AU724942B2 - Transgenic potatoes having reduced levels of alpha glucan L- or H-type tuber phosphorylase activity with reduced cold-sweetening - Google Patents

Description

WO 98/35051 PCT/CA98/00055 1 2 3 4 TRANSGENIC POTATOES HAVING REDUCED LEVELS OF ALPHA GLUCAN L- OR H-TYPE TUBER PHOSPHO- RYLASE ACTIVITY WITH REDUCED COLD-SWEETENING 6 7 8 This application claims the benefit of U.S. Provisional Patent Application No.

9 60/036,946 filed February 10, 1997, which is incorporated in its entirety by reference herein.

11 FIELD OF THE INVENTION 12 13 The invention relates to the inhibition of the accumulation of sugars in potatoes by 14 reducing the level of a glucan L-type tuber phosphorylase or a glucan H-type tuber phosphorylase enzyme activity in the potato plant.

16 17 BACKGROUND OF THE INVENTION 18 19 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.

21 Dormancy periods between harvesting and sprouting are critical to maintaining quality 22 potatoes. Processing potatoes are usually stored between 7 and 12°C. Cold storage at 2 to 23 6 0 C, versus storage at 7 to 12 0 C, provides the greatest longevity by reducing respiration, 24 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 26 resulting high sugar levels contribute to an unacceptable brown or black color in the fried 27 product (Coffin et al., 1987, Weaver et al., 1978). The sugars that accumulate are 28 predominantly glucose, fructose, and sucrose. It is primarily the glucose and fructose 29 (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 31 pigments (Burton, 1989, Shallenberger et al., 1959). Sucrose produces a black colouration 32 when fried due to caramelization and charring. The ideal reducing sugar content is generally 33 accepted to be 0.1% of tuber fresh weight with 0.33% as the upper limit and higher levels of -WO 98/35051 PCTCA98/00055 1 reducing sugars are sufficient to cause the formation of brown and black pigments that 2 results in an unacceptable fried product (Davies and Viola, 1992). Although the 3 accumulation of reducing sugars can be slowed in higher temperature (7 to 12'C) storage, 4 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 6 resistant to pesticides, and the fact that use of current sprout inhibitors may soon be 7 prohibited, a need exists for potato varieties that can withstand stress and long-term cold 8 storage without the use of chemicals, without the accumulation of reducing sugars, and with 9 greater retention of starch.

Carbohydrate metabolism is a complex process in plant cells. Manipulation of a 11 number of different enzymatic processes may potentially affect the accumulation of reducing 12 sugars during cold storage. For example, inhibition of starch breakdown would reduce the 13 buildup of free sugar. Other methods may also serve to enhance the cold storage properties of 14 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 16 other forms that would not participate in the Maillard reaction. However, many of the 17 enzymatic processes are reversible, and the role of most of the enzymes involved in 18 carbohydrate metabolism is poorly understood. The challenge remains to identify an enzyme 19 that will deliver the desired result, achieve function at low temperatures, and still retain the product qualities desired by producers, processors, and consumers.

21 It has been suggested that phosphofructokinase (PFK) has an important role in the 22 cold-induced sweetening process (Kruger and Hammond, 1988, ap Rees et al., 1988, Dixon et 23 al., 1981, Claassen et al., 1991). ap Reese et al. (1988) suggested that cold treatment had a 24 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 26 would then lead to an increased availability of hexose-phosphates for sucrose production. It 27 was disclosed in European Patent 0438904 (Burrell et al., July 31, 1991) that increasing PFK 28 activity reduces sugar accumulation during storage by removing hexoses through glycolysis 29 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 31 harvest. However it has been shown that pyrophosphate:fructose 6-phosphate WO 98/35051 PCT/CA98/00055 1 phosphotransferase (PFP) remains active at low temperatures (Claassen et al., 1991). PFP 2 activity can supply fructose 6-phosphate for glycolysis just as PFK can, since the two 3 enzymes catalyse the same reaction. Therefore, the efficacy of this strategy for improving 4 cold storage quality of potato tubers remains in doubt. Furthermore, removal of sugars through glycolysis and further metabolism would not be a preferred method of enhancing 6 storage properties of potato tubers because of the resultant loss of valuable dry matter through 7 respiration.

8 It has also been suggested that ADPglucose pyrophosphorylase (ADPGPP) has an 9 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 11 activity reduces sugar accumulation during storage by re-synthesising starch using reducing 12 sugars. An ADPGPP enzyme from E. coli was expressed in potato tubers under the control of 13 a cold-induced promoter and the report claimed to increase ADPGPP activity and lower 14 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 16 starch resynthesis. Thus, catabolism of sugars through glycolysis and respiration occurs and 17 re-incorporation into starch is limited. Up regulation of ADPGPP would not be a preferred 18 method of enhancing storage properties of potato tubers because of the resultant loss of 19 valuable dry matter through respiration. Again, a method involving the reduction of catabolism of starch would be preferable as dry matter would be retained.

21 The degradation of starch is believed to involve several enzymes including a-amylase 22 (endoamylase), P-amylase (exoamylase), amyloglucosidase, and c-glucan phosphorylase 23 (starch phosphorylase). By slowing starch catabolism, accumulation of reducing sugars 24 should be prevented and the removal of sugars through glycolysis and further metabolism would be minimized.

26 Three different isozymes of a glucan phosphorylase have been described. The tuber 27 L-type a1,4 glucan phosphorylase (EC 2.4.1.1) isozyme (GLTP) (Nakano and Fukui, 1986) 28 has a low affinity for highly branched glucans, such as glycogen, and is localized in 29 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, 31 51% and 40% amino acids, respectively. The nucleotide sequence of the GLTP gene and the WO 98/35051 PCT/CA98/00055 1 amino acid sequence of the GLTP enzyme are shown in SEQ ID NO: 1 and SEQ ID NO: 2, 2 respectively. The H-type tuber a-glucan phosphorylase isozyme H (GHTP) (Mori et al., 3 1991) has a high affinity for glycogen and is localized in the cytoplasm. The gene encodes 4 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- 6 type polypeptide. The nucleotide sequence of the GHTP gene and the amino acid sequence of 7 the GHTP enzyme are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. A third 8 isozyme has been reported (Sonnewald et al., 1995) that consists of 974 amino acids and is 9 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 11 highly diverse. This isozyme is referred to as the leaf L-type phosphorylase since the mRNA 12 accumulates equally in leaf and tuber, whereas the mRNA of the tuber L-type phosphorylase 13 accumulates strongly in potato tubers and only weakly in leaf tissues. The tuber L-type 14 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 16 phosphorylase gene and the amino acid sequence of the leaf L-type phosphorylase enzyme are 17 shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

18 The role of the various starch degrading enzymes is not clear, however, and 19 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 21 starch accumulation. Sonnewald et al. (1995) reported that constitutive expression of an 22 antisense RNA specific for the leaf L-type gene resulted in a strong reduction of a glucan 23 phosphorylase L-type activity in leaf tissue, but had no effect in potato tuber tissue. Since the 24 antisense repression of the a glucan phosphorylase activity had no significant influence on starch accumulation in leaves of transgenic potato plants, the authors concluded that starch 26 breakdown was not catalysed by phosphorylases. Considering the high level of sequence 27 homology between identified a glucan phosphorylase isozymes, a similar negative response 28 would be expected with the H-type (GHTP) and L-type tuber (GLTP) isozymes.

29 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 31 ambient and reduced temperatures, particularly at temperatures below 7 C.

WO 98/35051 PCT/CA98/00055 1 SUMMARY OF THE INVENTION 2 3 The inventors have found that surprisingly, reduction of the level of a glucan L-type 4 tuber phosphorylase (GLTP) or a glucan H-type tuber phosphorylase (GHTP) enzyme activity within the potato tuber results in a substantial reduction in the accumulation of sugars in the 6 tuber during propagation and storage, relative to wildtype potatoes, particularly at storage 7 temperatures below 10 0 C, and specifically at 4'C. It is remarkable that, given the complexity 8 of carbohydrate metabolism in the tuber, reduction in the activity of a single enzyme is 9 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 11 reported that reduced expression of the leaf L-type phosphorylase had no significant influence 12 on starch accumulation in leaves of potato plants.

13 The present invention provides tremendous commercial advantages. Tubers in which 14 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 16 fried potato products. Cold storage of tubers storage results in longer storage life, prolonged 17 dormancy by limiting respiration and delaying sprouting, and lower incidence of disease.

18 Reduction in GLTP or GHTP activity in potato plants and tubers can be accomplished 19 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 21 GHTP genes, chemical or protein inhibition, or plant breeding programs.

22 Thus, in broad terms, the invention provides modified potato plants having a reduced 23 level of a glucan L-type tuber phosphorylase (GLTP) or a glucan H-type tuber phosphorylase 24 (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 26 transformed with an expression cassette having a plant promoter sequence operably linked to 27 a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous 28 GLTP gene or GHTP gene. As will be discussed in detail hereinafter, the aforementioned 29 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 WO 98/35051 PCT/CA98/00055 1 one of GLTP or GHTP independently, or could have reduced activity levels of both GLTP 2 and GHTP.

3 As discussed above, the inventors have found that reduction of activity levels of 4 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.

6 Therefore, the invention further extends to methods for reducing sugar production in tubers 7 produced by a potato plant comprising reducing the level of activity of GLTP or GHTP in the 8 potato plant. In a preferred embodiment, such methods involve introducing into the potato 9 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 11 gene or GHTP gene. As above, the DNA sequence may be inserted in the expression cassette 12 in either a sense or antisense orientation.

13 As described in detail in the examples herein, improvements in cold-storage 14 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 16 reduction in the level of GLTP or GHTP enzyme activity detected in potatoes after harvest 17 and cold-storage. Transformed potato varieties have been developed wherein the total v 18 glucan phosphorylase activity measured as tmol NADPH produced mg protein h 1 in tubers 19 of plants stored at 4°C for 189 days is as much as 70% lower than the total ca glucan phosphorylase activity in tubers of untransformed plants stored under the same conditions.

21 Another relatively direct measure of improved cold-storage characteristics is a 22 reduction in sweetening of potatoes observed after a period of cold-storage. Transformed 23 potato varieties have been developed wherein the sum of the concentrations of glucose and 24 fructose in tubers stored at 4 0 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.

26 Yet another measure of improved cold-storage characteristics, demonstrating a 27 practical advantage of the present invention, is a reduction in darkening of a potato chip 28 during processing (cooking). As discussed hereinbefore, the accumulation of sugars in 29 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 3 score, of the fried potato chip. Techniques for measuring chip scores are discussed herein.

WO 98/35051 PCTICA98/00055 1 Transformed potato varieties of the present invention have been developed wherein the chip 2 score for tubers of plants stored at 4°C for 124 days was as much as 89% higher than the chip 3 scores for tubers of untransformed plants stored under the same conditions.

4 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 6 of potatoes at cooler temperatures than would be possible with wildtype potatoes of the same 7 cultivar. As discussed above, storage of potatoes at cooler temperatures than those 8 traditionally used could result in increased storage life, increased dormancy through reduced 9 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 11 characteristics and may be measured and quantified by known techniques.

12 13 BRIEF DESCRIPTION OF THE DRAWINGS 14 In drawings illustrating embodiments of the invention: 16 Figure 1 is a schematic diagram of the tuber L-type a glucan phosphorylase antisense 17 sequence inserted into the pBI121 transformation vector; 18 Figure 2 is a schematic diagram of the tuber H-type a glucan phosphorylase antisense 19 sequence inserted into the pBI121 transformation vector; Figure 3 shows the basic structure of the three isolated isoforms of glucan 21 phosphorylase. The transit peptide (TS) and insertion sequence (IS) are characteristic of the 22 L-type phosphorylases and are not found in the H-type phosphorylase. The percentages 23 indicate the nucleic acid sequence homologies between the isoforms; 24 Figure 4 is a schematic diagram of carbohydrate interconversions in potatoes (Sowokinos 1990); 26 Figure 5 is a comparison of the amino acid sequences of the three isoforms of 27 phosphorylase found in potato for the region targeted by the antisense GLTP construct used in 28 the Examples herein. Highlighted amino acids are identical. The leaf L-type a glucan 29 phosphorylase amino acid sequence is on top (amino acids 21 238 of SEQ ID NO: the tuber L-type a glucan phosphorylase amino acid sequence is in the middle (amino acids 49 WO 98/35051 PCT/CA98/00055 1 266 of SEQ ID NO: and tuber H-type a glucan phosphorylase amino acid sequence is on 2 the bottom (amino acids 46 264 of SEQ ID NO: 4); 3 Figure 6A and 6B are a comparison of the nucleotide sequences of the three isoforms 4 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 a glucan 6 phosphorylase nucleotide sequence is on top (nucleotides 389 1045 of SEQ ID NO: the 7 tuber L-type a glucan phosphorylase nucleotide sequence is in the middle (nucleotides 338 8 993 of SEQ ID NO: and tuber H-type a glucan phosphorylase nucleotide sequence is on 9 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 11 type and lines 3,4,5, and 9 transformed with the tuber L-type a glucan phosphorylase. The 12 blot was probed with a radiolabelled probe specific for the tuber L-type a glucan 13 phosphorylase; 14 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 a-glucan phosphorylase. The blot was probed with 16 a radio labelled probe specific for the H-type a-glucan phosphorylase; 17 Figure 9 shows the fried product obtained from wild type and tuber L-type a 18 glucan phosphorylase transformants ATL1 ATL3 ATL4 ATL5 ATL9 field 19 grown tubers following 86 days storage at 4°C ("ATL" antisense tuber L-type transformant); 21 Figure 10 shows the activity gel and western blot of L-type and H-type isozymes of a 22 1,4 glucan phosphorylase extracted from wild type tubers and tubers transformed with the 23 antisense construct for the L-type isoform; and 24 Figure 11 shows the activity gel and western blot of L-type and H-type isozymes of a 1,4 glucan phosphorylase extracted from wild type tubers and transformed with the antisense 26 construct for the H-type isoform..

27 28 DESCRIPTION OF THE PREFERRED EMBODIMENT 29 Potato plants having a reduced level of a glucan L-type tuber phosphorylase (GLTP) 31 or a glucan H-type tuber phosphorylase (GHTP) activity in tubers produced by the plants WO 98/35051 PCT/CA98/00055 1 relative to that of tubers produced by unmodified potato plants are provided. In the 2 exemplified case, reduction in a glucan phosphorylase activity is accomplished by 3 transforming a potato plant with an expression cassette having a plant promoter sequence 4 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 6 sequence is inserted in the expression cassette in the antisense orientation, a reduction in a 7 glucan phosphorylase activity can be achieved with the DNA sequence inserted in the 8 expression cassette in either a sense or antisense orientation.

9 1 Homology Dependent Silencing 11 The control of gene expression using sense or antisense gene fragments is standard 12 laboratory practice and is well documented in the literature. Antisense and sense suppression 13 are both gene sequence homology-dependent phenomena that may be described as 14 "homology-dependent silencing" phenomena.

A review of scientific research articles published during 1996 reveals several hundred 16 reports of homology-dependent silencing in transgenic plants. The mechanisms underlying 17 homology-dependent silencing are not fully understood, but the characteristics of the 18 phenomena have been studied in many plant genes and this body of work has been 19 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 21 general phenomenon that may be used to control the activity of many endogenous genes.

22 Examples of genes exhibiting reduced expression after the introduction of homologous 23 sequences include dihydroflavanol reductase (Van der Krol 1990), polygalacturonidase 24 (Smith et al 1990), phytoene synthase (Fray and Grierson 1993), pectinesterase (Seymour et al. 1993), phenylalanine ammonia-lyase (De Carvalho et al. 1992), 3 -1,3-glucanase (Hart et 26 al. 1992), chitinase (Dorlhac et al. 1994) nitrate reductase (Napoli et al. 1990), and chalcone 27 synthase Transformation of Russet Burbank potato plants with either sense- or 28 antisense- constructs of the potato leafroll virus coat protein gene has been reported to confer 29 resistance to potato leafroll virus infection (Kawchuk et al. 1991). The transfer of a homologous sense or antisense sequence usually generates transformants with reduced 31 endogenous gene expression. As discussed in detail in the examples herein, transformed WO 98/35051 PCT/CA98/00055 1 potato plants exhibiting phenotypes indicating reduced GLTP or GHTP expression can be 2 readily identified.

3 In the antisense suppression technique, a gene construct or expression cassette is 4 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 6 complementary RNA sequences form a duplex thereby inhibiting translation to protein. The 7 theory underlying both sense and antisense inhibition has been discussed in the literature, 8 including in Antisense Research and Applications (CRC Press, 1993) pp. 125-148. The 9 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, 11 Cannon et al. (1990) reveals that an antisense sequence as short as 41 base pairs is sufficient 12 to achieve gene inhibition. United States Patent No. 5,585,545 (Bennett et al., December 17, 13 1996) describes gene inhibition by an antisense sequence of only 20 base pairs. There are 14 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 16 an organism, including, for example, United States Patent No. 5,545,815 (Fischer et al., 17 August 13, 1996) and United States Patent No. 5,387,757 (Bridges et al., February 7, 1995).

18 Sense-sequence homology-dependent silencing is conducted in a similar manner to 19 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 21 5,034,323, 5,231,020 and 5,283,184, disclose the introduction of sense sequences leading to 22 suppression of gene expression.

23 Both forms of homology-dependent silencing, sense- and antisense-suppression, are 24 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.

