AU775714B2 - Cloned plant P-hydroxyphenyl pyruvic acid dioxygenase - Google Patents

Description

S&F Ref: 428459D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Arizona Board of Regents on Behalf of University of Arizona 888 North Euclid Avenue, Main Gate 515 P.O. Box 210158 Tucson Arizona 85721-0158 United States of America Dean Dellapenna, Susan R. Norris Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 Cloned Plant P-hydroxyphenyl Pyruvic Acid Dioxygenase The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 28. JUN. 2004 11:C1 SPRUSCN FERGUSON NO. 0982 P. 1 CLONED PLANT P-HYDROXYPHENYL PYRUVIC ACID

DIOXYGENASE

Field Of The Invention The present invention relates to a molecular approach for modifying the synthesis of vitamin E, plastoquinone, and carotenoids in plants by use of a full-length cloned cDNA which encodes a phydroxyphenyl pyruvic acid dioxygenase enzyme.

Background Of The Invention 1 0 The chloroplasts of higher plants contain many unique, interconnected biochemical pathways that produce an array of secondary metabolite compounds which not only perform vital functions within the plant but are also important from agricultural and nutritional perspectives. Three such secondary metabolites are the lipid soluble, chloroplastically synthesized compounds vitamin E (a-tocopherol or a-toc), plastoquinones and carotenoids, which together perform many crucial biochemical functions in the chloroplast. PQ and vitamin E are quinone compounds synthesized by a common pathway in the 20 plastid; carotenoids are tetraterpenoids synthesized by a separate plastid-localieed pathway.

Plastoquinone (PQ) often accounts for up to of the total plastidic quinone pool in green tissues.

The primary function of PQ is as a fundamental component of the photosynthetic electron transport 25 chain, acting as an electron carrier between COMS ID No: SBMI-00806247 Received by IP Australia: Time 11:04 Date 2004-06-28 photosystem II and the cytochrome b 6 f complex. PQ likely has other less well studied functions in plastids, namely in acting as a direct or intermediate electron carrier for a variety of other biosynthetic reactions in the chloroplast.

Vitamin E is the second major class of chloroplastic quinones, accounting for up to 40% of the quinone pool in plastids. The essential nutritional value of tocopherols was recognized around 1925, and the compound responsible for Vitamin E activity was first identified as a-tocopherol in 1936.

cy-Toc has a well-documented role in mammals as an antioxidant, and a similar, though less well understood antioxidant role in plants. Liebler, et al., Toxicology 23:147-169, 1993; Hess, Anti-oxidants in Higher Plants, CRC Press: 111-134, 1993.

Carotenoids are a separate, diverse group of lipophilic pigments synthesized in plants, fungi, and bacteria. In photosynthetic tissues, carotenoids 20 function as accessory pigments in light harvesting and play important roles in photo-protection by quenching free radicals, singlet oxygen, and other reactive species. Siefermann-Harms, Physiol. Plantarum. 69:561- 568, 1987. In the plastids of non-photosynthetic tissues, high levels of carotenoids often accumulate providing the intense orange, yellow, and red coloration of many fruits, vegetables, and flowers (Pfander, Methods in Enzvm., 213A, 3-13, 1992). In addition to their many functions in plants, 30 carotenoids and their metabolites also have important functions in animals, where they serve as the major source of Vitamin A (retinol), and have been identified as providing protection from some forms of cancer due to their antioxidant activities. Vitamin E's antioxidant activities are also thought to protect against some forms of cancer, and may act synergistically with carotenoids in this regard.

Liebler, et al., Toxicology 23:147-169, 1993; Krinsky, J. Nutr. 119:123-126, 1989.

Tocopherol and Plastoquinone Synthesis a-Tocopherol and plastoquinone are the most abundant quinones in the plastid and are synthesized by the common pathway shown in Figure 1. The precursor molecule for both compounds, homogentisic acid (HGA), is produced in the chloroplast from the shikimic acid pathway intermediate p-hydroxyphenyl pyruvic acid (pOHPP), in an oxidation/decarboxylation reaction catalyzed by the enzyme p-hydroxyphenyl pyruvic acid dioxygenase (pOHPP dioxygenase) Homogentisic acid is subject to phytylation/prenylation (phytylpyrophosphate and 15 solanylpyrophosphate, C 2 and C4s, respectively) coupled ~to a simultaneous decarboxylation by a phytyl/prenyl transferase to form the first true tocopherol and plastoquinone intermediates, 2-demethylphytylplastoquinol and 20 2-demethylplastoquinol-9, respectively. A single ring methylation occurs on 2-demethylplastocruinol to yield plastoquinol-9 (PQH,) which is then oxidized to plastoquinone-9 This oxidation is reversible and is the basis of electron transport by 25 plastoquinone in the chloroplast.

The preferred route, as established in spinach, for a-tocopherol formation from ~2-demethylphytylplastocruinol appears to be 1) ring methylation of the intermediate, 2-ademethylphytylplastoquinol, to yield phytylplastoquinol, 2) cyclization to yield d-tocopherol and, finally, 3) a second ring methylation to yield a-tocopherol. Ring methylation in both tocopherol and Dlastocuinone synthesis is carried out by a single enzyme that is specific for the site of methylation on the ring, but has -3relatively broad substrate specificity and accommodates both classes of quinone compounds. This methylation enzyme is the only enzyme of the pathway that has been purified from plants to date.

d'Harlingue, et al., J.Biol.Chem. 26:15200, 1985. All enzymatic activities of the c-toc/PQ pathway have been localized to the inner chloroplast envelope by cell fractionation studies except for pOHPP dioxygenase and the tocopherol cyclase enzyme. Difficulties with cell fractionation methods, low activities for some of the enzymes, substrate stability and availability and assay problems, make studying the pathway biochemically difficult.

Vitamin E and PQ levels, ratios, and total amounts vary by orders of magnitude in different plants, tissues and developmental stages. Such variations indicate that the vitamin E and PQ pathway is both highly regulated and has the potential for manipulation to modify the absolute levels and ratios 20 of the two end products. The pathway in Figure 1 makes it clear that production of homogentisic acid by pOHPP dioxygenase is likely to be a key regulatory point for bulk flow through the pathway, both because HGA production is the first committed step in a-toc/PQ synthesis, and also because the reaction is essentially irreversible. Therefore modifying the levels of HGA by modifying pOHPP dioxygenase activity should have a direct impact on the total a-toc/PQ :biosynthetic accumulation in plant tissues, and, as 30 described below, because of the connection of PQ and carotenoid synthesis, should also affect carotenoid synthesis in plant tissues.

Carotenoid Biosynthesis; Quinones as Electron Carriers In plants, carotenoids are synthesized and accumulate exclusively in plastids via the pathway shown on the left-hand side of Figure 1. The first committed step in carotenoid synthesis is the condensation of two molecules of the C 20 hydrocarbon geranylgeranyl pyrophosphate (GGDP) by the enzyme phytoene synthase, to form the colorless C 40 hydrocarbon, phytoene. In oxygenic photosynthetic organisms plants, algae, and cyanobacteria), phytoene undergoes two sequential desaturation reactions, catalyzed by phytoene desaturase, to produce C-carotene through the intermediate phytofluene. Subsequently, C-carotene undergoes two further desaturations, catalyzed by C-carotene desaturase, to yield the red pigment lycopene.

Lycopene is cyclized to produce either a-carotene or /-carotene, both of which are subject to various hydroxylation and epoxidation reactions to yield the carotenoids and xanthophylls most abundant in photosynthetic tissues of plants, lutein, /-carotene, violaxanthin and neoxanthin.

The genes encoding the first two enzymes of the 20 carotenoid pathway (phytoene synthase and phytoene desaturase) have been isolated and studied from a number of plant and bacterial sources in recent years.

