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WO1993015599A1 - Polyphenol oxydase - Google Patents

Polyphenol oxydase Download PDF

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WO1993015599A1
WO1993015599A1 PCT/US1993/000869 US9300869W WO9315599A1 WO 1993015599 A1 WO1993015599 A1 WO 1993015599A1 US 9300869 W US9300869 W US 9300869W WO 9315599 A1 WO9315599 A1 WO 9315599A1
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PCT/US1993/000869
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John C. Steffens
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Cornell Research Foundation, Inc.
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Priority to JP5514128A priority Critical patent/JPH07503374A/ja
Priority to EP93904786A priority patent/EP0625005A4/fr
Publication of WO1993015599A1 publication Critical patent/WO1993015599A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/18—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with another compound as one donor, and incorporation of one atom of oxygen (1.14.18)
    • C12Y114/18001—Tyrosinase (1.14.18.1)

Definitions

  • PPO Polyphenol oxidases
  • 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]).
  • cresolase activity 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 )].
  • 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
  • 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.
  • 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)].
  • 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.
  • PPO 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.
  • PPO-generated quinones 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].
  • 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
  • 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 ⁇ g) was reconstituted in 500 ⁇ l 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 ⁇ g 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 -
  • 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 NaHCO ⁇ , 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].
  • 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 ⁇ l of 10% heat-killed Staphlococcus a ⁇ reus 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 ⁇ l of anti-PPO antiserum at 4° C overnight. Then 10 ⁇ l of 10% heat-killed Staphlococcus aureus cells was added and incubated at 4° C for one h with intermittent shaking.
  • 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.
  • 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.
  • the DNA sequence of tomato PPO cDNA pPPO-T1 is as follows:
  • 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.
  • a downstream portion was also sequenced according to the following:
  • the DNA sequence of tomato PPO cDNA pPPO-T2 is as follows:
  • downstream portion was also sequenced according to the following:
  • 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.
  • 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 ⁇ g) were translated separately in vitro in the presence of 35 S-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.
  • Glandular trichomes were harvested (see above) from foliage of Lycopersicon esculentum cv.s VFNT, Freedom, Lycopersicon cheesmanii, L. chmielewskii, and Soian ⁇ m 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 ⁇ l 0.5% SDS (w/v), 1.25% ⁇ - 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,
  • L. esculentum cv.VFNT Ala Pro He Pro Pro Pro Pro Asp Leu Lys Ser Gly Gly Thr Ala
  • 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 ⁇ 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).
  • the 0.7 kb truncated tomato PPO cDNA was used to probe a potato leaf cDNA library, also constructed in ⁇ 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 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 is as follows:
  • the DNA sequence of potato PPO cDNA pPPO-P2, according to the present invention is as follows:
  • TAAGTCCTCA TGAGTTGGTG GCTATGCTAC
  • CAAATTTTAT GTTTAATTAG 50 TATTAATGTG TGTATCTGTT GATTATGTTT CGGGTAAAAT GTATCAGCTG 100 GATAGCTGAT TACTAGCCTT GCCAGTTGTT AATGCTATCT ATGAAATAAA 150
  • This cDNA sequence provides the following deduced amino acid sequence for the pPPO-P2 peptide:
  • 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.
  • 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.
  • 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.
  • 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).
  • leaf samples of non- transformed potato plants were homogenized in buffer containing 1.25 % ⁇ -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.
  • the cloning of tomato and potato PPO cDNAs provides a unique opportunity to investigate what have previously been the most intractable questions regarding 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.
  • 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
  • MOLECULE TYPE cDNA
  • 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

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Abstract

L'invention décrit le clonage et la mise en séquence des ADNc de polyphénol oxydase (PPO) de plantes, ce qui a permis de les utiliser afin de transformer génétiquement des plantes, de façon à réaliser une variété de phénotypes souhaités.
PCT/US1993/000869 1992-01-31 1993-01-29 Polyphenol oxydase WO1993015599A1 (fr)

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JP5514128A JPH07503374A (ja) 1992-01-31 1993-01-29 ポリフェノールオキシダーゼ
EP93904786A EP0625005A4 (fr) 1992-01-31 1993-01-29 Polyphenol oxydase.

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US833,839 1992-01-31

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WO1994003607A1 (fr) * 1992-07-30 1994-02-17 Keygene N.V. Structures d'adn, cellules et plantes derivees de celles-ci
DE4423022C1 (de) * 1994-06-30 1995-05-24 Lutz Prof Dr Heide Transgene Pflanzen mit erhöhtem Gehalt an Sekundärstoffen
WO1995021929A1 (fr) * 1994-02-09 1995-08-17 Mogen International N.V. Proteines antifongiques, adn les codant, et hotes renfermant l'adn
WO1996037617A1 (fr) * 1995-05-23 1996-11-28 Commonwealth Scientific And Industrial Research Organisation Genes de polyphenoloxidase provenant de la laitue et de la banane
WO1998011205A3 (fr) * 1996-09-11 1998-08-20 Genesis Res & Dev Corp Ltd Materiaux et procedes permettant de modifier le contenu de plantes en lignine
WO2000047726A3 (fr) * 1999-02-10 2001-03-08 Du Pont Homologues de la polyphenol oxydase de plante
US6204434B1 (en) 1996-09-11 2001-03-20 Genesis Research & Development Corporation Limited Materials and methods for the modification of plant lignin content
US6410718B1 (en) 1996-09-11 2002-06-25 Genesis Research & Development Corporation Ltd. Materials and methods for the modification of plant lignin content
WO2002061101A3 (fr) * 2000-11-03 2003-08-21 Monsanto Technology Llc Procede pour conferer une resistance aux maladies a des plantes en reduisant l'activite polyphenoloxydase
US6627794B1 (en) 1995-05-23 2003-09-30 Commonwealth Scientific And Industrial Research Organisation Polyphenyl oxidase genes from banana
US6680185B1 (en) 1999-02-10 2004-01-20 E. I. Du Pont De Nemours And Company Plant polyphenol oxidase homologs
US6703542B1 (en) 1991-07-17 2004-03-09 Commonwealth Scientific And Industrial Research Organisation Polyphenol oxidase genes
EP1015598A4 (fr) * 1996-02-05 2004-05-19 Commw Scient Andindustrial Res Clones d'opp du genome
US7087426B2 (en) 1996-09-11 2006-08-08 Agrigenesis Biosciences Ltd. Materials and methods for the modification of plant lignin content
US7910326B2 (en) 1996-09-11 2011-03-22 Arborgen, Inc. Materials and methods for the modification of plant lignin content

