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WO1999007841A2 - Regulatory mutants of adp-glucose pyrophosphorylase and related compositions and methods - Google Patents

Regulatory mutants of adp-glucose pyrophosphorylase and related compositions and methods Download PDF

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WO1999007841A2
WO1999007841A2 PCT/US1998/016551 US9816551W WO9907841A2 WO 1999007841 A2 WO1999007841 A2 WO 1999007841A2 US 9816551 W US9816551 W US 9816551W WO 9907841 A2 WO9907841 A2 WO 9907841A2
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nucleic acid
adpg
enzyme
mutant
wild
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PCT/US1998/016551
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French (fr)
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WO1999007841A3 (en
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Thomas Okita
Thomas W. Greene
Mary Laughlin
Michael Kahn
Hiroyuki Ito
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Washington State University Research Foundation
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Publication of WO1999007841A3 publication Critical patent/WO1999007841A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates to starch biosynthesis in plants, particularly to regulatory mutants of ADPglucose pyrophosphorylase .
  • ADPG-PP ADPglucose pyrophosphorylase
  • 3 -PGA 3-phosphoglycerate
  • I inorganic phosphate
  • Chlamydomonas starch mutant appears to contain an ADPG-PP that is defective in its activation by 3-PGA (Ball et al . , Planta 185:17-
  • ADPG-PPs The structure of higher plant ADPG-PPs.
  • Salmonella typhimurium are encoded by a single gene locus, glgC (Preiss et al . , In: Biocatalysis in
  • the large and small subunit sequences display about 30-55% amino acid identity.
  • the primary sequences of the small subunits are more conserved between species (>90% identity) than the primary sequences between large and small subunits within a species (Nakata et al . , Plant Mol . Biol .
  • ADP-glucose pyrophosphorylase Using chemical labeling and photoaffinity labeling strategies, three residues in the bacterial enzyme have been identified to be important in binding of the allosteric effectors or substrates. Lys 39 is located near the activator binding site (Parsons and Preiss, J. Biol . Chem . , 253:7638-7645,
  • Lys 195 participates in binding of the substrate glucose-1-phosphate (Glc 1-P) (Hill et al . , J " . Biol .
  • Lys 39 and Lys 195 are conserved in all small subunits of the plant enzyme, while a Phe is present in the corresponding Tyr 114 position in the plant's large subunit. The conservation of the two Lys residues suggest that they play a similar role in the plant enzymes.
  • ADPG-PP When pyridoxal phosphate is covalently bound, ADPG-PP no longer requires 3-PGA for maximal enzyme activity, suggesting that pyridoxal phosphate is bound at the activator site. Only a single reactive Lys, located near the C-terminus, is observed for the small subunit (Morell et al . , J. Biol . Chem.
  • Lys residues are located near the C-terminus. One Lys is aligned with the conserved Lys labeled in the small subunit. A second reactive Lys residue is positioned 38 residues away from the former on the N-terminal side of the primary sequence. The third reactive Lys is located about 115-120 residues from the N-terminus of the large subunit sequence and is not conserved in the small subunit sequence.
  • the small subunit has a Lys at the equivalent position of Lys 39 , while the large subunit has an Arg, indicating a conservation of charge.
  • the PAV motif also appears to be important for the allosteric response in the bacterial enzyme. When the Ala residue is replaced by a Thr, the resulting mutated enzyme has a greatly reduced affinity for the activator fructose-1, 6-diphosphate . Overall, the results from chemical labeling and random mutagenesis studies indicate that sequences located at both the N- and C-terminal regions are required for allosteric regulatory behavior of higher plant ADPG-PP.
  • the homotetrameric small subunit enzyme exhibits values of K m for glucose 1-phosphate, ATP and Mg 2+ ions that are similar to those of the wild-type heterotetrameric enzyme.
  • the homotetrameric small subunit enzyme requires at least 15-fold greater amounts of 3-PGA for 50% activation than the wild-type enzyme.
  • the large subunit alone is unable to assemble into a catalytically active enzyme.
  • ADPG-PPs of Seeds and Tubers In addition to the genes that encode the leaf ADPG-PP, cereals and peas possess a second set of genes that encode a seed-specific form.
  • the maize endosperm-specific ADPG-PP is composed of two distinct subunits encoded by the Bt2 and Sh2 loci. Immunoblot analysis of protein extracts revealed that the bt 2 mutant lacked a 55-kD species, while the sh 2 mutant lacked a 60- kD species (Preiss et al . , Plant Physiol . 92:881-885,
  • Bt2 and Sh2 contain the structural gene sequences for the maize endosperm ADPG-PP small and large subunits, respectively.
  • ADPG-PPs from storage tissues display variable allosteric responses in vi tro .
  • ADPG-PPs from maize endosperm (Plaxton and Preiss, Plant
  • the lack of allosteric response exhibited by the barley, wheat and pea seed enzymes may not be an intrinsic property of these enzymes but rather a result of a post-translational modification of the enzyme.
  • Post-translational-induced changes in allosteric properties of these ADPG-PPs is a likely possibility, as suggested by studies of the maize endosperm enzyme.
  • maize endosperm extracts are prepared in the absence of proteinase inhibitors, a substantial level of enzyme activity is observed even in the absence of the activator 3-PGA.
  • the sensitivity to allosteric effectors was observed to be much less than the leaf form. Only a two- to three-fold activation is detected under optimal conditions as opposed to the 20- to 30-fold evident for the leaf enzyme (Dickinson and Preiss, Arch . Biochem . Biophys .
  • ADPG-PP enzyme from maize endosperm has been found to be highly susceptible to proteolysis (Plaxton and Preiss, Plant Physiol . 83:105-112, 1987).
  • ADPG-PP may obstruct starch synthesis as the levels of the activator 3-PGA are likely to be much lower than the inhibitor Pi in amyloplasts due to the unique biochemistry of this organelle.
  • the amyloplast is dependent on the cytoplasm for these metabolic requirements .
  • the ADPglucose is then transported to the plastid where it is then utilized by starch synthase .
  • tuber disks incorporate 14 C-labeled sucrose into starch at 50% higher rates in the presence of mannose, which sequesters the inhibitor Pi (Hnilo and Okita, Plant and Cell Physiol . 30:1007-1010, 1989).
  • ADPG-PP results in an enhancement of starch synthesis in tubers (Stark et al . , Science 258:287-292, 1992).
  • the resulting variant ADPG-PP enzyme appears to be resistant to Pi inhibition, suggesting that the altered allosteric response may be responsible for increase seed weight.
  • these results support the view that starch synthesis has not reached it highest potential in developing harvestable sink organs, i.e.
  • ADPG-PP activity may also have an important role in increasing overall productivity of the plant by maximizing rates of carbon dioxide (C0 2 ) fixation and utilization in photosynthetic tissues such as leaves, stems etc.
  • C0 2 fixation rates in photosynthetic tissues is affected by the capacity of converting fixed C0 2 into carbohydrates such as sucrose and starch.
  • Recent evidence indicates that leaf starch plays a broader role than simply serving as a diurnal reserve of carbon and energy to enable the plant to survive during the dark period. Ludewig et al . (FEBS)
  • ADPG-PP up-regulated mutants of ADPG-PP, i.e., allosterically regulated mutant ADPG-PPs having enzymatic activity that is significantly higher than an otherwise similar wild-type enzyme at physiological concentrations (i.e., about equimolar levels) of 3-PGA and Pi (Heldt et al . , Plant Physiol . 59:1146-1155,
  • the methods of the invention are more efficient than any previous empirical methods involving mutagenesis and mass biochemical screening of mutants.
  • Applicants have generated such ADPG-PP mutants and have analyzed the enzymatic activity of the mutant ADPG- PP enzyme.
  • the mutant enzyme differs in that it has: (1) higher sensitivity to the activator 3-PGA; (2) lower sensitivity to the inhibitor Pi; (3) increased starch production; (4) increased yield; (5) increased plant size (especially leaves) ; and, surprisingly, (6) increased growth rate and (7) increased number of seeds.
  • methods are provided for producing and identifying nucleic acids that encode up-regulated mutant ADPG-PP enzymes.
  • the plant ADPG-PP large and small subunits are co-expressed in a bacterial cell lacking ADPG-PP activity (for example a glgC " strain) .
  • ADPG-PP activity for example a glgC " strain
  • One subunit is unmutated while the other is mutated. If the mutation affects only the allosteric regulatory properties of the assembled enzyme, then cells expressing this mutant enzyme will not accumulate glycogen under normal physiological levels of the activator 3-PGA, although they will have normal levels of ADPG-PP activity if measured in the presence of saturating levels of 3-PGA.
  • a simple means for assessing glycogen levels, which can be correlated with ADPG-PP anzyme activity levels in a bacterial cell is by exposing the cells to iodine vapor. Glycogen-accumulating cells stain brown to purplish black while cells lacking this carbohydrate stain light yellow. Upon determining that the mutated subunit affects only the allosteric regulatory properties of the assembled enzyme, the mutated subunit is again mutagenized to produce a second mutation which will restore the allosteric regulatory properties of the assembled enzyme and the bacterial cell ' s capacity to accumulate glycogen which can be readily detected by iodine staining. A subunit that contains only the second mutation but not the first mutation is then studied to determine the effect of this second mutation on ADPG-PP function, for example, whether the second mutation is an up-regulatory mutation.
  • An alternative embodiment of a method for the generation and identification of up-regulated ADPG-PP mutants is by direct screening methods.
  • the plant ADPG-PP subunits must be expressed on low-copy-number plasmids.
  • high-copy-number plasmids can be used to co-express the plant ADPG-PP subunits; but, the bacterial cells must be grown on enriched media (Govons et al . , J " . Bacteriol . 97:970-972, 1969) containing low amounts of glucose (0 to 0.5%) as compared to the normal 2%.
  • enriched media When grown on enriched media containing 0.1% glucose, cells expressing up-regulated ADPG-PP readily stain with iodine vapor whereas cells expressing the wild-type unmutated enzyme do not.
  • a third representative method for generating and identifying up-regulated ADPG-PP mutants involves generation of segmented mutations. Nucleotides encoding ADPG-PP are truncated such that the expressed proteins are truncated at either the N- or C- terminus. These mutants are screened for altered allosteric function and up-regulated mutants are identified by methods described herein.
  • nucleic acids are provided that encode an up-regulated mutant ADPG-PP enzyme, for example, a plant mutant ADPG- PP enzyme.
  • a nucleic acid may be made and identified by any of the methods of the invention.
  • the nucleic acid may be made by chemical in vi tro synthesis, such as by using an automated polynucleotide synthesizer, or may be made using site-directed mutagenesis.
  • the mutant ADPG-PP nucleic acid codes for an enzyme that is up-regulated, and therefore has a biological activity (i.e., an enzyme activity) that is higher than the biological activity of the wild-type enzyme under physiological conditions.
  • enzymes are provided that are encoded by the nucleic acids of the invention, for example, a plant mutant ADPG-PP enzyme.
  • Such enzymes are up-regulated mutant enzymes.
  • a mutant enzyme may be more sensitive to an allosteric activator, such as 3-PGA, and may be more resistant (i.e., less sensitive) to an allosteric inhibitor such as Pi.
  • Up-regulated mutant enzymes may be produced by a number of methods including, but not limited to, mutagenesis with chemicals such as hydrozylamine, mutagenesis with radiation, site-directed mutagenesis, PCR mutagenesis, or genetic engineering techniques such as N-terminal truncation of the enzyme.
  • transgenic plants that comprise the mutant nucleic acids of the invention.
  • Expression of up- regulated ADPG-PP mutant genes in transgenic plants can result in, for example, increased starch production in at least a portion of the plant (compared with the wild- type) and can also increase productivity, yield, growth rate, and seed number.
  • SEQ ID NO:l shows the nucleotide sequence of the mutant ADPG-PP large subunit UpRegl that, when co- expressed with a wild-type ADPG-PP small subunit, forms an up-regulated ADPG-PP enzyme.
  • SEQ ID NO: 2 shows the primary amino acid sequence of UpRegl .
  • SEQ ID NO: 3 shows the nucleotide sequence of the mutant ADPG-PP large subunit UpReg2 , that when co- expressed with a wild-type ADPG-PP small subunit, forms an up-regulated ADPG-PP enzyme.
  • SEQ ID NO : 4 shows the primary amino acid sequence of UpReg2.
  • SEQ ID NO: 5 shows the nucleotide and amino acid sequences of ⁇ N17-LS, a mutant ADPG-PP large subunit that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up-regulated ADPG-PP enzyme.
  • SEQ ID NO: 6 shows the primary amino acid sequence of ⁇ N17-LS.
  • SEQ ID NO : 7 shows the nucleotide sequence of the mutant ADPG-PP large subunit R20 that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up- regulated ADPG-PP enzyme.
  • SEQ ID NO : 8 shows the primary amino acid sequence of R20.
  • SEQ ID NO: 9 shows the nucleotide sequence of the mutant ADPG-PP large subunit R32 that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up- regulated ADPG-PP enzyme.
  • SEQ ID NO: 10 shows the primary amino acid sequence of R32.
  • SEQ ID NO: 11 shows the nucleotide sequence of the mutant ADPG-PP large subunit R4 that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up- regulated ADPG-PP enzyme.
  • SEQ ID NO: 12 shows the primary amino acid sequence of R4.
  • SEQ ID NO: 13 shows the nucleotide sequence for an upstream primer 5 ' -GATATTGGTACCATTG-3 ' that is useful for introducing double-termination codons in an ADPG-PP small subunit cDNA sequence.
  • the primer includes a Kpnl site .
  • SEQ ID NO: 14 shows the nucleotide sequence for a downstream primer that is useful for introducing double termination codons in an ADPG-PP small subunit cDNA sequence.
  • the primer includes a Sad site. Termination codon sequences are underlined.
  • SEQ ID NO: 15 shows the nucleotide sequence for an upstream primer that is useful for introducing double termination codons in an ADPG-PP large subunit cDNA sequence.
  • the primer includes an Nhel site.
  • SEQ ID NO: 16 shows the nucleotide sequence for an downtream primer that is useful for introducing double- termination codons in an ADPG-PP large subunit cDNA sequence.
  • the primer includes a Sad site. Termination codon sequences are underlined.
  • SEQ ID NO: 17 shows the nucleotide sequence for an upstream primer that is useful for restoring the N-terminus of an ADPG-PP small subunit expression plasmid.
  • the primer includes an Ncol site.
  • SEQ ID NO: 18 shows the nucleotide sequence for a downstream primer that is useful for restoring the N- terminus of an ADPG-PP small subunit expression plasmid.
  • the primer includes a Kpnl site.
  • SEQ ID NO: 19 shows the nucleotide sequence for an upstream primer that is useful for deleting DNA sequences that code for 17 amino acids at the N-terminus of an ADPG-PP large subunit.
  • the primer includes an Ncol site.
  • SEQ ID NO: 20 shows the nucleotide sequence for a downstream primer that is useful for deleting DNA sequences that code for 17 amino acids at the N-terminus of an ADPG-PP large subunit.
  • the primer includes an Nhel site.
  • SEQ ID NO: 21 shows the upstream primer used to amplify the Arabidopsis ribulose bisphosphate small subunit (atslA) promoter and transit leader coding sequences .
  • SEQ ID NO: 22 shows the downstream primer used to amplify the Arabidopsis ribulose bisphosphate small subunit (atslA) promoter and transit leader coding sequences .
  • FIG. 1 shows the structure of the plasmid pML7.
  • FIG. 2 shows the structure of the plasmid pMLlO.
  • FIG. 3 shows a comparison of the I 2 staining patterns of AC70R1-504 cells expressing the wild-type (LS/SS) and the mutant UpReg-1, R20, and 345 ADPG-PP enzymes when grown on glucose-enriched media. Mutant 345, which contains the P52L mutation on the large subunit (LS) does not accumulate glycogen and stains a faint yellow. In contrast, wild-type (LS/SS) ADPG-PP and the mutants UpReg-1 and R20 accumulate large amounts of glycogen and stain darkly.
  • FIG. 4 shows a graphical comparison of I 0-5 (the amount of Pi required for 50% inhibition) values at different 3-PGA concentrations for the native, recombinant wild-type (LS+SS) , and ⁇ N17-LS ADPG-PP enzymes.
  • Data on the native enzyme were taken from Sowokinos and Preiss, Plant Physiol . 69:1459-1466, 1982, while data for the recombinant enzyme were taken from Ballicora et al . ( Plant Physiol . 109:245-251, 1995, and from Laughlin and Okita ( Phytochem 47:621-629, 1998) .
  • FIG. 5 is a table comparing substrate binding (Km) 3-PGA activator (Ao. 5 ) affinity properties of various native and mutant ADPG-PPs.
  • FIG. 6 is a table comparing Pi inhibition (I 0 . 5 ) of various native and mutant ADPG-PPs.
  • FIG. 7 is a table comparing 3-PGA activation for various native and mutant ADPG-PPs.
  • a Q . 5 is the amount of 3-PGA required to give 50% activation.
  • I 0 . 5 is the amount of Pi required to inhibit the enzyme 50% in the presence of a known amount of 3-PGA.
  • FIG. 8 is a table showing seed yields from eight greenhouse-grown plants of various plant lines (T3 generation) .
  • Fig. 9 shows a recombinant vector comprising the atslA-potato large subunit cassettes contained within a Xba I/Sac I DNA fragment cloned into the Xba I and Sac I sites of the T-DNA binary vector pHI-32, a derivative of pIG-121, to produce pHI-33 to pHI-39.
  • Fig. 10 shows third generation progeny of the transgenic Arabidopsis plants containing pHI-33 to pHI-
  • FIG. 11 shows a generalized schematic drawing of the plasmid used to construct a gene fusion between the atslA promoter and transit leader sequences to the potato ADPG-PP large subunit sequences.
  • the coding sequences from UpRegl, R4 , R20, R32, M27, M345 and wildtype large subunit sequences were removed from the plasmid DNA by digestion with Nco I and Sac I, and the resulting DNA fragment was cloned into the relevant restriction sites of pHI-10 to produce plasmids pHI-ll to pHI-17.
  • Fig. 12 shows the Arabidopsis ribulose bisphosphate small subunit astlA promoter and transit leader coding sequence fragment digested with Xho I and Sac I cloned into the Xho I and Sac I sites of pBluescript II.
  • Fig. 13 is a table comparing phenotypic properties of various transgenic plants .
  • ADPG-PP which controls the flux of carbon into starch, is allosterically regulated.
  • the level of ADPG-PP activity is controlled by the levels of the effectors 3-PGA and Pi, which activate and inhibit, respectively, the activity of ADPG-PP.
  • ADPG-PPs that display up-regulated allosteric or different physical properties, e.g., temperature resistance. These allosterically up-regulated mutants require substantially less 3-PGA to attain maximum catalytic activity and/or are less susceptible to Pi inhibition than the unmutated enzyme.
  • Mutant ADPG-PP genes are useful, for example, for altering starch production in a host organism, e.g., to increase starch production in cereal seeds, tubers, fruit, leaves and other plant organs and tissues. Yield is also increased.
  • certain mutant ADPG-PP genes when expressed in a plant, result in increased growth rate and increased seed production.
  • a first approach involved the following steps:
  • a native ADPG-PP gene was mutagenized to produce a single mutation.
  • Double mutant cells were then screened for staining by iodine vapor, i.e. a reversal of the iodine staining-minus phenotype that is mediated by the primary mutation.
  • Double mutants exhibiting at least partial complementation of glgC ⁇ cells were then sequenced to reveal the location and nature of the two mutations.
  • Site-directed mutagenesis was then used to create a DNA containing the second (but not the first) mutation.
  • the protein product of the DNA containing the secondary site mutation was then analyzed by standard enzyme-kinetics methods (Cornish-Bowden, 1995) in the absence of the initial primary mutation to determine whether the secondary site mutation modifies the allosteric properties of the enzyme by itself. Mutants were thus identified that had increased biological activity over the wild-type enzyme and were therefore identified as. "up-regulated" mutant enzymes.
  • a second approach involved the direct screening method to identify up-regulated allosteric mutants.
  • Cells containing an up-regulated mutant enzyme would be expected to have increased glycogen production.
  • up-regulated mutants from normal cells are not distinguishable because the iodine screening procedure is saturated. That is, although cells harboring the up-regulated mutant accumulate more glycogen than cells containing the wild-type enzyme, the up-regulated cells stain with iodine at the same rate as cells containing the wild-type enzyme.
  • the ADPG-PP sequences were placed on a low copy-number plasmid. The plamids used were pWSK28 (that contains the polylinker from pBluescript Skt) and pWSK30 (that contains the polylinker from pBluescript Kst) .
  • Low copy-number plasmids are discussed in standard texts (e.g., Sambrook et al . , 1989) and may be obtained commercially.
  • Low copy-number plasmids other than pWSK28 and pWSK30 include pBHOl (Jefferson et al . , EMBO
  • cells expressing up- regulatory mutant ADPG-PP can be distinguished from cells containing the wild-type enzyme by altering the glucose concentration in the enriched media (Govons et al., J " . Bacteriol . 97:970-972, 1969).
  • enriched media containing 0.1% glucose instead of the usual 2% glucose level, up-regulatory mutants will stain darkly with I 2 , whereas normal cells stain very lightly.
  • a third approach involved the generation of allosteric mutants by segmental mutations.
  • DNA corresponding to N- and or C-terminal portions of the ADPG-PP polypeptide were deleted by recombinant DNA techniques (Sambrook et al . , 1989) .
  • Deletions may include the removal of nucleotides corresponding to 5, 10, 15, 17, 25, 30, 40 or more amino acid residues from the N- or C- terminus.
  • Escherichia coli expression systems were used to express the large and small subunit cDNAs of ADPG-PP under the control of bacterial promoters.
  • the large subunit cDNA was cloned into a pACYC-based cloning vector with expression of the large subunit cDNA being driven by a tac promoter
  • the small subunit cDNA was cloned in a pBR325-based cloning vector with expression of the small subunit cDNA driven by a recA promoter.
  • Examples of plasmid vectors useful for the expression of the large and small subunit cDNAs can be found, for example, in Iglesias et al . (J. Biol . Chem. 268:1081-1086, 1993).
  • the Agrobacterium system may be used to carry out transformation using pHI-32 or pCAMBIA T-DNA plasmids, both of which are ⁇ derived from pBHOl.