Sandmann, Eur. J. Biochem. 223:7-24, 1994. Phytoene .desaturase has been the most intensively studied, both because it is a target for numerous commercially important herbicides, and also because the phytoene desaturation reaction is thought to be a rate limiting step in carotenoid synthesis. Molecular and biochemical studies suggest that two types of phytoene 30 desaturase enzymes have evolved by independent evolution: the crtl-type found in anoxygenic photosynthetic organisms Rhodobacter and Erwinia), and the pds-type found in oxygenic photosynthetic organisms. Despite their differences in primary amino acid sequence, all phytoene desaturase enzymes contain a dinucleotide binding domain (FAD or NAD/NADP), which in Capsicum annum has been shown to be FAD. Hugueney et al., Eur. J.

Biochem. 209:399-407, 1992. Presumably, the bound dinucleotide in both types of phytoene desaturase enzymes is reduced during desaturation and reoxidized by an unknown reductant present in the plastid or bacterium.

Several lines of evidence have suggested a role for quinones in the phytoene desaturation reaction in higher plants. Using isolated daffodil chromoplasts, Mayer and co-workers demonstrated that in an anaerobic environment, oxidized artificial quinones were required for the desaturation of phytoene while reduced quinones were ineffective. Mayer et al., Eur.

J. Biochem. 191:359-363, 1990. Further supporting evidence comes from studies with the triketone class of herbicides Sulcotrione), which cause phytoene accumulation in treated tissues but unlike the wellstudied pyridazone class Norflorazon (NFZ)) do not directly affect the phytoene desaturase enzyme.

20 Rather, triketone herbicides competitively inhibit pOHPP dioxygenase, an enzyme common to the synthesis of both plastoquinone and tocopherols, suggesting that one or more classes of quinones may play a role in carotenoid desaturation reactions. Schulz et al., FEBS 318:162-166, 1993; Secor, Plant Phvsiol. 106: 1429-1433; Beyer et al., IUPAC Pure and Applied Chemistry 66:1047-1056, 1994.

Despite the well-studied, wide-spread importance of vitamin E, plastoquinone, and carotenoids to human 30 nutrition, agriculture, and biochemical processes within plant cells, much remains unclear about their biosynthesis and accumulation in plant tissues. This uncertainty has in turn limited the potential for manipulation of the synthesis and levels of these important compounds in plants.

S28. JUN. 2004 11:02 SPRUSON FERGUSON NO. 0982 P. 6 7 Summary of the Invention Accordirg to a first embodiment of the invention, there is provided a biologically pure sample of DNA, the DNA comprising a DNA sequence coding for the expression of a plant p-hydroxyphenyl pyruvic acid dioxygenase, wherein the DNA sequence encodes an amino acid sequence at least s 80% identical to the amino acid sequence of SEQ ID NO:2.

According to a second embodiment of the invention, there is provided a vector containing the DNA in accordance with the first embodiment of the present invention.

According to a third embodiment of the Invention, there is provided a microbial host transformed by the vector in accordance with the second embodiment of the present invention.

1o According to a fourth embodiment of the invention, there Is provided a transgenic tomato plant transformed with a DNA construct including the DNA in accordance with the first embodiment of the present invention.

According to a fifth embodiment of the invention, there is provided an isolated phydroxyphenyl pyruvic acid dioxygenase comprising SEQ ID NO:2.

According to a sixth embodiment of the invention, there is provided a DNA plant gene expression construct comprislng: a) a DNA sequence coding for the expression of a plant p-hydroxyphenyl pyruvic acid dioxygenase; b) a promoter effective In plant cells located 5' to the DNA coding sequence; and 20 c) a 3' termination sequence effective in plant cells.

According to a seventh embodiment of the invention, there is provided a DNA construct comprising: a) a promoter capable of expressing a downstream coding sequence in a tomato plant; b) a DNA sequence coding for the expression of a phydroxyphenyl pyruvic acid dioxygenase of plant origin; and c) a 3' termination sequence, the construction capable of expressing a p-hydroxyphenyl pyruvic acid dioxygenase gene when transformed into tomato plants.

According to an eighth embodiment of the invention, there is provided a transgenic tomato plant comprising in Its genome a foreign genetic construction comprising, 5' to a promoter 3o effective in tomato, a DNA coding region encoding p-hydroxyphenyl pyruvic add dioxygenase, and a transcriptional terminator, the genetic construction effective in vivo in tomato olants to stimulate expression of p-hydroxyphenyl pyruvic acid dioxygenase.

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COMS ID No: SBMI-00806247 Received by IP Australia: Time 11:04 Date 2004-06-28 2H:.JUN. 2004 11:02 SPRUSON FERGUSON NO. 10982 P. 7 According to a ninth embodiment of the invention, there is provided a method of suppressing the production of vitamin E and plastoquinones in a plant, the method comprising the steps of:, a) Isolating a DNA sequence encoding a p-hydroxyphe'yl pyruvic acid dioxygenase of plant origin; b) creating a genetic construction including, 5' to a promoter effective in the plant's cells, a coding sequence, and a transcriptional terminator, the coding region being derived from the DNA sequence, wherein the DNA sequence from step has been altered so that expression of phydroxyphenyl pyruvic acid dioxygenase Is suppressed; and c) transforming a cell of the plant with the genetic construction, whereby the plant's cell produces lowered levels of vitamin E and plastoquinones.

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*5 (R.'lZlEO13639q=eAocooc COMS ID No: SBMI-00806247 Received by IP Australia: Time 11:04 Date 2004-06-28 2. JUN. 2004 11:C2 SPRUSON FERGUSON NO. 0982 P. 8 7b In one embodiment, this invention provides a biologically pure sample of DNA which DNA comprises a sequence coding for the expression of Arabidopsis thaliana p-hydroxyphenyl pyruvic acid dioxygenase.

In other embodiments, this invention provides a vector and microbial host containing a DNA sequence sufficiently homologous to SEQ ID NO:1 so as to code for the expression of Arabidopsis thaliana phydroxyphenyl pyruvic acid dioxygenase, and a genetic construct containing a DNA sequence sufficiently homologous to SEQ ID NO:1 so as to code for the expression of Arabidopsis thaliana p-hydroxyphenyl pyruvic acid dioxygenase, together with a promoter located S' to the DNA coding sequence and a 3' termination sequence.

In another embodiment, this invention provides a method of creating a-tranagenic plant in which the levels of the pOHPP dioxygenase enzyme are elevated sufficient such that production of plastoquinones, vitamin E, and carotenoids are modified.

S: It is an object of the present invention to genetically engineer higher plants to modify the production of plastoquinones, vitamin E, and 25 carotenoids.

It is another object of the invention to provide transgenic plants that would express elevated levels of the pOHPP dioxygenase enzyme which would have resultant elevated resistance to the triketone class 30 of herbicides sulcotrione).

*It is another object of the present invention to provide a method for the preparation of the enzyme phydroxyphenyl pyruvic acid dioxygenase (pOHPP 9. dioxygenase), an enzyme which can be used to identify

S.

35 new pOHPPdioxygenase-inhibiting herbicides.

•oOther fgatil,,-r 0 oA o ther ftrs ad advantages of the invention will be apparent from the following description of the COMS ID No: SBMI-00806247 Received by IP Australia: Time 11:04 Date 2004-06-28 preferred embodiments thereof and from the claims.

Brief Description of the Drawings Fig. 1 is a diagram of the pathways for synthesis of carotenoids, vitamin E (tocopherol), and plastoquinone.

Fig. 2 is a diagram of the interconnections of the pathways illustrated in Fig. 1.

Fig. 3A-3E are graphs of pigment analyses of wild-type, NFZ-wt, and pdsl tissues.

Fig. 4 is a physical map of the pdsl mutation relative to visible markers.

Figs. 5A-5C present the results of C18 HPLC separation of lipid soluble pigments from wild-type plants on MS2 media, homozygous pdsl mutants on MS2 media supplemented with pOHPP, and homozygous pdsl mutants on MS2 media supplemented with homogentistic acid (HGA).

