TRANSGENIC POTATOES HAVING REDUCED LEVELS OF ALPHA GLUCAN L- OR H-TYPE TUBER PHOSPHORYLASE ACTIVITY WITH REDUCED COLD-SWEETENING

Abstract: Potato plants which exhibit reduced levels of alpha glucan L-type tuber phosphorylase (GLTP) or alpha glucan H-type tuber phosphorylase (GHTP) enzyme activity within the potato tuber are provided. The conversion of starches to sugars in potato tubers, particularly when stored at temperatures below 7 DEG C, is reduced in tubers exhibiting reduced GLTP or GHTP enzyme activity. Reducing cold-sweetening in potatoes allows for potato storage at cooler temperatures, resulting in prolonged dormancy, reduced incidence of disease, and increased storage life. Methods for producing potato plants which produce tubers exhibiting reduced GLTP or GHTP enzyme activity are also provided. Reduction of GLTP or GHTP activity within the potato tuber may be accomplished by such techniques as suppression of gene expression using homologous antisense RNA, the use of co-suppression, regulatory silencing sequences, chemical and protein inhibitors, or the use of site-directed mutagenesis or the isolation of alternative alleles to obtain GLTP or GHTP variants with reduced starch affinity or activity.

Description: TRANSGENIC POTATOES HAVING REDUCED LEVELS OF ALPHA GLUCAN L- OR H-TYPE TUBER PHOSPHORYLASE ACTIVITY WITH REDUCED COLD-SWEETENING This application claims the benefit of U.S. Provisional Patent Application No. 60/036,946 , filed February 10, 1997, which is incorporated in its entirety by reference herein. FIELD OF THE INVENTION The invention relates to the inhibition of the accumulation of sugars in potatoes by reducing the level of α glucan L-type tuber phosphorylase or glucan H-type tuber phosphorylase enzyme activity in the potato plant. BACKGROUND OF THE INVENTION Plant stresses caused by a wide variety of factors including disease, environment, and storage of potato tubers (Solanum tuberosum) represent major determinants of tuber quality. Dormancy periods between harvesting and sprouting are critical to maintaining quality potatoes. Processing potatoes are usually stored between 7 and 12°C. Cold storage at 2 to 6°C, versus storage at 7 to 12°C, provides the greatest longevity by reducing respiration, moisture loss, microbial infection, heating costs, and the need for chemical sprout inhibitors (Burton, 1989). However, low temperatures lead to cold-induced sweetening, and the resulting high sugar levels contribute to an unacceptable brown or black color in the fried product (Coffin et al., 1987, Weaver et al., 1978). The sugars that accumulate are predominantly glucose, fructose, and sucrose. It is primarily the glucose and fructose (reducing sugars) that react with free amino groups when heated during the various cooking processes such as frying via the Maillard reaction, resulting in the formation of brown pigments (Burton, 1989, Shallenberger et al., 1959). Sucrose produces a black colouration when fried due to caramehzation and charring. The ideal reducing sugar content is generally accepted to be 0.1% of tuber fresh weight with 0.33% as the upper limit and higher levels of reducing sugars are sufficient to cause the formation of brown and black pigments that results in an unacceptable fried product (Davies and Viola, 1992). Although the accumulation of reducing sugars can be slowed in higher temperature (7 to 12°C) storage, this increases microbial infection and the need to use sprout inhibitors. Given the negative environmental and health risks associated with chemical use, development of pathogens resistant to pesticides, and the fact that use of current sprout inhibitors may soon be prohibited, a need exists for potato varieties that can withstand stress and long-term cold storage without the use of chemicals, without the accumulation of reducing sugars, and with greater retention of starch. Carbohydrate metabolism is a complex process in plant cells. Manipulation of a number of different enzymatic processes may potentially affect the accumulation of reducing sugars during cold storage. For example, inhibition of starch breakdown would reduce the buildup of free sugar. Other methods may also serve to enhance the cold storage properties of potatoes through reduction of sugar content, including the resynthesis of starch using reducing sugars, removal of sugars through glycolysis and respiration, or conversion of sugars into other forms that would not participate in the Maillard reaction. However, many of the enzymatic processes are reversible, and the role of most of the enzymes involved in carbohydrate metabolism is poorly understood. The challenge remains to identify an enzyme that will deliver the desired result, achieve function at low temperatures, and still retain the product qualities desired by producers, processors, and consumers. It has been suggested that phosphofructokinase (PFK) has an important role in the cold-induced sweetening process (Kruger and Hammond, 1988, ap Rees et al., 1988, Dixon et al., 1981, Claassen et al., 1991). ap Reese et al. (1988) suggested that cold treatment had a disproportionate effect on different pathways in carbohydrate metabolism in that glycolysis was more severely reduced due to the cold-sensitivity of PFK. The reduction in PFK activity would then lead to an increased availability of hexose-phosphates for sucrose production. It was disclosed in European Patent 0438904 (Burrell et al., July 31, 1991) that increasing PFK activity reduces sugar accumulation during storage by removing hexoses through glycolysis and further metabolism. A PFK enzyme from E. coli was expressed in potato tubers and the report claimed to increase PFK activity and to reduce sucrose content in tubers assayed at harvest. However it has been shown that pyrophosphate:fructose 6-phosphate phosphotransferase (PFP) remains active at low temperatures (Claassen et al., 1991). PFP activity can supply fructose 6-phosphate for glycolysis just as PFK can, since the two enzymes catalyse the same reaction. Therefore, the efficacy of this strategy for improving cold storage quality of potato tubers remains in doubt. Furthermore, removal of sugars through glycolysis and further metabolism would not be a prefe ed method of enhancing storage properties of potato tubers because of the resultant loss of valuable dry matter through respiration. It has also been suggested that ADPglucose pyrophosphorylase (ADPGPP) has an important role in the cold-induced sweetening process. It was disclosed in International Application WO 94/28149 (Barry, et al, filed May 18, 1994) that increasing ADPGPP activity reduces sugar accumulation during storage by re-synthesising starch using reducing sugars. An ADPGPP enzyme from E. coli was expressed in potato tubers under the control of a cold-induced promoter and the report claimed to increase ADPGPP activity and lower reducing sugar content in tubers assayed at harvest and after cold temperature storage. However, this strategy does not eliminate starch catabolism but instead increases the rate of starch resyn thesis. Thus, catabolism of sugars through glycolysis and respiration occurs and re-incorporation into starch is limited. Up regulation of ADPGPP would not be a preferred method of enhancing storage properties of potato tubers because of the resultant loss of valuable dry matter through respiration. Again, a method involving the reduction of catabolism of starch would be preferable as dry matter would be retained. The degradation of starch is believed to involve several enzymes including -amylase (endoamylase), β-amylase (exoamylase), amyloglucosidase, and -glucan phosphorylase (starch phosphorylase). By slowing starch catabolism, accumulation of reducing sugars should be prevented and the removal of sugars through glycolysis and further metabolism would be minimized. Three different isozymes of glucan phosphorylase have been described. The tuber L-type c l,4 glucan phosphorylase (EC 2.4.1.1) isozyme (GLTP) (Nakano and Fukui, 1986) has a low affinity for highly branched glucans, such as glycogen, and is localized in amyoplasts. The monomer consists of 916 amino acids and sequence comparisons with phosphorylases from rabbit muscle and Escherichia coli revealed a high level of homology, 51% and 40% amino acids, respectively. The nucleotide sequence of the GLTP gene and the amino acid sequence of the GLTP enzyme are shown in SEQ LD NO: 1 and SEQ ID NO: 2, respectively. The H-type tuber -glucan phosphorylase isozyme H (GHTP) (Mori et al., 1991) has a high affinity for glycogen and is localized in the cytoplasm. The gene encodes for 838 amino acids and shows 63% sequence homology with the tuber L-type phosphorylase but lacks the 78-residue insertion and 50-residue amino-terminal extension found in the L- type polypeptide. The nucleotide sequence of the GHTP gene and the amino acid sequence of the GHTP enzyme are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. A third isozyme has been reported (Sonnewald et al., 1995) that consists of 974 amino acids and is highly homologous to the tuber L-type phosphorylase with 81 % identity over most of the polypeptide. However, the regions containing the transit peptide and insertion sequence are highly diverse. This isozyme is referred to as the leaf L-type phosphorylase since the mRNA accumulates equally in leaf and tuber, whereas the mRNA of the tuber L-type phosphorylase accumulates strongly in potato tubers and only weakly in leaf tissues. The tuber L-type phosphorylase is mainly present in the tubers and the leaf L-type phosphorylase is more abundunt in the leaves (Sonnewald et al., 1995). The nucleotide sequence of the leaf L-type phosphorylase gene and the amino acid sequence of the leaf L-type phosphorylase enzyme are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The role of the various starch degrading enzymes is not clear, however, and considerable debate has occurred over conflicting results. For example, reduced expression of the leaf L-type phosphorylase (Sonnewald et al., 1995) had no significant influence on starch accumulation. Sonnewald et al. (1995) reported that constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of glucan phosphorylase L-type activity in leaf tissue, but had no effect in potato tuber tissue. Since the antisense repression of the glucan phosphorylase activity had no significant influence on starch accumulation in leaves of transgenic potato plants, the authors concluded that starch breakdown was not catalysed by phosphorylases. Considering the high level of sequence homology between identified glucan phosphorylase isozymes, a similar negative response would be expected with the H-type (GHTP) and L-type tuber (GLTP) isozymes. In view of the foregoing, there remains a need for potato plants which produce tubers exhibiting reduced conversion of starches to sugars during propagation and during storage at ambient and reduced temperatures, particularly at temperatures below 7 °C. SUMMARY OF THE INVENTION The inventors have found that surprisingly, reduction of the level of α glucan L-type tuber phosphorylase (GLTP) or glucan H-type tuber phosphorylase (GHTP) enzyme activity within the potato tuber results in a substantial reduction in the accumulation of sugars in the tuber during propagation and storage, relative to wildtype potatoes, particularly at storage temperatures below 10°C, and specifically at 4°C. It is remarkable that, given the complexity of carbohydrate metabolism in the tuber, reduction in the activity of a single enzyme is effective in reducing sugar accumulation in the tuber. The inventors' discovery is even more surprising in light of the previously discussed work of Sonnewald et al. ( 1995) wherein it was reported that reduced expression of the leaf L-type phosphorylase had no significant influence on starch accumulation in leaves of potato plants. The present invention provides tremendous commercial advantages. Tubers in which cold-induced sweetening is inhibited or reduced may be stored at cooler temperatures without producing high levels of reducing sugars in the tuber which cause unacceptable darkening of fried potato products. Cold storage of tubers storage results in longer storage life, prolonged dormancy by limiting respiration and delaying sprouting, and lower incidence of disease. Reduction in GLTP or GHTP activity in potato plants and tubers can be accomplished by any of a number of known methods, including, without limitation, antisense inhibition of GLTP or GHTP mRNA, co-suppression, site-directed mutagenesis of wildtype GLTP or GHTP genes, chemical or protein inhibition, or plant breeding programs. Thus, in broad terms, the invention provides modified potato plants having a reduced level of α glucan L-type tuber phosphorylase (GLTP) or glucan H-type tuber phosphorylase (GHTP) activity in tubers produced by the plants, relative to that of tubers produced by an unmodified potato plant. In a preferred embodiment, the invention provides a potato plant transformed with an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene. As will be discussed in detail hereinafter, the aforementioned DNA sequence may be inserted in the expression cassette in either a sense or antisense orientation. Potato plants of the present invention could have reduced activity levels of either one of GLTP or GHTP independently, or could have reduced activity levels of both GLTP and GHTP. As discussed above, the inventors have found that reduction of activity levels of GLTP or GHTP enzymes in potato plants results in potato tubers in which sugar accumulation, particularly over long storage periods at temperatures below 10°C, is reduced. Therefore, the invention further extends to methods for reducing sugar production in tubers produced by a potato plant comprising reducing the level of activity of GLTP or GHTP in the potato plant. In a preferred embodiment, such methods involve introducing into the potato plant an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene. As above, the DNA sequence may be inserted in the expression cassette in either a sense or antisense orientation. As described in detail in the examples herein, improvements in cold-storage characteristics have been observed in the potato variety Desiree transformed by the methods of the present invention. A direct measure of improved cold-storage characteristics is a reduction in the level of GLTP or GHTP enzyme activity detected in potatoes after harvest and cold-storage. Transformed potato varieties have been developed wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h "1 in tubers of plants stored at 4°C for 189 days is as much as 70% lower than the total glucan phosphorylase activity in tubers of untransformed plants stored under the same conditions. Another relatively direct measure of improved cold-storage characteristics is a reduction in sweetening of potatoes observed after a period of cold-storage. Transformed potato varieties have been developed wherein the sum of the concentrations of glucose and fructose in tubers stored at 4°C for 91 days is 39% lower than the sum of the concentrations of glucose and fructose in tubers of an untransformed plant stored under the same conditions. Yet another measure of improved cold-storage characteristics, demonstrating a practical advantage of the present invention, is a reduction in darkening of a potato chip during processing (cooking). As discussed hereinbefore, the accumulation of sugars in potatoes during cold-storage contributes to unacceptable darkening of the fried product. Reduced darkening upon frying can be quantified as a measure of the reflectance, or chip score, of the fried potato chip. Techniques for measuring chip scores are discussed herein. Transformed potato varieties of the present invention have been developed wherein the chip score for tubers of plants stored at 4°C for 124 days was as much as 89% higher than the chip scores for tubers of untransformed plants stored under the same conditions. By reducing GLTP and/or GHTP activity in tubers of potato plants, thereby inhibiting sugar accumulation during cold-temperature storage, the present invention allows for storage of potatoes at cooler temperatures than would be possible with wildtype potatoes of the same cultivar. As discussed above, storage of potatoes at cooler temperatures than those traditionally used could result in increased storage life, increased dormancy through reduced respiration and sprouting, and reduced incidence of disease. It will be apparent to those skilled in the art that such additional benefits also constitute improved cold-storage characteristics and may be measured and quantified by known techniques. BRIEF DESCRJ TION OF THE DRAWINGS In drawings illustrating embodiments of the invention: Figure 1 is a