HK1020071A - Plant sugar sensors and uses thereof - Google Patents

Description

plant sugar sensing protein and application thereof

no marking

Background

The present application relates to plant sugar metabolism; in particular to an enzyme for transducing sugar sensing signals, a coding gene and application thereof.

Sugars are considered regulatory molecules that control physiology, metabolism, cell cycle, development, and gene expression. During the life cycle of higher plants, sugars affect growth and development from germination to flowering to senescence. Recently, it has been recognized that sugars are physiological signals capable of inhibiting or activating plant genes, which signals are involved in a number of important processes, including photosynthesis, glyoxylate cycle, respiration, starch and sucrose synthesis and degradation, nitrogen metabolism and storage, pathogen defense, wound response, cell cycle progression, pigmentation and senescence (Sheen, research on photosynthesis 39,427 (1994); Thomas and Rodriguez, plant physiology 106,1235 (1994); Knight and Gray molecular genetics 242,586 (1994); Lam et al, plant physiology 106,1347 (1994); Chen et al, plant journal 6,625 (1994); Reynolds and Smith, plant molecular biology 29,885 (1995); Herbers et al, plant molecular biology 29,1027 (1995); Mita et al, plant physiology 107,895 (1995)). Studies on various plant species have also shown that sugar homeostasis appears to be tightly regulated. Elevated sugar concentrations lead to growth disturbances, reduced photosynthesis, leaf rolling, chlorosis, necrotic leaf and anthocyanin accumulation (Casper et al, Plant physiology 79,11 (1985); Von Schaewen et al, EMBO J.9,3033 (1990); Dickinson et al, Plant physiol.95,420 (1991); Tsukaya et al, Plant physiology 97,1414 (1991); Sonnwald et al, Plant journal 1,95 (1991); Huberet and Hanson, Plant physiology 99,1449 (1992); Sonnwald et al, Plant response to sugar accumulation in transgenic tobacco plants, pp.246-257, see: M.A.Madore, W.J.Lucas, carbon partitioning and source pool interactions in plants, American Plant physiology society, Rockville, M D, (1995, M D)). In addition, proposals have been made such as CO₂Environmental factors with increased concentrations and inherent genetic variations such as different invertase content can influence photosynthetic capacity through sugar regulation (Stitt, plant cell environment 14,741 (1991); Stitt et al, plants 183,40 (1991); Van Oosten et al, plantsCellular environment 17,913 (1994); nie et al, plant physiology 108,975 (1995); goldschmidt and Huber, plant physiology 99,1443 (1992)).

Summary of the invention

By manipulating the expression of the plant hexokinase protein (HXK), we have found that this protein is a sensor protein that mediates multiple sugar responses in plants. Specifically, we have produced by genetic engineering methods transgenic plants that either (a) express lower levels of hexokinase protein due to the expression of an antisense hexokinase gene and thus exhibit lower sensitivity to sugars; or (b) express higher levels of hexokinase protein and thus exhibit higher sensitivity to sugars. Our findings have broadened the implications of manipulating crops for improving crop yield and quality and reducing production costs.

In general, the invention features a method for reducing the level of a plant hexokinase protein in a transgenic plant cell, the method comprising expressing in the transgenic plant cell (e.g., a cell derived from a monocot, a dicot, or a gymnosperm) an antisense hexokinase nucleic acid sequence. Thereby producing a transgenic plant that is less sensitive to sugars (e.g., glucose, sucrose, fructose, or mannose).

In a preferred embodiment, the antisense hexokinase nucleic acid sequence is encoded by a transgene integrated into the genome of the transgenic plant cell; the antisense hexokinase nucleic acid sequence comprises a plant antisense hexokinase DNA sequence (e.g., a sequence based on the AtHXK1 nucleotide sequence of FIG. 1F (SEQ ID NO:3) or the AtHXK2 nucleotide sequence of FIG. 1G (SEQ ID NO: 4)); moreover, the method further comprises culturing a transgenic plant derived from the transgenic plant cell, thereby reducing the level of hexokinase protein in the transgenic plant.

In a related aspect, the invention also features a plant cell (e.g., a plant cell derived from a monocot, dicot, or gymnosperm) that expresses an antisense hexokinase nucleic acid sequence; and a plant expression vector comprising an antisense hexokinase nucleic acid sequence, wherein said sequence is operably linked to an expression control region.

In another aspect, the invention also features a method for increasing the level of a hexokinase protein in a transgenic plant cell, the method comprising expressing a hexokinase nucleic acid sequence in the transgenic plant cell. In a preferred embodiment, the hexokinase nucleic acid sequence is derived from a plant (e.g., a DNA sequence identical to the AtHXK1 nucleotide sequence of FIG. 1F (SEQ ID NO:3), or a DNA sequence substantially identical to the AtHXK2 nucleic acid sequence of FIG. 1G (SEQ ID NO: 4)). The method can produce transgenic plants with enhanced sensitivity to sugars.

In a related aspect, the invention features a substantially pure plant HXK polypeptide that includes an amino acid sequence substantially identical to that of AtHXK1(SEQ ID NO:1) or AtHXK2(SEQ ID NO: 2). In preferred embodiments of both aspects, the hxhk polypeptide is obtained from a plant including, but not limited to, a monocotyledonous plant (e.g., rice, maize, wheat or barley), a dicotyledonous plant (e.g., a member of the solanaceae family (e.g., potato) or a member of the brassicaceae family (e.g., arabidopsis, broccoli, cabbage, brussel sprout, canola, kale, cabbage, cauliflower or horseradish)), and a gymnosperm.

In yet another related aspect, the invention features substantially pure DNA encoding a plant HXK polypeptide that includes an amino acid sequence substantially identical to that of AtHXK1(SEQ ID NO:1) or AtHXK2(SEQ ID NO: 2). In a preferred embodiment, the DNA comprises the nucleotide sequence shown in FIG. 1F (SEQ ID NO:3) or comprises a nucleotide sequence substantially identical to the sequence shown in FIG. 1G (SEQ ID NO: 4). The above DNA may be obtained from any plant, including, but not limited to, monocotyledonous plants (e.g., rice, maize, wheat and barley), dicotyledonous plants (e.g., a member of the Solanaceae family (e.g., potato) or a member of the Brassicaceae family (e.g., Arabidopsis, broccoli, cabbage, brussel sprout, canola, kale, cabbage mustard, cauliflower or horseradish), and gymnosperms.

In yet another related aspect, the invention features a vector that includes any one of the substantially pure DNAs of the invention, the vector being capable of directing expression of a protein encoded by the DNA in a cell containing the vector; a cell, such as a prokaryotic cell (e.g., an e.coli cell) or a eukaryotic cell (e.g., a plant cell) containing any one of the DNAs of the present invention; and a transgenic plant (or a cell or seed derived from the above transgenic plant) containing any one of the DNAs of the present invention integrated into the genome of the plant, wherein the DNA is expressed in the transgenic plant.

In various preferred embodiments, said plant cell contains said DNA in a sense orientation and has a higher sensitivity to sugars; the plant cells contain DNA in an antisense orientation and are less sensitive to sugars; the expression of the DNA is under the control of a constitutive or regulated promoter.

In two further aspects, the invention features a method of producing a plant HXK polypeptide, the method comprising: (a) providing a cell transformed with a gene encoding a polypeptide comprising either an amino acid sequence substantially identical to the amino acid sequence of AtHXK1(SEQ ID NO:1) or an amino acid sequence substantially identical to the amino acid sequence of AtHXK2(SEQ ID NO:2), the gene being positioned for expression in the cell; (b) expressing the plant HXK polypeptide; and (c) recovering the plant HXK polypeptide.

The "hexokinase" or "HXK" refers to a polypeptide that catalyzes the conversion of an ATP-dependent hexose to hexose-6-phosphate. Methods for determining the above enzymatic activities are well known in the art, for example, the method proposed by Renz and Stitt (plant 190,166(1993)) described herein.

The phrase "reducing the level of a plant hexokinase protein" means that the level of the plant hexokinase protein is reduced by at least 30-50%, preferably by 50-80%, more preferably by 80-95%, relative to the level in a control plant (e.g., a wild-type plant). The reduction of the hexokinase protein content can be achieved by expression of an antisense plant hexokinase nucleotide sequence in a transgenic plant. Plant hexokinase protein content can be monitored by any standard method, including but not limited to immunoblotting (e.g., as described herein). In addition, the content of plant hexokinase protein can be quantified by standard hexose phosphorylation assays (e.g., as described herein).

The phrase "increasing the level of a plant hexokinase protein" means that the level of the plant hexokinase protein is increased by at least 50%, preferably by 100%, more preferably by more than 200%, relative to the level in a control plant (e.g., a wild-type plant). The content of plant hexokinase protein can be monitored by any standard method, including but not limited to immunoblotting (e.g., as described herein). In addition, the content of plant hexokinase protein can be quantified by standard hexose phosphorylation assays (e.g., as described herein).

"antisense hexokinase sequence" refers to a nucleotide sequence that is complementary to a plant hexokinase messenger RNA. Generally, such antisense sequences are generally at least 15 nucleotides in length, preferably about 15-200 nucleotides, more preferably 200-2,000 nucleotides. The antisense sequence may be complementary to all or a portion of the plant hexokinase mRNA nucleotide sequence (e.g., the AtHXK1 and AtHXK2 antisense constructs described herein, and as will be appreciated by those skilled in the art, the specific binding site of the antisense sequence and the length of the antisense sequence may vary depending on the degree of inhibition desired and the uniqueness of the antisense sequence. Nature 377,495 (1995); cheung et al, cell 82,383 (1995); and U.S. Pat. No. 5,107,065.

By "less sensitive to sugars" is meant that developmental, physiological, or molecular processes, usually regulated or controlled by internal or external sugar concentrations, exhibit reduced response to the presence of sugars (e.g., glucose, fructose, mannose, or sucrose). For example, a plant with reduced sensitivity to sugars can activate a class of genes that are normally inhibited by the presence of sugars (e.g., photosynthetic genes), or the plant can develop through its normal developmental pathway even at sugar concentrations that would otherwise impede or inhibit its development. Analysis of the sensitivity of plants to sugars is achieved with a variety of bioassays (e.g., as described herein). These assays include, but are not limited to, evaluating and monitoring gene expression, seed germination, cotyledon development (e.g., cotyledon elongation), cotyledon greening, leaf development, radicle development, hypocotyl elongation, anthocyanin accumulation, starch accumulation, and the time required for flowering. By comparing the phenotype of the wild-type plant and the candidate plant (e.g., a plant expressing an antisense hexokinase gene), one can readily determine whether the candidate transgenic plant has reduced sensitivity to sugar. For example, sugars have been found to inhibit the expression of photosynthetic genes (e.g., the small subunit of rubisco and the photoplethysme a/b binding protein) and non-photosynthetic genes (e.g., alpha-amylase, sucrose synthase, malate synthase, and asparagine synthase). Thus, in plants that are less sensitive to sugars, the above-mentioned genes that can be inhibited by sugars have a reduced, attenuated or mitigated degree of sugar-mediated inhibition.

By "enhanced sensitivity" is meant that a developmental, physiological, or molecular process, usually regulated or controlled by internal or external sugar concentrations, exhibits an enhanced or increased response to the presence of a sugar (e.g., glucose, fructose, mannose, or sucrose). For example, a plant with increased sensitivity to sugars can increase, potentiate, or promote the activation of a class of genes that are normally activated by the presence of sugars (e.g., nutrient storage proteins). The analysis of the sensitivity of plants to sugars is carried out using a variety of bioassays. These assays include, but are not limited to, evaluating and monitoring gene expression, seed germination, cotyledon development (e.g., cotyledon elongation), cotyledon greening, leaf development, radicle development, hypocotyl elongation, anthocyanin accumulation, starch accumulation, and the time required for flowering. By comparing the phenotype of the wild-type plant and the candidate plant (e.g., a plant expressing at least one additional copy of the hexokinase gene), one can readily determine whether the candidate transgenic plant has increased sensitivity to sugar. For example, sugars have been found to activate the expression of genes such as nitrate reductase, beta-amylase, sucrose synthase, and potato storage protein. Thus, the above-described sugar-inducible genes have enhanced, elevated or elevated sugar-mediated expression levels in plants with enhanced sensitivity to sugar.

"polypeptide" or "protein" refers to any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

"substantially identical to AtHXK 1" refers to a plant hexokinase polypeptide comprising an N-terminus that is at least 50%, preferably 75%, more preferably 85-90%, most preferably 95% identical to the N-terminus of AtHXK1 (amino acids 1-61 of FIG. 1B; SEQ ID NO: 1). The length of comparison is typically at least 15 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids, and most preferably 60 amino acids.

"substantially identical to AtHXK 2" refers to a sequence of a plant hexokinase polypeptide or nucleic acid that is at least 86%, preferably 90%, more preferably 95%, and most preferably 99% identical to the amino acid or nucleic acid sequence of AtHXK2 (FIGS. 1B and 1G; SEQ ID Nos. 2 and 4).

Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package of the university of Wisconsin Biotechnology center, university of 1710 Dada, Madison, Wis. 53705), BLAST, or PILEUP/PRETTYBOX programs). The software described above pairs similar sequences by confirming the degree of homology to various substitutions, deletions, substitutions and other modifications. Conservative substitutions typically include substitutions that occur within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By "substantially pure polypeptide" is meant a plant hexokinase polypeptide (e.g., AtHXK1 or AtHXK2) that has been isolated from components with which it naturally occurs. Typically, a polypeptide is substantially pure when it is at least 60% free, by weight, of the protein and the naturally occurring organic molecule with which it is naturally associated. Desirably, the formulation contains at least 75%, more preferably at least 90%, most preferably at least 99% by weight of the plant hexokinase polypeptide. For example, a substantially pure plant hexokinase polypeptide can be obtained by extraction from a natural source (e.g., a plant cell); obtained by expressing a recombinant nucleic acid encoding a plant hexokinase polypeptide; or by chemical synthesis of the protein. Purity can be determined by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

"substantially pure DNA" refers to DNA that: the DNA does not contain a gene flanking the gene in the naturally occurring genome of the organism producing the DNA of the invention. Thus, for example, the term includes recombinant DNA incorporated into a vector; recombinant DNA incorporated into an autonomously replicating plasmid or virus; or a recombinant DNA incorporated into the genomic DNA of a prokaryote or eukaryote; or as a separate molecule (e.g., a cDNA or genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. The term also includes a recombinant DNA that is part of a hybrid gene encoding other polypeptide sequences.

"transformed cell" refers to a cell into which (or into an ancestor of which) a DNA molecule encoding a HXK polypeptide (e.g., AtHXK1 or AtHXK2), as used in the present invention, has been introduced by recombinant DNA techniques.

By "located in a position suitable for expression" is meant that the DNA molecule is located in proximity to a DNA sequence that directs the transcription and translation of the sequence (i.e., for example, to facilitate production of a plant hexokinase polypeptide, recombinant protein, or RNA molecule, such as AtHXK1 or AtHXK 2).

"promoter" refers to a minimal sequence sufficient to direct transcription. In the present invention, promoter elements sufficient to allow promoter-dependent gene expression to be controllable for cell-, tissue-or organ-specific gene expression, or elements inducible by external signals or agents (e.g., light, pathogen, wound or hormone-inducible elements); the elements may be located in the 5 'or 3' region of the native gene.

By "operably linked" is meant that a gene and a regulatory sequence are linked in such a way that the gene is capable of being expressed when an appropriate molecule (e.g., a transcriptional activator protein) is bound to the regulatory sequence.

