leu
gly
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EP96940757A
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German (de)
English (en)
Inventor
Christine K. Shewmaker
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Monsanto Co
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Calgene LLC
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Priority claimed from US08/549,016 external-priority patent/US5914446A/en
Application filed by Calgene LLC filed Critical Calgene LLC
Publication of EP0858513A2 publication Critical patent/EP0858513A2/fr
Withdrawn legal-status Critical Current

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Definitions

  • 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.
  • 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 sucrose phosphate synthase
  • 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 grow ⁇ jg tissue, therefore the study of SPS and its activity is of special interest.
  • Walker and Huber Plant Phys. (1989) 59:518-524, the purification and preliminary characterization of spinach (Spinachia oleracea) SPS was reported.
  • 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).
  • additional purified sources of SPS enzyme are needed for effective characterization of this factor.
  • 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).
  • sucrose phosphate synthase (SPS) activity and/or invertase activity in plant tissues are manipulated
  • nucleic acid constructs, vectors, plant cells, plant pans and plants containing at least one exogenously supplied copy of an SPS gene are provided.
  • 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.
  • 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.
  • 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.
  • FIG. 2 shows the results of a Western analysis of SPS using monoclonal antibodies.
  • 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.
  • 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.
  • Figure 4 shows oligonucleotides used for the PCR reactions CD3 (SEQ ID NOS:
  • 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).
  • SPS 90, SPS 61 and SPS 3 were fused at the points indicated in Fig. 2.
  • 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
  • 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 ;
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • preferred promoter regions are tiiose which provide for leaf, fruit and/or root specific expression of SPS.
  • 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.
  • sucrose synthesis is a key metabolic product that affects the interface between end-product synthesis and carbon partitioning for most plant systems.
  • 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.
  • 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.
  • 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.
  • altering SPS activity provides a means for altering plant growth and yield of specific plant cells, plant tissues, plant parts and plants.
  • 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.
  • endogenous gene expression and/or enzyme activity 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.
  • 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.
  • protein 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • PEG polyethyleneglycol
  • 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
  • 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.
  • 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.
  • 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 ⁇ mole (micromole) of sucrose formed per minute per mg of protein at 37°C.
  • 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.
  • 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.
  • mice 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.
  • 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.
  • cDNA comprising a nucleotide sequence represented in Figure 7 (SEQ ID NO: 6).
  • 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.
  • the DNA sequence is cDNA.
  • the DNA sequence is obtainable from com or is derived from the corn DNA sequence.
  • 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.
  • 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.
  • cDNA library construction (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 1.5xl0 6 plaques.
  • 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' .
  • a SPS 61 clone extending further 5' widiout having the 5' end of die reading frame is obtained (Fig. 6).
  • 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).
  • plants of the nightshade family Solanaceae including the genetically similar but physiologically disparate plants potato (Solanium tuberosum) and tomato (Hycopersicon esculentum).
  • the sequence When provided in a DNA construct for integration into a plant genome, the sequence can encode a sense strand or an anti-sense strand.
  • 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.
  • 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.
  • fruit having a modified sweetness phenotype is a modified sweetness phenotype.
  • sucrose 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.
  • glucose and fructose are produced from sucrose by a vacuolar acid invertase that is active during fruit ripening.
  • 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.
  • fruit of die plant family Solanaceae 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.
  • 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.
  • decreasing sucrose 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • expression constructs include endogenous plant transcription termination regions which will be located 3 ' to me desired sucrose metabolizing enzyme encoding sequence.
  • 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.
  • enzymes which may prove useful in modifying soluble solids ratios is the acid invertase enzyme.
  • 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.
  • a convenient cloning vector e.g. a plasmid
  • 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.
  • the construct 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.
  • 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.
  • the constructs of this invention may be further manipulated to include plant selectable markers.
  • 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.
  • 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.
  • 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).
  • PCR polymerase chain reaction
  • the gene sequences may be synmesized, either completely or in pan, especially where it is desirable to provide plant-prefened sequences.
  • 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.
  • 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.
  • 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).
  • Nos nopaline syndiase
  • Mos mannopine syndiase
  • Ocs octopine syndiase
  • 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.
  • 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.
  • 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.
  • the prefened SPSs are those obtainable from co .
  • 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.
  • nucleic acid probes DNA and RNA
  • die like can be prepared and used to screen odier plant sources for SPS and recover it.
  • 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.
  • 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.
  • 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.
  • probes are typically labeled in a detectable manner (for example with 32 P- 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.
  • 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.
  • d e SPS cDNA 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.
  • 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.
  • 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.
  • SSU tobacco ribulose biphosphate carboxylase small subunit
  • CaMV die cauliflower mosaic virus
  • E8 die tomato fruit-specific E8 transcriptional initiation region
  • those associated widi T-DNA such as the opine synthase transcriptional initiation region, e.g., o
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • E. coli 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.
  • 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.
  • terminal repeats of transposons may be used as borders in conjunction widi a transposase.
  • 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.
  • 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.
  • 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.
  • the expression cassette may be transferred to d e A. tumefaciens and die resulting transformed organism used for transforming plant cells.
  • 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.
  • the cell tissue for example protoplasts, explants or cotyledons
  • a regeneration medium such as Murashige-Skoog (MS) medium for plant tissue and cell culture, for formation of a callus.
  • MS Murashige-Skoog
  • 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.
  • transgenic plants which contain and express a given SPS transgene are compared to control plants.
  • 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.
  • 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.
  • plant part morphology including 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.
  • 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.
  • 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.