  • Such plasmids can be used to transform plants such as potato, rice, wheat, barley, maize, and tomato.
  • the mutant ADPG-PP subunits generated by this invention can be used to increase yield and productivity of many crop plants that use starch as their principal reserve in their photosynthetic tissues and/or storage organs.
  • mutant subunits and subsequent formation of ADPG-PP enzymes with up- regulatory properties can be used to increase starch production in storage organs such as potato tubers, cassava roots, and cereal seeds including those from wheat, maize, rice, barley, rye, and sorghum.
  • the mutant subunits can be used to increase starch production during the early development of the storage organs in oil-accumulating seed plants such as soybean, rape, and sunflower and in developing fruits of tomato, apple, pear, peach, etc.
  • starch is used as a transient reserve of carbon and energy which is then re- utilized for the formation of other molecules.
  • starch In oil- accumulating seed plants, starch accumulates during the early phase of seed development and is then re-utilized for the production of oils. In fruiting plants, accumulated starch is metabolized to reducing sugars, a preferred trait for tomatoes especially those used for processing ketchup, paste and the like and for the sweetness of apples, pears and other sweet fruits.
  • mutant ADPG-PP subunits generated by this invention can be used to increase overall productivity of all C 3 plants (defined as those plants that use the Calvin cycle to fixed C0 2 into 3-PGA as their first stable three-carbon intermediate) .
  • C 3 plants include potato, cassava, wheat, rice, barley, rye, soybean, rape, sunflower, flax, cotton, alfalfa, celery, cauliflower, and carrot.
  • C 3 plants typically exhibit C0 2 -limited photosynthesis at ambient and sub-ambient C0 2 partial pressures. Under ideal conditions, the rate of C 3 photosynthesis increases with increasing C0 2 as photorespiration is suppressed. However, under many environment conditions, e.g. low temperatures, high light, and increasing C0 2 (see Leegood and Edwards, Photosynthesis and the Environment, N.R. Baker, ed. ,
  • triose phosphate utilization can be overcome if starch synthesis is elevated in photosynthetic-competent tissues such as leaves and photosynthetic-competent tissues such as stems and seed pods, or grains. In turn, this increased photosynthesis will result in higher biomass production (productivity) and, in turn, higher yields of harvestable organs such as tuber, seeds, fruit etc.
  • the mutant ADPG-PP subunits can be transferred into crop plants by either Agrrojbacterium-mediated transformation or by mechanical introduction, e.g., biolistics bombardment.
  • Agrojbacterium-mediated transformation is the preferred method because of the smaller frequency of introducing multiple copies of the transgene and subsequent problems of gene expression instability due to co-suppression effects in offspring of transgenic plants (Kumpatla et al . , Plant Phsyiol . 115:361-373, 1997).
  • Agrobacterium has been used to introduce transgenes into rice (Hiei et al., lant J. 6:271-282, 1994), maize (Ishida et al . Nature Biotechnol . 14:745-750, 1996; Gould, Plant Physiol . 95:426-434, 1991), soybean (Hinchee et al .
  • Two previous approaches to manipulating ADPG-PP activity have been utilized.
  • One approach (referred to herein as the "Monsanto approach"), utilizes a mutant bacterial ADPG-PP, glgC16, that codes for a form of ADPG-PP that lacks allosteric regulation.
  • the glgC16 enzyme possesses 60% of the activity levels of the fully activated wild-type enzyme in the absence of the activator fructose-1, 6-diphosphate and is also less sensitive to the inhibitor adenosine monophosphate (AMP) . Transfer of this gene and expression in tubers and tomatoes results in potatoes with higher starch content (see Stark et al . , Science
  • a second approach utilizes the transposable element system Ac-Ds in maize to generate ADPG-PP variants.
  • One mutant was obtained by excision of a Ds element that was initially located in the Shrunken-2 gene, which encodes the endosperm- specific form of the large subunit gene of ADPG-PP.
  • a six-nucleotide "footprint" was left behind, resulting in a large subunit having two additional amino acids.
  • the resulting enzyme appears to be resistant to Pi inhibition.
  • a modified potato large subunit containing these two additional residues formed an enzyme resistant to Pi inhibition (Giroux et al . , Proc . Na tl . Acad . Sci .
  • the present invention differs from the Monsanto approach in that it utilizes (1) isolated plant genes for ADPG-PP instead of a bacterial gene and (2) an enzyme that is subject to allosteric regulation, although its sensitivity to activation by 3-PGA and inhibition by Pi is 20- to 40-fold higher than the wild- type enzyme.
  • the up-regulatory mutant ADPG-PPs described herein have less than 5% of the activity levels exhibited by the fully activated enzyme.
  • the E. coli glgC16 enzyme is essentially unregulated. Even in the total absence of the activator, it has 60% of the activity exhibited by the fully activated wild-type enzyme.
  • the bacterial enzyme is encoded by a single gene, glgC, which codes for a subunit of 50 kD molecular weight that assembles to form a homotetramer.
  • the higher plant enzyme is composed of two large subunits and two small subunits. Each subunit type is encoded by a distinct gene. As described above, it is believed that the large and small subunits play different roles in enzyme catalysis and allosteric regulation.
  • the small subunit is able to form a homotetrameric enzyme but requires more than 24 -fold greater levels of 3-PGA for activation than the normal heterotetrameric enzyme, which is composed of two large subunits and two small subunits.
  • the large subunit is not capable of forming an active enzyme by itself.
  • Yield refers to the amount of harvestable material, for instance, seeds or tubers. Yield is generally defined in units of mass.
  • Processivity refers to the total biomass produced by a plant, both harvestable and non-harvestable .
  • the word "increased” means that the mean value of a characteristic in a population, when compared with another (wild-type) population is measurably and statistically significantly greater than the mean value of the characteristic in question for the characteristic in the wild-type population.
  • ADPG-PP nucleic acid refers to a native (or wild-type) nucleic acid that encodes an ADPG-PP polypeptide (including, but not limited to, cDNA and genomic sequences) and fragments thereof .
  • ADPG-PP Polypeptide refers to a polypeptide encoded by an ADPG- PP nucleic acid.
  • An ADPG-PP polypeptide can be produced by the expression of a recombinant ADPG-PP nucleic acid or can be chemically synthesized. Techniques for chemical synthesis of polypeptides are described, for example, in Merrifield, J. Amer. Chem. Soc . 85:2149-
  • “Mutant” or “mutated” ADPG-PP nucleic acid include a change in at least one base of the protein-coding region of a native ADPG-PP nucleic acid that results in a corresponding change in an amino acid of an ADPG-PP polypeptide encoded by the ADPG-PP nucleic acid. Included are insertions, deletions (including deletions of one or more nucleotides internal to the protein- coding region or deletions (or truncations) from one or both ends of the protein-coding region) , and substitutions. Such mutated nucleic acids can be produced by any standard mutagenesis technique, including, but not limited to, those described in Molecular Cloning: A Laboratory Manual , 2nd ed. , vol. 1-
  • mutagenized is synonymous with the word “mutated” and means being altered from the native, natural state by any means including by chemical mutagens, electromagnetic radiation, genetic engineering, or molecular biology techniques.
  • a nucleic acid may be mutagenized by insertion, substitution, or deletion of a nucleic acid
  • a peptide may be mutagenized by insertion, substitution, or deletion of an amino acid residue such that the nucleic acid or peptide, respectively, is altered from its natural, native state.
  • “Native” refers to a naturally- occurring ( “wild-type” ) nucleic acid or polypeptide.
  • the native nucleic acid or protein may have been physically derived from a particular organism in which it is naturally occurring or may be a synthetically constructed nucleic acid or protein that is identical to the naturally-occurring nucleic acid or protein.
  • isolated nucleic acid is one that has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, by conventional nucleic acid-purification methods.
  • isolated nucleic acid also embraces recombinant nucleic acids and chemically synthesized nucleic acids.
  • a fragment of an ADPG-PP nucleic acid is less than full length and is capable of hybridizing specifically with a native ADPG- PP nucleic acid under stringent hybridization conditions.
  • the length of such a fragment is at least 15, 20, 30, 40, or 50 nucleotides of a native ADPG-PP nucleic acid sequence or another target sequence, e.g., sequences flanking the cloning site of a vector in which an ADPG-PP sequence is cloned.
  • a "probe” is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, e . g. , a radioactive isotope, ligand, chemiluminescent agent, or enzyme.
  • Primer pairs are isolated nucleic acids that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between' the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e . g. , a DNA polymerase. Primer pairs can be used for amplification of a nucleic acid sequence, e . g. , by the polymerase chain reaction
  • Probes and primers are generally 15 nucleotides or more in length, and maybe 20, 25, or 30 nucleotides or more. Such probes and primers hybridize specifically to a target nucleic acid under high-stringency hybridization conditions. Probes and primers have complete sequence similarity with the target sequence in at least about 7- 15 consecutive nucleotides to permit hybridization under high stringency conditions, although probes differing from a target sequence and that retain the ability to hybridize to the target sequence may be designed by conventional methods and are useful for introducing nucleotide sequence mutations and corresponding amino acid sequence mutations.
  • PCR-primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5 ® ,
  • portion is used to indicate a number of contiguous nucleotides, from either the 3' or the 5' terminus of the DNA, corresponding to 5, 10, 15, 17, 25, 30, 40 or more amino acid residues from the N- or C- terminal. Deletion may be performed by standard molecular biology techniques (Sambrook et al . , 1989) "Substantial Similarity".
  • a first nucleic acid is "substantially similar” to a second nucleic acid if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand) , there is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity.
  • Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using sequence analysis software such as the BLAST sequence analysis software available from the NCBI .
  • sequence analysis software such as the BLAST sequence analysis software available from the NCBI .
  • a particularly useful tool is BLAST 2.0 program "gapped blastn" set to default parameters.
  • Another useful software product is the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI .
  • nucleic acids are substantially similar if they hybridize under stringent conditions, as defined below.
  • a first nucleic-acid sequence is “operably” linked with a second nucleic-acid sequence when the first nucleic-acid sequence is placed in a functional relationship with the second nucleic-acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame .
  • Recombinant nucleic acid is made by an artificial combination of two otherwise separated segments of sequence, e . g. , by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • nucleic acids can be synthesized, for example, on commercial automated oligonucleotide synthesizers. Preparation of Recombinant or Chemically
  • Natural or synthetic nucleic acids according to the present invention can be incorporated into recombinant nucleic-acid constructs, typically DNA constructs, capable of introduction into and replication in a host cell .
  • a construct may be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • conventional compositions and methods for preparing and using vectors and host cells are employed, as discussed, inter alia, in Sambrook, 1989, or Ausubel, 1994.
  • a cell, tissue, organ, or organism into which a foreign nucleic acid has been introduced is considered
  • transgenic or transformed cell or organism also includes progeny of the cell or organism, including progeny produced from a sexual cross that includes the ADPG-PP transgene from one or both parents.
  • Nucleic-acid constructs, or vectors, for use with prokaryotic or eukaryotic hosts include a nucleic acid sequence that encodes an ADPG-PP polypeptide or a portion thereof and other vector sequences known in the art and appropriate for a given host cell, including, but not limited to, well known transcription and translation-initiation sequences; an origin of replication or autonomously replicating sequence (ARS) ; expression control sequences, including, but not limited to, promoter and enhancer sequences; processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences; secretion, transit, and other peptide sequences that allow the protein to cross and/or lodge in a cell membrane or be secreted from a cell; selectable or screenable marker genes, etc.
  • ARS autonomously replicating sequence
  • Nucleic-acid constructs can be introduced into a host cell by any suitable conventional method, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent; etc.) See, e.g., Sambrook, 1989, and Ausubel, 1994.
  • Nucleic acid constructs that express a mutated ADPG-PP according to the invention can be introduced into a variety of host cells or organisms in order to alter starch biosynthesis by the cell or organism, particularly higher plant cells, but also including other prokaryotic or eukaryotic host cells that synthesize starch.
  • Any well known vector suitable for stable transformation of plant cells and/or for the establishment of transgenic plants may be used, including those described in, e . g. , Pouwels et al . , Cloning Vectors : A Laboratory Manual , 1985, supp. 1987);
  • Such plant expression vectors can include expression control sequences (e . g. , inducible or constitutive, environmentally or developmentally regulated, or cell- or tissue-specific expression- control sequences) .
  • constitutive plant promoters useful for expressing ADPG-PP in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter (see, e . g. , Odel et al . , Nature 313:810, 1985; Dekeyser et al . , Plant Cell 2:591, 1990; and Terada and
  • Plant expression vectors can include regulatory sequences from the 3 ' -untranslated region of plant genes (Thornburg et al . , Proc . Na tl . Acad . Sci . USA 84:744
  • a 3' terminator region to increase mRNA stability of the mRNA such as the PI-II terminator region of potato or the octopine or nopaline synthase 3' terminator regions.
  • Useful dominant selectable marker genes for expression in plant cells include, but are not limited to: genes encoding antibiotic resistance genes (e . g. , resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) ; and herbicide resistance genes (e . g. , phosphinothricin acetyltransferase) .
  • Useful screenable markers include, but are not limited to, ⁇ -glucuronidase and green fluorescent protein.
  • Nucleic-Acid Hybridization The nucleic-acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Nucleic acid hybridization is discussed in Sambrook, 1989, at 9.52-9.55. See also, Sambrook, 1989 at 9.47-9.52, 9.56-9.58; Kanehisa, Nucl . Acids Res .
  • Nucleic acid and protein sequence similarity. homology, and identity. The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology) ; the higher the percentage, the more similar the two sequences.
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • BLAST National Center for Biotechnology Information
  • a description of how to determine sequence identity using this program is available at http : //www . ncbi . nlm . nih . gov/BLAST/blast_help . html .
  • Nucleic acid sequence similarity can be determined by using the NCBI BLAST 2.0 "gapped blastn” program set to default parameters. Amino acid sequence similarity can be compared using NCBI 2.0 "gapped blastP” set to default parameters .
  • the "gapped” feature of these programs allows gaps (deletions and insertions) to be introduced into the sequences to be aligned. Allowing such gaps means that similar regions are not broken into several separate segments, allowing a truer determination of sequence identity (and therefore similarity) to be made.
  • PSI-BLAST Position-Specific Iterated BLAST
  • the program first performs a gapped BLAST database search.
  • the PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching.
  • PSI-BLAST may be iterated until no new significant alignments are found.
  • Protein Orthologs are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the amino acid sequence of the gene in question using the NCBI Blast 2.0, gapped blastp set to default parameters. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95%, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at http : //www . ncbi . nlm . nih . gov/BLAST/blast_FAQs . html .
  • sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • the present invention provides not only the peptide homologs are described above, but also nucleic acid molecules that encode such homologs.
  • Hybridization and Stringent Conditions An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions.
  • Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al . (1989) and Tijssen (1993) .
  • Nucleic acid molecules that hybridize under stringent conditions to the target sequences will typically hybridize to a probe based on either the entire target cDNA or selected portions of the cDNA under wash conditions of 0.2x SSC, 0.1% SDS at 65°C.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code.
  • nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequence that all encode substantially the same protein.
  • stringent conditions are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product.
  • the term "specific for (a target sequence)" indicates that a probe or primer hybridizes under given hybridization conditions only to the target sequence in a sample comprising the target sequence. Nucleic-Acid Amplification. As used herein,
  • amplified DNA refers to the product of nucleic-acid amplification of a target nucleic-acid sequence. Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including the polymerase chain reaction (PCR) . A variety of amplification methods are known in the art and are described, inter alia , in U.S. Patent Nos.
  • PCR polymerase chain reaction
  • ADPG-PP nucleic Acids Using ADPG- PP nucleic acids that are known in the art or that are isolated using such well-known nucleic acids, any conventional mutagenesis method can be used to mutagenize (i.e., artificially alter from its wild-type form) ADPG-PP nucleic acids, including chemical mutagenesis, oligonucleotide site-directed mutagenesis, chemical synthesis of a mutant ADPG-PP sequence, etc., resulting in substitutions, insertions, deletions, or combinations thereof. Nucleic acids so produced are called "mutant" nucleic acids.
  • a mutant ADPG-PP polypeptide is a polypeptide produced by the expression of a mutant nucleic acid, or a peptide that has been intentionally artificially altered from its wild-type form.
  • "Silent” mutations include substitutions of one or more base pairs that result in no change in the amino acid sequence of the polypeptide encoded by the sequence.
  • "Conservative” mutations result in a conservative amino acid substitution in one or more amino acid residues of the polypeptide encoded by the nucleic-acid sequence.
  • ADPG-PP polypeptides encompassed by the present invention are at least about 70%, 80%, 90%, or 95% homologous to a native ADGP-PP polypeptide.
  • Polypeptide homology can be analyzed by conventional methods, e.g., using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI , or the NCBI BLAST 2.0 blast software. Polypeptide sequence analysis software matches homologous sequences using measures of homology assigned to various substitutions, deletions, substitutions, and other modifications.
  • sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI , or the NCBI BLAST 2.0 blast software.
  • Polypeptide sequence analysis software matches homologous sequences using measures of homology assigned to various substitutions, deletions, substitutions, and other modifications.
  • Isolated "Purified,” “Homogeneous” Polypeptides.
  • An “isolated” polypeptide is separated from the cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that naturally accompany it.
  • Such a polypeptide can also be referred to as “pure” or “homogeneous” or “substantially” pure or homogeneous.
  • a polypeptide which is chemically synthesized or recombinant i.e., the product of the expression of a recombinant nucleic acid, even if expressed in a homologous cell type
  • a monomeric polypeptide is isolated when at least 60% by weight of a sample is composed of the polypeptide.
  • polypeptide by weight in a sample.
  • Protein purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high performance liquid chromatography; or other conventional methods.
  • Polypeptides according to the present invention can be purified by any conventional method. See, e . g. , Guide to Protein
  • ADPG-PP "Biological Activity” or "Enzyme Activity”.
  • biological activity refers primarily to the characteristic biological activity or activities of a native ADPG-PP polypeptide, including, but not limited to, catalyzing the initial step in ⁇ - glucan and starch synthesis: glucose-1-phosphate + ATP --> ADP-glucose + PPi .
  • Other activities include allosteric regulation by 3-PGA and Pi. "Allosteric". The word allosteric literally means “another site”.
  • An enzyme is said to be "allosteric” or “allosterically regulated” if its enzymatic activity is regulated by the binding of non-substrate molecules at a site other than the active site of the enzyme, i.e., an "allosteric site".
  • the binding of non-substrate molecules at allosteric sites effects the binding kinetics of the substrate-binding (active) site.
  • a molecule that increases binding or decreases dissociation of an enzyme and a substrate complex, thus increasing enzyme activity is called an “activator” molecule.
  • a molecule that decreases binding or increases dissociation of an enzyme and a substrate complex, thus decreasing enzyme activity is called an "repressor” or “inhibitor” molecule.
  • Up-regulated (or “up-regulatory”) .
  • a mutant enzyme is “up-regulated” or “up-regulatory” when that enzyme has a higher biological activity than the wild-type enzyme under physiological conditions.
  • the up-regulated enzyme may have at least about 10, 20, 40, 60, 80, or 100% higher biological activity than wild-type. Under some circumstances, the up-regulated mutant may have greater than 100% higher biological activity than the wild-type.
  • An up-regulated mutant exhibits increased biological activity by virtue of its mutation (s) .
  • the effect of the mutation (s) may be to inherently increase biological activity independent of allosteric regulation (for instance by increasing binding or decreasing dissociation of the enzyme-substrate complex) , to increase sensitivity to an allosteric activator (e.g., 3-PGA) , and/or to decrease sensitivity to an allosteric inhibitor (e.g., Pi) . Any such effect brought about by a mutation will result in an increase in enzyme activity making the mutant enzyme up-regulated.
  • an allosteric activator e.g., 3-PGA
  • an allosteric inhibitor e.g., Pi
  • Fusion Polypeptides The present invention also provides fusion polypeptides including, for example, heterologous fusion polypeptides in which an ADPG-PP sequence is joined to a well-known fusion partner. Such fusion polypeptides can exhibit biological properties (such as substrate or ligand binding, enzymatic activity, antigenic determinants, etc.) derived from each of the fused sequences. Fusion polypeptides are preferably made by standard recombinant DNA techniques.
  • ADPG-PP nucleic acids can be expressed in plants or plant cells under the control of a suitable operably linked promoter that is capable of expression in a cell of a particular plant. Any well-known method can be employed for plant cell transformation, culture, and regeneration in the practice of the present invention with regard to a particular plant species. Conventional methods for introduction of foreign DNA into plant cells include, but are not limited to: (1) Agrobacterium- mediated transformation (Lichtenstein and Fuller In:
  • plant encompasses any higher plant and progeny thereof, including monocots (e.g., corn, rice, wheat, barley, etc.), dicots ( e . g. , potato, tomato, etc.), and includes parts of plants, including seeds, fruit, tubers, etc.
  • monocots e.g., corn, rice, wheat, barley, etc.
  • dicots e. g. , potato, tomato, etc.
  • parts of plants including seeds, fruit, tubers, etc.
  • Bacterial mutants that lack functional ADPG-PP activity e.g., glgC E. coli strains such as AC70R1-504
  • the large subunit is mutagenized, e.g., using hydroxylamine, and cells co- expressing a mutagenized large subunit and a wild-type small subunit were screened for mutants defective in glycogen accumulation by I 2 staining (Greene et al . ,
  • Mutant ADPG- PP enzymes defective for allosteric regulation were then identified by assaying crude extracts of non-staining cells under saturating conditions of 3-PGA (10 mM, or 100 -fold greater levels than required to activate the enzyme by 50%) . Hydroxylamine causes G/C-to-A/T transitions. Mutations can also be introduced, for example, by various other conventional mutagenesis techniques, e.g., by using other chemical mutagens or by PCR performed under limiting substrate and co-factor conditions.
  • Site-directed mutagenesis may also be used to introduce insertions, deletions or substitutions.
  • Various standard techniques are known to carry out site-directed mutagenesis (Sambrook et al . , 1989), and commercial kits are also available such as the QUICKCHANGETM mutagenesis site-directed mutagenesis kit (STRATEGENETM , CA) .
