EP0625005A1 - Polyphenol oxidase - Google Patents

Abstract

The invention describes the cloning and sequencing of polyphenol oxidase (PPO) cDNAs from plants, which has made it possible to use them in order to genetically transform plants, so as to produce a variety of desired phenotypes.

Description

Polyphenol Oxidase

Polyphenol oxidases (PPO) are ubiquitous copper metalloenzymes of angiosperms which catalyze the oxidation of phenols to quinones at the expense of O2. More specifically these enzymes catalyze the 0- hydroxylation of a monophenol followed by its oxidation to the 0- diquinone (cresolase activity [E.C. 1.14.18.1]), or the oxidation of an 0- dihydroxyphenol to the o-diquinone (catecholase activity [E.C. 1.10.3.2]). Although PPOs may possess both catecholase and cresolase activities, typically the cresolase activity is absent, labile, or requires priming with reducing agent or small amounts of an o-dihydroxyphenol. The quinonoid reaction products formed by PPO are highly reactive, electrophilic molecules which undergo secondary reactions with themselves or act to covalently modify and crosslink a variety of cellular nucleophiles, including nucleophiles of proteins such as sulfhydryl, amine, amide, indole and imidazole substituents. The formation of quinone adducts (usually brown or black colored) represents the primary detrimental effect of PPO in post harvest physiology and food processing and is the primary reason for the interest in PPOs in food technology. [See Adv. Food Res 19:75 (1971 )]. In the cultivated potato alone, melanization driven by PPO is responsible for significant losses each year in potato processing (prepeel blackening, blackspot, pressure bruising and blackheart). Sulfite or ascorbate additives used in the food, wine and beverage industry are frequently employed to inhibit activity of PPO. Conversely, the ability of quinones to covalently modify and reduce the nutritive value of plant proteins has generated interest in PPO for increasing the herbivore resistance of plants [see Naturally Occurring Pest Bioregulators, pp 166-197, ACE Books, Washington (1991)]. Despite the intense study of PPO since its first description in

1895, a large number of biochemical and physiological studies have provided few answers to the question of PPO function and expression. The primary obstacle to understanding PPO function is the formation of artifactual protein species and enzyme inactivation due to quinone adduct formation and PPO crosslinking during isolation and purification. Thus, rapid quinone formation makes it exceptionally difficult to isolate an unmodified PPO, and this problem is a significant factor contributing to the high and varying estimates of the number and properties of higher plant PPOs. In addition, the difficulty of obtaining PPO-null plants has thus far minimized the contribution of genetics to understanding the function and expression of these enzymes.

A variety of hypotheses concerning the function of PPO have been proposed since the first recognition of its activity in 1895. These proposed functions relate to the oxygen reduction activity of PPO as well as its ability to oxidize phenolics to quinones. PPO has been proposed to be involved in buffering of plastid oxygen levels, biosynthesis of phenolics, wound healing, and anti-nutritive modifications of plant proteins to discourage herbivory [see Naturally Occurring Pest Bioregulators, pp 166-197, ACS Books, Washington, DC

(1991 )].

PPO is present in many organs and tissues. It is often abundant in leaves, tubers, storage roots, floral parts and fruits. The abundance of PPO in tubers and fruits at early stages of development along with high levels of phenolic substrates has led to the suggestion that PPO makes the unripe fruit and storage organs inedible to predators [see Recent Advances in Biochemistry of Fruit and Vegetables, pp 159-180, Academic Press (1981)]. PPO has been detected in root plastids, potato amyloplasts, leucoplasts, etioplasts and chromoplasts, as well as in plastid-like particles isolated from sugar beet leaves [see Israel J. Bot.

12:74 (1964)].

Most studies indicate that PPO is membrane-bound in plastids of non-senescing tissues [see Phytochemistry 26:1 (1987)]. PPO activity is frequently latent, requiring activation by proteolysis, detergent, or Ca⁺⁺. It has been suggested that the enzyme is located exclusively in plastids preventing its interaction with phenolics until the cell is disrupted in some way. Thus, PPO is only released to the cytosol upon wounding, senescence or deterioration of the organelle [see Photobiochem. Photobiophys 3:69 (1981 ), and Physiol Plant 72:659 (1988)].

Although PPO is encoded by nuclear genes [see J. Heredity 81 :475 (1990)], very little is known about the targeting and import of PPO to organelles, its insertion into organellar membranes and the spatial arrangement of the enzyme within the membrane.

In Sorghum (a C4 plant) leaves, PPO has been detected only in mesophyll cells; it was absent from bundle sheath cells. Considering the distribution of thylakoid grana stacks and PSII activity, this observation suggests a possible functional association of PPO with photosynthetic activity. However, PPO is also present in cells of many non-photosynthetic organs, such as roots, tubers, fruit, etc. In non- photosynthetic plastids, or plastids treated with tentoxin, PPO has been detected in vesicles which appeared to be attached to the plastid envelope. These observations, along with latency phenomena and the marked changes often observed in PPO levels during development, suggest that the regulation of PPO expression is quite complex and may operate on several levels.

Several functions have been proposed to explain the role of PPOs in plants. Based on its location on the thylakoid membrane and high K_m for O2, PPO has been proposed to function in pseudocyclic photophosphorylation (ATP production with PPO as a terminal electron acceptor rather than NADP+), and regulation of plastidic oxygen levels. However, there is no evidence for a suitable substrate for PPO in this compartment which could allow a PPO-based oxygen reduction cycle to operate.

In contrast, the role of PPO in polymerization of trichome exudate and insect entrapment is relatively well established. [see Insects and The Plant Surface, pp 151 -172, Edward Arnold, London (1986)]. In various solanaceous plants, PPO is the dominant protein and oxidative enzyme of glandular trichomes (ca. 40-80% of total trichome protein) and appears to be responsible for the O2-requiring polymerization of trichome exudate which results in insect entrapment,) and therefore, resistance to insect feeding [see PANS 23:272 (1977) and Naturally Occurring Pest Bioregulators, pp 166-197, ACS Books, Washington, DC (1991)].

A third possible function for PPO in plant tissues is that sequestration of PPO in the thylakoid prevents its interaction with phenolics until the cell is disrupted by herbivores, pathogens, senescence, or other injury. The quinones thus generated by PPO activity on phenolics cross-link with themselves and protein to reduce the palatability, digestibility, and nutritive value of the plant tissue. This view suggests that the primary targets of quinones formed by PPO are the nucleophilic amino acids: histidine, cysteine, methionine, tryptophan, and lysine. The low abundance of these essential amino acids in plant proteins limits insect growth on plant diets. Covalent modification of these essential amino acids by PPO-generated quinones further decreases their nutritional availability to herbivores and may result in poorer insect performance, in addition, quinone-modified protein is thought to be less attractive or palatable to herbivores, thereby discouraging feeding. The ability of PPOs to covalently modify plant proteins upon wounding has led to their designation as being "antinutritive enzymes" which function in plant pest resistance in a manner complementary to the inducible proteinase inhibitors of plants.

PPO cDNAs can be ligated into an array of vectors used for transformation of plants (for example, vectors based on Agrobacterium tumefaciens [see Nature 310:115]) to achieve either overexpression or down-regulation of PPO. Overexpression is achieved by ligation of the PPO cDNA in a "sense" orientation using promoters such as the 35S promoter of cauliflower mosaic virus [see Plant Cell 2:7]. Alternatively, down-regulation of PPO can be obtained by ligating the PPO cDNA, or some fragment of this sequence, to a similar promoter in an "antisense" orientation [see Plant Molecular Biology 11 :301 ; Gene 72:45; Plant Molecular Biology 14:457; Cell 55:673; and Nature

334:724]. Down-regulation may also be obtained from the "sense" constructs by the phenomenon known as co-suppression [see Plant Cell 2:279-289; Plant Cell 2:291-299]. Tissue explants from plants are transformed using these vectors, subjected to selection for the presence of the integrated cassette, regenerated into plants [see Plant Cell Reports 8: 325], and analyzed for altered PPO expression . The following examples are provided for a more thorough understanding of the present invention. These examples are provided to demonstrate broad and various aspects of the present invention and are not meant to, nor should they be considered as to, limit the present invention to the specific conditions exhibited therein.

EXAMPLE I (Cloning of Polyphenol Oxidases) To clone PPO cDNAs by selection with antibodies, polyclonal antibodies were raised from purified PPO. A convenient source of PPO is Solarium berthaultii (wild potato) or Lycopersicon esculentum (tomato) which possess high densities of foliar glandular trichomes containing PPO as the dominant protein constituent (ca. 60%) of these organs [see J. Heredity 81 :475]. PPO was obtained from foliar glandular trichomes of Solarium berthaultii by wiping leaflets {ca. 4000) with a cotton swab saturated in 200 mM dithiothreitol. The crude trichome extract was squeezed from the swab using a syringe, and centrifuged for 10 min at 12,000 x g and 4° C. The supernatant was brought up to a volume of 30 ml with 2% pH 4-6 ampholyte and electrofocused in a preparative isoelectric focusing cell (Bio Rad Rotofor). Fractions from pH 4.5 to 6 were analyzed by electrophoresis on SDS-PAGE on a 10% polyacrylamide gel, followed by Coomassie staining to locate the protein. Fractions containing the purified 59 kD PPO were pooled and dialysed against two changes of 1 M NaCI (to remove ampholytes). The protein was then dialyzed against two changes of H2O, and lyophilized. The purified, lyophilized PPO (300 μg) was reconstituted in 500 μl H2O, emulsified with an equal volume of complete Freund's adjuvant, and injected into multiple subcutaneous sites on the back of two New Zealand White rabbits. Subsequent injections of 100 μg PPO in incomplete Freund's adjuvant were made at 15 and 21 d. The animals were exsanguinated and the antiserum collected 30 d after the final injection of PPO. Antiserum was stored at -80° C. - 0 -

Specificity of the antibodies were demonstrated by immunoblotting SDS-PAGE or isoelectric focusing gels. Prior to transblotting to nitrocellulose membranes, gels were equilibrated 20 min in a transfer buffer of 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3. Electrotransfer was accomplished at 100 V, 0.25 A for one hr. Non-specific binding to membranes was blocked, and the membranes washed, using conventional procedures [see Current Protocols in Molecular Biology (1987), ed F.M. Ausubel, John Wiley & Sons, New York; pp. 10.8.1-10.8.6]. Antibody was used at a dilution of 1 :4000, and the blots were developed with goat anti-rabbit alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate, 367 mN nitroblue tetzazolium, 0.1 M NaHCOβ, pH 9.8 [see Current Protocols in Molecular Biology (1987), ed F.M. Ausubel, John Wiley & Sons, New York; pp. 10.8.1 -10.8.6]. When PPO from potato or tomato trichomes, leaves or other organs were electrophoresed on SDS-PAGE or isoelectric focusing gels, electroblotted onto nitrocellulose and probed with these polyclonal rabbit anti-PPO antibodies, the anti-PPO serum detected tomato and potato PPO equally well.