schematic diagram of the tuber L-type α glucan phosphorylase antisense sequence inserted into the pBI121 transformation vector; Figure 2 is a schematic diagram of the tuber H-type α glucan phosphorylase antisense sequence inserted into the pBI121 transformation vector; Figure 3 shows the basic structure of the three isolated isoforms of glucan phosphorylase. The transit peptide (TS) and insertion sequence (IS) are characteristic of the L-type phosphorylases and are not found in the H-type phosphorylase. The percentages indicate the nucleic acid sequence homologies between the isoforms; Figure 4 is a schematic diagram of carbohydrate interconversions in potatoes (Sowokinos 1990); Figure 5 is a comparison of the amino acid sequences of the three isoforms of phosphorylase found in potato for the region targeted by the antisense GLTP construct used in the Examples herein. Highlighted amino acids are identical. The leaf L-type glucan phosphorylase amino acid sequence is on top (amino acids 21 - 238 of SEQ ID NO: 6), the tuber L-type a glucan phosphorylase amino acid sequence is in the middle (amino acids 49 - 266 of SEQ ID NO: 2), and tuber H-type glucan phosphorylase amino acid sequence is on the bottom (amino acids 46 - 264 of SEQ ID NO: 4); Figure 6A and 6B are a comparison of the nucleotide sequences of the three isoforms of phosphorylase found in potato for the region targeted by the antisense GLTP construct used in the Examples herein. Highlighted nucleotides are identical. The leaf L-type glucan phosphorylase nucleotide sequence is on top (nucleotides 389 - 1045 of SEQ ID NO: 5), the tuber L-type α glucan phosphorylase nucleotide sequence is in the middle (nucleotides 338 - 993 of SEQ ID NO: 1), and tuber H-type glucan phosphorylase nucleotide sequence is on the bottom (nucleotides 147 - 805 of SEQ ID NO: 3); Figure 7 is a northern blot of polyadenylated RNA isolated from potato tubers of wild type and lines 3,4,5, and 9 transformed with the tuber L-type α glucan phosphorylase. The blot was probed with a radiolabelled probe specific for the tuber L-type glucan phosphorylase; Figure 8 is a northern blot of total RNA isolated from potato tubers of wild type and lines 1 and 2 transformed with the H-type α-glucan phosphorylase. The blot was probed with a radio labelled probe specific for the H-type α-glucan phosphorylase; Figure 9 shows the fried product obtained from (A) wild type and tuber L-type α glucan phosphorylase transformants (B) ATLl (C) ATL3 (D) ATL4 (E) ATL5 (F) ATL9 field grown tubers following 86 days storage at 4°C ("ATL" = antisense tuber L-type transformant); Figure 10 shows the activity gel and western blot of L-type and H-type isozymes of α 1,4 glucan phosphorylase extracted from wild type tubers and tubers transformed with the antisense construct for the L-type isoform; and Figure 11 shows the activity gel and western blot of L-type and H-type isozymes of α 1 ,4 glucan phosphorylase extracted from wild type tubers and transformed with the antisense construct for the H-type isoform.. DESCRIPTION OF THE PREFERRED EMBODIMENT Potato plants having a reduced level of α glucan L-type tuber phosphorylase (GLTP) or α glucan H-type tuber phosphorylase (GHTP) activity in tubers produced by the plants O 98/35051 relative to that of tubers produced by unmodified potato plants are provided. In the exemplified case, reduction in α glucan phosphorylase activity is accomplished by transforming a potato plant with an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene. Although, in the exemplified case, the DNA sequence is inserted in the expression cassette in the antisense orientation, a reduction in α glucan phosphorylase activity can be achieved with the DNA sequence inserted in the expression cassette in either a sense or antisense orientation. 1 Homology Dependent Silencing The control of gene expression using sense or antisense gene fragments is standard laboratory practice and is well documented in the literature. Antisense and sense suppression are both gene sequence homology-dependent phenomena that may be described as "homology-dependent silencing" phenomena. A review of scientific research articles published during 1996 reveals several hundred reports of homology-dependent silencing in transgenic plants. The mechanisms underlying homology-dependent silencing are not fully understood, but the characteristics of the phenomena have been studied in many plant genes and this body of work has been extensively reviewed (Meyer and Saedler 1996, Matzke and Matzke 1995, Jorgensen 1995, Weintraub 1990, Van der Krol et al. 1988) Homology-dependent silencing appears to be a general phenomenon that may be used to control the activity of many endogenous genes. Examples of genes exhibiting reduced expression after the introduction of homologous sequences include dihydroflavanol reductase (Van der Krol 1990), polygalacturonidase (Smith et al 1990), phytoene synthase (Fray and Grierson 1993), pectinesterase (Seymour et al. 1993), phenylalanine ammonia-lyase (De Carvalho et al. 1992), β-l,3-glucanase (Hart et al. 1992), chitinase (Dorlhac et al. 1994) nitrate reductase (Napoli et al. 1990), and chalcone synthase (14). Transformation of Russet Burbank potato plants with either sense- or antisense- constructs of the potato leafroll virus coat protein gene has been reported to confer resistance to potato leafroll virus infection (Kawchuk et al. 1991 ). The transfer of a homologous sense or antisense sequence usually generates transformants with reduced endogenous gene expression. As discussed in detail in the examples herein, transformed potato plants exhibiting phenotypes indicating reduced GLTP or GHTP expression can be readily identified. In the antisense suppression technique, a gene construct or expression cassette is assembled which, when inserted into a plant cell, results in expression of an RNA which is of complementary sequence to the mRNA produced by the target gene. It is theorized that the complementary RNA sequences form a duplex thereby inhibiting translation to protein. The theory underlying both sense and antisense inhibition has been discussed in the literature, including in Antisense Research and Applications (CRC Press, 1993) pp. 125-148. The complementary sequence may be equivalent in length to the whole sequence of the target gene, but a fragment is usually sufficient and is more convenient to work with. For instance, Cannon et al. (1990) reveals that an antisense sequence as short as 41 base pairs is sufficient to achieve gene inhibition. United States Patent No. 5,585,545 (Bennett et al., December 17, 1996) describes gene inhibition by an antisense sequence of only 20 base pairs. There are many examples in the patent literature of patents including descriptions and claims to methods for suppressing gene expression through the introduction of antisense sequences to an organism, including, for example, United States Patent No. 5,545,815 (Fischer et al, August 13, 1996) and United States Patent No. 5,387,757 (Bridges et al, February 7, 1995). Sense-sequence homology-dependent silencing is conducted in a similar manner to antisense suppression, except that the nucleotide sequence is inserted in the expression cassette in the normal sense orientation. A number of patents, including United States patents 5,034,323, 5,231,020 and 5,283,184, disclose the introduction of sense sequences leading to suppression of gene expression. Both forms of homology-dependent silencing, sense- and antisense-suppression, are useful for accomplishing the down-regulation of GLTP or GHTP of the present invention. It is recognized in the art that both techniques are equally useful strategies for gene suppression. For instance, both US Patent No. 5,585,545 (Bennett et al., December 17, 1996) and US Patent No. 5,451,514 (Boudet et al, September 15, 1995) claim methods for inhibiting gene expression or recombinant DNA sequences useful in methods for suppressing gene expression drawn to both sense- and antisense-suppression techniques. 2 Alternate Techniques for Reducing GHTP and/or GLTP Activity in Tubers Although homology-dependent silencing is a preferred technique for the down- regulation of GLTP or GHTP in potato plants of the present invention, there are several commonly used alternative strategies available to reduce the activity of a specific gene product which will be understood by those skilled in the art to bear application in the present invention. Insertion of a related gene or promoter into a plant can induce rapid turnover of homologous endogenous transcripts, a process referred to as co-suppression and believed to have many similarities to the mechanism responsible for antisense RNA inhibition (Jorgensen, 1995; Brusslan and Tobin, 1995). Various regulatory sequences of DNA can be altered (promoters, polyadenylation signals, post-transcriptional processing sites) or used to alter the expression levels (enhancers and silencers) of a specific mRNA. Another strategy to reduce expression of a gene and its encoded protein is the use of ribozymes designed to specifically cleave the target mRNA rendering it incapable of producing a fully functional protein (Hasseloff and Gerlach, 1988). Identification of naturally occurring alleles or the development of genetically engineered alleles of an enzyme that have been identified to be important in determining a particular trait can alter activity levels and be exploited by classical breeding programs (Oritz and Huaman, 1994). Site-directed mutagenesis is often used to modify the activity of an identified gene product. The structural coding sequence for a phosphorylase enzyme can be mutagenized in E. coli or another suitable host and screened for reduced starch phosphorolysis. Alternatively, naturally occurring alleles of the phosphorylase with reduced affinity and/or specific activity may be identified. Additionally, the activity of a particular enzyme can be altered using various inhibitors. These procedures are routinely used and can be found in text books such as Sambrook et al. (1989). 3 Variants of GLTP and GHTP Enzymes and Sequences Used for Homology Dependent Silencing As discussed in the background of the invention, and in greater detail by Nakano et al. (1986), Mori et al. (1991), and Sonnewald et al. (1990), there are three known α glucan phosphorylase isozymes that occur in potato plants. The present invention relates to down- regulation of the GLTP and/or GHTP isozymes. While it is believed that the GLTP and GHTP genes of all known commercial potato varieties are substantially identical, it is expected that the principles and techniques of the present invention would be effective in potato plants having variant full length polynucleotide sequences or subsequences which encode polypeptides having the starch catabolizing enzymatic activity of the described GLTP and GHTP enzymes. The terms "GLTP" and "GHTP", as used herein and in the claims, are intended to cover the variants described above. The foregoing variants may include GLTP and GHTP nucleotide sequence variants that differ from those exemplified but still encode the same polypeptide due to codon degeneracy, as well as variants which encode proteins capable of recognition by antibodies raised against the GLTP and GHTP amino acid sequences set forth in SEQ ID NO's. 2 and 4. Similarly, those skilled in the art will recognize that homology dependent silencing of GLTP and/or GHTP in potato plants may be accomplished with sense or antisense sequences other than those exemplified. First, the region of the GLTP or GHTP cDNA sequence from which the antisense sequence is derived is not essential. Second, as described hereinabove, the length of the antisense sequence used may vary considerably. Further, the sense or antisense sequence need not be identical to that of the target GLTP or GHTP gene to be suppressed. As described in the Examples herein, the inventors have observed that transformation of potato plants with antisense DNA sequences derived from the GHTP gene not only substantially suppresses GHTP gene activity, but causes some degree of suppression of GLTP gene activity. The GHTP and GLTP genes antisense sequences have 56.8% sequence identity. The sequence identity between the GLTP antisense sequence and the corresponding leaf type α glucan phosphorylase squence described by Sonnewald et al. (1990) is 71.3%. In the inventors' research to date, the same phenomenon of cross- downregulation has not been observed when potato plants are transformed with antisense DNA sequences derived from the GLTP gene. Nevertheless, these results clearly indicate that absolute sequence identity between the target endogenous α glucan phosphorylase gene and the recombinant DNA is not essential given that GLTP activity was suppressed by an antisense sequence having about 57% sequence identity with the target GLTP sequence. Thus, it will be understood by those skilled in the art that sense or antisense sequences other than those exemplified herein and other than those having absolute sequence identity with the target endogenous GLTP or GHTP gene will be effective to cause suppression of the endogenous GLTP or GHTP gene when introduced into potato plant cells. Useful sense or antisense sequences may differ from the exemplified antisense sequences or from other sequences derived from the endogenous GHTP or GLTP gene sequences by way of conservative amino acid substitutions or differences in the percentage of matched nucleotides or amino acids over portions of the sequences which are aligned for comparison purposes. United States Patent 5,585,545 (Bennett et al., December 17, 1996) provides a helpful discussion regarding techniques for comparing sequence identity for polynucleotides and polypeptides, conservative amino acid substitutions, and hybridization conditions indicative of degrees of sequence identity. Relevant parts of that discussion are summarized herein. Percentage of sequence identity for polynucleotides and polypeptides may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and, (c) multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFTT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection. Polypeptides which are substantially similar share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Another indication that nucleotide sequences are substantially identical is if two molecules specifically hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The T m of a hybrid, which is a function of both the length and the base composition of the probe, can be calculated as described in Sambrook et al. (1989). Typically, stringent conditions for a Southern blot protocol involve washing at 65 °C with 0.2XSSC. For preferred oligonucleotide probes, washing conditions are typically about at 42°C in 6XSSC. 4 General Methods Various methods are available to introduce and express foreign DNA sequences in plant cells. In brief, the steps involved in preparing antisense α glucan phosphorylase cDNAs and introducing them into a plant cell include: (1) isolating mRNA from potato plants and preparing cDNA from the mRNA; (2) screening the cDNA for the desired sequences; (3) linking a promoter to the desired cDNAs in the opposite orientation for expression of the phosphorylase genes; (4) transforming suitable host plant cells; and (5) selecting and regenerating cells which transcribe the inverted sequences. In the exemplified case, DNA derived from potato GLTP and GHTP genes is used to create expression cassettes having a plant promoter sequence operably linked to an antisense DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous GLTP gene or GHTP gene. Agrobacterium tumefaciens is used as a vehicle for transmission of the DNA to stem explants of potato plant shoots. A plant regenerated from the transformed explants transcribes the antisense DNA which inhibits activity of the enzyme. The recombinant DNA technology described herein involves standard laboratory techniques that are well known in the art and are described in standard references such as Sambrook et al. (1989). Generally, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. 