26 For instance, both US Patent No. 5,585,545 (Bennett et al., December 17, 1996) and US 27 Patent No. 5,451,514 (Boudet et al., September 15, 1995) claim methods for inhibiting gene 28 expression or recombinant DNA sequences useful in methods for suppressing gene 29 expression drawn to both sense- and antisense-suppression techniques.

31 -WO 98/35051 PCT/CA98/00055 1 2 Alternate Techniques for Reducing GHTP and/or GLTP Activity in Tubers 2 Although homology-dependent silencing is a preferred technique for the down- 3 regulation of GLTP or GHTP in potato plants of the present invention, there are several 4 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 6 invention. Insertion of a related gene or promoter into a plant can induce rapid turnover of 7 homologous endogenous transcripts, a process referred to as co-suppression and believed to 8 have many similarities to the mechanism responsible for antisense RNA inhibition 9 (Jorgensen, 1995; Brusslan and Tobin, 1995). Various regulatory sequences of DNA can be altered (promoters, polyadenylation signals, post-transcriptional processing sites) or used to 11 alter the expression levels (enhancers and silencers) of a specific mRNA. Another strategy to 12 reduce expression of a gene and its encoded protein is the use of ribozymes designed to 13 specifically cleave the target mRNA rendering it incapable of producing a fully functional 14 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 16 important in determining a particular trait can alter activity levels and be exploited by 17 classical breeding programs (Oritz and Huaman, 1994). Site-directed mutagenesis is often 18 used to modify the activity of an identified gene product. The structural coding sequence for a 19 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 21 with reduced affinity and/or specific activity may be identified. Additionally, the activity of 22 a particular enzyme can be altered using various inhibitors. These procedures are routinely 23 used and can be found in text books such as Sambrook et al. (1989).

24 3 Variants of GLTP and GHTP Enzymes and Sequences Used for Homology Dependent 26 Silencing 27 As discussed in the background of the invention, and in greater detail by Nakano et al.

28 (1986), Mori et al. (1991), and Sonnewald et al. (1990), there are three known a glucan 29 phosphorylase isozymes that occur in potato plants. The present invention relates to downregulation of the GLTP and/or GHTP isozymes. While it is believed that the GLTP and 31 GHTP genes of all known commercial potato varieties are substantially identical, it is WO 98/35051 PCT/CA98/00055 1 expected that the principles and techniques of the present invention would be effective in 2 potato plants having variant full length polynucleotide sequences or subsequences which 3 encode polypeptides having the starch catabolizing enzymatic activity of the described GLTP 4 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 6 and GHTP nucleotide sequence variants that differ from those exemplified but still encode 7 the same polypeptide due to codon degeneracy, as well as variants which encode proteins 8 capable of recognition by antibodies raised against the GLTP and GHTP amino acid 9 sequences set forth in SEQ ID NO's. 2 and 4.

Similarly, those skilled in the art will recognize that homology dependent silencing of 11 GLTP and/or GHTP in potato plants may be accomplished with sense or antisense sequences 12 other than those exemplified. First, the region of the GLTP or GHTP cDNA sequence from 13 which the antisense sequence is derived is not essential. Second, as described hereinabove, 14 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 16 suppressed. As described in the Examples herein, the inventors have observed that 17 transformation of potato plants with antisense DNA sequences derived from the GHTP gene 18 not only substantially suppresses GHTP gene activity, but causes some degree of suppression 19 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 21 corresponding leaf type a glucan phosphorylase squence described by Sonnewald et al.

22 (1990) is 71.3%. In the inventors' research to date, the same phenomenon of cross- 23 downregulation has not been observed when potato plants are transformed with antisense 24 DNA sequences derived from the GLTP gene. Nevertheless, these results clearly indicate that absolute sequence identity between the target endogenous a glucan phosphorylase gene and 26 the recombinant DNA is not essential given that GLTP activity was suppressed by an 27 antisense sequence having about 57% sequence identity with the target GLTP sequence.

28 Thus, it will be understood by those skilled in the art that sense or antisense sequences 29 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 31 endogenous GLTP or GHTP gene when introduced into potato plant cells. Useful sense or WO 98/35051 PCT/CA98/00055 1 antisense sequences may differ from the exemplified antisense sequences or from other 2 sequences derived from the endogenous GHTP or GLTP gene sequences by way of 3 conservative amino acid substitutions or differences in the percentage of matched nucleotides 4 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 6 discussion regarding techniques for comparing sequence identity for polynucleotides and 7 polypeptides, conservative amino acid substitutions, and hybridization conditions indicative 8 of degrees of sequence identity. Relevant parts of that discussion are summarized herein.

9 Percentage of sequence identity for polynucleotides and polypeptides may be determined by comparing two optimally aligned sequences over a comparison window, 11 wherein the portion of the polynucleotide or polypeptide sequence in the comparison window 12 may include additions or deletions gaps) as compared to the reference sequence (which 13 does not comprise additions or deletions) for optimal alignment of the two sequences. The 14 percentage is calculated by: 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 16 matched positions; dividing the number of matched positions by the total number of 17 positions in the window of comparison; and, multiplying the result by 100 to yield the 18 percentage of sequence identity. Optimal alignment of sequences for comparison may be 19 conducted by computerized implementations of known algorithms GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer 21 Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the 22 National Center for Biotechnology Information), or by inspection.

23 Polypeptides which are substantially similar share sequences as noted above 24 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 26 having similar side chains. For example, a group of amino acids having aliphatic side chains 27 is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having 28 aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having 29 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 WO 98/35051 PCT/CA98/00055 1 having basic side chains is lysine, arginine, and histidine; and a group of amino acids having 2 sulfur-containing side chains is cysteine and methionine.

3 Another indication that nucleotide sequences are substantially identical is if two 4 molecules specifically hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances.

6 Generally, stringent conditions are selected to be about 10°C lower than the thermal melting 7 point for the specific sequence at a defined ionic strength and pH. The Tm is the 8 temperature (under defined ionic strength and pH) at which 50% of the target sequence 9 hybridizes to a perfectly matched probe. The Tm 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 11 al. (1989). Typically, stringent conditions for a Southern blot protocol involve washing at 12 65 C with 0.2XSSC. For preferred oligonucleotide probes, washing conditions are typically 13 about at 42°C in 6XSSC.

14 4 General Methods 16 Various methods are available to introduce and express foreign DNA sequences in 17 plant cells. In brief, the steps involved in preparing antisense a glucan phosphorylase cDNAs 18 and introducing them into a plant cell include: isolating mRNA from potato plants and 19 preparing cDNA from the mRNA; screening the cDNA for the desired sequences; (3) linking a promoter to the desired cDNAs in the opposite orientation for expression of the 21 phosphorylase genes; transforming suitable host plant cells; and selecting and 22 regenerating cells which transcribe the inverted sequences.

23 In the exemplified case, DNA derived from potato GLTP and GHTP genes is used to 24 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 26 GLTP gene or GHTP gene. Agrobacterium tumefaciens is used as a vehicle for transmission 27 of the DNA to stem explants of potato plant shoots. A plant regenerated from the 28 transformed explants transcribes the antisense DNA which inhibits activity of the enzyme.

29 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 31 Sambrook et al. (1989). Generally, enzymatic reactions involving DNA ligase, DNA WO 98/35051 PCT/CA98/00055 1 polymerase, restriction endonucleases and the like are performed according to the 2 manufacturer's specifications.

3 4 5 Preparation of GHTP and GLTP cDNA cDNA is prepared from isolated potato tuber mRNA by reverse transcription. A 6 primer is annealed to the mRNA, providing a free 3' end that can be used for extension by the 7 enzyme reverse transcriptase. The enzyme engages in the usual elongation, as directed 8 by complementary base pairing with the mRNA template to form a hybrid molecule, 9 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 11 complementary DNA strand to convert the single-stranded cDNA into a duplex DNA.

12 After DNA amplification, the double stranded cDNA is inserted into a vector for 13 propagation in E. coli. Typically, identification of clones harbouring the desired cDNA's 14 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 16 simplified in that the DNA sequences of the GLTP and GHTP genes are known, as are the 17 sequences of suitable primers (Brisson et al., 1990; Fukui et al., 1991). The primers used 18 hybridize within the GLTP and GHTP genes. Thus, it is expected that the amplified cDNA's 19 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 21 color selection. Appropriate E. coli strains transformed with plasmids which do not carry 22 inserts grow as blue colonies. Strains transformed with pBluescript plasmids carrying inserts 23 grow as white colonies. Plasmids isolated from transformed E. coli were sequenced to 24 confirm the sequence of the phosphorylase inserts.

26 6 Vector Construction 27 The cDNAs prepared can be inserted in the antisense or sense orientation into 28 expression cassette in expression vectors for transformation of potato plants to inhibit the 29 expression of the GLTP and/or GHTP genes in potato tubers.

As in the exemplified case, which involves antisense suppression, the desired 31 recombinant vector will comprise an expression cassette designed for initiating transcription WO 98/35051 PCT/CA98/00055 1 of the antisense cDNAs in plants. Additional sequences are included to allow the vector to be 2 cloned in a bacterial or phage host.

3 The vector will preferably contain a prokaryote origin of replication having a broad 4 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 6 antibiotics such ampicillin.

7 Other DNA sequences encoding additional functions may also be present in the 8 vector, as is known in the art. For instance, in the case of Agrobacterium transformations, 9 T-DNA sequences will also be included for subsequent transfer to plant chromosomes.

For expression in plants, the recombinant expression cassette will contain in 11 addition to the desired sequence, a plant promoter region, a transcription initiation site (if 12 the sequence to be transcribed lacks one), and a transcription termination sequence. Unique 13 restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for 14 easy insertion into a pre-existing vector. Sequences controlling eukaryotic gene expression are well known in the art.

16 Transcription of DNA into mRNA is regulated by a region of DNA referred to as the 17 promoter. The promoter region contains sequence of bases that signals RNA polymerase to 18 associate with the DNA, and to initiate the transcription of mRNA using one of the DNA 19 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 21 20 to 30 base pairs (bp) upstream (by convention -30 to -20 bp relative to the transcription 22 start site) of the transcription start site. In most instances the TATA box is required for 23 accurate transcription initiation. The TATA box is the only upstream promoter element that 24 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 26 that vary considerably from the start point and in either orientation.

27 Another common promoter element is the GC box at -90 which contains the 28 consensus sequence GGGCGG. It may occur in multiple copies and in either orientation.

29 Other sequences conferring tissue specificity, response to environmental signals, or maximum efficiency of transcription may also be found in the promoter region. Such 31 sequences are often found within 400 bp of transcription initiation size, but may extend as far WO 98/35051 PCT/CA98/00055 1 as 2000 bp or more. In heterologous promoter/structural gene combinations, the promoter is 2 preferably positioned about the same distance from the heterologous transcription start site as 3 it is from the transcription start site in its natural setting. However, some variation in this 4 distance can be accommodated without loss of promoter function.

The particular promoter used in the expression cassette is not critical to the 6 invention. Any of a number of promoters which direct transcription in plant cells is suitable.

7 The promoter can be either constitutive or inducible.

8 A number of promoters which are active in plant cells have been described in the 9 literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumour-inducing plasmids of Agrobacterium tumefaciens), 11 the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S and 12 the figwort mosaic virus 3 5 S-promoters, the light-inducible promoter from the small subunit 13 of ribulose-l,5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), 14 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., 16 PCT W08402913.

17 The CaMV 35S promoter used in the Examples herein, has been shown to be highly 18 active and constitutively expressed in most tissues (Bevan et al., 1986). A number of other 19 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 21 contemplated to be useful in this invention include those that show enhanced or specific 22 expression in potato tubers, that are promoters normally associated with the expression of 23 starch biosynthetic or modification enzyme genes, or that show different patterns of 24 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 26 starch synthases, the branching enzymes (Blennow et al., 1991; WO 9214827; WO 9211375), 27 disproportionating enzyme (Takaha et al., 1993) debranching enzymes, amylases, starch 28 phosphorylases (Nakano et al., 1989; Mori et al., 1991), pectin esterases (Ebbelaar et al., 29 1993), the 40 kD glycoprotein; ubiquitin, aspartic proteinase inhibitor (Stukerlj et al., 1990), the carboxypeptidase inhibitor, tuber polyphenol oxidases (Shahar et al., 1992; GenBank 31 Accession Numbers M95196 and M95197), putative trypsin inhibitor and other tuber cDNAs WO 98/35051 PCTICA98/00055 1 (Stiekema et al., 1988), and for amylases and sporamins (Yoshida et al., 1992; Ohta et al., 2 1991).

3 In addition to a promoter sequence, the expression cassette should also contain a 4 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 6 sequence or may be obtained from different genes. In the exemplified case the nopaline 7 synthase NOS 3' terminator sequence (Bevan et al. 1983) was used.

8 Polyadenylation sequences are also commonly added to the vector construct if the 9 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 11 sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen 12 et al., 1984) or the nopaline synthase signal (Depicker et al., 1982).

13 The vector will also typically contain a selectable marker gene by which transformed 14 plant cells can be identified in culture. Typically, the marker gene encodes antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, 16 and gentamycin. In the exemplified case, the marker gene confers resistance to kanamycin.

17 After transforming the plant cells, those cells containing the vector will be identified by their 18 ability to grow in a medium containing the particular antibiotic.

19 7 Transformation of Plant Cells 21 Although in the exemplified case potato plant shoot stem explants were transformed 22 via inoculation with Agrobacterium tumefaciens carrying the antisense sequence linked to a 23 binary vector, direct transformation techniques which are known in the art can also be used to 24 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 26 penetration by small particles having the nucleic acid of interest embedded within the matrix 27 of the particles or on the surface. Fusion of protoplasts with lipid-surfaced bodies such as 28 minicells, cells or lysosomes carrying the DNA of interest can be used. The DNA may also 29 be introduced into plant cells by electroporation, wherein plant protoplasts are electroporated in the presence of plasmids carrying the expression cassette.

-WO 98/35051 PCT/CA98/00055 1 In contrast to direct transformation methods, the exemplified case uses vectored 2 transformation using Agrobacterium tumefaciens. Agrobacterium tumefaciens is a Gram- 3 negative soil bacteria which causes a neoplastic disease known as crown gall in 4 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 6 are used as a source of carbon and/or nitrogen by the Agrobacteria.

7 The bacterium does not enter the plant cell, but transfers only part of the Ti plasmid, a 8 portion called T-DNA, which is stably integrated into the plant genome, where it expresses 9 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- 11 DNA. The vir region, however, is not transferred. In fact, the vir region, although required 12 for T-DNA transfer, need not be physically linked to the T-DNA and may be provided on a 13 separate plasmid.

14 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 16 cells may be produced which have lost all properties of tumour cells, but still harbour and 17 express certain parts of T-DNA, particularly the T-DNA border regions. Therefore, modified 18 Ti plasmids, in which the disease causing genes have been deleted, may be used as vectors for 19 the transfer of the sense and antisense gene constructs of the present invention into potato plants (see generally Winnacker, 1987).

21 Transformation of plants cells with Agrobacterium and regeneration of whole plants 22 typically involves either co-cultivation of Agrobacterium with cultured isolated protoplasts or 23 transformation of intact cells or tissues with Agrobacterium. In the exemplified case, stem 24 explants from potato shoot cultures are transformed with Agrobacterium.

Alternatively, cauliflower mosaic virus (CaMV) may be used as a vector for 26 introducing sense or antisense DNA into plants of the Solanaceae family. For instance, 27 United States Patent No. 4,407,956 (Howell, October 4, 1983) teaches the use of cauliflower 28 mosaic virus DNA as a plant vehicle.

29 WO 98/35051 PCT/CA98/00055 1 8 Selection and Regeneration of Transformed Plant Cells 2 After transformation, transformed plant cells or plants carrying the antisense or sense 3 DNA must be identified. A selectable marker, such as antibiotic resistance, is typically used.

4 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 6 the art. For instance, the presence of opines can be used to identify transformants if the plants 7 are transformed with Agrobacterium.

8 Expression of the foreign DNA can be confirmed by detection of RNA encoded by the 9 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 11 reaction, as well (See, generally, Sambrook et al. (1989)).

12 Generally, after it is determined that the transformed plant cells carry the recombinant 13 DNA, whole plants are regenerated. In the exemplified case, stem and leaf explants of potato 14 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 16 kanamycin-containing growth medium. After transfer to a suitable medium for shoot 17 induction, shoots were transferred to a medium suitable for rooting. Plants were then 18 transferred to soil and hardened off. The plants regenerated in culture were transplanted and 19 grown to maturity under greenhouse conditions.

21 9 Analysis of GHTP and GLTP Activity Levels in Transformed Tubers 22 Following regeneration of potato plants transformed with antisense DNA sequences 23 derived from the GHTP and GLTP genes, the biochemistry of transformed tuber tissue was 24 analyzed several ways. The in vitro activity of a glucan phosphorylase in the phosphorolytic direction was assayed according to the methods of Steup (1990) (Table The activity of the 26 enzyme in the synthetic direction and the amount of enzyme protein were compared after 27 electrophoretic separation of the enzyme isoforms on a glycogen-containing, polyacrylamide 28 gel (Figure Starch synthesis by the tuber L-type and H-type isoforms was determined by 29 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 31 unincubated native gel to nitrocellulose and probing with polyclonal antibodies specific for 1 tuber type L and type H glucan phosphorylase isoforms. Levels of reducing sugars (glucose,' 2 and fructose) in tuber tissues were quantified by HPLC (Tables 2, 3 and The extent of 3 Maillard reaction, which is proportional to the concentration of reducing sugars in tubers was 4 examined by determining chip scores after frying (Table 5 and Figure 9).