By "plant cell" is meant any autonomously propagating cell surrounded by a semi-permeable membrane and containing plastids. The cell also requires a cell wall if further propagation is required. Plant cells as used herein include, but are not limited to, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, stems, gametophytes, sporophytes, pollen, and microspores.

"transgene" refers to any segment of DNA that is artificially inserted into a cell and becomes part of the genome of an organism that develops from the cell. The transgene may include a gene that is partially or wholly heterologous (i.e., exogenous) to the transgenic organism, or may represent a gene that is homologous to an endogenous gene of the organism.

"transgenic" refers to any cell that includes a DNA sequence that is artificially inserted into the cell and becomes part of the genome of the organism being developed by the cell. In this context, transgenic organisms generally refer to transgenic plants, and the DNA (transgene) is inserted artificially into the genome of their nuclei or plastids.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.

Detailed Description

The drawings will be described first. Drawings

FIGS. 1A-E are schematic diagrams showing various aspects of the molecular characteristics of the Arabidopsis thaliana (Arabidopsis thaliana) HXK gene. FIG. 1A shows the functional complementation of HXK catalytic activity obtained with a yeast HXK1/HXK2 double mutant strain (named DBY2219) carrying the Arabidopsis thaliana HXK homologs AtHXK1 and AtHXK 2. pFL61 is shown as a control vector for the complementation study described above. FIG. 1B shows Arabidopsis thaliana hexokinase AtHXK1(SEQ ID NO: 1); schematic representation of amino acid sequence comparison of Arabidopsis thaliana hexokinase AtHXK2(SEQ ID NO:2), human GLK (SEQ ID NO:5), rat GLK (SEQ ID NO:6), Saccharomyces cerevisiae HXK1 (Yeast 1) (SEQ ID NO:7), Saccharomyces cerevisiae HXK2 (Yeast 2) (SEQ ID NO:8), and Kluyveromyces lactis (Kluyveromyces lactis) RAG5 (Yeast 3) (SEQ ID NO: 9). Underlined regions 1 and 2 indicate conserved phosphate 1 and 2 regions; the underlined shaded areas indicate adenosine interaction regions. Underlined amino acids with asterisks indicate conserved sugar-binding domains. Sequence analysis was performed using the PILEUP/PRETTYBOX program set as standard parameters. Identical and similar residues refer to boxed and highlighted portions, respectively. FIG. 1C is a schematic representation of the loci of AtHXK1 and AtHXK2 on chromosomes IV and II, respectively, of Arabidopsis. FIG. 1D is a photograph showing that AtHXK is a southern blot analysis encoded by a multigene family. The blot was hybridized with full-length cDNA probes of either athxhk 1 (designated as athxkk 1, shown on the left) or athxkk (designated as athxkk 2, shown on the right). The numbers indicated on the left side of the blot refer to molecular size markers in kilobases. FIG. 1E is a photograph of a southern blot analysis of Arabidopsis genomic DNA digested with HindIII, separated by gel electrophoresis, transferred to a nylon membrane, and hybridized with an AtHXK1 full-length cDNA probe under low stringency conditions. FIG. 1F is a schematic diagram showing the nucleotide sequence (SEQ ID NO:3) of AtHXK 1. FIG. 1G is a schematic diagram showing the nucleotide sequence (SEQ ID NO:4) of AtHXK 2.

FIGS. 2A-B are a series of schematic diagrams showing the expression of HXK in Arabidopsis. Fig. 2A is a photograph of a northern blot showing that athxhk 1 and athxhk 2 are expressed in leaf (flower node leaf and cauline leaf), stem, flower, silique and root tissues. Fig. 2B is a photograph showing that the expression of athxhk 1 and athxkk 2 is light-and sugar-induced northern blotting.

FIGS. 3A-F are color photographs of Arabidopsis seedlings illustrating HXK as a sugar sensor protein in plants. FIG. 3A is a photograph of transgenic Arabidopsis seedlings germinated on 1/2MS plates containing 6% glucose, with enhanced expression of either sense-AtHXK1 (left) or antisense-AtHXK 1 (middle) constructs. Wild type (control) plants are shown on the right. FIG. 3B is a photograph of transgenic Arabidopsis seedlings that germinated on 1/2MS plates containing 0.8mM2-dGlc with enhanced expression of either the sense-AtHXK1 (left) or antisense-AtHXK 1 (middle) constructs. Wild type (control) plants are shown on the right. Fig. 3C is a photograph showing T3 homozygous populations of sense-AtHXK1 (left) and antisense-AtHXK 1 (right) plants germinated on 1/2MS plates containing 6% glucose. FIG. 3D is a photograph showing T3 homozygous populations of sense-AtHXK1 (left) and antisense-AtHXK 1 (right) plants germinated on 1/2MS plates containing 0.8mM 2-dGlc. FIG. 3E is a photograph showing sense-AtHXK1 (left), antisense-AtHXK 1 (center) and control (right) plants germinated on 1/2MS plates containing 6% mannitol. FIG. 3F is a photograph showing sense-AtHXK1 (left), antisense-AtHXK 1 (center), and control (right) plants germinated on 1/2 plates containing 6% 3-MeGlc.

FIGS. 4A-C are a series of color photographs showing the effect of AtHXK-mediated sugar on seedling growth and development. Seedlings were grown in the dark for 6 days followed by 12 hours of light, growth being achieved on media containing different glucose concentrations (shown as increasing glucose concentration (Glc%) 2,3, 4, 5 or 6%). FIG. 4A is a photograph showing that higher concentrations of glucose inhibited hypocotyl elongation and enlargement, and cotyledon greening in wild-type (control) plants. FIG. 4B is a photograph of sense-AtHXK1 plants that are hypersensitive to glucose, as shown by the strong inhibitory effect on seedling development. FIG. 4C is a photograph of antisense-AtHXK 1 plants that are less sensitive to glucose, as shown by the reduced inhibitory effect on seedling development when compared to wild type plants.

FIGS. 5A-F are a series of schematic diagrams showing the expression of various genes in control-, sense-, and antisense-plants. FIG. 5A is a photograph of a northern blot analysis performed on illuminated yellowed seedlings germinated on 1/2MS plates containing 6% glucose. UBQ expression was monitored as a control. FIG. 5B is a photograph of a northern blot analysis of RNA prepared from light-grown green plants (light) (which were propagated without exogenous sugars) and light-grown green plants (dark) (which were adapted to dark for 3 days, then light for 4 hours). FIG. 5C is a photograph of several northern blots showing expression of sense and antisense constructs in transgenic plants. RNA was extracted from yellow seedlings grown on 1/2MS plates containing 6% glucose. Gene and strand specific probes were used to reveal sense-AtHXK1 (sense-1), sense-AtHXK2 (sense-2), antisense-AtHXK 1 (antisense-1), and antisense-AtHXK 2 (antisense-2) transcripts. The blot showing expression of sense-AtHXK1 (sense-1) was exposed for a longer period of time than the other blots. Wild type plants were used as controls. Fig. 5D is a photograph of a western blot analysis showing expression of AtHXK 1. sense-AtHXK1 plants showed increased expression of AtHXK1, while antisense-AtHXK 1 plants showed reduced expression. Wild type plants were used as controls. FIG. 5E is a histogram showing the phosphorylation activity of total hexoses in etiolated seedlings. FIG. 5F is a histogram showing the phosphorylation activity of total hexoses in light-grown plants. Error bars indicate standard deviation.

FIGS. 6A-C are series of schematic diagrams showing that sugar signaling is decoupled from sugar metabolism. FIG. 6A is a photograph showing that growth of wild type strains, but not the double mutant strain hxk1/hxk2, was inhibited on 2-dGlc/raffinose plates (left). Enhanced expression of AtHXK1 or AtHXK2 in strain hxk1/hxk2 did not restore glucose repression, as indicated by the level of growth of this strain on 2-dGlc/raffinose plates, similar to the double mutant strain (right) transformed with vector alone (pFL 61). FIG. 6B is a color photograph showing the dominant interference of enhanced yeast HXK2 expression in transgenic Arabidopsis seedlings (i.e., YHXK2 plants) grown for 7 days on 1/2MS plates containing 6% glucose. FIG. 6C is a histogram showing total hexose phosphorylation activity in yellow or green YHXK2, sense-AtHXK1, and control plants. Error bars indicate standard deviation.

The following is a description of the cloning and identification of two Arabidopsis HXK-encoding cDNAs that can be used in the present invention, as well as their ability to modulate sugar metabolism. These examples are provided for the purpose of illustrating the invention and are not to be construed as limiting the invention. Molecular identification of Arabidopsis HXK gene

To reveal the role of HXK as a sugar sensor protein, the Arabidopsis HXK gene was cloned by functional complementation, using a yeast strain lacking HXK activity-Saccharomyces cerevisiae HXK1/HXK2 double mutant strain (designated DBY2219) (Ma and Botstein, molecular cell biology 6,4046 (1986)). We used this method to identify two cDNAs, namely AtHXK1 (FIG. 1F; SEQ ID NO:3, GenBank accession No. U28214) and AtHXK2 (FIG. 1G; SEQ ID NO:4, GenBank accession No. U28215). The cDNA, which was 2.0 and 1.9kb in length, was found to reproducibly complement the yeast double mutant and was allowed to grow on selective plates containing fructose as the sole carbon source. Typical results are shown in FIG. 1A; mutant yeast cells transformed with either the AtHXK1 or AtHXK2 cDNAs were able to grow on selection medium, indicating that these genes were complementary to the double mutant. In contrast, the mutant transformed with plasmid vector pF61 alone could not grow on the same selection medium (FIG. 1A) (Minet et al, plant J2, 417 (1992)).

DNA sequence analysis of AtHXK1 (FIG. 1F, SEQ ID NO:3) and AtHXK2 (FIG. 1F, SEQ ID NO:4) predicted open reading frames of 496 and 502 amino acids, respectively (FIG. 1B). These genes have been found to possess 82% nucleotide identity and 85% amino acid identity. In addition, database searches and sequence comparisons revealed that the above-described AtHXKs share 34-35% sequence identity with human and rat GLKs (Nishi et al, diabetes 35,743 (1992); Magnus et al, Proc. Natl. Acad. Sci. USA 86,4838(1989)), and 36-38% sequence identity with several yeast HXKs (Stachelek et al, nucleic acid research 14,945 (1986); Prior et al, molecular cell biology 13,3882 (1993)). Conserved ATP-and sugar-binding domains were also identified in the predicted amino acid sequences of the two AtHXK genes. As shown in FIG. 1B, 3 functional domains involved in ATP binding were identified (Bork et al, protein science 2,31 (1993)). Also shown in FIG. 1B is a carbohydrate binding domain that is similar to the glucose binding site found in mammalian GLK (Bork et al, protein science 2,31 (1993)). In general, our sequence comparisons indicate that the entire sequence and constructs of Arabidopsis HXKs are similar to those of mammalian GLK and yeast HXKs, but different from those of plant fructokinases (Smith et al, plant physiology 102,1043 (1993)).

Subsequently, we determined the chromosomal mapping of the AtHXK1 and AtHXK2 genes by standard isolation analysis of Restriction Fragment Length Polymorphisms (RFLPs) in recombinant inbred lines (Nam et al, plant cell 1,699 (1989); Lister and Dean, plant journal 4, 745 (1993); Hauge et al, plant journal 3,745 (1993); Schmidt et al, science 270,480 (1995); Zachgo et al, genomic research 6,19 (1996)). From the above analysis, we found that AtHXK1 is located on chromosome 4 flanked by chromosome markers mi232 and g8300 (FIG. 1C), while AtHXK2 is located on chromosome 2 flanked by chromosome markers mi148 and mi238 (FIG. 1C).

The copy number of the AtHXK gene was determined by genomic DNA (southern) blot analysis. Genomic DNA was prepared from Arabidopsis thaliana by standard methods (Landsberg er), digested with Bgl II, Eco RI, Hind III, or Xba I, separated by gel electrophoresis, transferred to nylon membranes, and hybridized with full-length cDNA random primer probes of AtHXK1 or AtHXK2 (Ausubel et al, see below). Genomic southern blot analysis showed that AtHXK1 could hybridize to two DNA fragments corresponding to two AtHXK genes under highly stringent conditions (FIG. 1D, blot designated AtHXK 1). In addition, when AtHXK2 was used as a probe under the same hybridization conditions, at least one additional fragment was visible on the same blot (FIG. 1D, the blot was designated as AtHXK 2). A third cDNA (AtHXK3) was also identified using this same approach, further supporting the hypothesis that there are 3 homologous HXK genes in arabidopsis. Under low stringency conditions, a number of other bands were also detected, indicating that more than 3 genes share sequence similarity with AtHXK1 (FIG. 1E). AtHXK Gene expression

To detect the expression of the AtHXK gene, northern blot experiments were performed in the following manner. RNA was extracted from flower node leaves, cauline leaves, stems, flowers, siliques and roots using standard methods. The extracted RNAs are gel separated and transferred to a nylon membrane (e.g., as described in Ausubel et al, infra). The blot was then hybridized with AtHXK1, AtHXK2 or Ubiquitin (UBQ) probe (Greenberg et al, cell 77,551(1994)) using standard techniques. In the above experiment, a UBQ probe was used as a control. Equal amounts of RNA were added to each lane.

Northern blot analysis showed that both AtHXK1 and AtHXK2 probes detected RNA bands approximately 2kb in length. As shown in fig. 2A, the content of the athxhk 1 and athxhk 2 transcripts was greatest in siliques, moderate in flowers and flower nodes, and lowest in stems and cauline leaves. In roots, the expression of AtHXK1 was higher than that of AtHXK 2. The different levels of expression of AtHXK1 and AtHXK2 reflect the diversity of their physiological roles, e.g., feedback regulation of photosynthesis in source tissues such as flower nodes (i.e., sugar supply tissues), and sugar metabolism in sink tissues such as siliques and flowers (i.e., sugar receptors).

Since light is essential for plants to produce sugars through photosynthesis, we investigated the effect of light on the expression of the AtHXK gene. Northern blot analysis was performed as described above with total RNA prepared from dark-grown etiolated plants and light-grown, dark-adapted wild-type plants, with or without light illumination (indicated in FIG. 2B by dark and light, respectively). Specifically, dark-grown etiolated seedlings were germinated and grown on plates containing 1/2Murashige-Skoog (MS) medium (represented in FIG. 2B as + and-sugars, respectively) with or without 6% glucose. Plants were grown in the dark for 6 days and then irradiated with white light (120. mu. Em-2S-1) for 4 hours. Light-grown, dark-adapted plants are 15-day-old light-grown green plants that are dark-adapted for 3 days and remain in the dark, or are light-adapted and then cast with 3% glucose (indicated as light + sugar in fig. 2B). Growth conditions were as described by Cheng et al (proceedings of the national academy of sciences of the United states of America USA 89,1861 (1992)).