5 Preparation of GHTP and GLTP cDNA cDNA is prepared from isolated potato tuber mRNA by reverse transcription. A primer is annealed to the mRNA, providing a free 3' end that can be used for extension by the enzyme reverse transcriptase. The enzyme engages in the usual 5'-3' elongation, as directed by complementary base pairing with the mRNA template to form a hybrid molecule, consisting of a template RNA strand base-paired with the complementary cDNA strand. After degradation of the original mRNA, a DNA polymerase is used to synthesize the complementary DNA strand to convert the single-stranded cDNA into a duplex DNA. After DNA amplification, the double stranded cDNA is inserted into a vector for propagation in E. coli. Typically, identification of clones harbouring the desired cDNA's would be performed by either nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used. In the exemplified case, the matter is simplified in that the DNA sequences of the GLTP and GHTP genes are known, as are the sequences of suitable primers (Brisson et al., 1990; Fukui et al., 1991). The primers used hybridize within the GLTP and GHTP genes. Thus, it is expected that the amplified cDNA's prepared represent portions of the GLTP and GHTP genes without further analysis. E. coli transformed with pUC19 plasmids carrying the phosphorylase DNA insert were detected by color selection. Appropriate E. coli strains transformed with plasmids which do not carry inserts grow as blue colonies. Strains transformed with pBluescript plasmids carrying inserts grow as white colonies. Plasmids isolated from transformed E. coli were sequenced to confirm the sequence of the phosphorylase inserts. ' 6 Vector Construction The cDNAs prepared can be inserted in the antisense or sense orientation into expression cassette in expression vectors for transformation of potato plants to inhibit the expression of the GLTP and/or GHTP genes in potato tubers. As in the exemplified case, which involves antisense suppression, the desired recombinant vector will comprise an expression cassette designed for initiating transcription of the antisense cDNAs in plants. Additional sequences are included to allow the vector to be cloned in a bacterial or phage host. The vector will preferably contain a prokaryote origin of replication having a broad host range. A selectable marker should also be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers include resistance to antibiotics such ampicillin. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, in the case of Agrobacterium transformations, T-DNA sequences will also be included for subsequent transfer to plant chromosomes. For expression in plants, the recombinant expression cassette will contain in addition to the desired sequence, a plant promoter region, a transcription initiation site (if the sequence to be transcribed lacks one), and a transcription termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector. Sequences controlling eukaryotic gene expression are well known in the art. Transcription of DNA into mRNA is regulated by a region of DNA referred to as the promoter. The promoter region contains sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complimentary strand of RNA. Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs (bp) upstream (by convention -30 to -20 bp relative to the transcription start site) of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. The TATA box is the only upstream promoter element that has a relatively fixed location with respect to the start point. The CAAT box consensus sequence is centered at -75, but can function at distances that vary considerably from the start point and in either orientation. Another common promoter element is the GC box at -90 which contains the consensus sequence GGGCGG. It may occur in multiple copies and in either orientation. Other sequences conferring tissue specificity, response to environmental signals, or maximum efficiency of transcription may also be found in the promoter region. Such sequences are often found within 400 bp of transcription initiation size, but may extend as far as 2000 bp or more. In heterologous promoter/structural gene combinations, the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. However, some variation in this distance can be accommodated without loss of promoter function. The particular promoter used in the expression cassette is not critical to the invention. Any of a number of promoters which direct transcription in plant cells is suitable. The promoter can be either constitutive or inducible. A number of promoters which are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumour-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S and the figwort mosaic virus 35S-promoters, the light-inducible promoter from the small subunit of ribulose-l,5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), and the chlorophyll a/b binding protein gene promoter, etc. All of these promoters have been used to create various types of DNA constructs which have been expressed in plants; see, e.g., PCT WO8402913. The CaMV 35S promoter used in the Examples herein, has been shown to be highly active and constitutively expressed in most tissues (Bevan et al., 1986). A number of other genes with tuber-specific or enhanced expression are known, including the potato tuber ADPGPP genes, large and small subunits (Muller et al., 1990). Other promoters which are contemplated to be useful in this invention include those that show enhanced or specific expression in potato tubers, that are promoters normally associated with the expression of starch biosynthetic or modification enzyme genes, or that show different patterns of expression, for example, or are expressed at different times during tuber development. Examples of these promoters include those for the genes for the granule-bound and other starch synthases, the branching enzymes (Blennow et al., 1991 ; WO 9214827; WO 9211375), disproportionating enzyme (Takaha et al., 1993) debranching enzymes, amylases, starch phosphorylases (Nakano et al., 1989; Mori et al., 1991), pectin esterases (Ebbelaar et al., 1993), the 40 kD glycoprotein; ubiquitin, aspartic proteinase inhibitor (Stukerlj et al, 1990), the carboxypeptidase inhibitor, tuber polyphenol oxidases (Shahar et al, 1992; GenBank Accession Numbers M95196 and M95197), putative trypsin inhibitor and other tuber cDNAs (Stiekema et al., 1988), and for amylases and sporamins (Yoshida et al., 1992; Ohta et al, 1991). In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. In the exemplified case the nopaline synthase NOS 3' terminator sequence (Bevan et al. 1983) was used. Polyadenylation sequences are also commonly added to the vector construct if the mRNA encoded by the structural gene is to be efficiently translated (Alber and Kawasaki, 1982). Polyadenylation is believed to have an effect on stabilizing mRNAs. Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., 1984) or the nopaline synthase signal (Depicker et al., 1982). The vector will also typically contain a selectable marker gene by which transformed plant cells can be identified in culture. Typically, the marker gene encodes antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamycin. In the exemplified case, the marker gene confers resistance to kanamycin. After transforming the plant cells, those cells containing the vector will be identified by their ability to grow in a medium containing the particular antibiotic. 7 Transformation of Plant Cells Although in the exemplified case potato plant shoot stem explants were transformed via inoculation with Agrobacterium tumefaciens carrying the antisense sequence linked to a binary vector, direct transformation techniques which are known in the art can also be used to transfer the recombinant DNA. The vector can be microinjected directly into plant cells. Alternatively, nucleic acids may be introduced to the plant cell by high velocity ballistic penetration by small particles having the nucleic acid of interest embedded within the matrix of the particles or on the surface. Fusion of protoplasts with lipid-surfaced bodies such as minicells, cells or lysosomes carrying the DNA of interest can be used. The DNA may also be introduced into plant cells by electroporation, wherein plant protoplasts are electroporated in the presence of plasmids carrying the expression cassette. In contrast to direct transformation methods, the exemplified case uses vectored transformation using Agrobacterium tumefaciens. Agrobacterium tumefaciens is a Gram- negative soil bacteria which causes a neoplastic disease known as crown gall in dicotyledonous plants. Induction of tumours is caused by tumour-inducing plasmids known as Ti plasmids. Ti plasmids direct the synthesis of opines in the infected plant. The opines are used as a source of carbon and/or nitrogen by the Agrobacteria. The bacterium does not enter the plant cell, but transfers only part of the Ti plasmid, a portion called T-DNA, which is stably integrated into the plant genome, where it expresses the functions needed to synthesize opines and to transform the plant cell. Vir (virulence) genes on the Ti plasmid, outside of the T-DNA region, are necessary for the transfer of the T- DNA. The vir region, however, is not transferred. In fact, the vir region, although required for T-DNA transfer, need not be physically linked to the T-DNA and may be provided on a separate plasmid. The tumour-inducing portions of the T-DNA can be interrupted or deleted without loss of the transfer and integration functions, such that normal and healthy transformed plant cells may be produced which have lost all properties of tumour cells, but still harbour and express certain parts of T-DNA, particularly the T-DNA border regions. Therefore, modified Ti plasmids, in which the disease causing genes have been deleted, may be used as vectors for the transfer of the sense and antisense gene constructs of the present invention into potato plants (see generally Winnacker, 1987). Transformation of plants cells with Agrobacterium and regeneration of whole plants typically involves either co-cultivation of Agrobacterium with cultured isolated protoplasts or transformation of intact cells or tissues with Agrobacterium. In the exemplified case, stem explants from potato shoot cultures are transformed with Agrobacterium. Alternatively, cauliflower mosaic virus (CaMV) may be used as a vector for introducing sense or antisense DNA into plants of the Solanaceae family. For instance, United States Patent No. 4,407,956 (Howell, October 4, 1983) teaches the use of cauliflower mosaic virus DNA as a plant vehicle. 8 Selection and Regeneration of Transformed Plant Cells After transformation, transformed plant cells or plants carrying the antisense or sense DNA must be identified. A selectable marker, such as antibiotic resistance, is typically used. In the exemplified case, transformed plant cells were selected by growing the cells on growth medium containing kanamycin. Other selectable markers will be apparent to those skilled in the art. For instance, the presence of opines can be used to identify transformants if the plants are transformed with Agrobacterium. Expression of the foreign DNA can be confirmed by detection of RNA encoded by the inserted DNA using well known methods such as Northern blot hybridization. The inserted DNA sequence can itself be identified by Southern blot hybridization or the polymerase chain reaction, as well (See, generally, Sambrook et al. (1989)). Generally, after it is determined that the transformed plant cells carry the recombinant DNA, whole plants are regenerated. In the exemplified case, stem and leaf explants of potato shoot cultures were inoculated with a culture of Agrobacterium tumefaciens carrying the desired antisense DNA and a kanamycin marker gene. Transformants were selected on a kanamycin-containing growth medium. After transfer to a suitable medium for shoot induction, shoots were transferred to a medium suitable for rooting. Plants were then transferred to soil and hardened off. The plants regenerated in culture were transplanted and grown to maturity under greenhouse conditions. 9 Analysis of GHTP and GLTP Activity Levels in Transformed Tubers Following regeneration of potato plants transformed with antisense DNA sequences derived from the GHTP and GLTP genes, the biochemistry of transformed tuber tissue was analyzed several ways. The in vitro activity of α glucan phosphorylase in the phosphorolytic direction was assayed according to the methods of Steup (1990) (Table 1). The activity of the enzyme in the synthetic direction and the amount of enzyme protein were compared after electrophoretic separation of the enzyme isoforms on a glycogen-containing, polyacrylamide gel (Figure 7). Starch synthesis by the tuber L-type and H-type isoforms was determined by iodine staining of the gel after incubation with glucose- 1 -phosphate and a starch primer (Steup, 1990). Western analysis was performed by blotting the protein from an identical unincubated native gel to nitrocellulose and probing with polyclonal antibodies specific for tuber type L and type H glucan phosphorylase isoforms. Levels of reducing sugars (glucose and fructose) in tuber tissues were quantified by HPLC (Tables 2, 3 and 4). The extent of Maillard reaction, which is proportional to the concentration of reducing sugars in tubers was examined by determining chip scores after frying (Table 5 and Figure 6). 10 Definitions As used herein and in the claims, the term: - "about three months", "about four months" and "about six months" refer, respectively, to periods of time of three months plus or minus two weeks, four months plus or minus two weeks, and six months plus or minus two weeks; - "antisense orientation" refers to the orientation of nucleic acid sequence from a structural gene that is inserted in an expression cassette in an inverted manner with respect to its naturally occurring orientation. When the sequence is double stranded, the strand that is the template strand in the naturally occurring orientation becomes the coding strand, and vice versa; - "chip score" of a tuber means the reflectance measurement recorded by an Agtron model E-15-FP Direct Reading Abridged Spectrophotometer (Agtron Inc. 1095 Spice Island Drive #100, Sparks Nevada 89431) of a center cut potato chip fried at 205 °F in soybean oil for approximately 3 minutes until bubbling stops; - "cold storage" or "storage at reduced temperature" or variants thereof, shall mean holding at temperatures less than 10°C, that may be achieved by refrigeration or ambient temperatures; - "endogenous", as it is used with reference to α glucan phosphorylase genes of a potato plant, shall mean a naturally occurring gene that was present in the genome of the potato plant prior to the introduction of an expression cassette carrying a DNA sequence derived from an α glucan phosphorylase gene; - "expression" refers to the transcription and translation of a structural gene so that a protein is synthesized; - "heterologous sequence" or "heterologous expression cassette" is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form; - "improved cold-storage characteristics" includes, without limitation, improvements in chip score and reduction in sugar accumulation in tubers measured at harvest or after a period of storage below 10°C, and further includes improvements, advantages and benefits which may result from the storage of potatoes at cooler temperatures than those traditionally used, such as, without limitation, increased storage life of potatoes, increased dormancy through reduced respiration and sprouting of potatoes, and reduced incidence of disease. Unless further qualified by a specific measure or test, an improvement in a cold-storage characteristic refers to a difference in the described characteristic relative to that in a control, wildtype or unmodified potato plant; - "modified" or variants thereof, when used to describe potato plants or tubers, is used to distinguish a potato plant or tuber that has been altered from its naturally occurring state through: the introduction of a nucleotide sequence from the same or a different species, whether in a sense or antisense orientation, whether by recombinant DNA technology or by traditional cross-breeding methods including the introduction of modified structural or regulatory sequences; modification of a native nucleotide sequence by site -directed mutagenesis or otherwise; or the treatment of the potato plant with chemical or protein inhibitors. An "unmodified" potato plant or tuber means a control, wildtype or naturally occurring potato plant or tuber that has not been modified as described above; - "nucleic acid sequence" or "nucleic acid segment" refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA and non- functional DNA or RNA; - "operably linked" refers to functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates transcription of RNA corresponding to the second sequence; - "plant " includes whole plants, plant organs (e.g. leaves, stems, roots, etc.) seeds and plant cells; - "promoter" refers to a region of DNA upstream from the structural gene and involved in recognition and binding RNA polymerase and other proteins that initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells; - "reduced activity" or variants thereof, when used in reference to the level of GLTP or GHTP enzyme