6 10 Definitions 7 As used herein and in the claims, the term: 8 "about three months", "about four months" and "about six months" refer, respectively, 9 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; 11 "antisense orientation" refers to the orientation of nucleic acid sequence from a 12 structural gene that is inserted in an expression cassette in an inverted manner with respect to 13 its naturally occurring orientation. When the sequence is double stranded, the strand that is 14 the template strand in the naturally occurring orientation becomes the coding strand, and vice versa; 16 "chip score" of a tuber means the reflectance measurement recorded by an Agtron 17 model E-15-FP Direct Reading Abridged Spectrophotometer (Agtron Inc. 1095 Spice Island 18 Drive #100, Sparks Nevada 89431) of a center cut potato chip fried at 205 F in soybean oil 19 for approximately 3 minutes until bubbling stops; "cold storage" or "storage at reduced temperature" or variants thereof, shall mean 21 holding at temperatures less than 10 0 C, that may be achieved by refrigeration or ambient 22 temperatures; 23 "endogenous", as it is used with reference to a glucan phosphorylase genes of a potato 24 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 26 ct glucan phosphorylase gene; 27 "expression" refers to the transcription and translation of a structural gene so that a 28 protein is synthesized; 29 "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 31 form; MNE 21 -4 AMENDED SHEET WO 98/35051 PCTCA98/00055 1 "improved cold-storage characteristics" includes, without limitation, improvements in 2 chip score and reduction in sugar accumulation in tubers measured at harvest or after a period 3 of storage below 10°C, and further includes improvements, advantages and benefits which 4 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 6 reduced respiration and sprouting of potatoes, and reduced incidence of disease. Unless 7 further qualified by a specific measure or test, an improvement in a cold-storage characteristic 8 refers to a difference in the described characteristic relative to that in a control, wildtype or 9 unmodified potato plant; "modified" or variants thereof, when used to describe potato plants or tubers, is used 11 to distinguish a potato plant or tuber that has been altered from its naturally occurring state 12 through: the introduction of a nucleotide sequence from the same or a different species, 13 whether in a sense or antisense orientation, whether by recombinant DNA technology or by 14 traditional cross-breeding methods including the introduction of modified structural or regulatory sequences; modification of a native nucleotide sequence by site-directed 16 mutagenesis or otherwise; or the treatment of the potato plant with chemical or protein 17 inhibitors. An "unmodified" potato plant or tuber means a control, wildtype or naturally 18 occurring potato plant or tuber that has not been modified as described above; 19 "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 21 includes both self-replicating plasmids, infectious polymers of DNA or RNA and non- 22 functional DNA or RNA; 23 "operably linked" refers to functional linkage between a promoter and a second 24 sequence, wherein the promoter sequence initiates transcription of RNA corresponding to the second sequence; 26 "plant includes whole plants, plant organs leaves, stems, roots, etc.) seeds and 27 plant cells; 28 "promoter" refers to a region of DNA upstream from the structural gene and involved 29 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; WO 98/35051 PCT/CA98/00055 1 "reduced activity" or variants thereof, when used in reference to the level of GLTP or 2 GHTP enzyme activity in a potato tuber includes reduction of GLTP or GHTP enzyme 3 activity resulting from reduced expression of the GLTP or GHTP gene product, reduced 4 substrate affinity of the GLTP or GHTP enzyme, and reduced catalytic activity of the GLTP or GHTP enzyme; 6 "reduced" or variants thereof, may be used herein with reference to, without 7 limitation, activity levels of GLTP or GHTP enzyme in potato tubers, accumulation of sugars 8 in potato tubers and darkening of potato chips upon frying. Unless further qualified by a 9 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, 11 wildtype or unmodified potato plant; 12 "stress" or variants thereof, when used in relation to stresses experienced by potato 13 plants and tubers, includes the effects of environment, fertility, moisture, temperature, 14 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 16 stages of the growth and development cycle and during subsequent harvesting, transport, 17 storage and processing; 18 "stress resistance" or variants thereof, shall mean reduced effects of temperature, 19 aging, disease, atmosphere, physical handling, moisture, chemical residues, environment, pests and other stresses; 21 "suitable host" refers to a microorganism or cell that is compatible with a recombinant 22 plasmid, DNA sequence or recombinant expression cassette and will permit the plasmid to 23 replicate, to be incorporated into its genome, or to be expressed; and 24 "uninterrupted" refers to a DNA sequence cDNA) containing an open reading frame that lacks intervening, untranslated sequences.

26 27 EXAMPLE 1 28 This example describes the reduction of GHTP and/or GLTP activity in tubers of 29 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 31 antisense orientation.

WO 98/35051 PCT/CA98/00055 1 A Isolation of Potato Tuber mRNA 2 Potato total RNA was purified at 4°C using autoclaved reagents from Ig of tuber 3 tissue ground to a fine powder under liquid nitrogen with a mortar and pestle. The powder 4 was transferred to a 30ml corex tube and 3 volumes were added of 100 mM Tris-Cl, pH 100 mM NaCI, and 10 mM EDTA (10x TNE) containing 0.2% SDS and 0.5% 2- 6 mercaptoethanol. An equal volume of phenol-chloroform was added and the sample 7 gently vortexed before centrifugation at 4 OC in a SS34 rotor at 8,000 rpm for 5 min. The 8 organic phase was reextracted with 0.5 volume of 10x TNE containing 0.2% (w/v)SDS and 9 0.5% 2 -mercaptoethanol and the combined aqueous phases were extracted with chloroform. Nucleic acids were precipitated from the aqueous phase with sodium acetate and 11 absolute ethanol, pelleted by centrifugation, and resuspended in 3 ml of lx TNE. An equal 12 volume of 5 M LiC1 was added and the sample stored at -20°C for at 4 h before centrifuging 13 at 8,000 rpm in a SS34 rotor at 4'C for 10 min. The RNA pellet was washed with 14 ethanol, dried, and resuspended in DEPC-treated water.

Poly RNA was isolated using oligo (dT) cellulose (Boehringer Mannheim) 16 column chromatography. Poly RNA was isolated from total RNA resuspended in 17 RNAse free water. Columns were prepared using an autoclaved Bio-Rad Poly-Prep 10 ml 18 column to which was added 50 mg of oligo (dT) cellulose suspended in 1 ml of loading buffer 19 B which contains 20 mM Tris-Cl, pH 7.4, 0.1 M NaCI, 1 mM EDTA, and 0.1%

SDS.

The column was washed with 3 volumes of 0.1 M NaOH with 5 mM EDTA and then DEPC- 21 treated water until the pH of effluent was less than 8, as determined with pH paper. The 22 column was then washed with 5 volumes of loading buffer A containing 40 mM Tris-Cl, pH 23 7.4, 1 M NaCI, 1 mM EDTA, and 0.1%

SDS.

24 RNA samples were heated to 65 C for 5 min at which time 400 1l of loading buffer A, prewarmed to 65 C, was added. The sample was mixed and allowed to cool at room 26 temperature for 2 min before application to the column. Eluate was collected, heated to 65 0

C

27 for 5 min, cooled to room temperature for 2 min, and reapplied to the column. This was 28 followed by a 5 volume washing with loading buffer A followed by a 4 volume wash with 29 loading buffer B. Poly RNA was eluted with 3 volumes of 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, and 0.05% SDS. Fractions were collected and those containing RNA were 31 identified in an ethidium bromide plate assay, a petri dish with 1% agarose made with TAE WO 98/35051 PCTICA98/00055 1 containing EtBr. RNA was precipitated, resuspended in 10 c1, and a 1 p1 aliquot quantitated 2 with a spectrophotometer.

3 4 B Isolation of GLTP and GHTP DNA Sequences The nucleotide sequences utilized in the development of the antisense construct were 6 randomly selected from the 5' sequence of GLTP (SEQ ID NO: 1) and GHTP (SEQ ID NO: 7 DNA sequences used to develop the antisense constructs were obtained using reverse 8 transcription-polymerase chain reaction. GLTP (SPL1 and SPL2)- and GHTP (SPH1 and 9 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: 11 SPL1 Primer: 5'ATTCGAAAAGCTCGAGATTTGCATAGA3' (SEQ ID NO: 7) (additional 12 CG creates Xho I site); 13 SPL2 Primer: 5'GTGTGCTCTCGAGCATTGAAAGC3' (SEQ ID NO: 8) (changed C to G to 14 create Xho I site); SPH1 Primer: 5'GTTTATTTTCCATCGATGGAAGGTGGTG3' (SEQ ID NO: 9) (added 16 CGAT to create Cla I site); 17 SPH2 Primer: 5'ATAATATCCTGAATCGATGCACTGC3' (SEQ ID NO: 10) (changed G to 18 T to create Cla I site).

19 Reverse transcription was performed in a volume of 15 pl containing 1 x PCR buffer (10 mM Tris-Cl pH 8.2,50 mM KC1, 0.001% gelatin, 1.5 mM MgClI), 670 pM of each 21 dNTP, 6 pg of total potato tuber cv. Russet Burbank RNA, 1 mM each primer (SPH 1 and 22 SPL2, or SPH1 and SPH2) and 200 U of Maloney murine leukemia virus reverse 23 transcriptase (BRL). The reaction was set at 37°C for 30 minutes, then heat-killed at 94 0 C for 24 5 minutes and snap cooled on ice. To the reverse transcription reaction was added 2.5 U Taq DNA polymerase (BRL) in 35 pl of 1 x PCR buffer. DNA amplification was done in a Perkin 26 Elmer 480 programmed for 30 cycles with a 1 min 94°C denaturation step, a 1 min 56 °C 27 (SPL1 and SPL2) or 58 0 C (SPH1 and SPH2) annealing step, and a 2 min 72°C extension 28 step. PCR was completed with a final 10 min extension at 72 0

C.

29 -WO 98/35051 PCTCA98/00055 1 C Construction of SP Vectors for Phosphorylase Inhibition 2 To express the antisense constructs in plant cells, it was necessary to fuse the genes to 3 the proper plant regulatory regions. This was accomplished by cloning the antisense DNA 4 into a plasmid vector that contained the needed sequences.

Amplified DNA was blunt ended and cloned into a pUC19 vector at the Smal site.

6 The recombinant plasmid was transformed into sub-cloning efficiency E. coli DH5a cells 7 (BRL). The transformed cells were plated on LB (15 g/l Bactotyptone, 5 g/1 yeast extract, 8 g/1 NaC1, pH 7.3, and solidified with 1.5% agar) plates that contained ampicillin at 100 ug/ml.

9 Selection of bacteria containing plasmids with inserted plant phosphorylase sequence was accomplished using color selection. The polylinker and T3 and T7 RNA polymerase 11 promoter sequences are present in the N-terminal portion of the lacZ gene fragment. pUC19 12 plasmids without inserts in the polylinker grow as blue colonies in appropriate bacterial 13 strains such as DH5a. Color selection was made by spreading 100 pl of 2% X-gal (prepared 14 in dimethyl formamide) on LB plates containing 50 pg/ml ampicillin 30 minutes prior to plating the transformants. Colonies containing plasmids without inserts will be blue after 16 incubation for 12 to 18 hours at 37C and colonies with plasmids containing inserts will 17 remain white. An isolated plasmid was sequenced to confirm the sequence of the 18 phosphorylase inserts. Sequences were determined using the ABI Prism Dye Terminator 19 Cycle Sequencing Core Kit (Applied Biosystems, Foster City, CA), M13 universal and reverse primers, and an ABI automated DNA sequencer. The engineered plasmid was 21 purified by the rapid alkaline extraction procedure from a 5 ml overnight culture (Birboim 22 and Doly, 1979). Orientation of the SPL and SPH fragments in pUC19 was determined by 23 restriction enzyme digestion. The recombinant pUC19 vectors and the binary vector pBI121 24 (Clonetech) were restricted, run on a agarose gel and the fragments purified by gel separation as described by Thuring et al (1975).

26 Ligation fused the antisense sequence to the binary vector pBI121. The ligation 27 contained pBI121 vector that had been digested with BamHI and SacI, along with the SPL or 28 SPH phosphorylase DNA product, that had been cut from the pUC 19 subclone with BamHI 29 and SacI. Ligated DNA was transformed into SCE E. coli DH5a cells, and the transformed 3 0 cells were plated on LB plates containing ampicillin. The nucleotide sequences of the 31 antisense DNA SPL and SPH are nucleotides 338 to 993 of SEQ ID NO: 1 and nucleotides WO 98/35051 PCT/CA98/00055 1 147 to 799 of SEQ ID NO: 3, respectively. Selection of pBIl21 with phosphorlylase inserts 2 was done with CAMV and NOS specific primers.

3 Samples 1 and 2 representing the tuber L-type and tuber H-type phosphorylase

DNA

4 fragments were picked from a plate after overnight growth. These samples were inoculated into 5 ml of LB media and grown overnight at 37 0 C. Plasmids were isolated by the rapid 6 alkaline extraction procedure, and the DNA was electroporated into Agrobacterium 7 tumefaciens.

8 Constructs were engineered into the pBI121 vector that contains the CaMV 9 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 11 fragment isolated from transposon Tn7 which encodes bacterial spectinomycin/streptomycin 12 (Spc/Str) resistance and is a determinant for selection in E. coli and Agrobacterium 13 tumefaciens (Fling et al., 1985). This is joined to a chimeric kanamycin resistance gene 14 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., 16 1985), the 0.83 kb neomycin phosphotransferase type II gene (NPTII), and the 026 kb 3' non- 17 translated region of the nopaline synthase gene (NOS (Fraley et al., 1983). The next 18 segment is a 0.75 kb origin of replication from the RK2 plasmid (ori-V) (Stalker et al., 1981).

19 It is joined to a 3.1 kb Sal 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 21 in the Agrobacterium tumefaciens cells. Next is a 0.36 kb Pvul fragment from the pTiT37 22 plasmid which contains the nopaline-type T-DNA right border region (Fraley et al., 1985).

23 The antisense sequence was engineered for expression in the tuber by placing the gene under 24 the control of a constitutive tissue non-specific promoter.

26 D Plant Transformation/Regeneration 27 The SPL and SPH vectors were transformed into the Desiree potato cultivar according 28 to de Block (1988). To transform "Desiree" potatoes, sterile shoot cultures of "Desiree" were 29 maintained in test tubes containing 8 ml of S (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 31 plantlets reached approximately 5 cm in length, leaf pieces were excised with a single cut WO 98/35051 PCT/CA98/00055 1 along the base and inoculated with a 1:10 dilution of an overnight culture of Agrobacterium 2 tumefaciens. The stem explants were co-cultured for 2 days at 20 0 C on S medium (De 3 Block 1988). Following co-culture, the explants were transferred to S4 medium (MS medium 4 without sucrose, supplemented with 0.5 g/1 MES pH 5.7, 200 mg/1 glutamine, 0.5 g/1 PVP, g/1 mannitol, 20 g/1 glucose, 40 mg/1 adenine, 1 mg/l trans zeatin, 0.1 mg/1 NAA, 1 g/1 6 carbenicillin, 50 mg/l kanamycin, solidified with 6 g/1 phytagar) for 1 week and then 2 weeks 7 to induce callus formation.

8 After 3 weeks, the explants were transferred to S6 medium (S4 without NAA and with 9 half the concentration (500 mg/l) 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 11 acid, GA3) to promote shoot formation. Shoots began to develop approximately 2 weeks 12 after transfer to S8 shoot induction medium. These shoots were excised and transferred to 13 vials of S medium for rooting. After about 6 weeks of multiplication on the rooting 14 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 16 grown to maturity under greenhouse conditions. Tubers were harvested and allowed to 17 suberize at room temperature for two days. All tubers greater than 2 cm in length were 18 collected and stored at 4 0 C under high humidity.

19 E Field Trials 21 Untransformed controls, plants expressing the SPL construct, and plants expressing 22 the SPH construct were propagated in field trials in a single replicate randomized design. All 23 plants were grown side by side in the same field and exposed to similar pesticide, fertilizer, 24 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.

26 27 F Sugar Analysis 28 Tubers were stored at 4°C and were not allowed to recondition at room temperature 29 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 31 representing all of the tuber's tissues. At each harvest, the central slices from four tubers per WO 98/35051 PCT/CA98/00055 1 clone (3 replicates) were collectively diced into 1-cm cubes and 15 g was randomly selected 2 from the pooled tissue foranalysis. Glucan phosphorylase (see below) and sugars were 3 extracted with 15 mL of Tris buffer (50 mM, pH 7.0) containing 2 mM sodium bisulfite, 2 4 mM EDTA. 0.5 mM PMSF and 10% glycerol with a polytron homogenizer at 4 0

C.

The extracts were centrifuged at 4'C (30,000 g, 30 min) and reducing sugars (glucose and 6 fructose) were measured on a 10-fold dilution of the supernatant using a Spectra Physics high 7 performance liquid chromatograph interfaced to a refractive index detector. The separation 8 was performed at 80 0 C on a 30 x 0.78 cm Aminex HPX 87C column (Biorad) using 0.6 9 ml/min water as the mobile phase. Calibration of the instrument was via authentic standards of d-glucose and d-fructose.

11 12 G Analysis of ct-Glucan Phosphorylase Activity 13 Tubers stored at 4 0 C were not allowed to warm prior to extraction and analysis of a 14 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 16 extracts obtained for sugar analysis (see above) were added to a reaction medium which 17 coupled starch phosphorolysis to the reduction of NADP through the sequential actions of 18 phosphoglucomutase and glucose-6-phosphate dehydrogenase. The rate of reduction of 19 NADP during the reaction is stoichiometric with the rate of production of glucose- 1phosphate from the starch substrate. Reduction of NADP was followed at 340 nm in a Varian 21 Cary double-beam spectrophotometer. Protein levels in extracts were determined according 22 to Bradford (1976).