As shown in fig. 2B, it was found that athxhk 1 and athxhk 2 were expressed at extremely low levels in non-photosynthetic, etiolated seedlings, even after 4 hours of light exposure. However, its expression is induced by the addition of exogenous sugars. In dark-adapted green plants, both the transcript content of AtHXK1 and AtHXK2 was low, but was significantly induced when exposed to light and could be further enhanced by sugars (FIG. 2B). It has been found that expression of the UBQ gene is affected by both light and sugar. The above results indicate that the expression of AtHXK is limited by specific conditions in which sugar sensing and metabolism are necessary, suggesting that the homeostasis of plant sugars is controlled by the content of AtHXK through self-regulation mechanisms. AtHXK as sugar sensing protein in plants

To verify the hypothesis that AtHXKs act as sugar sensing proteins in whole plants, transgenic plant models were created by introducing sense and antisense genes to alter AtHXK content. Wild type (Bensheim) Arabidopsis plants were transformed by standard Agrobacterium-mediated root transformation methods (Czako et al, molecular genetics 235,33(1992)) with binary vectors carrying genes fused to the CaMV35S promoter and to the Sense AtHXK1(Sense-AtHXK1), Sense AtHXK2(Sense-AtHXK2), antisense AtHXK1(anti-AtHXK1) or antisense AtHXK2(anti-AtHXK 2). The presence of the transgene was determined by NPT ii expression and resultant kanamycin resistance and by southern blot analysis. For further analysis, several transgenic lines of T3 generation homozygous for the sense or antisense transgene were selected.

Transgenic plants were tested for sugar sensitivity by bioassay with 6% glucose or 0.8mM 2-deoxyglucose (2-dGlc), a non-metabolizable glucose analog. On 6% glucose plates, control (wild type) arabidopsis seedlings grown under constant light had green and enlarged cotyledons, initiation of true leaf development, and inhibited elongation of hypocotyls and roots (fig. 3A, right). These inhibitory effects caused by glucose were confirmed in 6 different Arabidopsis ecotypes including Bensheim (BE), C24, Columbia (Col), Landsberg erecta (Ler), RLD and Watslewskija (WS) (data not shown). Furthermore, the greening of the cotyledons was found to be inhibited by low concentrations of 2-dGlc in the control plants (FIG. 3B, right). This phenotype is consistent with the findings: i.e.2-dGlc can act as a potential sugar signal which can trigger the global repression of the gene encoding the photosynthetic protein.

sense-AtHXK1 plants showed hypersensitivity to 6% glucose compared to control plants as evidenced by the inhibition of growth of cotyledons, hypocotyls and roots (fig. 3A, left). In contrast, antisense-AtHXK 1 plants became green and elongated normally (FIG. 3A, middle), indicating that they were less sensitive to sugars. FIG. 3C shows that sugar hypersensitivity and insensitivity are consistent among the T3 transgenic plant population. As in the glucose assay, sense-AtHXK1 plants were hypersensitive to 2-dGlc, which was shown to severely inhibit cotyledon greening (FIG. 3B, left; FIG. 3D, left). antisense-AtHXK 1 plants were less sensitive to sugar and became green when germinated in the presence of 2-dGlc (FIG. 3B, center; FIG. 3D, right).

As shown in table 1 (see below), similar phenotypes were observed in the multiple independent transgenic lines generated with sense or antisense AtHXK1 or AtHXK 2. The counting phenotype shown in Table 1 was determined based on examination of light-grown 7-day-old seedlings that germinated on 1/2MS plates containing 6% glucose or 0.8mM 2-dGlc. The number of sugar insensitive (Ins), hypersensitive (Hyp) and amphoteric (a) phenotypes was recorded and tabulated. The results of this analysis are shown in table 1 (below).

TABLE 1

Sugar sensitivity of T3 homozygous transgenic plants

		6% glucose			0.8mM 2-dGlc
								Transgenosis	Assembly system	Is not sensitive	Chao Min	Amphoteric property	Is not sensitive	Chao Min	Amphoteric property
CaMV 35S: sense-AtHXK1	13	0	11	2	0	13	0
								CaMV 35S: sense-AtHXK2	13	2*	8	3	1*	12	0
CaMV 35S: antisense-AtHXK 1	14	9	3	2	13	0	1
								CaMV 35S: antisense-AtHXK 2	14	10	0	4	11	1	2

Sugar insensitivity is thought to be due to co-suppression.

To exclude the possibility that the difference in sugar sensing between transgenic and control plants is due to osmosis, mannitol and 3-O-methylglucose (3-Meglc) were used in the control experiments. When plated with 6% mannitol (FIG. 3E) or 6% 3-MeGlc (a glucose analog which is not phosphorylated by HXK) (FIG. 3F), no significant difference was seen between control and transgenic plants. In conclusion, the above results indicate that sugar sensing in transgenic plants is specific, since neither mannitol nor 3-MeGlc can replace glucose and interact with AtHXKs. AtHXK-mediated effects of sugars on plant growth and development

Subsequently, we compared the effect of sugar on hypocotyl and cotyledon development in wild type and transgenic plants. To perform the above experiments, Arabidopsis seedlings were grown in the dark for 6 days on plates containing 2-6% glucose. Since hypocotyl elongation occurs more easily in the dark, inhibition by sugars can be assessed visually. Since light can trigger cotyledons to swell and turn green, these dark-grown seedlings were subjected to 12 hours of light to determine the effect of sugars on cotyledon development. Our results are shown in figure 4.

Specifically, in control plants, the length of the hypocotyl is inversely proportional to the concentration of glucose (fig. 4A). Under similar growth conditions, sense AtHXK plants were hypersensitive to sugar as revealed by the decrease in length of the hypocotyl when grown in the presence of 3-6% glucose (FIG. 4B). In contrast, antisense AtHXK plants were able to elongate even in the presence of 5 or 6% glucose (FIG. 4C). While glucose concentrations below 2% in the presence of other nutrients promoted seedling growth (data not shown), the inhibition of the hypocotyl by glucose concentrations above 2% reflected sugar sensing mediated by AtHXK.

Glucose (at 4-6% concentration) inhibited cotyledon greening and swelling in control plants as opposed to light (fig. 4A). The damage caused in sense-AtHXK plants was greater, as indicated by etiolated cotyledons (FIG. 4B). However, antisense-AtHXK plants were found to be less sensitive to all concentrations of glucose and to normally turn green (FIG. 4C). The sugar-inhibiting effect of cotyledon development is explained by the ability of plants to convert to heterotrophic growth under conditions of abundant external sugar, rather than photoautotrophic growth, for which cotyledon enlargement and greening are essential. We also observed a lack of sugar inhibition in the roots and antisense seedlings of the dark grown controls (fig. 4A and 4C). This may be a result of low levels of AtHXK expression in roots in the dark, since ectopic AtHXK expression may produce glucose-dependent inhibition of root growth in sense plants (fig. 4B). The above results indicate that different sugar effects may occur in different tissues due to different expression of AtHXK (FIG. 2). AtHXK-mediated sugar suppression and activation of Gene expression

To determine whether HXK is involved in the sugar regulation of gene expression, we compared the expression levels of two sugar-repressing genes, i.e., the photophoroptel a/b binding protein (CAB1) and the rubisco small subunit (RBCS), and a sugar-inducible gene, i.e., the nitrate reductase (NR1) gene, in control, sense-and antisense-AtHXK plants (Sheen, research in photosynthesis, 39,427 (1994); Thomas and Rodriguez, plant physiology 106,1235 (1994); Cheng et al, Proc. Sci. 89,1861 (1992)).

As described above, we first examined light, dark-grown etiolated seedlings propagated on 6% glucose. Northern blot analysis was also performed with the 1.1kb CAB1, 0.5kb RBCS, and 3.2kb NR1 probes obtained from Arabidopsis, as described above.

In control plants, the transcriptional levels of CAB1 and RBCS were low, and were almost abolished in sense-AtHXK1 and sense-AtHXK2 plants that exhibited sugar hypersensitivity (fig. 5A). In contrast, in both antisense-AtHXK plants exhibiting sugar insensitivity, both genes were expressed at high levels. Consistent with the above findings, sense transgenic plants were hypersensitive to sugar, NR1 was activated in both sense-AtHXK1 and sense-AtHXK2 plants, but not in antisense-AtHXK or control plants (fig. 5A). Taken together, the above data indicate that AtHXK is a sensor protein that mediates both sugar-repressible and sugar-inducible gene expression in higher plants. In addition, the transcriptional levels of CAB1 and RBCS were similar in the sense-AtHXKs plants grown in the absence of sugar and in the control plants (data not shown), indicating that sugar sensing by AtHXKs is specific and that exogenous sugar is at least a sign of yellowing of seedlings for light.

To demonstrate that AtHXK regulates gene expression under physiological conditions, we examined the expression of CAB1 and RBCS (as described above) in green plants grown under light without the addition of exogenous sugars. The results of the above experiments show that transcript levels of both genes were nearly 5-fold lower in sense-AtHXK plants than in antisense-AtHXK or control plants (FIG. 5B). This difference in expression was probably due to photoinduced inhibition of endogenous sugars by enhancing the expression of AtHXK, since both genes were uniformly expressed at low levels in the dark in transgenic and control plants (FIG. 5B) (data on AtHXK2 not published). Altering expression of AtHXK in transgenic plants

To confirm that the sugar hypersensitivity or insensitivity observed in transgenic plants correlates with transgene expression, RNA and western blot analyses were performed as follows. Northern blot analysis was performed with probes specific for the genes and strands of the sense and antisense constructs, which were expressed in transgenic plants. The probe was synthesized using the Polymerase Chain Reaction (PCR) method described by Greenberg et al (cell 77,551 (1994)). The oligonucleotides used as PCR primers were designed from the sequence of AtHXK1(SEQ ID NO:3) and AtHXK2(SEQ ID NO:4) and were used to amplify the cDNA fragments corresponding thereto. Sense primers for AtHXK1 and AtHXK2 were 5'-ATGGGTAAAGTAGCTGTT-3' (SEQ ID NO:10) and 5'-ATGGGTAAAGTGGCAGTTGCAA-3' (SEQ ID NO:11), respectively. The antisense primers for AtHXK1 and AtHXK2 were 5'-TTAAGAGTCTTCAAGGTAGAG-3' (SEQ ID NO:12) and 5'-TTAACTTGTTTCAGAGTCATCTTC-3' (SEQ ID NO:13), respectively. A plasmid containing AtHXK1 or AtHXK2 full-length cDNA (pBluescriptTMKS +) was used as a template for PCR reaction. RNAs were extracted from light-yellow seedlings grown on plates containing 6% glucose, gel separated, blotted onto nylon membranes, and hybridized to each probe as described above. In light-lit etiolated seedlings, the content of AtHXK1 (sense-1) transcript in sense-AtHXK1 plants and the content of AtHXK2 (sense-2) transcript in sense-AtHXK2 plants was more than 20 times higher than in control plants (fig. 5C). In antisense plants, antisense RNA of AtHXK1 (antisense-1) and AtHXK2 (antisense-2) is expressed in their corresponding antisense transgenic plants. In contrast, endogenous transcripts of AtHXK1 were reduced to less than 20% of control content in antisense-AtHXK 1 and antisense-AtHXK 2 plants (FIG. 5C). Longer exposures to sense-2 blots showed that endogenous AtHXK2 expression was also reduced in antisense-AtHXK 1 and antisense-AtHXK 2 plants (data not shown). The above results indicate that RNA, either antisense to athxhk 1 or antisense to athxhk 2, reduces endogenous RNA levels of athxkk 1 and athxkk 2, probably due to their high sequence identity. Similar results were obtained when analyzing 15-day-old light-grown green transgenic plants (data not shown).

Western blot analysis was also performed on seedlings which germinated on plates containing 1/2MS medium with or without 6% glucose, grown in the dark for 6 days, and then grown with white light (120. mu. Em)^-2S^-1) The irradiation was carried out for 4 hours. The above analysis was also performed with proteins extracted from 15 day old light-grown green plants, which were allowed to acclimatize to the dark for 3 days and then light for 4 hours. The antibodies used in the above experiments were prepared as follows. AtHXK1 containing the entire open reading frame was subcloned into the plasmid vector pET-19b (Novagen) for overexpression in E.coli (Escherichia coli) according to standard procedures. The overexpressed AtHXK1 was then gel purified and used to produce rabbit polyclonal antibodies. The antibody was affinity purified and used with Photope^TMWestern blot analysis using Star Western blot detection kit (New England Biolabs). Proteins were extracted by conventional methods (Wei et al, cell 78,1994; Tots et al, EMBO J.6, 1843 (1987)).

The results of the western blot experiments showed that the expression of AtHXK1 was 5-10 times higher in the sense-AtHXK1 than in the control plants. In antisense-AtHXK 1 plants, the content of AtHXK1 was significantly lower than in control plants, however, it was not completely eliminated (FIG. 5D). Hexose phosphorylation activity in transgenic plants

To determine whether altered AtHXK expression could affect the overall catalytic activity of hexose phosphorylation in transgenic plants, we performed a series of standard hexose phosphorylation assays using the methods described by Renz and Stitt (plant 190,166 (1993)).

In light-lit etiolated seedlings (grown as described above), sense-AtHXK1 plants were found to have maximal hexose phosphorylation activity, while other plants showed lower activity (FIG. 5E). The higher activity measured in plants with enhanced expression of the AtHXK gene was consistent with the results of yeast transformation experiments, indicating that AtHXK1 has higher catalytic activity than AtHXK2 (data not shown).

We also performed enzymatic assays on green plants grown in 15-day-old light, which were acclimatized in the dark for 3 days and illuminated for 4 hours. As shown in fig. 5F, sense-AtHXK1 and sense-AtHXK2 plants had higher hexose phosphorylation activity compared to antisense-AtHXK and control plants. In summary, the above data provides evidence for the following conclusions: i.e., manipulation of the expression of AtHXK is sufficient to alter sugar sensing and sugar modulating activities in Arabidopsis. Thus, it was demonstrated that the specific interaction between sugars and hxhks (e.g. athxhk 1 and athxhk 2), rather than the overall catalytic activity of hxhks, is a key determinant of the sugar sensing mechanism of plants. Sugar signalling in plants is uncoupled from sugar metabolism

The above observations suggest that regulatory functions exist for HXK in plants and that sugar signaling is uncoupled from metabolism. To confirm the above hypothesis, we attempted to enhance the expression of heterologous hxhks, which could provide an excess of catalytic activity towards sugar metabolism, but without regulatory function. Yeast HXK2(YHXK2) has been proposed to have catalytic and regulatory functions and appears to be a good candidate for this experiment (Entian, molecular genetics 178,633 (1980); Entian and Frshlich, journal of bacteriology 158,29 (1984); Entian et al, molecular cell biology 5,3035 (1985)). First, we determined whether the putative regulatory functions of YHXK2 and AtHXK could be interchanged by examining the effect of enhancing the expression of AtHXK on glucose inhibition in the yeast hxk1/hxk2 double mutant strain (DBY 2219). The assay is based on YHXK 2-mediated growth inhibition of wild-type yeast cells (i.e., glucose inhibition) on 2-dGlc/raffinose plates. The glucose inhibition assay was performed according to the method described by Ma et al (molecular cell biology 9,5643(1989)) using YP plates containing 2% raffinose as carbon source in the presence of 2-deoxyglucose (0.02%). Glucose analog 2-dGlc mimics glucose by inducing strong inhibition of the transferase gene (SUC2), but cannot be used as a carbon source by itself. Thus, the wild-type yeast strain had glucose-inhibiting effect and could not grow under the above-described assay conditions; in contrast, in the double mutant hxk1/hxk2, expression of SUC2 was de-repressed, hydrolyzing raffinose and releasing fructose for growth on the test plates. As shown in FIG. 6A, DBY2219 can grow on 2-dGlc/raffinose plates due to lack of YHXK2 and de-repression of the invertase gene expression. However, the growth of this strain was inhibited after transformation with YHXK2 and restoration of glucose repression (FIG. 6A) (Ma and Botstein, molecular cell biology 6,4046 (1986); Ma et al, molecular cell biology 9,5643 (1989)).