activity in a potato tuber includes reduction of GLTP or GHTP enzyme activity resulting from reduced expression of the GLTP or GHTP gene product, reduced substrate affinity of the GLTP or GHTP enzyme, and reduced catalytic activity of the GLTP or GHTP enzyme; - "reduced" or variants thereof, may be used herein with reference to, without limitation, activity levels of GLTP or GHTP enzyme in potato tubers, accumulation of sugars in potato tubers and darkening of potato chips upon frying. Unless further qualified by a specific measure or test, reduced levels or reduced activity refers to a demonstrable statistically significant difference in the described characteristic relative to that in a control, wildtype or unmodified potato plant; - "stress" or variants thereof, when used in relation to stresses experienced by potato plants and tubers, includes the effects of environment, fertility, moisture, temperature, handling, disease, atmosphere and aging that impact upon plant or tuber quality and which may be experienced by potato plants through all stages of their life cycle and by tubers at all stages of the growth and development cycle and during subsequent harvesting, transport, storage and processing; - "stress resistance" or variants thereof, shall mean reduced effects of temperature, aging, disease, atmosphere, physical handling, moisture, chemical residues, environment, pests and other stresses; - "suitable host" refers to a microorganism or cell that is compatible with a recombinant plasmid, DNA sequence or recombinant expression cassette and will permit the plasmid to replicate, to be incorporated into its genome, or to be expressed; and - "uninterrupted" refers to a DNA sequence (e.g. cDNA) containing an open reading frame that lacks intervening, untranslated sequences. EXAMPLE 1 This example describes the reduction of GHTP and/or GLTP activity in tubers of potato plants by transforming potato plants with expression cassettes containing DNA sequences derived from the GLTP and GHTP gene sequences linked to the promoter in the antisense orientation. A Isolation of Potato Tuber mRNA Potato total RNA was purified at 4°C using autoclaved reagents from lg of tuber tissue ground to a fine powder under liquid nitrogen with a mortar and pestle. The powder was transferred to a 30ml corex tube and 3 volumes were added of 100 M Tris-Cl, pH 8.0, 100 mM NaCl, and 10 mM EDTA (lOx TNE) containing 0.2% (w/v) SDS and 0.5% (v/v) 2- mercaptoethanol. An equal volume of phenol-chloroform (1:1) was added and the sample gently vortexed before centrifugation at 4 °C in a SS34 rotor at 8,000 rpm for 5 min. The organic phase was reextracted with 0.5 volume of lOx TNE containing 0.2% (w/v)SDS and 0.5% (v/v) 2-mercaptoethanol and the combined aqueous phases were extracted with chloroform. Nucleic acids were precipitated from the aqueous phase with sodium acetate and absolute ethanol, pelleted by centrifugation, and resuspended in 3 ml of lx TNE. An equal volume of 5 M LiCl was added and the sample stored at -20 °C for at 4 h before centrifuging at 8,000 rpm in a SS34 rotor at 4°C for 10 min. The RNA pellet was washed with 70% ethanol, dried, and resuspended in DEPC-treated water. Poly (A + ) RNA was isolated using oligo (dT) cellulose (Boehringer Mannheim) column chromatography. Poly (A + ) RNA was isolated from total RNA resuspended in RNAse free water. Columns were prepared using an autoclaved Bio-Rad Poly-Prep 10 ml column to which was added 50 mg of oligo (dT) cellulose suspended in 1 ml of loading buffer B which contains 20 mM Tris-Cl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, and 0.1% (w/v) SDS. The column was washed with 3 volumes of 0.1 M NaOH with 5 mM EDTA and then DEPC- treated water until the pH of effluent was less than 8, as determined with pH paper. The column was then washed with 5 volumes of loading buffer A containing 40 mM Tris-Cl, pH 7.4, 1 M NaCl, 1 mM EDTA, and 0.1% (w/v) SDS. RNA samples were heated to 65 °C for 5 min at which time 400 μ\ of loading buffer A, prewarmed to 65 °C, was added. The sample was mixed and allowed to cool at room temperature for 2 min before application to the column. Eluate was collected, heated to 65 °C for 5 min, cooled to room temperature for 2 min, and reapplied to the column. This was followed by a 5 volume washing with loading buffer A followed by a 4 volume wash with loading buffer B. Poly (A + ) RNA was eluted with 3 volumes of 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, and 0.05% (w/v) SDS. Fractions were collected and those containing RNA were identified in an ethidium bromide plate assay, a petri dish with 1 % agarose made with TAE containing EtBr. RNA was precipitated, resuspended in 10 μl, and a 1 μl aliquot quantitated with a spectrophotometer. B Isolation of GLTP and GHTP DNA Sequences The nucleotide sequences utilized in the development of the antisense construct were randomly selected from the 5' sequence of GLTP (SEQ D NO: 1) and GHTP (SEQ ID NO: 3). DNA sequences used to develop the antisense constructs were obtained using reverse transcription-polymerase chain reaction. GLTP (SPL1 and SPL2)- and GHTP (SPH1 and SPH2)-specific primers were designed according to the published sequences (Brisson et al. 1990, Fukui et al. 1991) with minor modifications to facilitate restriction with enzymes: SPL1 Primer: 5ΑTTCGAAAAGCTCGAGATTTGCATAGA3 ' (SEQ ID NO: 7) (additional CG creates Xho I site); SPL2 Primer: 5'GTGTGCTCTCGAGCATTGAAAGC3' (SEQ ID NO: 8) (changed C to G to create Xho I site); SPH1 Primer: 5'GTTTATTTTCCATCGATGGAAGGTGGTG3' (SEQ ID NO: 9) (added CGAT to create Cla I site); SPH2 Primer: 5ΑTAATATCCTGAA1CGATGCACTGC3' (SEQ ID NO: 10) (changed G to T to create Cla I site). Reverse transcription was performed in a volume of 15 μl containing 1 x PCR buffer (10 mM Tris-Cl pH 8.2, 50 mM KC1, 0.001% gelatin, 1.5 mM MgCL), 670 μM of each dNTP, 6 μg of total potato tuber cv. Russet Burbank RNA, 1 mM each primer (SPH1 and SPL2, or SPH1 and SPH2) and 200 U of Maloney urine leukemia virus reverse transcriptase (BRL). The reaction was set at 37°C for 30 minutes, then heat-killed at 94°C for 5 minutes and snap cooled on ice. To the reverse transcription reaction was added 2.5 U Taq DNA polymerase (BRL) in 35 μl of 1 x PCR buffer. DNA amplification was done in a Perkin Elmer 480 programmed for 30 cycles with a 1 min 94°C denaturation step, a 1 min 56 °C (SPL1 and SPL2) or 58 °C (SPH1 and SPH2) annealing step, and a 2 min 72 °C extension step. PCR was completed with a final 10 min extension at 72°C. C Construction of SP Vectors for Phosphorylase Inhibition To express the antisense constructs in plant cells, it was necessary to fuse the genes to the proper plant regulatory regions. This was accomplished by cloning the antisense DNA into a plasmid vector that contained the needed sequences. Amplified DNA was blunt ended and cloned into a pUC19 vector at the Smal site. The recombinant plasmid was transformed into sub-cloning efficiency E. coli DH5α cells (BRL). The transformed cells were plated on LB (15 g/1 Bactotyptone, 5 g/1 yeast extract, 10 g/1 NaCl. pH 7.3, and solidified with 1.5% agar) plates that contained ampicillin at 100 ug/ml. Selection of bacteria containing plasmids with inserted plant phosphorylase sequence was accomplished using color selection. The polylinker and T3 and T7 RNA polymerase promoter sequences are present in the N-terminal portion of the lacZ gene fragment. pUC19 plasmids without inserts in the polylinker grow as blue colonies in appropriate bacterial strains such as DH5α. Color selection was made by spreading 100 μl of 2% X-gal (prepared in dimethyl formamide) on LB plates containing 50 μg/ml ampicillin 30 minutes prior to plating the transformants. Colonies containing plasmids without inserts will be blue after incubation for 12 to 18 hours at 37C and colonies with plasmids containing inserts will remain white. An isolated plasmid was sequenced to confirm the sequence of the phosphorylase inserts. Sequences were determined using the ABI Prism Dye Terminator Cycle Sequencing Core Kit (Applied Biosystems, Foster City, CA), M13 universal and reverse primers, and an ABI automated DNA sequencer. The engineered plasmid was purified by the rapid alkaline extraction procedure from a 5 ml overnight culture (Birnboim and Doly. 1979). Orientation of the SPL and SPH fragments in pUC19 was determined by restriction enzyme digestion. The recombinant pUC19 vectors and the binary vector pBI121 (Clonetech) were restricted, run on a agarose gel and the fragments purified by gel separation as described by Thuring et al (1975). Ligation fused the antisense sequence to the binary vector pBI121. The ligation contained pBI121 vector that had been digested with Bam I and Sαcl, along with the SPL or SPH phosphorylase DNA product, that had been cut from the pUC 19 subclone with JSαmHI and Sαcl. Ligated DNA was transformed into SCE E. coli DH5α cells, and the transformed cells were plated on LB plates containing ampicillin. The nucleotide sequences of the antisense DNA SPL and SPH are nucleotides 338 to 993 of SEQ ID NO: 1 and nucleotides 147 to 799 of SEQ D NO: 3, respectively. Selection of pBI121 with phosphorlylase inserts was done with CAMV and NOS specific primers. Samples 1 and 2 representing the tuber L-type and tuber H-type phosphorylase DNA fragments were picked from a plate after overnight growth. These samples were inoculated into 5 ml of LB media and grown overnight at 37 °C. Plasmids were isolated by the rapid alkaline extraction procedure, and the DNA was electroporated into Agrobacterium tumefaciens. Constructs were engineered into the pBI121 vector that contains the CaMV 35S promoter (Kay et al. 1987) and the NOS 3' terminator (Bevan et al. 1983) sequence. The pBI121 plasmid is made up of the following well characterized segments of DNA. A 0.93 kb fragment isolated from transposon Tn7 which encodes bacterial spectinomycin streptomycin (Spc/Str) resistance and is a determinant for selection in E. coli and Agrobacterium tumefaciens (Fling et al., 1985). This is joined to a chimeric kanamycin resistance gene engineered for plant expression to allow selection of the transformed tissue. The chimeric gene consists of the 0.35 kb cauliflower mosaic virus 35S promoter (P-35S) (Odell et al., 1985), the 0.83 kb neomycin phosphotransferase type II gene (NPTII), and the 026 kb 3' non- translated region of the nopaline synthase gene (NOS 3') (Fraley et al., 1983). The next segment is a 0.75 kb origin of replication from the RK2 plasmid (ori-V) (Stalker et al., 1981). It is joined to a 3.1 kb Sail to Pvul segment of pBR322 which provides the origin of replication for maintenance in E. coli (ori-322) and the bom site for the conjugational transfer in the Agrobacterium tumefaciens cells. Next is a 0.36 kb Pvul fragment from the pTiT37 plasmid which contains the nopaline-type T-DNA right border region (Fraley et al., 1985). The antisense sequence was engineered for expression in the tuber by placing the gene under the control of a constitutive tissue non-specific promoter. D Plant Transformation Regeneration The SPL and SPH vectors were transformed into the Desiree potato cultivar according to de Block (1988). To transform "Desiree" potatoes, sterile shoot cultures of "Desiree" were maintained in test tubes containing 8 ml of S 1 (Murashige and Skoog (MS) medium supplemented with 2% sucrose and 0.5 g/1 MES pH 5.7, solidified with 6 g/1 Phytagar). When plantlets reached approximately 5 cm in length, leaf pieces were excised with a single cut along the base and inoculated with a 1 : 10 dilution of an overnight culture of Agrobacterium tumefaciens. The stem explants were co-cultured for 2 days at 20° C on S 1 medium (De Block 1988). Following co-culture, the explants were transferred to S4 medium (MS medium without sucrose, supplemented with 0.5 g/1 MES pH 5.7, 200 mg/1 glutamine, 0.5 g/1 PVP, 20 g/1 mannitol, 20 g/1 glucose, 40 mg/1 adenine, 1 mg/1 trans zeatin, 0.1 mg/1 NAA, 1 g/1 carbenicillin, 50 mg/1 kanamycin, solidified with 6 g/1 phytagar) for 1 week and then 2 weeks to induce callus formation. After 3 weeks, the explants were transferred to S6 medium (S4 without NAA and with half the concentration (500 mg/1) of carbenicillin). After another two weeks, the explants were transferred to S8 medium (S6 with only 250 mg/1 carbenicillin and 0.01 mg/1 gibberellic acid, GA3) to promote shoot formation. Shoots began to develop approximately 2 weeks after transfer to S8 shoot induction medium. These shoots were excised and transferred to vials of S 1 medium for rooting. After about 6 weeks of multiplication on the rooting medium, the plants were transferred to soil and are gradually hardened off. Desiree plants regenerated in culture were transplanted in 1 gallon pots and were grown to maturity under greenhouse conditions. Tubers were harvested and allowed to suberize at room temperature for two days. All tubers greater than 2 cm in length were collected and stored at 4°C under high humidity. E Field Trials Untransformed controls, plants expressing the SPL construct, and plants expressing the SPH construct were propagated in field trials in a single replicate randomized design. All plants were grown side by side in the same field and exposed to similar pesticide, fertilizer, and irrigation regimes. Tubers were harvested and stored at 10°C for 2 weeks before randomly selecting a fraction of the tubers from each line to be placed in storage at 4°C. F Sugar Analysis Tubers were stored at 4°C and were not allowed to recondition at room temperature prior to sugar analysis. An intact longitudinal slice (1 cm thick, width variable and equal to the outside dimensions of the tuber) was cut from the central portion of each tuber, thus representing all of the tuber's tissues. At each harvest, the central slices from four tubers per clone (3 replicates) were collectively diced into 1-cm cubes and 15 g was randomly selected from the pooled tissue for analysis. Glucan phosphorylase (see below) and sugars were extracted with 15 mL of Tris buffer (50 mM, pH 7.0) containing 2 mM sodium bisulfite, 2 mM EDTA. 0.5 mM PMSF and 10% (w/w) glycerol with a polytron homogenizer at 4°C. The extracts were centrifuged at 4°C (30,000 g, 30 min) and reducing sugars (glucose and fructose) were measured on a 10-fold dilution of the supernatant using a Spectra Physics high performance liquid chromatograph interfaced to a refractive index detector. The separation was performed at 80°C on a 30 x 0.78 cm Aminex HPX 87C column (Biorad) using 0.6 ml/min water as the mobile phase. Calibration of the instrument was via authentic standards of d-glucose and d-fructose. G Analysis of α-Glucan Phosphorylase Activity Tubers stored at 4°C were not allowed to warm prior to extraction and analysis of α glucan phosphorylase activity and isozymes. The in vitro activity of glucan phosphorylase in the phosphorolytic direction was assayed as described by Steup (1990). Briefly, samples of extracts obtained for sugar analysis (see above) were added to a reaction medium which coupled starch phosphorolysis to the reduction of NADP through the sequential actions of phosphoglucomutase and glucose-6-phosphate dehydrogenase. The rate of reduction of NADP during the reaction is stoichiometric with the rate of production of glucose- 1- phosphate from the starch substrate. Reduction of NADP was followed at 340 nm in a Varian Cary double-beam spectrophotometer. Protein levels in extracts were determined according to Bradford (1976). Glucan phosphorylase activity gels were run essentially according to Steup (1990). Proteins were separated on native polyacrylamide gels (8.5 %) containing 1.5 % glycogen. Following electrophoresis at 80 V for 15 h (4°C), the gels were incubated (1-2 h) at 37 °C in 0.1 M citrate-NaOH buffer (pH 6.0) containing 20 mM glucose- 1 -P and 0.05% (w/v) hydrolyzed potato starch. Gels were then rinsed and stained with an iodine solution. For Western blot analysis, proteins were electrophoresed on glycogen-containing polyacrylamide gels as described above. The proteins were electroblotted to nitrocellulose and blots were probed with polyclonal antibodies raised against GHTP and GLTP. Immunoblots were developed with alkaline phosphatase conjugated anti-rabbit secondary antibodies (Sigma). H Chip Color Determination Five transgenic potato lines expressing the GLTP antisense sequence, two transgenic lines expressing the GHTP antisense sequence, non-transgenic Desiree control lines, and two control lines transformed with the pBI121 vector T-DNA, were grown under field conditions in Alberta, Canada. Tubers were harvested and stored at 10°C and 4°C. Chip color was determined for all potato lines by taking center cuts from representative samples from each line and frying at 