  • Plasmid DNA encoding the large subunit was mutagenized at 37°C for 24 h in the presence of 0.8 M hydroxylamine-HCl , 50 mM sodium phosphate, pH 6.0, and 1 mM EDTA (Isackson and Bertrand, Proc . Na tl . Acad . Sci .
  • ADPG-PP was purified by biochemical chromatography techniques, and the activity of the mutant enzyme was investigated. Cells were grown in 850 mL of LB in Fernbach flasks and induced by the addition of IPTG and nalidixic acid.
  • Enzyme was eluted from the column by a 200 mL linear gradient of buffer B (50 mM potassium phosphate, pH 6.0, 5 mM MgCl 2 , 1 mM EDTA, 5 mM DTT, and 400 mM potassium chloride) . Fractions containing enzyme activity were pooled, concentrated by addition of ammonium sulfate to 75%, resuspended in sucrose buffer, and desalted by dialysis. Aliquots of enzyme concentrate were stored at -80°C until needed for enzyme studies.
  • Kinetic parameters i.e., K m values for glucose 1-phosphate, ATP, and Mg 2+ and A 0 . 5 for 3-PGA and I c.5 for Pi, were defined with the synthesis (forward) assay, which measures the incorporation of 1 C-glucose- 1-phosphate into ADPglucose.
  • the mutated plasmid DNA coding for the large subunit coding for the large subunit was isolated and subjected to a second round of hydroxylamine mutagenesis and re-transformed into AC70R1-504 harboring a plasmid DNA containing the small subunit counterpart cDNA.
  • the mutated plasmid DNA was isolated then transformed into E. coli .
  • Cells containing the plasmid DNA of interest were then selected by growth on enriched media containing the appropriate antibiotic. After replica plating and growth overnight, revertant cells that accumulated glycogen were identified by I 2 staining. The enzyme activities from these revertant cells were partially purified and characterized kinetically as described above.
  • the nature of the second-site mutation (because hydroxylamine causes G/C to A/T transitions, the reversion event is caused by second- site mutation) was identified by DNA sequencing of the cDNA sequence.
  • This second-site mutation on enzyme function was then evaluated by introducing the second- site mutation by itself (i.e., without the original mutation) into a wild-type cDNA sequence for the appropriate subunit by site-directed mutagenesis (using a commercial site-directed mutagenesis kit (such as the QuickchangeTM kit from Stratagene, CA) according to the manufacturer's instructions).
  • site-directed mutagenesis using a commercial site-directed mutagenesis kit (such as the QuickchangeTM kit from Stratagene, CA) according to the manufacturer's instructions).
  • the resulting subunit cDNA including the second-site mutation was then transformed into and co-expressed in AC70R1-504 together with the counterpart wild-type subunit.
  • the resulting mutant ADPG-PP enzyme was then characterized.
  • PCR strategy or other conventional site- directed mutagenesis method can be employed.
  • the large subunit or small subunit sequences can be amplified under conditions (high Mn 2+ or limiting nucleotide concentrations) that result in the misincorporation of nucleotides (Erlich, In: PCR
  • amplified sequences can then be purified, cleaved with the appropriate restriction enzymes, cloned into the appropriate sites of the expression vectors, and expressed in E. coli .
  • Mutants defective in pyrophosphorylase activity can then be screened initially by I 2 staining, followed by ELISA and enzyme assays as described above.
  • up-regulated enzymes can be identified directly by using a variant of the mutagenesis and selection protocol described above.
  • the large or small subunit cDNA that will be mutagenized is cloned into a low copy number plasmid such as pWSK 28 or other plasmids described in Wang and Kushner, Gene
  • Low-copy-number vectors are generally larger than high-copy-number vectors, having sizes of 6 or 7 kilobases or more.
  • low-copy-number plasmids contain, for example, a pSClOl origin of replication, which results in substantially fewer copies (e.g., about 6 copies/cell) than a vector possessing a pBR325, Col El, or ACYC origin of replication.
  • a pSClOl origin of replication which results in substantially fewer copies (e.g., about 6 copies/cell) than a vector possessing a pBR325, Col El, or ACYC origin of replication.
  • cells expressing wild-type large subunit sequences that are carried on a low-copy-number plasmid stain very lightly with I 2 when co-expressed with the small subunit sequences, providing a cleaner background to identify up-regulated mutants. Screening several thousand cells results in the isolation of several putative up-regulated mutants.
  • mutations can produce similar phenotypes, including mutations that increase plasmid copy number, promoter activity, and ribosome-binding efficiency. These other mutations can be identified by evaluating cellular extracts by immunological techniques as described above, since it is expected that cells containing up-regulated mutants of ADPG-PPs contain the same amount of antigen as cells expressing the wild-type enzyme.
  • other types of mutations can be eliminated by subjecting the cDNA sequences alone to the chemical mutagen or PCR mutagenesis protocol (rather than the vector as a whole) , then inserting the mutated cDNA sequences into an appropriate expression vector.
  • a third strategy for the generation of up-regulatory mutants is to mutate specific DNA sequences that code for specific peptide regions of either the large or small subunit. For example, peptide sequences at both the N- and C-terminus are required for normal allosteric regulation. Mutations in this region alter the allosteric response of the resulting enzyme (Ball and Preiss, J " . Biol . Chem . 269:24706-24711, 1994;
  • Mutations can be generated randomly using PCR techniques under conditions that cause misincorporation of nucleotides or by using synthetic oligonucleotides that include random mutations. Mutations can also be generated by deletion of specific DNA sequences, especially those encoding amino acid residues located at the N- or C-terminus, for example .
  • the expression plasmids, pMON17335 and pMON17336 (Inglesias et al . , J. Biol . Chem . 268:1081-1086, 1993), that contain the small and large subunit cDNAs, respectively, were modified by incorporating double translation termination codons using a PCR approach.
  • the primers shown in SEQ ID Nos. 13 and 14 were used to amplify a 538 bp DNA fragment covering the 3' end of the small subunit coding sequence. After digestion with Kpnl and Sad, the amplified product was cloned into pMON17335 to produce pML5.
  • the primers shown in SEQ ID NOS. 15 and 16 were used to amplify a 608 bp DNA fragment covering the 3 ' end of the large subunit coding sequence. After digestion with Nhel and Hindlll, the amplified product was cloned into pMON17336 to produce pML7 (FIG. 1) .
  • pML5-encoded small subunit polypeptide lacked 10 amino acid residues of the N-terminus of the mature protein. These ten amino acids were restored, together with an additional Met-Ala, by PCR using the primer shown in SEQ ID No. 17, which introduces a unique Ncol site (underlined DNA sequences) at the Met codon.
  • the amplified DNA fragment was digested with Ncol and Kpnl and cloned into pML5 to give pMLlO (shown in FIG. 2) .
  • Fig. 5 shows substrate binding (K m s) and 3-PGA activator (A 0 5 ) affinity properties of various ADPG-PPs: native (Sowokinos and Preiss, Plant Physiol . 69:1459-1466,
  • mutant 345 Greene et al . , Proc . Natl . Acad. Sci . USA 93:1509-1513, 1996); R20, UpReg-1, and ⁇ 17-LS (R20, UpReg-1, and ⁇ N17-LS are described below) .
  • the partially purified enzyme from mutant 345 displayed normal binding constants (K m ) for glucose- 1 -phosphate, ATP and Mg2 + (Fig. 5) . However, the enzyme from mutant 345 required 28 -fold greater levels of the activator 3-PGA than the wild-type enzyme.
  • DNA sequence analysis indicated the presence of single base substitution that resulted in the replacement of a proline located at residue 52 by a leucine (Greene et al . , Proc . Na tl . Acad . Sci . USA 93:1509-1513, 1996) .
  • the large-subunit plasmid from mutant 345 was isolated and subjected to a second hydroxylamine treatment, co-expressed with pMLlO, and screened for glycogen production.
  • Eight genetic revertants were isolated that displayed varying levels of glycogen accumulation as determined by I 2 staining.
  • the remaining revertants showed either intermediate (Class II) or light staining (Class III) phenotypes.
  • R4 , RIO, R20, and R32 identified a single second-site mutation in close proximity to the primary mutation Pro52Leu in each of the revertants.
  • R4 contained a base-pair mutation that replaced Pro at position 66 with a Leu, resulting in the addition of a second structural modification.
  • RIO and R32 contained an identical second site mutation in which Gly at position 101 was replaced with Asn.
  • a negatively charged Glu residue fourteen amino acids upstream from P52L was replaced with a positively charged Lys residue (SEQ ID Nos. 7 and 8); note that Lys38 is at position 40 in the recombinant cDNA sequence due to the addition of Met and Ala residues. Introduction of the positively charged Lys residue potentially enhances the enzyme's ability to interact with the negatively charged activator 3-PGA.
  • the R20 enzyme was partially purified to a specific activity of about 20 ⁇ mol/min/mg by differential ammonium sulfate precipitation, heat treatment, amino-propyl (C 3 ) chromatography, and DEAE anion-exchange chromatography and was estimated to be about 40% pure.
  • SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified protein showed a dramatic increase in a broad band migrating in the 50-52 kD range.
  • Fig. 6 shows the results of studies to determine the Pi inhibition constants (I 0 . 5 ) of various ADPG-PPs : wild-type (Ballicora et al . , Plant Physiol . 109:245-251, 1995); mutant 345 (Greene et al . , Proc . Natl . Acad. Sci .
  • I 0.5 value determined for the R20 ADPG-PP at 0.25 mM 3-PGA was roughly 4.5- and 8 -fold higher than that of the native potato and wild-type recombinant enzymes, respectively (Fig. 6) .
  • the increase in positive charge in this region of the enzyme appears to give R20 ADPG-PP an increased affinity for 3-PGA, while decreasing its affinity for Pi.
  • Site-directed mutagenesis was conducted to introduce a Lys residue at position 38 (Glu38Lys) in pML7 that is independent of the P52L mutation. Of four putative Glu38Lys site-directed mutants (SEQ ID Nos. 1 and 2), all contained the specific mutation as determined by sequence analysis.
  • Glu38Lys large-subunit plasmid DNA was then transformed into AC70R1-504 containing pMLlO for purification and kinetic analysis.
  • the Glu38Lys site-directed mutant was partially purified to a specific activity of 19.7 ⁇ mol/min/mg.
  • Kinetic analysis identified an enzyme which was unchanged in its affinity for glucose- 1-phosphate, ATP, and Mg 2+ as compared to R20 (Fig. 5) .
  • Activation curves showed that the Glu38Lys mutant has an A 0.5 of 2 ⁇ M, a 58.5- and 80-fold increase in affinity for 3-PGA compared to R20 and wild-type recombinant ADPG-PP, respectively (Fig.
  • ADPG-PP containing the Glu38Lys mutant large subunit is an up-regulatory mutant. Henceforth, it is referred to as "UpReg-1" (SEQ ID Nos. 1 and 2) . Phenotypically, the UpReg-1 mutant, when co-expressed with pMLlO stains, is significantly darker on a glucose media compared to the wild-type recombinant and R20 (FIG. 3) .
  • R32 which contained a GlylOl Asn mutation (SEQ ID Nos. 9 and 10) .
  • Substrate and cofactor binding constants were also relatively unaffected for R32 AGPase (Fig. 5) .
  • R32 was more sensitive to 3-PGA activation, showing an A 0 . 5 of 414 ⁇ M, an 11-fold increase over the Pro52Leu mutant.
  • Pi sensitivity was also lower in R32 AGPase (Figs. 6 and 7) but the I 0 . 5 values for this second-site revertant were more similar to the values determined for the WT recombinant enzyme than for the other second-site revertants.
  • substitution of the polar amino acid Asn for the nonpolar Gly restored 3-PGA sensitivity in R32 while only slightly changing the enzyme's affinity for Pi .
  • UpReg-2 (SEQ ID Nos. 3 and 4), displays up-regulatory properties.
  • 3-PGA activation studies of the partially purified enzyme yielded an A 0 . 5 of 0.035 mM, an 11.8- and 4.6-fold enhancement over the parental revertant R32 and WT.
  • the increase in affinity for 3-PGA was less pronounced for UpReg-2 than for UpReg-1, the response to Pi was more dramatic for UpReg-2 than for UpReg-1 .
  • Pi inhibition studies in the presence of 0.1 and 0.001 mM 3-PGA revealed I 05 constants of 6.2 and 1.3 mM, respectively. Hence, UpReg-2 shows a higher resistance to Pi inhibition than UpReg-1.
  • a DNA fragment encoding a truncated N-terminal peptide missing the first 17 amino acids of the large subunit was amplified using PCR with the primers shown in FIGS. 51 and 5J.
  • the PCR product was purified, digested with Ncol and Nhel, subcloned into pML7 (Fig. 1) and expressed in the E. coli strain JM101
  • the resulting truncated large subunit was co-expressed with the wild-type small subunit in E. coli to form the ⁇ N17-LS enzyme.
  • the small subunit was expressed at 109% and the large subunit at 114% of the wild-type level, as quantified by ELISA.
  • the ⁇ N17-LS enzyme was partially purified to a final specific activity of 20 units/mg under standard assay conditions and estimated to be approximately 51% pure based on video image analysis of a purified protein fraction on Commassie Brilliant Blue-stained polyacrylamide gels.
  • Kinetic analysis of the partially purified ⁇ N17-LS enzyme showed no effect on the binding constants for the substrates ATP and glucose-1-P, while the enzyme's affinity for the co-factor Mg + increased slightly (Fig.
  • this 17 amino-acid N-terminal region of the large subunit is essential for proper allosteric regulation, since its removal increases the enzyme's sensitivity to the allosteric activator 3-PGA 10-fold and decreases its sensitivity to the allosteric inhibitor Pi 5- to 16-fold.
  • ADPG-PP up-regulatory allosteric large subunits of ADPG-PP in photosynthetic tissues of Arabidopsis increases vegetative growth rate and seed yield.
  • the allosteric regulatory properties of the ADPG-PP enzymes formed from the mutant large-subunit sequences together with normal small-subunit sequences are shown in Fig. 7.
  • Sensitivity to the activator 3-PGA ranges from 0.002 mM (upRegl) , 0.14 mM (wild-type: "W.T.”), to 4 mM (M345) . Since the enzyme is also inhibited by Pi, a more accurate view of the differences in the allosteric regulation of the mutant enzymes is the I 0.5 /[3-PGA] ratio, where I 0.5 is the amount of Pi required to produce 50% inhibition of enzyme activity (I 0 . 5 ) at a known concentration of 3-PGA.
  • I 0-5 /[3-PGA] ratios range from a value of 24 for UpRegl to 1.4 for the W.T. enzyme to 0.6 for the down-regulatory mutant M345.
  • the up-regulatory type enzymes have a higher affinity for the activator 3-phosphoglyceric acid (3-PGA) and/or higher resistance to the inhibitor inorganic phosphate (Pi) than the W.T. enzyme.
  • Expression of these up- regulatory ADPG-PP enzymes results in an increased production of glycogen (starch-like) in Escherichia coli , and it is expected that expression of these gene sequences in higher plants would increase starch biosynthesis and levels in cells capable of starch accumulation.
  • ADPG-PP up-regulated ADPG-PP in leaves may have deleterious consequences on plant growth and development because the allosteric regulatory properties of ADPG-PP are believed to be one of several important processes that are responsible for controlling the distribution of fixed carbon into sucrose and starch in leaves and other photosynthetic competent tissues.
  • One hypothesis (Eichelmann and Laisk, Plant Physiol . 106:679-687, 1994) is that the bulk of the fixed carbon is converted into sucrose and that starch is made only when the rate of sucrose synthesis is saturated. Under conditions where sucrose synthesis is saturated, the ratio of activator to inhibitor (3 -PGA/Pi) is high enough to activate ADPG-PP, thereby allowing starch synthesis to occur.
  • ADPG-PP In the dark, the 3 -PGA/Pi ratio is low, which suppresses the enzyme activity of ADPG-PP and, in turn, limits starch synthesis.
  • the normal allosteric regulatory properties of ADPG-PP account for the diurnal oscillation of starch synthesis during the day and net breakdown of starch at night. Based on this hypothesis regarding the critical role of ADPG-PP in leaf starch metabolism, it is expected that the expression of unregulated and up-regulated forms of ADPG-PP in leaf cells would drastically alter the normal partitioning of fixed carbon between sucrose and starch and cause much higher starch synthesis to occur. These events, in turn, would reduce sucrose availability to the remainder of the plant which could disrupt normal plant growth and development processes. Stark et al .
  • the 5' primer contained a XhoI-Xbal-BamHI sequence 5' (underlined) to the atslA nucleotide sequence beginning with base -1701 from the translation start of the atslA gene.
  • the 3' primer had a Ncol-Sacl sequence (underlined) at the complementary nucleotide located at +164 from the translation start.
  • the amplified 1897-nucleotide DNA fragment was ethanol precipitated, resuspended in TE, and digested with Xhol and Sacl .
  • the digested DNA was then resolved by agarose gel electrophoresis, and then purified by binding and elution on DEAE-membrane filters.
  • the purified DNA fragment was then collected by ethanol precipitation and then cloned into the Xhol and Sacl sites of pBluescript II to give pHI-10 (Fig. 6) :
  • the coding sequences from UpRegl, R4 , R20, R32, M27, M345, and wild-type large-subunit sequences were removed from the plasmid DNA (Iglesias et al . , J. Biol . Chem . 268:1081-1086, 1993) by digestion with Ncol and Sacl and the resulting DNA fragment cloned into the relevant restriction sites of pHI-10 to give pHI-11 to pHI-17 (Fig. 11) .
  • AtslA-potato large subunit cassettes contained within a Xbal/Sacl DNA fragment were then cloned into the Xbal and Sacl sites of the T-DNA binary vector pHI- 32, a derivative of pIG-121 (Ohta et al . , Plant Cell
  • EXAMPLE 6 Transformation into Arabidopsis TL46 The pHI-33 to -39 series of plasmid DNAs were transferred into Agrobacterium GV3101 using standard methods (An, Methods Enzymol . 153:292-305, 1987). These
  • TL-46 is a starch- deficient line (Lin et al . , Plant Physiol . 99:1175-1181,
  • Arabidopsis plants containing pHI-33 to pHI-39 were evaluated for their growth properties .
  • transgenic plants expressing up-regulatory AGPase (UpRegl and R4) and wildtype (R20 and WT) type AGPases grew considerably faster than control plants (see Fig. 10 and Fig. 13) .
  • UpReg-1 and R4 produced larger quantities of seed at the end of 8 weeks of growth than normal plants (Fig. 8) .
  • phenotypic traits expressed by these transgenic plants are striking (Figs. 8 and 13) . They include higher rates of development and growth, and variation in leaf size, seed weights, and seed yields. None of these phenotypes are strictly inter-related although there appears to be several general trends which can be summarized as follows:

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Abstract

Up-regulated allosteric mutants of plant ADPG-PP enzymes having (1) higher sensitivity to allosteric activators (2) lower sensitivity to allosteric inhibitors; (3) increased starch production; (4) increased yield; (5) increased plant size; (6) increased growth rate and (7) increased number of seeds, and methods for efficiently producing and identifying such mutants.

Description

REGULATORY MUTANTS OF ADP-GLUCOSE PYROPHOSPHORYLASE AND RELATED COMPOSITIONS AND METHODS
FIELD OF THE INVENTION This invention relates to starch biosynthesis in plants, particularly to regulatory mutants of ADPglucose pyrophosphorylase .
BACKGROUND The regulation of starch biosynthesis. The first committed step in the synthesis of starch is the formation of ADPglucose, the substrate utilized by starch synthase . ADPglucose formation is catalyzed by the enzyme ADPglucose pyrophosphorylase (ADPG-PP) . In many plant tissues, ADPG-PP is subject to allosteric control by small effector molecules, being activated by 3-phosphoglycerate (3 -PGA) and inhibited by inorganic phosphate (Pi) . It has been proposed that starch synthesis is controlled by the allosteric behavior of ADPG-PP (Preiss, In: The Biochemistry of Plants, Preiss, ed., New York: Academic Press, pp. 181-254, 1988; Preiss et al . , "Prospects for the production of cereals with improved starch properties, " In: Proceedings of the
Improvement of Cereal Quali ty by Genetic Engineering, Henry and Ronalds, eds., Plenum Press, 1994) .
Several lines of evidence support the allosteric regulation of ADPG-PP as the dominant control of starch biosynthesis in leaf tissue. Several studies have evaluated the effect of Pi levels or the ratio of 3 -PGA to Pi on the rate of starch biosynthesis in isolated chloroplasts or leaf discs (Heldt et al . , Plant Physiol .
59:1146-1155, 1977; Preiss, In: Oxford Surveys of Plant
Molecular and Cellular Biology, Vol. 7, Miflin, ed.,
Oxford: Oxford Univ. Press, pp. 59-114, 1992; Preiss, In: The Biochemistry of Plants, Preiss, ed. , New York: Academic Press, pp. 181-254, 1988; Preiss, Ann. Rev.
Plant Physiol . 33:432-454, 1982; Preiss, Biochem. Soc .
Trans . 19:539-547, 1991; Preiss et al., In: Tailoring
Genes for Crop Improvement : An Agricul tural Perspective, Kosuge et al . , eds., Plenum Press, pp. 133-152, 1987;
Preiss et al . , In: Biocatalysis in Agricul tural
Biotechnology, hitaker and Sonnet, eds., American
Chemical Society, pp. 84-92, 1989) . Theoretical modeling studies show a direct correlation between the relative levels of the activator 3 -PGA in modulating ADPG-PP activity and, in turn, the rate of starch synthesis (Petterson and Ryde-Petterson, J". Biochem
179:169-172, 1989). More recent studies on the biochemical responses of plant mutants that are defective in carbon metabolism also support a regulatory role for ADPG-PP in starch synthesis (Krukeberg et al . ,
Biochem. J. 261:457-467, 1989; Neuhaus, Planta 178:110-
112, 1989) . In particular, a Chlamydomonas starch mutant appears to contain an ADPG-PP that is defective in its activation by 3-PGA (Ball et al . , Planta 185:17-
26, 1991) .