To determine the effect of enhancing YHXK2 expression in plants, a transgenic construct pCaMV35S-YHXK2 expressing YHXK2 was introduced into arabidopsis using the agrobacterium-mediated method described above. Transgenic plants with enhanced YHXK2 expression (YHXK2 plants) were found to exhibit sugar insensitivity in multiple assays. For example, YHXK2 seedlings were less sensitive to 6% glucose than control and sense-AtHXK plants. Hypocotyl elongation, root growth and cotyledon greening were found to be inhibited in sense-AtHXK1 or control plants, but not in YHXK2 plants (FIG. 6B). Northern blot analysis confirmed that neither CAB1 nor RBCS transcripts were inhibited in YHXK2 plants, whether grown in the presence or absence of exogenous glucose (data not shown).

To ensure that YHXK2 produced hexose phosphorylation activity in plants, enzymatic assays were performed with yellowed and green transgenic plants. Fig. 6C shows that the total hexose phosphorylation activity of YHXK2 was somewhat higher than in control plants and was similar to or higher than that of sense-athxhk plants. However, YHXK2 plants were sugar insensitive, not hypersensitive (fig. 6B). This dominant interference effect of YHXK2 in transgenic plants could be due to enhanced YHXK2 expression, YHXK2 competed with athhxk for sugars, but it failed to transmit signals. Based on the normal expression of AtHXK1 and AtHXK2 in YHXK2 plants, we excluded the possibility of gene silencing effects (results not shown).

The above experiments show that the catalytic function of HXK is interchangeable between yeast and plant, but not the regulatory function of sugar signaling. Our recent results also confirm that the third AtHXK is unable to complement the regulatory function of HXK (data not shown). Thus, glucose signaling was not necessarily completely metabolized, and was attenuated when YHXK2 was overexpressed in plants.

In summary, based on analysis of transgenic plants that gain or lose athxhk function and dominant interference YHXK2, we found that HXK mediates sugar sensing in higher plants. Isolation of other HXKcDNAs and genomic DNAs

Based on the isolation of the HXK genes and polypeptides described above herein, additional plant HXK coding sequences can be isolated using standard methods and techniques well known in the art. For example, using all or part of the amino acid sequence of a HXK polypeptide, one can conveniently design HXK-specific oligonucleotide probes that include a HXK degenerate oligonucleotide probe (i.e., a mixture of all possible coding sequences for a particular amino acid sequence). The oligonucleotides can be based on any suitable portion of the sequence of the DNA strand and the HXK sequence (see, e.g., FIGS. 1F-G; SEQ ID NOS: 3 and 4, respectively). General methods for designing and preparing such probes are described, for example, in Ausubel et al (1996, methods in modern molecular biology, Wiley Interscience, New York), and Berger and Kimmel (molecular cloning guide, 1987, academic Press, New York). The oligonucleotides described above can be used for the isolation of the HXK gene, either by using them as probes capable of hybridizing to complementary sequences of HXK or as primers for various amplification techniques, such as Polymerase Chain Reaction (PCR) cloning methods.

Hybridization techniques and screening methods are well known to those skilled in the art and are described in, for example, the descriptions by Ausubel et al (supra), Berger and Kimmel (supra) and Sambrook et al (molecular cloning, A laboratory Manual, Cold spring harbor laboratory Press, N.Y.). If desired, a combination of different oligonucleotide probes can be used to screen a recombinant DNA library. The oligonucleotides can be detectably labeled by methods known in the art and used to probe replicating filters derived from recombinant DNA libraries. Recombinant DNA libraries are prepared by methods well known in the art, for example, as described by Ausubel et al (supra), or are commercially available.

For the detection or isolation of closely related HXK sequences with identity greater than 80%, it is preferred to use highly stringent conditions; the above conditions include: hybridization in formamide at a temperature of about 65 ℃; a first wash is performed in about 2 XSSC and 1% SDS at about 65 ℃ followed by a second wash in about 0.1% SDS and l XSSC at about 65 ℃. Less stringent conditions for detecting a HXK gene having about 30-50% sequence identity to the HXK gene described herein include: for example, hybridization at about 45 ℃ under formamide-free conditions; a first wash is performed in about 6 x SSC and about 1% SDS at a temperature of about 45 ℃, and a second wash is performed in about 6 x SSC and about 1% SDS at a temperature of about 50 ℃. The above stringent conditions are exemplary; other suitable conditions may be determined by one skilled in the art.

As mentioned above, HXK oligonucleotides can also be used as primers in amplification cloning methods, e.g.by PCR. PCR methods are well known in the art and are described in the following references: for example, PCR technology, Erlich, Stockton Press, London, 1989; the PCR method comprises the following steps: guidelines for methods and applications, Innis et al, academic press limited, new york, 1990; and Ausubel et al (supra). The primers are optionally designed to allow cloning of the amplification product into a suitable vector, for example, by including suitable restriction sites at the 5 'and 3' ends of the amplified fragments (as described herein). HXK can be isolated if necessary using PCR "RACE" techniques or rapid amplification of cDNA ends (see, e.g., Innis et al, supra). By this method, oligonucleotide primers based on HXK sequences are oriented in the 3 'and 5' directions and used to generate overlapping PCR fragments. The overlapping 3 '-and 5' -terminal RACE products described above were combined to generate the complete full-length cDNA. This method is described in the literature of Innis et al (supra) and Frohman et al (Proc. Natl. Acad. Sci. U.S. 85,8998 (1988)).

In addition, any plant cDNA expression library can be screened by functional complementation of the yeast hxk1/hxk2 double mutant strain, as described herein by Ma and Botstein (molecular cell biology 6,4046 (1986)).

Useful HXK sequences can be isolated from any suitable organism. Confirmation of sequence relatedness to the HXK family of polypeptides can be accomplished by a variety of conventional methods including, but not limited to, functional complementation assays and sequence comparisons. In addition, the activity of any HXK sequence can be assessed by any of the methods described herein. Polypeptide expression

The hxhk polypeptide can be produced by transforming a suitable host cell with all or part of an hxkkcdna (e.g., the cDNA described above) located on a suitable expression vector or with a plasmid construct designed by genetic engineering methods to enhance the expression of an hxhk polypeptide (supra) in vivo.

It will be appreciated by those skilled in the art of molecular biology that the recombinant protein may be produced using any of a variety of expression systems. The exact host cell used is not critical to the present invention. The hxhk protein may be produced in a prokaryotic host, such as e.coli, or in a eukaryotic host, such as saccharomyces cerevisiae, mammalian cells (e.g., COS1 or NIH3T3 cells), or any of a number of plant cells, including, but not limited to, algae, tree species, ornamental plant species, tropical fruit species, vegetable species, legume species, monocots, dicots, or in any plant of commercial or agricultural value. Specific examples of suitable plant hosts include, but are not limited to, conifers, petunias, tomatoes, potatoes, tobacco, arabidopsis, lettuce, sunflower, oilseed rape, flax, cotton, sugar beet, celery, soybean, alfalfa, lotus, cowpea, cucumber, carrot, eggplant, cauliflower, horseradish, morning glory, poplar, walnut, apple, asparagus, rice, maize, millet, onion, barley, orchard grass, oat, rye, and wheat.

Such cells may be obtained from a variety of sources, including: american type culture collection (Rockland, MD); or any of a number of seed companies, such as w.atlee burbee seed company (Warminster, PA), Park seed company (Greenwood, SC), Johnny seed company (Albion, ME), or Northrup King seed company (hardville, SC). Descriptions and sources of useful host cells can also be found in the following references: vasil i.k., plant cell culture and somatic genetics, vol.i, ii, iii, laboratory methods and uses thereof, academic press, new york, 1984; dixon.r.a., plant cell culture-practice method, IRL press, oxford university, 1985; green et al, plant tissue and cell culture, academic press, new york, 1987; and Gasser and Fraley, science 244,1293 (1989).

For prokaryotic expression, the DNA encoding the HXK polypeptide is carried on a vector operably linked to control signals capable of effecting expression in a prokaryotic host. If necessary, the coding sequence may contain at its 5' end a sequence encoding any known signal sequence which enables secretion of the expressed protein into the periplasmic space of the host cell in order to facilitate recovery of the protein and subsequent purification. The most commonly used prokaryotes are various strains of E.coli; but strains of other microorganisms may also be used. The plasmid vector used contains an origin of replication, a selectable marker and control sequences, the plasmid being derived from a species compatible with the microbial host. Examples of such vectors are described in Pouwels et al (supra) or Ausubel et al (supra). Commonly used eukaryotic control sequences (also referred to as "control elements") as defined herein include a promoter for initiating transcription, optionally with an operator, and a ribosome binding site sequence. Promoters commonly used to direct protein expression include beta-lactamase (penicillinase), lactose (lac) (Chang et al, Nature 198,1056(1977)), tryptophan (Trp) (Goeddel et al, Nucl. acids Res. 8,4057(1980)), and tac promoter systems, as well as the lambda-derived PL promoter and N-gene ribosome binding site (Simotake et al, Nature 292,128 (1981)).

One specific bacterial expression system for HXK polypeptide production is the E.coli pET expression system (Novagen, McDmadison, Wis.). According to this expression system, DNA encoding an HXK polypeptide is inserted into the pET vector in an orientation designed for expression. Since the hxhk gene is placed under the control of the T7 regulatory signal, expression of hxhk is induced by inducing expression of T7 RNA polymerase in the host cell. This is typically achieved using a host strain that expresses T7 RNA polymerase in response to IPTG induced production. Once the recombinant HXK polypeptide is produced, the polypeptide can be isolated using standard methods known in the art, such as those described herein.

Another bacterial expression system for HXK polypeptide production is the pGEX expression system (Pharmacia). The system uses a GST gene fusion system designed for high level expression of genes or gene fragments in the form of fusion proteins, allowing rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxy terminus of the Glutathione S-transferase protein obtained from Schistosoma japonicum (Schistosoma japonicum) and can be conveniently purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion protein can be recovered by elution with glutathione under mild conditions. Cleavage of the glutathione S-transferase domain of the fusion protein is facilitated by the presence of a site-specific protease recognition site located upstream of the domain. For example, proteins expressed in pGEX-2T plasmids can be cleaved with thrombin; while the protein expressed in pGEX-3X can be cleaved with element Xa.

For eukaryotic expression, the method of transformation or transfection, and the choice of vector for expressing the HXK polypeptide will depend on the host system chosen. For example, transformation and transfection methods are described in the following documents: ausubel et al (supra); weissbach and Weissbach, methods of plant molecular biology, academic press, 1989; gelvin et al, handbook of plant molecular biology, Kluwer academic Press, 1990; kindle, k., proceedings of the american academy of sciences 87,1228 (1990); potrykus, I, annual review of plant physiology in plant molecular biology 42,205 (1991); and BioRad (Hercules, Calif.) technical publication #1687(Biolistic particle delivery System). The expression vector may be selected from the vectors provided in the following documents: see, for example, cloning vectors: a laboratory manual (p.h. pouwels et al, 1985, supp.1987); gasser and Fraley (supra); clontech molecular biology catalog (catalog 1992/93 tools used by molecular biologists, Palo Alto, Calif.); and the references cited above.

One preferred eukaryotic expression system is a mouse 3T3 fibroblast host cell transfected with the pMAMneo expression vector (Clontech). pMAMneo has: an RSV-LTR enhancer linked to the dexamethasone inducible MMTV-LTR promoter, an SV40 origin of replication that replicates in mammalian systems, an alternative neomycin gene, and SV40 splice and polyadenylation sites. DNA encoding an HXK polypeptide is inserted into the pmameneo vector in an orientation designed for expression. The recombinant HXK protein was then isolated as follows. Other preferred host cells that may be used in combination with the pMAMneo expression vector include COS cells and CHO cells (ATCC accession numbers CRL1650 and CCL61, respectively).

In addition, if necessary, HXK polypeptides are produced by stably transfected mammalian cell lines. A variety of vectors suitable for stable transfection of mammalian cells are publicly available, see, e.g., Pouwels et al (supra); methods for constructing such cell lines are also publicly available, see, e.g., Ausubel et al (supra). In one example, a cDNA encoding the HXK polypeptide is cloned into an expression vector that includes a dihydrofolate reductase (DHFR) gene. Integration of the plasmid into the host cell chromosome is selected by inclusion of 0.01-300. mu.M methotrexate in the cell culture medium, so that the HXK-encoding gene is also integrated into the chromosome (as described by Ausubel et al, supra). This dominant selection can be achieved in most types of cells. Expression of the recombinant protein can be enhanced by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines with gene amplification are described in Ausubel et al (supra); the above methods generally involve culturing in a medium containing increasing concentrations of methotrexate for extended periods of time. Expression vectors containing DHFR commonly used for this purpose include pCVSE II-DHrF and pAdD26SV (AX is described in Ausubel et al, supra). Any of the above host cells, or preferably a DHFR-deficient CHO cell line (e.g., CHO DHFR cells, ATCC accession number CRL 9096), are among the host cells preferably used for DHFR selection or DHFR-mediated gene amplification of stably transfected cell lines.

Most desirably, the hxhk polypeptide is produced from a stably transfected plant cell line or from a transgenic plant. A variety of vectors are available to the public that are suitable for stably transfecting plant cells or for creating transgenic plants; such vectors are described in Pouwels et al (supra), Weissbach and Weissbach (supra), and Gelvin et al (supra). Methods for constructing such cell lines are described in, for example, Weissbach and Weissbach (supra), and Gelvin et al (supra). In general, plant expression vectors include (1) a cloned plant gene under the transcriptional control of 5 'and 3' regulatory sequences, and (2) a dominant selectable marker. If desired, the plant expression vector can also include a promoter regulatory region (e.g., a regulatory region that can produce inducible or constitutive, environmentally-or developmentally-regulated, or cell-or tissue-specific expression), a transcription initiation site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

Alternatively, the HXK polypeptide may be produced using a transient expression system (e.g., the maize transient expression system described by Sheen, plant cell 2,1027 (1990)).

Once the desired HXK nucleic acid sequence is obtained, it can be manipulated by a variety of methods well known in the art. For example, if the sequence involves a non-coding flanking region, the flanking region may be subjected to mutagenesis.

If desired, the HXKDNA sequence of the invention can be combined with other DNA sequences in a variety of ways. The HXK DNA sequences of the invention can be used with all or part of the gene sequences normally associated with the HXK protein. In its component parts, a DNA sequence encoding an HXK protein is combined with a DNA construct having a transcription initiation control region that facilitates transcription and translation in a host cell.

In general, the constructs may comprise regulatory regions capable of functioning in plants, which may provide for improved production of the HXK protein as described herein. The open reading frame encoding the HXK protein or a functional fragment thereof is joined at its 5 'end to a transcription initiation regulatory region, such as a sequence naturally present in the 5' upstream region of the HXK construct gene. Many other transcriptional initiation regions are available that provide constitutive or inducible regulation.

For applications where developmental, cellular, tissue or environmental expression is desired, suitable 5' upstream non-coding regions are obtained from other genes; for example, from genes regulated during seed development, embryo development or leaf development.

Regulatory transcription termination regions may also be present in the DNA constructs of the invention. The transcription termination region may be provided by a DNA sequence encoding said HXK protein or any of the usual transcription termination regions derived from different gene sources. The transcription termination region may contain a sequence of preferably at least 1-3kb at the 3' end of the gene of the construct from which the termination region is derived. Plant expression constructs (in sense or antisense orientation) having HXK as the DNA sequence of interest for expression are applicable to a variety of plant life, particularly those involved in the production of storage stocks (e.g., substances involved in carbon and nitrogen metabolism). The above-described genetically engineered plants can be used in a variety of industrial and agricultural applications as described below. Importantly, the present invention is applicable to both dicotyledonous and monocotyledonous plants, and can be conveniently applied to any new or improved transformation or regeneration method.