205 °F in soybean oil for approximately 3 minutes until bubbling stops. I Results All tubers were harvested from plants of the same cultivar (Desiree), the same age, and grown side by side under identical growth conditions. Northern analysis of tubers showed a considerable reduction of endogenous GLTP transcript in transgenic plants expressing the homologous antisense transcript (Figure 5). Glucan phosphorylase assays showed that activities (μmol NADPH mg "1 protein h '1 ) were reduced (Table 1) at harvest and for at least six months following harvest in transgenic plants expressing the GLTP antisense DNA. The results tabulated in Table 1 show that α glucan phosphorylase activity in tubers stored at 4°C for 189 days was reduced from approximately 16% to 70% in various transformed potato varieties relative to the wildtype control strain. Activity gels and western blot analysis showed specific reduced expression of homologous enzymes and lower reduction of expression for heterologous enzymes (Figure 8). This specificity for homologous products may result from differences between the phosphorylases (Figures 3 and 4). Analysis of tubers at harvest (0 days) shows that those expressing the antisense GLTP transcript have up to 5-fold less reducing sugars than control tubers (Table 2). Furthermore, after 91 days storage at 4°C, transformed tubers contained 28-39% lower reducing sugar concentrations than the wildtype control strain. Concentrations of glucose and fructose were reduced significantly in tubers expressing the antisense GLTP transcript (Tables 3 and 4). These results suggest that reduced GLTP activity slows the catabolism of starch into reducing sugars in tubers, while in the control tubers the sugars continue to accumulate. The correlation between total phosphorylase activity and the concentration of reducing sugars is not direct, suggesting that certain isozymes of phosphorylase may play a more important role in the catabolism of starch, that specific levels of reduced expression of particular phosphorylase isozymes may be more optimum than others, and/or that there may be unidentified interactions involved in the lower reducing sugar levels. Transgenic potato plants expressing the antisense GLTP or GHTP transcript have been grown under field conditions and their tubers stored at 4°C. Chip color, which correlated with sugar content, was determined prior to cold storage and after 86 and 124 days of cold storage. The chip color of tubers from all transgenic plants expressing the antisense GLTP transcript was significantly improved (lighter) relative to that of control tubers (darker) stored under identical conditions (Table 5 and Figure 7). Chip scores of tubers from "Desiree " potato plants expressing the GLTP transcript were improved by at least 4.3 points and 8.9 points as determined with an Agtron model E-15-FP Direct Reading Abridged Spectrophotometer (Agtron Inc. 1095 Spice Island Drive #100, Sparks Nevada 89431) following storage at 10°C and 4°C, respectively, for 86 days. Chip scores of GLTP transformants measured after 124 days of storage at 4°C were improved by 44% to 89% relative to wildtype (Table 5). The Desiree cultivar is not a commercially desirable potato for chipping due to its high natural sugar content and propensity to sweeten rapidly in cold storage. Nevertheless, significant improvements in fried chip color were noted with the transformed "Desiree" potatoes. It is expected that superior color lightening would be achieved if the methods of the invention were applied to commercial processing potato varieties. Analysis of tubers stored at 10°C and 4°C shows that those expressing the antisense GHTP transcript sometimes provided chips that fried lighter than control tubers, indicating a lower buildup of reducing sugars (Table 5). Results showing heterologous and homologous reduction in phosphorylase activity (Figure 8) indicate that the improvement may be a result of reducing one or both tuber phosphorylases. However, these results suggest that the L-type phosphorylase plays a more important role in the catabolism of starch into reducing sugars. Further, the results show that the difference in reducing sugar levels (Table 2) and chip scores (Table 5) between tubers wildtype plants and those expressing tuber phosphorylase antisense RNA, are sustained during long-term storage. As shown in Table 5, the chip scores are approximately the same at 86 days and 124 days. No further increases in reducing sugar concentrations were evident after 49 and 91 days storage at 4°C (Table 2). This equilibrium in sugar concentration was probably associated with the kinetics of the tuber phosphorylases. The capability of maintaining lower sugar levels has the potential of extending the period of storage by at least several months. Presently, processing potatoes are usually stored for a maximum of three to six months at 10°C to 12°C before the sugar accumulation reaches levels that reduce quality. Fresh product must be imported until the present season potatoes become available. Extending the storage period of potatoes by many months may reduce import costs. Table 6 provides a summary of the percentage improvement in various improved tuber cold-storage characteristics of tubers of potato plants transformed with antisense DNA derived from the GLTP gene sequence (ATL3 - ATL9), and from the GHTP gene sequence (ATH1 and ATH2) relative to untransformed control plants. It is apparent from the results summarized in Table 6 that substantial improvements in tuber cold-storage characteristics may be consistently obtained through the methods of the present invention. Particularly noteworthy are the percentage chip score improvements over wildtype observed after storage at 4 °C for approximately four months (124 days). Relative chip score improvements of up to 89% relative to wildtype were observed. Improved chip scores reflect the commercial utility of the invention. That is, by reducing cold-induced sweetening, tubers can be stored at cooler temperatures, without causing unacceptable darkening of fried potato products. The reduction in sugar accumulation of transformed potato lines relative to wildtype, both at harvest and after 91 day storage, also demonstrates significant advantages of the invention. Reduced sugar accumulation relates to the observed chip score improvements, and also reflects improved specific gravity of tubers, another important commercial measure of tuber quality. Even at harvest, substantial improvements in chip score and reduced sugar accumulation were noted for transformed lines relative to wildtype. Thus, the benefits of the invention are not limited to improvements that arise only after extended periods of cold storage, but that are present at the time of harvest. In this sense, the invention is not limited only to improvements in cold-storage characteristics but encompasses improvements in tuber quality characteristics such as chip score or sugar accumulation which are present at the time of harvest, resulting in earlier maturity. Turning to specific improvements summarized in Table 6, it can be seen that GLTP- type transformants (ATL3 - ATL9) exhibited up to a 66%, 70% and 69% reduction in α glucan phosphorylase activity relative to wildtype, at harvest, and after storage for 91 and 189 days, respectively. Most also exhibited improvements in excess of 10% and 30% relative to wildtype at harvest and after storage for 91 and 189 days. After storage for 91 and 189 days, the GHTP-type transformants (ATHl and ATH2) exhibited, respectively, up to 28% and 39% relative improvement over wildtype and generally showed at least 10% improvement. The GLTP-type transformants exhibited up to 80% and 39% reduction of sugar accumulation relative to wildtype at harvest and at 91 days, respectively. At harvest, all GLTP-type transformants exhibited at least 10% and at least 30% relative improvement. At 91 days, all GLTP-type transformants exhibited at least 10% and most exhibited at least 30% relative improvement. The GLTP-type transformants exhibited up to 46%, 89% and 89% chip score improvement relative to wildtype at harvest, and after storage for 86 days and 124 days, respectively. Almost all exhibited at least 10% and 30% relative improvement at harvest, and after storage for 86 and 124 days. At least one of the GHTP-type transformants exhibited at least 5% and at least 10% improvement relative to wildtype at harvest, and after storage for 86 and 124 days. After 124 days storage, at least one of the GHTP-type transformants exhibited up to 25% relative improvement in chip score. The results clearly demonstrate that substantial improvements in tuber cold-storage characteristics may be readily obtained through the methods of the invention. Results will vary due to, among other things, the location within the plant genome where the recombinant antisense or sense DNA is inserted, and the number of insertion events that occur. It is important to note that despite the variability in the results amongst the various transformed lines, there was little variation in the results amongst the samples within a single transformed potato line (see footnotes to Tables 1 to 5). Results are presented in Table 6 for all potato plant lines which were successfully transformed with the GHTP or GLTP antisense DNA. Therefore, all transformants show at least some improvement in one or more cold-storage characteristics such as increased chip score (lighter color on cooking) and reduced sugar accumulation, and most show very substantial improvements. Given the large proportion of positive transformants observed in the examples herein, it is expected that, using the cold- storage characteristic testing procedures described in the examples, potato plants transformed through the methods of the invention can be readily screened to identify transformed lines exhibiting significantly improved cold-storage characteristics. By applying the techniques disclosed herein to commercially important potato varieties, it will be possible to readily create and select transformants having significantly improved cold-storage characteristics. Those transformants showing the greatest relative improvements over wildtype controls can be used in the development of new commercial potato varieties. Table 1 Effects of an antisense transcript on glucan phosphorylase activity measured in enzyme extracts from field grown "Desiree" tubers. Glucan Phosphorylase Activity Storage Period at 4C (days) Clone 0 49 91 140 189 μmol NADPH i Jig "1 protein h "1 Wt a 10.50 11.83 9.94 11.90 13.04 ATL3 4.90 4.86 4.49 4.73 4.88 ATL4 11.45 7.17 8.09 11.32 10.99 ATL5 3.58 3.56 2.97 4.59 4.79 ATL9 3.59 3.88 3.84 4.72 3.98 kC o 5 1.97 2.94 1.59 2.34 2.58 I-k 0| 2.87 4.28 2.31 3.41 3.75 Clone c 0.01 d WT vs. ATL's 0.01 Days NS Clone x Days 0.05 WT 11.49 8.90 12.66 13.66 ATH-1 10.40 9.69 10.79 10.10 ATH-2 6.46 6.40 6.56 8.38 T I -" S- > D L -'θ.05 b 2.02 0.41 3.00 NS L D 001 4.78 0.95 NS NS Clone 0 0.01 WT vs. ATH's 0.01 Days 0.05 Clone x Days NS a WT, wild type untransformed tubers. b LSD, least significant difference at 0.05 or 0.01 level for each storage period. °Sources of variation i in factorial analysis. d Significance levels for indicated sources of variation Table 2 Effects of an antisense GLTP transcript on low temperature induced sweetening of field grown "Desiree" tubers Reducing Sugars (glucose + fructose) Storage Period at 4C (days) Clone 0 49 91 mg g " ' fresh weight Wt a 5.63 31.8 28.0 ATL3 1.88 17.3 17.3 ATL4 1.11 14.3 20.1 ATL5 1.51 18.3 17.0 ATL9 1.36 17.3 18.5 WT vs. ATL's b 0.01 0.01 0.05 Clone 0 0.0 l d Days 0.01 Clone x Days NS a WT, wild type untransformed tubers. b Orthogonal comparisons for ANOVA's at each storage period. c sources of variation in factorial analysis. d Significance levels for indicated sources of variation. Table 3 Effects of an antisense GLTP transcript on low temperature induced fructose accumulation of field grown "Desiree" tubers. Fructose Storage Period at 4C (days) Clone 0 49 91 m "1 ' fresh weight Wt a 3.53 15.10 12.20 ATL3 1.21 8.40 8.79 ATL4 0.79 7.22 8.56 ATL5 0.61 10.00 8.09 ATL9 0.54 8.38 8.72 WT vs. ATL's" 0.01 0.01 NS ■ Clone 0 0.01 d Days 0.01 Clone x Days NS a WT, wild type untransformed tubers. b Orthogonal comparisons for ANOVA's at each storage period. c Sources of variation in factorial analysis. d Significance levels for indicated sources of variation. Table 4 Effects of an antisense GLTP transcript on low temperature induced glucose accumulation of field grown "Desiree" tubers. Glucose Storage Period at 4C (days) Clone 0 49 91 mg g '1 fresh weight Wt a 2.10 16.60 15.90 ATL3 0.68 8.94 8.49 ATL4 0.32 7.07 11.06 ATL5 1.05 8.33 8.91 ATL9 0.83 8.87 9.78 WT vs. ATL's b 0.01 0.01 0.05 Clone 0 0.0 l d Days 0.01 Clone x Days NS WT, wild type untransformed tubers. "Orthogonal comparisons for ANOVA's at each storage period. c sources of variation in factorial analysis. d Significance levels for indicated sources of variation. Table 5 Average chip color of field grown "Desiree" tubers. The chip color rating was assigned using an Agtron meter similar to that used by industry to measure color of fried potatoes. In this index, the higher the number the lighter the chip product but color does not represent a linear relationship to the index. Storage Temperature, Period, and Agtron Reading 3 Harvest 86 days 86 days 124 days IOC 4C 4C Wt b 26 25.3 15.4 17.1 ATL3 C 25 37.4 26.7 30.8 ATL4 35 43.7 29.1 32.3 ATL5 36 29.6 24.7 24.6 ATL9 38 38.7 24.3 26.6 ATHl" 26 49.7 17.5 21.4 ATH2 29 31.2 15.6 15.9 GMPl e 31 15.7 15.7 GMP2 35 16.7 16.6 a Agtron Inc. 1095 Spice Island Drive #100, Sparks Nevada 89431. Agtron model E- 15-FP (Direct Reading Abridged Spectrophotometer). Measures ratio of reflectance in two spectral modes, near infrared and green. Results represent the measurement of 6 to 8 chips from 3 randomly selected tubers approximately 3 to 4 cm in diameter. b WT, negative control, wild type untransformed tubers. C ATL, tubers transformed with the tuber L-type<* glucan phosphorylase. d ATH, tubers transformed with the tuber H-type<* glucan phosphorylase. e GMP, negative control, tubers transformed with pBI121 T-DNA. Table 6 Summary of Results REFERENCES Alber and Kawasaki (1982) Mol. And Appl. Genet. 1:419-434. ap Rees et al. (1988) Symp. Soc. Exp. Biol. 42:377-393. Bevan et al. (1983) Nature (London) 304: 184-187. Bevan et al. (1986) Nucleic Acids Res. 14 (11):4625-4638. Birnboim et al. (1979) Nucleic Acids Res. 7: 1513-1523. Blennow et al. ( 1991 ) Phytochemistry 30:437-444. Bradford, M.M. 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 243-254. Brisson et al. (1989) The Plant Cell 1:559-566. 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Biol. 11:255-269. Stukerlj et al. (1990) Nucl. Acids Res. 18:46050. Takaha et al., (1993) J. Biol. Chem. 26 8: 1391-1396. Thuring et al. (1975) Analytical Biochemistry 66:213-220. Van der Krol et al (1988) Gene 72:45-50. Van der Krol (1990) Plant Cell 2:291-299. Weaver et al. (1978) Am. Pot. J. 55:83-93. Weintraub (1990) Scientific American 1:34-40. Winnacker, Ernst L. (1987) From Genes to Clones. VCH Verlagsgesellschaft mbH, Federal Replublic of Germany Yoshida et al. (1992) Geneg 10:255-259. All publications mentioned in this specification are indicative of the level of skill in the art to which this invention pertains. All publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practised within the scope of the appended claims. 