The structure of higher plant ADPG-PPs. The bacterial ADPG-PPs from both Escherichia coli and
Salmonella typhimurium are encoded by a single gene locus, glgC (Preiss et al . , In: Biocatalysis in
Agricul tural Biotechnology, Whitaker and Sonnet, eds.,
American Chemical Society, pp. 84-92, 1989), which encodes a subunit of 48 kD that aggregates to form a homotetramer of 200 kD (Ghosh and Preiss, J. Biol . Chem . 241:4491-4504, 1966) . The higher plant enzyme is comprised of two distinct subunits, large subunit and small subunit, encoded by unique genes (Bae et al . ,
Maydica 35:317-322, 1990; Bhave et al . , Plant Cell 2:581-588, 1990; Copeland and Preiss, Plant Physiol .
68:996-1001, 1981; Lin et al . , Plant Physiol . 99:1175-
1181, 1988; Lin et al . , Plant Physiol . 86:1131-1135,
1988; Morell et al . , Plant Physiol . 85:185-187, 1987; Okita et al . , Plant Physiol . 93:785-790, 1990; Plaxton and Preiss, Plant Physiol . 83:105-112, 1987; Smith-White and Preiss, J. Mol . Evol . 34:449-464, 1992).
A number of genes that code for the large and small subunits have been isolated from a diverse group of plants (Ainsworth, Plant Mol . Biol . 23:22-33, 1993,
Anderson et al . , J. Biol . Chem . 264:12238-12242, 1989;
Anderson et al . , Gene 97:199-205, 1991; Bae et al . ,
Maydica 35:317-322, 1990; Bhave et al . , Plant Cell
2:581-588, 1990; Jardin and Berhin, Plant Mol . Biol . 16:349-351, 1991; La Cognata et al . , Mol . Gen . Genet .
246:538-548, 1995; Muller-Rober et al . , Mol . Gen . Genet .
224:136-146, 1990; Muller-Rober et al . , Plant . Mol .
Biol . 27:191-197, 1995; Nakata et al . , J. Biol . Chem.
269:30798-30807, 1994; Nakata et al . , Plant Mol . Biol . 17:1089-1093, 1991; Shaw and Hannah, Plant Physiol .
69:1214, 1992; Smith-White and Preiss, J. Mol . Evol .
34:449-464, 1992; Villand et al . , Plant Mol . Biol .
19:381-389, 1992; Villand et al . , Plant Physiol .
100:1617-1618, 1992; Villand et al . , Plant Mol . Biol . 23:1279-1284, 1993). The large and small subunit sequences display about 30-55% amino acid identity. The primary sequences of the small subunits are more conserved between species (>90% identity) than the primary sequences between large and small subunits within a species (Nakata et al . , Plant Mol . Biol .
17:1089-1093, 1991; Smith-White and Preiss, J. Mol .
Evol . 34:449-464, 1992). This' is consistent with immunological data that demonstrated that antibodies raised against the spinach leaf small subunit but not to the large subunit cross-react with small subunits isolated from a number of different plants (Krishnan et al . , Plant Physiol . 81:642-645, 1986; Okita et al . , Plant Physiol . 93:785-790, 1990).
These subunits are encoded by multiple genes that are expressed in specific tissues of the plant (Krishnan et al . , Plant Physiol . 81:642-645, 1986; La Cognata et al . ,
Mol . Gen . Genet . 246:538-548, 1995; Olive et al . , Plant Mol . Biol . 12:525-538, 1989; Smith-White and Preiss, J.
Mol . Evol . 34:449-464, 1992; Villand et al . , Plant Mol .
Biol . 19:381-389, 1992; Villand et al . , Plant Physiol .
100:1617-1618, 1992; Villand et al . , Plant Mol . Biol .
23 :1279-1284, 1993). In potato, three large subunit sequences have been isolated that differ in their spatial and temporal patterns of expression (La Cognata et al . , Mol . Gen . Genet . 246:538-548, 1995). Two of the genes are expressed in leaves and tubers, while a third is expressed solely in tubers. The three large subunit sequences share from 68% to 75% identity at the amino acid level .
Structure-function relationships of the plant
ADP-glucose pyrophosphorylase. Using chemical labeling and photoaffinity labeling strategies, three residues in the bacterial enzyme have been identified to be important in binding of the allosteric effectors or substrates. Lys39 is located near the activator binding site (Parsons and Preiss, J. Biol . Chem . , 253:7638-7645,
1978) , Lys195 participates in binding of the substrate glucose-1-phosphate (Glc 1-P) (Hill et al . , J". Biol .
Chem. 266:12455-12460, 1991), while Tyr114 is located at or near the ATP/ADPglucose binding site (Lee and Preiss, J. Biol . Chem. 261:1058-1064, 1986). Lys39 and Lys195 are conserved in all small subunits of the plant enzyme, while a Phe is present in the corresponding Tyr114 position in the plant's large subunit. The conservation of the two Lys residues suggest that they play a similar role in the plant enzymes. This has been confirmed by replacement of the corresponding Lys195 residue in the potato small subunit with a glutamate by site-directed mutagenesis experiments (Preiss and Sivak, "Starch synthesis in sinks and sources," In: Photoassimila te
Distribu tion in Plants and Crops : Source-Sink
Relationships, Zamski and Schaffer, eds., Marcel Dekker, Inc., 1995) . This mutation increases the binding constant (Kra) of the enzyme for glucose-1-phosphate from 80 μM to over 45 mM without any effect on the kinetic parameters for the other substrates .
The role of specific amino-acid residues for allosteric regulation and catalysis for ADPG-PP from higher plants is being studied using a similar chemical labeling strategy. The chemical probe pyridoxal phosphate, which mimics the activator, 3-PGA, labels both the large (54 kD) and small (51 kD) subunits of the spinach leaf enzyme (Ball and Preiss, J. Biol . Chem.
269 : 24706-24711, 1994; Morrell et al . , J. Biol . Chem.
263:633-637, 1988). When pyridoxal phosphate is covalently bound, ADPG-PP no longer requires 3-PGA for maximal enzyme activity, suggesting that pyridoxal phosphate is bound at the activator site. Only a single reactive Lys, located near the C-terminus, is observed for the small subunit (Morell et al . , J. Biol . Chem.
263:633-637, 1988). In contrast, three Lys residues are phosphopyridoxylated in the spinach leaf large subunit (Ball and Preiss, J. Biol . Chem . 269:24706-24711, 1994).
Two Lys residues are located near the C-terminus. One Lys is aligned with the conserved Lys labeled in the small subunit. A second reactive Lys residue is positioned 38 residues away from the former on the N-terminal side of the primary sequence. The third reactive Lys is located about 115-120 residues from the N-terminus of the large subunit sequence and is not conserved in the small subunit sequence.
Using a random mutagenesis strategy, Green et al .
( Proc . Na tl . Acad . Sci . USA 93:1509-1513, 1996) have shown that a mutation at Pro52 of the large subunit, when co-expressed with the wild-type small subunit, results in the formation of an ADPG-PP enzyme that is defective in allosteric regulation, requiring 45-fold greater levels of the activator 3-PGA than the wild-type enzyme. Pro52 is part of the sequence motif, PAV, that is conserved in all known ADPG-PPs (Smith-White and Preiss, J. Mol . Evol . 34:449-464, 1992). Four residues away from the PAV in the E. coli enzyme is Lys39, a residue that is located at or near the activator binding site in the bacterial enzyme. The small subunit has a Lys at the equivalent position of Lys39, while the large subunit has an Arg, indicating a conservation of charge. The PAV motif also appears to be important for the allosteric response in the bacterial enzyme. When the Ala residue is replaced by a Thr, the resulting mutated enzyme has a greatly reduced affinity for the activator fructose-1, 6-diphosphate . Overall, the results from chemical labeling and random mutagenesis studies indicate that sequences located at both the N- and C-terminal regions are required for allosteric regulatory behavior of higher plant ADPG-PP.
Roles of the large and small subunits in enzyme function. Under certain growth conditions, the small subunit alone can assemble to form a homotetrameric enzyme when expressed in E. coli (Ballicora et al . ,
Plant Physiol . 109:245-251, 1995). The homotetrameric small subunit enzyme exhibits values of Km for glucose 1-phosphate, ATP and Mg2+ ions that are similar to those of the wild-type heterotetrameric enzyme. The homotetrameric small subunit enzyme, however, requires at least 15-fold greater amounts of 3-PGA for 50% activation than the wild-type enzyme. In contrast, the large subunit alone is unable to assemble into a catalytically active enzyme. These observations suggest that the two subunit types possess distinct roles in the functioning of this enzyme. The small subunit may play a more dominant role in catalysis, while the large subunit may increase the sensitivity of the small subunit to activation by 3-PGA. Results from random mutagenesis (Greene et al . , Proc . Na tl . Acad . Sci . USA 93:1509-1513, 1996; Greene et al . , Plant Physiol .
112:1315-1320, 1996; Laughlin et al . , Phytochem . 47:621-
629, 1998) of the large and small subunit sequences support this view. Most large subunit mutants that display high enzymatic activity are defective in allosteric regulation, while high-activity small subunit mutants are mainly defective in binding of the substrates glucose 1-phosphate and/or ATP.
ADPG-PPs of Seeds and Tubers. In addition to the genes that encode the leaf ADPG-PP, cereals and peas possess a second set of genes that encode a seed-specific form. For example, the maize endosperm-specific ADPG-PP is composed of two distinct subunits encoded by the Bt2 and Sh2 loci. Immunoblot analysis of protein extracts revealed that the bt2 mutant lacked a 55-kD species, while the sh2 mutant lacked a 60- kD species (Preiss et al . , Plant Physiol . 92:881-885,
1990) . The identification of the maize ADPG-PP subunits was substantiated by structural analysis of Sh2 and Bt2 genes, which revealed substantial homology to known ADPG-PP sequences from both plants and bacteria (Bae et al . , Maydica 35:317-322, 1990; Bhave et al . , Plant Cell
2:581-588, 1990; Smith-White and Preiss, J". Mol . Evol .
34:449-464, 1992). Based on these results, Bt2 and Sh2 contain the structural gene sequences for the maize endosperm ADPG-PP small and large subunits, respectively.
In contrast to the absolute dependence of the leaf ADPG-PP for the activator 3-PGA, ADPG-PPs from storage tissues display variable allosteric responses in vi tro .
ADPG-PPs from maize endosperm (Plaxton and Preiss, Plant
Physiol . 83:105-112, 1987) and potato tubers (Sowokinos and Preiss, Plant Physiol . 69:1459-1466, 1982) are absolutely dependent on 3-PGA for maximum enzyme activity. Substantial enzyme activity is evident only when the enzyme is assayed in the presence of 3-PGA, yielding more than a 25-fold activation over levels observed in the absence of activator. Likewise, the rice endosperm enzyme displays allosteric activation by 3-PGA, although the level of activation (5-fold) is much less than the maize enzyme. In contrast, ADPG-PPs from barley (Kleczkowski et al . , Plant Physiol . 101:179-186,
1993), wheat endosperm (Duffus, Biochem. Soc . Trans .
20:13-18, 1992; Olive et al . , Plant Mol . Biol . 12:525- 538, 1989) , and pea embryos (Hylton and Smith, Plant
Physiol . 99:1626-1634, 1992) display little or no allosteric regulation. In each instance, substantial enzyme activity was evident even in the absence of 3-PGA, and the enzyme was not significantly activated by 3-PGA or inhibited by Pi, suggesting that ADPG-PPs from these developing seeds may not be subject to allosteric control, unlike leaf ADPG-PPs.
Alternatively, the lack of allosteric response exhibited by the barley, wheat and pea seed enzymes may not be an intrinsic property of these enzymes but rather a result of a post-translational modification of the enzyme. Post-translational-induced changes in allosteric properties of these ADPG-PPs is a likely possibility, as suggested by studies of the maize endosperm enzyme. When maize endosperm extracts are prepared in the absence of proteinase inhibitors, a substantial level of enzyme activity is observed even in the absence of the activator 3-PGA. In addition, the sensitivity to allosteric effectors was observed to be much less than the leaf form. Only a two- to three-fold activation is detected under optimal conditions as opposed to the 20- to 30-fold evident for the leaf enzyme (Dickinson and Preiss, Arch . Biochem . Biophys .
130:119-128, 1981). ADPG-PP enzyme from maize endosperm has been found to be highly susceptible to proteolysis (Plaxton and Preiss, Plant Physiol . 83:105-112, 1987).
Incubation of crude extracts at 30°C resulted in the degradation of the Bt2 (55 kD) subunit to a 53 kD entity as viewed by immunoblot analysis using antibody raised against the spinach leaf enzyme. When the enzyme was purified in the presence of protease inhibitors, it was shown to have allosteric and physical properties similar to those of leaf ADPG-PPs. In contrast to the proteolytically cleaved enzyme which displays very little allosteric response, the intact enzyme was activated by about 25-fold by 3-PGA and this activation was suppressed by Pi .
An important conclusion drawn from the study of the maize endosperm enzyme (Plaxton and Preiss, Plant
Physiol . 83:105-112, 1987) is that small structural changes of the native enzyme result in significant changes in the catalytic and allosteric properties of enzyme function. Such proteolytic-induced changes are also evident for the recombinant potato enzyme expressed in E. Coli (Iglesias et al . , J". Biol . Chem . 268:1081- 1086, 1993) and are believed to be responsible for the distinct properties exhibited by the barley and wheat endosperm enzyme activities as well as those from pea embryos (Preiss et al . , "Prospects for the production of cereals with improved starch properties," In: Improvement of Cereal Quali ty by Genetic Engineering,
Henry and Ronalds, eds., Plenum Press, 1994) . Expression of a recombinant potato tuber enzyme whose small subunit is lacking about nine residues of the N-terminus results in an enzyme that is less stable to heat denaturation and is only sensitive to Pi inhibition in the presence of 3-PGA. Restoration of these nine residues on the small subunit results in the formation of a recombinant enzyme that is stable toward heat treatment at 60 °C and more sensitive to Pi inhibition. As mentioned above, the barley endosperm enzyme displays almost no activation by 3-PGA and is only weakly inhibited by Pi (Kleczkowski et al . , Plant
Physiol . 101:179-186, 1993). Immunoblot analysis of crude extracts indicated, however, that even at 4°C, the barley large subunit was degraded first to a 53 kD and then later to a 51 kD species with a half-life on the order of minutes. In view of the distinct changes in allosteric and catalytic properties mediated by the structural changes of the maize endosperm and the recombinant tuber enzymes, the distinct allosteric properties exhibited by the barley endosperm enzyme (and wheat and pea as well) are likely due to proteolysis of one or both subunits (Preiss et al . , "Prospects for the production of cereals with improved starch properties," In: Proceedings of th Improvement of Cereal Quali ty by Genetic Engineering, Henry and Ronalds, eds., Plenum
Press, 1994) .
Irrespective of the in vi tro allosteric properties displayed by the seed and tuber enzymes, questions have been raised regarding the functional significance of this control mechanism in vivo . Unlike leaf tissue, where starch metabolism must be tightly regulated so that synthesis occurs during the day and is degraded at night, synthesis and degradation of starch in seeds and tubers are temporally separated to distinct stages of plant development. Therefore, allosteric control of ADPG-PP during the diurnal cycle is not an essential requirement for starch synthesis in non-photosynthetic sink tissues. Indeed, the inherent allosteric property of ADPG-PP may obstruct starch synthesis as the levels of the activator 3-PGA are likely to be much lower than the inhibitor Pi in amyloplasts due to the unique biochemistry of this organelle. Unlike the more autonomous chloroplast, which generates its own carbon and energy via C02 fixation and photophosphorylation (which result in a production of 3-PGA and ATP and a corresponding reduction in Pi) , the amyloplast is dependent on the cytoplasm for these metabolic requirements .
These biochemical differences are also reflected in the permeability properties of chloroplasts and amyloplasts. In chloroplasts, the principal metabolite transport system between the chloroplast and cytoplasm is the Pi translocator, which transports triose phosphate and 3-PGA to the cytoplasm in exchange for Pi. In contrast, in non-photosynthetic sink organs, carbon is imported in the form of sucrose which is subsequently processed into hexose-phosphate, which is transported into the amyloplast to be directly utilized as a substrate (Glc-l-P) by ADPG-PP (Heldt et al . , Plant
Physiol . 95:341-343, 1991; Hill and Smith, Planta
185:91-96, 1991; Keeling et al . , Plant Physiol . 87:311- 319, 1988; Kosegarten and Mengel, Physiol . Plant .
91:111-120, 1994; Okita, Plant Physiol . 100:560-564,
1992; Tyson and ap Rees, Planta 175:33-38, 1988) or is converted into ADPglucose in the cytoplasm by a cytoplasmically-localized ADPG-PP (Denyer et al . , Plant Physiol . 112: 779-785, 1996; Thorbjonsen et al . , Plant
J. 10: 243-250, 1996). The ADPglucose is then transported to the plastid where it is then utilized by starch synthase .
These proposed pathways and the growing evidence that amyloplasts lack intact glycolytic and gluconeogenic pathways (Entwistle and ap Rees, Biochem. J. 271:467-472, 1990; Frehner et al . , Plant Physiol .
94:538-544, 1990) indicate a limited role for triose-phosphate in amyloplastic starch biosynthesis. Although 3-PGA is probably not a major metabolite in the amyloplasts, Pi is likely a major metabolite because it is a direct byproduct of the ADPG-PP reaction (in conjunction with inorganic pyrophosphatase) , resulting in low 3 -PGA/Pi ratios. Under these conditions, the allosteric properties of the maize and rice endosperm and tuber ADPG-PP enzymes suppress the catalytic activities of these enzymes and, in turn, constrain starch synthesis in these plants.
Evidence in support of this view has been obtained in tubers. First, tuber disks incorporate 14C-labeled sucrose into starch at 50% higher rates in the presence of mannose, which sequesters the inhibitor Pi (Hnilo and Okita, Plant and Cell Physiol . 30:1007-1010, 1989).
Second, expression of an allosteric E. coli mutant of
ADPG-PP results in an enhancement of starch synthesis in tubers (Stark et al . , Science 258:287-292, 1992).
Third, a maize line, Sh2 -mlRev6, which bears seeds with weight increases of 10-18% higher than normal lines, encodes a ADPG-PP large subunit that contains two additional amino acids near the C-terminus (Giroux et al . , Proc . Na tl . Acad . Sci . USA 93:5788-5792, 1996), a peptide region known to be essential for allosteric regulation. The resulting variant ADPG-PP enzyme appears to be resistant to Pi inhibition, suggesting that the altered allosteric response may be responsible for increase seed weight. Overall, these results support the view that starch synthesis has not reached it highest potential in developing harvestable sink organs, i.e. tubers, seeds and fruit, and that expression of allosteric mutant plant ADPG-PPs would increase starch synthesis and, in turn, plant yields. It would be desirable and advantageous to produce a mutant form of the ADPG-PP enzyme wherein a plant expressing such a gene would have increased yield and increased plant size. In addition to having a large impact on the degree of starch synthesis and, in turn, yield in developing storage organs such as developing seeds, tubers, fruit etc., ADPG-PP activity may also have an important role in increasing overall productivity of the plant by maximizing rates of carbon dioxide (C02) fixation and utilization in photosynthetic tissues such as leaves, stems etc. In general, C02 fixation rates in photosynthetic tissues is affected by the capacity of converting fixed C02 into carbohydrates such as sucrose and starch. Recent evidence indicates that leaf starch plays a broader role than simply serving as a diurnal reserve of carbon and energy to enable the plant to survive during the dark period. Ludewig et al . (FEBS
Lett . 429:147-151) have shown that there is a direct correlation between the capacity of starch synthesis and the rate of photosynthesis at elevated C02. Likewise, recent evidence from J. Sun, G.E. Edwards, and T.W. Okita (unpublished) showed that there are significant correlations between the rates of starch synthesis and C02 assimilation, and between the rates of starch synthesis and accumulative leaf area. These results indicate that leaf starch plays an important role as a transient "sink" in which synthesis can ameliorate potential reduction in photosynthesis due to feedback regulation.
SUMMARY OF THE INVENTION
Applicants have discovered methods for generating and identifying up-regulated mutants of ADPG-PP, i.e., allosterically regulated mutant ADPG-PPs having enzymatic activity that is significantly higher than an otherwise similar wild-type enzyme at physiological concentrations (i.e., about equimolar levels) of 3-PGA and Pi (Heldt et al . , Plant Physiol . 59:1146-1155,
1977) . The methods of the invention are more efficient than any previous empirical methods involving mutagenesis and mass biochemical screening of mutants. Applicants have generated such ADPG-PP mutants and have analyzed the enzymatic activity of the mutant ADPG- PP enzyme. Applicants have discovered that, in comparison to the wild-type enzyme, the mutant enzyme differs in that it has: (1) higher sensitivity to the activator 3-PGA; (2) lower sensitivity to the inhibitor Pi; (3) increased starch production; (4) increased yield; (5) increased plant size (especially leaves) ; and, surprisingly, (6) increased growth rate and (7) increased number of seeds.
According to one aspect of the invention methods are provided for producing and identifying nucleic acids that encode up-regulated mutant ADPG-PP enzymes. For example, in a first representative embodiment of the method, the plant ADPG-PP large and small subunits are co-expressed in a bacterial cell lacking ADPG-PP activity (for example a glgC" strain) . One subunit is unmutated while the other is mutated. If the mutation affects only the allosteric regulatory properties of the assembled enzyme, then cells expressing this mutant enzyme will not accumulate glycogen under normal physiological levels of the activator 3-PGA, although they will have normal levels of ADPG-PP activity if measured in the presence of saturating levels of 3-PGA. A simple means for assessing glycogen levels, which can be correlated with ADPG-PP anzyme activity levels in a bacterial cell, is by exposing the cells to iodine vapor. Glycogen-accumulating cells stain brown to purplish black while cells lacking this carbohydrate stain light yellow. Upon determining that the mutated subunit affects only the allosteric regulatory properties of the assembled enzyme, the mutated subunit is again mutagenized to produce a second mutation which will restore the allosteric regulatory properties of the assembled enzyme and the bacterial cell ' s capacity to accumulate glycogen which can be readily detected by iodine staining. A subunit that contains only the second mutation but not the first mutation is then studied to determine the effect of this second mutation on ADPG-PP function, for example, whether the second mutation is an up-regulatory mutation.