To obtain PPO mRNA for construction and screening of a cDNA expression library, mRNA was obtained and verified to encode PPO.

Forceps were used to peel 40 g of the epidermis and outer tissue layers from leaflets and petioles of tomato {Lycopersicon esculentum cv. VFNT Cherry). Peeled tissue was immediately frozen in liquid N2. RNA was then extracted [see Analytical Biochemistry 162:156] and purified by oligo-dT cellulose column chromatography [see Molecular Cloning: A

Laboratory Handbook, 2nd ed. (1989), ed. J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press; pp 197-198] to yield 30 μg polyadenylated RNA. 5 μg of the mRNA was translated in vitro in the presence of 35s-methionine using a reticulocyte lysate in vitro translation system (following the manufacturer's instructions [Promega

Biotec]). The products of translation, when separated on 10% SDS-PAGE and autoradiographed overnight using Kodak XAR-5 film, did not clearly demonstrate the presence of the 59 kD PPO expected to be translated from mRNA of this tissue. Therefore, the translation products were subjected to immunoprecipitation. The translation reaction was diluted with nine volumes of 1% Nonidet P-40, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 1% methionine, 0.01 M sodium phosphate, ph 7.2. Twenty μl of 10% heat-killed Staphlococcus aυreus cells was added to the translation mix and shaken at 4° C for 1 h. The sample was then centrifuged at 13,000 x g for five min. The supernatant was then incubated with 1 μl of anti-PPO antiserum at 4° C overnight. Then 10 μl of 10% heat-killed Staphlococcus aureus cells was added and incubated at 4° C for one h with intermittent shaking. After centrifugation at 13,000 x g for five min the pellet was resuspended in 0.5 ml 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 1% methionine, 0.01 M sodium phosphate, ph 7.2 and centrifuged three times through a 0.5 ml pad of 10% sucrose (in 0.5 ml 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 1% methionine, 0.01 M sodium phosphate, ph 7.2) at 13,000 x g for five min. The pellet was then washed three times with 0.5 ml 1 % Nonidet P-40, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 1% methionine, 0.01 M sodium phosphate, ph 7.2. The immunoprecipiate (pellet) was then loaded onto 10% SDS-PAGE, electrophoresed, and autoradiographed overnight. This experiment showed that a product of 67 kD was precipitated by the anti-PPO. There was no evidence for a protein of 59 kD as would be expected from the Mr of the PPO in the glandular trichome.

To determine whether the 67 kD protein immunoprecipitated was a precursor to the 59 kD PPO, a series of peptide mapping experiments were conducted using N-chlorsuccinimide and formic acid digestion [see Anal. Biochem. 122:298, and Anal. Biochem. 127:453]. Both N- chlorosuccinimide and formic acid were used to partially digest the mature 59 kD PPO from glandular trichomes and the ³⁵S-labelled, immunoprecipitated 67 kD band from the in vitro translation. When the digestion products were electrophoresed, both the mature 59 kD PPO and the 67 kD translation product were found to share common fragment polypeptides. This result strongly suggested that the 59 kD PPO is initially translated as a 67 kD precursor polypeptide, and then processed proteolytically to its mature Mr of 59,000. A cDNA library was then constructed in λ ZAP (following the manufacturer's instructions [Stratagene]) using 2 μg polyadenylated tomato RNA, and was screened using a 1 : 4000 dilution of polyclonal anti-S. berthaultii PPO following conventional techniques [see Molecular Cloning: A Laboratory Handbook, pp 12.16-12.20; Methods of Enzymology, Vol. 152)]. After screening of 3 X 10⁵ plaques with PPO antibody, 12 candidates remained positive after quaternary screening. Restriction mapping of the candidate cDNAs showed that all 12 clones belonged to one of two different cDNA classes. Both classes of cDNA hybridized to bands of ca. 2.0 Kb on Northern blots [see Molecular Cloning: A Laboratory Handbook, pp 7.43-7.45] of tomato leaf and epidermal mRNA. The longest cDNA, however, was only 1.7 kbp, and a second screening of the library did not yield longer clones. A second λ ZAP II (Stratagene) cDNA library was therefore constructed using a new mRNA preparation. The library was screened, following conventional techniques (see Molecular Cloning: A Laboratory Handbook, pp 2.108- 2.117, and the manufacturer's instructions), using a 0.7 kb truncated PPO cDNA obtained from the first library. After quaternary screening, two classes of PPO remained, (pPPO-T1 and pPPO-T2) both of a size approaching 2.0 kbp. Although both cDNAs hybridized with ca. 2.0 kb mRNA species on Northern blots made from epidermal or leaf mRNA, Northern blots conducted using tissue prints (see Plant Molecular Biology 12: 517-524) of stem sections showed that pPPO-T1 is expressed in trichome and epidermal cells, and pPPO-T2 is expressed in photosynthetic cells.

The DNA sequence of tomato PPO cDNA pPPO-T1 , according to the present invention, is as follows:

ATG TCT TCT TC CT TCT ATT ACT ACT ACT CTT CCT TTA 39 TGC ACC AAC AAA TCC CTC TCT TCT TCC TTC ACC ACC ACC 78 AAC TCA TCC TTG TTA TCA AAA CCC TCT CAA CTT TTC CTC 117

CAC GGA AGG CCT AAT CAA ACT TTC AAG GTT TCA TGC AAC 156 GCA AAC AAC CTT GAC AAA AAC CCT GAC GCT GTT GAT AGA 195 CGA AAC CTT CTT TTA GGG TTA GGA GCT CTT TAT GCT GCA 234 GCT AAT CTT GCA CCA TTA GCG ACT GCT GCA CCT ATA CCA 273 AAT ACT AAT CAT GTT ACC AGT GTT ACT TTC AAG CTG GCG 1638 ATA ACT GAA CTG TTG GAG GAT ATT GGA TTG GAA GAT GAA 1677 GAT ACT ATT GCG GTG ACT TTG GTT CCA AAA GCT GGC GCT 1716 GAA GAA GTG TCC ATT GAA AGT GTG GAG ATC AAG CTT GAG 1755 GAT TCT 1761

In addition to the reading frame given above, the pPPO-T1 gene was sequenced with an upstream portion of the following sequence immediately prior to the ATG start codon: GGAATTCGGC ACGAGCTCCA TCACAACACA 30. In addition to the upstream portion, a downstream portion was also sequenced according to the following:

TAAAGTCTGC ATGAGTTGGT GGCTATGGTG CCAAATTTTA TGTTTAATTA 50 GTATAATTAT GTGTGGTTTG AGTTATGTTT TGTTAAAA TGTATCAGCT 100 CGATCGATAG CTGATTGCTA GTTGTGTTAA TGCTATGTAT G 141 This cDNA sequence provides the following deduced amino acid sequence for the pPPO-T1 peptide:

Met Ser Ser Ser Ser Ser He Thr Thr Thr Leu Pro Leu Cys Thr

5 10 15

Asn Lys Ser Leu Ser Ser Ser Phe Thr Thr Thr Asn Ser Ser Leu 20 25 30

Leu Ser Lys Pro Ser Gin Leu Phe Leu His Gly Arg Arg Asn Gin

35 40 45

Ser Phe Lys Val Ser Cys Asn Ala Asn Asn Val Asp Lys Asn Pro

50 55 60 Asp Ala Val Asp Arg Arg Asn Val Leu Leu Gly Leu Gly Gly Leu

65 70 75

Tyr Gly Ala Ala Asn Leu^'Ala Pro Leu Ala Thr ALa Ala Pro He

80 85 90

Pro Pro Pro Asp Leu Lys Ser Cys Gly Thr Ala His Val Lys Glu 95 100 105

Gly Val Asp Val He Tyr Ser Cys Cys Pro Pro Val Pro Asp Glu

110 115 120

He Asp Ser Val Pro Tyr Tyr Lys Phe Pro Ser Met Thr Lys Leu

125 130 135 Arg He Arg Pro Pro Ala His Ala Ala Asp Glu Glu Tyr Val Ala

140 145 150

Lys Tyr Gin Leu Ala Thr Ser Arg Met Arg Glu Leu Asp Lys Asp

155 160 165

Pro Phe Asp Pro Leu Gly Phe Lys Gin Gin Ala Asn He His Cys

500 505 510

Asn Ala Asp Glu Leu Asp Lys Ala Glu Phe Ala Gly Ser Tyr Thr

515 520 525

Ser Leu Pro His Val His Gly Ser Asn Thr Asn His Val Thr Ser 530 535 540

Val Thr Phe Lys Leu Ala He Thr Glu Leu Leu Glu Asp He Gly

545 550 555

Leu Glu Asp Glu Asp Thr He Ala Val Thr Leu Val Pro Lys Ala

560 565 570 Gly Gly Glu Glu Val Ser He Glu Ser Val Glu He Lys Leu Glu

575 - 580 585

Asp Cys 587

The DNA sequence of tomato PPO cDNA pPPO-T2, according to the present invention, is as follows:

ATG GCA ACT GTA GTG TGC AAT AGT AGT AGT AGT ACT ACT 39

ACT ACA ACG CTC AAA ACT CCT TTT ACT TCT TTA GCT TCC 78

ACT CCT AAG CCC TCT CM CTT TTC CTT CAT GGA AAA CGT 117

AAC AAA ACA TTC AAA GTT TCA TGC AAG GTT ATC AAT AAT 156 AAC GCT AAC CAA GAT GAA ACG AAT TCT GTT GAT CGA AGG 195

AAT GTT CTT CTT GCT TTA GGA GCT CTT TAT GCT CTT GCT 234

AAT GCT ATA CCA TTA GCG GCA TCG GCT ACT CCT ATT CCA 273

TCC CCT GAT CTC AAA ACT TCT GCT AGA GCC ACC ATA TCG 312

GAT GCT CCA CTT CTA CCC TAT TCT TGT TGT CCC CCT CCT 351 ATG CCG ACT AAC TTT GAC ACC ATT CCA TAT TAC AAG TTC 390

CCT TCT ATG ACT AAA CTC CCT ATC CGT ACC CCT GCT CAT 429

GCT GTA GAT GAG GAG TAT ATC GCG AAG TAT AAT TTG GCC 468

ATA ACT CGA ATG AGG GAT CTT GAC AAG ACA GAA CCG TTA 507

AAC CCT CTA GGG TTT AAG CAA CAA GCT AAT ATA CAC TGT 546 GCT TAT TCT AAC GGT GCT TAT ATA ATT GGT GGC AAA GAG 585

TTA CAA GTT CAT AAC TCG TGG CTT TTC TTC CCG TTC CAT 624

CGA TGG TAC TTC TAC TTT TAC GAA AGA ATA TTG GGG AAA 663

CTC ATT GAT GAT CCA ACT TTC GCT TTA CCA TAC TGG AAT 702

TGG GAT CAT CCA AAG GGC ATG CGT TTA CCT CCC ATG TTC 741 GAT CCT GAA GCT TCT TCC CTC TAC GAT GAA AGG CGT AAT 780

CAA CAA GTC CCT AAT GGA ACG GTT TTG GAT CTT GGT TCA 819

TTT GGG GAT AAA GTT GAA ACA ACT CAA CTC CAG TTG ATG 858 AGC AAT AAT TTA ACC CTA ATG TAC CGT CAA ATG GTA ACT 897 AAT GCT CCA TCT CCT CTC TTG TTC TTC GGT GCG CCT TAC 936 GTT CTT GGG AAT AAC GTT GAA GCA CCG GGA ACC ATT GAA 975 ACC ATC CCT CAT ATT CCT CTA CAT ATT TGG GCT GGT ACT 1014 CTC CGT GGT TCA AAA TTT CCT AAC GCT GAT GTG TCC TAC 1053 GCT GAG GAT ATG GCT AAT TTC TAC TCA GCT GCT TTG GAC 1092 ^' CCG GTT TTC TAT TGC CAT CAC GGC AAT GTG GAC CGG ATG 1131 TGG AAC GAA TGG AAG GCA ATA GGA GGT AAA AGA AGA GAT 1170 ATA TCT GAA AAG GAT TGG TTG AAC TCC GAG TTC TTT TTC 1209 ^' TAC GAC GAA CAC AAA AAT CCT TAC CGT CTG AAA CTC AGG 1248 GAC TGT TTG GAC ACG AAG AAA ATG GGG TAT GAT TAC GCA 1287 CCA ATG CCA ACT CCA TGG CCT AAT TTC AAA CCA AAA TCA 1326 AAG GCG TCC GTA GGG AAA GTG AAT ACA AGT ACA CTC CCC 1365 CCA GCA AAC GAG GTA TTC CCA CTC GCG AAG ATG GAT AAG 1404 ACT ATT TCA TTT GCT ATC AAC AGG CCA GCT TCA TCG CGG 1443 ACT CAA CAA GAG AAA AAT GAA CAA GAG GAG ATG TTA ACG 1482 TTC AAT AAC ATA AGA TAT GAT AAC AGA GGG TAC ATA AGG 1521 TTC GAT GTG TTC CTG AAC GTG GAC AAC AAT GTG AAC GCG 1560 AAT GAG CTT GAT AAG GCA GAG TTC GCG GGG ACT TAT ACT 1599 - ACT TTG CCA CAT GTT CAC AGA GCT GGC GAG AAT GAT CAT 1638 ATC GCG AAG GTT AAT TTC CAG CTG GCG ATA ACA GAA CTG 1677 TTC GAG GAC ATT GCT TTG GAA GAT GAA GAT ACT ATC GCG 1716 GTG ACT CTG GTA CCA AAG AAA GGC GGT GAA GCT ATC TCC 1755 ATT GAG AAT GTG GAG ATC AAG CTT GTG GAT TGT 1788 In addition to the reading frame given above, cDNA pPPO-T2 was sequenced with an upstream portion of the following sequence immediately prior to the ATG start codon: TAATTCGGCA CGAGAGCA 18

In addition to the upstream portion, a downstream portion was also sequenced according to the following:

TAACTCTCAA TTG TTTGC TGAGATTACA ATTATGATGG ATGATGATAT 50 GTTTTTATCT TALTTITGTT CTGTTATCTA CTTTTGCTTT TCTCGTGTAA 100 CTΠTCCTGT TGAΆATCACC CTACATGCTT GATTTCCTTG GAGTTGTTAT 150 TCACTAATAA ATCAGTTAGG TTAAAAAAAA AAAAAAAAA 189 This cDNA sequence provides the following deduced amino acid sequence for the pPPO-T2 peptide:

Met Ala Ser Val Val Cys Asn Ser Ser Ser Ser Thr Thr Thr Thr

5 10 15 Thr Leu Lys Thr Pro Phe Thr Ser Leu Gly Ser Thr Pro Lys Pro

20 25 30

Ser Gin Leu Phe Leu His Gly Lys Arg Asn Lys Thr Phe Lys Val

35 40 45

Ser Cys Lys Val He Asn Asn Asn Gly Asn Gin Asp Glu Thr Asn 50 55 60

Ser Val Asp Arg Arg Asn Val Leu Leu Gly Leu Gly Gly Leu Tyr

65 70 75

Gly Val Ala Asn Ala He Pro Leu Ala Ala Ser Ala Thr Pro He

80 85 90 Pro Ser Pro Asp Leu Lys Thr Cys Gly Arg Ala Thr He Ser Asp

95 100 105

Gly Pro Leu Val Pro Tyr Ser Cys Cys Pro Pro Pro Met Pro Thr

110 115 120

Asn Phe Asp Thr He Pro Tyr Tyr Lys Phe Pro Ser Met Thr Lys 125 130 135

Leu Arg He Arg Thr Pro Ala His Ala Val Asp Glu Glu Tyr He 140 145 150

Ala Lys Tyr Asn Leu GCC He Ser Arg Met Arg Asp Leu Asp Lys 155 160 165 Thr Glu Pro Leu Asn Pro Leu Gly Phe Lys Gin Gin Ala Asn He

170 175 180

His Cys Ala Tyr Cys Asn Gly Ala Tyr He He Gly Gly Lys Glu

185 190 195

Leu Gin Val His Asn Ser Trp Leu Phe Phe Pro Phe His Arg Trp 200 205 210

Tyr Phe Tyr Phe Tyr Glu Arg He Leu Gly Lys Leu He Asp Asp 215 220 225

Pro Thr Phe Ala Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly 230 235 240 Met Arg Leu Pro Pro Met Phe Asp Arg Glu Gly Ser Ser Leu Tyr

245 250 255

Asp Glu Arg Arg Asn Gin Gin Val Arg Asn Gly Thr Val Leu Asp

260 265 270

Leu Gly Ser Phe Gly Asp Lys Val Glu Thr Thr Gin Leu Gin Leu 275 280 285

Met Ser Asn Asn Leu Thr Leu Met Tyr Arg Gin Met Val Thr Asn 290 295 300 Ala Pro Cys Pro Leu Leu Phe Phe Gly Ala Pro Tyr Val- Leu Gly

305 310 315

Asn Asn Val Glu Ala Pro Gly Thr He Glu Thr He Pro His He

320 325 330 Pro Val His He Trp Ala Gly Thr Val Arg Gly Ser Lys Phe Pro

335 340 345

Asn Gly Asp Val Ser Tyr Gly Glu Asp Met Gly Asn Phe Tyr Ser

350 355 360

Ala Gly Leu Asp Pro Val Phe Tyr Cys His His Gly Asn Val Asp 365 370 375

Arg Met Trp Asn Glu Trp Lys Ala He Gly Gly Lys Arg Arg Asp

380 385 390

He Ser Glu Lys Asp Trp Leu Asn Ser Glu Phe Phe Phe Tyr Asp

395 400 405 Glu His Lys Asn Pro Tyr Arg Val Lys Val Arg Asp Cys Leu Asp

410 415 420

Thr Lys Lys Met Gly Tyr Asp Tyr Ala Pro Met Pro Thr Pro Trp

425 430 435

Arg Asn Phe Lys Pro Lys Ser Lys Ala Ser Val Gly Lys Val Asn 440 445 450

Thr Ser Thr Leu Pro Pro Ala Asn Glu Val Phe Pro Leu Ala Lys

455 460 465

Met Asp Lys Thr He Ser Phe Ala He Asn Arg Pro Ala Ser Ser

470 475 480 Arg Thr Gin Gin Glu Lys Asn Glu Gin Glu Glu Met Leu Thr Phe

485 490 495

Asn Asn He Arg Tyr Asp Asn Arg Gly Tyr He Arg Phe Asp Val

500 505 510

Phe Leu Asn Val Asp Asn Asn Val Asn Ala Asn Glu Leu Asp Lys 515 520 525

Ala Glu Phe Ala Gly Ser Tyr Thr Ser Leu Pro His Val His Arg

530 535 540

Ala Gly Glu Asn Asp His He Ala Lys Val Asn Phe Gin Leu Ala

545 550 555 He Thr Glu Leu Leu Glu Asp He Gly Leu Glu Asp Glu Asp Thr

560 565 570

He Ala Val Thr Leu Val Pro Lys Lys Gly Gly Glu Gly He Ser

575 580 585

He Glu Asn Val Glu He Lys Leu Val Asp Cys 590 595 596

The pPPO-T1 and pPPO-T2 cDNAs were then subjected to in vitro transcription and translation to determine whether they were capable of encoding the 67 kD polypeptides expected for cDNAs encoding PPO. After linearization of the excised pBluescript-containing cDNAs with Xho I, RNA was transcribed using T3 RNA polymerase and capped (following the manufacturer's instructions [Stratagene]). The resulting pPPO-T1 and pPPO-T2 mRNA (0.5 μg) were translated separately in vitro in the presence of ³⁵S-methionine using a reticulocyte lysate in vitro translation system (see above). The products of translation were separated by SDS-PAGE on a 10% acrylamide gel. After electrophoresis the gel was dried and exposed for autoradiography overnight. The primary translation product of pPPO-T2 was the 67 kD polypeptide shown earlier to be the precusor to the 59 kD mature PPO seen in immunoprecipitations of mRNA from the outer tissues of tomato foliage.