23 Glucan phosphorylase activity gels were run essentially according to Steup (1990).

24 Proteins were separated on native polyacrylamide gels (8.5 containing 1.5 glycogen.

Following electrophoresis at 80 V for 15 h the gels were incubated (1-2 h) at 37°C in 26 0.1 M citrate-NaOH buffer (pH 6.0) containing 20 mM glucose-1-P and 0.05% (w/v) 27 hydrolyzed potato starch. Gels were then rinsed and stained with an iodine solution.

28 For Western blot analysis, proteins were electrophoresed on glycogen-containing 29 polyacrylamide gels as described above. The proteins were electroblotted to nitrocellulose and blots were probed with polyclonal antibodies raised against GHTP and GLTP.

1 Immunoblots were developed with alkaline phosphata e conjugated anti-rabbit secondary 2 antibodies (Sigma).

3 4 H Chip Color Determination Five transgenic potato lines expressing the GLTP antisense sequence, two transgenic 6 lines expressing the GHTP antisense sequence, non-transgenic Desiree control lines, and two 7 control lines transformed with the pB1121 vector T-DNA, were grown under field conditions 8 in Alberta, Canada. Tubers were harvested and stored at 10"C and 4°C. Chip color was 9 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.

11 12 I Results 13 All tubers were harvested from plants of the same cultivar (Desiree), the same age, 14 and grown side by side under identical growth conditions. Northern analysis of tubers showed a considerable reduction of endogenous GLTP transcript in transgenic plants 16 expressing the homologous antisense transcript (Figure Glucan phosphorylase assays 17 showed that activities (pimol NADPH mg" protein were reduced (Table 1) at harvest and 18 for at least six months following harvest in transgenic plants expressing the GLTP antisense 19 DNA. The results tabulated in Table I show that a glucan phosphorylase activity in tubers stored at 4°C for 189 days was reduced from approximately 16% to 70% in various 21 transformed potato varieties relative to the wildtype control strain. Activity gels and western 22 blot analysis showed specific reduced expression of homologous enzymes and lower 23 reduction of expression for heterologous enzymes (Figures 10 and 11). This specificity for 24 homologous products may result from differences between the phosphorylases (Figures 6A and 6B).

2 6 Analysis of tubers at harvest (0 days) shows that those expressing the antisense GLTP 27 transcript have up to 5-fold less reducing sugars than control tubers (Table Furthermore, 28 after 91 days storage at 4 0 C, transformed tubers contained 28-39% lower reducing sugar 29 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).

31 These results suggest that reduced GLTP activity slows the catabolism of starch into reducing AMENDED

SHEET

1 sugars in tubers, while in the control tubers the sugars continue to accumulate: The 2 correlation between total phosphorylase activity and the concentration of reducing sugars is 3 not direct, suggesting that certain isozymes of phosphorylase may play a more important role 4 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 6 unidentified interactions involved in the lower reducing sugar levels.

7 Transgenic potato plants expressing the antisense GLTP or GHTP transcript have 8 been grown under field conditions and their tubers stored at 4°C. Chip color, which 9 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 11 GLTP transcript was significantly improved (lighter) relative to that of control tubers (darker) 12 stored under identical conditions (Table 5 and Figure Chip scores of tubers from 13 "Desiree" potato plants expressing the GLTP transcript were improved by at least 4.3 points 14 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) 16 following storage at 10 0 C and 4 0 C, respectively, for 86 days. Chip scores of GLTP 17 transformants measured after 124 days of storage at 4 0 C were improved by 44% to 89% 18 relative to wildtype (Table 19 The Desiree cultivar is not a commercially desirable potato for chipping due to its 2 0 high natural sugar content and propensity to sweeten rapidly in cold storage. Nevertheless, 21 significant improvements in fried chip color were noted with the transformed "Desiree" 22 potatoes. It is expected that superior color lightening would be achieved if the methods of the 23 invention were applied to commercial processing potato varieties.

24 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 26 lower buildup of reducing sugars (Table Results showing heterologous and homologous 27 reduction in phosphorylase activity (Figures 10 and 11) indicate that the improvement may be 28 a result of reducing one or both tuber phosphorylases. However, these results suggest that the 29 L-type phosphorylase plays a more important role in the catabolism of starch into reducing sugars.

1Further, the results show that the difference at 1 reducing sugar levels (Table and 2 chip scores (Table 5) between tubers wildtype plants and those expressing tuber

RA(

1 31la WO 98/35051 PCT/CA98/00055 1 phosphorylase antisense RNA, are sustained during long-term storage. As shown in Table 2 the chip scores are approximately the same at 86 days and 124 days. No further increases in 3 reducing sugar concentrations were evident after 49 and 91 days storage at 4°C (Table 2).

4 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 6 extending the period of storage by at least several months. Presently, processing potatoes are 7 usually stored for a maximum of three to six months at 10°C to 12'C before the sugar 8 accumulation reaches levels that reduce quality. Fresh product must be imported until the 9 present season potatoes become available. Extending the storage period of potatoes by many months may reduce import costs.

11 Table 6 provides a summary of the percentage improvement in various improved tuber 12 cold-storage characteristics of tubers of potato plants transformed with antisense DNA 13 derived from the GLTP gene sequence (ATL3 ATL9), and from the GHTP gene sequence 14 (ATHI 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 16 may be consistently obtained through the methods of the present invention. Particularly 17 noteworthy are the percentage chip score improvements over wildtype observed after storage 18 at 4 0 C for approximately four months (124 days). Relative chip score improvements of up to 19 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 21 temperatures, without causing unacceptable darkening of fried potato products.

22 The reduction in sugar accumulation of transformed potato lines relative to wildtype, 23 both at harvest and after 91 day storage, also demonstrates significant advantages of the 24 invention. Reduced sugar accumulation relates to the observed chip score improvements, and also reflects improved specific gravity of tubers, another important commercial measure of 26 tuber quality.

27 Even at harvest, substantial improvements in chip score and reduced sugar 28 accumulation were noted for transformed lines relative to wildtype. Thus, the benefits of the 29 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 31 only to improvements in cold-storage characteristics but encompasses improvements in tuber WO 98/35051 PCT/CA98/00055 1 quality characteristics such as chip score or sugar accumulation which are present at the time 2 of harvest, resulting in earlier maturity.

3 Turning to specific improvements summarized in Table 6, it can be seen that GLTP- 4 type transformants (ATL3 ATL9) exhibited up to a 66%, 70% and 69% reduction in a glucan phosphorylase activity relative to wildtype, at harvest, and after storage for 91 and 189 6 days, respectively. Most also exhibited improvements in excess of 10% and 30% relative to 7 wildtype at harvest and after storage for 91 and 189 days. After storage for 91 and 189 days, 8 the GHTP-type transformants (ATH1 and ATH2) exhibited, respectively, up to 28% and 39% 9 relative improvement over wildtype and generally showed at least 10% improvement.

The GLTP-type transformants exhibited up to 80% and 39% reduction of sugar 11 accumulation relative to wildtype at harvest and at 91 days, respectively. At harvest, all 12 GLTP-type transformants exhibited at least 10% and at least 30% relative improvement. At 13 91 days, all GLTP-type transformants exhibited at least 10% and most exhibited at least 14 relative improvement.

The GLTP-type transformants exhibited up to 46%, 89% and 89% chip score 16 improvement relative to wildtype at harvest, and after storage for 86 days and 124 days, 17 respectively. Almost all exhibited at least 10% and 30% relative improvement at harvest, and 18 after storage for 86 and 124 days. At least one of the GHTP-type transformants exhibited at 19 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 21 exhibited up to 25% relative improvement in chip score.

22 The results clearly demonstrate that substantial improvements in tuber cold-storage 23 characteristics may be readily obtained through the methods of the invention. Results will 24 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 26 important to note that despite the variability in the results amongst the various transformed 27 lines, there was little variation in the results amongst the samples within a single transformed 28 potato line (see footnotes to Tables 1 to Results are presented in Table 6 for all potato 29 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 31 characteristics such as increased chip score (lighter color on cooking) and reduced sugar WO 98/35051 PCT/CA98/00055 1 accumulation, and most show very substantial improvements. Given the large proportion of 2 positive transformants observed in the examples herein, it is expected that, using the cold- 3 storage characteristic testing procedures described in the examples, potato plants transformed 4 through the methods of the invention can be readily screened to identify transformed lines exhibiting significantly improved cold-storage characteristics. By applying the techniques 6 disclosed herein to commercially important potato varieties, it will be possible to readily 7 create and select transformants having significantly improved cold-storage characteristics.

8 Those transformants showing the greatest relative improvements over wildtype controls can 9 be used in the development of new commercial potato varieties.

WO 98/35051 PCT/CA98/00055 1 Table 1 2 3 Effects of an antisense transcript on glucan phosphorylase activity measured in enzyme 4 extracts from field grown "Desiree" tubers.

6 Glucan Phosphorylase Activity 7 Storage Period at 4C (days) 8 9 Clone 0 49 91 140 189 11 12 pmol NADPH mg- protein h' 13 Wta 10.50 11.83 9.94 11.90 13.04 14 ATL3 4.90 4.86 4.49 4.73 4.88 ATLA 11.45 7.17 8.09 11.32 10.99 16 ATL5 3.58 3.56 2.97 4.59 4.79 17 ATL9 3.59 3.88 3.84 4.72 3.98 18 19 LSDo.osb 1.97 2.94 1.59 2.34 2.58 21 LSDo.o 0 2.87 4.28 2.31 3.41 3.75 22 23 Clonec 0.01d 24 WT vs. ATL's 0.01 Days

NS

26 Clone x Days 0.05 29 WT 11.49 8.90 12.66 13.66.

31 ATH-1 10.40 9.69 10.79 10.10 32 ATH-2 6.46 6.40 6.56 8.38 33 34

LSDO.

05 b 2.02 0.41 3.00

NS

36 LSDo.l 4.78 0.95 NS

NS

37 38 Clone' 0.01 39 WT vs. ATH's 0.01 Days 0.05 41 Clone x Days

NS

42 43 aWT, wild type untransformed tubers. bLSD, least significant difference at 0.05 or 0.01 level 44 for each storage period. cSources of variation in factorial analysis. dSignificance levels for indicated sources of variation.

WO 98/35051 -WO 9835051PCTICA98/00055 1 Table 2 2 3 Effects of an antisense GLTP transcript on low temperature induced sweetening of field 4 grown "Desiree" tubers.

6 Reducing Sugars (glucose fructose) 7 Storagye Period at 4C (days) 8 9 Clone 0 49 91 11 mg fresh weight 12 Wto 5.63 31.8 28.0 13 ATL3 1.88 17.3 17.3 14 ATM4 1.11 14.3 20.1 ATL5 1.51 18.3 17.0 16 ATL9 1.36 17.3 18.5 17 18 19 WT vs. ATL'sb 0.01 0.01 0.05 21 Clone'c.l 22 Days 0.01 23 Clone x Days

NS

24 26 wild type untransformed tubers. Orthogonal comparisons for ANO VA's at each 27 storage period. csources of variation in factorial analysis. 'Significance levels for indicated 28 sources of variation.

-WO 98/35051 PCTICA98/00055 Table 3 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 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 mg g' fresh weight Wta 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'sb 0.01 0.01

NS

Clone' 0.01d Days 0.01 Clone x Days

NS

"WT, wild type untransformed tubers. bOrthogonal comparisons for ANOVA's at each storage period. cSources of variation in factorial analysis. dSignificance levels for indicated sources of variation.

WO 98/35051 PCT/CA98/00055 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 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' fresh weight Wta 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'sb Clone' Days Clone x Days 0.01 0.01 0.05 0.01d 0.01

NS

"WT, wild type untransformed tubers. bOrthogonal comparisons for ANOVA's at each storage period. csources of variation in factorial analysis. dSignificance levels for indicated sources of variation.

WO 98/35051 PCT/CA98/00055 Table 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" Harvest 86 days 86 days 124 days 4C 4C Wtb ATL3c ATL4 ATL9 ATH 1d ATH2 GMP1l GMP2 25.3 37.4 43.7 29.6 38.7 49.7 31.2 15.4 26.7 29.1 24.7 24.3 17.5 15.6 15.7 16.7 17.1 30.8 32.3 24.6 26.6 21.4 15.9 15.7 16.6 aAtron Inc. 1095 Spice Island Drive #100, Sparks Nevada 89431. Agtron model E- (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.

bWT, negative control, wild type untransformed tubers.

CATL, tubers transformed with the tuber L-type- glucan phosphorylase.

dATH, tubers transformed with the tuber H-type- glucan phosphorylase.

eGMP, negative control, tubers transformed with pBI121 T-DNA.

WO 98/35051 PCT/CA98/00055 Table 6 Summary of Results Sample Reduction of a Reduction of Chip Score glucan phosphorylase Sugar Improvement relative to activity relative to Accumulation wildtype wildtype relative to wildtype at 91 189 at 91 at 86 124 harvest days days harvest days harvest days days ATL 3 53 55 63 67 38 -4 73 ATL 4 -9 19 16 80 28 35 89 89 ATL 5 66 70 63 73 39 38 60 44 ATL 9 66 61 69 76 34 46 58 56 ATH 1 n/a -9 26 n/a n/a 0 14 ATH 2 n/a 28 39 n/a n/a 12 1 -7 WO 98/35051 PCT/CA98/00055 1

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14 All publications mentioned in this specification are indicative of the level of skill in 16 the art to which this invention pertains. All publications are herein incorporated by reference 17 to the same extent as if each individual publication was specifically and individually indicated 18 to be incorporated by reference.

19 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 21 certain changes and modifications may be practised within the scope of the appended claims.

WO 98/35051 PCT/CA98/00055 SEQUENCE

LISTING

GENERAL

INFORMATION:

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: (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: McKay-Carey Company STREET: 2125 Commerce Place, 1 0155-102nd Street CITY: Edmonton STATE: Alberta COUNTRY: Canada ZIP: T5J 4G8 COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER:

WO

FILING DATE: 10-FEB-1998

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 60/036,946 FILING DATE: 10-FEB-1997 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 08/868,786 FILING DATE: 04-JUN-1997 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: McKay-Carey, Mary Jane REGISTRATION NUMBER: 3790 REFERENCE/DOCKET NUMBER: 24002WOO (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (403) 424-0222 TELEFAX: (403) 421-0834 INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 3101 base pairs TYPE: nucleic acid WO 98/35051 WO 9835051PCTCA98OOO55 STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL

SOURCE:

ORGANISM: Solanum tuberosum (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 44. .2944 OTHER INFORMATION: /product= "Potato alpha-glucan L-type tuber phosphorylase-, (ix) FEATURE: NAME/KEY: mat-peptide LOCATION: 194. .2941 (ix) FEATURE: NAME/KEY: Sig-peptide LOCATION: 44. .193 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATCACTCTCA TTC!GAAAAGC TAGATTTGCA

TAGAGAGCAC

A.AA ATO GCG ACT GCA Met Ala Thr Ala AAT GGA GCA CAC TTG TTC Asn Gly Ala His Leu Phe AAC CAT Asn His -40 TAC AGC TCC Tyr Ser Ser

AAT

Asn TCC AGA TTC ATC Ser Arg Phe Ile

CAT

His TTC ACT TCT AGA Phe Thr Ser Arg

AAC

Asn -25 ACA AGC TCC AAA Thr Ser Ser Lys

TTG

Leu TTC CTT ACC AAA Phe Leu Thr Lys

ACO

Thr 103 151 199 TCC CAT TTT CGG Ser His Phe Arg

AGA

Arg CCC AAA CGC TGT Pro Lys Arg Cys CAT GTC AAC AAT His Val Asn Asn ACC TTG Thr Leu AGT GAG AAA ATT CAC CAT CCC Ser Glu Lys Ile His His Pro

ATT

Ile ACT GAA CAA GGT Thr Glu Gin Gly GAG AGC GAC Glu Ser Asp CTG AGT Leu Ser TCT TTT GCT CCT Ser Phe Ala Pro GCC GCA TCT ATT Ala Ala Ser Ile

ACC

Thr TCA AGT ATC AAA Ser Ser Ile Lys WO 98/35051 WO 9835051PCT/CA98/00055 TAC CAT GCA GAA TTC ACA CCT GTA TTC Tlyr His Ala Giu Ph-e Pro Val Phe TCT CCT Ser Pro GAA AGG TTT GAG Glu Arg Phe Glu CCT AAG GCA TTC TTT GCA ACA GCT CAA Pro Lys Ala Phe Phe Ala Thr Ala Gln GTT CGT GAT TCG Val Arg Asp Ser CTC CTT Leu Leu ATT AAT TG Ile Asn Trop CAA GCG TAC Gln Ala Tyr

AAT

Asn GCT ACG TAT GAT Ala Thr Tyr Asp

ATT

Ile 75 TAT GAA AAG CTG Tyr Glu Lys Leu AAC ATG AAG Asn Met Lys GCA TTG TTA Ala Leu Leu TAT CTA TCC ATG Tyr Leu Ser Met

GAA

Glu 90 TTT CTG CAG GGT Phe Leu Gin Gly

AGA

Arg AAT GCA Asn Ala 100 ATT GGT AAT CTG Ile Gly Asn Leu CTT ACT GGT GCA Leu Thr Gly Ala