An example of a useful plant promoter of the present invention is a cauliflower mosaic virus (CaMV) promoter, for example, a cauliflower mosaic virus (CaMV) promoter. The promoters described above can be expressed at high levels in most plant tissues, and the activity of these promoters is not dependent on the protein encoded by the virus. CaMV is the source of the 35S and 19S promoters. In most tissues of transgenic plants, the CaMV35S promoter is a strong promoter (see, e.g., Odell et al, Nature 313,810 (1985)). The CaMV promoter is also very active in monocots (see, e.g., Dekeyser et al, plant cells 2,591 (1990); Terada and Shimamoto, molecular genetics 220,389, (1990)). Moreover, the activity of the CaMV35S promoter may be further enhanced (i.e., increased by 2-10 fold) by duplicate use of the promoter (see, for example, Kay et al, science 236,1299 (1987); Ow et al, Proc. Natl. Acad. Sci. USA 84,4870 (1987); and Fang et al, plant cell 1,141 (1989)).

Other useful plant promoters include, but are not limited to, the nopaline synthase promoter (An et al, plant physiology 88,547(1988)) and the octopine synthase promoter (Fromm et al, plant cell 1,977 (1989)).

For some applications, it may be desirable to produce the HXK gene product in an appropriate tissue, at an appropriate level, or at an appropriate developmental stage. To this end, there is a class of gene promoters, each of which has its unique characteristics embodied in its regulatory sequences, which have been shown to be regulated according to environmental, hormonal and/or developmental cues. Including gene promoters determining the expression of thermoregulatory genes (see, for example, Callis et al, plant physiology, 88,965 (1988); Takahashi and Komeda, molecular genetics 219,365 (1989); and Takahashi et al, journal of plants 2,751(1992)), gene promoters determining the expression of photoregulated genes (e.g., pea rbcS-3A described by Kuhlemieer et al, plant cell 1,471 (1989); maize rbc S promoter described by Schendeffner and Sheene, plant cell 3,997 (1991); or chlorophyll a/b-binding protein gene found in pea described by Sihlemson et al, EMBO journal 4,2723(1985)), gene promoters determining the expression of hormone regulated genes (e.g., abscisic acid (ABA) effector sequence derived from wheat Em gene described by Marcottte et al, (1981, 1989), barley 22A and barley 1 for HVauma induction, And rd29A promoter, see plant cell 6,617(1994), Shen et al, plant cell 7,295(1994), and Yamaguchi-Shinosaki et al, wound-induced gene expression (e.g., wun I as described by Siebertz et al, plant cell 1, 961(1989)), or gene promoters determining organ-specific gene expression (e.g., tuber-specific storage protein gene as described by Roshal et al, EMBO J. 6,1155 (1987); 23-kDa zein gene from maize as described by Schernthaner et al, see EMBO J. 7,1249 (1988); or Phaseolus vulgaris β -dolichol protein gene as described by Bustos et al, see plant cell 1,839 (1989)).

Plant expression vectors may also optionally include RNA processing signals, such as introns, which have been shown to play an important role in efficient RNA synthesis and accumulation (Callis et al, Gene and development 1,1183 (1987)). The position of the RNA splice sequence can significantly affect the expression level of the transgene in the plant. In view of the above, introns may be located upstream or downstream of the HXK polypeptide coding sequence in the transgene to modulate the expression level of the gene.

In addition to the 5 'regulatory control sequences described above, the expression vector may also include regulatory control regions that are typically present in the 3' region of plant genes (Thomburg et al, Proc. Natl. Acad. Sci. USA 84,744 (1987); An et al, plant cell 1,115 (1989)). For example, the 3' end region may be included in the expression vector to improve the stability of its mRNA. One such termination region may be derived from the Pi-II termination region of potato. In addition, other commonly used terminators are derived from the octopine or nopaline synthase signal.

The plant expression vector also typically contains a dominant selectable marker gene that is used to identify those cells that have been transformed. Selection genes useful in plant systems include genes encoding antibiotic resistance genes, for example, genes encoding resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin. Genes required for photosynthesis can also be used as selection markers for photosynthetic deficient strains. In addition, green fluorescent protein derived from jellyfish (Aequorea victoria) can be used as a selection marker (Sheen et al, J. planta 8: 777,1995; Chiu et al, modern biology 6,325 (1996)). Finally, genes encoding herbicide resistance can be used as selectable markers; useful herbicide resistance genes include genes encoding phosphinothricin acetyltransferase and conferring tolerance to the broad spectrum herbicide Basta^(Hoechst AG, Frankfurt, Germany).

Efficient use of selectable markers is facilitated by determining the susceptibility of a plant cell to a particular selection agent and determining the concentration of that agent that effectively kills most, if not all, of the transformed cells. Some useful concentrations of antibiotics for tobacco transformation include, for example, 75-100. mu.g/ml (kanamycin), 20-50. mu.g/ml (hygromycin), or 5-10. mu.g/ml (bleomycin). Useful methods for selecting transformants for herbicide resistance are described, for example, by Vasil et al (supra).

It is readily understood by those skilled in the art of molecular biology, particularly plant molecular biology, that the level of expression of a gene depends not only on the combination of promoter, RNA processing signal, and termination elements, but also on how these elements are used to increase the level of selectable marker gene expression. Plant transformation

In constructing plant expression vectors, several standard methods are available for introducing the vectors into plant hosts to produce transgenic plants. These methods include (1) Agrobacterium-mediated transformation (Agrobacterium tumefaciens (A. tumefaciens) or Agrobacterium rhizogenes (A. rhizogenes)) (see, e.g., Lichtenstein and Fuller: genetic engineering, Vol.6, PWJ Rigby, London, academic Press, 1987; and Lichtenstein, C.P., and Draper, J., see: DNA cloning, Vol.II, D.M.Glover, Oxford, IRI Press, 1985), (2) particle delivery systems (see, e.g., Gordon-Kamm et al, plant cell 2,603(1990), or BioRad technical bulletin 1687, supra), (3) microinjection methods (see, e.g., Green et al, supra), (4) polyethylene glycol (PEG) methods (see, e.g., Draper et al, plant cell physiology 23, 1982; or Zhan et al, theory of plant cell uptake (1985, Weman et al, 1985, W.76, W.g., Freeman et al, Liposome delivery systems (see, Freeton et al, 1985, Freeman et al, 1985, Liposome uptake methods, Liposome delivery systems, (6) electroporation methods (see, e.g., Gelvin et al, supra; Dekeyser et al, supra; Fromm et al, Nature 319,791 (1986); Sheen, plant cell 2,1027 (1990); or Jang and Sheen, plant cell 6,1665(1994)), and (7) vortexing methods (see, e.g., Kindle, supra). The method of transformation is not critical to the present invention. Any method that provides efficient transformation can be used. These methods can be used directly when there is a renewal method to transform a crop or other host cell.

The following is an example summarizing one particular technique, Agrobacterium-mediated plant transformation. By this technique, the general method for manipulating genes to be transferred into the genome of a plant cell is accomplished in two stages. First, cloning and DNA modification steps are performed in E.coli and plasmids containing the gene construct of interest are transferred to Agrobacterium by conjugation or electroporation. Secondly, the resulting Agrobacterium strain is used to transform plant cells. Thus, for non-specific plant expression vectors, the plasmid contains an origin of replication that allows it to replicate in Agrobacterium, and a high copy number origin of replication that can function in E.coli. This facilitates the production and testing of transgenes in E.coli and then transferred to Agrobacterium for subsequent introduction into plants. Multiple resistance genes may be carried on the vector, one for selection in bacteria, e.g., for streptomycin resistance, and another that will function in plants, e.g., a gene encoding kanamycin resistance or herbicide resistance. Restriction endonuclease sites for the addition of one or several transgenes and targeting of T-DNA border sequences are also present on the vector, which, after recognition by the transfer function of Agrobacterium, delimit the DNA region to be transferred into the plant.

In another example, a plant cell may be transformed by shooting into the cell tungsten microparticles having cloned DNA deposited thereon. In the Biolistic apparatus for shooting (Bio-Rad), plastic macroparticles are driven through a gun barrel by a batch of powder (22 gauge Power stone Tool Charge) or air-driven explosions. An aliquot of the tungsten particle suspension was placed in front of the plastic macroparticle, on which the DNA had been deposited. Plastic macroparticles are shot onto an acrylic baffle where there is a hole through the baffle that is too small for the macroparticles to pass through. As a result, the plastic macroparticles are crashed against the baffle, while the tungsten microparticles continue to advance toward their target through the holes in the plate. For the purposes of the present invention, the target may be any plant cell, tissue, seed or embryo. The DNA on the microparticles introduced into the cells is integrated into their nuclei or into the chloroplasts.

In general, the transfer and expression of transgenes in plant cells has become a routine practice for those skilled in the art, and has become a major tool for gene expression studies in plants and for the production of improved plant varieties of agricultural or commercial value. Regeneration of transgenic plants

For example, plant cells transformed with a plant expression vector can be regenerated from single cells, callus tissue, or leaf discs according to standard plant tissue culture techniques. It is well known in the art that a variety of cells, tissues and organs derived from almost all plants can be successfully cultured to regenerate whole plants; the above techniques are described, for example, by Vasil (supra); green, et al (supra); weissbach and Weissbach (supra); and Gelvin et al (supra).

In one embodiment, a cloned HXK polypeptide or antisense construct is transformed into Agrobacterium under the control of the 35SCaMV promoter and the nopaline synthase terminator and with a selectable marker (e.g., kanamycin resistance). Transformation of leaf discs (e.g.tobacco leaf discs) with Agrobacterium containing the vector was carried out as described by Horsch et al (science 227,1229 (1985)). Several weeks later (e.g., 3-5 weeks), putative transformants were selected on plant tissue culture medium containing kanamycin (e.g., 100. mu.g/ml). Kanamycin-resistant shoots were then placed on hormone-free plant tissue culture medium for rooting. Kanamycin resistant plants were then selected for greenhouse growth. If necessary, seeds from the flower fertilized transgenic plants can then be sown in soil-less medium and grown in the greenhouse. Kanamycin resistant progeny were selected by sowing surface sterilized seeds on hormone-free kanamycin-containing medium. Analysis of transgene integration is accomplished by standard techniques (see, e.g., Ausubel et al, supra; Gelvin et al, supra).

Transgenic plants expressing the selectable marker are then screened for delivery of the transgenic DNA by standard immunoblotting and DNA detection techniques. Each positive transgenic plant and its transgenic progeny are unique compared to other transgenic plants constructed with the same transgene. Integration of transgenic DNA into plant genomic DNA is random in most cases, and the site of integration can significantly affect the level of transgene expression and the manner of organization and development. Thus, a number of transgenic lines are typically screened for each transgene in order to identify and select plants with optimal expression characteristics.

Transgenic expression levels of the transgenic lines were evaluated. Expression at the RNA level was first determined in order to identify and quantify expression-positive and plants. Standard techniques for RNA analysis are employed, including PCR amplification assays with oligonucleotide primers designed to amplify only transgenic RNA templates, and solution hybridization assays with transgene-specific probes (see, e.g., Ausubel et al, supra). Protein expression in RNA-positive plants is then analyzed by Western immunoblot analysis using HXK-specific antibodies (see, e.g., Ausubel et al, supra). In addition, in situ hybridization and immunocytochemistry can be performed using transgene-specific nucleotide probes and antibodies, respectively, according to standard methods to locate expression sites in transgenic tissues.

Once the recombinant HXK protein is expressed in any cell or transgenic plant (e.g., as described above), it can be isolated using methods such as affinity chromatography. In one example, an anti-HXK antibody (e.g., produced by the method described in Ausubel et al, supra, or by any standard method) can be bound to a column and used to isolate the polypeptide. Lysis and isolation of HXK-producing cells prior to affinity chromatography can be accomplished by standard methods (see, e.g., Ausubel et al, supra). Once the recombinant protein has been isolated, it may be further purified, if necessary, for example, by high performance liquid chromatography (see, for example, Fisher, Biochemical and molecular biology laboratory techniques, Work and Burdon eds., Elsevier, 1980).

The general methods for polypeptide expression and purification described above can also be used to produce and isolate useful HXK fragments or analogs. Use of

The invention described herein can be applied to a variety of agricultural and commercial purposes, including, but not limited to, increasing crop yield, improving the quality of crops and ornamental plants, and reducing the cost of agricultural production. For example, the methods, DNA constructs, proteins, and transgenic plants described herein can be used to improve the characteristics of fruits and vegetables, including: taste, texture, size, color, acidity or sweetness; the nutrient content; disease resistance; and the maturation process.

Our results provided above demonstrate that expression of a hexokinase gene in transgenic plants can be regulated by providing transcription of a hexokinase sequence that is complementary to an endogenous plant hexokinase mRNA. In this way, various plant processes can be modified, controlled or manipulated, resulting in increased yield of carbohydrate (e.g., sucrose and starch) products, altered plant growth, cellular differentiation and development, altered plant phenotype, and altered carbon/nitrogen partitioning and accumulation. In addition, as described above, antisense expression can be controlled in a cell-, tissue-, organ-or development-specific manner, if necessary. Thus, the use of antisense control can provide substantial inhibition or varying degrees of attenuation of hexokinase gene expression. In this way, the phenotype of the cell can be improved without producing additional proteins and specifically targeting specific genes.

For example, transgenic plants expressing antisense hexokinase RNA constructs can be used to abrogate feedback inhibition of photosynthesis (e.g., inhibition of photosynthetic genes induced by sugars) resulting from the accumulation of sugar metabolites (e.g., sucrose and glucose, the end products of photosynthesis). As demonstrated herein, transgenic plants expressing antisense hexokinase genes are less sensitive to sugars and are no longer subject to growth limitations and limitations that result from sugar suppression (e.g., reduced expression of photosynthetic genes). In particular, we have found that transgenic plants expressing the antisense hexokinase gene can develop and thrive normally under conditions that would normally limit and restrict plant growth due to feedback inhibition (e.g., shoot development in wild-type plants is inhibited by high hexose concentrations, but shoots of transgenic plants expressing the antisense hexokinase can develop normally). Thus, transgenic plants expressing antisense hexokinase can be used for a variety of agricultural purposes, including, but not limited to, promoting growth rate and development, seed germination, stimulating flowering, and increasing crop yield, particularly under adverse environmental conditions, e.g., high light, high temperature, and high CO2 conditions.

In addition, the results provided above indicate that the sensitivity of plants to sugars can be modulated by increasing the level of hexokinase protein. In particular, we have found that increasing the concentration of hexokinase protein can be used to promote enhanced expression of sugar activated genes (e.g., NRI). Thus, by increasing the level of hexokinase protein in a particular plant cell, tissue or organ, various plant processes that are controlled, modulated or activated by sugars can be modulated or manipulated. Genetic engineering of the above gene expression can be used to enhance accumulation of storage proteins and nitrogen, improve wound response and disease prevention mechanisms of plants, and improve pigmentation (e.g., anthocyanin) formation of plant tissues (e.g., fruits and flowers) for ornamental and horticultural purposes. For example, enhancing expression of hexokinase can be used to manipulate or facilitate expression of a variety of sugar-activated genes encoding a class of proteins including, but not limited to, patatin, soybean dystrophin, sporamin, proteinase inhibitor II, sucrose phosphate synthase, sucrose synthase from rice and corn, chalcone synthase, and nitrate reductase.