98/35051 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Her Majesty the Queen in Right of Canada as Represented by the Department of Agriculture and Agri-Food Canada (ii) TITLE OF INVENTION: Potatoes Having Improved Quality Characteristics and Methods for Their Production (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: McKay-Carey & Company (B) STREET: 2125 Commerce Place, 10155-102nd Street (C) CITY: Edmonton (D) STATE: Alberta (E) COUNTRY: Canada (F) ZIP: T5J 4G8 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: WO (B) FILING DATE: 10-FEB-1998 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/036,946 (B) FILING DATE: 10-FEB-1997 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/868,786 (B) FILING DATE: 04-JUN-1997 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: McKay-Carey, Mary Jane (B) REGISTRATION NUMBER: 3790 (C) REFERENCE/DOCKET NUMBER: 24002WO0 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (403) 424-0222 (B) TELEFAX: (403) 421-0834 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3101 base pairs (B) TYPE: nucleic acid O 98/35051 (C) STRANDEDNESS : double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosu (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 44..2944 (D) OTHER INFORMATION: /product= "potato alpha-glucan L-type tuber phosphorylase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 194..2941 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 44..193 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATCACTCTCA TTCGAAAAGC TAGATTTGCA TAGAGAGCAC AAA ATG GCG ACT GCA 55 Met Ala Thr Ala -50 AAT GGA GCA CAC TTG TTC AAC CAT TAC AGC TCC AAT TCC AGA TTC ATC 103 Asn Gly Ala His Leu Phe Asn His Tyr Ser Ser Asn Ser Arg Phe lie -45 -40 -35 CAT TTC ACT TCT AGA AAC ACA AGC TCC AAA TTG TTC CTT ACC AAA ACC 151 His Phe Thr Ser Arg Asn Thr Ser Ser Lys Leu Phe Leu Thr Lys Thr -30 -25 -20 -15 TCC CAT TTT CGG AGA CCC AAA CGC TGT TTC CAT GTC AAC AAT ACC TTG 199 Ser His Phe Arg Arg Pro Lys Arg Cys Phe His Val Asn Asn Thr Leu -10 -5 1 AGT GAG AAA ATT CAC CAT CCC ATT ACT GAA CAA GGT GGT GAG AGC GAC 247 Ser Glu Lys lie His His Pro lie Thr Glu Gin Gly Gly Glu Ser Asp 5 10 15 CTG AGT TCT TTT GCT CCT GAT GCC GCA TCT ATT ACC TCA AGT ATC AAA 295 Leu Ser Ser Phe Ala Pro Asp Ala Ala Ser lie Thr Ser Ser lie Lys 20 25 30 TAC CAT GCA GAA TTC ACA CCT GTA TTC TCT CCT GAA AGG TTT GAG CTC 343 Tyr His Ala Glu Phe Thr Pro Val Phe Ser Pro Glu Arg Phe Glu Leu 35 40 45 50 CCT AAG GCA TTC TTT GCA ACA GCT CAA AGT GTT CGT GAT TCG CTC CTT 391 Pro Lys Ala Phe Phe Ala Thr Ala Gin Ser Val Arg Asp Ser Leu Leu 55 60 65 ATT AAT TGG AAT GCT ACG TAT GAT ATT TAT GAA AAG CTG AAC ATG AAG 439 lie Asn Trp Asn Ala Thr Tyr Asp lie Tyr Glu Lys Leu Asn Met Lys 70 75 80 CAA GCG TAC TAT CTA TCC ATG GAA TTT CTG CAG GGT AGA GCA TTG TTA 487 Gin Ala Tyr Tyr Leu Ser Met Glu Phe Leu Gin Gly Arg Ala Leu Leu 85 90 95 AAT GCA ATT GGT AAT CTG GAG CTT ACT GGT GCA TTT GCG GAA GCT TTG 535 Asn Ala lie Gly Asn Leu Glu Leu Thr Gly Ala Phe Ala Glu Ala Leu 100 105 110 AAA AAC CTT GGC CAC AAT CTA GAA AAT GTG GCT TCT CAG GAA CCA GAT 583 Lys Asn Leu Gly His Asn Leu Glu Asn Val Ala Ser Gin Glu Pro Asp 115 120 125 130 GCT GCT CTT GGA AAT GGG GGT TTG GGA CGG CTT GCT TCC TGT TTT CTG 631 Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu 135 140 145 GAC TCT TTG GCA ACA CTA AAC TAC CCA GCA TGG GGC TAT GGA CTT AGG 679 Asp Ser Leu Ala Thr Leu Asn Tyr Pro Ala Trp Gly Tyr Gly Leu Arg 150 155 160 TAC AAG TAT GGT TTA TTT AAG CAA CGG ATT ACA AAA GAT GGT CAG GAG 727 Tyr Lys Tyr Gly Leu Phe Lys Gin Arg lie Thr Lys Asp Gly Gin Glu 165 170 175 GAG GTG GCT GAA GAT TGG CTT GAA ATT GGC AGT CCA TGG GAA GTT GTG 775 Glu Val Ala Glu Asp Trp Leu Glu He Gly Ser Pro Trp Glu Val Val 180 185 190 AGG AAT GAT GTT TCA TAT CCT ATC AAA TTC TAT GGA AAA GTC TCT ACA 823 Arg Asn Asp Val Ser Tyr Pro He Lys Phe Tyr Gly Lys Val Ser Thr 195 200 205 210 GGA TCA GAT GGA AAG AGG TAT TGG ATT GGT GGA GAG GAT ATA AAG GCA 871 Gly Ser Asp Gly Lys Arg Tyr Trp He Gly Gly Glu Asp He Lys Ala 215 220 225 GTT GCG TAT GAT GTT CCC ATA CCA GGG TAT AAG ACC AGA ACC ACA ATC 919 Val Ala Tyr Asp Val Pro He Pro Gly Tyr Lys Thr Arg Thr Thr He 230 235 240 AGC CTT CGA CTG TGG TCT ACA CAG GTT CCA TCA GCG GAT TTT GAT TTA 967 Ser Leu Arg Leu Trp Ser Thr Gin Val Pro Ser Ala Asp Phe Asp Leu 245 250 255 TCT GCT TTC AAT GCT GGA GAG CAC ACC AAA GCA TGT GAA GCC CAA GCA 1015 Ser Ala Phe Asn Ala Gly Glu His Thr Lys Ala Cys Glu Ala Gin Ala 260 265 270 AAC GCT GAG AAG ATA TGT TAC ATA CTC TAC CCT GGG GAT GAA TCA GAG 1063 Asn Ala Glu Lys He Cys Tyr He Leu Tyr Pro Gly Asp Glu Ser Glu 275 280 285 290 GAG GGA AAG ATC CTT CGG TTG AAG CAA CAA TAT ACC TTA TGC TCG GCT 1111 Glu Gly Lys He Leu Arg Leu Lys Gin Gin Tyr Thr Leu Cys Ser Ala 295 300 305 TCT CTC CAA GAT ATT ATT TCT CGA TTT GAG AGG AGA TCA GGT GAT CGT 1159 Ser Leu Gin Asp He He Ser Arg Phe Glu Arg Arg Ser Gly Asp Arg 310 315 320 ATT AAG TGG GAA GAG TTT CCT GAA AAA GTT GCT GTG CAG ATG AAT GAC 1207 He Lys Trp Glu Glu Phe Pro Glu Lys Val Ala Val Gin Met Asn Asp 325 330 335 ACT CAC CCT ACA CTT TGT ATC CCT GAG CTG ATG AGA ATA TTG ATA GAT 1255 Thr His Pro Thr Leu Cys He Pro Glu Leu Met Arg He Leu He Asp 340 345 350 CTG AAG GGC TTG AAT TGG AAT GAA GCT TGG AAT ATT ACT CAA AGA ACT 1303 Leu Lys Gly Leu Asn Trp Asn Glu Ala Trp Asn He Thr Gin Arg Thr 355 360 365 370 GTG GCC TAC ACA AAC CAT ACT GTT TTG CCT GAG GCA CTG GAG AAA TGG 1351 Val Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu Lys Trp 375 380 385 AGT TAT GAA TTG ATG CAG AAA CTC CTT CCC AGA CAT GTC GAA ATC ATT 1399 Ser Tyr Glu Leu Met Gin Lys Leu Leu Pro Arg His Val Glu He He 390 395 400 GAG GCG ATT GAC GAG GAG CTG GTA CAT GAA ATT GTA TTA AAA TAT GGT 1447 Glu Ala He Asp Glu Glu Leu Val His Glu He Val Leu Lys Tyr Gly 405 410 415 TCA ATG GAT CTG AAC AAA TTG GAG GAA AAG TTG ACT ACA ATG AGA ATC 1495 Ser Met Asp Leu Asn Lys Leu Glu Glu Lys Leu Thr Thr Met Arg He 420 425 430 TTA GAA AAT TTT GAT CTT CCC AGT TCT GTT GCT GAA TTA TTT ATT AAG 1543 Leu Glu Asn Phe Asp Leu Pro Ser Ser Val Ala Glu Leu Phe He Lys 435 440 445 450 CCT GAA ATC TCA GTT GAT GAT GAT ACT GAA ACA GTA GAA GTC CAT GAC 1591 Pro Glu He Ser Val Asp Asp Asp Thr Glu Thr Val Glu Val His Asp 455 460 465 AAA GTT GAA GCT TCC GAT AAA GTT GTG ACT AAT GAT GAA GAT GAC ACT 1639 Lys Val Glu Ala Ser Asp Lys Val Val Thr Asn Asp Glu Asp Asp Thr 470 475 480 GGT AAG AAA ACT AGT GTG AAG ATA GAA GCA GCT GCA GAA AAA GAC ATT 1687 Gly Lys Lys Thr Ser Val Lys He Glu Ala Ala Ala Glu Lys Asp He 485 490 495 GAC AAG AAA ACT CCC GTG AGT CCG GAA CCA GCT GTT ATA CCA CCT AAG 1735 Asp Lys Lys Thr Pro Val Ser Pro Glu Pro Ala Val He Pro Pro Lys 500 505 510 AAG GTA CGC ATG GCC AAC TTG TGT GTT GTG GGC GGC CAT GCT GTT AAT 1783 Lys Val Arg Met Ala Asn Leu Cys Val Val Gly Gly His Ala Val Asn 515 520 525 530 GGA GTT GCT GAG ATC CAT AGT GAA ATT GTG AAG GAG GAG GTT TTC AAT 1831 Gly Val Ala Glu He His Ser Glu He Val Lys Glu Glu Val Phe Asn 535 540 545 GAC TTC TAT GAG CTC TGG CCG GAA AAG TTC CAA AAC AAA ACA AAT GGA 1879 Asp Phe Tyr Glu Leu Trp Pro Glu Lys Phe Gin Asn Lys Thr Asn Gly 550 555 560 GTG ACT CCA AGA AGA TGG ATT CGT TTC TGC AAT CCT CCT CTT AGT GCC 1927 Val Thr Pro Arg Arg Trp He Arg Phe Cys Asn Pro Pro Leu Ser Ala 565 570 575 ATC ATA ACT AAG TGG ACT GGT ACA GAG GAT TGG GTC CTG AAA ACT GAA 1975 He He Thr Lys Trp Thr Gly Thr Glu Asp Trp Val Leu Lys Thr Glu 580 585 590 AAG TTG GCA GAA TTG CAG AAG TTT GCT GAT AAT GAA GAT CTT CAA AAT 2023 Lys Leu Ala Glu Leu Gin Lys Phe Ala Asp Asn Glu Asp Leu Gin Asn 595 600 605 610 GAG TGG AGG GAA GCA AAA AGG AGC AAC AAG ATT AAA GTT GTC TCC TTT 2071 Glu Trp Arg Glu Ala Lys Arg Ser Asn Lys He Lys Val Val Ser Phe 615 620 625 CTC AAA GAA AAG ACA GGG TAT TCT GTT GTC CCA GAT GCA ATG TTT GAT 2119 Leu Lys Glu Lys Thr Gly Tyr Ser Val Val Pro Asp Ala Met Phe Asp 630 635 640 ATT CAG GTA AAA CGC ATT CAT GAG TAC AAG CGA CAA CTG TTA AAT ATC 2167 He Gin Val Lys Arg He His Glu Tyr Lys Arg Gin Leu Leu Asn He 645 650 655 TTC GGC ATC GTT TAT CGG TAT AAG AAG ATG AAA GAA ATG ACA GCT GCA 2215 Phe Gly He Val Tyr Arg Tyr Lys Lys Met Lys Glu Met Thr Ala Ala 660 665 670 GAA AGA AAG ACT AAC TTC GTT CCT CGA GTA TGC ATA TTT GGG GGA AAA 2263 Glu Arg Lys Thr Asn Phe Val Pro Arg Val Cys He Phe Gly Gly Lys 675 680 685 690 GCT TTT GCC ACA TAT GTG CAA GCC AAG AGG ATT GTA AAA TTT ATC ACA 2311 Ala Phe Ala Thr Tyr Val Gin Ala Lys Arg He Val Lys Phe He Thr 695 700 705 GAT GTT GGT GCT ACT ATA AAT CAT GAT CCA GAA ATC GGT GAT CTG TTG 2359 Asp Val Gly Ala Thr He Asn His Asp Pro Glu He Gly Asp Leu Leu 710 715 720 AAG GTA GTC TTT GTG CCA GAT TAC AAT GTC AGT GTT GCT GAA TTG CTA 2407 Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val Ala Glu Leu Leu 725 730 735 ATT CCT GCT AGC GAT CTA TCA GAA CAT ATC AGT ACG GCT GGA ATG GAG 2455 He Pro Ala Ser Asp Leu Ser Glu His He Ser Thr Ala Gly Met Glu 740 745 750 GCC AGT GGA ACC AGT AAT ATG AAG TTT GCA ATG AAT GGT TGT ATC CAA 2503 Ala Ser Gly Thr Ser Asn Met Lys Phe Ala Met Asn Gly Cys He Gin 755 760 765 770 ATT GGT ACA TTG GAT GGC GCT AAT GTT GAA ATA AGG GAA GAG GTT GGA 2551 He Gly Thr Leu Asp Gly Ala Asn Val Glu He Arg Glu Glu Val Gly 775 780 785 GAA GAA AAC TTC TTT CTC TTT GGT GCT CAA GCT CAT GAA ATT GCA GGG 2599 Glu Glu Asn Phe Phe Leu Phe Gly Ala Gin Ala His Glu He Ala Gly 790 795 800 CTT AGA AAA GAA AGA GCT GAC GGA AAG TTT GTA CCT GAT GAA CGT TTT 2647 Leu Arg Lys Glu Arg Ala Asp Gly Lys Phe Val Pro Asp Glu Arg Phe 805 810 815 GAA GAG GTG AAG GAA TTT GTT AGA AGC GGT GCT TTT GGC TCT TAT AAC 2695 Glu Glu Val Lys Glu Phe Val Arg Ser Gly Ala Phe Gly Ser Tyr Asn 820 825 830 TAT GAT GAC CTA ATT GGA TCG TTG GAA GGA AAT GAA GGT TTT GGC CGT 2743 Tyr Asp Asp Leu He Gly Ser Leu Glu Gly Asn Glu Gly Phe Gly Arg 835 840 845 850 GCT GAC TAT TTC CTT GTG GGC AAG GAC TTC CCC AGT TAC ATA GAA TGC 2791 Ala Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Ser Tyr He Glu Cys 855 860 865 CAA GAG AAA GTT GAT GAG GCA TAT CGC GAC CAG AAA AGG TGG ACA ACG 2839 Gin Glu Lys Val Asp Glu Ala Tyr Arg Asp Gin Lys Arg Trp Thr Thr 870 875 880 ATG TCA ATC TTG AAT ACA GCG GGA TCG TAC AAG TTC AGC AGT GAC AGA 2887 Met Ser He Leu Asn Thr Ala Gly Ser Tyr Lys Phe Ser Ser Asp Arg 885 890 895 ACA ATC CAT GAA TAT GCC AAA GAC ATT TGG AAC ATT GAA GCT GTG GAA 2935 Thr He His Glu Tyr Ala Lys Asp He Trp Asn He Glu Ala Val Glu 900 905 910 ATA GCA TAA GAGGGGGAAG TGAATGAAAA ATAACAAAGG CACAGTAAGT 2984 He Ala * 915 AGTTTCTCTT TTTATCATGT GATGAAGGTA TATAATGTAT GTGTAAGAGG ATGATGTTAT 3044 TACCACATAA TAAGAGATGA AGAGTCTCAT TTTGCTTCAA AAAAAAAAAA AAAAAAA 3101 (2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 967 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : Met Ala Thr Ala Asn Gly Ala His Leu Phe Asn His Tyr Ser Ser Asn -50 -45 -40 -35 Ser Arg Phe He His Phe Thr Ser Arg Asn Thr Ser Ser Lys Leu Phe -30 -25 -20 Leu Thr Lys Thr Ser His Phe Arg Arg Pro Lys Arg Cys Phe His Val -15 -10 -5 Asn Asn Thr Leu Ser Glu Lys He His His Pro He Thr Glu Gin Gly 1 5 10 Gly Glu Ser Asp Leu Ser Ser Phe Ala Pro Asp Ala Ala Ser He Thr 15 20 25 30 Ser Ser He Lys Tyr His Ala Glu Phe Thr Pro Val Phe Ser Pro Glu 35 40 45 Arg Phe Glu Leu Pro Lys Ala Phe Phe Ala Thr Ala Gin Ser Val Arg 50 55 60 Asp Ser Leu Leu He Asn Trp Asn Ala Thr Tyr Asp He Tyr Glu Lys 65 70 75 Leu Asn Met Lys Gin Ala Tyr Tyr Leu Ser Met Glu Phe Leu Gin Gly 80 85 90 Arg Ala Leu Leu Asn Ala He Gly Asn Leu Glu Leu Thr Gly Ala Phe 95 100 105 110 Ala Glu Ala Leu Lys Asn Leu Gly His Asn Leu Glu Asn Val Ala Ser 115 120 125 Gin Glu Pro Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala 130 135 140 Ser Cys Phe Leu Asp Ser Leu Ala Thr Leu Asn Tyr Pro Ala Trp Gly 145 150 155 Tyr Gly Leu Arg Tyr Lys Tyr Gly Leu Phe Lys Gin Arg He Thr Lys 160 165 170 Asp Gly Gin Glu Glu Val Ala Glu Asp Trp Leu Glu He Gly Ser Pro 175 180 185 190 Trp Glu Val Val Arg Asn Asp Val Ser Tyr Pro He Lys Phe Tyr Gly 195 200 205 Lys Val Ser Thr Gly Ser Asp Gly Lys Arg Tyr Trp He Gly Gly Glu 210 215 220 Asp He Lys Ala Val Ala Tyr Asp Val Pro He Pro Gly Tyr Lys Thr 225 230 235 Arg Thr Thr He Ser Leu Arg Leu Trp Ser Thr Gin Val Pro Ser Ala 240 245 250 Asp Phe Asp Leu Ser Ala Phe Asn Ala Gly Glu His Thr Lys Ala Cys 255 260 265 270 Glu Ala Gin Ala Asn Ala Glu Lys He Cys Tyr He Leu Tyr Pro Gly 275 280 285 Asp Glu Ser Glu Glu Gly Lys He Leu Arg Leu Lys Gin Gin Tyr Thr 290 295 300 Leu Cys Ser Ala Ser Leu Gin Asp He He Ser Arg Phe Glu Arg Arg 305 310 315 Ser Gly Asp Arg He Lys Trp Glu Glu Phe Pro Glu Lys Val Ala Val 320 325 330 Gin Met Asn Asp Thr His Pro Thr Leu Cys He Pro Glu Leu Met Arg 335 340 345 350 He Leu He Asp Leu Lys Gly Leu Asn Trp Asn Glu Ala Trp Asn He 355 360 365 Thr Gin Arg Thr Val Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala 370 375 • 380 Leu Glu Lys Trp Ser Tyr Glu Leu Met Gin Lys Leu Leu Pro Arg His 385 390 395 Val Glu He He Glu Ala He Asp Glu Glu Leu Val His Glu He Val 400 405 410 Leu Lys Tyr Gly Ser Met Asp Leu Asn Lys Leu Glu Glu Lys Leu Thr 415 420 425 430 Thr Met Arg He Leu Glu Asn Phe Asp Leu Pro Ser Ser Val Ala Glu 435 440 445 Leu Phe He Lys Pro Glu He Ser Val Asp Asp Asp Thr Glu Thr Val 450 455 460 Glu Val His Asp Lys Val Glu Ala Ser Asp Lys Val Val Thr Asn Asp 465 470 475 Glu Asp Asp Thr Gly Lys Lys Thr Ser Val Lys He Glu Ala Ala Ala 480 485 490 Glu Lys Asp He Asp Lys Lys Thr Pro Val Ser Pro Glu Pro Ala Val 495 500 505 510 He Pro Pro Lys Lys Val Arg Met Ala Asn Leu Cys Val Val Gly Gly 515 520 525 His Ala Val Asn Gly Val Ala Glu He His Ser Glu He Val Lys Glu 530 535 540 Glu Val Phe Asn Asp Phe Tyr Glu Leu Trp Pro Glu Lys Phe Gin Asn 545 550 555 Lys Thr Asn Gly Val Thr Pro Arg Arg Trp He Arg Phe Cys Asn Pro 560 565 570 Pro Leu Ser Ala He He Thr Lys Trp Thr Gly Thr Glu Asp Trp Val 575 580 585 590 Leu Lys Thr Glu Lys Leu Ala Glu Leu Gin Lys Phe Ala Asp Asn Glu 595 600 605 Asp Leu Gin Asn Glu Trp Arg Glu Ala Lys Arg Ser Asn Lys He Lys 610 615 620 Val Val Ser Phe Leu Lys Glu Lys Thr Gly Tyr Ser Val Val Pro Asp 625 630 635 Ala Met Phe Asp He Gin Val Lys Arg He His Glu Tyr Lys Arg Gin 640 645 650 Leu Leu Asn He Phe Gly He Val Tyr Arg Tyr Lys Lys Met Lys Glu 655 660 665- 670 Met Thr Ala Ala Glu Arg Lys Thr Asn Phe Val Pro Arg Val Cys He 675 680 685 Phe Gly Gly Lys Ala Phe Ala Thr Tyr Val Gin Ala Lys Arg He Val 690 695 700 Lys Phe He Thr Asp Val Gly Ala Thr He Asn His Asp Pro Glu He 705 710 715 Gly Asp Leu Leu Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val 720 725 730 Ala Glu Leu Leu He Pro Ala Ser Asp Leu Ser Glu His He Ser Thr 735 740 745 750 Ala Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe Ala Met Asn 755 760 765 Gly Cys He Gin He Gly Thr Leu Asp Gly Ala Asn Val Glu He Arg 770 775 780 Glu Glu Val Gly Glu Glu Asn Phe Phe Leu Phe Gly Ala Gin Ala His 785 790 795 Glu He Ala Gly Leu Arg Lys Glu Arg Ala Asp Gly Lys Phe Val Pro 800 805 810 Asp Glu Arg Phe Glu Glu Val Lys Glu Phe Val Arg Ser Gly Ala Phe 815 820 825 830 Gly Ser Tyr Asn Tyr Asp Asp Leu He Gly Ser Leu Glu Gly Asn Glu 835 840 845 Gly Phe Gly Arg Ala Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Ser 850 855 860 Tyr He Glu Cys Gin Glu Lys Val Asp Glu Ala Tyr Arg Asp Gin Lys 865 870 875 Arg Trp Thr Thr Met Ser He Leu Asn Thr Ala Gly Ser Tyr Lys Phe 880 885 890 Ser Ser Asp Arg Thr He His Glu Tyr Ala Lys Asp He Trp Asn He 895 900 905 910 Glu Ala Val Glu He Ala * 915 ( 2 ) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2655 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME/KEY: CDS O 98/35051 (B) LOCATION: 12..2528 (D) OTHER INFORMATION: /product= "potato alpha-glucan H-type tuber phosphorylase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 12..2525 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : GTTTATTTTC C ATG GAA GGT GGT GCA AAA TCG AAT GAT GTA TCA GCA GCA 50 Met Glu Gly Gly Ala Lys Ser Asn Asp Val Ser Ala Ala 1 5 10 CCT ATT GCT CAA CCA CTT TCT GAA GAC CCT ACT GAC ATT GCA TCT AAT 98 Pro He Ala Gin Pro Leu Ser Glu Asp Pro Thr Asp He Ala Ser Asn 15 20 25 ATC AAG TAT CAT GCT CAA TAT ACT CCT CAT TTT TCT CCT TTC AAG TTT 146 He Lys Tyr His Ala Gin Tyr Thr Pro His Phe Ser Pro Phe Lys Phe 30 35 40 45 GAG CCA CTA CAA GCA TAC TAT GCT GCT ACT GCT GAC AGT GTT CGT GAT 194 Glu Pro Leu Gin Ala Tyr Tyr Ala Ala Thr Ala Asp Ser Val Arg Asp 50 55 60 CGC TTG ATC AAA CAA TGG AAT GAC ACC TAT CTT CAT TAT GAC AAA GTT 242 Arg Leu He Lys Gin Trp Asn Asp Thr Tyr Leu His Tyr Asp Lys Val 65 70 75 AAT CCA AAG CAA ACA TAC TAC TTA TCA ATG GAG TAT CTC CAG GGG CGA 290 Asn Pro Lys Gin Thr Tyr Tyr Leu Ser Met Glu Tyr Leu Gin Gly Arg 80 85 90 GCT TTG ACA AAT GCA GTT GGA AAC TTA GAC ATC CAC AAT GCA TAT GCT 338 Ala Leu Thr Asn Ala Val Gly Asn Leu Asp He His Asn Ala Tyr Ala 95 100 105 GAT GCT TTA AAC AAA CTG GGT CAG CAG CTT GAG GAG GTC GTT GAG CAG 386 Asp Ala Leu Asn Lys Leu Gly Gin Gin Leu Glu- Glu Val Val Glu Gin 110 115 120 125 GAA AAA GAT GCA GCA TTA GGA AAT GGT GGT TTA GGA AGG CTC GCT TCA 434 Glu Lys Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser 130 135 140 TGC TTT CTT GAT TCC ATG GCC ACA TTG AAC CTT CCA GCA TGG GGT TAT 482 Cys Phe Leu Asp Ser Met Ala Thr Leu Asn Leu Pro Ala Trp Gly Tyr 145 150 155 GGC TTG AGG TAC AGA TAT GGA CTT TTT AAG CAG CTT ATC ACA AAG GCT 530 Gly Leu Arg Tyr Arg Tyr Gly Leu Phe Lys Gin Leu He Thr Lys Ala 160 165 170 GGG CAA GAA GAA GTT CCT GAA GAT TGG TTG GAG AAA TTT AGT CCC TGG 578 Gly Gin Glu Glu Val Pro Glu Asp Trp Leu Glu Lys Phe Ser Pro Trp 175 180 185 GAA ATT GTA AGG CAT GAT GTT GTC TTT CCT ATC AGG TTT TTT GGT CAT 626 Glu He Val Arg His Asp Val Val Phe Pro He Arg Phe Phe Gly His 190 195 200 205 GTT GAA GTC CTC CCT TCT GGC TCG CGA AAA TGG GTT GGT GGA GAG GTC 674 Val Glu Val Leu Pro Ser Gly Ser Arg Lys Trp Val Gly Gly Glu Val 210 215 220 CTA CAG GCT CTT GCA TAT GAT GTG CCA ATT CCA GGA TAC AGA ACT AAA 722 Leu Gin Ala Leu Ala Tyr Asp Val Pro He Pro Gly Tyr Arg Thr Lys 225 230 235 AAC ACT AAT AGT CTT CGT CTC TGG GAA GCC AAA GCA AGC TCT GAG GAT 770 Asn Thr Asn Ser Leu Arg Leu Trp Glu Ala Lys Ala Ser Ser Glu Asp 240 245 250 TTC AAC TTG TTT CTG TTT AAT GAT GGA CAG TAT GAT GCT GCT GCA CAG 818 Phe Asn Leu Phe Leu Phe Asn Asp Gly Gin Tyr Asp Ala Ala Ala Gin 255 260 265 CTT CAT TCT AGG GCT CAG CAG ATT TGT GCT GTT CTC TAC CCT GGG GAT 866 Leu His Ser Arg Ala Gin Gin He Cys Ala Val Leu Tyr Pro Gly Asp 270 275 280 285 GCT ACA GAG AAT GGA AAA CTC TTA CGG CTA AAG CAA CAA TTT TTT CTG 914 Ala Thr Glu Asn Gly Lys Leu Leu Arg Leu Lys Gin Gin