Figs. 6A-6B present the results of C8 HPLC analyses of quinones in NFZ-wt and pdsl tissues.

Detailed Descriotion Of The Invention e*e *As described above, both Vitamin E, plastoquinones and carotenoids are synthesized and accumulated in plastids by the pathways shown in :*Figure 1. This specification describes the 25 identification, isolation, characterization and functional analysis of a higher plant pOHPP dioxygenase cDNA, its role in a-toc, PQ and carotenoid synthesis, and the use of this cDNA to modify pOHPP dioxygenase activity in plant tissues and hence the accumulation of one or more of the compounds plastoquinones, vitamin E, and carotenoids in plant tissues. The overexpression of pOHPP dioxygenase in transgenic plants will modify the enzyme-to-inhibitor ratio of plant tissues exposed to triketone herbicides, as compared to non-transgenic plants, resulting in increased herbicide resistance. The present specification also describes a genetic construct for use in the production of pOHPP dioxygenase, an enzyme useful in identifying new pOHPP dioxygenase-inhibiting herbicides.

By genetic analysis the present inventors have shown that the vitamin E, plastoquinone, and carotenoid biosynthetic pathways are interconnected and share common elements as shown in Figure 2. From mutational studies in Arabidopsis thaliana, the present inventors identified one genetic locus, designated pdsl (pds= phytoene desaturation), the disruption of which results in accumulation of the first carotenoid of the carotenoid biosynthetic pathway, phytoene. Surprisingly, though this mutation disrupts carotenoid synthesis and was originally identified on this basis, it does not map to the locus encoding the phytoene desaturase enzyme. Evidence o indicates that pdsl defines a second gene product in 20 addition to the phytoene desaturase enzyme, necessary for phytoene desaturation and hence carotenoid synthesis in higher plants. This gene product proved to be pOHPP dioxygenase.

e* To provide a molecular mechanism for manipulating synthesis and accumulation of the compounds plastoquinone, vitamin E, and carotenoids, the present inventors used a molecular genetic approach, taking advantage of the model plant system Arabidopsis thaliana to define, isolate and study genes required 30 for synthesis of the compounds in plants. The flowering plant Arabidopsis thaliana has come into wide use as a model system to explore the molecular biology and genetics of plants. Arabidopsis offers many advantages for genetic analysis: it can be selfed and very large numbers of progeny can be obtained (up to 10,000 seeds from a single plant). Furthermore, Arabidopsis has a short generation time of five to six weeks, so crosses can be set up and the progeny analyzed within reasonable periods of time. Mutation screens have identified thousands of mutations affecting many aspects of basic plant biology, including morphogenesis, photosynthesis, fertility, starch and lipid metabolism, mineral nutrition, an so on. In addition, its haploid genome is only about 108 base pairs.

An important aspect of the successful approach used here is that essential components were first functionally defined genetically, prior to their isolation, analysis and molecular maniDulation.

Briefly, potential mutants were identified by a combination of phenotypic and biochemical screening, characterized at the genetic and molecular levels, loci of interest selected, and the corresponding genes then cloned and studied further. By this approach, the inventors genetically defined and isolated cDNAs for one gene, pdsl, whose mutation disrupts synthesis 20 of all three classes of compounds in the plastid, tocopherols, plastoquinones and carotenoids. Based on biochemical analysis of the pdsl mutant, the pdsl gene was identified as affecting the activity of pOHPP dioxygenase, a crucial enzyme of the plastidic quinone pathway in plants (Figure that is directly required for the synthesis of plastoquinone and a-tocopherol and indirectly for carotenoid synthesis.

In particular, the deduced function of the pdsl mutant and pOHPP dioxygenase enzyme are noted in Figure 2.

30 The present inventors demonstrated by biochemical complementation that the pdsl mutation affects the enzyme p-hydroxyphenyl pyruvic acid dioxygenase (pOHPP dioxygenase), because pdsl plants can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid (HGA) and p-hydroxyphenylpyruvate (pOHPP), respectively. pOHPP dioxygenase is the key branch point enzyme and committed step in the synthesis of both Vitamin E and plastoquinones and several independent lines of biochemical evidence confirm pdsl affects this enzyme (Figures 1, 5, These results provide the first genetic evidence that plastoquinones are essential components for carotenoid synthesis in higher plants, most likely as an electron carrier/redox element in the desaturation reaction (Figure The Arabidopsis pOHPP dioxygenase gene/cDNA thus provides a basis for modifying the production of plastoquinones, c-tocopherol and carotenoids in all higher plants.

Specifically, the specification describes the genetic identification of the Arabidopsis pOHPP dioxygenase gene by mutational analysis, the physical isolation and functional confirmation of an Arabidopsis pOHPP dioxygenase cDNA, its nucleotide sequence and its use to isolate pOHPP dioxygenase genes and cDNAs from other plant species. Also included in the specification is a description of the 20 use of the Arabidopsis pOHPP dioxygenase cDNA, and related cDNAs from other plants, to positively or negatively modify the expression/activity of pOHPP dioxygenase by recombinant techniques (overexpression, cosuppression, antisense, etc.) in any and all plant tissues, especially leaf and fruit tissues, to positively or negatively affect the production of S-toc, PQ and carotenoids.

Elevating pOHPP dioxygenase protein levels increases the amount of homogentisic acid (HGA) 30 synthesized in plant tissues. Because HGA is the limiting precursor molecule for a-toc and PQ synthesis (the end products of the pathway), increasing HGA synthesis increases the levels of c-toc (Vitamin E) and PQ in plant tissues. The increase in PQ indirectly increases the synthesis of carotenoids, which require PQ for their synthesis. In addition, the increase in PQ increases photosynthetic efficiency -11by increasing electron flow between photosystem II and photosystem I, because PQ is the primary electron transporter between the two photosystems. The increase in a-toc, a well-studied antioxidant in mammals, increases the ability of plants to withstand oxidative stresses, such as that caused by high light, high temperature, water stress, ozone stress, UV stress or other abiotic or biotic stresses. Elevating the levels of pOHPP dioxygenase will modify the dose response curve of herbicides targeting pOHPP dioxygenase, thus increasing the relative resistance to such herbicides in transgenic plants as compared to native plants of the same species. Inhibiting the expression of pOHPP dioxygenase is expected to have the opposite effect.

Genetic Construct To express pOHPP dioxygenase in a plant, it is .*oo required that a DNA sequence containing the pOHPP dioxygenase coding sequence be combined with 20 regulatory sequences capable of expressing the coding sequence in a plant. A number of effective plant promoters, both constitutive and developmentally or tissue specific, are known to those of skill in the art. A transcriptional termination sequence 25 (polyadenylation sequence) may also be added. Plant expression vectors, or plasmids constructed for expression of inserted coding sequences in plants, are .widely used in the art-to assemble chimeric plant expression constructs including the coding sequence, and to conveniently transfer the constructs into plants. A sequence which codes for pOHPP dioxygenase includes, for example, SEQ ID NO:1, or versions of the designated sequence sufficient to effect coding for the expression of pOHPP dioxygenase. Commonly used methods of molecular biology well-known to those of skill in the art may be used to manipulate the DNA -12sequences.

By "genetic construct" we mean any of a variety of ways of combining the protein-encoding sequences with a promoter sequence (and termination sequence, if necessary) in a manner that operably connects the promoter sequence (and termination sequence, if present) with the protein-encoding sequences.

Typically, the promoter sequence will be "upstream" of a protein-encoding sequence, while the termination sequence, if used, will be "downstream" of the protein-encoding sequences.

The protein-encoding, promoter and termination sequences may be combined on a plasmid or viral vector, and inserted into a microbial host. Other functional sequences may be added to the gene construct. Alternatively, the protein-encoding, o promoter, and termination sequence, if added, may be combined with any other needed functional sequences and used without a vector.