The in vitro transcription/translation results from the pPPO-T1 cDNA were less clear, as it translated several products. This cDNA was later shown, by sequencing of its genomic counterpart, to be slightly less than full-length. Absence of a starting methionine codon in the truncated cDNA apparently led to initiation of translation at downstream Met codons, accounting for the multiple PPO products.

The identification of the cDNA candidates as polyphenoloxidases was further confirmed by comparision of their deduced amino acid sequences to those obtained from sequencing PPO proteins from various sources. Glandular trichomes were harvested (see above) from foliage of Lycopersicon esculentum cv.s VFNT, Freedom, Lycopersicon cheesmanii, L. chmielewskii, and Soianυm berthaultii by wiping leaves with a cotton swab saturated in 200 mM dithiothreitol. The trichome extract was squeezed from the swab using a syringe, and 1/10 volume of ice-cold trichloacetic acid was added to precipitate the proteins. The precipitate was suspended in 200 μl 0.5% SDS (w/v), 1.25% β- mercaptoethanol in 25 mM Tris, pH 7.5, and electrophoresed on a 10% SDS-PAGE gel system. After electrophoresis the gel was transblotted onto PVDF membrane (Immobilon-P) in 10 mM CAPS, pH 11 , 10% MeOH. After transfer the membrane was stained with Coomassie Blue R-250 and destained. The PPO-containing bands, located by mol wt, were excised and the N-terminal amino acid sequence was obtained by microsequencing (see Guide to Protein Purification, ed. M.P. Deutscher,

Academic Press, San Diego; pp.602-613). The N-terminal sequences were as follows:

L. esculentum cv.VFNT: Ala Pro He Pro Pro Pro Asp Leu Lys Ser Gly Gly Thr Ala

5 10 14

L. esculentum cv. Freedom:

Ala Pro He Pro Pro Pro Asp Leu Lys Ser Asp Xaa Thr Ala

5 10 14 (Xaa= unable to assign residue)

L. cheesmanii:

Ala Pro He Pro Pro Pro Asp Leu Lys Ser Gin Gly Thr Ala His

5 10 15

L. chmielewskii: Ala Pro He Pro Pro Pro Asp Leu Lys Ser Gin Gly Thr Ala His

5 10 15

S. berthaultii:

Ser Pro He Pro Pro Pro Asp Leu Lys Ser Xaa Gly Val Ala His

5 10 15 Tyr Lys Glu Pro

19

(Xaa= unable to assign residue)

The N-terminal amino acid sequence of the mature tomato (L. esculentum cv.VFNT) 59 kD PPO from glandular trichomes is also present within the deduced amino acid sequence of the pPPO-T1 cDNA. Similar deduced amino acid sequence is present in the tomato cDNA pPPO-T2 and the potato cDNAs pPPO-P1 and pPPO-P2 (see below). The position of this sequence within the clones leads to deduced masses for the precursor protein and mature PPO polypeptide of ca. 67 and 59 kD, respectively. pPPO-T1 and pPPO-T2 show 78% nucleic acid sequence identity, and when compared over the length of the 67 kD precursor polypeptide possess 84% deduced amino acid similarity and 76% deduced amino acid identity. The genomic clones representing these cDNAs were recovered from a λ Ch35 library constructed from tomato {Lycopersicon esculentum cv. VFNT Cherry) and screened with tomato PPO cDNA, using conventional methods (see Molecular Cloning: A Laboratory Handbook, pp 2.108-2.1 13).

In addition, the 0.7 kb truncated tomato PPO cDNA was used to probe a potato leaf cDNA library, also constructed in λ ZAP II. Screening of 2 x 10^ plaques led to the purification of three PPO cDNA candidates, two of which were about 2.0 Kbp in length, and a third truncated at about 1.2 Kbp. Northern analysis of a number of potato tissue explants including leaves, roots, flowers, tubers and petioles showed the presence of a single PPO class of 2.0 kb. DNA sequencing and comparison of the two longest potato PPO cDNAs (pPPO-P1 and pPPO-P2) showed 96% nucleic acid and deduced amino acid identity, and high sequence similarity to the tomato PPO cDNAs at both nucleic acid and deduced amino acid levels.

The DNA sequence of potato PPO cDNA pPPO-P1 , according to the present invention, is as follows:

TCT TCT TCT ACT ACT ACT ACT ATT CCA TTA TGC ACC AAC 39 AAA TCC CTC TCT TCT TCC TTC ACC ACC AAC AAC TCA TCT 78 TTC TTA TCA AAA CCC TCT CAA CTT TTC CTC CAC GGA AGG 117 CCT AAT CAA ACT TTC AAG GTT TCA TGC AAC GCC AAC AAT 156 AAT GTT GGC GAG CAT GAC AAA AAC CTT GAC ACT GTT GAT 195 AGG CGA AAT GTT CTT TTA GGG TTA GGA GGT CTT TAT GCT 234 GCT GCT AAT CTT GCA CCA TTA GCC TCT GCT TCT CCT ATA 273 CCA CCT CCT GAT CTA AAA TCT TGT GCT GTT GCC CAT GTA 312 ACA GAA GCT GTT GAT GTG ACA TAT ACT TGT TGC CCA CCT 351 GTA CCC GAT GAT ATC GAT AGC GTT CCG TAC TAC AAG TTC 390

CCT CCT ATG ACT AAA CTC CGC ATC CGC CCC CCT GCT CAT 429 GCG GCG GAT GAG GAG TAT GTA GCC AAG TAT CAA TTG GCT 468 ACG ACT CGA ATG AGG GAA CTT GAT AAA GAC TCT TTT GAC 507 CCT CTT GGG TTT AAA CAA CAA GCT AAT ATT CAT TGT GCT 546 TAT TCT C GCT GCT TAT AAA GTT GCT GCT AAA GAG TTG 585 CAA GTT CAT TTC TCG TGG CTT TTC TTT CCG TTT CAT AGA 624 TGG TAC TTG TAT TTC TAT GAA AGA ATA TTG GGA TCA CTT 663 ATT AAT GAT CCA ACT TTT GCT TTA CCA TAT TGG AAT TGG 702 GAT CAT CCA AAA GCT ATG CCT ATA CCT CCC ATG TTT GAT 741 CGT GAG GGG TCA TCT CTT TAC GAT GAT AAA CCT AAC CAA 780 AAC CAT CGC AAT GGA ACT ATT ATT GAT CTT GCT CAT TTT 819 GGT CAG GAA GTT GAC ACA CCT CAG CTT CAG ATA ATG ACT 858 AAT AAT TTA ACA CTA ATG TAC CCT CAA ATG GTC ACT AAT 897 GCT CCT TCT CCG TCC CAA TTC TTC GGT GCT GCT TAC CTC 936 TGG GGA CTC AAC CM GTC CAG G TGG GTA CTA TTG AGA 975 ACA TCC CTC ATA CCC CCT GCC ATA TCT GGA CTG CTG ATA 1014 GTC CTA GAC AAA AAA ACG CTG AAA ACA TGG GTA ATT TCT 1053 ATT CAG CAC GCT TTA GAC CCG ATT TTT TAC TCT CAC CAC 1092 GCA AAT GTG GAC CGG ATG TGG GAT GAA TGG AAA TTA ATT 1131 GGC GGG AAA AGA AGG GAT CTA TCA AAT AAA GAT TGG TTG 1170 AAC TCA GAA TTC TTT TTC TAC GAT GAA AAT CGC AAC CCT 1209 TAC CCT GTG AAA CTC CCT GAC TCT TTG GAC ACT AAA AAA 1248 ATG GGA TTC ACT TAC GCT CCA ATG CCA ACT CCA TGG CCT 1287 AAT TTT AAA CCA ATC AGA AAA ACT ACA GCA GGA AAA GTG 1326

AAT ACA GCG TCA ATT GCA CCA GTC ACC AAG GTC TTC CCA 1365 CTA GCG AAG CTG GAC CCT GCA ATT TCG TTC TCT ATC ACC 1404 AGA CCA GCT TCG TCA AGG ACT ACA CAG GAG AAA AAT GAG 1443 CAA GAG GAG ATA CTG ACA TTC AAC AAA CTA GCC TAT GAT 1482 GAT ACT AAG TAT GTA AGG TTC GAT GTG TTC CTG AAC GTT 1521

GAC AAG ACT GTG AAT GCG GAT GAG CTT GAT AAG GCG GAG 1560 TTT GCG GGG ACT TAT ACT AGC TTG CCG CAT GTT CAT GGA 1599 AAT AAT ACT AAT CAT GTT ACG ACT GTT ACT TTC AAG CTG 1638 GCG ATA ACA GAA CTG TTG GAG GAT AAT GGA TTG GAA GAT 1677 GAA GAT ACT ATT GCG GTA ACT TTG GTT CCA AAA GTT GCT 1716

GCT GAA GCT GTA TCC ATT GAA ACT GTG GAG ATC AAG CTT 1755 GAG GAT TCT 1764

In addition, a downstream portion was also sequenced according to the following: TAAGTCCTCA TGAGTTGGTC GCTATGCTAC CA TTTTAT GTTTAATTAT 50

AmATGTCT GTGTTTGATT ATGTTTCGCT TAAAATGTAT CAGCTGGATA 100 GCTGATTACT AGCCTTCCCA GTTGTTAATC CTATCTATGA AATAAATAAA 150 TAAATGGTTG TCTTCCATTT AATTTTAAAA AAAAAAAAAA AAAAAAAAAA 200 AAAAAAAAAA AAAAAAAAAA AA 222 This cDNA sequence provides the following deduced amino acid sequence for the pPPO-P1 peptide:

Ser Ser Ser Ser Thr Thr Thr He Pro Leu Try Thr Asn Lys Ser

5 10 15 Leu Ser Ser Ser Phe Thr Thr Asn Asn Ser Ser Phe Leu Ser Lys

20 25 30

Pro Ser Gin Leu Phe Leu His Gly Arg Arg Asn Gin Ser Phe Lys

35 40 45

Val Ser Try Asn Ala Asn Asn Asn Val Gly Glu His Asp Lys Asn 50 55 60

Leu Asp Thr Val Asp Arg Arg Asn Val Leu Leu Gly Leu Gly Gly

65 70 75

Leu Try Gly Ala Ala Asn Leu Ala Pro Leu Ala Ser Ala Ser Pro

80 85 90 He Pro Pro Pro Asp Leu Lys Ser Cys Gly Val Ala His Val Thr

95 100 105

Glu Gly Val Asp Val Thr Try Ser Cys Try Pro Pro Val Pro Asp

110 115 120

Asp He Asp Ser Val Pro Tyr Tyr Lys Phe Pro Pro Met Thr Lys 125 130 135

Leu Arg He Arg Pro Pro Ala His Ala Ala Asp Glu Glu Try Val

140 145 150

Ala Lys Try Gin Leu Ala Thr Ser Arg Met Arg Glu Leu Asp Lys

155 160 165 Asp Ser Phe Asp Pro Leu Gly Phe Lys Gin Gin Ala Asn He His

170 175 180

Cys Ala Try Cys Asn Gly Ala Try Lys Val Gly Gly Lys Glu Leu

185 190 195

Gin Val His Phe Ser Trp Leu Phe Phe Pro Phe His Arg Trp Tyr 200 205 210

Leu Try Phe Try Glu Arg He Leu Gly Ser Leu He Asn Asp Pro 215 220 225

Thr Phe Ala Leu Pro Try Trp Asn Trp Asp His Pro Lys Gly Met 230 235 240 Arg He Pro Pro Met Phe Asp Arg Glu Gly Ser Ser Leu Tyr Asp

245 250 255

Asp Lys Arg Asn Gin Asn His Arg Asn Gly Thr He He Asp Leu

260 265 270

Gly His Phe Gly Gin Glu Val Asp Thr Pro Gin Leu Gin He Met 275 280 285

Thr Asn Asn Leu Thr Leu Met Tyr Arg Gin Met Val Thr Asn Ala 290 295 300 Pro Cys Pro Ser Gin Phe Phe Gly Ala Ala Tyr Leu Trp Gly Leu

305 310 315

Asn Gin Val Gin Glu Trp Val Leu Leu Arg Thr Ser Leu He Pro

320 325 330 Arg Ala He Ser Gly Leu Val He Val Leu Asp Lys Lys Thr Val

335 340 345

Lys Thr Trp Val He Ser He Gin His Gly Leu Asp Pro He Phe

350 355 360

Tyr Cys His His Ala Asn Val Asp Arg Met Trp Asp Glu Trp Lys 365 370 375

Leu He Gly Gly Lys Arg Arg Asp Leu Ser Asn Lys Asp Trp Leu

380 385 390

Asn Ser Glu Phe Phe Phe Tyr Asp Glu Asn Arg Asn Pro Tyr Arg

395 400 405 Val Lys Val Arg Asp Cys Leu Asp Ser Lys Lys Met Gly Phe Ser

410 415 420

Tyr Ala Pro Met Pro Thr Pro Trp Arg Asn Phe Lys Pro He Arg

425 430 435

Lys Thr Thr Ala Gly Lys Val Asn Thr Ala Ser He Ala Pro Val 440 445 450

Thr Lys Val Phe Pro Leu Ala Lys Leu Asp Arg Ala He Ser Phe 455 460 465

Ser He Thr Arg Pro Ala Ser Ser Arg Thr Thr Gin Glu Lys Asn 470 475 480 Glu Gin Glu Glu He Leu Thr Phe Asn Lys Val Ala Try Asp Asp

485 490 495

Thr Lys Try Val Arg Phe Asp Val Phe Leu Asn Val Asp Lys Thr

500 505 510

Val Asn Ala Asp Glu Leu Asp Lys Ala Glu Phe Ala Gly Ser Try 515 520 525

Thr Ser Leu Pro His Val His Gly Asn Asn Thr Asn His Val Thr

530 535 540

Ser Val Thr Phe Lys Leu Ala He Thr Glu Leu Leu Glu Asp Asn

545 550 555 Gly Leu Glu Asp Glu Asp Thr He Ala Val Thr Leu Val Pro Lys

560 565 570

Val Gly Gly Glu Gly Val Ser He Glu Ser Val Glu He Lys Leu

575 580 585

Glu Asp Cys 588

The DNA sequence of potato PPO cDNA pPPO-P2, according to the present invention, is as follows:

ACT ACT CTT CCA TTA TGC MC AAC AAA TCC CTC TCT TCT 39 CCT GCG ATT TCA TTC TCT ATC ACC AGA CCA GCT TCG TCA 1404 AGG ACT ACG CAG GAG AAA AAT GAG CAA GAG GAG ATA CTG 1443 ACA TTC AAA AAG ATA GCC .TAT GAT GAT ACT CAG TAT GTA 1482 AGG TTC GAT GTG TTC CTG AAC GTT GAC AAG ACT GTG AAT 1521 GCG GAT GAG CTT GAT AAG GCA GAG TTT GCG GGG AGT TAT 1560 ACT AGC TTG CCG CAT GTT CAT GGA AAT AAT ACT AAT CAT 1599 GCT ACG AGT GTT ACT TTC ACA GCT GGC ATA ACA GAA CTG 1638 TTG GAG GAT ATT GGA TTG GAA GAT GM GAT ACT ATT GCG 1677 GTA ACT TTG GTT CCA AAA GTA GCT GGT GAA GCT CTA TCC 1716 ATT GAA AGT GTG GAG ATC AAG CTT GAG GAT TGT 1749

In addition, a downstream portion was also sequenced according to the following:

TAAGTCCTCA TGAGTTGGTG GCTATGCTAC CAAATTTTAT GTTTAATTAG 50 TATTAATGTG TGTATCTGTT GATTATGTTT CGGGTAAAAT GTATCAGCTG 100 GATAGCTGAT TACTAGCCTT GCCAGTTGTT AATGCTATCT ATGAAATAAA 150

TAAATAAAAA AAAAAAAAAA AAA 173

This cDNA sequence provides the following deduced amino acid sequence for the pPPO-P2 peptide:

Thr Thr Leu Pro Leu Cys Asn Asn Lys Ser Leu Ser Ser Ser Phe 5 10 15

Thr Thr Asn Asn Ser Ser Phe Leu Ser Lys Pro Ser Gin Leu Phe

20 25 30

Leu His Gly Arg Arg Asn Gin Ser Phe Lys Val Ser Cys Asn Ala

35 40 45 Asn Asn Asn Val Gly Glu His Asp Lys Asn Leu Asp Ala Val Asp

50 55 60

Arg Arg Asn Val Leu Leu Gly Leu Gly Gly Leu Tyr Gly Ala Ala

65 70 75

Asn Leu Ala Pro Leu Ala Ser Ala Ser Pro He Pro Pro Pro Asp 80 85 90

Leu Lys Ser Cys Gly Val Ala His Val Lys Glu Gly Val Asp Val

95 100 105

Ser Tyr Ser Cys Cys Pro Pro Val Pro Asp Asp He Asp Ser Val

110 115 120 Pro Tyr Tyr Lys Phe Pro Ser Met Thr Lys Leu Arg He Arg Pro

125 130 135

Pro Ala His Ala Ala Asp Glu Glu Tyr Val Ala Lys Tyr Gin Leu

140 145 150

Leu Thr Phe Lys Lys He Ala Tyr Asp Asp Thr Gin Tyr Val Arg

485 490 495

Phe Asp Val Phe Leu Asn Val Asp Lys Thr Val Asn Ala Asp Glu

500 505 510 Leu Asp Lys Ala Glu Phe Ala Gly Ser Tyr Thr Ser Leu Pro His

515 520 525

Val His Gly Asn Asn Thr Asn His Ala Thr Ser Val Thr Phe Thr

530 535 540

Ala Gly He Thr Glu Leu Leu Glu Asp He Gly Leu Glu Asp Glu 545 550 555

Asp Thr He Ala Val Thr Leu Val Pro Lys Val Gly Gly Glu Gly

560 565 570

Val Ser He Glu Ser Val Glu He Lys Leu Glu Asp Cys

575 580 583 The sequence for pPPO-T1 given above includes several bp determined from sequencing its genomic clone. In both tomato and potato all PPO cDNAs map to chromosome 8. PPOs are encoded by a family of genes which typically specify the synthesis of precursor proteins of Mr ca. 67,000 which are then processed to a mature Mr of ca. 59,000. A number of posttranslational modification events such as crosslinking via quinones, glycosylation, and proteolysis (see Phytochemistry 18: 193-215, and Phytochemistry 26: 11-20) give rise to multiple Mr and pi species observed for mature PPOs.

EXAMPLE II (transformation of Potato to Achieve Altered PPO Expression)

The potato PPO cDNA (pPPO-P1) was excised from pBluescript using Sma I and Xho I. The resulting 5' overhang from the Xho I restriction digest was filled in using the Klenow fragment of DNA Pol I. The transformation construct pBI121 was restriction digested with Sma I and Sst I. The resulting 3' overhang was treated with T4 DNA polymerase to produce a blunt end. This procedure removed the endogenous GUS gene and prepared the vector for pPPO-P1 integration. The PPO insert and pBI121 prepared as described above were ligated overnight using T4 DNA ligase in a reaction employing a 2:1 ratio of insert to vector. The resulting recombinant pBI121/PPO plasmids were electroporated into £ coli strain DH5α. Plasmid was isolated from kan^r colonies and restriction digested with Bam HI/ Kpn I and Bam HI/ Pst I, to positively identify sense and antisense constructs, respectively. For both constructs, Hind lll/Xba I restriction digests were carried out to confirm the integrity of the CaMV 35S promoter of the vector. Sense and antisense PPO-containing plasmids were electroporated into Agrobacterium tumefaciens (PC2760) (Nucleic Acids Research 16: 6127-6145; Molecular and General Genetics 216:175-177; Nucleic Acids Research 17: 6747). Microtuber discs of potato {Solanum tuberosum L. cv. Atlantic) were inoculated for 15 min and then co-cultivated for two days with Agrobacterium cultures harboring either the pPPO-P1 sense or antisense constructs and then transferred to medium containing kanamycin. Plantlets regenerating from the tuber discs were removed and transferred to larger containers for growth (see Plant Cell Reports 8:325-328).