  • SPS 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.
  • Fructose 6-P or F6P Fructose 6-Phosphate
  • Sucrose 6-P Sucrose 6-Phosphate
  • Optical Density (O.DJ 520 nm).
  • 25 ⁇ l (microliter) of enzymatic preparation 25 ⁇ l 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
  • NAD formed or 1 mole of NADH consumed corresponds to 1 mole of sucrose 6 P formed.
  • HEPES buffered 50 mM, MgCl 2 10 mM, KC1 20 mM pH 7.5, - 250 ⁇ l of a mixture of substrates PEP (1.6 mM NADH 0.6 mM, ATP 4 mM UDPG 112 mM),
  • 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.
  • the SPS activity is eluted at about 0J7 M KC1.
  • the proteins adsorbed are eluted in an isocratic way by means of a 10 mM CAPS, 10 mM MgCl 2 , 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
  • the heparin fraction is diluted by adding one diird volume of 20 mM Triethanolamine, 10 mM MgCl 2 , 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).
  • 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.
  • 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").
  • the Mono Q fraction is loaded on an HPLC column of hydroxyapatite 4 mm x 75 mm neutralized with 20 mM KH 2 PO 4 /K 2 HPO 4 , 3% EG, 0.3% Tween 80, 5 mM DTT, pH
  • buffer A see above
  • buffer B the same as buffer A additionally containing but 500 mM Phosphate of K
  • the flow rate applied to die column is 60 ml/h.
  • 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.
  • Triethanolamine 10 mM MgCl 2 , 1 mM EDTA, 3% EG, 2.5 mM DTT, 2% betaine, pH
  • the flow rate applied to the column is 60 ml/h.
  • the SPS activity is eluted with about 0.3M NaCl.
  • 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.
  • 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.
  • FIG. 1 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.
  • 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.
  • 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.
  • 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).
  • FCA Mono Q pool emulsified volume for volume widi Freund's Complete Adjuvant
  • 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).
  • 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.
  • 70 ⁇ l of serum or of supernatant of hybridoma culture diluted in a suitable way was mixed widi 70 ⁇ l 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.
  • This method is based on die precipitation of die antibody-SPS complex by goat anti-mouse IgG coupled to sepharose beads (GAM sepharose).
  • GAM sepharose goat anti-mouse IgG coupled to sepharose beads
  • 60 ⁇ l of serum or supernatant of hybridoma culture diluted in any suitable manner were added to 60 ⁇ l of SPS preparation (Heparin fraction). After 2 hours of incubation at ambient temperature, the mixture was added to 50 ⁇ l of 25% GAM-Sepharose previously washed diree times with a buffer of 50 mM HEPES, 10 mM MgCl 2 , 1 mM EDTA, 10% EG, 5 mM DTT, pH 7.5. The mixture was incubated overnight at 4°C wid strong agitation.
  • 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.
  • 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. ⁇ ahk ⁇
  • mice 1 and 4 The spleens of mice 1 and 4 were used for the fusion with myeloma cells.
  • mice 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.
  • 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).
  • 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 H 2 0 2 /ABTS system. All the anti-SPS monoclonal antibodies were found to be of IgG, type, b) Inhibition of SPS activity
  • 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 125 I diluted in a washing buffer (50,000 cpm per cm 2 of membrane).
  • a washing buffer 0.1 % Casein, 0.5% Tween 20, in PBS
  • 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.
  • 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.
  • 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).
  • 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 .
  • 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.
  • Total Corn Leaf Library cDNA synthesis was performed according to die manufacturer's instructions (RiboClone cDNA Synthesis System by Promega, Madison, WI), using five ⁇ g 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 10 6 transformants.
  • 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.
  • 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.
  • the 1200 bp PCR-generated fragment was labeled with 32 P (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 32 P by random priming (Random Primed DNA Labeling Kit) and used as a probe to re-screen the library.
  • the library was screened widi die 32 P-labeled EcoRI insert from SPS#61 , and 16 positive clones were obtained.
  • 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.
  • 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 B ⁇ mHI/H. ⁇ dlll 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).
  • 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.
  • PEPcase phosphoenolpyruvate carboxylase
  • 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.
  • 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 It ⁇ mHI 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.
  • 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 £ ⁇ >RJ digestion, the 3403 bp BamHl (filled in) EcoRI SPS cDNA fragment (see, Example 7.1.) was inserted.
  • 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.
  • 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 Ec ⁇ RI.
  • the Ec ⁇ RI/blunted-Hmdi ⁇ fragment containing the SSU promoter region was ligated widi Smal/Ec ⁇ RI-digested pUC18 to yield pCGN627.
  • 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 Ec ⁇ R 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-Ec ⁇ 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.
  • 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.
  • 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/Hp ⁇ I and me resulting EcoRI-Hphl fragment containing the 35S promoter was ligated into EcoRl-Smal digested M13mp8 (Vieira and Messing, supra) to create pCGN164.
  • 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.
  • 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 pCGN971 ⁇ , 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
  • pCGN526 was made by inserting die BamHl fragment 19 of pTiA6 (Thomashow et al.
  • pCGN528 was obtained by deleting die small Xh ⁇ l and religating.
  • pCGN149a was made by cloning the ftamHI kanamycin gene fragment from pMB9KanXXI into the BamHl site of ⁇ CGN148a.
  • pMB9KanXXI is a ⁇ UC4K variant (Vieira and Messing, Gene (1982) 79:259-268) which has die Xh ⁇ l 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 di ⁇ 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 fl ⁇ mHI-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.
  • 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).
  • Figures 10A through IOC A schematic summary of the construction of the various plasmids is shown in Figures 10A through IOC.
  • 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.
  • SPS encoding sequences pCGN3812, pCGN3815, pCGN3342, and pCGN3343
  • Agrobacterium tumefaciens mediated transformation Feratti, et al , Bio/Technology (1987) 5:726-730
  • 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 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 Ap ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ mol 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% .
  • 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.
  • LSD least significant difference
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ mols sucrose/gram fresh weight/hour.
  • Activity was much higher in the transgenic lines, with die 3343-6 event having a rate of 67.5 ⁇ mols sucrose/grown fresh weight/hour and die 3342-11 event measured at 36.6 ⁇ mols sucrose/gram fresh weight/hour.
  • 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.
  • Potato is adapted to cool weadier and has a large vegetative sink, whereas the genetically similar tomato has a large reproductive sink.
  • oxygen sensitivity was examined in potatoes expressing the com SPS gene.
  • Potatoes expressing the co SPS exhibited a higher capacity for photosynthesis in elevated CO 2 when die plants were three weeks old compared to controls.
  • die potato com SPS expressing plants were six to seven weeks old wid developing tubers, they showed die acclimation to elevated CO 2 found in many plants and the controls (Fig. 12).
  • 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.
  • 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.
  • 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, CO 2 and/or O 2 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.
  • sucrose synthesis and conversion to other sugars in sink tissue such as glucose and fructose
  • sink tissue such as glucose and fructose
  • 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 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.
  • T4 lines 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.
  • 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 reffractive index
  • the transgenic A and B lines consistently had a higher R/I than the control UC82-B.
  • 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.
  • 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.
  • 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
  • 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
  • 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
  • MOLECULE TYPE protein