20 The DNA sequence described by SEQ ID NO:I is S"sufficient to effect coding for the expression of pOHPP dioxygenase. However, it is envisioned that the above sequence could be truncated and still confer the same properties. It is not known at present which specific deletions would be successful, but it is S•likely that some deletions to the protein would still result in effective enzymatic activity. One skilled in the art of molecular biology would be able to take the designated seqcuence and perform deletional 30 analysis experiments to determine what portions of the designated sequence are essential to effect coding for the expression of pOHPP dioxygenase. One could create a genetic construct with the candidate deletion mutations and perform experiments as described below in the Examples, to test whether such deletion mutation sequences effect coding for the enzyme.

Expression of the enzyme activity indicates a -13successful deletion mutant or mutants. In this manner, one could determine which parts of the designated sequence is essential for expression of the enzyme.

It is also known that the genetic code is degenerate, meaning that more than one codon, or set of three nucleotides, codes for each amino acid. Thus it is possible to alter the DNA coding sequence to a protein, such as the sequence for pOHPP dioxygenase described here, without altering the sequence of the protein produced. Selection of codon usage may affect expression level in a particular host. Such changes in codon usage are also contemplated here.

It is further contemplated that using the Arabidopsis pOHPP gene coding sequence described here, that the homologous pOHPP dioxygenase sequences from oo* other higher plants can be readily recovered.

Oligonucleotides can be made from the sequence set forth below to either hybridize against cDNA or 20 genomic libraries or used for PCR amplification of homologous pOHPP dioxygenase sequences from other plants.

*oo ~Once a pOHPP gene is in hand, whether from Arabidopsis or from some other plant species, it then becomes possible to insert a chimeric plant expression genetic construct into any plant species of interest.

Suitable plant transformation methods exist to insert such genetic constructs into most, if not all, commercially important plant species. Presently known 30 methods include Agrobacterium-mediated transformation, coated-particle gene delivery (Biolistics) and electroporation, in which an electric voltage is used to facilitate gene insertion. All these methods, and others, can insert the genetic construct into the genome of the resulting transgenic plant in such a way that the genetic construct becomes an inheritable trait, transmitted to progeny of the original -14transgenic plant by the normal rules of Mendelian inheritance. Thus, once a genetic construct expressing a pOHPP gene is inserted into a plant, it can become a part of a plant breeding program for transfer into any desired genetic background.

To over-express pOHPP dioxygenase, a genetic construct may be used with a higher strength promoter.

To inhibit expression of endogenous pOHPP dioxygenase, an antisense genetic construct can be made, as is known by those of skill in the art, to reduce the level of pOHPP dioxygenase present in the plant tissues.

EXAMPLES

Isolation of pdsl, a mutant defective in carotenoid 15 synthesis To further understand carotenoid biosynthesis and its integration with other pathways in the chloroplast in higher plants, the present inventors studied the pathway by isolating Arabidopsis thaliana mutants that are blocked in carotenoid synthesis.

Plants homozygous for defects in the early stages of carotenoid synthesis prior to production of -carotene) are lethal when grown in soil and the isolation of such mutations requires the design of 25 screening procedures to identify plants heterozygous for soil lethal mutations. The present inventors found that most soil lethal, homozygous pigment-deficient Arabidopsis mutants can be grown to near maturity in tissue culture on Murashige and Skoog basal media (Murashige and Skoog, Physiol. Plant.

15:473-497, 1962) supplemented with sucrose (MS2 media). Under these conditions, photosynthesis and chloroplast development are essentially dispensable and all the energy and nutritional needs of the plant are supplied by the media.

Greater than 500 lines from the 10,000 member Feldmann T-DNA tagged Arabidopsis thaliana population (Forsthoefel et al., Aust. J. Plant Physiol. 19:353- 366, 1992) were selected for pigment analysis based on their segregation for lethal pigment mutations. Seed from plants heterozygous for lethal pigment mutations were surface sterilized, grown on MS2 media, the segregating pigment mutants identified, tissue harvested from individual plants, and HPLC pigment analysis performed. Although numerous mutant lines with severe pigment deficiencies were identified, only two were found to be carotenoid biosynthetic mutants.

One mutant line isolated from this group, pdsl, is described in detail here.

The hallmark phenotype for disruption of a biosynthetic pathway is the accumulation of an intermediate compound prior to the site of blockage.

Such blockage of the carotenoid pathway can be mimicked chemically by treatment of wild-type plants S•with the herbicide NFZ, an inhibitor of the phytoene 20 desaturase enzyme (Figure 1) which has been reported t c o cause accumulation of phytoene in treated tissues.

Britton, Z. Naturforsch 34c:979-985, 1979. Figs. 3A- *3E present the results of pigment analysis of wildtype, NFZ-wt, and pdsl tissues. Abbreviations in Figs. 3A-3E are as follows: N, neoxanthin;

V,

violaxanthin; L, lutein; Cb, chlorophyll b; Ca, chlorophyll a; 0, (-carotene.

Figure 3A shows. C, Reverse Phase HPLC analysis of the carotenoids that accumulate in wild-type 30 Arabidopsis thaliana leaves. In comparison, Figure 3B shows the pigment profile for NFZ treated wild-type (NFZ-Wt). Spectral analysis of the strongly absorbing 296nm peak at 33 minutes in NFZ-Wt tissue shows absorbance maxima at 276, 286, and 298nm, indicative of phytoene (Figure 3D). Figure 3C shows pigment analysis of tissue culture grown homozygous pdsl mutant plants. The low absorbance at 440nm in Figures -16- 3B and C demonstrates that like NFZ-Wt, pdsl mutants lack all chlorophylls and carotenoids that normally accumulate in wild-type tissue (compare to Figure 3A) However, unlike wild-type, pdsl mutants contain a peak with a retention time at approximately 33 minutes that absorbs strongly at 296nm. The retention time and absorbance of the 33-minute peak in the pdsl mutant corresponds to the phytoene peak in pigment extracts of NFZ-Wt tissue (Figure 3B). Spectral analysis of the 33-minute peak from pdsl is shown in Figure 3E and is virtually identical to the spectra of phytoene from NFZ-Wt tissue (Figure 3D) as well as to the published spectra for phytoene. These results confirm the chemical identity of the accumulating compound in pdsl as phytoene and conclusively demonstrate that the pdsl mutation disrupts carotenoid biosynthesis.

Carotenoid Analysis For quantitative and qualitative carotenoid analysis, plant tissue is placed in a microfuge tube and ground with a micropestle in 200gl of 80% acetone.

120A1 of ethyl acetate is added and the mixture vortexed. 14041 of water is added and the mixture centrifuged for 5 minutes. The carotenoid containing upper phase is then transferred to a fresh tube and 25 vacuum dried in a Jouan RC1010 Centrifugal Evaporator.

The dried extract is resuspended in ethyl acetate at a concentration of 0.5mg fresh weight of tissue per p! Sand either analyzed immediately by HPLC or stored at -80'C under nitrogen.

Carotenoids were separated by reverse-phase HPLC analysis on a Spherisorb ODS2 5 micron column, cm in length (Phase Separations Limited, Norwalk, CT) using a 45 minute gradient of Ethyl Acetate (0-100%) in Acetonitrile/water/triethylamine (9:1:0.01 v/v) at a flow rate of 1 ml per minute (Goodwin and Britton, 1988). Carotenoids were identified by retention time -17relative to known standards with detection at both 296nm and 440nm. When needed, absorption spectra for individual peaks were obtained with a Hewlett Packard 1040A photodiode array detector and compared with published spectra or available standards.

Quinone analysis Quinones were extracted from tissue using a method modified from that described in Bligh et al., Can. J. Biochem. Physiol. 37:911-917, 1959. Frozen plant tissue was ground in a mortar with 3 volumes of chloroform and 6 volumes of methanol and transferred to a test tube. Water and additional chloroform were added until a biphasic mixture was obtained. The quinone containing chloroform phase was then collected. To increase yields, the aqueous phase was back-extracted with chloroform, the two chloroform phases pooled, and then filtered through Whatman #3 filter paper. The resulting filtrate was dried under a constant stream of nitrogen. Once dried, the pellet 20 was resuspended in methanol at a concentration of fresh weight per ml and immediately analyzed by HPLC.