To determine endogenous PPO levels, leaf samples of non- transformed potato plants were homogenized in buffer containing 1.25 % β-mercaptoethanol and 0.5% SDS, boiled for 5 min, and loaded on 10% SDS-PAGE. Following electrophoresis, gels were eiectroblotted onto nitrocellulose and probed with polyclonal rabbit anti-PPO antibodies (see above), then developed with goat anti-rabbit alkaline phosphatase conjugate. Similar leaf extracts were made from regenerated plants transformed with the sense and antisense PPO constructs. Equal amounts of protein from non-transformed, PPO transformants and control plants (transformed with pBI121 alone) were loaded in each well of 10% SDS-PAGE gels. After electrotransfer and immunoblotting with polyclonal rabbit anti-PPO antibodies as described above, the results showed that those plants transformed with constructs containing PPO in either sense or antisense orientations possessed a range of altered PPO expression relative to non-transformed plants or control transformed with the pBI121 vector alone. The range of expression present in transformed plants was from several fold overexpression to nearly undetectable levels of PPO.

The cloning of tomato and potato PPO cDNAs, according to the present invention, provides a unique opportunity to investigate what have previously been the most intractable questions regarding PPO. As a result of the present invention, it is now possible to address the function and expression of PPOs by using transgenic plants over-and under-expressing PPO. Transgenic plants with altered PPO expression can be used to alter the economic impacts of PPO on the postharvest physiology of food plants and are similarly important in efforts to exploit PPO to increase the pest tolerance of crop plants.

The ability to alter PPO levels in commercially important plants should have utility in both increasing the pest tolerance of plants and decreasing the deleterious effect of PPO on crop quality. Increased PPO levels in foliage (or other non-food components) of crop plants should thus increase pest tolerance. Such an approach may have utility as a general method for crop protection. Alternatively, selective down- regulation of PPO expression in various plant organs could decrease the economic impact of an array of PPO-catalyzed browning syndromes and eliminate or reduce the need for antioxidant additives to preserve and/or process these commodities.

The sequence listing for the amino acid and nucleic acid sequences described herein is as follows:

SEQUENCE LISTING (1 ) GENERAL INFORMATION:

(i) APPLICANT: John C. Steffens

(ii) TITLE OF INVENTION: Polyphenol Oxidase

(iii)NUMBEROFSEQUENCES: 19

(2) INFORMATIONFORSEQIDNO:1: (i)SEQUENCECHARACTERISTICS:

(A) LENGTH: 1761 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

ATG TCT TCT TCT TCT TCT ATT ACT ACT ACT CTT CCT TTA 39

TGC ACC AAC AAA TCC CTC TCT TCT TCC TTC ACC ACC ACC 78

AAC TCA TCC TTG TTA TCA AAA CCC TCT CAA CTT TTC CTC 117 CAC GGA AGG CCT AAT CAA ACT TTC AAG GTT TCA TGC AAC 156

GCA AAC AAC GTT GAC AAA AAC CCT GAC GCT GTT GAT AGA 195 CGA AAC GH CH HA GGG TTA GGA GGT CLT TAT GGT GCA 234 GCT AAT CTT GCA CCA TTA GCG ACT GCT GCA CCT ATA CCA 273 CCT CCT GAT CTC AAG TCT TCT GGT ACT GCC CAT CTA AAA 312 GAA GCT GTT GAT GTA ATA TAC ACT TGT TGC CCT CCT GTA 351 CCC GAT GAG ATC GAT ACT CTT CCG TAC TAC AAG TTC CCT 390 TCT ATG ACT AAA CTC CGC ATC CGC CCC CCT GCT CAT GCG 429 GCG GAT GAG GAG TAC GTA GCC AAG TAT CAA TTG GCT ACG 468 ACT CGA ATG AGG GAA CTT GAT AAA GAC CCC TTT GAC CCT 507 CTT GGC TTT AAA CAA CAA GCT AAT ATT CAT TCT GCT TAT 546 TGC AAC GCT GCT TAC AM GTT GCT GGT AM G TTG CM 585 GTT CAT TTC TCG TGG CTT TTC TTT CCC TTT CAT AGA TGG 624 TAC TTG TAC TTT TAC GM AGA ATT TTG GGA TCA CTT ATT 663 AAT GAT CCA ACT TTT GCT TTA CCT TAC TGG AAT TGG GAT 702 CAT CCA AM GGC ATG CCT ATA CCT CCC ATG TTT GAT CCT 741 GAG GGA TCA TCT CTT TAC GAT GAG AM CGT AAC CM AAT 780

CAT CGC AAT GGA ACT ATT ATT GAT CTT GCT CAT TTT GCT 819 AAG GAA GTT GAC ACA CCT CAG CTA CAG ATA ATG ACT AAT 858 AAT TTA ACC CTA ATG TAC CCT CAA ATG GTT ACT MT GCT 897 CCT TGC CCT TCC CM TTC TTC GCT GCT GCT TAC CCT CTC 936 GCT TCT GM CCA ACT CCG GCT CAG GGT ACT ATT GM MC 975 ATC CCT CAT ACT CCG GTT CAC ATC TGG ACC GCT GAC AM 1014 CCT CCT CM AM AAC GCT GM GAC ATG GCT AAT TTC TAC 1053 TCA CCC GCT TTA GAT CCG ATT TTT TAC TGC CAC CAT GCC 1092 AAT GTG GAC AGG ATG TGG MT GAA TGG AM TTA ATT GGC 1131 GGG AAA AGA AGG GAT TTA ACA GAT AM GAT TGG TTG AAC 1170

TCT GAA TTC TTT TTC TAC GAT GM AAT CCT AAC CCT TAC 1209 CCT GTG AAG TCC GTA GAC TGT TTG GAC AGT AM MA ATG 1248 GGA TTC GAT TAC GCG CCA ATG CCC ACT CCA TGG CCT MT 1287 TTT AM CCA ATC AGA AAG TCA TCA TCA GGA AM CTG AAT 1326 ACA GCG TCA ATT GCA CCA GTT AGC MG GTG TTC CCA TTG 1365

GCG AAG CTC GAC CCT GCG ATT TCG TTC TCT ATC ACG CGG 1404 CCA GCC TCG TCA AGG ACA ACA CM GAG AM AAT GAG CM 1443 GAG GAG ATA CTC ACA TTC AAT AM ATA TCG TAT GAT GAT 1482 AGG AAC TAT GTA AGG TTC GAT GTC TTT CTG AAC GTG GAC 1521 AAG ACT GTG AAT GCA GAT GAG CTT GAT AAG GCG GAG TTT 1560

GCA GGG ACT TAT ACT AGC TTG CCG CAT GTT CAT GGA ACT 1599 AAT ACT AAT CAT GTT ACC AGT GTT ACT TTC AAG CTG GCG 1638 ATA ACT GM CTC TTG GAG GAT ATT GGA TTG GM GAT GM 1677 GAT ACT ATT GCG GTC ACT TTC GTT CCA AM GCT GGC GCT 1716 GAA GAA GTG TCC ATT GAA AGT GTG GAG ATC AAG CTT GAG 1755

GAT TCT 1761

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGMTTCGGC ACGAGCTCCA TCACMCACA 30 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 141 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TAAAGTCTGC ATGAGTTGCT GGCTATGGTG CCAMTTTTA TGTTTAATTA 50 GTATAATTAT GTGTCGTTTC AGTTATCTTT TATCTTMM TGTATCAGCT 100 CGATCGATAG CTGATTGCTA GTTCTGTTAA TGCTATGTAT G 141

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 587 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ser Ser Ser Ser Ser He Thr Thr Thr Leu Pro Leu Cys Thr

5 10 15

Asn Lys Ser Leu Ser Ser Ser Phe Thr Thr Thr Asn Ser Ser Leu 20 25 30

Leu Ser Lys Pro Ser Gin Leu Phe Leu His Gly Arg Arg Asn Gin

35 40 45

Ser Phe Lys Val Ser Cys Asn Ala Asn Asn Val Asp Lys Asn Pro

50 55 60 Asp Ala Val Asp Arg Arg Asn Val Leu Leu Gly Leu Gly Gly Leu

65 70 75

Tyr Gly Ala Ala Asn Leu Ala Pro Leu Ala Thr Ala Ala Pro He

80 85 90

- 3 '

Ala Pro Met Pro Thr Pro Trp Arg Asn Phe Lys Pro He Arg Lys

425 430 435

Ser Ser Ser Gly Lys Val Asn Thr Ala Ser He Ala Pro Val Ser

440 445 450 Lys Val Phe Pro Leu Ala Lys Leu Asp Arg Ala He Ser Phe Ser

455 460 465

He Thr Arg Pro Ala Ser Ser Arg Thr Thr Gin Glu Lys Asn Glu

470 475 480

Gin Glu Glu He Leu Thr Phe Asn Lys He Ser Tyr Asp Asp Arg 485 490 495

Asn Tyr Val Arg Phe Asp Val Phe Leu Asn Val Asp Lys Thr Val

500 505 510

Asn Ala Asp Glu Leu Asp Lys Ala Glu Phe Ala Gly Ser Tyr Thr

515 520 525 Ser Leu Pro His Val His Gly Ser Asn Thr Asn His Val Thr Ser

530 535 540

Val Thr Phe Lys Leu Ala He Thr Glu Leu Leu Glu Asp He Gly

545 550 555

Leu Glu Asp Glu Asp Thr He Ala Val Thr Leu Val Pro Lys Ala 560 565 570

Gly Gly Glu Glu Val Ser He Glu Ser Val Glu He Lys Leu Glu

575 580 585

Asp Cys

587 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1788 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATG GCA ACT CTA GTG TGC AAT ACT AGT ACT ACT ACT ACT 39