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WO1994003607A1 (fr) * 1992-07-30 1994-02-17 Keygene N.V. Structures d'adn, cellules et plantes derivees de celles-ci

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Annual Review of Genetics, Vol. 22, issued 1988, WEISING et al., "Foreign Genes in Plants: Transfer, Structure, Expression, and Applications", pages 421-477, see pages 436-437. *
Biological Abstracts, Vol. 18, No. 4, issued 1985, BATISTUTI et al., "Isolation and Purification of Polyphenol Oxidase from a New Variety of Potato Solanum-Tuberosum Cultivar Tebere", Abstract, FOOD CHEMISTRY. *
Biological Abstracts, Vol. 5, No. 2, issued 1985, SAINT-PAUL et al., "Tomato Lycopersicon Esculentum Peroxidases Purification and Some Properties", Abstract, SCIENCES DES ALIMENTS. *
Proceedings of the National Academy of Sciences USA, Vol. 85, issued June 1988, DELAUNEY et al., "A Stable Bifunctional Antisense Transcript Inhibiting Gene Expression in Transgenic Plants", pages 4300-4304, see page 4300. *
See also references of EP0625005A4 *

Cited By (21)

* Cited by examiner, † Cited by third party
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US6936748B1 (en) 1991-07-17 2005-08-30 Commonwealth Scientific And Industrial Research Organisation Polyphenol oxidase genes from potato tuber, grape, apple and broad bean
US6703542B1 (en) 1991-07-17 2004-03-09 Commonwealth Scientific And Industrial Research Organisation Polyphenol oxidase genes
WO1994003607A1 (fr) * 1992-07-30 1994-02-17 Keygene N.V. Structures d'adn, cellules et plantes derivees de celles-ci
WO1995021929A1 (fr) * 1994-02-09 1995-08-17 Mogen International N.V. Proteines antifongiques, adn les codant, et hotes renfermant l'adn
US6291647B1 (en) 1994-02-09 2001-09-18 Syngenta Mogen B.V. Antifungal proteins, DNA coding therefor, and hosts incorporating same
DE4423022C1 (de) * 1994-06-30 1995-05-24 Lutz Prof Dr Heide Transgene Pflanzen mit erhöhtem Gehalt an Sekundärstoffen
WO1996000788A1 (fr) * 1994-06-30 1996-01-11 Lutz Heide Vegetaux transgeniques a teneur elevee en substances secondaires
US6627794B1 (en) 1995-05-23 2003-09-30 Commonwealth Scientific And Industrial Research Organisation Polyphenyl oxidase genes from banana
WO1996037617A1 (fr) * 1995-05-23 1996-11-28 Commonwealth Scientific And Industrial Research Organisation Genes de polyphenoloxidase provenant de la laitue et de la banane
US7381810B2 (en) 1995-05-23 2008-06-03 Commonwealth Scientific And Industrial Research Organistion Polyphenol oxidase genes from lettuce
EP1015598A4 (fr) * 1996-02-05 2004-05-19 Commw Scient Andindustrial Res Clones d'opp du genome
WO1998011205A3 (fr) * 1996-09-11 1998-08-20 Genesis Res & Dev Corp Ltd Materiaux et procedes permettant de modifier le contenu de plantes en lignine
US6410718B1 (en) 1996-09-11 2002-06-25 Genesis Research & Development Corporation Ltd. Materials and methods for the modification of plant lignin content
US6204434B1 (en) 1996-09-11 2001-03-20 Genesis Research & Development Corporation Limited Materials and methods for the modification of plant lignin content
US7087426B2 (en) 1996-09-11 2006-08-08 Agrigenesis Biosciences Ltd. Materials and methods for the modification of plant lignin content
US5952486A (en) * 1996-09-11 1999-09-14 Genesis Research & Development Corporation Limited Materials and methods for the modification of plant lignin content
US7910326B2 (en) 1996-09-11 2011-03-22 Arborgen, Inc. Materials and methods for the modification of plant lignin content
US6680185B1 (en) 1999-02-10 2004-01-20 E. I. Du Pont De Nemours And Company Plant polyphenol oxidase homologs
WO2000047726A3 (fr) * 1999-02-10 2001-03-08 Du Pont Homologues de la polyphenol oxydase de plante
WO2002061101A3 (fr) * 2000-11-03 2003-08-21 Monsanto Technology Llc Procede pour conferer une resistance aux maladies a des plantes en reduisant l'activite polyphenoloxydase
US7122719B2 (en) 2000-11-03 2006-10-17 Monsanto Technology Llc Method of imparting disease resistance to plants by reducing polyphenol oxidase activities

Also Published As

Publication number Publication date
JPH07503374A (ja) 1995-04-13
EP0625005A1 (fr) 1994-11-23
EP0625005A4 (fr) 1995-07-26
AU3602593A (en) 1993-09-03

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