An alternative embodiment of a method for the generation and identification of up-regulated ADPG-PP mutants is by direct screening methods. However, in order to directly detect up-regulated mutants, the plant ADPG-PP subunits must be expressed on low-copy-number plasmids. Alternatively, high-copy-number plasmids can be used to co-express the plant ADPG-PP subunits; but, the bacterial cells must be grown on enriched media (Govons et al . , J". Bacteriol . 97:970-972, 1969) containing low amounts of glucose (0 to 0.5%) as compared to the normal 2%. When grown on enriched media containing 0.1% glucose, cells expressing up-regulated ADPG-PP readily stain with iodine vapor whereas cells expressing the wild-type unmutated enzyme do not.
A third representative method for generating and identifying up-regulated ADPG-PP mutants involves generation of segmented mutations. Nucleotides encoding ADPG-PP are truncated such that the expressed proteins are truncated at either the N- or C- terminus. These mutants are screened for altered allosteric function and up-regulated mutants are identified by methods described herein.
According to another aspect of the invention, nucleic acids are provided that encode an up-regulated mutant ADPG-PP enzyme, for example, a plant mutant ADPG- PP enzyme. Such a nucleic acid may be made and identified by any of the methods of the invention.
Alternatively, once a particular mutant sequence has been identified, the nucleic acid may be made by chemical in vi tro synthesis, such as by using an automated polynucleotide synthesizer, or may be made using site-directed mutagenesis. The mutant ADPG-PP nucleic acid codes for an enzyme that is up-regulated, and therefore has a biological activity (i.e., an enzyme activity) that is higher than the biological activity of the wild-type enzyme under physiological conditions.
According to yet another aspect of the invention, enzymes are provided that are encoded by the nucleic acids of the invention, for example, a plant mutant ADPG-PP enzyme. Such enzymes are up-regulated mutant enzymes. For instance, such a mutant enzyme may be more sensitive to an allosteric activator, such as 3-PGA, and may be more resistant (i.e., less sensitive) to an allosteric inhibitor such as Pi. Up-regulated mutant enzymes may be produced by a number of methods including, but not limited to, mutagenesis with chemicals such as hydrozylamine, mutagenesis with radiation, site-directed mutagenesis, PCR mutagenesis, or genetic engineering techniques such as N-terminal truncation of the enzyme.
According to yet another aspect of the invention transgenic plants are provided that comprise the mutant nucleic acids of the invention. Expression of up- regulated ADPG-PP mutant genes in transgenic plants can result in, for example, increased starch production in at least a portion of the plant (compared with the wild- type) and can also increase productivity, yield, growth rate, and seed number.
According to yet another aspect of the invention specific nucleic acids as recited in the specification are provided.
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings .
SEQUENCE LISTING
SEQ ID NO:l shows the nucleotide sequence of the mutant ADPG-PP large subunit UpRegl that, when co- expressed with a wild-type ADPG-PP small subunit, forms an up-regulated ADPG-PP enzyme.
SEQ ID NO: 2 shows the primary amino acid sequence of UpRegl . SEQ ID NO: 3 shows the nucleotide sequence of the mutant ADPG-PP large subunit UpReg2 , that when co- expressed with a wild-type ADPG-PP small subunit, forms an up-regulated ADPG-PP enzyme.
SEQ ID NO : 4 shows the primary amino acid sequence of UpReg2.
SEQ ID NO: 5 shows the nucleotide and amino acid sequences of ΔN17-LS, a mutant ADPG-PP large subunit that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up-regulated ADPG-PP enzyme. SEQ ID NO : 6 shows the primary amino acid sequence of ΔN17-LS.
SEQ ID NO : 7 shows the nucleotide sequence of the mutant ADPG-PP large subunit R20 that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up- regulated ADPG-PP enzyme.
SEQ ID NO : 8 shows the primary amino acid sequence of R20.
SEQ ID NO: 9 shows the nucleotide sequence of the mutant ADPG-PP large subunit R32 that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up- regulated ADPG-PP enzyme.
SEQ ID NO: 10 shows the primary amino acid sequence of R32.
SEQ ID NO: 11 shows the nucleotide sequence of the mutant ADPG-PP large subunit R4 that, when co-expressed with a wild-type ADPG-PP small subunit, forms an up- regulated ADPG-PP enzyme.
SEQ ID NO: 12 shows the primary amino acid sequence of R4. SEQ ID NO: 13 shows the nucleotide sequence for an upstream primer 5 ' -GATATTGGTACCATTG-3 ' that is useful for introducing double-termination codons in an ADPG-PP small subunit cDNA sequence. The primer includes a Kpnl site .
SEQ ID NO: 14 shows the nucleotide sequence for a downstream primer that is useful for introducing double termination codons in an ADPG-PP small subunit cDNA sequence. The primer includes a Sad site. Termination codon sequences are underlined.
SEQ ID NO: 15 shows the nucleotide sequence for an upstream primer that is useful for introducing double termination codons in an ADPG-PP large subunit cDNA sequence. The primer includes an Nhel site.
SEQ ID NO: 16 shows the nucleotide sequence for an downtream primer that is useful for introducing double- termination codons in an ADPG-PP large subunit cDNA sequence. The primer includes a Sad site. Termination codon sequences are underlined.
SEQ ID NO: 17 shows the nucleotide sequence for an upstream primer that is useful for restoring the N-terminus of an ADPG-PP small subunit expression plasmid. The primer includes an Ncol site.
SEQ ID NO: 18 shows the nucleotide sequence for a downstream primer that is useful for restoring the N- terminus of an ADPG-PP small subunit expression plasmid. The primer includes a Kpnl site. SEQ ID NO: 19 shows the nucleotide sequence for an upstream primer that is useful for deleting DNA sequences that code for 17 amino acids at the N-terminus of an ADPG-PP large subunit. The primer includes an Ncol site. SEQ ID NO: 20 shows the nucleotide sequence for a downstream primer that is useful for deleting DNA sequences that code for 17 amino acids at the N-terminus of an ADPG-PP large subunit. The primer includes an Nhel site. SEQ ID NO: 21 shows the upstream primer used to amplify the Arabidopsis ribulose bisphosphate small subunit (atslA) promoter and transit leader coding sequences . SEQ ID NO: 22 shows the downstream primer used to amplify the Arabidopsis ribulose bisphosphate small subunit (atslA) promoter and transit leader coding sequences .
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structure of the plasmid pML7. FIG. 2 shows the structure of the plasmid pMLlO. FIG. 3 shows a comparison of the I2 staining patterns of AC70R1-504 cells expressing the wild-type (LS/SS) and the mutant UpReg-1, R20, and 345 ADPG-PP enzymes when grown on glucose-enriched media. Mutant 345, which contains the P52L mutation on the large subunit (LS) does not accumulate glycogen and stains a faint yellow. In contrast, wild-type (LS/SS) ADPG-PP and the mutants UpReg-1 and R20 accumulate large amounts of glycogen and stain darkly. Note the more intense staining by UpReg-1 cells as compared to the wild-type (LS/SS) and R20 cells. FIG. 4 shows a graphical comparison of I0-5 (the amount of Pi required for 50% inhibition) values at different 3-PGA concentrations for the native, recombinant wild-type (LS+SS) , and ΔN17-LS ADPG-PP enzymes. Data on the native enzyme were taken from Sowokinos and Preiss, Plant Physiol . 69:1459-1466, 1982, while data for the recombinant enzyme were taken from Ballicora et al . ( Plant Physiol . 109:245-251, 1995, and from Laughlin and Okita ( Phytochem 47:621-629, 1998) .
FIG. 5 is a table comparing substrate binding (Km) 3-PGA activator (Ao.5) affinity properties of various native and mutant ADPG-PPs.
FIG. 6 is a table comparing Pi inhibition (I0.5) of various native and mutant ADPG-PPs.
FIG. 7 is a table comparing 3-PGA activation for various native and mutant ADPG-PPs. AQ.5 is the amount of 3-PGA required to give 50% activation. I0.5 is the amount of Pi required to inhibit the enzyme 50% in the presence of a known amount of 3-PGA.
FIG. 8 is a table showing seed yields from eight greenhouse-grown plants of various plant lines (T3 generation) .
Fig. 9 shows a recombinant vector comprising the atslA-potato large subunit cassettes contained within a Xba I/Sac I DNA fragment cloned into the Xba I and Sac I sites of the T-DNA binary vector pHI-32, a derivative of pIG-121, to produce pHI-33 to pHI-39.
Fig. 10 shows third generation progeny of the transgenic Arabidopsis plants containing pHI-33 to pHI-
39 germinated and cultured on MS media. Transgenic plants expressing up-regulatory AGPase (left and right) grew considerably faster than control plants (center) . Fig. 11 shows a generalized schematic drawing of the plasmid used to construct a gene fusion between the atslA promoter and transit leader sequences to the potato ADPG-PP large subunit sequences. The coding sequences from UpRegl, R4 , R20, R32, M27, M345 and wildtype large subunit sequences were removed from the plasmid DNA by digestion with Nco I and Sac I, and the resulting DNA fragment was cloned into the relevant restriction sites of pHI-10 to produce plasmids pHI-ll to pHI-17.
Fig. 12 shows the Arabidopsis ribulose bisphosphate small subunit astlA promoter and transit leader coding sequence fragment digested with Xho I and Sac I cloned into the Xho I and Sac I sites of pBluescript II. Fig. 13 is a table comparing phenotypic properties of various transgenic plants .
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Overall yield or productivity is governed not only by a plant's capacity to fix carbon dioxide and produce energy but also by the ability of the plant to utilize this fixed carbon efficiently. For many crop plants that accumulate starch in their reserve organs, productivity is limited by the capacity of the plant to convert sugar into starch. ADPG-PP, which controls the flux of carbon into starch, is allosterically regulated. The level of ADPG-PP activity is controlled by the levels of the effectors 3-PGA and Pi, which activate and inhibit, respectively, the activity of ADPG-PP. Because there are significant levels of Pi in the cell and levels of this effector molecule are not expected to diurnally oscillate as in photosynthetic tissue, the net enzyme catalytic activity and, in turn, the net rate of starch synthesis in developing sink organs such as tubers and developing seeds, is much lower than the maximum potential rate. In addition to the above-summarized biochemical events in developing non-photosynthetic sink organs such as tubers, developing seeds, and fruits, etc., starch synthesis in photosynthetic tissues such as leaves may also impact productivity. Recent evidence by Ludewig et al. { FEBS Lett . 429:147-151) and unpublished results
(Jindong Sun, G.E. Edward and T.W. Okita) indicate that there is a direct correlation between starch synthesis and C02 fixation. These observations indicate that starch can serve as a transient "sink" to store fixed carbon in a chemical form that does not inhibit C02 fixation and photosynthesis. Because ADPG-PP is a key regulatory enzyme in controlling starch synthesis, allosteric regulatory mutant forms of this enzyme may increase leaf starch formation and, in turn, productivity.
Three approaches have now been discovered for generating and identifying variant ADPG-PPs that display up-regulated allosteric or different physical properties, e.g., temperature resistance. These allosterically up-regulated mutants require substantially less 3-PGA to attain maximum catalytic activity and/or are less susceptible to Pi inhibition than the unmutated enzyme. Mutant ADPG-PP genes are useful, for example, for altering starch production in a host organism, e.g., to increase starch production in cereal seeds, tubers, fruit, leaves and other plant organs and tissues. Yield is also increased. Surprisingly, certain mutant ADPG-PP genes, when expressed in a plant, result in increased growth rate and increased seed production.
A first approach involved the following steps:
(1) A native ADPG-PP gene was mutagenized to produce a single mutation.
(2) Mutants that are defective in enzyme function were identified by their inability to complement a mutation in the bacterial glgC gene. Cells harboring these mutant sequences do not produce glycogen and hence are not stained with iodine vapor.
(3) The mutant cells were cultured and the mutant ADPG-PP enzyme was purified. Enzyme kinetics were determined by standard methods (Cornish-Bowden, Analysis of Enzyme Kineti c Da ta , Oxford University Press, 1995) ; thus, enzymes were identified that are defective in allosteric function (Green et al . , 1996).
(4) Having been identified, the allosteric mutants were then subjected to a second round of mutagenesis to produce a double mutant . (5) Double mutant cells were then screened for staining by iodine vapor, i.e. a reversal of the iodine staining-minus phenotype that is mediated by the primary mutation.
(6) Double mutants exhibiting at least partial complementation of glgC~ cells were then sequenced to reveal the location and nature of the two mutations.
(7) Site-directed mutagenesis was then used to create a DNA containing the second (but not the first) mutation. (8) The protein product of the DNA containing the secondary site mutation was then analyzed by standard enzyme-kinetics methods (Cornish-Bowden, 1995) in the absence of the initial primary mutation to determine whether the secondary site mutation modifies the allosteric properties of the enzyme by itself. Mutants were thus identified that had increased biological activity over the wild-type enzyme and were therefore identified as. "up-regulated" mutant enzymes.
A second approach involved the direct screening method to identify up-regulated allosteric mutants. Cells containing an up-regulated mutant enzyme would be expected to have increased glycogen production.
However, using the iodine vapor staining method to assess mutations, up-regulated mutants from normal cells are not distinguishable because the iodine screening procedure is saturated. That is, although cells harboring the up-regulated mutant accumulate more glycogen than cells containing the wild-type enzyme, the up-regulated cells stain with iodine at the same rate as cells containing the wild-type enzyme. To circumvent this barrier, the ADPG-PP sequences were placed on a low copy-number plasmid. The plamids used were pWSK28 (that contains the polylinker from pBluescript Skt) and pWSK30 (that contains the polylinker from pBluescript Kst) .
Low copy-number plasmids are discussed in standard texts (e.g., Sambrook et al . , 1989) and may be obtained commercially. Low copy-number plasmids other than pWSK28 and pWSK30 include pBHOl (Jefferson et al . , EMBO
6:3901-3907, 1987) which has a wide host-range replicon.
Under normal growth conditions, the level of enzyme produced is insufficient to completely complement the glgC mutation and the cells stain only lightly with iodine vapor. On the other hand, cells containing an up-regulated mutant enzyme are more catalytically active and greater glycogen levels will be produced. As a result, cells with an up-regulated mutant enzyme stain darker with iodine.
Alternatively, instead of changing the copy number of the expression plasmids, cells expressing up- regulatory mutant ADPG-PP can be distinguished from cells containing the wild-type enzyme by altering the glucose concentration in the enriched media (Govons et al., J". Bacteriol . 97:970-972, 1969). When cultured in enriched media containing 0.1% glucose instead of the usual 2% glucose level, up-regulatory mutants will stain darkly with I2, whereas normal cells stain very lightly.
A third approach involved the generation of allosteric mutants by segmental mutations. DNA corresponding to N- and or C-terminal portions of the ADPG-PP polypeptide were deleted by recombinant DNA techniques (Sambrook et al . , 1989) . Deletions may include the removal of nucleotides corresponding to 5, 10, 15, 17, 25, 30, 40 or more amino acid residues from the N- or C- terminus.
In one embodiment of the invention, Escherichia coli expression systems were used to express the large and small subunit cDNAs of ADPG-PP under the control of bacterial promoters. For example, the large subunit cDNA was cloned into a pACYC-based cloning vector with expression of the large subunit cDNA being driven by a tac promoter, while the small subunit cDNA was cloned in a pBR325-based cloning vector with expression of the small subunit cDNA driven by a recA promoter. Examples of plasmid vectors useful for the expression of the large and small subunit cDNAs can be found, for example, in Iglesias et al . (J. Biol . Chem. 268:1081-1086, 1993).
Many suitable expression systems are commercially available, from, for instance, Invitrogen, Pharmacia and New England Biolabs.
Those skilled in the art will recognize that co- expression of the large and small subunit cDNAs can be accomplished with a wide variety of compatible hosts and cloning vectors and that both the large and small subunit genes can be cloned and expressed together on a single vector. The Examples below discuss up-regulated mutant ADPG-PP enzymes that require less than one-tenth of the level of 3-PGA than wild-type ADPG-PP for complete activation. These mutant enzymes enhance the rate of glycogen synthesis in bacteria and can increase starch synthesis in other organisms such as plants if expressed in the appropriate tissue. Expression systems that can be used to express genes in plants are widely known and commercially available. For instance, the Agrobacterium system may be used to carry out transformation using pHI-32 or pCAMBIA T-DNA plasmids, both of which are ■ derived from pBHOl. Such plasmids can be used to transform plants such as potato, rice, wheat, barley, maize, and tomato. The mutant ADPG-PP subunits generated by this invention can be used to increase yield and productivity of many crop plants that use starch as their principal reserve in their photosynthetic tissues and/or storage organs. Expression of these mutant subunits and subsequent formation of ADPG-PP enzymes with up- regulatory properties can be used to increase starch production in storage organs such as potato tubers, cassava roots, and cereal seeds including those from wheat, maize, rice, barley, rye, and sorghum. The mutant subunits can be used to increase starch production during the early development of the storage organs in oil-accumulating seed plants such as soybean, rape, and sunflower and in developing fruits of tomato, apple, pear, peach, etc. In both oil-accumulating seed plants and in fruiting plants, starch is used as a transient reserve of carbon and energy which is then re- utilized for the formation of other molecules. In oil- accumulating seed plants, starch accumulates during the early phase of seed development and is then re-utilized for the production of oils. In fruiting plants, accumulated starch is metabolized to reducing sugars, a preferred trait for tomatoes especially those used for processing ketchup, paste and the like and for the sweetness of apples, pears and other sweet fruits.
In addition to the engineering of storage organs, mutant ADPG-PP subunits generated by this invention can be used to increase overall productivity of all C3 plants (defined as those plants that use the Calvin cycle to fixed C02 into 3-PGA as their first stable three-carbon intermediate) . Examples of C3 plants include potato, cassava, wheat, rice, barley, rye, soybean, rape, sunflower, flax, cotton, alfalfa, celery, cauliflower, and carrot. C3 plants typically exhibit C02-limited photosynthesis at ambient and sub-ambient C02 partial pressures. Under ideal conditions, the rate of C3 photosynthesis increases with increasing C02 as photorespiration is suppressed. However, under many environment conditions, e.g. low temperatures, high light, and increasing C02 (see Leegood and Edwards, Photosynthesis and the Environment, N.R. Baker, ed. ,
Kluwer Academic Publ . , pp. 191-221, 1996) the rate of photosynthesis is much lower than predicted because of the limitations in triose phosphate utilization. An excellent example that illustrates this "feedback" of photosynthesis is rice. Rice grown at a photosynthetic photon flux of 1000 μM πf2 s"1 and 26°C day, 24 °C night exhibits rates of photosynthesis significantly lower than expected when shifted to low levels of 02 under moderate temperatures and ambient levels of C02 (Sun, Edwards and Okita, unpublished data) , a response indicative of photosynthetic feedback. The limitations in triose phosphate utilization can be overcome if starch synthesis is elevated in photosynthetic-competent tissues such as leaves and photosynthetic-competent tissues such as stems and seed pods, or grains. In turn, this increased photosynthesis will result in higher biomass production (productivity) and, in turn, higher yields of harvestable organs such as tuber, seeds, fruit etc. The mutant ADPG-PP subunits can be transferred into crop plants by either Agrrojbacterium-mediated transformation or by mechanical introduction, e.g., biolistics bombardment. Agrojbacterium-mediated transformation is the preferred method because of the smaller frequency of introducing multiple copies of the transgene and subsequent problems of gene expression instability due to co-suppression effects in offspring of transgenic plants (Kumpatla et al . , Plant Phsyiol . 115:361-373, 1997). Agrobacterium has been used to introduce transgenes into rice (Hiei et al., lant J. 6:271-282, 1994), maize (Ishida et al . Nature Biotechnol . 14:745-750, 1996; Gould, Plant Physiol . 95:426-434, 1991), soybean (Hinchee et al . Biotechnology 6:915-922, 1988; Stewart et al . , Plant Physiol . 112:121- 129, 1996), rape (Falco et al . , Biotechnology 13:577- 582, 1995), potato (Stark et al . , Science 258:287-292, 1992; Tu et al . Plant Mol . Biol . 37:829-838, 1998), tomato (McGarvey et al . , Biotechnology 13:1484-1487, 1995), sunflower (Bidney et al . , Plant Mol . Biol .
18:301-313, 1992), cotton (Hansen et al . , Proc . , Natl . Acad . Sci . USA 91:7603-7607, 1994), cassava (Li et al . , Nature Biotechnol . 14:736-740, 1996), apple (Maximova et al., Plant Mol . Biol . 37:549-559, 1998). Pea (Lurquin et al., Mol . Biotechnol . 9:175-179, 1998), and chickpea
(Ramana et al . , Indian J". Exp . Biol . 34:716-718, 1996) as well as lettuce, sugarbeet, celery, cucumber, alfalfa, carrot, cauliflower, horseradish, poplar, walnut and asparagus (Gasser and Fraley, Science 244:1293-1299, 1989). Wheat and barley have also been transformed by Agrobacterium (D. von Wettstein, personal communication) . An important consideration for the engineering of these plants with the mutant ADPG-PP subunits is the judicious use of promoters to drive expression of the introduced transgene at high levels in targeted tissues. Examples include the use of the patatin promoter to drive expression in potato tubers (Stark et al . , Science
258:287-292, 1992), the vicilin or kunitz trypsin inhibitor promoter to drive expression during the early stages of seed development of soybeans and other legumes (Walling et al . , Proc . Na tl . Acad. Sci . USA 83:2123-
2127, 1986; Wandelt et al . , Plant J. 2:181-192, 1992) as well as chickpea, pea, and rape, the glutelin Gtl (Okita et al., J. Biol . Chem . 264:12573-12581, 1989), the wheat high molecular weight glutenin and its related hor D gene in barley (Blechl and Anderson, Nature Biotechnol .
14:875-879, 1996), or Shrunken 2 (Shaw and Hannah, Plant
Physiol . 69: 1214-1219, 1992) promoters to drive expression in developing cereal seeds, the atslA promoter to drive expression in leaves and other photosynthetic competent tissues (Krebbers et al . , Plant
Mol . Biol . 11:745-759, 1988), the E4 or E8 promoters to drive expression in developing tomato fruit (Deikman et al., Plant Cell 1:1025-1034, 1989), and the AX92 promoter to drive expression in root cortex (Dietrich et al., Plant Cell 4:1371-1382, 1992).