Quinones were resolved by reversed-phase HPLC analysis oo** on a LiChrosorb RP-8, 5 micron column, 25cm in length, (Alltech, San Jose, CA) using an isocratic solvent of 25 10% H20 in Methanol for the first 14 minutes, at which time the solvent was switched to 100% methanol for the remainder of the run .(modified from the method described in Lichtenthaler, Handbook of Chromatoaraphv, CRC Press, 115-159, 1984). The flow rate was iml per minute for the duration. Peaks were identified based upon the retention time of known standards with detection at 280nm for a-tocopherol and 260nm for plastoquinone and ubiquinone as well as by absorption spectra from a Hewlett Packard 1040A photodiode array detector. When needed, fractions represented by individual chromatographic peaks were -18collected, and submitted to the Southwest Environmental Health Science Center, Analytical Core laboratory for mass spectral analysis. Results were obtained using a TSQ7000 tandem mass spectrometer (Finnigan Corp., San Jose, CA) equipped with an atmospheric pressure chemical ionization source operated in the positive ion mode. The instrument was set to unit resolution and the samples were introduced into the source in a 0.3 ml/minute methanol stream and ionized using a 5kV discharge.

Genetic analysis of pdsl The genetic nature of the pdsl mutation was determined by analyzing seeds resulting from selfing pdsl heterozygous plants. Prior to desiccation, Fl 15 seeds were scored as either green (wild-type or heterozygous) or white (homozygous). A 3:1 segregation ratio was observed (146 green seeds: 48 white seeds), indicating that pdsl is inherited as single recessive nuclear mutations (X 2 =0.01, p 0.90) Because pdsl mutants are inhibited in the desaturation of phytoene, the inventors believed that it might be a mutation in the phytoene desaturase enzyme, which had previously been mapped to chromosome 4, between ag and bp. Wetzel et al., Plant J. 6:161-175, 1994. To test 25 this hypothesis, the pdsl mutation was mapped relative to visible markers. The pds2 mutation was found to map to chromosome 1, approximately 7 cM from disl toward clv2. Franzmann et al., Plant J. 7:341-350, 1995. These data points are summarized in Figure 4 and establish that pdsl does not map to the phytoene desaturase enzyme locus, thus proving that the pdsl mutation is not in the phytoene desaturase enzyme.

This data provided important insight for characterization of the pdsl mutant.

-19- Homozygous pdsl mutants can be rescued by Homogentisic Acid, an intermediate in plastoquinone and tocopherol biosynthesis As described earlier, previous research suggesting a role for quinones and pOHPP dioxygenase in phytoene desaturation lead the present inventors to investigate the quinone biosynthetic pathway in the pdsl mutant. The early stages of plastoquinone/tocopherol synthesis were functionally analyzed by growth in the presence of two intermediate compounds in the pathway, p-hydroxyphenylpyruvate (pOHPP) and homogentisic acid (HGA) (refer to Figures 1 and Albino pdsl homozygous plants were first germinated on MS2 media and then transferred to MS2 media supplemented with 100yM of either pOHPP or HGA.

pdsl plants remained albino when transferred to media containing pOHPP but greening occurred when pdsl plants were transferred to media containing HGA.

Figs. 5A-5C present the results of complementation of 20 the pdsl mutation with homogentisic acid. Each profile represents pigments extracted from 10mg fresh weight of tissue. Abbreviations used in Figs. are as described in Figs. 3A-3E. HPLC analysis with detection at 440nm of the carotenoids extracted from pdsl plants grown on pOHPP and HGA are shown in Figures 5B and C, respectively. The pigment profiles of pdsl mutants grown on pOHPP are similar to the profiles of pdsl plants grown on MS2 media shown in Figure 3B. Comparison-of the pigment profiles for 30 pdsl HGA tissue and wild-type tissue (Figures 5A and S1S 5C) indicates that growth in the presence of HGA is able to qualitatively restore a wild-type carotenoid profile to albino, homozygous pdsl plants. These results indicate that the pdsl mutation affects the enzyme pOHPP dioxygenase, because pdsl mutants are not altered by .gowth the substrate of this enzyme, pOHPP, but rather, are restored qualitatively to wild-type pigmentation by growth on the product of this enzyme, HGA (refer to Figures 1 and The complementation of pdsl with HGA also indicates that intermediates or end products of this pathway (plastoquinone and/or tocopherols, refer to Figures 1 and 2) are necessary components for phytoene desaturation in plants and confirms the observation of Schultz et al. in FEBS where inhibitors of pOHPP dioxygenase were shown to cause accumulation of phytoene.

HPLC analysis conclusively demonstrates that pdsl is a mutation in the plastoquinone/ tocopherol biosynthetic pathway that also affects carotenoid synthesis In addition to biochemical complementation of pdsl mutants, the plastoquinone/tocopherol pathway was 15 also directly analyzed in pdsl tissue by utilizing C, HPLC to resolve total lipid extracts and identify :three separate classes of quinones: ubiquinone, plastoquinone, and a-tocopherol (Vitamin E) (Figures and Ubiquinone and plastoquinone perform analogous electron transport functions in the mitochondria and chloroplast, respectively, but are synthesized by different pathways in separate subcellular compartments (Goodwin et al., Introduction to Plant Biochemistry, Oxford, Pergamon Press, 1983), 25 making ubiquinone an ideal internal control in these analyses. Figure 6 shows the C, HPLC analysis of lipid soluble extracts from NFZ-Wt tissue and pdsl tissue.

In NFZ-Wt tissue (Figure 6A), peaks 3 and 4 were identified as ubiquinone and plastoquinone, respectively, based on retention time (26 and 27 minutes), optical spectra, and mass spectra (results not shown). NFZ-Wt tissue contained a peak with a retention time of 13.5 minutes which was identified as a-tocopherol based upon the retention time of a standard However, optical specLroscopy and mass spectrometry demonstrated that peak 1 was composed of two major components: a-tocopherol (la) and an -21unidentified compound The mass of a-tocopherol was determined to be 430 as indicated by the presence of the 431 protonated molecule while the molecular mass of the unidentified compound was 412, as indicated by the presence of the 413 protonated molecule (data not shown), clearly demonstrating the presence of two compound in peak 1. This quinone analysis demonstrates that the herbicide NFZ, which specifically inhibits the phytoene desaturase enzyme, does not affect synthesis of homogentisate derived quinones. pdsl tissue (Figure 6B) contain ubiquinone (peak 3) but lack plastoquinone (peak 4).

Additionally, though pdsl contains a peak at 13.5 minutes, optical spectroscopy and mass spectrometry 15 data demonstrate that this peak lacks c-tocopherol and is composed solely of the compound lb (data not shown). Therefore, homozygous pdsl plants accumulate ubiquinone but lack both plastoquinone and a-tocopherol. This is consistent with the pdsl mutation affecting pOHPP dioxygenase (refer to Figures 1 and as suggested by the rescue of the mutation by HGA, and provide additional evidence that the pdsl mutation disrupts pOHPP dioxygenase.

Isolation of a truncated, putative pOHPP dioxygenase 25 Arabidopsis cDNA The observation of Schultz et al. demonstrating that inhibitors of pOHPP dioxygenase activity disrupt carotenoid synthesis and cause accumulation of phytoene provided important insight for the characterization of the pdsl mutant which in turn provided the present inventors with important insight for the isolation of a putative cDNA for the pdsl locus. In animals, genetic defects which inhibit the activity of pOHPP dioxygenase lead to tyrosinemia type I, a fatal inherited disease in aromatic amino acid catabolism characterized by the presence of high levels of pOHPP in the urine.