Phe Phe Leu 290 295 300 TGC AGT GCA TCG CTT CAG GAT ATT ATT GCC AGA TTC AAA GAG AGA GAA 962 Cys Ser Ala Ser Leu Gin Asp He He Ala Arg Phe Lys Glu Arg Glu 305 310 315 GAT GGA AAG GGT TCT CAC CAG TGG TCT GAA TTC CCC AAG AAG GTT GCG 1010 Asp Gly Lys Gly Ser His Gin Trp Ser Glu Phe Pro Lys Lys Val Ala 320 325 » 330 ATA CAA CTA AAT GAC ACA CAT CCA ACT CTT ACG ATT CCA GAG CTG ATG 1058 He Gin Leu Asn Asp Thr His Pro Thr Leu Thr He Pro Glu Leu Met 335 340 345 CGG TTG CTA ATG GAT GAT GAA GGA CTT GGG TGG GAT GAA TCT TGG AAT 1106 Arg Leu Leu Met Asp Asp Glu Gly Leu Gly Trp Asp Glu Ser Trp Asn 350 355 360 365 ATC ACT ACT AGG ACA ATT GCC TAT ACG AAT CAT ACA GTC CTA CCT GAA 1154 He Thr Thr Arg Thr He Ala Tyr Thr Asn His Thr Val Leu Pro Glu 370 375 380 GCA CTT GAA AAA TGG TCT CAG GCA GTC ATG TGG AAG CTC CTT CCT AGA 1202 Ala Leu Glu Lys Trp Ser Gin Ala Val Met Trp Lys Leu Leu Pro Arg 385 390 395 O 98/35051 CAT ATG GAA ATC ATT GAA GAA ATT GAC AAA CGG TTT GTT GCT ACA ATA 1250 His Met Glu He He Glu Glu He Asp Lys Arg Phe Val Ala Thr He 400 405 410 ATG TCA GAA AGA CCT GAT CTT GAG AAT AAG ATG CCT AGC ATG CGC ATT 1298 Met Ser Glu Arg Pro Asp Leu Glu Asn Lys Met Pro Ser Met Arg He 415 420 425 TTG GAT CAC AAC GCC ACA AAA CCT GTT GTG CAT ATG GCT AAC TTG TGT 1346 Leu Asp His Asn Ala Thr Lys Pro Val Val His Met Ala Asn Leu Cys 430 435 440 445 GTT GTC TCT TCA CAT ACG GTA AAT GGT GTT GCC CAG CTG CAT AGT GAC 1394 Val Val Ser Ser His Thr Val Asn Gly Val Ala Gin Leu His Ser Asp 450 455 460 ATC CTG AAG GCT GAG TTA TTT GCT GAT TAT GTC TCT GTA TGG CCC ACC 1442 He Leu Lys Ala Glu Leu Phe Ala Asp Tyr Val Ser Val Trp Pro Thr 465 470 475 AAG TTC CAG AAT AAG ACC AAT GGT ATA ACT CCT CGT AGG TGG ATC CGA 1490 Lys Phe Gin Asn Lys Thr Asn Gly He Thr Pro Arg Arg Trp He Arg 480 485 490 TTT TGT AGT CCT GAG CTG AGT CAT ATA ATT ACC AAG TGG TTA AAA ACA 1538 Phe Cys Ser Pro Glu Leu Ser His He He Thr Lys Trp Leu Lys Thr 495 500 505 GAT CAA TGG GTG ACG AAC CTC GAA CTG CTT GCT AAT CTT CGG GAG TTT 1586 Asp Gin Trp Val Thr Asn Leu Glu Leu Leu Ala Asn Leu Arg Glu Phe 510 515 520 525 GCT GAT AAT TCG GAG CTC CAT GCT GAA TGG GAA TCA GCC AAG ATG GCC 1634 Ala Asp Asn Ser Glu Leu His Ala Glu Trp Glu Ser Ala Lys Met Ala 530 535 540 AAC AAG CAG CGT TTG GCA CAG TAT ATA CTG CAT GTG ACA GGT GTG AGC 1682 Asn Lys Gin Arg Leu Ala Gin Tyr He Leu His Val Thr Gly Val Ser 545 550 555 ATC GAT CCA AAT TCC CTT TTT GAC ATA CAA GTC AAA CGT ATC CAT GAA 1730 He Asp Pro Asn Ser Leu Phe Asp He Gin Val Lys Arg He His Glu 560 565 570 TAC AAA AGG CAG CTT CTA AAT ATT CTG GGC GTC ATC TAT AGA TAC AAG 1778 Tyr Lys Arg Gin Leu Leu Asn He Leu Gly Val He Tyr Arg Tyr Lys 575 580 585 AAG CTT AAG GGA ATG AGC CCT GAA GAA AGG AAA AAT ACA ACT CCT CGC 1826 Lys Leu Lys Gly Met Ser Pro Glu Glu Arg Lys Asn Thr Thr Pro Arg 590 595 600 605 ACA GTC ATG ATT GGA GGA AAA GCA TTT GCA ACA TAC ACA AAT GCA AAA 1874 Thr Val Met He Gly Gly Lys Ala Phe Ala Thr Tyr Thr Asn Ala Lys 610 615 620 CGA ATT GTC AAG CTC GTG ACT GAT GTT GGC GAC GTT GTC AAT AGT GAC 1922 Arg He Val Lys Leu Val Thr Asp Val Gly Asp Val Val Asn Ser Asp 625 630 635 CCT GAC GTC AAT GAC TAT TTG AAG GTG GTT TTT GTT CCC AAC TAC AAT 1970 Pro Asp Val Asn Asp Tyr Leu Lys Val Val Phe Val Pro Asn Tyr Asn 640 645 650 GTA TCT GTG GCA GAG ATG CTT ATT CCG GGA AGT GAG CTA TCA CAA CAC 2018 Val Ser Val Ala Glu Met Leu He Pro Gly Ser Glu Leu Ser Gin His 655 660 665 ATC AGT ACT GCA GGC ATG GAA GCA AGT GGA ACA AGC AAC ATG AAA TTT 2066 He Ser Thr Ala Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe 670 675 680 685 GCC CTT AAT GGA TGC CTT ATC ATT GGG ACA CTA GAT GGG GCC AAT GTG 2114 Ala Leu Asn Gly Cys Leu He He Gly Thr Leu Asp Gly Ala Asn Val 690 695 700 GAA ATT AGG GAG GAA ATT GGA GAA GAT AAC TTC TTT CTT TTT GGT GCA 2162 Glu He Arg Glu Glu He Gly Glu Asp Asn Phe Phe Leu Phe Gly Ala 705 710 715 ACA GCT GAT GAA GTT CCT CAA CTG CGC AAA GAT CGA GAG AAT GGA CTG 2210 Thr Ala Asp Glu Val Pro Gin Leu Arg Lys Asp Arg Glu Asn Gly Leu 720 725 730 TTC AAA CCT GAT CCT CGG TTT GAA GAG GCA AAA CAA TTT ATT AGG TCT 2258 Phe Lys Pro Asp Pro Arg Phe Glu Glu Ala Lys Gin Phe He Arg Ser 735 740 745 GGA GCA TTT GGG ACG TAT GAT TAT AAT CCC CTC CTT GAA TCA CTG GAA 2306 Gly Ala Phe Gly Thr Tyr Asp Tyr Asn Pro Leu Leu Glu Ser Leu Glu 750 755 760 765 GGG AAC TCG GGA TAT GGT CGT GGA GAC TAT TTT CTT GTT GGT CAT GAT 2354 Gly Asn Ser Gly Tyr Gly Arg Gly Asp Tyr Phe Leu Val Gly His Asp 770 775 780 TTT CCG AGC TAC ATG GAT GCT CAG GCA AGG GTT' GAT GAA GCT TAC AAG 2402 Phe Pro Ser Tyr Met Asp Ala Gin Ala Arg Val Asp Glu Ala Tyr Lys 785 790 795 GAC AGG AAA AGA TGG ATA AAG ATG TCT ATA CTG AGC ACT AGT GGG AGT 2450 Asp Arg Lys Arg Trp He Lys Met Ser He Leu Ser Thr Ser Gly Ser 800 805 810 GGC AAA TTT AGT AGT GAC CGT ACA ATT TCT CAA TAT GCA AAA GAG ATC 2498 Gly Lys Phe Ser Ser Asp Arg Thr He Ser Gin Tyr Ala Lys Glu He 815 820 825 TGG AAC ATT GCC GAG TGT CGC GTG CCT TGA GCACACTTCT GAACCTGGTA 2548 Trp Asn He Ala Glu Cys Arg Val Pro * 830 835 TCTAATAAGG ATCTAATGTT CATTGTTTAC TAGCATATGA ATAATGTAAG TTCAAGCACA 2608 ACATGCTTTC TTATTTCCTA CTGCTCTCAA GAAGCAGTTA TTTGTTG 2655 (2) INFORMATION FOR SEQ ID NO : 4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 839 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : Met Glu Gly Gly Ala Lys Ser Asn Asp Val Ser Ala Ala Pro He Ala 1 5 10 15 Gin Pro Leu Ser Glu Asp Pro Thr Asp He Ala Ser Asn He Lys Tyr 20 25 30 His Ala Gin Tyr Thr Pro His Phe Ser Pro Phe Lys Phe Glu Pro Leu 35 40 45 Gin Ala Tyr Tyr Ala Ala Thr Ala Asp Ser Val Arg Asp Arg Leu He 50 55 60 Lys Gin Trp Asn Asp Thr Tyr Leu His Tyr Asp Lys Val Asn Pro Lys 65 70 75 80 Gin Thr Tyr Tyr Leu Ser Met Glu Tyr Leu Gin Gly Arg Ala Leu Thr 85 90 95 Asn Ala Val Gly Asn Leu Asp He His Asn Ala Tyr Ala Asp Ala Leu 100 105 110 Asn Lys Leu Gly Gin Gin Leu Glu Glu Val Val Glu Gin Glu Lys Asp 115 120 125 Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu 130 135 140 Asp Ser Met Ala Thr Leu Asn Leu Pro Ala Trp Gly Tyr Gly Leu Arg 145 150 155 160 Tyr Arg Tyr Gly Leu Phe Lys Gin Leu He Thr Lys Ala Gly Gin Glu 165 170 175 Glu Val Pro Glu Asp Trp Leu Glu Lys Phe Ser Pro Trp Glu He Val 180 185 190 Arg His Asp Val Val Phe Pro He Arg Phe Phe Gly His Val Glu Val 195 200 205 Leu Pro Ser Gly Ser Arg Lys Trp Val Gly Gly Glu Val Leu Gin Ala 210 215 220 Leu Ala Tyr Asp Val Pro He Pro Gly Tyr Arg Thr Lys Asn Thr Asn 225 230 235 240 Ser Leu Arg Leu Trp Glu Ala Lys Ala Ser Ser Glu Asp Phe Asn Leu 245 250 255 Phe Leu Phe Asn Asp Gly Gin Tyr Asp Ala Ala Ala Gin Leu His Ser 260 265 270 Arg Ala Gin Gin He Cys Ala Val Leu Tyr Pro Gly Asp Ala Thr Glu 275 280 285 Asn Gly Lys Leu Leu Arg Leu Lys Gin Gin Phe Phe Leu Cys Ser Ala 290 295 300 Ser Leu Gin Asp He He Ala Arg Phe Lys Glu Arg Glu Asp Gly Lys 305 310 315 320 Gly Ser His Gin Trp Ser Glu Phe Pro Lys Lys Val Ala He Gin Leu 325 330 335 Asn Asp Thr His Pro Thr Leu Thr He Pro Glu Leu Met Arg Leu Leu 340 345 350 Met Asp Asp Glu Gly Leu Gly Trp Asp Glu Ser Trp Asn He Thr Thr 355 360 365 Arg Thr He Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu 370 375 380 Lys Trp Ser Gin Ala Val Met Trp Lys Leu Leu Pro Arg His Met Glu 385 390 395 400 He He Glu Glu He Asp Lys Arg Phe Val Ala Thr He Met Ser Glu 405 410 415 Arg Pro Asp Leu Glu Asn Lys Met Pro Ser Met Arg He Leu Asp His 420 425 ' 430 Asn Ala Thr Lys Pro Val Val His Met Ala Asn Leu Cys Val Val Ser 435 440 445 Ser His Thr Val Asn Gly Val Ala Gin Leu His Ser Asp He Leu Lys 450 455 460 Ala Glu Leu Phe Ala Asp Tyr Val Ser Val Trp Pro Thr Lys Phe Gin 465 470 475 480 Asn Lys Thr Asn Gly He Thr Pro Arg Arg Trp He Arg Phe Cys Ser 485 490 495 Pro Glu Leu Ser His He He Thr Lys Trp Leu Lys Thr Asp Gin Trp 500 505 510 Val Thr Asn Leu Glu Leu Leu Ala Asn Leu Arg Glu Phe Ala Asp Asn 515 520 525 Ser Glu Leu His Ala Glu Trp Glu Ser Ala Lys Met Ala Asn Lys Gin 530 535 540 Arg Leu Ala Gin Tyr He Leu His Val Thr Gly Val Ser He Asp Pro 545 550 555 560 Asn Ser Leu Phe Asp He Gin Val Lys Arg He His Glu Tyr Lys Arg 565 570 575 Gin Leu Leu Asn He Leu Gly Val He Tyr Arg Tyr Lys Lys Leu Lys 580 585 590 Gly Met Ser Pro Glu Glu Arg Lys Asn Thr Thr Pro Arg Thr Val Met 595 600 605 He Gly Gly Lys Ala Phe Ala Thr Tyr Thr Asn Ala Lys Arg He Val 610 615 620 Lys Leu Val Thr Asp Val Gly Asp Val Val Asn Ser Asp Pro Asp Val 625 630 635 640 Asn Asp Tyr Leu Lys Val Val Phe Val Pro Asn Tyr Asn Val Ser Val 645 650 655 Ala Glu Met Leu He Pro Gly Ser Glu Leu Ser Gin His He Ser Thr 660 665 670 Ala Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe Ala Leu Asn 675 680 685 Gly Cys Leu He He Gly Thr Leu Asp Gly Ala Asn Val Glu He Arg 690 695 700 Glu Glu He Gly Glu Asp Asn Phe Phe Leu Phe Gly Ala Thr Ala Asp 705 710 715- 720 Glu Val Pro Gin Leu Arg Lys Asp Arg Glu Asn Gly Leu Phe Lys Pro 725 730 735 Asp Pro Arg Phe Glu Glu Ala Lys Gin Phe He Arg Ser Gly Ala Phe 740 745 750 Gly Thr Tyr Asp Tyr Asn Pro Leu Leu Glu Ser Leu Glu Gly Asn Ser 755 760 765 Gly Tyr Gly Arg Gly Asp Tyr Phe Leu Val Gly His Asp Phe Pro Ser 770 775 780 Tyr Met Asp Ala Gin Ala Arg Val Asp Glu Ala Tyr Lys Asp Arg Lys 785 790 795 800 Arg Trp He Lys Met Ser He Leu Ser Thr Ser Gly Ser Gly Lys Phe 805 810 815 Ser Ser Asp Arg Thr He Ser Gin Tyr Ala Lys Glu He Trp Asn He 820 825 830 Ala Glu Cys Arg Val Pro * 835 (2) INFORMATION FOR SEQ ID NO : 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3171 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear ' (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 87..3011 (D) OTHER INFORMATION: /product= "potato alpha-glucan L-type leaf phosphorylase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 330..3008 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 87..329 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : TTTTTTTTTT CAACATGCAC AACAATTATT TTGATTAAAT TTTGTATCTA AAAATTTAGC 60 ATTTTGAAAT TCAGTTCAGA GACATC ATG GCA ACT TTT GCT GTC TCT GGA TTG 113 Met Ala Thr Phe Ala Val Ser Gly Leu -81 -80 -75 AAC TCA ATT TCA AGT ATT TCT AGT TTT AAT AAC AAT TTC AGA AGC AAA 161 Asn Ser He Ser Ser He Ser Ser Phe Asn Asn Asn Phe Arg Ser Lys -70 -65 -60 AAC TCA AAC ATT TTG TTG AGT AGA AGG AGG ATT TTA TTG TTC AGT TTT 209 Asn Ser Asn He Leu Leu Ser Arg Arg Arg He Leu Leu Phe Ser Phe -55 -50 -45 AGA AGA AGA AGA AGA AGT TTC TCT GTT AGC AGT GTT GCT AGT GAT CAA 257 Arg Arg Arg Arg Arg Ser Phe Ser Val Ser Ser Val Ala Ser Asp Gin -40 -35 -30 -25 AAG CAG AAG ACA AAG GAT TCT TCC TCT GAT GAA GGA TTT ACA TTA GAT 305 Lys Gin Lys Thr Lys Asp Ser Ser Ser Asp Glu Gly Phe Thr Leu Asp -20 -15 -10 GTT TTT CAG CCG GAC TCC ACG TCT GTT TTA TCA AGT ATA AAG TAT CAC 353 Val Phe Gin Pro Asp Ser Thr Ser Val Leu Ser Ser He Lys Tyr His -5 1 5 GCT GAG TTC ACA CCA TCA TTT TCT CCT GAG AAG TTT GAA CTT CCC AAG 401 Ala Glu Phe Thr Pro Ser Phe Ser Pro Glu Lys Phe Glu Leu Pro Lys 10 15 20 GCA TAC TAT GCA ACT GCA GAG AGT GTT CGA GAT ACG CTC ATT ATA AAT 449 Ala Tyr Tyr Ala Thr Ala Glu Ser Val Arg Asp Thr Leu He He Asn 25 30 35 40 TGG AAT GCC ACA TAC GAA TTC TAT GAA AAG ATG AAT GTA AAG CAG GCA 497 Trp Asn Ala Thr Tyr Glu Phe Tyr Glu Lys Met Asn Val Lys Gin Ala 45 50 55 TAT TAC TTG TCT ATG GAA TTT CTT CAG GGA AGA GCT TTA CTC AAT GCT 545 Tyr Tyr Leu Ser Met Glu Phe Leu Gin Gly Arg Ala Leu Leu Asn Ala 60 65 70 ATT GGT AAC TTG GGG CTA ACC GGA CCT TAT GCA GAT GCT TTA ACT AAG 593 He Gly Asn Leu Gly Leu Thr Gly Pro Tyr Ala Asp Ala Leu Thr Lys 75 80 85 CTC GGA TAC AGT TTA GAG GAT GTA GCC AGG CAG GAA CCG GAT GCA GCT 641 Leu Gly Tyr Ser Leu Glu Asp Val Ala Arg Gin Glu Pro Asp Ala Ala 90 95 100 TTA GGT AAT GGA GGT TTA GGA AGA CTT GCT TCT' TGC TTT CTG GAC TCA 689 Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu Asp Ser 105 110 115 120 ATG GCG ACA CTA AAC TAC CCT GCA TGG GGC TAT GGA CTT AGA TAC CAA 737 Met Ala Thr Leu Asn Tyr Pro Ala Trp Gly Tyr Gly Leu Arg Tyr Gin 125 130 135 TAT GGC CTT TTC AAA CAG CTT ATT ACA AAA GAT GGA CAG GAG GAA GTT 785 Tyr Gly Leu Phe Lys Gin Leu He Thr Lys Asp Gly Gin Glu Glu Val 140 145 150 GCT GAA AAT TGG CTC GAG ATG GGA AAT CCA TGG GAA ATT GTG AGG AAT 833 Ala Glu Asn Trp Leu Glu Met Gly Asn Pro Trp Glu He Val Arg Asn 155 160 165 GAT ATT TCG TAT CCC GTA AAA TTC TAT GGG AAG GTC ATT GAA GGA GCT 881 Asp He Ser Tyr Pro Val Lys Phe Tyr Gly Lys Val He Glu Gly Ala 170 175 180 GAT GGG AGG AAG GAA TGG GCT GGC GGA GAA GAT ATA ACT GCT GTT GCC 929 Asp Gly Arg Lys Glu Trp Ala Gly Gly Glu Asp He Thr Ala Val Ala 185 190 195 200 TAT GAT GTC CCA ATA CCA GGA TAT AAA ACA AAA ACA ACG ATT AAC CTT 977 Tyr Asp Val Pro He Pro Gly Tyr Lys Thr Lys Thr Thr He Asn Leu 205 210 215 CGA TTG TGG ACA ACA AAG CTA GCT GCA GAA GCT TTT GAT TTA TAT GCT 1025 Arg Leu Trp Thr Thr Lys Leu Ala Ala Glu Ala Phe Asp Leu Tyr Ala 220 225 230 TTT AAC AAT GGA GAC CAT GCC AAA GCA TAT GAG GCC CAG AAA AAG GCT 1073 Phe Asn Asn Gly Asp His Ala Lys Ala Tyr Glu Ala Gin Lys Lys Ala 235 240 245 GAA AAG ATT TGC TAT GTC TTA TAT CCA GGT GAC GAA TCG CTT GAA GGA 1121 Glu Lys He Cys Tyr Val Leu Tyr Pro Gly Asp Glu Ser Leu Glu Gly 250 255 260 AAG ACG CTT AGG TTA AAG CAG CAA TAC ACA CTA TGT TCT GCT TCT CTT 1169 Lys Thr Leu Arg Leu Lys Gin Gin Tyr Thr Leu Cys Ser Ala Ser Leu 265 270 275 280 CAG GAC ATT ATT GCA CGG TTC GAG AAG AGA TCA GGG AAT GCA GTA AAC 1217 Gin Asp He He Ala Arg Phe Glu Lys Arg Ser Gly Asn Ala Val Asn 285 290 295 TGG GAT CAG TTC CCC GAA AAG GTT GCA GTA CAG ATG AAT GAC ACT CAT 1265 Trp Asp Gin Phe Pro Glu Lys Val Ala Val Gin Met Asn Asp Thr His 300 305 310 CCA ACA CTT TGT ATA CCA GAA CTT TTA AGG ATA TTG ATG GAT GTT AAA 1313 Pro Thr Leu Cys He Pro Glu Leu Leu Arg He Leu Met Asp Val Lys 315 320 325 GGT TTG AGC TGG AAG CAG GCA TGG GAA ATT ACT CAA AGA ACG GTC GCA 1361 Gly Leu Ser Trp Lys Gin Ala Trp Glu He Thr Gin Arg Thr Val Ala 330 335 340 TAC ACT AAC CAC ACT GTT CTA CCT GAG GCT CTT GAG AAA TGG AGC TTC 1409 Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu Lys Trp Ser Phe 345 350 355 360 ACA CTT CTT GGT GAA CTG CTT CCT CGG CAC GTG GAG ATC ATA GCA ATG 1457 Thr Leu Leu Gly Glu Leu Leu Pro Arg His Val Glu He He Ala Met 365 370 375 ATA GAT GAG GAG CTC TTG CAT ACT ATA CTT GCT GAA TAT GGT ACT GAA 1505 He Asp Glu Glu Leu Leu His Thr He Leu Ala Glu Tyr Gly Thr Glu 380 385 390 GAT CTT GAC TTG TTG CAA GAA AAG CTA AAC CAA ATG AGG ATT CTG GAT 1553 Asp Leu Asp Leu Leu Gin Glu Lys Leu Asn Gin Met Arg He Leu Asp 395 400 405 AAT GTT GAA ATA CCA AGT TCT GTT TTG GAG TTG CTT ATA AAA GCC GAA 1601 Asn Val Glu He Pro Ser Ser Val Leu Glu Leu Leu He Lys Ala Glu 410 415 420 GAA AGT GCT GCT GAT GTC GAA AAG GCA GCA GAT GAA GAA CAA GAA GAA 1649 Glu Ser Ala Ala Asp Val Glu Lys Ala Ala Asp Glu Glu Gin Glu Glu 425 430 435 440 GAA GGT AAG GAT GAC AGT AAA GAT GAG GAA ACT GAG GCT GTA AAG GCA 1697 Glu Gly Lys Asp Asp Ser Lys Asp Glu Glu Thr Glu Ala Val Lys Ala 445 450 455 GAA ACT ACG AAC GAA GAG GAG GAA ACT GAG GTT AAG AAG GTT GAG GTG 1745 Glu Thr Thr Asn Glu Glu Glu Glu Thr Glu Val Lys Lys Val Glu Val 460 465 470 GAG GAT AGT CAA GCA AAA ATA AAA CGT ATA TTC GGG CCA CAT CCA AAT 1793 Glu Asp Ser Gin Ala Lys He Lys Arg He Phe Gly Pro His Pro Asn 475 480 485 AAA CCA CAG GTG GTT CAC ATG GCA AAT CTA TGT GTA GTT AGC GGG CAT 1841 Lys Pro Gin Val Val His Met Ala Asn Leu Cys Val Val Ser Gly His 490 495 500 GCA GTT AAC GGT GTT GCT GAG ATT CAT AGT GAA ATA GTT AAG GAT GAA 1889 Ala Val Asn Gly Val Ala Glu He His Ser Glu He Val Lys Asp Glu 505 510 515 520 GTT TTC AAT GAA TTT TAC AAG TTA TGG CCA GAG AAA TTC CAA AAC AAG 1937 Val Phe Asn Glu Phe Tyr Lys Leu Trp Pro Glu Lys Phe Gin Asn Lys 525 530 535 ACA AAT GGT GTG ACA CCA AGA AGA TGG CTA AGT TTC TGT AAT CCA GAG 1985 Thr Asn Gly Val Thr Pro Arg Arg Trp Leu Ser Phe Cys Asn Pro Glu 540 545 550 TTG AGT GAA ATT ATA ACC AAG TGG ACA GGA TCT GAT GAT TGG TTA GTA 2033 Leu Ser Glu He He Thr Lys Trp Thr Gly Ser Asp Asp Trp Leu Val 555 560 565 AAC ACT GAA AAA TTG GCA GAG CTT CGA AAG TTT GCT GAT AAC GAA GAA 2081 Asn Thr Glu Lys Leu Ala Glu Leu Arg Lys Phe Ala Asp Asn Glu Glu 570 575 580 CTC CAG TCT GAG TGG AGG AAG GCA AAA GGA AAT AAC AAA ATG AAG ATT 2129 Leu Gin Ser Glu Trp Arg Lys Ala Lys Gly Asn Asn Lys Met Lys He 585 590 595 600 GTC TCT CTC ATT AAA GAA AAA ACA GGA TAC GTG GTC AGT CCC GAT GCA 2177 Val Ser Leu He Lys Glu Lys Thr Gly Tyr Val Val Ser Pro Asp Ala 605 610 615 ATG TTT GAT GTT CAG ATC AAG CGC ATC CAT GAG TAT AAA AGG CAG CTA 2225 Met Phe Asp Val Gin He Lys Arg He His Glu Tyr Lys Arg Gin Leu 620 625 630 TTA AAT ATA TTT GGA ATC GTT TAT CGC TAT AAG AAG ATG AAA GAA ATG 2273 Leu Asn He Phe Gly He Val Tyr Arg Tyr Lys Lys Met Lys Glu Met 635 640 645 AGC CCT GAA GAA CGA AAA GAA AAG TTT GTC CCT CGA GTT TGC ATA TTT 2321 Ser Pro Glu Glu Arg Lys Glu Lys Phe Val Pro Arg Val Cys He Phe 650 655 660 GGA GGA AAA GCA TTT GCT ACA TAT GTT CAG GCC AAG AGA ATT GTA AAA 2369 Gly Gly Lys Ala Phe Ala Thr Tyr Val Gin Ala Lys Arg He Val Lys 665 670 675 680 TTT ATC ACT GAT GTA GGG GAA ACA GTC AAC CAT GAT CCC GAG ATT GGT 2417 Phe He Thr Asp Val Gly Glu Thr Val Asn His Asp Pro Glu He Gly 685 690 695 GAT CTT TTG AAG GTT GTA TTT GTT CCT GAT TAC AAT GTC AGT GTA GCA 2465 Asp Leu Leu Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val Ala 700 