Comparison with previous methods for manipulating ADPG-PP activity. Two previous approaches to manipulating ADPG-PP activity have been utilized. One approach (referred to herein as the "Monsanto approach"), utilizes a mutant bacterial ADPG-PP, glgC16, that codes for a form of ADPG-PP that lacks allosteric regulation. The glgC16 enzyme possesses 60% of the activity levels of the fully activated wild-type enzyme in the absence of the activator fructose-1, 6-diphosphate and is also less sensitive to the inhibitor adenosine monophosphate (AMP) . Transfer of this gene and expression in tubers and tomatoes results in potatoes with higher starch content (see Stark et al . , Science
258:287-292, 1992) and tomatoes with higher solids content .
A second approach utilizes the transposable element system Ac-Ds in maize to generate ADPG-PP variants. One mutant was obtained by excision of a Ds element that was initially located in the Shrunken-2 gene, which encodes the endosperm- specific form of the large subunit gene of ADPG-PP. During excision of Ds, a six-nucleotide "footprint" was left behind, resulting in a large subunit having two additional amino acids. The resulting enzyme appears to be resistant to Pi inhibition. A modified potato large subunit containing these two additional residues formed an enzyme resistant to Pi inhibition (Giroux et al . , Proc . Na tl . Acad . Sci .
USA 93:5824-5829, 1996).
The present invention differs from the Monsanto approach in that it utilizes (1) isolated plant genes for ADPG-PP instead of a bacterial gene and (2) an enzyme that is subject to allosteric regulation, although its sensitivity to activation by 3-PGA and inhibition by Pi is 20- to 40-fold higher than the wild- type enzyme. In the absence of the activator 3-PGA, the up-regulatory mutant ADPG-PPs described herein have less than 5% of the activity levels exhibited by the fully activated enzyme. In contrast, the E. coli glgC16 enzyme is essentially unregulated. Even in the total absence of the activator, it has 60% of the activity exhibited by the fully activated wild-type enzyme. In addition to differences in allosteric regulation, there are differences in the structures of the bacterial and higher plant ADPG-PPs. The bacterial enzyme is encoded by a single gene, glgC, which codes for a subunit of 50 kD molecular weight that assembles to form a homotetramer. The higher plant enzyme is composed of two large subunits and two small subunits. Each subunit type is encoded by a distinct gene. As described above, it is believed that the large and small subunits play different roles in enzyme catalysis and allosteric regulation. Under certain conditions the small subunit is able to form a homotetrameric enzyme but requires more than 24 -fold greater levels of 3-PGA for activation than the normal heterotetrameric enzyme, which is composed of two large subunits and two small subunits. The large subunit is not capable of forming an active enzyme by itself. These results indicate that the small subunit plays more of a catalytic role, while the large subunit regulates the activity of the small subunit . The present invention includes procedures for efficiently generating and identify mutations in the large subunit or small subunit of ADPG-PP that result in the formation of up-regulated ADPG-PPs. When such up- regulated mutant ADPG-PPs are expressed, for example, in leaves and/or the appropriate developing organs of transgenic cereal, potato, tomato, and other starch- accumulating plants, both starch production and yield are increased. By contrast, the Monsanto invention is only concerned with increase starch production. The Monsanto approach involves the use of random mutagenesis techniques and screening for mutants by iodine staining to generate unregulated mutants. However, the Monsanto approach would not identify unregulated mutant enzymes because the approach provides no means to distinguish unregulated mutant enzymes from the wild-type enzyme based on iodine staining. Moreover, the Monsanto approach cannot generate unregulated enzymes by mutagenesis of plant ADPG-PP sequences, since the plant enzyme is composed of two subunit types, with the large subunit increasing the sensitivity of the small subunit to the allosteric effector molecules. Definitions and Methods
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art . Definitions of common terms in molecular biology may also be found in Rieger et al . , Glossary of Genetics : Classical and Molecular, 5th edition, Springer-Verlag:
New York, 1991; and Lewin, Genes VI, Oxford University
Press: New York, 1997. The nomenclature for DNA bases as set forth at 37 C.F.R. § 1.822 is used. The standard one- and three-letter nomenclature for amino acid residues is used.
"Yield" refers to the amount of harvestable material, for instance, seeds or tubers. Yield is generally defined in units of mass.
"Productivity" refers to the total biomass produced by a plant, both harvestable and non-harvestable .
"Increased ("characteristic! compared to a wild-type plant cell" . When used in the context of plant characteristics (for instance, increased yield compared to the wild-type plant cell, increased productivity compared to the wild-type plant cell, increased starch production compared to the wild-type plant cell, increased size compared to the wild-type plant cell, increased rate of growth compared to the wild-type plant cell, or increased number of seeds compared to the wild- type plant cell) the word "increased" means that the mean value of a characteristic in a population, when compared with another (wild-type) population is measurably and statistically significantly greater than the mean value of the characteristic in question for the characteristic in the wild-type population.
"ADPG-PP nucleic acid". The term "ADPG-PP nucleic acid" refers to a native (or wild-type) nucleic acid that encodes an ADPG-PP polypeptide (including, but not limited to, cDNA and genomic sequences) and fragments thereof .
"ADPG-PP Polypeptide". The term "ADPG-PP polypeptide" refers to a polypeptide encoded by an ADPG- PP nucleic acid. An ADPG-PP polypeptide can be produced by the expression of a recombinant ADPG-PP nucleic acid or can be chemically synthesized. Techniques for chemical synthesis of polypeptides are described, for example, in Merrifield, J. Amer. Chem. Soc . 85:2149-
2156, 1963.
"Mutant" or "mutated" ADPG-PP nucleic acid. "Mutated" or "mutant" ADPG-PP nucleic acids include a change in at least one base of the protein-coding region of a native ADPG-PP nucleic acid that results in a corresponding change in an amino acid of an ADPG-PP polypeptide encoded by the ADPG-PP nucleic acid. Included are insertions, deletions (including deletions of one or more nucleotides internal to the protein- coding region or deletions (or truncations) from one or both ends of the protein-coding region) , and substitutions. Such mutated nucleic acids can be produced by any standard mutagenesis technique, including, but not limited to, those described in Molecular Cloning: A Laboratory Manual , 2nd ed. , vol. 1-
3, ed. Sambrook et al . , Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989 (hereinafter, "Sambrook, 1989"); Current Protocols in Molecular
Biology, ed. Ausubel et al . , Greene Publishing and Wiley- Interscience, New York, 1994 (with periodic updates) (hereinafter, "Ausubel, 1994").
"Mutagenized". The word "mutagenized" is synonymous with the word "mutated" and means being altered from the native, natural state by any means including by chemical mutagens, electromagnetic radiation, genetic engineering, or molecular biology techniques. Thus a nucleic acid may be mutagenized by insertion, substitution, or deletion of a nucleic acid, and a peptide may be mutagenized by insertion, substitution, or deletion of an amino acid residue such that the nucleic acid or peptide, respectively, is altered from its natural, native state.
"Native" . The term "native" refers to a naturally- occurring ( "wild-type" ) nucleic acid or polypeptide. The native nucleic acid or protein may have been physically derived from a particular organism in which it is naturally occurring or may be a synthetically constructed nucleic acid or protein that is identical to the naturally-occurring nucleic acid or protein.
"Isolated" . An "isolated" nucleic acid is one that has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, by conventional nucleic acid-purification methods. The term also embraces recombinant nucleic acids and chemically synthesized nucleic acids.
"Fragments, Probes, and Primers". A fragment of an ADPG-PP nucleic acid is less than full length and is capable of hybridizing specifically with a native ADPG- PP nucleic acid under stringent hybridization conditions. The length of such a fragment is at least 15, 20, 30, 40, or 50 nucleotides of a native ADPG-PP nucleic acid sequence or another target sequence, e.g., sequences flanking the cloning site of a vector in which an ADPG-PP sequence is cloned. A "probe" is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, e . g. , a radioactive isotope, ligand, chemiluminescent agent, or enzyme. "Primers" are isolated nucleic acids that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between' the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e . g. , a DNA polymerase. Primer pairs can be used for amplification of a nucleic acid sequence, e . g. , by the polymerase chain reaction
(PCR) or other conventional nucleic-acid amplification methods . Probes and primers are generally 15 nucleotides or more in length, and maybe 20, 25, or 30 nucleotides or more. Such probes and primers hybridize specifically to a target nucleic acid under high-stringency hybridization conditions. Probes and primers have complete sequence similarity with the target sequence in at least about 7- 15 consecutive nucleotides to permit hybridization under high stringency conditions, although probes differing from a target sequence and that retain the ability to hybridize to the target sequence may be designed by conventional methods and are useful for introducing nucleotide sequence mutations and corresponding amino acid sequence mutations.
Methods for preparing and using probes and primers are described, for example, in Sambrook, 1989; Ausubel, 1994; and Innis et al . , PCR Protocols : A Guide to
Methods and Applica tions, Academic Press: San Diego,
1990. PCR-primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5®,
1991, Whitehead Institute for Biomedical Research, Cambridge, MA) .
"Portions" of DNA. In relation to methods of the invention directed to deletion of DNA sequences that correspond to parts of the N- or C- terminus of the
ADPG-PP protein, the term "portion" is used to indicate a number of contiguous nucleotides, from either the 3' or the 5' terminus of the DNA, corresponding to 5, 10, 15, 17, 25, 30, 40 or more amino acid residues from the N- or C- terminal. Deletion may be performed by standard molecular biology techniques (Sambrook et al . , 1989) "Substantial Similarity". A first nucleic acid is "substantially similar" to a second nucleic acid if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand) , there is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity. Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using sequence analysis software such as the BLAST sequence analysis software available from the NCBI . A particularly useful tool is BLAST 2.0 program "gapped blastn" set to default parameters. Another useful software product is the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI .
Alternatively, two nucleic acids are substantially similar if they hybridize under stringent conditions, as defined below.
"Operably Linked" . A first nucleic-acid sequence is "operably" linked with a second nucleic-acid sequence when the first nucleic-acid sequence is placed in a functional relationship with the second nucleic-acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame .
"Recombinant" . A "recombinant" nucleic acid is made by an artificial combination of two otherwise separated segments of sequence, e . g. , by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
Techniques for nucleic-acid manipulation are well- known (see, e . g. , Sambrook, 1989, and Ausubel, 1994).
Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra . Letts . 22:1859-1862, 1981, and Matteucci et al . ,
J. Am. Chem . Soc . 103:3185, 1981. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers. Preparation of Recombinant or Chemically
Synthesized Nucleic acids; Vectors. Transformation, Host cells . Natural or synthetic nucleic acids according to the present invention can be incorporated into recombinant nucleic-acid constructs, typically DNA constructs, capable of introduction into and replication in a host cell . Such a construct may be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. For the practice of the present invention, conventional compositions and methods for preparing and using vectors and host cells are employed, as discussed, inter alia, in Sambrook, 1989, or Ausubel, 1994.
A cell, tissue, organ, or organism into which a foreign nucleic acid has been introduced is considered
"transformed", "transfected", or "transgenic." A "transgenic" or "transformed" cell or organism also includes progeny of the cell or organism, including progeny produced from a sexual cross that includes the ADPG-PP transgene from one or both parents.
Nucleic-acid constructs, or vectors, for use with prokaryotic or eukaryotic hosts, include a nucleic acid sequence that encodes an ADPG-PP polypeptide or a portion thereof and other vector sequences known in the art and appropriate for a given host cell, including, but not limited to, well known transcription and translation-initiation sequences; an origin of replication or autonomously replicating sequence (ARS) ; expression control sequences, including, but not limited to, promoter and enhancer sequences; processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences; secretion, transit, and other peptide sequences that allow the protein to cross and/or lodge in a cell membrane or be secreted from a cell; selectable or screenable marker genes, etc.
Nucleic-acid constructs can be introduced into a host cell by any suitable conventional method, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent; etc.) See, e.g., Sambrook, 1989, and Ausubel, 1994.
Nucleic acid constructs that express a mutated ADPG-PP according to the invention can be introduced into a variety of host cells or organisms in order to alter starch biosynthesis by the cell or organism, particularly higher plant cells, but also including other prokaryotic or eukaryotic host cells that synthesize starch.
Any well known vector suitable for stable transformation of plant cells and/or for the establishment of transgenic plants may be used, including those described in, e . g. , Pouwels et al . , Cloning Vectors : A Laboratory Manual , 1985, supp. 1987);
Weissbach and Weissbach, Methods for Plant Molecular
Biology, Academic Press, 1989; and Gelvin et al . , Plant
Molecular Biology Manual , Kluwer Academic Publishers,
1990. Such plant expression vectors can include expression control sequences ( e . g. , inducible or constitutive, environmentally or developmentally regulated, or cell- or tissue-specific expression- control sequences) .
Examples of constitutive plant promoters useful for expressing ADPG-PP in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter ( see, e . g. , Odel et al . , Nature 313:810, 1985; Dekeyser et al . , Plant Cell 2:591, 1990; and Terada and
Shimamoto, Mol . Gen . Genet . 220:389, 1990); the nopaline synthase promoter (An et al . , Plant Physiol . 88:547,
1988) and the octopine synthase promoter (Fromm et al . , Plant Cell 1:977, 1989).
A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals, also can be used for protein expression in plant cells, including promoters regulated by (1) heat (Callis et al . , Plant
Physiol . 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al . , Plant Cell 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell
3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpson et al . , EMBO J. 4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et al . , Plant
Cell 1:969, 1989), (4) wounding ( e . g. , wunl, Siebertz et al . , Plant Cell 1:961, 1989); or (5) chemicals such as methyl jasminate, salicylic acid, or a safener. It may also be advantageous to employ (6) organ-specific promoters (e.g., Roshal et al . , EMBO J. 6:1155, 1987;
Schernthaner et al . , EMBO J. 7:1249, 1988; Bustos et al . , Plant Cell 1:839, 1989; Zheng et al . , Plan t J.
4:357-366, 1993) . Plant expression vectors can include regulatory sequences from the 3 ' -untranslated region of plant genes (Thornburg et al . , Proc . Na tl . Acad . Sci . USA 84:744
(1987); An et al . , Plant Cell 1:115 (1989), e . g. , a 3' terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3' terminator regions.
Useful dominant selectable marker genes for expression in plant cells include, but are not limited to: genes encoding antibiotic resistance genes ( e . g. , resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) ; and herbicide resistance genes ( e . g. , phosphinothricin acetyltransferase) .
Useful screenable markers include, but are not limited to, β-glucuronidase and green fluorescent protein. Nucleic-Acid Hybridization. The nucleic-acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Nucleic acid hybridization is discussed in Sambrook, 1989, at 9.52-9.55. See also, Sambrook, 1989 at 9.47-9.52, 9.56-9.58; Kanehisa, Nucl . Acids Res .
12:203-213, 1984; and Wetmur and Davidson, J. Mol . Biol .
31:349-370, 1968.
Nucleic acid and protein sequence, similarity. homology, and identity. The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology) ; the higher the percentage, the more similar the two sequences.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms may be found, for instance, in: Smith and Waterman (1981) ; Needleman and Wunsch (1970) ; Pearson and Lipman (1988) ; Higgins and Sharp (1988) ;
Higgins and Sharp (1989); Corpet et al . (1988); Huang et al. (1992); and Pearson et al . (1994). Altschul et al .
(1994) presents a detailed consideration of sequence alignment methods and homology calculations. A particularly useful tool is the NCBI Basic Local
Alignment Search Tool (BLAST) (Altschul et al . , J. Mol .
Biol . 215:403-410, 1990) which is available from the
National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet. It can be accessed at htp: //www. ncbi .nlm.nih.gov/BLAST/ . A description of how to determine sequence identity using this program is available at http : //www . ncbi . nlm . nih . gov/BLAST/blast_help . html .
Nucleic acid sequence similarity can be determined by using the NCBI BLAST 2.0 "gapped blastn" program set to default parameters. Amino acid sequence similarity can be compared using NCBI 2.0 "gapped blastP" set to default parameters . The "gapped" feature of these programs allows gaps (deletions and insertions) to be introduced into the sequences to be aligned. Allowing such gaps means that similar regions are not broken into several separate segments, allowing a truer determination of sequence identity (and therefore similarity) to be made.
Another useful BLAST tool is the Position-Specific Iterated BLAST (PSI-BLAST) that provides a sensitive way to look for sequence homologues . The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. PSI-BLAST may be iterated until no new significant alignments are found.
Protein Orthologs . Orthologs of a particular protein are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the amino acid sequence of the gene in question using the NCBI Blast 2.0, gapped blastp set to default parameters. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95%, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at http : //www . ncbi . nlm . nih . gov/BLAST/blast_FAQs . html .
One of ordinary skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. The present invention provides not only the peptide homologs are described above, but also nucleic acid molecules that encode such homologs. Hybridization and Stringent Conditions. An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al . (1989) and Tijssen (1993) . Nucleic acid molecules that hybridize under stringent conditions to the target sequences will typically hybridize to a probe based on either the entire target cDNA or selected portions of the cDNA under wash conditions of 0.2x SSC, 0.1% SDS at 65°C. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequence that all encode substantially the same protein. Regarding the amplification of a target nucleic-acid sequence ( e . g. , by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product.
The term "specific for (a target sequence) " indicates that a probe or primer hybridizes under given hybridization conditions only to the target sequence in a sample comprising the target sequence. Nucleic-Acid Amplification. As used herein,
"amplified DNA" refers to the product of nucleic-acid amplification of a target nucleic-acid sequence. Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including the polymerase chain reaction (PCR) . A variety of amplification methods are known in the art and are described, inter alia , in U.S. Patent Nos.
4,683,195 and 4,683,202, and in PCR Protocols : A Guide to Methods and Applications, ed. Innis et al . , Academic Press, San Diego, 1990.
Mutagenesis of ADPG-PP Nucleic Acids. Using ADPG- PP nucleic acids that are known in the art or that are isolated using such well-known nucleic acids, any conventional mutagenesis method can be used to mutagenize (i.e., artificially alter from its wild-type form) ADPG-PP nucleic acids, including chemical mutagenesis, oligonucleotide site-directed mutagenesis, chemical synthesis of a mutant ADPG-PP sequence, etc., resulting in substitutions, insertions, deletions, or combinations thereof. Nucleic acids so produced are called "mutant" nucleic acids.
Mutant ADPG-PP Polypeptides . A mutant ADPG-PP polypeptide is a polypeptide produced by the expression of a mutant nucleic acid, or a peptide that has been intentionally artificially altered from its wild-type form. "Silent" mutations include substitutions of one or more base pairs that result in no change in the amino acid sequence of the polypeptide encoded by the sequence. "Conservative" mutations result in a conservative amino acid substitution in one or more amino acid residues of the polypeptide encoded by the nucleic-acid sequence. Examples of conservative amino acid substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys ; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for lie; lie or Val for Leu; Arg, Gin, or Glu for Lys; Leu or lie for Met; Met, Leu, or Tyr for Phe; Thr for Ser; Ser for Tyr; Tyr for Trp; Trp or Phe for Tyr; and lie or Leu for Val . Polypeptide Sequence Homology. ADPG-PP polypeptides encompassed by the present invention are at least about 70%, 80%, 90%, or 95% homologous to a native ADGP-PP polypeptide.
Polypeptide homology can be analyzed by conventional methods, e.g., using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI , or the NCBI BLAST 2.0 blast software. Polypeptide sequence analysis software matches homologous sequences using measures of homology assigned to various substitutions, deletions, substitutions, and other modifications.
"Isolated." "Purified," "Homogeneous" Polypeptides. An "isolated" polypeptide is separated from the cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that naturally accompany it. Such a polypeptide can also be referred to as "pure" or "homogeneous" or "substantially" pure or homogeneous. Thus, a polypeptide which is chemically synthesized or recombinant (i.e., the product of the expression of a recombinant nucleic acid, even if expressed in a homologous cell type) is considered to be isolated. A monomeric polypeptide is isolated when at least 60% by weight of a sample is composed of the polypeptide. In some cases it is advantageous to have 90%, 95%, or 99% polypeptide by weight in a sample. Protein purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high performance liquid chromatography; or other conventional methods.
Protein Purification. Polypeptides according to the present invention can be purified by any conventional method. See, e . g. , Guide to Protein
Purification, ed. Deutscher, Meth . Enzymol . 185,
Academic Press, San Diego, 1990; and Scopes, Protein
Purification : Principles and Practice, ■ Springer Verlag, New York, 1982.
ADPG-PP "Biological Activity" or "Enzyme Activity". The terms "biological activity", "enzyme activity", "biologically active", "activity", and "active" refer primarily to the characteristic biological activity or activities of a native ADPG-PP polypeptide, including, but not limited to, catalyzing the initial step in α- glucan and starch synthesis: glucose-1-phosphate + ATP --> ADP-glucose + PPi . Other activities include allosteric regulation by 3-PGA and Pi. "Allosteric". The word allosteric literally means "another site". An enzyme is said to be "allosteric" or "allosterically regulated" if its enzymatic activity is regulated by the binding of non-substrate molecules at a site other than the active site of the enzyme, i.e., an "allosteric site". The binding of non-substrate molecules at allosteric sites effects the binding kinetics of the substrate-binding (active) site. A molecule that increases binding or decreases dissociation of an enzyme and a substrate complex, thus increasing enzyme activity, is called an "activator" molecule. A molecule that decreases binding or increases dissociation of an enzyme and a substrate complex, thus decreasing enzyme activity, is called an "repressor" or "inhibitor" molecule.
"Up-regulated" (or "up-regulatory") . A mutant enzyme is "up-regulated" or "up-regulatory" when that enzyme has a higher biological activity than the wild-type enzyme under physiological conditions. The up-regulated enzyme may have at least about 10, 20, 40, 60, 80, or 100% higher biological activity than wild-type. Under some circumstances, the up-regulated mutant may have greater than 100% higher biological activity than the wild-type. An up-regulated mutant exhibits increased biological activity by virtue of its mutation (s) . The effect of the mutation (s) may be to inherently increase biological activity independent of allosteric regulation (for instance by increasing binding or decreasing dissociation of the enzyme-substrate complex) , to increase sensitivity to an allosteric activator (e.g., 3-PGA) , and/or to decrease sensitivity to an allosteric inhibitor (e.g., Pi) . Any such effect brought about by a mutation will result in an increase in enzyme activity making the mutant enzyme up-regulated.