ACT ACA ACG CTC AM ACT CCT TTT ACT TCT TTA GGT TCC 78 ACT CCT AAG CCC TCT CAA CTT TTC CTT CAT GGA AM CCT 117

AAC AM ACA TTC AM GTT TCA TGC AAG GTT ATC AAT AAT 156

AAC GCT AAC CAA GAT GAA ACG AAT TCT CTT GAT CGA. AGG 195

AAT GTT CTT CTT GCT TTA GGA GCT CTT TAT GCT GTT GCT 234

AAT GCT ATA CCA TTA GCG GCA TCG GCT ACT CCT ATT CCA 273 TCC CCT GAT CTC AAA ACT TGT GCT AGA GCC ACC ATA TCG 312

GAT GGT CCA CTT CTA CCC TAT TCT TCT TGT CCC CCT CCT 351

ATG CCG ACT AAC TTT GAC ACC ATT CCA TAT TAC AAG TTC 390 CCT TCT ATG ACT AM CTC CGT ATC CGT ACC CCT GCT CAT 429 GCT GTA GAT GAG GAG TAT ATC GCG MG TAT AAT TTG GCC 468 ATA ACT CGA ATC AGG GAT CTT GAC AAG ACA GM CCG TTA 507 AAC CCT CTA GGG TTT AAG CM CM GCT MT ATA CAC TGT 546 GCT TAT TCT AAC GCT GCT TAT ATA ATT GGT GGC AM GAG 585 TTA CM CTT CAT MC TCG TGG CTT TTC TTC CCG TTC CAT 624 CGA TCG TAC TTC TAC TTT TAC GM AGA ATA TTC GGG AM 663 CTC ATT GAT GAT CCA ACT TTC GCT TTA CCA TAC TGG AAT 702 TGG GAT CAT CCA MG GGC ATG CCT TTA CCT CCC ATG TTC 741 GAT CCT GM GCT TCT TCC CTC TAC GAT GM AGG CGT MT 780 CM CAA CTC CCT MT GGA ACG GTT TTG GAT CTT GGT TCA 819 TTT GGG GAT AM GTT GM ACA ACT CM CTC CAG TTG ATC 858 AGC AAT AAT TTA ACC CTA ATG TAC CCT C ATG GTA ACT 897 AAT GCT CCA TCT CCT CTC TTG TTC TTC GGT GCG CCT TAC 936 GTT CTT GGG AAT MC GTT GAA GCA CCG GGA ACC ATT GM 975 ACC ATC CCT CAT ATT CCT GTA CAT ATT TGG GCT GCT ACT 1014 GTC CCT GGT TCA AAA TTT CCT AAC GGT GAT GTC TCC TAC 1053 GCT GAG GAT ATC GCT AAT TTC TAC TCA GCT GGT TTG GAC 1092 CCG GTT TTC TAT TGC CAT CAC GGC AAT GTC GAC CGG ATG 1131 TGG AAC GAA TGG MG GCA ATA GGA GGT AM AGA AGA GAT 1170 ATA TCT GM AAG GAT TGG TTC AAC TCC GAG TTC TIT TTC 1209 TAC GAC GM CAC AM AAT CCT TAC CCT CTG AM CTC AGG 1248 GAC TCT TTC GAC ACG AAG AM ATG GGG TAT GAT TAC GCA 1287 CCA ATG CCA ACT CCA TGG CCT AAT TTC AM CCA AM TCA 1326 AAG GCG TCC GTA GGG AM GTC AAT ACA ACT ACA CTC CCC 1365

CCA GCA AAC GAG CTA TTC CCA CTC GCG MG ATG GAT AAG 1404 ACT ATT TCA TTT GCT ATC MC AGG CCA GCT TCA TCG CGG 1443 ACT CM CM GAG AM AAT GM CM GAG GAG ATG TTA ACG 1482 TTC AAT MC ATA AGA TAT GAT AAC AGA GGG TAC ATA AGG 1521 TTC GAT GTC TTC CTC AAC CTG GAC AAC MT GTC MC GCG 1560

AAT GAG CTT GAT MG GCA GAG TTC GCG GGG ACT TAT ACT 1599 ACT TTG CCA CAT CTT CAC AGA GCT GGC GAG AAT GAT CAT 1638 ATC GCG MG GTT MT TTC CAG CTC GCG ATA ACA GM CTC 1677 TTG GAG GAC ATT GCT TTC GM GAT GM GAT ACT ATC GCG 1716 GTC ACT CTG GTA CCA MG MA GGC GGT GM GGT ATC TCC 1755

ATT GAG MT GTG GAG ATC AAG CTT CTG GAT TCT 1788

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TAATTCGGCA CGAGAGCA 18 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 189 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

TMGTCTCM TTGAATTTGC TGAGATTACA ATTATGATGG ATGATGATAT 50 GTTTTTATGT CTTTTCTT CTGTTATCTA CTTTTGCTTT TCTCCTGTAA 100 CTTTTCCTCT TGAMTCACC CTACATGCTT GATTTCCTTG GAGTTGTTAT 150 TCACTAATAA ATCACTTAGG TTAAAAAMA AAAMAAM 189 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 596 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Ala Ser Val Val Cys Asn Ser Ser Ser Ser Thr Thr Thr Thr

5 10 15 Thr Leu Lys Thr Pro Phe Thr Ser Leu Gly Ser Thr Pro Lys Pro

20 25 30

Ser Gin Leu Phe Leu His Gly Lys Arg Asn Lys Thr Phe Lys Val

35 40 45

Ser Cys Lys Val He Asn Asn Asn Gly Asn Gin Asp Glu Thr Asn 50 55 60

Ser Val Asp Arg Arg Asn Val Leu Leu Gly Leu Gly Gly Leu Tyr

65 70 75

Gly Val Ala Asn Ala He Pro Leu Ala Ala Ser Ala Thr Pro He

80 85 90 Pro Ser Pro Asp Leu Lys Thr Cys Gly Arg Ala Thr He Ser Asp

95 100 105

Gly Pro Leu Val Pro Tyr Ser Cys Cys Pro Pro Pro Met Pro Thr

110 115 120

Asn Phe Asp Thr He Pro Tyr Tyr Lys Phe Pro Ser Met Thr Lys 125 130 135

Met Asp Lys Thr He Ser Phe Ala He Asn Arg Pro Ala Ser Ser

470 475 480

Arg Thr Gin Gin Glu Lys Asn Glu Gin Glu Glu Met Leu Thr Phe

485 490 495 Asn Asn He Arg Tyr Asp Asn Arg Gly Tyr He Arg Phe Asp Val

500 505 • 510

Phe Leu Asn Val Asp Asn Asn Val Asn Ala Asn Glu Leu Asp Lys

515 520 525

Ala Glu Phe Ala Gly Ser Tyr Thr Ser Leu Pro His Val His Arg 530 535 540

Ala Gly Glu Asn Asp His He Ala Lys Val Asn Phe Gin Leu Ala

545 550 555

He Thr Glu Leu Leu Glu Asp He Gly Leu Glu Asp Glu Asp Thr

560 565 570 He Ala Val Thr Leu Val Pro Lys Lys Gly Gly Glu Gly He Ser

575 580 585

He Glu Asn Val Glu He Lys Leu Val Asp Cys

590 595 596

(2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Ala Pro He Pro Pro Pro Asp Leu Lys Ser Gly Gly Thr Ala

5 10 14

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Ala Pro He Pro Pro Pro Asp Leu Lys Ser Asp Xaa Thr Ala

5 10 14

(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11 :

Ala Pro He Pro Pro Pro Asp Leu Lys Ser Gin Gly Thr Ala His

5 10 15

(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Ala Pro He Pro Pro Pro Asp Leu Lys Ser Gin Gly Thr Ala His

5 10 15

(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Ser Pro He Pro Pro Pro Asp Leu Lys Ser Xaa Gly Val Ala His

5 10 15

Tyr Lys Glu Pro 19

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1764 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCT GM GCT CTA TCC ATT GM AGT GTG GAG ATC AAG CTT 1755 GAG GAT TCT 1764

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 222 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

TMCTCCTCA TCACTTGGTC GCTATGCTAC CAMTTTTAT GTTTMTTAT 50 ATTAATGTGT GTGTTTGATT ATGTTTCGGT TAAMTCTAT CAGCTGGATA 100 GCTGATTACT AGCCTTCCCA GTTGTTMTG CTATGTATGA AATAMTAM 150 TAAATGCTTG TCTTCCATTT TFm_* h AAAAAAAAM AAAAAAAAAA 200 AAAAAAAAM AAAAAAAAM AA 222

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 588 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Ser Ser Ser Ser Thr Thr Thr He Pro Leu Try Thr Asn Lys Ser 5 10 15

Leu Ser Ser Ser Phe Thr Thr Asn Asn Ser Ser Phe Leu Ser Lys

20 25 30

Pro Ser Gin Leu Phe Leu His Gly Arg Arg Asn Gin Ser Phe Lys

35 40 45 Val Ser Try Asn Ala Asn Asn Asn Val Gly Glu His Asp Lys Asn

50 55 60

Leu Asp Thr Val Asp Arg Arg Asn Val Leu Leu Gly Leu Gly Gly

65 70 75

Leu Try Gly Ala Ala Asn Leu Ala Pro Leu Ala Ser Ala Ser Pro 80 85 90

He Pro Pro Pro Asp Leu Lys Ser Cys Gly Val Ala His Val Thr

95 100 105

Glu Gly Val Asp Val Thr Try Ser Cys Try Pro Pro Val Pro Asp

110 115 120 Asp He Asp Ser Val Pro Tyr Tyr Lys Phe Pro Pro Met Thr Lys

125 130 135

Ser He Thr Arg Pro Ala Ser Ser Arg Thr Thr Gin Glu Lys Asn

470 475 480

Glu Gin Glu Glu He Leu Thr Phe Asn Lys Val Ala Try Asp Asp

485 490 495 Thr Lys Try Val Arg Phe Asp Val Phe Leu Asn Val Asp Lys Thr

500 505 510

Val Asn Ala Asp Glu Leu Asp Lys Ala Glu Phe Ala Gly Ser Try

515 520 525

Thr Ser Leu Pro His Val His Gly Asn Asn Thr Asn His Val Thr 530 535 540

Ser Val Thr Phe Lys Leu Ala He Thr Glu Leu Leu Glu Asp Asn

545 550 555

Gly Leu Glu Asp Glu Asp Thr He Ala Val Thr Leu Val Pro Lys

560 565 570 Val Gly Gly Glu Gly Val Ser He Glu Ser Val Glu He Lys Leu

575 580 585

Glu Asp Cys 58 8

(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1749 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