705 710 GAA GTG CTA ATT CCT GGT AGT GAG TTG TCC CAG CAT ATT AGT ACT GCT 2513 Glu Val Leu He Pro Gly Ser Glu Leu Ser Gin His He Ser Thr Ala 715 720 725 GGT ATG GAG GCT AGT GGA ACC AGC AAC ATG AAA TTT TCA ATG AAT GGC 2561 Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe Ser Met Asn Gly 730 735 740 TGC CTC CTC ATC GGG ACA TTA GAT GGT GCC AAT GTT GAG ATA AGA GAG 2609 Cys Leu Leu He Gly Thr Leu Asp Gly Ala Asn Val Glu He Arg Glu 745 750 755 760 GAA GTT GGA GAG GAC AAT TTC TTT CTT TTC GGA GCT CAG GCT CAT GAA 2657 Glu Val Gly Glu Asp Asn Phe Phe Leu Phe Gly Ala Gin Ala His Glu 765 770 775 ATT GCT GGC CTA CGA AAG GAA AGA GCC GAG GGA AAG TTT GTC CCG GAC 2705 He Ala Gly Leu Arg Lys Glu Arg Ala Glu Gly Lys Phe Val Pro Asp 780 785 790 CCA AGA TTT GAA GAA GTA AAG GCG TTC ATT AGG ACA GGC GTC TTT GGC 2753 Pro Arg Phe Glu Glu Val Lys Ala Phe He Arg Thr Gly Val Phe Gly 795 800 805 ACC TAC AAC TAT GAA GAA CTC ATG GGA TCC TTG GAA GGA AAC GAA GGC 2801 Thr Tyr Asn Tyr Glu Glu Leu Met Gly Ser Leu Glu Gly Asn Glu Gly 810 815 820 TAT GGT CGT GCT GAC TAT TTT CTT GTA GGA AAG GAT TTC CCC GAT TAT 2849 Tyr Gly Arg Ala Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Asp Tyr 825 830 835 840 ATA GAG TGC CAA GAT AAA GTT GAT GAA GCA TAT CGA GAC CAG AAG AAA 2897 He Glu Cys Gin Asp Lys Val Asp Glu Ala Tyr Arg Asp Gin Lys Lys 845 850 855 TGG ACC AAA ATG TCG ATC TTA AAC ACA GCT GGA TCG TTC AAA TTT AGC 2945 Trp Thr Lys Met Ser He Leu Asn Thr Ala Gly Ser Phe Lys Phe Ser 860 865 870 AGT GAT CGA ACA ATT CAT CAA TAT GCA AGA GAT ATA TGG AGA ATT GAA 2993 Ser Asp Arg Thr He His Gin Tyr Ala Arg Asp He Trp Arg He Glu 875 880 885 CCT GTT GAA TTA CCT TAA AAGTTAGCCA GTTAAAGGAT GAAAGCCAAT 3041 Pro Val Glu Leu Pro * 890 TTTTTCCCCC TGAGGTTCTC CCATACTGTT TATTAGTACA TATATTGTCA ATTGTTGCTA 3101 CTGAAATGAT AGAAGTTTTG AATATTTACT GTCAATAAAA TACAGTTGAT TCCATTTGAA 3161 AAAAAAAAAA 3171 (2) INFORMATION FOR SEQ ID NO : 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 975 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : Met Ala Thr Phe Ala Val Ser Gly Leu Asn Ser He Ser Ser He Ser -81 -80 -75 -70 Ser Phe Asn Asn Asn Phe Arg Ser Lys Asn Ser Asn He Leu Leu Ser -65 -60 -55 -50 Arg Arg Arg He Leu Leu Phe Ser Phe Arg Arg. Arg Arg Arg Ser Phe -45 -40 -35 Ser Val Ser Ser Val Ala Ser Asp Gin Lys Gin Lys Thr Lys Asp Ser -30 -25 -20 Ser Ser Asp Glu Gly Phe Thr Leu Asp Val Phe Gin Pro Asp Ser Thr -15 -10 -5 Ser Val Leu Ser Ser He Lys Tyr His Ala Glu Phe Thr Pro Ser Phe 1 5 10 15 Ser Pro Glu Lys Phe Glu Leu Pro Lys Ala Tyr Tyr Ala Thr Ala Glu 20 25 30 Ser Val Arg Asp Thr Leu He He Asn Trp Asn Ala Thr Tyr Glu Phe 35 40 45 Tyr Glu Lys Met Asn Val Lys Gin Ala Tyr Tyr Leu Ser Met Glu Phe 50 55 60 Leu Gin Gly Arg Ala Leu Leu Asn Ala He Gly Asn Leu Gly Leu Thr 65 70 75 Gly Pro Tyr Ala Asp Ala Leu Thr Lys Leu Gly Tyr Ser Leu Glu Asp 80 85 90 95 Val Ala Arg Gin Glu Pro Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly 100 105 110 Arg Leu Ala Ser Cys Phe Leu Asp Ser Met Ala Thr Leu Asn Tyr Pro 115 120 125 Ala Trp Gly Tyr Gly Leu Arg Tyr Gin Tyr Gly Leu Phe Lys Gin Leu 130 135 140 He Thr Lys Asp Gly Gin Glu Glu Val Ala Glu Asn Trp Leu Glu Met 145 150 155 Gly Asn Pro Trp Glu He Val Arg Asn Asp He Ser Tyr Pro Val Lys 160 165 170 175 Phe Tyr Gly Lys Val He Glu Gly Ala Asp Gly Arg Lys Glu Trp Ala 180 185 190 Gly Gly Glu Asp He Thr Ala Val Ala Tyr Asp Val Pro He Pro Gly 195 200 205 Tyr Lys Thr Lys Thr Thr He Asn Leu Arg Leu Trp Thr Thr Lys Leu 210 215 220 Ala Ala Glu Ala Phe Asp Leu Tyr Ala Phe Asn Asn Gly Asp His Ala 225 230 235 Lys Ala Tyr Glu Ala Gin Lys Lys Ala Glu Lys He Cys Tyr Val Leu 240 245 250 255 Tyr Pro Gly Asp Glu Ser Leu Glu Gly Lys Thr Leu Arg Leu Lys Gin 260 265 270 Gin Tyr Thr Leu Cys Ser Ala Ser Leu Gin Asp He He Ala Arg Phe 275 280 285 Glu Lys Arg Ser Gly Asn Ala Val Asn Trp Asp Gin Phe Pro Glu Lys 290 295 300 Val Ala Val Gin Met Asn Asp Thr His Pro Thr Leu Cys He Pro Glu 305 310 315 Leu Leu Arg He Leu Met Asp Val Lys Gly Leu Ser Trp Lys Gin Ala 320 325 330 335 Trp Glu He Thr Gin Arg Thr Val Ala Tyr Thr Asn His Thr Val Leu 340 345 350 Pro Glu Ala Leu Glu Lys Trp Ser Phe Thr Leu Leu Gly Glu Leu Leu 355 360 365 Pro Arg His Val Glu He He Ala Met He Asp Glu Glu Leu Leu His 370 375 380 Thr He Leu Ala Glu Tyr Gly Thr Glu Asp Leu Asp Leu Leu Gin Glu 385 390 395 Lys Leu Asn Gin Met Arg He Leu Asp Asn Val Glu He Pro Ser Ser 400 405 410 415 Val Leu Glu Leu Leu He Lys Ala Glu Glu Ser Ala Ala Asp Val Glu 420 425 430 Lys Ala Ala Asp Glu Glu Gin Glu Glu Glu Gly Lys Asp Asp Ser Lys 435 440 445 Asp Glu Glu Thr Glu Ala Val Lys Ala Glu Thr Thr Asn Glu Glu Glu 450 455 460 Glu Thr Glu Val Lys Lys Val Glu Val Glu Asp Ser Gin Ala Lys He 465 470 475 Lys Arg He Phe Gly Pro His Pro Asn Lys Pro Gin Val Val His Met 480 485 490 495 Ala Asn Leu Cys Val Val Ser Gly His Ala Val Asn Gly Val Ala Glu 500 505 510 He His Ser Glu He Val Lys Asp Glu Val Phe Asn Glu Phe Tyr Lys 515 520 525 Leu Trp Pro Glu Lys Phe Gin Asn Lys Thr Asn Gly Val Thr Pro Arg 530 535 • 540 Arg Trp Leu Ser Phe Cys Asn Pro Glu Leu Ser Glu He He Thr Lys 545 550 555 Trp Thr Gly Ser Asp Asp Trp Leu Val Asn Thr Glu Lys Leu Ala Glu 560 565 570 575 Leu Arg Lys Phe Ala Asp Asn Glu Glu Leu Gin Ser Glu Trp Arg Lys 580 585 590 Ala Lys Gly Asn Asn Lys Met Lys He Val Ser Leu He Lys Glu Lys 595 600 605 Thr Gly Tyr Val Val Ser Pro Asp Ala Met Phe Asp Val Gin He Lys 610 615 620 Arg He His Glu Tyr Lys Arg Gin Leu Leu Asn He Phe Gly He Val 625 630 635 Tyr Arg Tyr Lys Lys Met Lys Glu Met Ser Pro Glu Glu Arg Lys Glu 640 645 650 655 Lys Phe Val Pro Arg Val Cys He Phe Gly Gly Lys Ala Phe Ala Thr 660 665 670 Tyr Val Gin Ala Lys Arg He Val Lys Phe He Thr Asp Val Gly Glu 675 680 685 Thr Val Asn His Asp Pro Glu He Gly Asp Leu Leu Lys Val Val Phe 690 695 700 Val Pro Asp Tyr Asn Val Ser Val Ala Glu Val Leu He Pro Gly Ser 705 710 715 Glu Leu Ser Gin His He Ser Thr Ala Gly Met Glu Ala Ser Gly Thr 720 725 730 735 Ser Asn Met Lys Phe Ser Met Asn Gly Cys Leu Leu He Gly Thr Leu 740 745 750 Asp Gly Ala Asn Val Glu He Arg Glu Glu Val Gly Glu Asp Asn Phe 755 760 765 Phe Leu Phe Gly Ala Gin Ala His Glu He Ala Gly Leu Arg Lys Glu 770 775 780 Arg Ala Glu Gly Lys Phe Val Pro Asp Pro Arg Phe Glu Glu Val Lys 785 790 795 Ala Phe He Arg Thr Gly Val Phe Gly Thr Tyr Asn Tyr Glu Glu Leu 800 805 810 815 Met Gly Ser Leu Glu Gly Asn Glu Gly Tyr Gly Arg Ala Asp Tyr Phe 820 825 ' 830 Leu Val Gly Lys Asp Phe Pro Asp Tyr He Glu Cys Gin Asp Lys Val 835 840 845 Asp Glu Ala Tyr Arg Asp Gin Lys Lys Trp Thr Lys Met Ser He Leu 850 855 860 Asn Thr Ala Gly Ser Phe Lys Phe Ser Ser Asp Arg Thr He His Gin 865 870 875 Tyr Ala Arg Asp He Trp Arg He Glu Pro Val Glu Leu Pro * 880 885 890 ( 2 ) INFORMATION FOR SEQ ID NO : 7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..27 (D) OTHER INFORMATION: /function= "primer" /label= SPL1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : ATTCGAAAAG CTCGAGATTT GCATAGA 27 (2) INFORMATION FOR SEQ ID NO : 8 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) AN I-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..27 (D) OTHER INFORMATION: /function= "primer" /label= SPL2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : GTTTATTTTC CATCGATGGA AGGTGGT 27 (2) INFORMATION FOR SEQ ID NO : 9 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..23 (D) OTHER INFORMATION: /function= "primer" /label= SPH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GTGTGCTCTC GAGCATTGAA AGC 23 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum tuberosum (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..25 (D) OTHER INFORMATION: /function= "primer" / label= SPH2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATAATATCCT GAATCGATGC ACTGC 25

Claims: AIM: A potato plant having improved tuber cold-storage characteristics, comprising a modified potato plant having a reduced level of activity of an α glucan phosphorylase enzyme selected from the group consisting of α glucan L-type tuber phosphorylase (GLTP) and glucan H-type phosphorylase (GHTP) in tubers produced by the plant relative to that of tubers produced by an unmodified potato plant. The potato plant of claim 1 transformed with an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous glucan phosphorylase gene selected from the group consisting of a GLTP gene and a GHTP gene. A potato plant having improved cold-storage characteristics, comprising a potato plant transformed with an expression cassette having a plant promoter sequence operably linked to a DNA sequence comprising at least 20 nucleotides of a gene encoding an α glucan phosphorylase selected from the group consisting of α glucan L-type tuber phosphorylase (GLTP) and glucan H-type phosphorylase (GHTP). The potato plant of claim 3, wherein the encoded glucan phosphorylase is GLTP. The potato plant of claim 3, wherein the encoded glucan phosphorylase is GHTP. The potato plant of claim 3, wherein the DNA sequence comprises nucleotides 338 to 993 of SEQ LD NO: 1. The potato plant of claim 3, wherein the DNA sequence comprises nucleotides 147 to 799 of SEQ LD NO: 3. The potato plant of any one of claims 2, 3, 4, 5, 6 or 7, wherein the DNA sequence is linked to the promoter sequence in an antisense orientation. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant measured at harvest is at least 10% lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant measured at harvest is at least 30% lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant measured at harvest is at least 80% lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant stored at 4 C C for about three months is at least 10% lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant stored at 4°C for about three months is at least 30% lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant stored at 4°C for about three months is at least 39% lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the total α glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h "1 in tubers of the plant measured at harvest is at least 10% lower than the total α glucan phosphorylase activity in tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg '1 protein "1 h "1 in tubers of the plant measured at harvest is at least 30% lower than the total glucan phosphorylase activity in tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h "1 in tubers of the plant measured at harvest is at least 66% lower than the total glucan phosphorylase activity in tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg '1 protein "1 h "1 in tubers of the plant stored at 4°C for about three months is at least 10% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg '1 protein "1 h "1 in tubers of the plant stored at 4°C for about three months is at least 30% lower than the total glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein '1 h "1 in tubers of the plant stored at 4°C for about three months is at least 70% lower than the total glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h "1 in tubers of the plant stored at 4°C for about three months is at least 10% lower than the total glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein the total α glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h '1 in tubers of the plant stored at 4°C for about three months is at least 28% lower than the total glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the total α glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein '1 h "1 in tubers of the plant stored at 4°C for about six months is at least 10% lower than the total glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the total α glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein '1 h "1 in tubers of the plant stored at 4°C for about six months is at least 30% lower than the total α glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h "1 in tubers of the plant stored at 4°C for about six months is at least 69% lower than the total glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein the total α glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h "1 in tubers of the plant stored at 4°C for about six months is at least 10% lower than the total glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein the total glucan phosphorylase activity measured as μmol NADPH produced mg "1 protein "1 h "1 in tubers of the plant stored at 4°C for about six months is at least 39% lower than the total α glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein a chip score for tubers of the plant measured at harvest is at least 5% higher than the chip scores for tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein a chip score for tubers of the plant measured at harvest is at least 30% higher than the chip scores for tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein a chip score for tubers of the plant measured at harvest is at least 46% higher than the chip scores for tubers of an untransformed plant measured at harvest. The potato plant of claim 5, wherein a chip score for tubers of the plant measured at harvest is at least 5% higher than the chip scores for tubers of an untransformed plant measured at harvest. The potato plant of claim 5, wherein a chip score for tubers of the plant measured at harvest is at least 10% higher than the chip scores for tubers of an untransformed plant measured at harvest. The potato plant of claim 4, wherein a chip score for tubers of the plant stored at 4°C for about three months is at least 5% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein a chip score for tubers of the plant stored at 4°C for about three months is at least 30% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein a chip score for tubers of the plant stored at 4°C for about three months is at least 89% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein a chip score for tubers of the plant stored at 4 °C for about three months is at least 5% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein a chip score for tubers of the plant stored at 4°C for about three months is at least 10% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein a chip score for tubers of the plant stored at 4 °C for about four months is at least 5% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein a chip score for tubers of the plant stored at 4°C for about four months is at least 30% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 4, wherein a chip score for tubers of the plant stored at 4°C for about four months is at least 89% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein a chip score for tubers of the plant stored at 4 °C for about four months is at least 5% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein a chip score for tubers of the plant stored at 4°C for about four months is at least 25% higher than the chip scores for tubers of an untransformed plant stored under the same conditions. A method for improving the cold-storage characteristics of a potato tuber, comprising providing a potato plant which has been modified to reduce the level of activity in the tubers of an α glucan phosphorylase enzyme selected from the group consisting of glucan L-type tuber phosphorylase (GLTP) and glucan H-type phosphorylase (GHTP). The method of claim 43, comprising: introducing into the potato plant an expression cassette having a plant promoter sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous α glucan phosphorylase gene selected from the group consisting of a GLTP gene and a GHTP gene. A method for improving the cold-storage characteristics of a potato tuber, comprising: introducing into a potato plant an expression cassette having a plant promoter sequence operably linked to a DNA sequence comprising at least 20 nucleotides of a gene encoding an α glucan phosphorylase selected from the group consisting of GLTP and GHTP. The method of claim 45, wherein the encoded glucan phosphorylase is GLTP. The method of claim 45, wherein the encoded α glucan phosphorylase is GHTP. The method of claim 45, wherein the DNA sequence comprises nucleotides 338 to 993 of SEQ ID. NO: 1. The method of claim 45, wherein the DNA sequence comprises nucleotides 147 to 799 of SEQ ID. NO: 3. The method of any one of claims 44, 45, 46, 47, 48 or 49 wherein the DNA sequence is linked to the promoter sequence in an antisense orientation.

Inventors: ARMSTRONG JOHN DAVID, KAWCHUK LAWRENCE MICHAEL, KNOWLES NORMAN RICHARD, LYNCH DERMOT ROBORG, ARMSTRONG, JOHN, DAVID, KAWCHUK, LAWRENCE, MICHAEL, KNOWLES, NORMAN, RICHARD, LYNCH, DERMOT, ROBORG
Applicants: ARMSTRONG JOHN DAVID, CANADA AGRICULTURE, KAWCHUK LAWRENCE MICHAEL, KNOWLES NORMAN RICHARD, LYNCH DERMOT ROBORG, ARMSTRONG, JOHN, DAVID, HER MAJESTY THE QUEEN IN RIGHT OF CANADA AS REPRESENTED BY THE DEPARTMENT OF AGRICULTURE AND AGRI-FOOD CANADA, KAWCHUK, LAWRENCE, MICHAEL, KNOWLES, NORMAN, RICHARD, LYNCH, DERMOT, ROBORG
Application year: 1998
Main classification: C12N15
IPC codes: A01H5, C12N9, C12N15