-22- In an effort to further understand the nature of this disease, pOHPP dioxygenase cDNAs have been cloned from several mammalian and bacterial sources (summarized in Ruetschi et al., Eur. J. Biochem.

205:459-466, 1992). Amino acid identity between various mammalian pOHPP dioxygenase enzymes is in comparison, their identity to bacterial homologs is very low, less than 28%. By using mammalian and bacterial sequences to search the Expressed Sequence Tags (ESTs) computer DNA database (Newman et al., Plant Physiol. 106:1241-1255, 1994), one partial length Arabidopsis EST was identified and used as a probe. The partial length Arabidopsis probe corresponds to base pairs 1072 through 1500 of SEQ ID 15 NO:1.

This cDNA contained only 99 amino acids of the carboxyl terminal portion of the protein coding region. The deduced protein sequence of this putative Arabidopsis pOHPP dioxygenase cDNA shows similar homology identity) to both the mammalian and bacterial pOHPP dioxygenases. Interestingly, the partial Arabidopsis sequence also contains a 15 amino acid insertion not found in the human or bacterial enzymes. Finally, alignment of six pOHPP dioxygenase 25 sequences from mammals and bacteria identified three regions of high conservation, the highest being a 16 amino acid region near the carboxy end of pOHPP dioxygenases that shows 62.5% identity across all phyla. Ruetschi et al., Eur. J. Biochem. 205:459-466, 1992. This region is also present in the truncated Arabidopsis sequence. The lines of evidence suggest that the partial length Arabidopsis cDNA described above encodes a pOHPP dioxygenase, most likely the pdsl locus.

-23- Isolation and Characterization of a full length ArabidopsispOHPP dioxygenase cDNA Utilizing the partial length Arabidopsis cDNA probe, an Arabidopsis cDNA library was screened by nucleic acid hybridization for full length cDNAs. A large number of hybridizing cDNAs were isolated, and one of the longest, pHPP1.5, containing a 1,520 bp insertion, was sequenced completely; the insert is presented as SEQ ID NO: 1. pHPP1.5 encodes a 446 amino acid protein (presented as SEQ ID NO:2), which is slightly larger in size than mammalian and bacterial pOHPP dioxygenases. pHPP1.5 shows 34-40% identity at the amino acid level to pOHPP dioxygenases from various mammals and bacteria. In comparing four 15 bacterial pOHPP dioxygenases and one mammalian pOHPP dioxygenases (pig) which ranged in size from 346-404 amino acids, Denoya et al. identified 69 amino acids that were conserved between all five DOHPP dioxygenases. Denoya et al., J. Bacteriol. 176:5312- 20 5319, 1994. The pHPPI.5 coding region contains 52 of these 69 conserved amino acids.

Demonstration that pHPP1.5 encodes an active pOHPP dioxygenase protein and complements the pdsl mutation In order to definitively demonstrate that 25 is the gene product encoded by the pdsl locus and that it encodes a functional pOHPP dioxygenase protein, the pHPP1.5 cDNA was cloned into a plant transformation vector for molecular complementation experiments with the pdsl. The full length wild-type pOHPP dioxygenase cDNA will be subcloned into a plant transformation vector driven by the Cauliflower Mosaic Virus (CaMV) promoter and containing all necessary termination cassettes and selectable markers (Kan) The CaMV promoter is a strong constitutive promoter.

This single nstruct and the vector without the insert (as a control) will be used in vacuum infiltration transformation which uses whole soil -24grown plants and will be done on plants that are heterozygous for the pdsl mutation. Bouchez et al., CR Acad. Sci. Paris, Sciences de la vie 316, 1993.

In the standard procedure, 20-30 soil grown plants will be independently transformed and analyzed separately. In this case homozygous plants containing the pdsl mutation would be lethal while heterozygous plants containing the pdsl mutation would be segregating 2:1 for the pdsl mutation in their siliques. The inventors will use a similar number of wild type plants in a parallel transformation as a control. After transformation of the pdsl segregating plant population, as the plants are setting seed the inventors can easily identify those heterozygous for 15 the pdsl mutation in retrospect by inspection of their siliques which would contain green:white embryos in a 3:1 ratio.

Seed harvested from individually transformed heterozygous pdsl plants will be germinated on kanamycin and resistant seedlings transferred to soil.

Segregation analysis of seed from these primary transformants (T2 seed) and T3 seed for segregation of the pdsl phenotype (albino and phytoene accumulating) and the T-DNA encoded kanamycin resistance marker 00. 25 (wild type pOHPP dioxygenase cDNA) will conclusively demonstrate complementation of the pdsl mutation with the pOHPP dioxygenase cDNA. To provide additional :proof that pHPPl.5 is encoded by the pdsl locus, the cDNA has been mapped relative to the pdsl locus using recombinant inbred lines, as described in Lister et al., Plant J. 4:745-750, 1993. The cDNA mapped to the region of chromosome 1 containing the pdsl mutation (Figure Finally, the cDNA will be overexpressed in E. coli and the activity of the protein determined Modification of pOHPP Expression From the genetic and biochemical studies described above it is clear that only one pOHPP dioxygenase gene product is involved in chloroplastic quinone synthesis, that the pdsl mutation defines this gene, that the pHPP1.5 cDNA is the product encoded by the pdsl locus and that disruption of its function completely eliminates Vitamin E production and plastoquinone and carotenoid synthesis in plant tissues. Modification of pOHPP dioxygenase expression in plants by molecular techniques using pHPPi.5 can therefore be used to positively or negatively affect the production of tocopherols, plastoquinones directly and carotenoids indirectly (refer to Figures 1 and 2) 15 Specifically, overexpression of the pOHPP dioxygenase enzyme will result in increased levels of one or more of these compounds in the tissues of transgenic plants. Alternatively, using antisense techniques, it is possible to lower the level of enzyme activity to decrease the levels of these compounds in plants.

Additionally, overexpression of the pOHPP dioxygenase will enable a transgenic plant to withstand elevated levels of herbicides that target this enzyme, providing agrinomically significant herbicide S" 25 resistance relative to normal plants.

Two different plant systems, Arabidopsis and tomato, are being used to demonstrate the effects of Smodified pOHPP dioxygenase in plant tissues.

Constitutive overexpression of pOHPP dioxygenase will be done in both plant systems utilizing the CaMV promoter and the pHPP1.5 cDNA. The consequences of this altered expression on tocopherol, plastoquinone and carotenoid levels and profiles in various plant tissues will be determined as described below. In tomato, tissue specific overexpression of pOHPP dioxygenase (pHPP1.5) will be driven by the fruit specific promoter derived from the tomato /subunit -26gene, which is expressed specifically in developing, but not ripening tomato fruit. This will determine the potential for modifying the levels of tocopherol, plastoquinone and carotenoids specifically in developing and ripening fruit for nutritional purposes without affecting their production in other plant tissues. These combined experiments will determine whether pOHPP dioxygenase is a rate limiting step in chloroplastic homogentisic acid derived auinone synthesis and the potential for manipulating chloroplastic homogentisic acid derived auinones (tocopherols and plastoquinones) and compounds that require quinones for their synthesis (carotenoids, etc) by increasing pOHPP dioxygenase activity.

15 Multiple independent transformants will be produced for each construct and plant species used.

The integration and gene copy number of each chimeric gene in each line will be confirmed by southern analysis, the level of pOHPP mRNA determined by Northern blot analysis, pOHPP dioxygenase activity determined as described in Schulz et al., FEBS 318:162-166, 1993, and the effects on individual chloroplastic components of interest analyzed (ocopherols, plastoquinones and carotenoids). In 25 green tissue containing constitutively expressing constructs this analysis can occur relatively soon after transformants are put into soil. Analysis of fruit specific construct lines will require much more time for fruit set to occur. Analysis of tocopherols, plastoquinones and carotenoids will be by a combination of HPLC, optical and mass spectra as described in Norris et al. (1995, in press). Analysis of tocopherol levels is performed by HPLC and when needed by GC:mass spectroscopy in selected ion mode.