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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

Des procédés permettent d'utiliser des séquences codant la phosphate synthase de saccharose pour modifier des solides solubles dans des tissus végétaux de destination. On décrit aussi des plantes, tissus et parties de végétaux qui ont été modifiés. Ces procédés permettent par exemple de modifier l'édulcoration de parties de végétaux telles que fruits et tubercules.
EP96940757A 1995-10-27 1996-10-25 Modification de solides solubles a l'aide d'une sequence codant la phosphate synthase de saccharose Withdrawn EP0858513A2 (fr)

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PCT/US1996/017351 WO1997015678A2 (fr) 1995-01-15 1996-10-25 Modification de solides solubles a l'aide d'une sequence codant la phosphate synthase de saccharose

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AU662111B2 (en) * 1991-03-18 1995-08-24 Roussel-Uclaf Sucrose phosphate synthase (SPS), its process for preparation, its cDNA, and utilization of cDNA to modify the expression of SPS in the plant cells
GB9117159D0 (en) * 1991-08-08 1991-09-25 Cambridge Advanced Tech Modification of sucrose accumulation
WO1994028146A2 (fr) * 1993-05-24 1994-12-08 Hoechst Schering Agrevo Gmbh Sequences d'adn et plasmides destines a la production d'une betterave a concentration de sucre modifiee

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