TTT

Phe 110 GCG GAA GCT TTG Ala Glu Ala Leu 535 583

AAA

Lys 115 AAC CTT GGC CAC Asn Leu Gly His CTA GAA AAT GTG Leu Glu Asn Val

GCT

Ala 125 TCT CAG GAA CCA Ser Gin Oiu Pro OCT GCT CTT GGA AAT GGG GOT TTG GGA Ala Ala Leu Gly Asn Gly Gly Leu Gly CTT GCT TCC TGT Leu Ala Ser Cys TTT CTG Phe Leu GAC TCT TTG Asp Ser Leu TAC AAG TAT Tyr Lys Tyr 165

GCA

Ala 150 ACA CTA AAC TAC Thr Leu Asn Tyr

CCA

Pro 155 OCA TGG GGC TAT Ala Trp Giy Tyr OGA CTT AGG Gly Leu Arg 160 GGT CAG GAG Gly Gin Giu GGT TTA TTT AAG Gly Leu Phe Lys

CAA

Gin 170 CGG ATT A CA AAA Arg Ile Thr Lys

GAT

Asp 175 GAG GTG Giu Val 180 GCT GAA GAT TGG Ala Giu Asp Trp

CTT

Leu 185 GAA ATT GGC AGT Glu Ile Gly Ser

CCA

Pro 190 TGG GAA GTT GTG Trp, Giu Val Val 775 823

AGO

Arg 195 AAT GAT OTT TCA Asn Asp Val Ser CCT ATC AAA TTC Pro Ile Lys Phe

TAT

Tyr 205 GGA AAA GTC TCT Gly Lys Val Ser GGA TCA OAT GGA Gly Ser Asp Oly AGG TAT TGG ATT Arg Tyr Trp Ile OGA GAG GAT ATA Oiy Giu Asp Ile AAG OCA Lys Ala OTT GCO TAT Val Ala Tyr

GAT

Asp 230 OTT CCC ATA CCA Val Pro Ile Pro 000 Oly 235 TAT AAG ACC AGA Tyr Lys Thr Arg ACC ACA ATC Thr Thr Ile 240 TTT GAT TTA Phe Asp Leu 919 AGC CTT CGA CTG TGG TCT ACA CAG Ser Leu Arg Leu Trp Ser Thr Gin 245 250 OTT CCA TCA GCG Val Pro Ser Ala

GAT

Asp 255 -WO 98/35051 PCT/CA98/00055 TCT GCT Ser Ala 260 TTC AAT GCT GGA Phe Asn Ala Gly CAC ACC AAA GCA His Thr Lys Ala

TGT

Cys 270 GAA GCC CAA GCA Glu Ala Gin Ala

AAC

Asn 275 GCT GAG AAG ATA Ala Glu Lys Ile TAC ATA CTC TAC Tyr Ile Leu Tyr GGG GAT GAA TCA Gly Asp Glu Ser

GAG

Glu 290 1015 1063 1111 1159 GAG GGA AAG ATC Glu Gly Lys Ile

CTT

Leu 295 CGG TTG AAG CAA Arg Leu Lys Gin

CAA

Gin 300 TAT ACC TTA TGC Tyr Thr Leu Cys TCG GCT Ser Ala 305 TCT CTC CAA Ser Leu Gin ATT AAG TGG Ile Lys Trp 325 ATT ATT TCT CGA Ile Ile Ser Arg GAG AGG AGA TCA GGT GAT CGT Glu Arg Arg Ser Gly Asp Arg 320 GAA GAG TTT CCT Glu Glu Phe Pro

GAA

Glu 330 AAA GTT GCT GTG Lys Val Ala Val

CAG

Gin 335 ATG AAT GAC Met Asn Asp 1207 ACT CAC Thr His 340 CCT ACA CTT TGT Pro Thr Leu Cys CCT GAG CTG ATG Pro Glu Leu Met

AGA

Arg 350 ATA TTG ATA GAT Ile Leu Ile Asp 1255 1303

CTG

Leu 355 AAG GGC TTG AAT Lys Gly Leu Asn AAT GAA GCT TGG Asn Glu Ala Trp ATT ACT CAA AGA Ile Thr Gin Arg

ACT

Thr 370 GTG GCC TAC ACA Val Ala Tyr Thr

AAC

Asn 375 CAT ACT GTT TTG His Thr Val Leu

CCT

Pro 380 GAG GCA CTG GAG Glu Ala Leu Glu AAA TGG Lys Trp 385 1351 AGT TAT GAA Ser Tyr Glu GAG GCG ATT Glu Ala Ile 405 ATG CAG AAA CTC Met Gin Lys Leu

CTT

Leu 395 CCC AGA CAT GTC Pro Arg His Val GAA ATC ATT Glu Ile Ile 400 AAA TAT GGT Lys Tyr Gly 1399 1447 GAC GAG GAG CTG Asp Glu Glu Leu

GTA

Val 410 CAT GAA ATT GTA His Glu Ile Val

TTA

Leu 415 TCA ATG Ser Met 420 GAT CTG AAC AAA Asp Leu Asn Lys GAG GAA AAG TTG Glu Glu Lys Leu

ACT

Thr 430 ACA ATG AGA ATC Thr Met Arg Ile 1495 1543

TTA

Leu 435 GAA AAT TTT GAT Glu Asn Phe Asp CCC AGT TCT GTT Pro Ser Ser Val

GCT

Ala 445 GAA TTA TTT ATT Glu Leu Phe Ile

AAG

Lys 450 CCT GAA ATC TCA GTT GAT GAT GAT ACT Pro Glu Ile Ser Val Asp Asp Asp Thr 455 ACA GTA GAA GTC Thr Val Glu Val CAT GAC His Asp 465 1591 AAA GTT GAA Lys Val Glu

GCT

Ala 470 TCC GAT AAA GTT Ser Asp Lys Val

GTG

Val 475 ACT AAT GAT GAA Thr Asn Asp Glu GAT GAC ACT Asp Asp Thr 480 1639 -WO 98/35051 PCT/CA98/00055 GGT AAG AAA ACT AGT GTG AAG ATA GAA GCA GCT Gly Lys Lys Thr Ser Val Lys Ile Glu Ala Ala 485 490 GCA GAA Ala Glu 495 AAA GAC ATT Lys Asp Ile 1687 GAC AAG Asp Lys 500 AAA ACT CCC GTG Lys Thr Pro Val

AGT

Ser 505 CCG GAA CCA GCT GTT ATA CCA CCT AAG Pro Glu Pro Ala Val Ile Pro Pro Lys 510 1735

AAG

Lys 515 GTA CGC ATG GCC Val Arg Met Ala

AAC

Asn 520 TTG TGT GTT GTG Leu Cys Val Val

GGC

Gly 525 GGC CAT GCT GTT Gly His Ala Val

AAT

Asn 530 1783 GGA GTT GCT GAG Gly Val Ala Glu CAT AGT GAA ATT GTG AAG GAG GAG GTT His Ser Glu Ile Val Lys Glu Glu Val 540 TTC AAT Phe Asn 545 1831 GAC TTC TAT Asp Phe Tyr GTG ACT CCA Val Thr Pro 565

GAG

Glu 550 CTC TGG CCG GAA Leu Trp Pro Glu

AAG

Lys 555 TTC CAA AAC AAA Phe Gin Asn Lys ACA AAT GGA Thr Asn Gly 560 CTT AGT GCC Leu Ser Ala 1879 1927 AGA AGA TGG ATT Arg Arg Trp Ile

CGT

Arg 570 TTC TGC AAT CCT Phe Cys Asn Pro

CCT

Pro 575 ATC ATA Ile Ile 580 ACT AAG TGG ACT Thr Lys Trp Thr

GGT

Gly 585 ACA GAG GAT TGG Thr Glu Asp Trp

GTC

Val 590 CTG AAA ACT GAA Leu Lys Thr Glu

AAG

Lys 595 TTG GCA GAA TTG Leu Ala Glu Leu

CAG

Gin 600 AAG TTT GCT GAT Lys Phe Ala Asp

AAT

Asn 605 GAA GAT CTT CAA Glu Asp Leu Gin

AAT

Asn 610 1975 2023 2071 GAG TGG AGG GAA Glu Trp Arg Glu AAA AGG AGC AAC Lys Arg Ser Asn ATT AAA GTT GTC Ile Lys Val Val TCC TTT Ser Phe 625 CTC AAA GAA Leu Lys Glu ATT CAG GTA Ile Gin Val 645

AAG

Lys 630 ACA GGG TAT TCT Thr Gly Tyr Ser

GTT

Val 635 GTC CCA GAT GCA Val Pro Asp Ala ATG TTT GAT Met Phe Asp 640 TTA AAT ATC Leu Asn Ile 2119 2167 AAA CGC ATT CAT Lys Arg Ile His

GAG

Glu 650 TAC AAG CGA CAA Tyr Lys Arg Gin TTC GGC Phe Gly 660 ATC GTT TAT CGG Ile Val Tyr Arg

TAT

Tyr 665 AAG AAG ATG AAA Lys Lys Met Lys

GAA

Glu 670 ATG ACA GCT GCA Met Thr Ala Ala

GAA

Glu 675 AGA AAG ACT AAC Arg Lys Thr Asn

TTC

Phe 680 GTT CCT CGA GTA Val Pro Arg Val ATA TTT GGG GGA Ile Phe Gly Gly 2215 2263 2311 GCT TTT GCC Ala Phe Ala ACA TAT Thr Tyr 695 GTG CAA GCC AAG Val Gin Ala Lys ATT GTA AAA TTT Ile Val Lys Phe ATC ACA Ile Thr 705 -WO 98/35051 -WO 9835051PCT/CA98/00055 GAT CTT GGT Asp Val Gly AAG GTA GTC Lys Val Val 725

GCT

Ala 710 ACT ATA AAT CAT Thr Ile Asn His

GAT

Asp 715 CCA GAA ATC GGT Pro Glu Ile Gly GAT CTG TTG Asp Leu Leu 720 GAA TTG CTA Giu Leu Leu 2359 2 407 TTT GTG CCA GAT Phe Val Pro Asp AAT GTC ACT GTT Asn Val Ser Val

GCT

Ala 735 ATT CCT Ile Pro 740 GCT AGC CAT CTA Ala Ser Asp Leu

TCA

Ser 745 CAA CAT ATC ACT Giu His Ile Ser

ACG

Thr 750 GCT CCA ATC CAC Ala Cly Met Giu 2455 2503 CCC ACT GCA ACC ACT Ala Ser Cly Thr Ser 755

AAT

Asn 760 ATG AAC TTT CCA Met Lys Phe Ala

ATG

Met 765 AAT CGT TCT ATC Asn Cly Cys Ile

CAA

Gin 770 ATT GCT ACA TTG Ile Gly Thr Leu CCC CCT AAT CTT GAA ATA AGG GAA GAG Cly Ala Asn Val Clu Ile Arg Clu Glu 780 CTT CGA Val Gly 785 2551 CAA GAA AAC Ciu Giu Asn CTT AGA AAA Leu Arg Lys 805

TTC

Phe 790 TTT CTC TTT CCT Phe Leu Phe Gly

CCT

Ala 795 CAA CCT CAT CAA Gin Ala His Clu ATT CCA CCC Ile Ala Cly 800 CAA CCT TTT Ciu Arg Phe 2599 2647 GAA ACA CCT GAC Glu Arg Ala Asp

GCA

Cly 810 AAG TTT GTA CCT Lys Phe Val Pro

CAT

Asp 815 GAA CAG Giu Ciu 820 GTG AAC GAA TTT Val Lys Clu Phe

GTT

Val1 825 ACA ACC GCT CCT Arg Ser Gly Ala

TTT

Phe 830 CCC TCT TAT AAC Gly Ser Tyr Asn 2695 2743

TAT

Tyr 835 CAT CAC CTA ATT Asp Asp Leu Ile

GGA

C ly 840 TCC TTC CAA GCA Ser Leu Ciu Cly

AAT

Asn 845 CAA GCT TTT GC Clu Cly Phe Cly CCT CAC TAT TTC Ala Asp Tyr Phe GTG CCC AAG CAC Val Cly Lys Asp CCC AGT TAC ATA Pro Ser Tyr Ile CAA TC Ciu Cys 865 2791 CAA GAG AAA Gin Giu Lys ATG TCA ATC Met Ser Ile 885

GTT

Val 870 GAT GAG CCA TAT Asp Ciu Ala Tyr

CC

Arg 875 GAC CAG AAA AGG Asp Gin Lys Arg TCG ACA ACG Trp Thr Thr 880 ACT CAC AGA Ser Asp Arg 2839 2887 TTC AAT ACA C Leu Asn Thr Ala

GCA

C ly 890 TCC TAC AAG TTC Ser Tyr Lys Phe

AC

Ser 895 ACA ATC Thr Ile 900 CAT GAA TAT GCC His Giu Tyr Ala GAC ATT TGG AAC ATT GAA OCT CTG GAA Asp Ile Trp Asn Ile Ciu Ala Val Ciu 910 2935 ATA GCA TAA GAGGGGCAAG TGAATGAAAA ATAACAGG CACAGTAACT Ile Ala 915 2984 WO 98/35051 WO98/3051PCTICA98/00055 AGTTTCTCTT TTTATCATGT GATGAAGGTA TATAATGTAT GTGTAAGAGG ATGATGTTAT TACCACATAA TAAGAGATGA' AGAGTCTCAT TTTGCTTCAA A kAAAAAAJA, AAAAAAA INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 967 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2z 3044 3101 Met Ala Thr Ala Asn Gly Ala His Leu Phe Asn His Ser Leu Asn G ly Ser Arg Asp Leu Arg Ala Gln Arg Thr Asn Glu Ser Phe Ser Asn Ala Glu Glu Phe Lys Thr 1 Ser Ile Glu Leu Met Leu Al a Pro Ile His Thr Ser Leu Ser Asp Leu Lys Tyr Leu Pro Leu Ile Lys Gln Leu Asn Leu Lys 115 Asp Ala 130 -45 Phe His Giu Ser 20 His Lys Asn Ala Ala 100 Asn Ala Thr Phe Lys 5 Ser Ala Al a Trp Tyr 85 Ile Leu Leu Ser Arg Ile Phe Giu Phe Asn 70 Tyr Gly Gly Gly Arg Arg His Ala Phe Phe Ala Leu Asn His Asn Asn -25 Pro His Pro Thr 40 Ala Thr Ser Leu Asn 120 Gly -40 Thr Lys Pro Asp 25 Pro Thr Tyr Met Glu 105 Leu Gly Ser Arg Ile Ala Val Ala Asp Giu Leu Glu Leu Tyr Ser Cys Thr Ala Phe Gin Ile Phe Thr Asn Gly Pro 155 Ser Leu His Gin Ile Pro Val1 Giu Gin Ala Ala 125 Leu Asn Phe Val Gly Thr Giu Arg Lys Gly Phe 110 Ser Ala 135 Ser Cys Phe Leu Asp Ser Leu Ala Thr Leu Asn Tyr Ala Trp Gly WO 98/35051 WO 9835051PCTCA98OOO55 Tyr Asp 175 Trp Lys Asp Arg Asp 255 Giu Asp Leu Ser Gin 335 Ile Thr Leu Val Leu 415 Gly 160 Gly G lu Val Ile Thr 240 Phe Ala Glu Cys Gly 320 Met Leu Gin Giu Giu 400 Lys Leu Gin Val1 Ser Lys 225 Thr Asp Gin Ser Ser 305 Asp Asn le Arg Lys 385 Ilie Pyr Arg Tyr Lys Tyr Gly Leu Phe Lys Gin Arg Ile Thr Lys 165c 170 Giu Val1 Thr 210 Ala Ile Leu Ala Glu 290 Ala Arg Asp Asp Thr 370 Trp Ile G ly Giu Arg 195 Gly Val1 Ser Ser Asn 275 Giu Ser Ile Thr Leu 355 Val Ser Giu Ser Val1 180 Asn Ser Ala Leu Ala 260 Ala Gly Leu Lys His 340 Lys Al a Tyr PDia Mlet 420 Ala Asp Asp Tyr Arg 245 Phe Giu Lys Gin Trp 325 Pro Gly Tyr Giu Ile 405 Asp Giu Val1 Gly Asp 230 Leu Asn Lys Ile Asp 310 Giu Thr Leu Thr Leu 390 Asp Leu Asp Ser Lys 215 Val1 Trp Ala Ile Leu 295 Ile Giu Leu Asn Asn 375 Met Glu Asn Trp Tyr 200 Arg Pro Ser Gly Cys 280 Arg Ile Phe Cys Trp 360 His Gin Glu Lys Leu 18S Pro Tyr le Thr Glu 265 Tyr Leu Ser Pro Ile 345 Asn Thr Lys Leu Leu 425 Giu Ile Trp Pro Gin 250 His Ile Lys Arg Giu 330 Pro G-lu.