Fusion Polypeptides. The present invention also provides fusion polypeptides including, for example, heterologous fusion polypeptides in which an ADPG-PP sequence is joined to a well-known fusion partner. Such fusion polypeptides can exhibit biological properties (such as substrate or ligand binding, enzymatic activity, antigenic determinants, etc.) derived from each of the fused sequences. Fusion polypeptides are preferably made by standard recombinant DNA techniques.
Transgenic Plants; Plant Transformation and Regeneration
Various nucleic acid constructs that include a mutant ADPG-PP nucleic acid are useful for producing transgenic plants with altered starch biosynthesis, increased starch production, and increased yield. Such plants may also grow faster and produce more seeds. ADPG-PP nucleic acids can be expressed in plants or plant cells under the control of a suitable operably linked promoter that is capable of expression in a cell of a particular plant. Any well-known method can be employed for plant cell transformation, culture, and regeneration in the practice of the present invention with regard to a particular plant species. Conventional methods for introduction of foreign DNA into plant cells include, but are not limited to: (1) Agrobacterium- mediated transformation (Lichtenstein and Fuller In:
Genetic Engineering, Vol 6, Rigby, ed. , London, Academic
Press, 1987; and Lichtenstein and Draper, in: DNA
Cloning, Vol II, Glover, ed. , Oxford, IRI Press, 1985);
(2) particle delivery ( see, e . g. , Gordon-Kamm et al . , Plant Cell 2:603, 1990; or BioRad Technical Bulletin 1687); (3) microinjection ( see, e . g. , Green et al . , Plant Tissue and Cell Cul ture, Academic Press, New York, 1987); (4) polyethylene glycol (PEG) procedures (see, e.g., Draper et al . , Plant Cell Physiol . 23:451, 1982); Zhang and Wu, Theor. Appl . Genet . 76:835, 1988); (5) liposome-mediated DNA uptake ( see, e . g. , Freeman et al . , Plant Cell Physiol . 25:1353, 1984); (6) electroporation ( see, e . g. , Fromm et a . , Nature 319:791 (1986)); and (7) vortexing methods ( see, e . g. , Kindle, Proc . Na tl . Acad . Sci . USA 87:1228 (1990)).
The term "plant" encompasses any higher plant and progeny thereof, including monocots (e.g., corn, rice, wheat, barley, etc.), dicots ( e . g. , potato, tomato, etc.), and includes parts of plants, including seeds, fruit, tubers, etc.
The invention will be better understood by reference to the following Examples. The scope of the invention is not to be considered limited thereto. EXAMPLES
EXAMPLE 1: Generation and Identification of ADPG-PP
Mutants
Bacterial mutants that lack functional ADPG-PP activity, e.g., glgC E. coli strains such as AC70R1-504
(Leung et al . , J. Bacteriol . 167:82-87, 1986) are useful for studying the function of mutant plant ADPG-PP. Co-expression of cDNAs encoding the large and small subunits of plant ADGP-PP in such bacterial mutants results in production of a plant ADPG-PP enzyme that is capable of complementing the glgC~ mutation, restoring glycogen production. Glycogen accumulation can be easily scored by exposing the cells to I2 vapor. Within 30 sec, cells co-expressing wild-type plant ADGP-PP stain purplish-black, whereas the untransformed glgC~ host strain stains light yellow.
The ability of the plant enzyme to complement complement glgC' E. coli mutants was exploited in several related mutagenesis approaches that were developed to generate and identify up-regulated and other ADPG-PP mutants. One of the plant ADPG-PP subunit cDNA sequences was mutagenized then co-expressed with the unmutated counterpart subunit.
For example, in one approach, the large subunit is mutagenized, e.g., using hydroxylamine, and cells co- expressing a mutagenized large subunit and a wild-type small subunit were screened for mutants defective in glycogen accumulation by I2 staining (Greene et al . ,
Proc . Natl . Acad. Sci . USA 93:1509-1513, 1996; Greene et al . , Plant Physiol . 112:1315-1320, 1996). Mutant ADPG- PP enzymes defective for allosteric regulation were then identified by assaying crude extracts of non-staining cells under saturating conditions of 3-PGA (10 mM, or 100 -fold greater levels than required to activate the enzyme by 50%) . Hydroxylamine causes G/C-to-A/T transitions. Mutations can also be introduced, for example, by various other conventional mutagenesis techniques, e.g., by using other chemical mutagens or by PCR performed under limiting substrate and co-factor conditions.
Site-directed mutagenesis may also be used to introduce insertions, deletions or substitutions. Various standard techniques are known to carry out site-directed mutagenesis (Sambrook et al . , 1989), and commercial kits are also available such as the QUICKCHANGE™ mutagenesis site-directed mutagenesis kit (STRATEGENE™ , CA) .
Plasmid DNA encoding the large subunit was mutagenized at 37°C for 24 h in the presence of 0.8 M hydroxylamine-HCl , 50 mM sodium phosphate, pH 6.0, and 1 mM EDTA (Isackson and Bertrand, Proc . Na tl . Acad . Sci .
USA 82:6226-6230, 1985). The reaction was terminated by the addition of 50 μL of 1 M Tris base, ethanol precipitated, and resuspended in 10 mM Tris-Cl, pH 8.0, and 1 mM EDTA, pH 8.0. DNA was then transformed into E. coli AC70R1-504 (Leung et al . , J. Bacteriol . 167:82-87,
1986), a glgC strain, carrying pML7 , the wild-type small subunit cDNA (the construction of pML7 is described below) . After overnight growth on Luria broth (LB) media, colonies were then screened for the production of glycogen by replica plating the colonies onto Kornberg media containing 2% glucose (Greene et al . , Proc . Natl .
Acad . Sci . USA 93:1509-1513, 1996; Greene et al . , Plant Physiol . 112:1315-1320, 1996), growing the cells overnight, then exposing the plates to I2 vapor. Glycogen-deficient cells stain a faint yellow, whereas glycogen-positive cells stain a purplish-black color. Cells that were deficient for glycogen accumulation were grown overnight in 1 mL of LB media. Cells were collected by brief centrifugation, lysed in 100 μL of sucrose lysis buffer by repeated freeze/thawing, then clarified by centrifugation. The cell extracts were analyzed by ELISA using rabbit monospecific anti-large subunit antibodies and goat anti-rabbit antibodies linked to horseradish peroxidase (Greene et al . , Proc .
Na tl . Acad. Sci . USA 93:1509-1513, 1996). Cells that accumulated normal antigen levels for both the large and small subunit were then analyzed by enzyme assay. Cells were grown overnight on an enriched solid media at 37°C then lysed by sonication in 250 μL of a sucrose buffer (50 M Hepes, pH 8.0, 20% sucrose, 5 mM magnesium chloride (MgCl2) , 1 mM EDTA, 5 mM dithiothreitol (DTT) , 5 mM sodium/potassium phosphate buffer, pH 8.0, containing 500 μg/mL lysozyme, 0.5 μg/mL pepstatin, 0.5 μg/mL leupeptin, 0.5 mM benzamidine, and 1 mM phenyl methyl sulfonyl fluoride. After centrifugation, crude extracts were assayed for ADPG-PP activity in the pyrophosphorylysis direction as described (Greene et al . , Proc . Na tl . Acad . Sci . USA 93:1509-1513, 1996;
Greene et al . , Plant Physiol . 112:1315-1320, 1996), except that the reaction contained 20 mM 3-PGA. Under these conditions, several mutant lines could be identified that contained normal levels of ADPG-PP enzyme activity when assayed under saturating activator conditions (Greene et al . , Proc . Na tl . Acad . Sci . USA
93:1509-1513, 1996; Greene et al . , Plant Physiol . 112:1315-1320, 1996). This was verified for mutant 345, which displayed an A0.5 (defined as the amount of 3-PGA required for 50% activation) of 4.0 mM or about 30- to 40-fold greater than the wild-type enzyme (Greene et al . , Proc . Na tl . Acad . Sci . USA 93:1509-1513, 1996). To substantiate that the mutation affects only the allosteric regulatory properties of the enzyme, ADPG-PP was purified by biochemical chromatography techniques, and the activity of the mutant enzyme was investigated. Cells were grown in 850 mL of LB in Fernbach flasks and induced by the addition of IPTG and nalidixic acid.
Large scale crude extracts were obtained by lysing and clarifying as described above . The crude extract was then subjected to a 33-55% ammonium sulfate fractionation. The resulting precipitate was resuspended in sucrose buffer and desalted by dialysis. The desalted sample (30 mg) was diluted to 30 mL in buffer A (5 mM potassium phosphate, pH 7.5 , 50 mM glycylglycine, pH 7.5 , 5 mM MgCl2, 1 mM EDTA, and 5 mM DTT) and was applied to a Millipore MEMSEP 1010 DEAE-chromatography column previously equilibrated with buffer A. Enzyme was eluted from the column by a 200 mL linear gradient of buffer B (50 mM potassium phosphate, pH 6.0, 5 mM MgCl2, 1 mM EDTA, 5 mM DTT, and 400 mM potassium chloride) . Fractions containing enzyme activity were pooled, concentrated by addition of ammonium sulfate to 75%, resuspended in sucrose buffer, and desalted by dialysis. Aliquots of enzyme concentrate were stored at -80°C until needed for enzyme studies. Kinetic parameters, i.e., Km values for glucose 1-phosphate, ATP, and Mg2+ and A0.5 for 3-PGA and Ic.5 for Pi, were defined with the synthesis (forward) assay, which measures the incorporation of 1C-glucose- 1-phosphate into ADPglucose.
Once a mutant enzyme was identified as defective in allosteric regulation, the mutated plasmid DNA coding for the large subunit coding for the large subunit was isolated and subjected to a second round of hydroxylamine mutagenesis and re-transformed into AC70R1-504 harboring a plasmid DNA containing the small subunit counterpart cDNA. The mutated plasmid DNA was isolated then transformed into E. coli . Cells containing the plasmid DNA of interest were then selected by growth on enriched media containing the appropriate antibiotic. After replica plating and growth overnight, revertant cells that accumulated glycogen were identified by I2 staining. The enzyme activities from these revertant cells were partially purified and characterized kinetically as described above. The nature of the second-site mutation (because hydroxylamine causes G/C to A/T transitions, the reversion event is caused by second- site mutation) was identified by DNA sequencing of the cDNA sequence.
The effect of this second-site mutation on enzyme function was then evaluated by introducing the second- site mutation by itself (i.e., without the original mutation) into a wild-type cDNA sequence for the appropriate subunit by site-directed mutagenesis (using a commercial site-directed mutagenesis kit (such as the Quickchange™ kit from Stratagene, CA) according to the manufacturer's instructions). The resulting subunit cDNA including the second-site mutation was then transformed into and co-expressed in AC70R1-504 together with the counterpart wild-type subunit. The resulting mutant ADPG-PP enzyme was then characterized.
Because of its specificity towards deoxycytidine nucleosides, the spectrum of mutations generated by hydroxylamine is limited. Mutations that result in the replacement of Lys and Tyr residues, which are known to play a prominent role in the bacterial enzyme, are not expected. To generate as wide a spectrum of mutations as possible, a PCR strategy or other conventional site- directed mutagenesis method can be employed. For example, the large subunit or small subunit sequences can be amplified under conditions (high Mn2+ or limiting nucleotide concentrations) that result in the misincorporation of nucleotides (Erlich, In: PCR
Technology -- Principles and Applications for DNA
Amplification, New York: Stockton Press, 1989; Slepak et al . , J. Biol . Chem . 268:1414-1423, 1993). The amplified sequences can then be purified, cleaved with the appropriate restriction enzymes, cloned into the appropriate sites of the expression vectors, and expressed in E. coli . Mutants defective in pyrophosphorylase activity can then be screened initially by I2 staining, followed by ELISA and enzyme assays as described above. In addition to the protocol described above, up-regulated enzymes can be identified directly by using a variant of the mutagenesis and selection protocol described above. Cells expressing wild-type ADPG-PP enzyme on medium or high copy number plasmids are close to saturating the amount of glycogen that can be accumulated under normal culture conditions. Therefore, cells expressing an up-regulated ADPG-PP cannot be easily distinguished from cells expressing the wild-type enzyme by iodine staining.
To distinguish the expression of an up-regulated enzyme from the expression of the wild-type enzyme, the large or small subunit cDNA that will be mutagenized is cloned into a low copy number plasmid such as pWSK 28 or other plasmids described in Wang and Kushner, Gene
100:195-199, 1991 (Other low copy-number plasmids may be obtained commercially from such companies as Invitrogen, Pharmacia and New England Biolabs. Low-copy-number vectors are generally larger than high-copy-number vectors, having sizes of 6 or 7 kilobases or more.
These low-copy-number plasmids contain, for example, a pSClOl origin of replication, which results in substantially fewer copies (e.g., about 6 copies/cell) than a vector possessing a pBR325, Col El, or ACYC origin of replication. As a result of the decrease in copy number, cells expressing wild-type large subunit sequences that are carried on a low-copy-number plasmid stain very lightly with I2 when co-expressed with the small subunit sequences, providing a cleaner background to identify up-regulated mutants. Screening several thousand cells results in the isolation of several putative up-regulated mutants.
In addition to up-regulated mutations, other mutations can produce similar phenotypes, including mutations that increase plasmid copy number, promoter activity, and ribosome-binding efficiency. These other mutations can be identified by evaluating cellular extracts by immunological techniques as described above, since it is expected that cells containing up-regulated mutants of ADPG-PPs contain the same amount of antigen as cells expressing the wild-type enzyme. Alternatively, other types of mutations can be eliminated by subjecting the cDNA sequences alone to the chemical mutagen or PCR mutagenesis protocol (rather than the vector as a whole) , then inserting the mutated cDNA sequences into an appropriate expression vector. An alternative approach to identify up-regulatory mutants directly is by altering the level of glucose in the Kornberg media (normally 2%) to a level at which glycogen accumulation is approximately directly proportional to ADPG-PP activity (e.g., 0-0.5%). Cells expressing an up-regulated ADPG-PP mutant enzyme accumulate more glycogen at 0.1% glucose than cells expressing wild-type enzyme ( see FIG. 3) .
A third strategy for the generation of up-regulatory mutants is to mutate specific DNA sequences that code for specific peptide regions of either the large or small subunit. For example, peptide sequences at both the N- and C-terminus are required for normal allosteric regulation. Mutations in this region alter the allosteric response of the resulting enzyme (Ball and Preiss, J". Biol . Chem . 269:24706-24711, 1994;
Greene et al . , Proc . Na tl . Acad . Sci . USA 93:1509-1513,
1996; Greene et al . , Plant Physiol . 112:1315-1320, 1996;
Morell et al . , J. Biol . Chem . 263:633-637, 1988). Other peptide regions of the large and small subunits may also be mutagenized to identify residues that play a role in allosteric regulation. Mutations can be generated randomly using PCR techniques under conditions that cause misincorporation of nucleotides or by using synthetic oligonucleotides that include random mutations. Mutations can also be generated by deletion of specific DNA sequences, especially those encoding amino acid residues located at the N- or C-terminus, for example .
EXAMPLE 2 : Generation and Identification of Up-Regulatory Mutants of the Higher Plant ADPG-PP
The expression plasmids, pMON17335 and pMON17336 (Inglesias et al . , J. Biol . Chem . 268:1081-1086, 1993), that contain the small and large subunit cDNAs, respectively, were modified by incorporating double translation termination codons using a PCR approach.
The primers shown in SEQ ID Nos. 13 and 14 were used to amplify a 538 bp DNA fragment covering the 3' end of the small subunit coding sequence. After digestion with Kpnl and Sad, the amplified product was cloned into pMON17335 to produce pML5. The primers shown in SEQ ID NOS. 15 and 16 were used to amplify a 608 bp DNA fragment covering the 3 ' end of the large subunit coding sequence. After digestion with Nhel and Hindlll, the amplified product was cloned into pMON17336 to produce pML7 (FIG. 1) . pML5-encoded small subunit polypeptide lacked 10 amino acid residues of the N-terminus of the mature protein. These ten amino acids were restored, together with an additional Met-Ala, by PCR using the primer shown in SEQ ID No. 17, which introduces a unique Ncol site (underlined DNA sequences) at the Met codon. The oligonucleotide primer shown in SEQ ID. No. 18, which spans a unique Kpn I site (DNA sequences underlined) located about 850 nucleotides from the newly created AUG codon, was employed together with the upstream oligonucleotide (SEQ ID No. 17) for PCR amplification. The amplified DNA fragment was digested with Ncol and Kpnl and cloned into pML5 to give pMLlO (shown in FIG. 2) .
Random Mutagenesis. The large subunit plasmid, pMON17336, was subjected to hydroxylamine treatment and then transformed into the glgC~ strain AC70R1-504 containing pMON17335 as described above. After overnight growth, cells were replica-plated onto Kornberg media containing 2% glucose, grown for at least 12 hours, then exposed to iodine vapor. The glycogen-deficient cells were then analyzed by ELISA (Greene et al . , Proc . Natl . Acad . Sci . USA 93:1509-1513,
1996) and enzyme assays ( id. ) under saturating substrate and activator conditions. One glycogen-deficient mutant, mutant 345, which contained normal levels of enzyme activity, was further studied kinetically ( id. ) . Fig. 5 shows substrate binding (Kms) and 3-PGA activator (A0 5) affinity properties of various ADPG-PPs: native (Sowokinos and Preiss, Plant Physiol . 69:1459-1466,
1992); wild-type (Ballicora et al . , Plant Physiol .
109: 245 -251, 1995); mutant 345 (Greene et al . , Proc . Natl . Acad. Sci . USA 93:1509-1513, 1996); R20, UpReg-1, and ΔΝ17-LS (R20, UpReg-1, and ΔN17-LS are described below) . The partially purified enzyme from mutant 345 displayed normal binding constants (Km) for glucose- 1 -phosphate, ATP and Mg2+ (Fig. 5) . However, the enzyme from mutant 345 required 28 -fold greater levels of the activator 3-PGA than the wild-type enzyme. DNA sequence analysis indicated the presence of single base substitution that resulted in the replacement of a proline located at residue 52 by a leucine (Greene et al . , Proc . Na tl . Acad . Sci . USA 93:1509-1513, 1996) .
The large-subunit plasmid from mutant 345 was isolated and subjected to a second hydroxylamine treatment, co-expressed with pMLlO, and screened for glycogen production. Eight genetic revertants were isolated that displayed varying levels of glycogen accumulation as determined by I2 staining. Four of the eight revertants, including R20, demonstrated wild-type staining phenotypes (Fig. 3) and higher than wild-type ADPG-PP activities (Class I) . The remaining revertants showed either intermediate (Class II) or light staining (Class III) phenotypes. Molecular characterization of the large subunit plasmid DNAs of Class I revertants R4 , RIO, R20, and R32 identified a single second-site mutation in close proximity to the primary mutation Pro52Leu in each of the revertants. R4 contained a base-pair mutation that replaced Pro at position 66 with a Leu, resulting in the addition of a second structural modification. RIO and R32 contained an identical second site mutation in which Gly at position 101 was replaced with Asn. In R20, a negatively charged Glu residue fourteen amino acids upstream from P52L was replaced with a positively charged Lys residue (SEQ ID Nos. 7 and 8); note that Lys38 is at position 40 in the recombinant cDNA sequence due to the addition of Met and Ala residues. Introduction of the positively charged Lys residue potentially enhances the enzyme's ability to interact with the negatively charged activator 3-PGA.
The R20 enzyme was partially purified to a specific activity of about 20 μmol/min/mg by differential ammonium sulfate precipitation, heat treatment, amino-propyl (C3) chromatography, and DEAE anion-exchange chromatography and was estimated to be about 40% pure. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified protein showed a dramatic increase in a broad band migrating in the 50-52 kD range.
Immunoblot analysis showed that this band cross-reacted with monospecific IgG directed against the recombinant potato ADPG-PP large subunit or small subunit. Kinetic analysis of R20 enzyme showed that the values of Kra for glucose-1-phosphate, ATP, and Mg2+ were identical to the
Km of the wild-type recombinant enzyme (Fig. 5) . The affinity of R20 enzyme for 3-PGA was increased about 38-fold compared to the P52L mutant, which displayed an A0.5 of 117 μM, which is very close to that of the wild- type recombinant enzyme (Fig. 5) .
Fig. 6 shows the results of studies to determine the Pi inhibition constants (I0.5) of various ADPG-PPs : wild-type (Ballicora et al . , Plant Physiol . 109:245-251, 1995); mutant 345 (Greene et al . , Proc . Natl . Acad. Sci .
USA 93:1509-1513, 1996); R20, UpReg-1, and ΔN17-LS. The
I0.5 value determined for the R20 ADPG-PP at 0.25 mM 3-PGA was roughly 4.5- and 8 -fold higher than that of the native potato and wild-type recombinant enzymes, respectively (Fig. 6) . The increase in positive charge in this region of the enzyme appears to give R20 ADPG-PP an increased affinity for 3-PGA, while decreasing its affinity for Pi. Site-directed mutagenesis was conducted to introduce a Lys residue at position 38 (Glu38Lys) in pML7 that is independent of the P52L mutation. Of four putative Glu38Lys site-directed mutants (SEQ ID Nos. 1 and 2), all contained the specific mutation as determined by sequence analysis. Glu38Lys large-subunit plasmid DNA was then transformed into AC70R1-504 containing pMLlO for purification and kinetic analysis. The Glu38Lys site-directed mutant was partially purified to a specific activity of 19.7 μmol/min/mg. Kinetic analysis identified an enzyme which was unchanged in its affinity for glucose- 1-phosphate, ATP, and Mg2+ as compared to R20 (Fig. 5) . Activation curves showed that the Glu38Lys mutant has an A0.5 of 2 μM, a 58.5- and 80-fold increase in affinity for 3-PGA compared to R20 and wild-type recombinant ADPG-PP, respectively (Fig. 5) and a dramatic 2,250-fold increase in affinity for 3-PGA compared to the Pro52Leu mutant. The Glu38Lys site-directed mutant was also significantly less sensitive to Pi. Inhibition curves showed I0-5 values of 0.6, 3.1, and 4.7 mM in the presence of 25, 125, and 250 μM 3-PGA. When compared to the I0.5 values of the native potato and wild-type recombinant enzymes, the Glu38Lys mutant displayed a dramatic reduction in affinity for Pi (Fig. 6) . The Glu38Lys mutation alone in the large subunit, when co-expressed with the unmutated small subunit, produced an up-regulated form of ADPG-PP by dramatically increasing the affinity for the activator and dramatically decreasing the affinity for the inhibitor Pi. ADPG-PP containing the Glu38Lys mutant large subunit is an up-regulatory mutant. Henceforth, it is referred to as "UpReg-1" (SEQ ID Nos. 1 and 2) . Phenotypically, the UpReg-1 mutant, when co-expressed with pMLlO stains, is significantly darker on a glucose media compared to the wild-type recombinant and R20 (FIG. 3) .