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. Other embodiments

It will be appreciated from the foregoing description that changes and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

General data in sequence listing (1): the applicant (i): the General Hospital Corporation (ii) entitled: number of plant sugar-sensing proteins and their use (iii) sequence: 13 (iv) communication address:

(A) the addressee: clark & Elbin LLP

(B) Street:

(C) city: boston

(D) State: MA (MA)

(E) The state is as follows: united states of America

(F) And E, postcode: (v) a computer-readable form:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patent In Releaes #1.0, Version #1.30 (vi) current application data:

(A) application No.:

(B) application date:

(C) and (4) classification: (vii) prior application data:

(A) application No.: US 08/622, 191

(B) Application date: 3 month and 25 days 1996

(C) And (4) classification: (viii) attorney/agent profile:

(A) name: lech, karen.

(B) Registration number: 35,238

(C) Reference/profile No.: 00786/307 WO1 (ix) telecommunication data:

(A) telephone: 617/723-6777

(B) Faxing: 617/723-8962

(C) Electric transmission: (2) data of SEQ ID NO:1: sequence characteristics:

(A) length: 453 amino acids

(B) Type (2): amino acids

(C) Chain type: is not related

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: SEQ ID NO 1: Met Gly Lys Val Ala Val Gly Ala Thr Val Val Cys Thr Ala Ala Val 151015 Cys Ala Val Ala Val Leu Val Val Arg Arg Arg Met Gln Ser Ser Gly

20 25 30Lys Trp Gly Arg Val Leu Ala Ile Leu Lys Ala Phe Glu Glu Asp Cys

35 40 45Ala Thr Pro Ile Ser Lys Leu Arg Gln Val Ala Asp Ala Met Thr Val

50 55 60Glu Met His Ala Gly Leu Ala Ser Asp Gly Gly Ser Lys Leu Lys Met65 70 75 80Leu Ile Ser Tyr Val Asp Asn Leu Pro Ser Gly Asp Glu Lys Gly Leu

85 90 95Phe Tyr Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Met Arg Val

100 105 110Leu Leu Gly Gly Lys Gln Glu Arg Val Val Lys Gln Glu Phe Glu Glu

115 120 125Val Ser Ile Pro Pro His Leu Met Thr Gly Gly Ser Asp Glu Leu Phe

130 135 140Asn Phe Ile Ala Glu Ala Leu Ala Lys Phe Val Ala Thr Glu Cys Glu145 150 155 160Asp Phe His Leu Pro Glu Gly Arg Gln Arg Glu Leu Gly Phe Thr Phe

165 170 175Ser Phe Pro Val Lys Gln Thr Ser Leu Ser Ser Gly Ser Leu Ile Lys

180 185 190Trp Thr Lys Gly Phe Ser Ile Glu Glu Ala Val Gly Gln Asp Val Val

195 200 205Gly Ala Leu Asn Lys Ala Leu Glu Arg Val Gly Leu Asp Met Arg Ile

210 215 220Ala Ala Leu Val Asn Asp Thr Val Gly Thr Leu Ala Gly Gly Arg Tyr225 230 235 240Tyr Asn Pro Asp Val Val Ala Ala Val Ile Leu Gly Thr Gly Thr Asn

245 250 255Ala Ala Tyr Val Glu Arg Ala Thr Ala Ile Pro Lys Trp His Gly Leu

260 265 270Leu Pro Lys Ser Gly Glu Met Val Ile Asn Met Glu Trp Gly Asn Phe

275 280 285Arg Ser Ser His Leu Pro Leu Thr Glu Phe Asp His Thr Leu Asp Phe

290 295 300Glu Ser Leu Asn Pro Gly Glu Gln Ile Leu Glu Lys Ile Ile Ser Gly305 310 315 320Met Tyr Leu Gly Glu Ile Leu Arg Arg Val Leu Leu Lys Met Ala Glu

325 330 335Asp Ala Ala Phe Phe Gly Asp Thr Val Pro Ser Lys Leu Arg Ile Pro

340 345 350Phe Ile Ile Arg Thr Pro His Met Ser Ala Met His Ash Asp Thr Ser

355 360 365Pro Asp Leu Lys Ile Val Gly Ser Lys Ile Lys Asp Ile Leu Glu Val

370 375 380Pro Thr Thr Ser Leu Lys Met Arg Lys Val Val Ile Ser Leu Cys Asn385 390 395 400Ile Ile Ala Thr Arg Gly Ala Arg Leu Ser Ala Ala Gly Ile Tyr Gly

405 410 415Ile Leu Lys Lys Leu Gly Arg Asp Thr Thr Lys Asp Glu Glu Val Gln

420 425 430Lys Ser Val Ile Ala Met Asp Gly Gly Leu Phe Glu His Tyr Thr Gln

435 440 445Phe Ser Glu Cys Met

450(2) data of SEQ ID NO: sequence characteristics:

(A) length: 502 amino acids

(B) Type (2): amino acids

(C) Chain type: is not related

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: 2 SEQ ID NO: Met Gly Lys Val Ala Val Ala Thr Thr Val Val Cys Ser Val Ala Val 151015 Cys Ala Ala Ala Ala Leu Ile Val Arg Arg Arg Met Lys Ser Ala Gly

20 25 30Lys Trp Ala Arg Val Ile Glu Ile Leu Lys Ala Phe Glu Glu Asp Cys

35 40 45Ala Thr Pro lle Ala Lys Leu Arg Gln Val Ala Asp Ala Met Thr Val

50 55 60Glu Met His Ala Gly Leu Ala Ser Glu Gly Gly Ser Lys Leu Lys Met65 70 75 80Leu Ile Ser Tyr Val Asp Asn Leu Pro Ser Gly Asp Glu Thr Gly Phe

85 90 95Phe Tyr Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Met Arg Val

100 105 110Leu Leu Gly Gly Lys His Asp Arg Val Val Lys Arg Glu Phe Lys Glu

115 120 125Glu Ser Ile Pro Pro His Leu Met Thr Gly Lys Ser His Glu Leu Phe

130 135 140Asp Phe Ile Val Asp Val Leu Ala Lys Phe Val Ala Thr Glu Gly Glu145 150 155 160Asp Phe His Leu Pro Pro Gly Arg Gln Arg Glu Leu Gly Phe Thr Phe

165 170 175Ser Phe Pro Val Lys Gln Leu Ser Leu Ser Ser Gly Thr Leu Ile Asn

180 185 190Trp Thr Lys Gly Phe Ser Ile Asp Asp Thr Val Asp Lys Asp Val Val

195 200 205Gly Glu Leu Val Lys Ala Met Glu Arg Val Gly Leu Asp Met Leu Val

210 215 220Ala Ala Leu Val Asn Asp Thr Ile Gly Thr Leu Ala Gly Gly Arg Tyr225 230 235 240Thr Asn Pro Asp Val Val Val Ala Val Ile Leu Gly Thr Gly Thr Asn

245 250 255Ala Ala Tyr Val Glu Arg Ala His Ala Ile Pro Lys Trp His Gly Leu

260 265 270Leu Pro Lys Ser Gly Glu Met Val Ile Asn Met Glu Trp Gly Asn Phe

275 280 285Arg Ser Ser His Leu Pro Leu Thr Glu Tyr Asp His Ser Leu Asp Val

290 295 300Asp Ser Leu Asn Pro Gly Glu Gln Ile Leu Glu Lys Ile Ile Ser Gly305 310 315 320Met Tyr Leu Gly Glu Ile Leu A rg Arg Val Leu Leu Lys Met Ala Glu

325 330 335Glu Ala Ala Phe Phe Gly Asp Ile Val Pro Pro Lys Leu Lys Ile Pro

340 345 350Phe Ile Ile Arg Thr Pro Asn Met Ser Ala Met His Ser Asp Thr Ser

355 360 365Pro Asp Leu Lys Val Val Gly Ser Lys Leu Lys Asp Ile Leu Glu Val

370 375 380Gln Thr Ser Ser Leu Lys Met Arg Lys Val Val Ile Ser Leu Cys Asn

385 390 395 400Ile lle Ala Ser Arg Gly Ala Arg Leu Ser Ala Ala Gly Ile Tyr Gly

405 410 415Ile Leu Lys Lys Ile Gly Arg Asp Ala Thr Lys Asp Gly Glu Ala Gln

420 425 430Lys Ser Val Ile Ala Met Asp Gly Gly Leu Phe Glu His Tyr Thr Gln

435 440 445Phe Ser Glu Ser Met Lys Ser Ser Leu Lys Glu Leu Leu Gly Asp Glu

450 455 460Val Ser Glu Ser Val Glu Val Ile Leu Ser Asn Asp Gly Ser Gly Val465 470 475 480Gly Ala Ala Leu Leu Ala Ala Ser His Ser Gln Tyr Leu Glu Leu Glu

485 490 495Asp Asp Ser Glu Thr Ser

500(2) data of SEQ ID NO. 3: sequence characteristics:

(A) length: 2023 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linear (ii) molecular type: DNA (xi) sequence description: 3: CAGTGTGAGT AATTTAGATC GGTATTAGAT CCATCTTAGG TTTCTCTAAT TTCTCTCAAT 60TCACTCCAAA ATTTTGATTA TTTCTTCTTT CTGGCTTGTC AATTTTAGTC ATTTGTAATC 120CTTGCTTTTG CGATCGGAAT CGTAAAAATC CGATCTTTCT TTTAGATTCG TTTTGTTTTT 180GATTCCAAAT CGGAAAAATG GGTAAAGTAG CTGTTGGAGC GACTGTTGTT TGCACGGCGG 240CGGTTTGTGC GGTGGCTGTT TTGGTTGTTC GACGACGGAT GCAGAGCTCA GGGAAGTGGG 300GACGTGTTTT GGCTATCCTC AAGGCCTTTG AAGAGGATTG TGCGACTCCG ATCTCGAAAC 360TGAGACAAGT GGCTGATGCT ATGACCGTTG AGATGCATGC TGGTCTTGCA TCCGACGGTG 420GTAGCAAACT CAAGATGCTT ATCAGCTACG TTGATAATCT TCCTTCCGGG GATGAAAAGG 480GTCTCTTTTA TGCATTGGAC CTAGGGGGGA CAAACTTCCG TGTCATGCGT GTGCTTCTTG 540GCGGGAAGCA AGAGCGTGTT GTTAAACAAG AATTCGAAGA AGTTTCGATT CCTCCTCATT 600TGATGACTGG TGGTTCAGAT GAGTTGTTCA ATTTTATAGC TGAAGCTCTT GCGAAGTTTG 660TCGCTACAGA ATGCGAAGAC TTTCATCTTC CAGAAGGTAG ACAGAGGGAA TTAGGTTTCA 720CTTTCTCGTT TCCTGTTAAG CAGACTTCTC TGTCCTCTGG TAGTCTCATC AAATGGACAA 780AAGGCTTTTC CATCGAAGAA GCAGTTGGAC AAGATGTTGT TGGAGCACTT AATAAGGCTC 840TGGAAAGAGT TGGTCTTGAC ATGCGAATCG CAGCACTTGT TAATGATACC GTTGGAACAC 900TAGCCGGTGG TAGATACTAT AACCCGGATG TTGTTGCTGC TGTTATTTTA GGCACTGGGA 960CAAACGCAGC CTATGTTGAG CGTGCAACCG CGATCCCTAA ATGGCATGGT CTGCTTCCAA 1020AATCAGGAGA AATGGTTATA AACATGGAAT GGGGAAACTT CAGGTCATCA CATCTTCCAT 1080TAACCGAGTT TGATCACACG CTGGATTTCG AGAGTCTGAA TCCAGGCGAA CAGATTCTTG 1140AGAAAATCAT TTCCGGTATG TACTTGGGAG AGATTTTGCG AAGAGTTCTT CTAAAGATGG 1200CTGAAGATGC TGCTTTCTTT GGCGATACAG TCCCATCTAA GCTGAGAATA CCATTCATCA 1260TTAGGACTCC TCACATGTCG GCTATGCACA ACGACACTTC TCCAGACTTG AAGATTGTTG 1320GGAGCAAGAT TAAGGATATA TTGGAGGTCC CTACAACTTC TCTGAAAATG AGAAAAGTTG 1380TGATCAGTCT CTGCAACATC ATAGCAACCC GAGGAGCTCG TCTCTCTGCT GCTGGAATCT 1440ATGGTATTCT GAAGAAACTG GGAAGAGATA CTACTAAAGA CGAGGAGGTG CAGAAATCGG 1500TTATAGCCAT GGATGGTGGA TTGTTTGAGC ATTACACTCA GTTTAGTGAG TGTATGGAGA 1560GCTCACTAAA AGAGTTGCTT GGAGATGAAG CTTCAGGAAG CGTTGAAGTC ACTCACTCCA 1620ATGATGGATC AGGCATTGGA GCTGCGCTTC TTGCTGCTTC TCACTCTCTC TACCTTGAAG 1680ACTCTTAAAA CCTACCCAAA GAGCGCCATT TTTCGGTAAT TTACTGAAAG CTTTTCGCTA 1740TCAGAAAACG CCTAAGCCAA GTTCTAAGGC GTCATAAAAG AAAGCATTCC ATGTTTTTAC 1800TCTTCCCCAA GACTTTCTTT GTAGCAAATA AGTTTCCTTG GGAGAAATAT TTGTTTTCAT 1860GTTCTTCAAA AATAAAAGAC TCAGTTCTTC AGATTCTGGG ATTTTATTAT AACCAGATAT 1920GTTGTAAAAA CTACAAATTC AAAGCTCACT TCACTGGAGT TCTGAGTATA TAAAGATTTC 1980ATTTTTCCTA AAAAAAAAAA AAAAAACTAA ATTACTCACA CTC

2023(2) data of SEQ ID NO. 4: i) sequence characteristics:

(A) length: 1883 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: linear (ii) molecular type: dna (xi) sequence description: SEQ ID NO 4: CAGTGTGAGT AATTTAGATC ATCTCTAGCG TTCTTAAAGT TTCCAACTTT TTTTTTTTAT 60TAATTTGGGC CAACTTTTTT GTTTTATTAA TTTGGGCCAA CCTTTTTTGG TTTGAGAATT 120GGGCGAGGGA GAAAGATGGG TAAAGTGGCA GTTGCAACGA CGGTAGTGTG TTCGGTGGCG 180GTATGTGCGG CGGCGGCGTT GATAGTACGG AGGAGAATGA AAAGCGCAGG GAAATGGGCA 240AGAGTGATAG AGATATTGAA AGCCTTTGAA GAAGATTGTG CAACGCCAAT TGCCAAATTG 300AGACAAGTGG CTGATGCTAT GACTGTTGAG ATGCATGCTG GTCTTGCTTC TGAAGGTGGC 360AGCAAGCTTA AGATGCTTAT TAGCTACGTT GATAATCTTC CTTCTGGGGA TGAGACTGGT 420TTTTTCTATG CGTTGGATCT AGGCGGAACA AACTTCCGTG TTATGCGTGT GCTTCTTGGT 480GGGAAGCACG ACCGTGTTGT TAAACGAGAA TTCAAAGAAG AATCTATTCC TCCTCATTTG 540ATGACCGGGA AGTCACATGA ATrATTCGAT TTTATCGTTG ATGTTCTTGC CAAGTTTGTC 600GCTACAGAAG GCGAGGACTT TCATCTCCCA CCTGGTAGAC AACGGGAACT AGGTTTCACT 660TTCTCATTTC CGGTTAAGCA GCTATCTTTA TCCTCTGGCA CTCTCATCAA CTGGACAAAG 720GGCTTTTCCA TTGACGATAC AGTTGATAAA GATGTTGTTG GAGAACTTGT TAAAGCTATG 780GAAAGAGTTG GGCTGGACAT GCTTGTCGCA GCGCTTGTTA ATGATACCAT TGGAACACTT 840GCGGGTGGTA GATACACTAA CCCGGATGTC GTTGTCGCAG TTATTTTGGG CACCGGCACA 900AATGCAGCCT ATGTCGAACG TGCACATGCA ATTCCCAAAT GGCATGGTTT GCTACCCAAA 960TCAGGAGAAA TGGTGATCAA CATGGAATGG GGAAACTTCA GGTCATCACA TCTTCCATTG 1020ACAGAGTACG ACCACTCTCT AGATGTCGAT AGTTTGAATC CTGGTGAACA GATTCTTGAG 1080AAAATCATTT CCGGAATGTA TCTGGGAGAA ATCTTGCGTA GAGTTCTTCT GAAGATGGCT 1140 53926 1140GAAGAAGCTG CCTTCTTTGG CGATATCGTC CCACCTAAGC TGAAAATACC ATTCATCATA 1200AGGACCCCCA ACATGTCTGC TATGCACAGT GATACTTCCC CGGATTTGAA GGTTGTAGGA 1260AGCAAGTTAA AAGACATATT GGAGGTCCAG ACTAGTTCTC TGAAGATGAG GAAAGTTGTG 1320ATCAGCCTAT GTAACATCAT TGCAAGCCGA GGAGCTCGTT TATCTGCTGC GGGGATCTAT 1380GGAATCCTCA AGAAAATAGG AAGAGACGCA ACAAAAGATG GAGAAGCTCA GAAATCTGTG 1440ATAGCGATGG ACGGTGGGCT ATTCGAGCAT TACACTCAGT TCAGTGAGTC GATGAAGAGT 1500TCATTGAAAG AGTTGCTTGG AGATGAAGTT TCAGAGAGTG TTGAAGTGAT ACTGTCGAAT 1560GATGGTTCAG GTGTTGGAGC TGCATTACTT GCTGCTTCTC ACTCTCAGTA TCTCGAACTT 1620GAAGATGACT CTGAAACAAG TTAATTTAAA GCTTTTTTGT GTTTAACCTT CTTCTTGTTG 1680CGTAGGTTAA CAATAAAAGT AGAGGTAAAT GCCTTTGGGA AATTTTATTT TTGACAATTT 1740TCAGGAACAA TAAAACCTGG ATTCTTCATC AAAGCTCTGG GAAATTCAAA CGACCAGCCA 1800ATGTTGTAGA ACTATACATA TATATTCGAG TTCTTTCTAT GAAAAAAAAA AAAAAAAAAA 1860AACCTTAAAT TACTCACACT GGC 1883(2) data of SEQ ID NO:5: sequence characteristics:

(A) length: 465 amino acids

(B) Type (2): amino acids

(C) Chain type: is not related

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: 5: Met Leu Asp Asp Arg Ala Arg Met Glu Ala Ala Lys Lys Glu Lys Val 151015 Glu Gln Ile Leu Ala Glu Phe Gln Leu Gln Glu Glu Asp Leu Lys Lys SEQ ID NO

20 25 30Val Met Arg Arg Met Gln Lys Glu Met Asp Arg Gly Leu Arg Leu Glu

35 40 45Thr His Glu Glu Ala Ser Val Lys Met Leu Pro Thr Tyr Val Arg Ser

50 55 60Thr Pro Glu Gly Ser Glu Val Gly Asp Phe Leu Ser Leu Asp Leu Gly65 70 75 80Gly Thr Asn Phe Arg Val Met Leu Val Lys Val Gly Glu Gly Glu Glu

85 90 95Gly Gln Trp Ser Val Lys Thr Lys His Gln Met Tyr Ser Ile Pro Glu

100 105 110Asp Ala Met Thr Gly Thr Ala Glu Met Leu Phe Asp Tyr Ile Ser Glu

115 120 125Cys Ile Ser Asp Phe Leu Asp Lys His Gln Met Lys His Lys Lys Leu

130 135 140Pro Leu Gly Phe Thr Phe Ser Phe Pro Val Arg His Glu Asp Ile Asp145 150 155 160Lys Gly Ile Leu Leu Asn Gln Thr Lys Gly Phe Lys Ala Ser Gly Ala

165 170 175Glu Gly Asn Asn Val Val Gly Leu Leu Arg Asp Ala Ile Lys Arg Arg

180 185 190Gly Asp Phe Glu Met Asp Val Val Ala Met Val Asn Asp Thr Val Ala

195 200 205Thr Met Ile Ser Cys Tyr Tyr Glu Asp His Gln Cys Glu Val Gly Met210 215 220 225lle Val Gly Thr Gly Cys Asn Ala Cys Tyr Met Glu Glu Met Gln Asn

230 235 240Val Glu Leu Val Glu Gly Asp Glu Gly Arg Met Cys Val Asn Thr Glu

245 250 255Gln Gly Ala Phe Gly Asp Ser Gly Glu Leu Asp Glu Phe Leu Leu Glu

260 265 270Tyr Asp Arg Met Val Asp Glu Ser Ser Ala Asn Pro Gly Gln Gln Leu

275 280 285Tyr Glu Lys Leu lle Gly Gly Lys Thr Met Gly Glu Leu Val Arg Leu290 295 300 305Val Leu Leu Arg Leu Val Asp Glu Asn Leu Leu Phe His Gly Glu Ala

310 315 320Ser Glu Gln Leu Arg Thr Arg Gly Ala Phe Glu Thr Arg Phe Val Ser

325 330 335Gln Val Glu Ser Asp Thr Gly Asp Arg Lys Gln Ile Tyr Asn Ile Leu

340 345 350Ser Thr Leu Gly Leu Arg Pro Ser Thr Thr Asp Cys Asp Ile Val Arg

355 360 365Arg Ala Cys Glu Ser Val Ser Thr Arg Ala Ala His Met Cys Set Ala370 375 380 385Gly Leu Ala Gly Val Ile Asn Arg Met Arg Glu Ser Arg Ser Glu Asp

390 395 400Val Met Arg Ile Thr Val Gly Val Asp Gly Ser Val Tyr Lys Leu His

405 410 415Pro Ser Phe Lys Glu Arg Phe His Ala Ser Val Arg Arg Leu Thr Pro

420 425 430Ser Cys Glu Ile Thr Phe Ile Glu Ser Glu Glu Gly Ser Gly Arg Gly

435440445 Ala Ala Leu Val Ser Ala Val Ala Cys Lys Lys Ala Cys Met Leu Gly 450455460465 Gln (2) data of SEQ ID NO:6: sequence characteristics:

(A) length: 465 amino acids

(B) Type (2): amino acids

(C) Chain type: is not related

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: 6: Met Ala Met Asp Thr Thr Arg Cys Gly Ala Gln Leu Leu Thr Leu Val 151015 Glu Gln Ile Leu Ala Glu Phe Gln Leu Gln Glu Glu Asp Leu Lys Lys SEQ ID NO

20 25 30Val Met Ser Arg Met Gln Lys Glu Met Asp Arg Gly Leu Arg Leu Glu

35 40 45Thr His Glu Glu Ala Ser Val Lys Met Leu Pro Thr Tyr Val Arg Ser

50 55 60Thr Pro Glu Gly Ser Glu Val Gly Asp Phe Leu Ser Leu Asp Leu Gly65 70 75 80Gly Thr Asn Phe Arg Val Met Leu Val Lys Val Gly Glu Gly Glu Ala

85 90 95Gly Gln Trp Ser Val Lys Thr Lys His Gln Met Tyr Ser Ile Pro Glu

100 105 110Asp Ala Met Thr Gly Thr Ala Glu Met Leu Phe Asp Tyr Ile Ser Glu

115 120 125Cys Ile Ser Asp Phe Leu Asp Lys His Gln Met Lys His Lys Lys Leu

130 135 140Pro Leu Gly Phe Thr Phe Ser Phe Pro Val Arg His Glu Asp Leu Asp145 150 155 160Lys Gly Ile Leu Leu Asn Trp Thr Lys Gly Phe Lys Ala Ser Gly Ala

165 170 175Glu Gly Asn Asn Ile Val Gly Leu Leu Arg Asp Ala Ile Lys Arg Arg

180 185 190Gly Asp Phe Glu Met Asp Val Val Ala Met Val Asn Asp Thr Val Ala

195 200 205Thr Met Ile Ser Cys Tyr Tyr Glu Asp Arg Gln Cys Glu Val Gly Met

210 215 220Ile Val Gly Thr Gly Cys Asn Ala Cys Tyr Met Glu Glu Met Gln Asn225 230 235 240Val Glu Leu Val Glu Gly Asp Glu Gly Arg Met Cys Val Asn Thr GIu

245 250 255Trp Gly Ala Phe Gly Asp Ser Gly Glu Leu Asp Glu Phe Leu Leu Glu

260 265 270Tyr Asp Arg Met Val Asp Glu Ser Ser Ala Asn Pro Gly Gln Gln Leu

275 280 285Tyr Glu Lys Ile Ile Gly Gly Lys Tyr Met Gly Glu Leu Val Arg Leu

290 295 300Val Leu Leu Lys Leu Val Asp Glu Asn Leu Leu Phe His Gly Glu Ala305 310 315 320Ser Glu Gln Leu Arg Thr Arg Gly Ala Phe Glu Thr Arg Phe Val Ser

325 330 335Gln Val Glu Ser Asp Ser Gly Asp Arg Lys Gln Ile His Asn Ile Leu

340 345 350Ser Thr Leu Gly Leu Arg Pro Ser Val Thr Asp Cys Asp Ile Val Arg

355 360 365Arg Ala Cys Glu Ser Val Ser Thr Arg Ala Ala His Met Cys Ser Ala

370 375 380Gly Lcu Ala Gly Val Ile Asn Arg Met Arg Glu Ser Arg Ser Glu Asp385 390 395 400Val Met Arg Ile Thr Val Gly Val Asp Gly Ser Val Tyr Lys Leu His

405 410 415Pro Ser Phe Lys Glu Arg Phe His Ala Ser Val Arg Arg Leu Thr Pro

420 425 430Asn Cys Glu Ile Thr Phe Ile Glu Ser Glu Glu Gly Ser Gly Arg Gly

435 440 445Ala Ala Leu Val Ser Ala Val Ala Cys Lys Lys Ala Cys Met Leu Ala

450455260 Gln465(2) data of SEQ ID NO:7: sequence characteristics:

(A) length: 486 amino acids

(B) Type (2): amino acids

(C) Chain type: is not related

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: 7: Met Val His Leu Gly Pro Lys Lys Pro Gln Ala Arg Lys Gly Ser Met 151015 Ala Asp Val Pro Lys Glu Leu Met Asp Glu Ile His Gln Leu Glu Asp SEQ ID NO

20 25 30Met Phe Thr Val Asp Ser Glu Thr Leu Arg Lys Val Val Lys His Phe

35 40 45Ile Asp Glu Leu Asn Lys Gly Leu Thr Lys Lys Gly Val Asn Ile Pro

50 55 60Met Ile Pro Gly Trp Val Met Glu Phe Pro Thr Gly Lys Glu Ser Gly65 70 75 80Asn Tyr Leu Ala Ile Asp Leu Gly Gly Thr Asn Leu Arg Val Val Leu

85 90 95Val Lys Leu Ser Gly Asn Arg Thr Phe Asp Thr Thr Gln Ser Lys Tyr

100 105 110Lys Leu Pro His Asp Met Arg Thr Thr Lys His Gln Glu Glu Leu Trp

115 120 125Ser Phe Ile Ala Asp Ser Leu Lys Asp Phe Met Val Glu Gln Glu Leu

130 135 140Leu Asn Thr Lys Asp Thr Leu Pro Leu Gly Phe Thr Phe Ser Tyr Pro145 150 155 160Ala Ser Gln Asn Lys Ile Asn Glu Gly Ile Leu Gln Arg Trp Thr Lys

165 170 175Gly Phe Asp Ile Pro Asn Val Glu Gly His Asp Val Val Pro Leu Leu

180 185 190Gln Lys Glu Ile Ser Lys Arg Glu Leu Pro Ile Glu Ile Val Ala Leu

195 200 205Ile Asn Asp Thr Val Gly Thr Leu Ile Ala Ser Tyr Tyr Thr Asp Pro

210 215 220Glu Thr Lys Met Gly Val Ile Phe Gly Thr Gly Val Asn Gly Ala Phe225 230 235 240Tyr Asp Val Cys Ser Asp Ile Glu Lys Leu Glu Gly Lys Leu Ala Asp

245 250 255Asp Ile Pro Ser Asn Ser Pro Met Ala Ile Asn Cys Glu Tyr Gly Ser

260 265 270Phe Asp Asn Glu His Leu Val Leu Pro Arg Thr Lys Tyr Asp Val Ala

275 280 285Val Asp Glu Gln Ser Pro Arg Pro Gly Gln Gln Ala Phe Glu Lys Met

290 295 300Thr Ser Gly Tyr Tyr Leu Gly Glu Leu Leu Arg Leu Val Leu Leu Glu305 310 315 320Leu Asn Glu Lys Gly Leu Met Leu Lys Asp Gln Asp Leu Ser Lys Leu

325 330 335Lys Gln Pro Tyr Ile Met Asp Thr Ser Tyr Pro Ala Arg Ile Glu Asp

340 345 350Asp Pro Phe Glu Asn Leu Glu Asp Thr Asp Asp Met Phe Gln Lys Asp

355 360 365Phe Gly Val Lys Thr Thr Leu Pro Glu Arg Lys Leu Ile Arg Arg Leu

370 375 380Cys Glu Leu Ile Gly Thr Arg Ala Ala Arg Leu Ala Val Cys Gly Ile385 390 395 400Ala Ala Ile Cys Gln Lys Arg Gly Tyr Lys Thr Gly His Ile Ala Ala

405 410 415Asp Gly Ser Val Tyr Asn Lys Tyr Pro Gly Phe Lys Glu Ala Ala Ala

420 425 430Lys Gly Leu Arg Asp Ile Tyr Gly Trp Thr Gly Glu Asn Ala Ser Lys

435 440 445Asp Pro Ile Thr Ile Val Pro Ala Glu Asp Gly Ser Gly Ala Gly Ala

450 455 460Ala Val Ile Ala Ala Leu Ser Glu Lys Arg Ile Ala Glu Gly Lys Val465 470 475 480Ser Gly Ile Ile Gly Ala

485(2) data of SEQ ID NO:8: sequence characteristics:

(A) length: 486 amino acids

(B) Type (2): amino acids

(C) Chain type: is not related

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: 8: Met Val His Leu Gly Pro Lys Lys Pro Gln Ala Arg Lys Gly Ser Met 151015 Ala Asp Val Pro Lys Glu Leu Met Gln Gln Ile Glu Asn Phe Glu Lys SEQ ID NO

20 25 30Ile Phe Thr Val Pro Thr Glu Thr Leu Gln Ala Val Thr Lys His Phe

35 40 45Ile Ser Glu Leu Glu Lys Gly Leu Ser Lys Lys Gly Gly Asn Ile Pro

50 55 60Met Ile Pro Gly Trp Val Met Asp Phe Pro Thr Gly Lys Glu Ser Gly65 70 75 80Asn Tyr Leu Ala Ile Asp Leu Gly Gly Thr Asn Leu Arg Val Val Leu