Val Leu2 Val 410 Glu Ile Lys Ile G ly 235 Val1 Thr L eu Gin Phe 315 Lys Giu Al.a Leu Leu 395 His5 Glu Giy Phe Gly 220 Tyr Pro Lys Tyr Gin 300 Giu Val1 Leu Trp Pro 380 Pro Giu Lys *Ser Tyr 205 Gly Lys Ser Ala Pro 285 Tyr Arg Ala Met As n 365 Giu Arg Ile Leu *Pro 190 Gly Giu Thr Ala Cys 270 Gly Thr Arg Vali Arg 350 Ile Aia His Val1 Thr 430 Thr Met Arg Ile Leu Giu Asn Phe Asp Leu Pro Ser Ser Val Ala Giu WO 98/35051 WO 9835051PCT/CA98/00055 Leu Phe Ile Lys Pro Giu Ile 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 Ile Glu Ala Ala Ala 480 485 490 Giu Lys Asp Ile Asp Lys Lys Thr Pro Val Ser Pro Glu Pro Ala Val 495 500 505 510 Ile 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 Giu Ile His Ser Glu Ile Val Lys Giu 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 Ile Arg Phe Cys Asn Pro 560 565 570 Pro Leu Ser Ala Ile Ile Thr Lys Trp, Thr Gly Thr Glu Asp Trp Val 575 580 585 590 Leu Lys Thr Giu Lys Leu Ala Glu Leu Gin Lys Phe Ala Asp Asn Giu 595 600 605 Asp Leu Gin Asn Giu Trp Arg Giu Ala Lys Arg Ser Asn Lys Ile Lys 610 615 620 Val Val Ser Phe Leu Lys Glu Lys Thr Gly Tyr Ser Val Vai Pro Asp 625 630 635 Ala Met Phe Asp Ile Gin Val Lys Arg Ile His Glu Tyr Lys Arg Gin 640 645 650 Leu Leu Asn Ilie Phe Giy Ile Val Tyr Arg Tyr Lys Lys Met Lys Glu 655 660 665- 670 Met Thr Ala Ala Giu Arg Lys Thr Asn Phe Val Pro Arg Val Cys Ile 675 680 685 Phe Gly Gly Lys Ala Phe Ala Thr Tyr Val Gin Ala Lys Arg Ile Val 690 695 700 Lys Phe Ile Thr Asp Val Gly Ala Thr Ile Asn His Asp Pro Glu Ile 705 710 715 Gly Asp Leu Leu Lys Val Vai Phe Val Pro Asp Tyr Asn Val Ser Val 720 725 730 WO 98/35051 PCT/CA98/00055 Ala Glu Leu Leu Ile Pro Ala Ser Asp Leu Ser Glu His Ile 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 Ile Gln Ile Gly Thr Leu Asp Gly Ala Asn Val Glu Ile Arg 770 775 780 Glu Glu Val Gly Glu Glu Asn Phe Phe Leu Phe Gly Ala Gin Ala His 785 790 795 Glu Ile 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 Ile 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 Ile Glu Cys Gin Glu Lys Val Asp Glu Ala Tyr Arg Asp Gin Lys 865 870 875 Arg Trp Thr Thr Met Ser Ile Leu Asn Thr Ala Gly Ser Tyr Lys Phe 880 885 890 Ser Ser Asp Arg Thr Ile His Glu Tyr Ala Lys Asp Ile Trp Asn Ile 895 900 905 910 Glu Ala Val Glu Ile Ala 915 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 2655 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum (ix) FEATURE: NAME/KEY: CDS WO 98/35051 PCT/CA98/00055 LOCATION: 12..2528 OTHER INFORMATION: /product= "potato alpha-glucan H-type tuber phosphorylase" (ix) FEATURE: NAME/KEY: matpeptide 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 Met Glu Gly Gly Ala Lys Ser Asn Asp Val Ser Ala Ala 1 5 CCT ATT GCT 'Pro Ile Ala CAA CCA CTT TCT GAA GAC CCT ACT GAC ATT GCA TCT AAT Gin Pro Leu Ser Glu Asp Pro Thr Asp Ile Ala Ser Asn ATC AAG TAT CAT GCT CAA TAT ACT Lys Tyr His Ala Gin Tyr Thr CCT CAT Pro His TTT TCT CCT TTC AAG TTT Phe 40 GAG CCA CTA CAA Glu Pro Leu Gin TAC TAT GCT GCT ACT GCT Tyr Tyr Ala Ala Thr Ala Ser Pro Phe Lys Phe GAC AGT GTT CGT GAT Asp Ser Val Arg Asp CGC TTG ATC Arg Leu Ile AAT CCA AAG Asn Pro Lys CAA TGG AAT GAC Gin Trp Asn Asp TAT CTT CAT TAT Tyr Leu His Tyr GAC AAA GTT Asp Lys Val CAG GGG CGA Gin Gly Arg CAA ACA TAC TAC Gin Thr Tyr Tyr

TTA

Leu 85 TCA ATG GAG TAT Ser Met Glu Tyr

CTC

Leu GCT TTG ACA AAT GCA GTT Ala Leu Thr Asn Ala Val

GGA

Gly 100 AAC TTA GAC ATC Asn Leu Asp Ile

CAC

His 105 AAT GCA TAT GCT Asn Ala Tyr Ala

GAT

Asp 110 GCT TTA AAC AAA Ala Leu Asn Lys

CTG

Leu 115 GGT CAG CAG CTT Gly Gin Gin Leu GAG GAG Glu Glu 120 GTC GTT GAG Val Val Glu GAA AAA GAT GCA GCA TTA GGA AAT GGT GGT TTA GGA AGG CTC Glu Lys Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu 1 -in GCT TCA Ala Ser 140 TGC TTT CTT GAT TCC ATG GCC ACA Cys Phe Leu Asp Ser Met Ala Thr 145 GGC TTG AGG TAC AGA TAT GGA CTT Gly Leu Arg Tyr Arg Tyr Gly Leu 160 Icc

TTG

Leu 150 AAC CTT CCA GCA TGG GGT TAT Asn Leu Pro Ala Trp Gly Tyr 434 482 530 TTT AAG CAG CTT ATC ACA AAG GCT Phe Lys Gin Leu Ile Thr Lys Ala WO 98/35051 WO 9835051PCT/CA98/00055 GGG CAA Gly Gin -175 GAA GAA GTT CCT Giu Glu Val Pro

GAA

Giu 180 GAT TGG TTG Asp Trp Leu GAG AAA TTT AGT CCC Glu Lys 185 Phe Ser Pro

TGG

Trp

CAT

His 205

GAA

Giu 190 ATT GTA AGG CAT Ile Val Arg His

GAT

Asp 195 GTT GTC TTT CCT Val Val Phe Pro AGG TTT TTT GGT Arg Phe Phe Gly GTT GAA GTC CTC Val Giu Val Leu CCT TCT Pro Ser 210 GGC TCG CGA Gly Ser Arg

AAA

Lys 215 TGG GTT GGT GGA Trp Val Gly Gly GAG GTC Giu Val 220 CTA CAG GCT Leu Gin Ala AAC ACT AAT Asn Thr Asn 240 GCA TAT GAT GTG Ala Tyr Asp Val

CCA

Pro 230 ATT CCA GGA TAC Ile Pro Gly Tyr AGA ACT AAA Arg Thr Lys 235 TCT GAG GAT Ser Giu Asp ACT CTT CGT CTC Ser Leu Arg Leu

TGG

Trp 245 GAA CCC AAA GCA Giu Ala Lys Ala

AGC

Ser 250 TTC AAC Phe Asn 255 TT G TTT CTG TTT Leu Phe Leu Phe

AAT

Asn 260 GAT GGA CAG TAT Asp Gly Gin Tyr GCT GCT OCA CAG Ala Ala Ala Gin

CTT

Leu 270 CAT TCT AGG GCT His Ser Arg Ala CAG ATT TGT CCT Gin Ile Cys Ala

CTT

Val 280 CTC TAC CCT GGG Leu Tyr Pro Gly

CAT

Asp 285 CCT ACA GAG AAT Ala Thr Giu Asn

GGA

Gly 290 AAA CTC TTA CGG Lys Leu Leu Arg AAG CAA CAA TTT Lys Gin Gin Phe TTT CTG Phe Leu 300 914 TGC AGT GCA Cys Ser Ala GAT GGA AAG Asp Gly Lys 320 CTT CAG GAT ATT Leu Gin Asp Ile

ATT

Ile 310 GCC AGA TTC AAA Ala Arg Phe Lys GAG AGA GAA Clu Arg Giu 315 AAG GTT GCG Lys Val Ala 962 GGT TCT CAC CAG Gly Ser His Gin TCT GAA TTC CCC Ser Giu Phe Pro

AAG

Lys 330 1010 ATA CAA Ile Gin 335 CTA AAT GAC ACA Leu Asn Asp Thr

CAT

His 340 CCA ACT CTT ACG Pro Thr Leu Thr CCA GAG CTG ATG Pro Giu Leu Met TTC CTA ATG GAT Leu Leu Met Asp GAA GGA CTT GGG Giu Gly Leu Gly GAT GAA TCT TGG Asp Giu Ser Trp

AAT

Asn 365 1058 1106 1154 ATC ACT ACT AGG Ile Thr Thr Arg

ACA

Thr 370 ATT GCC TAT ACG Ile Ala Tyr Thr CAT ACA GTC CTA His Thr Val Leu CCT GAA Pro Giu 380 GCA CTT GAA Ala Leu Giu

AAA

Lys 385 TGG TCT CAG GCA Trp Ser Gin Ala

GTC

Val1 390 ATG TGG AAG CTC Met Trp Lys Leu CTT CCT AGA Leu Pro Arg 395 1202 -WO 98/35051 CAT ATG GAA His Met Glu 400 PCTCA98OOO55 ATC ATT GAA Ile Ile Giu GAA ATT Glu Ile 405 GAC AAA CGG TTT GTT GCT ACA ATA Asp Lys Arg Phe Val Ala Thr Ile 410 1250 ATG TCA Met Ser 415 GAA AGA CCT GAT Glu Arg Pro Asp

CTT

Leu 420 GAG AAT AAG ATG CCT AOC ATG CGC ATT 01u Asn Lys Met Pro Ser Met Arg Ile 425 1298

TTG

Leu 430 GAT CAC AAC GCC Asp His Asn Ala

ACA

Thr 435 AAA CCT GTT GTG Lys Pro Val Val

CAT

His 440 ATG GCT AAC TTG Met Ala Asn Leu GTT GTC TCT TCA Val Val Ser Ser ACG GTA AAT GGT Thr Val Asn Gly

OTT

Val 455 GCC CAG CTG CAT Ala Gin Leu His AGT GAC Ser Asp 1346 1394 1442 1490 ATC CTO AAG Ile Leu Lys AAO TTC CAG Lys Phe Gin 480

OCT

Ala 465 GAO TTA TTT GCT Olu Leu Phe Ala

GAT

Asp 470 TAT OTC TCT OTA Tyr Val Ser Val TOO CCC ACC Trp Pro Thr 475 TOO ATC CGA Trp Ile Arg AAT AAO ACC AAT Asn Lys Thr Asn

GGT

Gly 485 ATA ACT CCT COT Ile Thr Pro Arg

AGO

Arg TTT TOT Phe Cys 495 ACT CCT GAO CTG Ser Pro Olu Leu CAT ATA ATT ACC His Ile Ilie Thr

AAO

Lys TOG TTA AAA ACA Trp Leu Lys Thr

OAT

Asp 510 CAA TOO OTO ACO Gin Trp Val Thr

AAC

Asn 515 CTC OAA CTG CTT Leu Glu Leu Leu

OCT

Ala 520 AAT CTT COG GAO Asn Leu Arg Olu

TTT

Phe 1538 1586 1634 OCT OAT AAT TCG Ala Asp Asn Ser GAG CTC CAT OCT GAA Glu Leu His Ala Glu 530

TG

Trp 535 GAA TCA GCC AAG Glu Ser Ala Lys ATO 0CC Met Ala AAC AAG CAG Asn Lys Gin ATC OAT CCA Ile Asp Pro 560

CGT

Arg 545 TTG OCA CAG TAT Leu Ala Gin Tyr CTG CAT GTG ACA Leu His Val Thr GOT OTO AOC Gly Val Ser 555 ATC CAT GAA Ilie His Glu 1682 1730 AAT TCC CTT TTT Asn Ser Leu Phe ATA CAA GTC AAA Ile Gin Val Lys

COT

Arg TAC AAA Tyr Lys 575 AAG CTT Lys Leu 590 AGO CAG CTT CTA Arg Gin Leu Leu

AAT

Asn 580 ATT CTG 0CC OTC Ile Leu Gly Val

ATC

Ile TAT AGA TAC AAG Tyr Arg Tyr Lys 1778 1826 AAG OGA ATO Lys Gly Met

AOC

Ser 595 CCT GAA OAA AGO Pro Glu Olu Arg AAT ACA ACT CCT Asn Thr Thr Pro ACA GTC ATO ATT OGA OGA AAA OCA TTT Thr Val Met Ile Oly Gly Lys Ala Phe 610

OCA

Ala 615 ACA TAC ACA AAT Thr Tyr Thr Asn OCA AAA Ala Lys 620 1874 -WO 98/35051 -WO 8/355 1PCTCA98OOO55 CGA ATT GTC Arg Ile Val CCT GAC GTC Pro Asp Val 640 AAG CTC GTG ACT CAT GTT GGC GAC CTT GTC AAT ACT 1922 Lys 625 Leu Val Thr Asp Val1 630 Gly Asp Val Val Asn 635

GAC

Ser Asp AAT GAC TAT TTG Asn Asp Tyr Leu

AAG

Lys 645 CTG GTT TTT GTT Val Val Phe Val

CCC

Pro 650 AAC TAC AAT Asn Tyr Asn 1970 GTA TCT Val Ser 655 CTG GCA GAG ATC Val Ala Giu Met

CTT

Leu 660 ATT CCC GGA AGT Ile Pro Cly Ser

GAG

Glu 665 CTA TCA CAA CAC Leu Ser Gin His

ATC

Ile 670 ACT ACT GCA GGC Ser Thr Ala Gly GAA GCA ACT OGA Ciu Ala Ser Ciy

ACA

Thr 680 AGC AAC ATG AAA Ser Asn Met Lys 2018 2066 2114 CCC CTT AAT CGA Ala Leu Asn Cly CTT ATC ATT GGC Leu Ile Ile ly CTA GAT GGG CC Leu Asp Gly Ala AAT GTG Asn Val 700 GAA ATT ACC Glu Ile Arg ACA CCT GAT Thr Ala Asp 720 CAA ATT GCA CAA Glu Ile Gly Clu

CAT

Asp 710 AAC TTC TTT CTT Asn Phe Phe Leu TTT CCT GCA Phe Gly Ala 715 AAT CCA CTG Asn Gly Leu 2162 2210 CAA GTT CCT CAA Ciu Val Pro Cmn

CTG

Leu 725 CCC AAA CAT CGA Arg Lys Asp Arg

GAG

Glu 730 TTC AAA Phe Lys 735 CCT CAT CCT CCC Pro Asp Pro Arg

TTT

Phe 740 GAA GAG GCA AAA Clu Clu Ala Lys

CAA

Gin 745 TTT ATT ACC TCT Phe Ile Arg Ser

OGA

Cly 750 CCA TTT CCC ACG Ala Phe Gly Thr

TAT

Tyr 755 CAT TAT AAT CCC Asp Tyr Asn Pro

CTC

Leu 760 CTT GAA TCA CTC Leu Clu Ser Leu 2258 2306 2354 CCC AAC TCC CCA Cly Asn Ser Cly OCT CGT CCA GAC Cly Arg Cly Asp TTT CTT CTT CGT Phe Leu Val Gly CAT CAT His Asp 780 TTT CCC AC Phe Pro Ser CAC AGO AAA Asp Arg Lys 800

TAC

Tyr 785 ATO CAT CCT CAG Met Asp Ala Gin AGC GTT CAT CAA Arg Val Asp Clu GCT TAC AAG Ala Tyr Lys 795 ACT CCC ACT Ser Cly Ser 2402 2450 AGA TGG ATA AAC Arg Trp Ile Lys

ATC

Met 805 TCT ATA CTC AC Ser Ile Leu Ser

ACT

Thr 810 CCC AAA Gly Lys 815 TTT ACT ACT GAC Phe Ser Ser Asp ACA ATT TCT CAA Thr Ile Ser Gin

TAT

Tyr 825 OCA AAA GAG ATC Ala Lys Olu Ile 2498

TGG

Trp 830 AAC ATT CCC GAG TCT CCC GTC CCT TCA Asn Ile Ala Clu Cys Arg Val Pro 835 CCACACTTCT

GAACCTGGTA

2548 WO 98/35051 WO 9835051PCT/CA98/00055 TCTAATAAGG ATCTAATGTT CATTGTTTAC TAGCATATGA ATAATGTAAG

TTCAAGCACA

ACATGCTTTC TTATTTCCTA CTGCTCTCAA. GAAGCAGTTA

TTTGTTG

INFORMATION FOR SEQ ID NO:4: SEQUENCE

CHARACTERISTICS:

LENGTH: 839 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 2608 2655 Met Glu Gly Gly Ala Lys Ser Asn Asp Val Gin His Gin Lys Gin Asn Asn Ala Asp 145 Tyr Glu Pro Al a Ala Gin Thr Al a Lys Ala 130 Ser Arg Val L eu Gin Tyr Trp Tyr Val1 Leu 115 Leu Met Tyr Pro Ser Tyr Tyr Asn Tyr Gly 100 Gly Gly Ala Gly Glu 180 Giu Thr Ala Asp Leu Asn Gin Asn Thr Leu 165 Asp Asp Pro Ala Thr 70 Ser Leu Gin Gly Leu 150 Phe Trp Pro His Thr 55 Tyr Met Asp Leu Gly 135 Asn Lys Leu Thr Phe 40 Ala Leu Giu Ile Glu 120 Leu Leu Gin Glu Asp 25 Ser Asp His Tyr His 105 Glu Gly Pro Leu Lys 185 10 Ile Pro Ser Tyr Leu 90 Asn Val Arg Ala Ile 170 Phe Ser Ala Phe Vai Asp 75 Gin Ala Val1 Leii Trp 155 Thr Ser Ala S er Lys Arg Lys G ly Tyr Glu Ala 140 Gly Lys Pro Ala Asn Phe Asp Val1 Ar g Al a Gin 125 Ser Tyr Al a Trp Pro Ile Giu Arg Asn Ala Asp 110 Glu Cys Gly Gly Giu 190 Ile Lys Pro Leu Pro Leu Ala Lys Phe Leu Gin 175 Ile Ala Tyr Leu Ile Lys Thr Leu Asp Leu Arg 160 Giu Val1 Arg His Asp 195 Val Val Phe Pro Ile Arg Phe Phe Gly His Val Giu Val 200 205 WO 98/35051 PCT/CA98/00055 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 Ile 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 Ile 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 Ile Ile 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 Ile Gin Leu 325 330 335 Asn Asp Thr His Pro Thr Leu Thr Ile Pro Glu Leu Met Arg Leu Leu 340 345 350 Met Asp Asp Glu Gly Leu Gly Trp Asp Glu Ser Trp Asn Ile Thr Thr 355 360 365 Arg Thr Ile 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 Ile Ile Glu Glu Ile Asp Lys Arg Phe Val Ala Thr Ile Met Ser Glu 405 410 415 Arg Pro Asp Leu Glu Asn Lys Met Pro Ser Met Arg Ile 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 Ile 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 Ile Thr Pro Arg Arg Trp Ile Arg Phe Cys Ser 485 490 495 -WO 98/35051 WO 9835051PCT/CA98/00055 Pro Val1 Ser Arg 545 Asn Gin G ly Ile Lys 625 Asn Ala Ala Gly Giu 705 Glu Asp Gly Glu Thr Glu 530 Leu S er Leu Met Gly 610 Leu Asp Giu dly Cys 690 Glu Val1 Pro Thr Leu Asn 515 Leu Ala Leu Leu Ser 595 Gly Val Tyr Met Met 675 Leu Ile Pro Arg Tyr 755 Ser 500 Leu His Gin Phe Asn 580 Pro Lys Thr Leu Leu 660 Giu Ile Gly Gin Phe 740 Asp His Giu Ala Tyr Asp 565 Ile Giu Ala Asp Lys 645 Ile Ala Ile Glu Leu 725 Glu Tyr Ile Leu Giu Ile 550 Ile Leu Giu Phe Val 630 Val1 Pro Ser Gly Asp 710 Arg Glu 1\5n Ile Leu Trp 535 Leu Gin Gly Arg Ala 615 Gly Val Gly Gly Thr 695 Asn Lys Ala Pro Thr Ala 520 Giu His Val1 Val1 Lys 600 Thr Asp Phe Ser Thr 680 Leu Phe Asp Lys Leu 760 Lys 505 Asn Ser Val1 Lys Ile 585 Asn Tyr Val1 Val1 Glu 665 Ser Asp Phe Arg Gin 745 Leu Trp Leu Ala Thr Arg 570 Tyr Thr Thr Val Pro 650 Leu Asn Giy Leu Giu 730 Phe Glu Leu Lys Arg Glu Lys Met 540 Gly Val 555 Ile His Arg Tyr Thr Pro Asn Ala 620 Asi Ser 635 Asn Tyr Ser Gin Met Lys Ala Asn 700 Phe Gly 715 Asn Gly Ile Arg Ser Leu Thr Phe 525 Ala Ser Giu Lys Arg 605 Lys Asp Asn His Phe 685 Val Ala Leu Ser Glu 765 Asp 510 Ala Asn Ile Tyr Lys 590 Thr Arg Pro Val1 Ile 670 Al a Giu Thr Phe Gly 750 Gly Gin Asp Lys Asp Lys 575 Leu Vali Ile Asp Ser 655 Ser Leu Ile Ala Lys 735 Ala Asn Trp Asn Gin Pro 560 Arg Lys Met Val Val1 640 Val1 Thr Asn Arg Asp 720 Pro Phe S er Gly Tyr 770 Gly Arg Gly Asp Tyr Phe Leu Val Gly His Asp Phe Pro Ser 775 780 WO 98/35051 PCT/CA98/00055 Tyr Met Asp Ala Gin Ala Arg Val Asp Glu Ala Tyr Lys Asp Arg Lys 785 790 795 800 Arg Trp Ile Lys Met Ser Ile Leu Ser Thr Ser Gly Ser Gly Lys Phe 805 810 815 Ser Ser Asp Arg Thr Ile Ser Gin Tyr Ala Lys Glu Ile Trp Asn Ile 820 825 830 Ala Glu Cys Arg Val Pro 835 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 3171 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum (ix) FEATURE: NAME/KEY: CDS LOCATION: 87..3011 OTHER INFORMATION: /product= "potato alpha-glucan L-type leaf phosphorylase" (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 330..3008 (ix) FEATURE: NAME/KEY: sig-peptide LOCATION: 87..329 (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTTTTTTT CAACATGCAC AACAATTATT TTGATTAAAT TTTGTATCTA AAAATTTAGC ATTTTGAAAT TCAGTTCAGA GACATC ATG GCA ACT TTT GCT GTC TCT GGA TTG 113 Met Ala Thr Phe Ala Val Ser Gly Leu -81 -80 AAC TCA ATT TCA AGT ATT TCT AGT TTT AAT AAC AAT TTC AGA AGC AAA 161 Asn Ser Ile Ser Ser Ile Ser Ser Phe Asn Asn Asn Phe Arg Ser Lys -65 -WO 98/35051 -WO 8/355 1PCTCA98OOO55 AAC TCA AAC Asn Ser Asn ATT TTG TTG AGT AGA AGG AGG ATT Ile Leu Leu Ser Arg Arg Arg Ile

TTA

Leu TTG TTC AGT TTT Leu Phe Ser Phe

AGA

Arg AGA AGA AGA AGA Arg Arg Arg Arg

AGT

Ser -35 TTC TCT GTT AGC Phe Ser Val Ser

AGT

Ser GTT GCT AGT GAT Val Ala Ser Asp AAG CAG AAG ACA Lys Gin Lys Thr

AAG

Lys GAT TCT TCC TCT Asp Ser Ser Ser GAA GGA TTT ACA Giu Gly The Thr TTA GAT Leu Asp GTT TTT CAG Val Phe Gin GAC TCC ACG TCT GTT TTA TCA AGT Asp Ser Thr Ser Val Leu Ser Ser 1

ATA

Ile AAG TAT CAC Lys Tyr His GCT GAG TTC ACA CCA TCA Ala Glu Phe Thr Pro Ser

TTT

Phe TCT CCT GAG AAG Ser Pro Glu Lys

TTT

Phe GAA CTT CCC AAG Glu Leu Pro Lys

GCA

Ala TAC TAT GCA ACT Tyr Tyr Ala Thr GAG AGT GTT CGA Glu Ser Val Arg ACG CTC ATT ATA Thr Leu Ile Ile

AAT

Asn TGG AAT GCC ACA Trp, Asn Ala Thr GAA TTC TAT GAA Glu Phe Tyr Glu ATG AAT GTA AAG Met Asn Val Lys CAG GCA Gin Ala TAT TAC TTG Tyr Tyr Leu ATT GGT AAC Ile Gly Asn ATG GAA TTT CTT Met Giu Phe Leu GGA AGA GCT TTA Giy Arg Ala Leu CTC AAT GCT Leu Asn Ala TTA ACT AAG Leu Thr Lys TTG GGG CTA ACC Leu Gly Leu Thr

GGA

Gly 80 CCT TAT GCA GAT Pro Tyr Ala Asp

GCT

Al a CTC GGA Leu Gly TAC AGT TTA GAG GAT GTA CCC AGG CAG Tyr Ser Leu Glu Asp Val Ala Arg Gin 95

GAA

Giu 100 CCG CAT GCA GCT Pro Asp Ala Ala

TTA

Leu 105 GGT AAT GGA GGT Gly Asn Cly Gly

TTA

Leu 110 GGA AGA CTT CCT Ciy Arg Leu Ala

TCT'TGC

Ser Cys 115 TTT CTG GAC Phe Leu Asp 689 ATG CC ACA CTA Met Ala Thr Leu

AAC

Asn 125 TAC CCT GCA TGG GGC TAT CGA CTT ACA TAC CAA Tyr Pro Ala Trp Cly Tyr Cly Leu Arg Tyr Gin TAT CCC CTT Tyr Gly Leu GCT GAA AAT Ala Giu Asn 155

TTC

Phe 140 AAA CAG CTT ATT Lys Gin Leu Ile

ACA

Thr 145 AAA CAT CGA CAG GAG GAA CTT Lys Asp Gly Gin Glu Giu Val 150 TGG CTC GAG ATC GGA AAT CCA TGG GAA ATT GTG AGG AAT Trp Leu Glu Met Gly Asn Pro Trp Ciu Ile Val Arg Asn WO 98/35051 WO 9835051PCTCA98OO55 GAT ATT Asp Ile 170.

TCG TAT CCC GTA Ser Tyr Pro Val

AAA

Lys 175 TTC TAT GGG AAG Phe Tyr Gly 4ys

GTC

Val1 180 ATT GAA GGA GCT Ile Glu Gly Ala

GAT

Asp 185 GGG AGG AAG GAA Gly Arg Lys Glu

TGG

Trp 190 GCT GGC GGA GAA Ala Gly Gly Glu ATA ACT GCT GTT Ile Thr Ala Val

GCC

Ala 200 TAT GAT GTC CCA Tyr Asp Val Pro CCA GGA TAT AAA Pro Gly Tyr Lys

ACA

Thr 210 AAA ACA ACG ATT Lys Thr Thr Ile AAC CTT Asn Leu 215 CGA TTG TGG Arg Leu Trp TTT AAC AAT Phe Asn Asn 235

ACA

Thr 220 ACA AAG CTA GCT Thr Lys Leu Ala GAA GCT TTT GAT Glu Ala Phe Asp TTA TAT GCT Leu Tyr Ala 230 AAA AAG GCT Lys Lys Ala 1025 1073 GGA GAC CAT GCC Gly Asp His Ala

AAA

Lys 240 GCA TAT GAG GCC Ala Tyr Glu Ala

CAG

Gin 245 GAA AAG Glu Lys 250 ATT TGC TAT GTC Ile Cys Tyr Val

TTA

Leu 255 TAT CCA GGT GAC Tyr Pro Gly Asp

GAA

Glu 260 TCG CTT GMA GGA Ser Leu Giu Gly 1121 1169

AAG

Lys 265 ACG CTT AGG TTA Thr Leu Arg Leu

AAG

Lys 270 CAG CAA TAC ACA Gin Gin Tyr Thr TGT TCT GCT TCT Cys Ser Ala Ser

CTT

Leu 280 CAG GAC ATT ATT Gin Asp Ile Ile

GCA

Ala 285 CGG TTC GAG AAG Arg Phe Glu Lys

AGA

Arg 290 TCA GGG ALAT GCA Ser Gly Asn Ala GTA AAC Val Asn 295 1217 1265 TGG GAT CAG Trp Asp Gin CCA ACA CTT Pro Thr Leu 315 CCC GAA AAG GTT Pro Giu Lys Val GTA CAG ATG AAT GAC ACT CAT Vai Gin Met Asn Asp Thr His 310 TGT ATA CCA GAA Cys Ile Pro Giu

CTT

Leu 320 TTA AGG ATA TTG Leu Arg Ile Leu

ATG

Met 325 GAT GTT A Asp Val Lys 1313 GGT TTG Gly Leu 330 AGC TGG AAG CAG Ser Trp Lys Gin

GCA

Ala 335 TGG GMA ATT ACT Trp Glu Ile Thr

CAA

Gin 340 AGA ACG GTC GCA Arg Thr Val Ala 1361 1409

TAC

Tyr 345 ACT MAC CAC ACT Thr Asn His Thr CTA CCT GAG GCT Leu Pro Giu Ala GAG AAA TGG AGC Glu Lys Trp Ser

TTC

Phe 360 ACA CTT CTT GGT Thr Leu Leu Gly

GMA

Glu 365 CTG CTT CCT CGG Leu Leu Pro Arg GTG GAG ATO ATA Val Gu Ile Ile GCA ATG Ala Met 375 1457 1505 ATA GAT GAG Ile Asp Giu

GAG

Glu 380 CTC TTG CAT ACT Leu Leu His Thr CTT GCT GMA TAT GGT ACT GMA Leu Ala Glu Tyr Gly Thr Glu 390 -WO 98/35051 -WO 9835051PCTCA98OOO55 GAT CTT GAC Asp Leu Asp 395 AAT GTT GAA Asn Val Giu 410 TTG TTG CAA GAA Leu Leu Gin Giu

AAG

Lys 400 CTA AAC CAA ATG Leu Asn Gin Met ATT CTG GAT Ile Leu Asp 1553 ATA CCA AGT TCT GTT TTG GAG TTG Ile Pro Ser Ser Val Leu Giu Leu

CTT

Leu 420 ATA AAA GCC GAA Ile Lys Ala Glu 1601 1649

GAA

Giu 425 AGT GCT GCT GAT Ser Ala Aia Asp GAA AAG GCA GCA Glu Lys Aia Ala GAA GAA CAA GAA GAA Giu Giu Gin Giu Giu 440 CAA GGOT AAG GAT GAC AGT AAA CAT GAG Giu Giy Lys Asp Asp Ser Lys Asp Giu 445 GAA ACT ACG AAC GAA GAG GAG GAA ACT Giu Thr Thr Asn Giu Giu Giu Clu Thr 460 Ar

GAA

Giu 450 ACT GAG GCT GTA Thr Giu Ala Val AAG GCA Lys Ala 455 1697 GAG GTT AAG AAG Giu Val Lys Lys GTT GAG CTC Vai Giu Val 470 CAT CCA AAT His Pro Asn 1745 GAG GAT ACT Giu Asp Ser 475 CPA GCA AAA ATA Gin Ala Lys Ile

MA

Lys 480 CGT ATA TTC GGG Arg Ile Phe Giy 1793 AAA CCA Lys Pro 490 CAG GTG GTT CAC Gin Val Val His

ATG

Met 495 GCA AAT CTA TGT Ala Asn Leu Cys

GTA

Val 500 GTT AGC GGG CAT Vai Ser Gly His

GCA

Ala 505 GTT AAC GGT GTT Val Asn Giy Val GAG ATT CAT AGT Giu Ile His Ser ATA GTT AAG GAT Ile Vai Lys Asp 1841 1889 1937 GTT TTC AAT GAA Val Phe Asn Glu TAC AAG TTA TGG Tyr Lys Leu Trp

CCA

Pro 530 GAG AAA TTC CAA Giu Lys Phe Gin AAC AAG Asn Lys 535 ACA AAT GOT Thr Asn Giy TTG ACT GAA Leu Ser Ciu 555 ACA CCA AGA AGA Thr Pro Arg Arg

TGG

Trp 545 CTA AGT TTC TGT Leu Ser Phe Cys AAT CCA GAG Asn Pro Clu 550 TGC TTA GTA Trp Leu Val 1985 2033 ATT ATA ACC AAG Ile Ile Thr Lys

TCG

Trp 560 ACA CGA TCTI GAT Thr Gly Ser Asp

GAT

Asp 565 AAC ACT Asn Thr 570 CAA AAA TTG GCA Ciu Lys Leu Ala

GAG

Giu 575 CTT CGA AAG TTT Leu Arg Lys Phe CAT AAC GAA CAA Asp Asn Giu Giu

CTC

Leu 585 CAG TCT GAG TGG Gin Ser Glu Trp

AGG

Arg 590 AAG GCA AAA GGA Lys Ala Lys Cly

MAT

Asn 595 AAC AMA ATG MAG Asn Lys Met Lys

ATT

Ile 600 2081 2129 2177 CTC TCT CTC ATT Val Ser Leu Ile GMA AAA ACA GGA Clu Lys Thr Cly

TAC

Tyr 610 GTC GTC ACT CCC Val Vai Ser Pro CAT CCA Asp Ala 615 WO 98/35051 -WO 9835051PCTICA98/00055 ATG TTT GAT Met Phe Asp TTA AAT ATA Leu Asn Ile 635

GTT

Val 620 CAG ATC AAG CGC Gin Ile Lys Arg

ATC

Ile 625 CAT GAG TAT AAA His Giu Tyr Lys AGG CAG CTA Arg Gin Leu 630 2225 TTT GGA ATC GTT Phe Gly Ile Vai CGC TAT AAG AAG ATG AAA GAA ATG Arg Tyr Lys Lys Met Lys Giu Met 645 2273 AGC CCT Ser Pro 650 GAA GAA CGA AAA Glu Giu Arg Lys AAG TTT GTC CCT Lys Phe Vai Pro GTT TGC ATA TTT Val Cys Ile Phe

GGA

Gly 665 GGA AAA GCA TTT Giy Lys Ala Phe

GCT

Aia 670 ACA TAT GTT CAG Thr Tyr Val Gin

GCC

Al a 675 AAG AGA ATT GTA Lys Arg Ile Val 2321 2369 2417 TTT ATC ACT CAT Phe Ile Thr Asp

GTA

Val1 685 CCC GAA ACA CTC Giy Glu Thr Val

AAC

Asn 690 CAT GAT CCC GAG ATT GGT His Asp Pro Glu Ile Gly 695 GAT CTT TTG Asp Leu Leu GAA GTG CTA Glu Val Leu 715