ACT ACT CTT CCA TTA TGC AAC MC AM TCC CTC TCT TCT 39

TCC TTC ACC ACC AAC AAC TCA TCT TTC TTA TCA AM CCC 78

TCT CAA CTT TTC CTC CAC GGA AGG CCT AAT CM AGT TTC 117 AAG GTT TCA TGC MC GCC MC AAT AAT GTT GGC GAG CAT 156

GAC AM AAC CTT GAC GCT CTT GAT AGG CGA AAT GTT CTT 195

TTA GGG T GGA GCT CTT TAT GCT GCT GCT AAT CTT GCA 234

CCA TTA GCC TCT GCT TCT CCT ATA CCA CCT CCT GAT CTA 273

AM TCT TCT GCT GTT GCC CAT GTA AM GM GCT GTT GAT 312 GTC TCA TAC ACT TCT TGC CCT CCT CTA CCC GAT GAT ATC 351

GAT AGC GTT CCG TAC TAC MG TTC CCT TCT ATG ACT AM 390

CTC CGC ATC CGC CCC CCT GCT CAT GCG GCG GAT GAG GAG 429

TAT GTA GCC AAG TAT CM TTG GCT ACG AGT CGA ATG AGG 468

GM CTT GAT AM GAC TCT TTT GAC CCT CTT GGG TTT AM 507 CAA CAA GCT AAT ATT CAT TCT GCT T GT MC GCT GCT 546

TAT AM CTT GCT GCT AM GAG TTG CAA GTT CAT TTC TCG 585

TGG CTC TTC TTT CCG TTT CAT AGA TGG TAC TTG TAC TTC 624 TAC GAA AGA ATT TTG GGA TCA CTT ATT AAT GAT CCA ACT 663 TTT GCT TTA CCA TAT TGG AAT TGG GAT CAT CCA AAA GCT 702 ATG CGT ATA CCT CCC ATC TTT GAT CGT GAG GGG TCA TCT 741 CTT TAC GAT GAT AM CCT MC CM MC CAT CGC MT GGA 780 ACT ATT ATT GAT CTT GCT CAT TTT GCT AAG GAA CTT GAC 819 ACA CCT CAG CTC CAG ATA ATG ACT AAT AAT TTA ACA CTA 858 ATC TAC CCT CAA ATG GTC ACT AAT GCT CCT TCT CCG TCC 897 CAA TTC TTC GCT GCT GCT TAC CTC TGG GGA CTC AAC CAA 936 GTC CGG GAC AGG CTA CTA TTG AGA ACA TCC CTC ATA CTC 975 CGG TTC ACA TCT GGA CCG GTG ACA AAC CTC GAC AM AM 1014 ACG GTC AM ACA TCG GTA ATT TCT ATT CAG CAC GCT TTA 1053 GAC CCG CTT TTT TAC TGT CAC CAT GCA AAT GTC GAC CGG 1092 ATG TGG GAT GM TGG AM TTA ATT GCT GGG AM AGA AGG 1131 GAT CTA TCA AAT AM GAT TGG TTC AAC TCA GAA TTC TTT 1170 TTC TAC GAT GAA AAT CGC AAC CCT TAC CCT GTG AM GTC 1209 CCT GAC AGT TTG GAC ACT AM AM ATG GGA TTC ACT TAC 1248 GCT CCA ATG CCA ACT CCA TGG CCT AAT TTT AM CCA ATC 1287 AGA AM ACT ACA GCA GGA ATA GTG AAT ACA GCG TCA ATT 1326 GCA CCA CTC ACC AAG GTC CTC CCA CTC GCG AAG CTG GAC 1365 CCT GCG ATT TCA TTC TCT ATC ACC AGA CCA GCT TCG TCA 1404 AGG ACT ACG CAG GAG AM AAT GAG CAA GAG GAG ATA CTC 1443 ACA TTC AM AAG ATA GCC TAT GAT GAT ACT CAG TAT GTA 1482 AGG TTC GAT GTG TTC CTG AAC GTT GAC AAG ACT CTG AAT 1521 GCG GAT GAG CTT GAT AAG GCA GAG TTT GCG GGG AGT TAT 1560 ACT AGC TTG CCG CAT GTT CAT GGA AAT AAT ACT AAT CAT 1599 GCT ACG AGT GTT ACT TTC ACA GCT GGC ATA ACA GM CTC 1638 TTG GAG GAT ATT GGA TTG GM GAT GM GAT ACT ATT GCG 1677 GTA ACT TTG GTT CCA AM GTA GCT GGT GM GCT CTA TCC 1716 ATT GM ACT GTG GAG ATC AAG CTT GAG GAT TCT 1749 (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 173 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

TAAGTCCTCA TGAGTTGGTC GCTATGGTAC CAMTTTTAT GTTTAATTAG 50 TATTAATGTG TGTATGTGTT GATTATGTTT CGGGTAAMT GTATCAGCTC 100 GATAGCTGAT TACTAGCCTT GCCAGTTGTT AATGCTATGT ATGAAATAM 150

TAMTAAMA AAAAAAAAM AM 173

(2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 633 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Thr Thr Leu Pro Leu Cys Asn Asn Lys Ser Leu Ser Ser Ser Phe

5 10 15 Thr Thr Asn Asn Ser Ser Phe Leu Ser Lys Pro Ser Gin Leu Phe

20 25 30

Leu His Gly Arg Arg Asn Gin Ser Phe Lys Val Ser Cys Asn Ala

35 40 45

Asn Asn Asn Val Gly Glu His Asp Lys Asn Leu Asp Ala Val Asp 50 55 60

Arg Arg Asn Val leu Leu Gly Leu Gly Gly Leu Tyr Gly Ala Ala

65 70 75

Asn Leu Ala Pro Leu Ala Ser Ala Ser Pro He Pro Pro Pro Asp

80 85 90 Leu Lys Ser Cys Gly Val Ala His Val Lys Glu Gly Val Asp Val

95 100 105

Ser Tyr Ser Cys Cys Pro Pro Val Pro Asp Asp He Asp Ser Val

110 115 120

Pro Tyr Tyr Lys Phe Pro Ser Met Thr Lys Leu Arg He Arg Pro 125 130 135

Pro Ala His Ala Ala Asp Glu Glu Tyr Val Ala Lys Tyr Gin Leu 140 145 150

Ala Thr Ser Arg Met Arg Glu Leu Asp Lys Asp Ser Phe Asp Pro 155 160 165 Leu Gly Phe Lys Gin Gin Ala Asn He His Cys Ala Tyr Cys Asn

170 175 180

Gly Ala Tyr Lys Val Gly Gly Lys Glu Leu Gin Val His Phe Ser

185 190 195

Trp Leu Phe Phe Pro Phe His Arg Trp Tyr Leu Tyr Phe Tyr Glu 200 205 210

Arg He Leu Gly Ser Leu He Asn Asp Pro Thr Phe Ala Leu Pro 215 220 225

Tyr Trp Asn Trp Asp His Pro Lys Gly Met Arg He Pro Pro Met 230 235 240 Phe Asp Arg Glu Gly Ser Ser Leu Tyr Asp Asp Lys Arg Asn Gin

245 250 255

Asn His Arg Asn Gly Thr He He Asp Leu Gly His Phe Gly Lys

260 265 270 Glu Val Asp Thr Pro Gin Leu Gin He Met Thr Asn Asn Leu Thr

275 280 285

Leu Met Tyr Arg Gin Met Val Thr Asn Ala Pro Cys Pro Ser Gin

290 295 300 Phe Phe Gly Ala Ala Tyr Leu Trp Gly Leu Asn Gin Val Arg Asp

305 310 315

Arg Val Leu Leu Arg Thr Ser Leu He Leu Arg Phe Thr Ser Gly

320 325 330

Pro Val Thr Asn Leu Asp Lys Lys Thr Val Lys Thr Trp Val He 335 340 345

Ser He Gin His Gly Leu Asp Pro Leu Phe Tyr Cys His His Ala

350 355 360

Asn Val Asp Arg Met Trp Asp Glu Trp Lys Leu He Gly Gly Lys

365 370 375 Arg Arg Asp Leu Ser Asn Lys Asp Trp Leu Asn Ser Glu Phe Phe

380 385 390

Phe Tyr Asp Glu Asn Arg Asn Pro Tyr Arg Val Lys Val Arg Asp

395 400 405

Ser Leu Asp Ser Lys Lys Met Gly Phe Ser Tyr Ala Pro Met Pro 410 415 420

Thr Pro Trp Arg Asn Phe Lys Pro He Arg Lys Thr Thr Ala Gly

425 430 435

He Val Asn Thr Ala Ser He Ala Pro Val Thr Lys Val Phe Pro

440 445 500 Leu Ala Lys Leu Asp Arg Ala He Ser Phe Ser He Thr Arg Pro

505 510 515

Ala Ser Ser Arg Thr Thr Gin Glu Lys Asn Glu Gin Glu Glu He

520 525 530

Leu Thr Phe Lys Lys He Ala Tyr Asp Asp Thr Gin Tyr Val Arg 535 540 545

Phe Asp Val Phe Leu Asn Val Asp Lys Thr Val Asn Ala Asp Glu

550 555 560

Leu Asp Lys Ala Glu Phe Ala Gly Ser Tyr Thr Ser Leu Pro His

565 570 575 Val His Gly Asn Asn Thr Asn His Ala Thr Ser Val Thr Phe Thr

580 585 590

Ala Gly He Thr Glu Leu Leu Glu Asp He Gly Leu Glu Asp Glu

595 600 605

Asp Thr He Ala Val Thr Leu Val Pro Lys Val Gly Gly Glu Gly 610 615 620

Val Ser He Glu Ser Val Glu He Lys Leu Glu Asp Cys

625 630 633

Thus, while I have illustrated and described the preferred embodiment of my invention, it is to be understood that this invention is capable of variation and modification, and I therefore do not wish to be limited to the precise terms set forth, but desire to avail myself of such changes and alterations which may be made for adapting the invention to various usages and conditions. Such changes and alterations to the present invention include, without limitation, single base substitutions, and deletions, insertions or translations of the DNA sequences presented herein providing that such alterations do not significantly affect the properties of the altered sequence from that of the cDNA sequences given above. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims. Having thus described my invention and the manner and a process of making and using it in such full, clear, concise and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same; I claim:

Claims

Claims

1. An isolated cDNA from the group of DNA for the expression of PPO-T1 , PPO-T2, PPO-P1 , and PP0-P2 proteins.

2. A transgenic plant having a genome which provides for the increase of PPO production by said plant, said plant genome having inserted in the sense direction therein, a DNA sequence capable of the expression of PPO.

3. A transgenic plant having a genome which provides for the decrease of PPO production by said plant under normal non-transgenic conditions, said plant genome having inserted in the antisense direction therein, a DNA sequence capable of the expression of PPO.