In MS analysis the absollute l of tcc-herl will be quantified by isotopic dilution with a known, "heavy carbon" tocopherol standard added at the start -27of the extraction. Determination based on fresh weight of tissue can also be performed. Plastoquinone levels will be quantified by C8 HPLC and optical spectra as described in Norris et al. (1995, in press). Total carotenoid levels are determined spectrophotometrically and the levels of individual carotenes quantified by C18 HPLC and optical spectra quantified to standards. In the course of these experiments we will identify high expressing lines with simple insertions that segregate as single genetic loci in progeny. This will facilitate analysis of the inheritance of the gene and phenotype in future generations.

Overexoression of DOHPP for in vitro Herbicide ooo 15 Analysis pOHPP dioxygenase will be overexpressed in E coli or other prokaryotic or eukaryotic protein production systems and purified in large amounts for use in enzymatic assays for identifying new herbicide compounds (pOHPP inhibitors) and optimizing existing chemistries through detailed kinetic analysis.

a-- *r -28- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: DellaPenna, Dean Norris, Susan (ii) TITLE OF INVENTION: Cloned Plant P-Hydroxyphenyl Pyruvic Acid Dioxygenase (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Quarles Brady STREET: PO Box 2113 CITY: Madison STATE: WI COUNTRY: USA ZIP: 53701-2113 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version i1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

25 (viii) ATTORNEY/AGENT INFORMATION: NAME: Seay, Nicholas J REGISTRATION NUMBER: 27,386 REFERENCE/DOCKET NUMBER: 920214.90158 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 608-251-5000 TELEFAX: 608-251-9166 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1519 base pairs 35 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA ORIGINAL SOURCE: 40 ORGANISM: Arabidopsis thaliana (vii) IMMEDIATE SOURCE: CLONE: (ix) FEATURE: NAME/KEY: CDS LOCATION: 37..1374 -29- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCACGCGTCC GAGTTTTAGC AGAGTTGGTG AAATCA ATG CCC CAC CAA AAC GCC 54 Met Gly His Gin Asn Ala 1 GCC GTT TCA GAG AAT CAA AAC CAT GAT CAC GGC GCT GCG TCG TCG CCG 102 Ala Val Ser Clu Asn Gin Asn His Asp Asp Cly Ala Ala Ser Ser Pro 15 GGA TTC AAG CTC CTC CGA TTT TCC AAG TTC GTA AGA AAG AAT CCA AAC 150 Cly Phe Lys Leu Val Cly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys 25 30 TCT GAT AAA TTC AAC GTT AAC CCC TTC CAT CAC ATC GAG TTC TCC TGC 198 Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Ciu Phe Trp Cys 45 CGC GAC CCA ACC AAC GTC GCT CGT CGC TTC TCC TCC GCT CTG GCG ATC 246 Gly Asp Ala Thr Asn Val Ala Arg Arg Phe Ser Trp Cly Leu Gly Met 60 65 AGA TTC TCC GCC AAA TCC GAT CTT TCC ACC GGA AAC ATG GTT CAC GCC 294 Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala 80 TCT TAC CTA CTC ACC TCC CCT CAC CTC CGA TTC CTT TTC ACT CCT CCT 342 Ser Tyr Leu Leu Thr Ser Cly Asp Leu Arg Phe Leu Phe Thr Ala Pro 95 100 TAC TCT CCC TCT CTC TCC GCC CGA GAG ATT AAA CCC ACA ACC ACA GCT 390 Tyr Ser Pro Ser Leu Ser Ala Cly Glu Ile Lys Pro Thr Thr Thr Ala 105 110 115 *TCT ATC CCA ACT TTC CAT CAC CCC TCT TCT CGT TCC TTC TTC TCT TCA 438 :Ser Ile Pro Ser Phe Asp His Cly Ser Cys Arg Ser Phe Phe Ser Ser 120 125 130 CAT CCT CTC CGT GTT ACA CCC GTT GCG ATT GAA GTA CAA GAC CCA GAG 486 His Cly Leu Cly Val Arg Ala Val Ala Ile Clu Val Clu Asp Ala Glu 135 140 145 150 TCA CCT TTC TCC ATC ACT CTA GCT AAT CCC CCT ATT CCT TCC TCC CCT 534 Ser Ala Phe Ser Ile Ser Val Ala Asn Cly Ala Ile Pro Ser Ser Pro 155 160 165 CCT ATC GTC CTC AAT GAA GCA CTT ACG ATC GCT GAG CTT AAA CTA TAC 582 Pro Ile Val Leu Asn Giu Ala Val Thr Ile Ala Giu Val Lys Leu Tyr 170 175 180 GGC GAT GTT GTT CTC CGA TAT GTT ACT TAC AAA CCA GAA GAT ACC GAA 630 4 ly Asp Vai Val Leu Arg Tyr-Val Ser Tyr Lys Ala Clu Asp Thr Clu 40185 190 195 AAA TCC CAA TTC TTC CCA CCC TTC GAG CCT GTA GAG GAT GCC TCC TCC 678 Lys Ser Clu Phe Leu Pro Cly Phe Clu Arg Val Glu Asp Ala Ser Ser 200 205 210 TTC CCA TTC CAT TAT COT ATC CCC CCC CTT CAC CAC CCC CTC GGA AAC 726 Phe Pro Leu Asp Tyr Cly Ile Arg Arg Leu Asp His Ala Vai Gly Asn 215 220 225 230 CTT CCT GAG CTT GOT CCC OCT TTA ACT TAT GTA CC CCC TTC ACT GCT 774 Val Pro Gl T.eu 21 y Pro Ala~ Lcu Thr TIyr Val Ala GIL y Phie Thr Gly 235 240 245 TTT CAC CAA TTC GCA GAG TTC ACA Phe His Gin Phe Ala Giu Phe Thr 250