Additional site-directed mutagenesis of Glu 38 was conducted. An Ala residue is highly conserved at the analogous position on the small subunit. Substitution of Ala for Glu at position 38 generates an enzyme that is insensitive to activation by 3-PGA, similar to the Pro52Leu mutation (Greene et al . , Proc . Natl . Acad . Sci .
USA 93:1509-1513, 1996). An Arg residue resides at an analogous position in the E. coli ADPG-PP enzyme.
Introduction of an Arg residue at position 38 generates an enzyme that remains as insensitive to 3-PGA activation as the Pro52Leu mutant. At high concentrations of 3-PGA (50 mM) , an enzyme activity comparable to that of the wild-type recombinant enzyme can be achieved with the E38A and E38R mutants. The lack of activation in the E38R mutant at the lower concentrations of 3-PGA indicates that activation may not be strictly charge-dependent, but may also be affected by size constraints.
Similar studies were also conducted for R32 which contained a GlylOl Asn mutation (SEQ ID Nos. 9 and 10) . Substrate and cofactor binding constants were also relatively unaffected for R32 AGPase (Fig. 5) . R32 was more sensitive to 3-PGA activation, showing an A0.5 of 414 μM, an 11-fold increase over the Pro52Leu mutant. Pi sensitivity was also lower in R32 AGPase (Figs. 6 and 7) but the I0.5 values for this second-site revertant were more similar to the values determined for the WT recombinant enzyme than for the other second-site revertants. Thus, substitution of the polar amino acid Asn for the nonpolar Gly restored 3-PGA sensitivity in R32 while only slightly changing the enzyme's affinity for Pi .
Site-directed studies were conducted to introduce an Asn at position 101 in the large subunit which was then co-expressed with the wildtype small subunit in bacterial cells. The resulting enzyme, UpReg-2 (SEQ ID Nos. 3 and 4), displays up-regulatory properties. 3-PGA activation studies of the partially purified enzyme (specific activity of 9.1 μmol/min/mg ) yielded an A0.5 of 0.035 mM, an 11.8- and 4.6-fold enhancement over the parental revertant R32 and WT. Although the increase in affinity for 3-PGA was less pronounced for UpReg-2 than for UpReg-1, the response to Pi was more dramatic for UpReg-2 than for UpReg-1 . Pi inhibition studies in the presence of 0.1 and 0.001 mM 3-PGA revealed I05 constants of 6.2 and 1.3 mM, respectively. Hence, UpReg-2 shows a higher resistance to Pi inhibition than UpReg-1.
EXAMPLE 3: Generation and Identification of
Up-Reσulatory ADPG-PP by N-Terminal Deletions of the
Large Subunit
A DNA fragment encoding a truncated N-terminal peptide missing the first 17 amino acids of the large subunit was amplified using PCR with the primers shown in FIGS. 51 and 5J. The PCR product was purified, digested with Ncol and Nhel, subcloned into pML7 (Fig. 1) and expressed in the E. coli strain JM101
(Messing, Meth . Enzymol . 101:20-78, 1983) containing pMLlO (Fig. 2) .
The resulting truncated large subunit was co-expressed with the wild-type small subunit in E. coli to form the ΔN17-LS enzyme. The small subunit was expressed at 109% and the large subunit at 114% of the wild-type level, as quantified by ELISA. The ΔN17-LS enzyme was partially purified to a final specific activity of 20 units/mg under standard assay conditions and estimated to be approximately 51% pure based on video image analysis of a purified protein fraction on Commassie Brilliant Blue-stained polyacrylamide gels. Kinetic analysis of the partially purified ΔN17-LS enzyme showed no effect on the binding constants for the substrates ATP and glucose-1-P, while the enzyme's affinity for the co-factor Mg+ increased slightly (Fig. 5) . A marked change in allosteric regulatory behavior of the ΔN17-LS enzyme was observed. The ΔN17-LS enzyme required only 0.015 mM 3-PGA for 50% activation, reflecting a ten-fold greater sensitivity to 3-PGA than the wild-type enzyme (Fig. 5) . The ΔN17-LS enzyme was also less sensitive to inhibition by Pi (Fig. 6) . In the presence of 0.25 mM 3-PGA, the ΔN17-LS enzyme had an I0.5 of 2.0 mM (Fig. 5), a value more than 28-fold greater than that exhibited by the wild-type enzyme (Ballicora et al . , Plant Physiol . 109:245-251, 1995).
Even at 0.025 mM 3-PGA, 0.13 mM Pi was required to inhibit the ΔN-L17 enzyme activity by 50%. Comparing the calculated I0.5s of the native and recombinant enzymes, this decrease in sensitivity to Pi is dramatic. In contrast to the shallow linear increases in the ratio of I05/3-PGA exhibited by the native and recombinant wild-type enzymes, the ΔN17-LS enzyme showed an increasing resistance to Pi inhibition as 3-PGA levels increase (Fig. 4) . Thus, this 17 amino-acid N-terminal region of the large subunit is essential for proper allosteric regulation, since its removal increases the enzyme's sensitivity to the allosteric activator 3-PGA 10-fold and decreases its sensitivity to the allosteric inhibitor Pi 5- to 16-fold.
EXAMPLE 4; Transfer and Expression of Reσulatory
Mutants and Wildtype Potato Adp-glucose Pyrophosphorylase in Arabidopsis
Expression of up-regulatory allosteric large subunits of ADPG-PP in photosynthetic tissues of Arabidopsis increases vegetative growth rate and seed yield.
The allosteric regulatory properties of the ADPG-PP enzymes formed from the mutant large-subunit sequences together with normal small-subunit sequences are shown in Fig. 7. Sensitivity to the activator 3-PGA ranges from 0.002 mM (upRegl) , 0.14 mM (wild-type: "W.T."), to 4 mM (M345) . Since the enzyme is also inhibited by Pi, a more accurate view of the differences in the allosteric regulation of the mutant enzymes is the I0.5 /[3-PGA] ratio, where I0.5 is the amount of Pi required to produce 50% inhibition of enzyme activity (I0.5) at a known concentration of 3-PGA. I0-5 /[3-PGA] ratios range from a value of 24 for UpRegl to 1.4 for the W.T. enzyme to 0.6 for the down-regulatory mutant M345. Specifically, the up-regulatory type enzymes have a higher affinity for the activator 3-phosphoglyceric acid (3-PGA) and/or higher resistance to the inhibitor inorganic phosphate (Pi) than the W.T. enzyme. Expression of these up- regulatory ADPG-PP enzymes results in an increased production of glycogen (starch-like) in Escherichia coli , and it is expected that expression of these gene sequences in higher plants would increase starch biosynthesis and levels in cells capable of starch accumulation. The effect of this increased starch production on plant growth and development will depend on the organs of the plant in which these up-regulated enzymes are expressed. In non-photosynthetic storage organs such as tubers, developing seeds, fruits and roots, expression of unregulated or up-regulated forms of ADPG-PP would not be expected to have a serious effect on plant growth and development because starch serves as a primary form of fixed carbon.
In contrast, expression of up-regulated ADPG-PP in leaves may have deleterious consequences on plant growth and development because the allosteric regulatory properties of ADPG-PP are believed to be one of several important processes that are responsible for controlling the distribution of fixed carbon into sucrose and starch in leaves and other photosynthetic competent tissues. One hypothesis (Eichelmann and Laisk, Plant Physiol . 106:679-687, 1994) is that the bulk of the fixed carbon is converted into sucrose and that starch is made only when the rate of sucrose synthesis is saturated. Under conditions where sucrose synthesis is saturated, the ratio of activator to inhibitor (3 -PGA/Pi) is high enough to activate ADPG-PP, thereby allowing starch synthesis to occur. In the dark, the 3 -PGA/Pi ratio is low, which suppresses the enzyme activity of ADPG-PP and, in turn, limits starch synthesis. The normal allosteric regulatory properties of ADPG-PP account for the diurnal oscillation of starch synthesis during the day and net breakdown of starch at night. Based on this hypothesis regarding the critical role of ADPG-PP in leaf starch metabolism, it is expected that the expression of unregulated and up-regulated forms of ADPG-PP in leaf cells would drastically alter the normal partitioning of fixed carbon between sucrose and starch and cause much higher starch synthesis to occur. These events, in turn, would reduce sucrose availability to the remainder of the plant which could disrupt normal plant growth and development processes. Stark et al . ( Science 258:287-292, 1992) showed that expression of the allosteric unregulated glgC16 gene by the constitutive CaMV35S promoter produced a transgenic potato plant that could not survive without sucrose . More recent evidence indicates that starch does not merely serve as a transient reserve to support the metabolic activities of the plant during the night, but also as a transient sink to accommodate excess photosynthate. Ludewig et al . (FEBS Lett . 429: 147-151) have shown that there is a direct correlation between the capacity of starch synthesis and the rate of photosynthesis at elevated C02. Likewise, recent data from J. Sun, G.E. Edwards, and T.W. Okita (unpublished) exhibited a significant correlation between the rates of starch synthesis and C02 assimilation, and between the rates of starch synthesis and accumulative leaf area. These results indicate that leaf starch plays an important role as a transient "sink" for excess carbon formed during photosynthesis and thereby alleviates any potential feedback of photosynthesis. If this view is correct, increased starch production in leaf tissue should lead to increased photosynthesis and, in turn, higher productivity. This was tested by transforming Arabidopsis plants with the various up-regulated potato large subunit sequences shown in Fig. 5 under the control of the Arabidopsis ribulose bisphosphate carboxylase small -subunit promoter.
EXAMPLE 5; Construction of Plant Expression Plasmids Containing The Wild-Type and Mutant ADPG-PP Large Subunits The Arabidopsis ribulose bisphosphate small -subunit
(atslA) promoter (Krebbers et al . , Plant Mol . Biol .
11:745-759, 1989)) and transit leader coding sequences were amplified using synthetic primers using Taq DNA polymerase. The 5' primer contained a XhoI-Xbal-BamHI sequence 5' (underlined) to the atslA nucleotide sequence beginning with base -1701 from the translation start of the atslA gene. The 3' primer had a Ncol-Sacl sequence (underlined) at the complementary nucleotide located at +164 from the translation start. The amplified 1897-nucleotide DNA fragment was ethanol precipitated, resuspended in TE, and digested with Xhol and Sacl . The digested DNA was then resolved by agarose gel electrophoresis, and then purified by binding and elution on DEAE-membrane filters. The purified DNA fragment was then collected by ethanol precipitation and then cloned into the Xhol and Sacl sites of pBluescript II to give pHI-10 (Fig. 6) :
5' primer GCTCGAGTCTAGAGGATCCGTGGTCGAGATTGTGTATTATTCTTTAG
3 ' primer CGAGCTCGCCATGGCAGTTAACTCTTCCGCCGTTGCTTG
To form a gene fusion between the atslA promoter and transit leader sequences to the potato ADPG-PP large- subunit sequences, the coding sequences from UpRegl, R4 , R20, R32, M27, M345, and wild-type large-subunit sequences were removed from the plasmid DNA (Iglesias et al . , J. Biol . Chem . 268:1081-1086, 1993) by digestion with Ncol and Sacl and the resulting DNA fragment cloned into the relevant restriction sites of pHI-10 to give pHI-11 to pHI-17 (Fig. 11) .
The atslA-potato large subunit cassettes contained within a Xbal/Sacl DNA fragment were then cloned into the Xbal and Sacl sites of the T-DNA binary vector pHI- 32, a derivative of pIG-121 (Ohta et al . , Plant Cell
Physiol . 31:805-813, 1990) to give pHI-33 to pHI-39
(Fig. 9) .
EXAMPLE 6: Transformation into Arabidopsis TL46 The pHI-33 to -39 series of plasmid DNAs were transferred into Agrobacterium GV3101 using standard methods (An, Methods Enzymol . 153:292-305, 1987). These
Agrobacterium lines were then used to transformed
Arabidopsis Columbia line TL-46. TL-46 is a starch- deficient line (Lin et al . , Plant Physiol . 99:1175-1181,
1988) which is defective for the leaf ADPG-PP large subunit . Transformation was accomplished by co- cultivation of leaf sections (discs) of Arabidopsis TL- 46, selection of kanamycin- and hygromycin-resistant calli and the regeneration of plants by methods described by van der Graaf and Hooykaas ( Plant Cell Reports 15:572-577, 1966). Alternatively, transgenic
Arabidopsis plants containing pHI-33 to pHI-39 were obtained by the vacuum infiltration method (Bechtold et al., C R . Acad. Sci . Paris 316:1194-1199, 1993). Third generation progeny of the transgenic
Arabidopsis plants containing pHI-33 to pHI-39 were evaluated for their growth properties . When germinated and cultured on MS media with or without 2% sucrose, transgenic plants expressing up-regulatory AGPase (UpRegl and R4) and wildtype (R20 and WT) type AGPases grew considerably faster than control plants (see Fig. 10 and Fig. 13) . Moreover, when transferred to soil, several of the transgenic plants bearing UpReg-1 and R4 produced larger quantities of seed at the end of 8 weeks of growth than normal plants (Fig. 8) .
The observed phenotypic traits expressed by these transgenic plants are striking (Figs. 8 and 13) . They include higher rates of development and growth, and variation in leaf size, seed weights, and seed yields. None of these phenotypes are strictly inter-related although there appears to be several general trends which can be summarized as follows:
1. Most of the plants containing the up- regulatory (UpRegl and R4) to wild-type (R20 and W.T.) potato AGPase LS grew faster than normal Columbia plants (Fig. 13) . In contrast, plants containing the down- regulatory AGPase LS genes, R32 and M345, grew at the same rate as Columbia plants suggesting that increased growth rates are due to an increase in enzyme activity levels in these transgenic plants.
2. Increased seed yield but not seed weight is correlated with the presence of up-regulatory allosteric potato AGPase UpRegl and R4 (Fig. 8) . This observation suggests that the increase in seed yield is due to increased enzyme activity via the up-regulatory response .
3. There is little correlation between increased leaf size and allosteric regulatory behavior and, in turn, enzyme activity of AGPase (Fig. 13) . Large leaves are not only evident in the up-regulatory plants such as UpRegl but also in the down-regulatory plants such as R32 and M345. Alternatively, the up-regulatory plant, R , which grows faster than normal Columbia plants, has smaller leaves. These findings suggest that the variation in leaf size is not due to enzyme function but due to some other non-catalytic interaction by the transgene AGPase LS .
4. There is little correlation between larger seed weight and allosteric regulatory behavior and, in turn, enzyme activity of AGPase (Fig. 13) . Larger seed weights are observed in plants containing either up-regulatory or down regulatory AGPase LS genes. Larger seed weights, however, are correlated with a reduction in seed yield. The reduction in seed yield is due to the reduced number of seeds per pod.
Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. The invention encompasses all modifications that are within the spirit and scope of the appended claims .

Claims

WHAT IS CLAIMED IS:
1. A method for producing a nucleic acid encoding an up-regulated mutant ADPG-PP enzyme, comprising the steps of :
(i) providing a nucleic acid that encodes a large subunit of an allosterically regulated ADPG-PP enzyme, the ADPG-PP enzyme consisting of the large subunit and a small subunit; (ii) introducing a first mutation to said nucleic acid to produce a first mutant nucleotide;
(iii) identifying a first mutant nucleic acid, a protein product of which, when co-expressed in a cell with a wild-type ADPG-PP small subunit, is defective in allosteric regulation;
(iv) introducing a second mutation to said nucleic acid to produce a second mutant nucleic acid;
(v) identifying a second mutant nucleic acid, a protein product of which, when co-expressed in a cell with a wild-type ADPG-PP small subunit, at least partially restores allosteric regulation;
(vi) sequencing the second mutant nucleic acid, the protein product of which at least partially restores allosteric regulation so as to characterize the first and second mutations;
(vii) producing a third mutant nucleic acid having the second, but not the first mutation; and
(viii) identifying a third mutant nucleic acid, a protein product of which has greater biological activity than a wild-type ADPG-PP enzyme, thereby producing and identifying a nucleic acid encoding an up-regulated mutant ADPG-PP enzyme.
2. The method of claim 1, wherein the nucleic acid that encodes the large subunit of ADPG-PP is native to a plant .
3. A nucleic acid molecule produced by the method of claim 1.
4. A peptide encoded by the nucleic acid molecule of claim 3.
5. A cell comprising the nucleic acid molecule of claim 3.
6. A method for producing a nucleic acid encoding an up-regulated mutant ADPG-PP enzyme, comprising the steps of :
(i) providing a nucleic acid that encodes a large subunit of an allosterically regulated ADPG-PP enzyme, the ADPG-PP enzyme consisting of the large subunit and a small subunit;
(ii) introducing a first mutation to said nucleic acid to produce a first mutant nucleic acid;
(iii) cloning said first mutant nucleic acid into a low copy-number vector;
(iv) transforming said cloned first mutant nucleic acid into a population of cells defective in ADPG-PP function;
(v) expressing said cloned first mutant nucleic acid in said cells; and
(vi) screening said transformed cells to identify a nucleic acid coding for an up-regulated mutant ADPG-PP enzyme, thereby producing and identifying a nucleic acid encoding an up-regulated mutant ADPG-PP enzyme.
7. The method of claim 6, wherein the nucleic acid that encodes the large subunit of ADPG-PP is native to a plant .
8. A nucleic acid molecule produced by the method of claim 6.
9. A peptide encoded by the nucleic acid molecule of claim 8.
10. A cell comprising the nucleic acid molecule of claim 8.
11. A method for producing a nucleic acid encoding an up-regulated mutant ADPG-PP enzyme, comprising the steps of: (i) providing a nucleic acid that encodes a large subunit of an allosterically regulated ADPG-PP enzyme, the ADPG-PP enzyme consisting of the large subunit and a small subunit;
(ii) deleting a terminal portion of said of the nucleic acid molecule to produce a first mutant nucleic acid;
(iii) cloning said first mutant nucleic acid into a low copy-number vector;
(iv) transforming said cloned first mutant nucleic acid into a population of cells defective in ADPG-PP function;
(v) expressing said cloned first mutant nucleic acid in said cells; and
(vi) screening said cells to identify a nucleic acid encoding an up-regulated mutant ADPG-PP enzyme, thereby producing and identifying a nucleic acid encoding an up-regulated mutant ADPG-PP enzyme.
12. The method of claim 11 wherein the nucleic acid that encodes the large subunit of ADPG-PP is native to a plant .
13. A nucleic acid molecule produced by the method of claim 11
14. A peptide encoded by the nucleic acid molecule of claim 13.
15. A cell comprising the nucleic acid molecule of claim 13.
16. A nucleic acid molecule, comprising a nucleic acid sequence selected from the group consisting of the sequences shown in SEQ. I.D. Nos. 1, 3, 5, 7, 9, and 11.
17. A polypeptide, comprising an amino acid sequence selected from the group consisting of the sequences shown in SEQ. I.D. Nos. 2, 4, 6, 8, 10, and 12.
18. A cell, comprising the nucleic acid molecule of claim 16.
19. The cell of claim 18, selected from a group consisting of a plant cell, a bacterial cell, and a fungal cell.
20. The cell of claim 19, exhibiting at least one characteristic selected from the group consisting of increased yield compared to a wild-type plant cell, increased productivity compared to a wild-type plant cell, increased starch production compared to a wild- type plant cell, increased size compared to a wild-type plant cell, increased rate of growth compared to a wild- type plant cell, and increased number of seeds compared to a wild-type plant cell.
PCT/US1998/016551 1997-08-07 1998-08-07 Regulatory mutants of adp-glucose pyrophosphorylase and related compositions and methods WO1999007841A2 (en)

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US7285703B2 (en) 1998-04-03 2007-10-23 Basf Plant Science Gmbh Plant like starches and the method of making them in hosts
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US7285703B2 (en) 1998-04-03 2007-10-23 Basf Plant Science Gmbh Plant like starches and the method of making them in hosts
FR2828694A1 (en) * 2001-08-17 2003-02-21 Agronomique Inst Nat Rech USE OF ASSOCIATIONS BETWEEN AT LEAST ONE NUCLEIC SEQUENCE POLYMORPHISM OF THE SH2 GENE AND AT LEAST ONE SEED QUALITY CHARACTERISTIC, IN PLANT SELECTION PROCESSES
WO2003016564A3 (en) * 2001-08-17 2004-02-19 Genoplante Valor S A S Use of associations between at least one nucleic sequence polymorphism of the sh2 gene and at least one seed quality characteristic in plant selection methods
EP1461431A4 (en) * 2001-12-03 2005-05-25 Univ Floridares Foundation Inc VARIANTS OF ADP-GLUCOSE PYROPHOSPHORYLASE AFFECTING PHOSPHATE SENSITIVITY AND OTHER PARAMETERS
US7173165B2 (en) 2001-12-03 2007-02-06 University Of Florida Research Foundation, Inc. Variants of ADP-glucose pyrophosphorylase affecting phosphate sensitivity and other parameters
EP2186900A3 (en) * 2001-12-03 2010-06-02 University Of Florida Research Foundation, Inc. Variants of ADP-Glucose pyrophosphorylase affecting phosphate sensitivity and other parameters
EP2275550A3 (en) * 2001-12-03 2011-07-06 University of Florida Research Variants of ADP-glucose pyrophosphorylase affecting phosphate sensitivity and other parameters
US8030540B2 (en) 2004-04-21 2011-10-04 Basf Plant Science Gmbh Transgenic corn having enhanced nutritional qualities
CN108728478A (en) * 2017-04-20 2018-11-02 中国科学院上海生命科学研究院 The method for improving yield and modified starch property using hydrogen ion pyrophosphorylase
CN108728478B (en) * 2017-04-20 2021-08-27 中国科学院分子植物科学卓越创新中心 Method for improving yield and starch property by using hydrogen ion pyrophosphorylase

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