AAG

Lys 700 GTT GTA TTT GTT Val Val Phe Val

CCT

Pro 705 GAT TAC AAT GTC Asp Tyr Asn Val ACT GTA GCA Ser Val Ala 710 2465 ATT CCT GGT AGT Ile Pro Gly Ser

GAG

Glu 720 TTG TCC CAG CAT ATT AGT ACT GCT Leu Ser Gin His Ile Ser Thr Ala 725 2513 GGT ATG Gly Met 730 GAG GCT AGT GGA Ciu Ala Ser Gly

ACC

Thr 735 AGC AAC ATG AAA Ser Asn Met Lys

TTT

Phe 740 TCA ATG AAT GCC Ser Met Asn Gly

TC

Cys 745 CTC CTC ATC GGG Leu Leu Ile Cly TTA GAT GGT GCC Leu Asp Gly Ala

AAT

Asn 755 GTT GAG ATA AGA Val Glu Ile Arg 2561 2609 2657 GALA GTT GGA GAG Glu Val Gly Glu AAT TTC TTT CTT Asn Phe Phe Leu

TTC

Phe 770 GGA GCT CAG GCT Gly Ala Gin Ala CAT GAA His Glu 775 ATT GCT GGC Ile Ala Gly CCA AGA TTT Pro Arg Phe 795

CTA

Leu 780 CGA AAC GAA AGA Arg Lys Giu Arg

GCC

Al a 785 GAG OGA AAG TTT Clu Cly Lys Phe GTC CCG GAC Val Pro Asp 790 2705 GAA GAA GTA AAG Clu Glu Val Lys TTC ATT AGO ACA GGC GTC TTT GCC Phe Ile Arg Thr Gly Val Phe Gly 805 2753 ACC TAC Thr Tyr 810 AAC TAT GAA OAA Asn Tyr Clu Clu

CTC

Leu 815 ATO GGA TCC TTG Met Gly Ser Leu GGA AAC GAA GGC Gly Asn Glu Gly 2801 2849

TAT

Tyr 825 OCT CGT OCT GAC Gly Arg Ala Asp

TAT

Tyr 830 TTT CTT GTA OGA Phe Leu Val Gly

KAG

Lys 835 GAT TTC CCC GAT Asp Phe Pro Asp wo 98/35051 WO 98/505 1PCTCA98OOO55 ATA GAG TGC CAA GAT AAA GTT GAT GAA GCA TAT Ile Giu Cys Gin Asp Lys Val Asp Glu Ala Tyr 845 850 TGG ACC AAA ATO TCG ATC TTA AAC ACA GCT GGA Trp Thr Lys Met Ser Ile Leu Asn Thr Ala Gly 860 865 AGT GAT CGA ACA ATT CAT CAA TAT GCA AGA GAT Ser Asp Arg Thr Ile His Gin Tyr Ala Arg Asp 875 880 CCT OTT GAA TTA CCT TAA AAGTTAGCCA

GTTAAAGG

Pro Val Giu Leu Pro* 890 TTTTTCCCCC TGAGGTTCTC CCATACTGTT

TATTAGTACA

CTGAAATGAT AGAAGTTTTG AATATTTACT

GTCAATA.AA

INFORMATION FOR SEQ ID NO:6: SEQUENCE

CHARACTERISTICS:

LENGTH: 975 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 Met Ala Thr Phe Ala Val Ser Gly Leu Asn Ser -81 -80 -75 Ser Phe Asn Asn Asn Phe Arg Ser Lys Asn Ser -60 -55 Arg Arg Arg Ile Leu Leu Phe Ser Phe Arg Arg.

-40 Ser Val Ser Ser Val Ala Ser Asp Gin Lys Gin -25 Ser Ser Asp Giu Gly Phe Thr Leu Asp Val Phe -10 Ser Val Leu Ser Ser Ile Lys Tyr His Ala Giu 1 5 10 Ser Pro Olu Lys Phe Giu Leu Pro Lys Ala Tyr 25 CGA GAC CAG AAG Arg Asp Gin Lys 855 TCG TTC AAA TTT Ser Phe Lys Phe 870 ATA TOG AGA ATT Ile Trp Arg Ile 885 AT GAAAGCCAAT 2897 2945 2993 3041 TATATTGTCA

ATTGTTOCTA

TACAGTTGAT TCCATTTGAA 3101 3161 3171 Ile Asn Arg Lys Gin Phe T'yr Ser Ser Ile Leu Arg Arg Thr Lys Pro Asp Thr Pro Ala Thr Ser Ser Phe Ser Thr Phe Glu WO 98/35051 WO98/3051PCT/CA98/00055 Ser Val Arg Tyr Glu L.ys Leu Gin Gly Gly Pro Tyr Val Ala Arg Arg Leu Ala Ala Trp Cly 130 Ile Thr Lys 145 Gly Asri Pro 160 Phe Tyr Gly Gly Gly Glu Tyr Lys Thr 210 Ala Ala Glu 225 Lys Ala Tyr 240 Tyr Pro Gly Gin Tyr Thr Glu Lys Arg 290 Asp Met Arg Ala Gin Ser 115 Tyr Asp Trp Lys Asp 195 Lys Ala Glu Asp Leu 275 Ser Leu Val1 Leu Ala 8S Pro Phe Leu Gin Ile 165 Ile Thr Thr Asp Gin 245 Ser Ser Asn Ile Lys Leu 70 Leu Asp Leu Arg Glu 150 Val1 Glu Ala Ile Leu 230 Lys Leu Ala Ala Asn Ala Ala Lys Ala Ser 120 Gin Val1 Asn Ala Al a 200 Leu Ala Ala Gly Leu 280 Asn Trp Tyr Ile Leu Leu 105 Met Tyr Ala Asp Asp 185 Tyr Arg Phe Giu Lys 265 Gin Trp As n Tyr G ly G ly 90 Gly Al a Gly Giu Ile 170 Gly Asp Leu Asn Lys 250 Thr Asp Asp Al a Leu As n Tyr Asn Thr Leu Asn 155 Ser Arg Val Trp Asn 235 Ile Leu Ile Gin Phe Phe Thr Asp Gly Pro Leu Met Lys 175 Ala Gly Leu Ala Leu 255 Gin Phe Lys Val Ala 305 Val Gin Met Asn Thr His Pro Thr Cys Ile Pro Glu WO 98/35051 WO 9835051PCTCA98OOO55 Leu Leu Arg le .Leu Met Asp Val Lys Gly Leu Ser Trp Lys Gin Ala 320 325 330 335 Trp Giu Ile Thr Gin Arg Thr Val Ala Tyr Thr Asn H-is Thr Val Leu 340 345 350 Pro Giu Ala Leu Giu Lys Trp Ser Phe Thr Leu Leu Gly Giu Leu Leu 355 360 365 Pro Arg His Val Giu Ile Ile Ala Met Ile Asp Giu Giu Leu Leu His 370 375 380 Thr Ile Leu Ala Giu Tyr Gly Thr Glu Asp Leu Asp Leu Leu Gin Giu 385 390 395 Lys Leu Asn Gin Met Arg Ile Leu Asp Asn Val Giu Ile Pro Ser Ser 400 405 410 415 Val Leu Giu Leu Leu Ile Lys Ala Giu Glu Ser Ala Ala Asp Val Giu 420 425 430 Lys Ala Ala Asp Giu Giu Gin Giu Giu Giu Gly Lys Asp Asp Ser Lys 435 440 445 Asp Giu Giu Thr Glu Ala Val Lys Ala Glu Thr Thr Asn Giu Giu Giu 450 455 460 Giu Thr Giu Val Lys Lys Val Giu Val Giu Asp Ser Gin Ala Lys Ile 465 470 475 Lys Arg Ile 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 Giu 500 505 510 Ile His Ser Giu Ile Val Lys Asp Giu Val Phe Asn Giu Phe Tyr Lys 515 520 525 Leu Trp Pro Giu Lys Phe Gin Asn Lys Thr Asn Gly Val Thr Pro Arg 530 535 540 Arg Trp Leu Ser Phe Cys Asn Pro Giu Leu Ser Gu Ile Ile Thr Lys 545 550 555 Trp Thr Gly Ser Asp Asp Trp Leu Val Asn Thr Giu Lys Leu Ala Glu 560 565 570 575 Leu Arg Lys Phe Ala Asp Asn Giu Giu Leu Gin Ser Giu Trp Arg Lys 580 585 590 Ala Lys Gly Asn Asn Lys Met Lys Ile Val Ser Leu Ile Lys Giu Lys 595 600 605 WO 98/35051 PCT/CA98/00055 Val Val Ser Pro Asp Ala Met Phe Asp Vai Gin Ile Lys Thr Arg Tyr 640 Lys Tyr Thr Val Giu 720 Ser Asp Phe Arg Ala 800 Met Leu Asp Asn Tyr 880 Gly Ile 625 Arg Phe Val1 Val1 Pro 705 Leu Asn Gly Leu Ala 785 Phe Gly Val1 Glu Thr 865 Ala 615 620 Glu Lys Pro Ala 675 His Tyr Gin Lys Asn 755 Gly Giy Arg Leu Lys 835 Tyr Gly Asp Lys Met 645 Vai Arg Pro Val1 Ile 725 Ser Giu Gin Phe Giy 805 Gly Phe Asp Phe Trp Arg 630 Lys Cys Ile Giu Ser 710 Ser Met Ile Ala Val 790 Val1 Asn Pro Gin Lys 870 Arg Gin Giu Ile Val1 Ile 695 Val1 Thr ASn Arg His 775 Pro Phe Glu Asp Lys 855 Phe Ile Leu Met Phe Lys 680 Gly Ala Ala Gly Glu 760 Giu Asp Gly Gly Tyr 840 Lys Ser Glu Leu Ser Gly 665 Phe Asp Giu Giy Cys 745 Giu Ile Pro Thr Tyr 825 Ile Trp Ser Pro Asn Pro 650 Gly Ile Leu Val1 Met 730 Leu Val1 Ala Arg Tyr 810 G ly Giu Thr Asp Val1 890 Ilie 635 Giu Lys Thr Leu Leu 715 Giu Leu Gly Gly Phe 795 Asn Arg Cys Lys Arg 875 Giu Phe Giu Ala Asp Lys 700 Ile Ala Ile Giu Leu 780 Giu Tyr Ala Gin Met 860 Thr Leu Gly Arg Phe Val1 685 Val1 Pro Ser Gly Asp 765 Arg G lu Giu Asp Asp 845 Ser Ile Pro Val Giu 655 Thr Glu Phe Ser Thr 735 Leu Phe Glu Lys Leu 815 Phe Val Leu Gin INFORMATION FOR SEQ ID NO:7: WO 98/35051 PCT/CA98/00055 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..27 OTHER INFORMATION: /function= "primer" /label= SPL1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATTCGAAAAG CTCGAGATTT GCATAGA 27 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..27 OTHER INFORMATION: /function= "primer" /label= SPL2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: WO 98/35051 PCT/CA98/00055 GTTTATTTTC CATCGATGGA AGGTGGT 27 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..23 OTHER INFORMATION: /function= "primer" /label= SPH1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTGTGCTCTC GAGCATTGAA AGC 23 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..25 OTHER INFORMATION: /function= "primer" WO 98/35051 PCT/CA98/00055 /label.= SPH2 (xi) SEQUENCE DESCRIPTION: SEQ TD NO:1O: ATAATATCCT GAATCGATCC ACTGC

Claims (40)

1. A potato plant having improved tuber cold-storage characteristics, comprising a modified potato plant having a reduced level of activity of an a glucan phosphorylase enzyme selected from the group consisting of a glucan L-type tuber phosphorylase (GLTP) and a glucan H-type tuber phosphorylase (GHTP) in tubers produced by the plant relative to that of tubers produced by an unmodified potato plant.

2. 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 a glucan phosphorylase gene selected from the group consisting of a GLTP gene and a GHTP gene.

3. 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 a glucan phosphorylase selected from the group consisting of a glucan L-type tuber phosphorylase (GLTP) and a glucan H-type tuber phosphorylase (GHTP). 4 The potato plant of claim 3, wherein the encoded a glucan phosphorylase is GLTP. 5 The potato plant of claim 3, wherein the encoded a glucan phosphorylase is GHTP.

6- The potato plant of claim 3, wherein the encoded a glucan phosphorylase comprises an amino acid sequence depicted in SEQ ID NO:2.

7. The potato plant of claim 3, wherein the DNA sequence comprises at least nucleotides of the gene encoding an a glucan phosphorylase as depicted in SEQ ID NO:1. 74

8. The potato plant of claim 3, wherein the encoded a glucan phosphorylase comprises an amino acid sequence depicted in SEQ ID NO:4.

9. The potato plant of claim 3, wherein the DNA sequence comprises at least nucletides of the gene encoding an a glucan phosphorylase as depicted in SEQ ID NO: 3. The potato plant of claim 3, wherein the DNA sequence comprises nucleotides 338 to 993 of SEQ ID NO: 1.

11. The potato plant of claim 3, wherein the DNA sequence comprises nucleotides 147 to 799 of SEQ ID NO: 3.

12. The potato plant of any one of claims 2 11, wherein the DNA sequence is linked to the promoter sequence in an antisense orientation.

13. 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 20 measured at harvest.

14. 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.

16. The potato plant of claim 4, wherein the sum of the concentration of glucose RA and fructose in tubers of the plant stored at 4 0 C for about three months is at least lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant stored under the same conditions.

17. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant stored at 4 0 C for about three months is at least lower than the sum of the concentration of glucose and fructose in tubers of an untransformed plant stored under the same conditions.

18. The potato plant of claim 4, wherein the sum of the concentration of glucose and fructose in tubers of the plant stored at 4 0 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.

19. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg' protein h' in tubers of the plant measured at harvest is at least 10% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant measured at harvest.

20. The potato plant of claim 4, wherein the total a glucan phosphorylase activity .20 measured as pmol NADPH produced mg-' protein-' h'in tubers of the plant measured at harvest is at least 30% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant measured at harvest.

21. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg-' protein' h 1 in tubers of the plant measured at harvest is at least 66% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant measured at harvest. 0 22. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg' protein-' h 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. 76

23. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg-' protein-' h' in tubers of the plant stored at 4°C for about three months is at least 30% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions.

24. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg-' protein-' in tubers of the plant stored at 4°C for about three months is at least 70% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg' protein-' h 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.

26. The potato plant of claim 5, wherein the total a glucan phosphorylase activity 20 measured as pmol NADPH produced mg-' protein-' h in tubers of the plant stored at 4 0 C for about three months is at least 28% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. 25 27. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg' protein-' h' in tubers of the plant stored at 4°C for about six months is at least 10% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions.

28. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg" protein-' h' in tubers of the plant stored at RA 4'C for about six months is at least 30% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. 77

29. The potato plant of claim 4, wherein the total a glucan phosphorylase activity measured as umol NADPH produced mg protein h in tubers of the plant stored at 4°C for about six months is at least 69% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions. The potato plant of claim 5, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg" protein"' h' in tubers of the plant stored at 4°C for about six months is at least 10% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions.

31. The potato plant of claim 5, wherein the total a glucan phosphorylase activity measured as pmol NADPH produced mg protein' h in tubers of the plant stored at 4°C for about six months is at least 39% lower than the total a glucan phosphorylase activity in tubers of an untransformed plant stored under the same conditions.

32. 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. Si0 33. 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.

34. 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.

35. 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. 78

36. 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.

37. 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.

38. 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.

39. 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.

41. 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.

42. 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. S43. 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. 79 4 4. 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.

45. 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. 4 6. 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.

47. 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 a glucan phosphorylase enzyme selected from the group consisting of a glucan L-type tuber phosphorylase (GLTP) and a glucan H-type tuber phosphorylase (GHTP).

48. The method of claim 47, comprising: .2'20 introducing into the potato plant an expression cassette having a plant promoter o• sequence operably linked to a DNA sequence which, when transcribed in the plant, inhibits expression of an endogenous a glucan phosphorylase gene selected from the group consisting of a GLTP gene and a GHTP gene. 25 49. 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 a glucan phosphorylase selected from th6 group consisting of a glucan L-type tuber phosphorylase (GLTP) and a glucan H-type tuber phosphorylase (GHTP). The method of claim 49, wherein the encoded a glucan phosphorylase is GLTP.

51. The method of claim 49, wherein the encoded a glucan phosphorylase is GHTP. 2. The method of claim 49, wherein the encoded a glucan phosphorylase comprises an amino acid sequence depicted in SEQ ID NO: 2.

53. The method of claim 49, wherein the DNA sequence comprises at least nucleotides of the gene encoding an a glucan phosphorylase as depicted in SEQ ID NO:1.

54. The method of claim 49, wherein the encoded a glucan phosphorylase comprises an amino acid sequence depicted in SEQ ID NO:4. The method of claim 49, wherein the DNA sequence comprises at least nucleotides of the gene encoding an a glucan phosphorylase as depicted in SEQ ID NO:3. *20 56. The method of claim 49, wherein the DNA sequence comprises nucleotides 338 to 993 of SEQ ID. NO: 1.

57. The method of claim 49, wherein the DNA sequence comprises nucleotides 147 to 799 of SEQ ID. NO: 3. ***25

58. The method of any one of claims 48 57 wherein the DNA sequence is linked to the promoter sequence in an antisense orientation. *s DATED this 14th day of March 2000. HER MAJESTY THE QUEEN IN RIGHT OF CANADA AS REPRESENTED BY THE DEPARTMENT OF AGRICULTURE AND AGRI-FOOD CANADA ABy their Patent Attorney A.P.T. Patent and Trade Mark Attorneys