AGC

Ser

CTA

Leu

CAG

Gin 295

CTG

Leu

AGC

Ser

TAC

Tyr

ATC

Ile

GGG

Gly 375

ATA

Ile

GAA

Giu

AAT

Asn

GAA

AAT

Asn

AAC

Asn

TTG

Leu

GAA

Giu

GGA

Gly 330

CTC

Leu

TGT

Cys

CTT

Leu

GAG

Giu

GCT

Ala 410

GAG

Giu

CAG

TCA

Ser

GAG

Giu

GAA

Giu

GAC

Asp 315

GGA

Giy

AAG

Lys

GAG

Giu

CAA

Gin

ATA

Ile 395

TAC

Tyr

CTC

Leu

TTA

GCG

Aila

CCA

Pro

CAT

His 300

ATA

Ile

TTC

Phe

AAA

Lys

GAA

Giu

ATC

Ile 380

ATC

Ile

CAG

Gin

TTC

Phe

GTG

GTC

Vali

GTG

Val 285

AAC

Asn

TTC

Phe

GAC

Asp

CGG

Arg

TTA

Leu 365

TTC

Phe

CAG

Gin

AGT

Ser

AAG

Lys

GGA

Gly 445

GCA

Ala 255

GCT

Ala

GGA

Gly

GGC

Gly

ACC

Thr

ATG

Met 335

GGC

Giy

ATT

Ile

AAA

Lys

GTA

Val

GGA

Gly 415

ATT

AGC

Ser

ACA

Thr

GCA

Al a

CTG

Leu 320

CCT

Pro

GAC

Asp

CTT

Leu

CCA

Pro

GGA

Gly 400

TGT

Cys

GAA

AAT

Asn

AAG

Lys

GGG

Gly 305

AGA

Arg

TCT

Ser

GTG

Val

GTA

Val1

CTA

Leu 385

TGC

Cys

GGT

Gly

GAA

GAT

Asp

AGG

Arg 290

CTA

Leu

GAG

Giu

CCT

Pro

CTC

Leu

GAC

Asp 370

GGT

Gly

ATG

Met

GGT

Gly

TAC

GAA

Giu 275

AAG

Lys

CAA

Gin

ATG

Met

CCG

Pro

AGC

Ser 355

AGA

Arg

GAC

Asp

ATG

Met

TTT

Phe

GAA

GAC GAC GTT GGA ACC GCC GAG Asp Asp Vai Giy Thr Ala Giu 260

ATG

Met

AGT

Ser

CAT

His

AGG

Arg

CCT

Pro 340

GAT

Asp

GAT

Asp

AGG

Arg

AAA

Lys

GGC

Giy 420

AAG

CTT

Leu

ATT

Ile

GCT

Ala 310

AGG

Arg

TAC

Tyr

CAG

Gin

CAA

Gin

ACG

Thr 390

GAG

Giu

GGC

Giy

CTT

870 918 966 1014 1062 1110 1158 1206 1254 1302 1350 1404 Ile Giu Giu Tyr Giu Lys Thr Leu 435 ACAAGAAGAA GAACCAACrA AAGGATTGTG Giu Ala Lys Gin Leu Val 440 TAATTAATGT AAAACTGTTT TATCTTATCA AAACAATGTT ATACAACATC TCATTTAAAA 1464 ACGAGATCAA TCAAAAAATA CAATCTTAAA TTCAAAACCA AAAAAAAAAA AAAAA 15.19 INFORMATION FOR SEQ ID NO:2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 446 amino acids TYPE: amino acid TOPOLOGY: linear -31- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Gly His Gin Asn Ala Ala Val Ser Giu Asn Gin Asn His Asp Asp 1 5 10 Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe 20 25 Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His 40 His Ile Glu Phe Trp, Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe so 55 Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu Ser Thr 70 75 Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg 90 Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Giu Ile 100 105 110 Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys 115 120 125 Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile 130 135 140 Giu Val Giu Asp Ala Giu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly 145 150 155 160 *Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Giu Ala Val Thr Ile *165 170 175 *.:Ala Giu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr 180 185 190 Lys Ala Glu Asp Thr Giu Lys Ser Giu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu 210 215 220 Asp His Ala Val Gly Asn Val Pro Giu Leu Gly Pro Ala Leu Thr Tyr 225 230 235 240 Val Ala Gly Phe Thr Gly Phe His Gin Phe Ala Giu Phe Thr Ala Asp 245 250 255 Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser 260 265 270 Asn Asp Giu Met Val Leu Leu Pro Ile Asn Giu Pro Val His Gly Thr 275 280 285 Lys Arg Lys Ser Gin Ile Gin Thr Tyr Leu Giu His Asn Giu Gly Ala 290 295 300 Gly Leu Gin His Leu Ala Leu Met Ser Giu Asp Ile Phe Arg Thr Leu 305 310 315 320 Arg Giu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gin Asn Leu Lys Lys Arg Val Gly Asp 340 345 350 -32- Val1 Val1 Leu 385 Cys Gly Giu Leu Asp 370 Gly Met Gly Tyr Lys 360 Thr Phe Gly Phe Al a 440 Glu Ile 380 Ile Gin Phe Val1 Leu 365 Phe Gin Ser Lys Gly 445 Giy Ile Leu Thr Lys Pro Arg Val Gly 400 Giy Gly Cys 415 Ser Ile Glu 430 -33-

Claims (5)

1. 2.JUN. 2004 111:22 SPRUSCN FERGUSONNO092 P9 34 The claims defining the invention are as follows: 1. A biologically pure sample of DNA, the DNA comprising a DNA sequence coding for the expression of a plant p-hydroxyphenyl pyruvic acid dioxygenase, wherein the DNA sequence encodes an amino acid sequence at least 80% Identical to the amino acid sequence of SEQ ID 5NO:2. 2. A vector containing the DNA sequence of claim 1.
2. 3. A microbial host transformed by the vector of claim 2.
3. 4. The DNA of claim 1, wherein the p-hydroxyphenyl pyruvic acid dioxygenase is from Arab/do ps/s thaine.
4. 105. A transgenic tomato plant transformed with a DNA construct Including the DNA of claim 1. 6. A transgenic Arabidopsis plant transformed with a DNA construct Including the DNA of claim 1. 7. The biologically pure DNA of claim 1 wherein the DNA is SEQ ID NO:1. is 8. An Isolated p-hydroxyphenyl pyruvic acid dioxygenase comprising SEQ ID NO:2 9. A DNA plant gene expression construct comprising:. a) a DNA sequence coding for the expression of a plant p-hydroxyphenyl pyruvic acid dioxygenase; a promoter effective in plant cells located 5' to the DNA coding sequence; and c) a 3'termination sequence effective In plant cells. 1.A DNA construct comprising: 0a) a promoter capable of expressing a downstream coding sequence in a tomato :0...plant; a DNA sequence coding for the expression of a p-hydroxyphenyl pyruvic acid 23 dioxygenase of plant origin; and C) a 3' termination sequence, the construction capable of expressing a p- hydroxyphenyl pyruvic acid dioxygenase gene when transformed into. tomato plants. i::11. A bacteria containing the construction of claim 12. A tomato plant cell containing the construction of claim 13. An Arabidopsis plant cell containing the construction of claim A trantagenic tomato plant comprising in Its genome a foreign genetic construction .comprising, 5- to a promoter effective in tomato, a DNA coding region encoding p-hydroxyphenyl pyruvic acid dioxygenase, and a transcriptional terminator, the genetic construction effective In vivo in tomato plants to stimulate expression of p-hydroxyphenyl pyruvic aold dioxygenase. COMS ID No: SBMI-00806247 Received by IP Australia: Time 11:04 Date 2004-06-28 28. JUN. 2L004 11 :03 SPRUSCN FERGUSON NO. '082 P. Transgenic seed of the tomato plant of cilm 14. 16. Fruit of the tomato plant of claim 14. 17. The transgenic tomato piant of claim 12 whereil the DNA coding region encoding p- hydroxyphenyl pyruvic acid dioxygenase is that set forth in SEQ ID NO:1.
5. 518. A method of suppressing the production of vitamin E and plastoquinones in a plant, the method comprising the steps of: a) isolating a DNA sequence encoding a p-hydroxyphenyl pyruvic acid dioxygenase of plant origin; b) creating a genetic construction including, 5' to a promoter effective in the plant's cells, a coding sequence, and a transcriptional terminator, the coding region being derived from the DNA sequence, wherein the DNA sequence from step has been altered so that expression of p-hydroxyphenyl pyruvic acid dioxygenase Is suppressed; and c) transforming a cell of the plant with the genetic construction, whereby the plant's cell produces lowered levels of vitamin E and plastoquinones, is 19. A biologically pure sample of DNA, the DNA omprising a DNA sequence coding for the expression of a plant p-hydmxyphenyl pyruvic acid dioxygenase, substantially as hereinbefore described with reference to any one of the examples. A transgenlc tomato0 plant transformed with a DNA construct, substantially as heroinbefore described with reference to any one of the examples. 21. A transgenic Arabidopsis plant transformed with a DNA construct substantially as hereinbefore described with reference to any one of the examples. 22. A DNA plant gene expression construct, substantially as hereinbefore described with reference to any one of the examples. Atransgenic tomato plant comprising in its genome a foreign genetic constructon, 25 substantially as hereinbefore described with reference to any one of the examples. 24. A method of suppressing the production of vitamin E and plastoquinones in a plant, substantially as hereinbefore described with reference to any one of the examples. .25. Transgenic seed of the plant of any one of claims 20, 21 and 23. Dated 28 June, 2004 Arizona Board of Regents on Behalf of University of Arizona Paen Atony foSh plcn/oiae eo SPUO FEGUO [kl* M9RS.o-C COMS ID No: SBMI-00806247 Received by IP Australia: Time 11:04 Date (Y-vl-d) 2004-06-28