85 90 95Val Lys Leu Gly Gly Asp Arg Thr Phe Asp Thr Thr Gln Ser Lys Tyr

100 105 110Arg Leu Pro Asp Ala Met Arg Thr Thr Gln Asn Pro Asp Glu Leu Trp

115 120 125Glu Phe Ile Ala Asp Ser Leu Lys Ala Phe lle Asp Glu Gln Phe Pro

130 135 140Gln Gly Ile Ser Glu Pro Ile Pro Leu Gly Phe Thr Phe Ser Tyr Pro145 150 155 160Ala Ser Gln Asn Lys Ile Asn Glu Gly Ile Leu Gln Arg Trp Thr Lys

165 170 175Gly Phe Asp Ile Pro Asn Val Glu Gly His Asp Val Val Pro Leu Leu

180 185 190Gln Lys Glu Ile Ser Lys Arg Glu Leu Pro Ile Glu Cys Cys Ala Leu

195 200 205Ile Asn Asp Thr Thr Gly Thr Leu Val Ala Ser Tyr Tyr Thr Asp Pro

210 215 220Glu Thr Lys Met Gly Val Ile Phe Gly Thr Gly Val Asn Gly Ala Tyr225 230 235 240Tyr Asp Val Cys Ser Asp Ile Glu Lys Leu Trp Gly Lys Leu Ser Asp

245 250 255Asp Ile Pro Pro Ser Ala Pro Met Ala Ile Asn Cys Glu Tyr Gly Ser

260 265 270Phe Asp Asn Glu His Val Val Leu Pro Arg Thr Lys Tyr Asp Ile Thr

275 280 285Ile Asp Glu Glu Ser Pro Arg Pro Gly Trp Trp Thr Phe Glu Lys Met

290 295 300Ser Ser Gly Tyr Tyr Leu Gly Glu Ile Leu Arg Leu Ala Leu Met Asp305 310 315 320Met Tyr Lys Gln Gly Phe Ile Phe Lys Asn Gln Asp Leu Ser Lys Phe

325 330 335Asp Lys Pro Phe Val Met Asp Thr Ser Tyr Pro Ala Arg Ile Glu Glu

340 345 350Asp Pro Phe Glu Asn Leu Glu Asp Thr Asp Asp Leu Phe Gln Asn Glu

355 360 365Phe Gly Ile Asn Thr Thr Val Gln Glu Arg Lys Leu Ile Arg Arg Leu

370 375 380Ser Glu Leu Ile Gly Ala Arg Ala Ala Arg Leu Ser Val Cys Gly Ile385 390 395 400Ala Ala Ile Cys Gln Lys Arg Gly Tyr Lys Thr Giy His Ile Ala Ala

405 410 415Asp Gly Ser Val Tyr Asn Arg Tyr Pro Gly Phe Lys Glu Lys Ala Ala

420 425 430Asn Ala Leu Lys Asp I1e Tyr Gly Trp Thr Gln Thr Ser Leu Asp Asp

435 440 445Tyr Pro Ile Lys Ile Val Pro Ala Glu Asp Gly Ser Gly Ala Gly Ala

450 455 460Ala Val Ile Ala Ala Leu Ala Gln Lys Arg Ile Ala Glu Gly Lys Ser465 470 475 480Val Gly Ile Ile Gly Ala

485(2) data of SEQ ID NO. 9: sequence characteristics:

(A) length: 485 amino acids

(B) Type (2): amino acids

(C) Chain type: is not related

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: SEQ ID NO 9: Met Val Arg Leu Gly Pro Lys Lys Pro Pro Ala Arg Lys Gly Ser Met 151015 Ala Asp Val Pro Ala Asn Leu Met Glu Gln Ile His Gly Leu Glu Thr

20 25 30Leu Phe Thr Val Ser Ser Glu Lys Met Arg Ser Ile Val Lys His Phe

35 40 45Ile Ser Glu Leu Asp Lys Gly Leu Ser Lys Lys Gly Gly Asn lle Pro

50 55 60Met Ile Pro Gly Trp Val Val Glu Tyr Pro Thr Gly Lys Glu Thr Gly65 70 75 80Asp Phe Leu Ala Leu Asp Leu Gly Gly Thr Asn Leu Arg Val Val Leu

85 90 95Val Lys Leu Gly Gly Asn His Asp Phe Asp Tyr Tyr Gln Asn Lys Tyr

100 105 110Arg Leu Pro Asp His Leu Arg Thr Gly Thr Ser Glu Gln Leu Trp Ser

115 120 125Phe Ile Ala Lys Cys Leu Lys Glu Phe Val Asp Glu Trp Tyr Pro Asp

130 135 140Gly Val Ser Glu Pro Leu Pro Leu Gly Phe Thr Phe Ser Tyr Pro Ala145 150 155 160Ser Gln Lys Lys Ile Asn Ser Gly Val Leu Gln Arg Trp Thr Lys Gly

165 170 175Phe Asp Ile Glu Gly Val Glu Gly His Asp Val Val Pro Met Leu Gln

180 185 190Glu Gln Ile Glu Lys Leu Asn Ile Pro Ile Asn Val Val Arg Leu Ile

195 200 205Asn Asp Thr Thr Gly Thr Leu Val Ala Ser Leu Tyr Thr Asp Pro Gln

210 215 220Thr Lys Met Gly Ile Ile Ile Gly Thr Gly Val Asn Gly Ala Tyr Tyr225 230 235 240Asp Val Val Ser Gly Ile Glu Lys Leu Glu Gly Leu Leu Pro Glu Asp

245 250 255Ile Gly Pro Asp Ser Pro Met Ala Ile Asn Cys Glu Tyr Gly Ser Phe

260 265 270Asp Asn Glu Gly Leu Val Leu Pro Arg Thr Lys Tyr Asp Val Ile Ile

275 280 285Asp Glu Glu Ser Pro Arg Pro Gly Gln Gln Ala Phe Glu Lys Met Thr

290 295 300Ser Gly Tyr Tyr Leu Gly Glu Ile Met Arg Leu Val Leu Leu Asp Leu305 310 315 320Tyr Asp Ser Gly Phe Ile Phe Lys Asp Gln Asp Ile Ser Lys Leu Lys

325 330 335Glu Ala Tyr Val Met Asp Thr Ser Tyr Pro Ser Lys Ile Glu Asp Asp

340 345 350Pro Phe Glu Asn Leu Glu Asp Thr Asp Asp Leu Phe Lys Thr Asn Leu

355 360 365Asn Ile Glu Thr Thr Val Val Glu Arg Lys Leu Ile Arg Lys Leu Ala

370 375 380Glu Leu Val Gly Thr Arg Ala Ala Arg Leu Thr Val Cys Gly Val Ser385 390 395 400Ala Ile Cys Asp Lys Arg Gly Tyr Lys Thr Ala His Ile Ala Ala Asp

405 410 415Gly Ser Val Phe Asn Arg Tyr Pro Gly Tyr Lys Glu Lys Ala Ala Gln

420 425 430Ala Leu Lys Asp Ile Tyr Asn Trp Asp Val Glu Lys Met Glu Asp His

435 440 445Pro Ile Gln Leu Val Ala Ala Glu Asp Gly Ser Gly Val Gly Ala Ala

450 455 460Ile Ile Ala Cys Leu Thr Trp Lys Arg Leu Ala Ala Gly Lys Ser Val465 470 475 480Gly Ile Lys Gly Glu

485(2) data of SEQ ID NO: sequence characteristics:

(A) length: 18 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (xi) sequence description: 10, SEQ ID NO:

ATGGGTAAAG TAGCTGTT 18(2) data of SEQ ID NO:11: sequence characteristics:

(A) length: 22 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (xi) sequence description: 11, SEQ ID NO:

ATGGGTAAAG TGGCAGTTGC AA 22(2) data of SEQ ID NO:12: sequence characteristics:

(A) length: 21 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (xi) sequence description: 12, SEQ ID NO:

TTAAGAGTCT TCAAGGTAGA G21 (2) data of SEQ ID NO:13: sequence characteristics:

(A) length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (xi) sequence description: 13 in SEQ ID NO:

TTAACTTGTT TCAGAGTCAT CTTC 24

Claims (72)

1. A method for reducing the level of a plant hexokinase protein in a transgenic plant cell, said method comprising expressing an antisense hexokinase nucleic acid sequence in said transgenic plant cell.

2. The method of claim 1, wherein the plant cell is derived from a monocot.

3. The method of claim 1, wherein the plant cell is derived from a dicot.

4. The method of claim 1, wherein the plant cell is derived from a gymnosperm.

5. The method of claim 1, wherein the hexokinase nucleic acid sequence is encoded by a transgene integrated into the genome of the transgenic plant cell.

6. The method of claim 1, further comprising growing a transgenic plant from said transgenic plant cell, thereby reducing the level of said hexokinase protein in said transgenic plant.

7. The method of claim 1, wherein the antisense hexokinase nucleic acid sequence is based on the AtHXK1 nucleotide sequence shown in figure IF (SEQ ID NO: 3).

8. The method of claim 1, wherein the antisense hexokinase nucleic acid sequence is based on the AtHXK2 nucleotide sequence shown in figure 1G (SEQ ID NO: 4).

9. The method of claim 6, wherein the transgenic plant is less sensitive to sugar.

10. A plant cell expressing an antisense hexokinase nucleic acid sequence.

11. The plant cell of claim 10, which is derived from a monocot.

12. The plant cell of claim 10, which is derived from a dicot.

13. The plant cell of claim 10, which is derived from a gymnosperm.

14. A plant expression vector comprising an antisense hexokinase nucleic acid sequence, wherein said sequence is operably linked to an expression control region.

15. A substantially pure plant HXK polypeptide, which contains substantially with the amino acid sequence of AtHXK1(SEQ ID NO:1) the same amino acid sequence.

16. The polypeptide of claim 15, wherein the polypeptide comprises the amino acid sequence shown in FIG. 1B (SEQ ID NO: 1).

17. The polypeptide of claim 15, wherein the polypeptide is from a monocot.

18. The polypeptide of claim 15, wherein the polypeptide is derived from a dicot.

19. The polypeptide of claim 18, wherein said dicotyledonous plant is a member of the solanaceae family.

20. The polypeptide of claim 18, wherein said dicot is a member of the family Brassicaceae.

21. The polypeptide of claim 20, wherein the crucifer is arabidopsis.

22. The polypeptide of claim 15, wherein the polypeptide is derived from a gymnosperm.

23. A substantially pure plant HXK polypeptide, which contains substantially with the amino acid sequence of AtHXK2(SEQ ID NO:2) the same amino acid sequence.

24. The polypeptide of claim 23, wherein the polypeptide comprises the amino acid sequence shown in FIG. 1B (SEQ ID NO: 2).

25. The polypeptide of claim 23, wherein the polypeptide is from a monocot.

26. The polypeptide of claim 23, wherein the polypeptide is derived from a dicotyledonous plant.

27. The polypeptide of claim 26, wherein said dicotyledonous plant is a member of the solanaceae family.

28. The polypeptide of claim 26, wherein said dicot is a member of the family Brassicaceae.

29. The polypeptide of claim 28, wherein the crucifer is arabidopsis.

30. The polypeptide of claim 23, wherein the polypeptide is derived from a gymnosperm.

31. Substantially pure DNA encoding a plant HXK polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of AtHXK1(SEQ ID NO: 1).

32. The DNA of claim 31, wherein the DNA comprises the nucleotide sequence set forth in FIG. 1F (SEQ ID NO: 3).

33. The DNA of claim 31, wherein said DNA is derived from a monocot.

34. The DNA of claim 31, wherein the DNA is derived from a dicot.

35. The DNA of claim 34, wherein the dicotyledonous plant is a member of the family Solanaceae.

36. The DNA of claim 34, wherein the dicot is a member of the family Brassicaceae.

37. The DNA of claim 36, wherein the crucifer is Arabidopsis.

38. The DNA of claim 31, wherein the DNA is derived from a gymnosperm.

39. Substantially pure DNA encoding a plant HXK polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of AtHXK2(SEQ ID NO: 2).

40. The DNA of claim 39, wherein the DNA comprises the nucleotide sequence set forth in FIG. 1F (SEQ ID NO: 4).

41. The DNA of claim 39, wherein the DNA is derived from a monocotyledonous plant.

42. The DNA of claim 39, wherein the DNA is derived from a dicot.

43. The DNA of claim 42, wherein the dicotyledonous plant is a member of the family Solanaceae.

44. The DNA of claim 42, where the dicotyledonous plant is a member of the family Brassicaceae.

45. The DNA of claim 44, wherein the crucifer is Arabidopsis.

46. The DNA of claim 39, wherein the DNA is derived from a gymnosperm.

47. The DNA of claim 31, wherein said DNA is operably linked to a constitutive or regulated promoter.

48. The DNA of claim 39, wherein said DNA is operably linked to a constitutive or regulated promoter.

49. A vector comprising the substantially pure DNA of claim 31, said vector being capable of directing the expression of a protein encoded by said DNA in a cell containing the vector.

50. A cell comprising the DNA of claim 31.

51. The cell of claim 50, which is a plant cell.

52. The cell according to claim 50, wherein said plant cell is hypersensitive to sugar.

53. A cell according to claim 50, wherein said plant cell is less sensitive to sugars.

54. A vector comprising the substantially pure DNA of claim 39, said vector being capable of directing the expression of a protein encoded by said DNA in a cell containing the vector.

55. A cell comprising the DNA of claim 39.

56. The cell of claim 55, wherein said cell is a plant cell.

57. The cell according to claim 55, wherein said plant cell is hypersensitive to sugar.

58. A cell according to claim 55, wherein said plant cell is less sensitive to sugars.

59. A transgenic plant comprising the DNA of claim 31 integrated into the genome of said plant, wherein said DNA is expressed in said transgenic plant.

60. The plant of claim 59 wherein said DNA is expressed under the control of a constitutive promoter.

61. The plant of claim 59 wherein said DNA is expressed under the control of a regulated promoter.

62. A seed obtained from the transgenic plant of claim 59.

63. A transgenic plant comprising the DNA of claim 39 integrated into the genome of said plant, wherein said DNA is expressed in said transgenic plant.

64. The plant of claim 63, wherein said DNA is expressed under the control of a constitutive promoter.

65. The plant of claim 63, wherein said DNA is expressed under the control of a regulated promoter.

66. A seed obtained from the transgenic plant of claim 63.

67. A method of producing a plant HXK polypeptide, comprising:

(a) providing a cell transformed with a gene encoding a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of AtHXK1(SEQ ID NO:1), the gene being positioned for expression in the cell;

(b) expressing the plant HXK polypeptide; and

(c) recovering the plant HXK polypeptide.

68. A method of producing a plant HXK polypeptide, comprising:

(a) providing a cell transformed with a gene encoding a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of AtHXK2(SEQ ID NO:2), the gene being positioned for expression in the cell;

(b) expressing the plant HXK polypeptide; and

(c) recovering the plant HXK polypeptide.

69. A method for increasing the level of a hexokinase protein in a transgenic plant cell, said method comprising expressing a hexokinase nucleic acid sequence in said transgenic plant cell.

70. The method of claim 69, wherein, the hexokinase nucleic acid sequence includes a and figure 1F shows the AtHXK1 nucleotide sequence (SEQ ID NO:3) substantially the same DNA sequence.

71. The method of claim 69, wherein, the hexokinase nucleic acid sequence includes a and figure 1G shows the AtHXK2 nucleotide sequence (SEQ ID NO:4) substantially the same DNA sequence.

72. The method of claim 69, wherein the transgenic plant has increased sensitivity to sugar.