CA2245003A1 - Cloned plant p-hydroxyphenyl pyruvic acid dioxygenase - Google Patents

Abstract

A cDNA clone from Arabidopsis thaliana, pHPP1.5, SEQ ID NO:1, which encodes the enzyme p-hydroxyphenyl pyruvic acid dioxygenase, is disclosed. A vector and microbial host containing a DNA sequence coding for the expression of Arabidopsis thaliana p-hydroxyphenyl pyruvic acid dioxygenase, and a genetic construct containing a DNA sequence coding for the expression of Arabidopsis thaliana p-hydroxyphenyl pyruvic acid dioxygenase, together with a promoter located 5' to the DNA coding sequence and a 3' termination sequence, are also disclosed. A method of creating a transgenic plant in which production of plastoquinones, vitamin E, and carotenoids has been modified, is also disclosed.

Description

CA 0224~003 1998-07-28 W 097/27285 PCT~US97/01384 CLONED PLANT P-~YDROXYPHENYL PYRWIC ACID DIOXYGENASE

This invention was made with Government support under Grant Number 93373069083 awarded by the U.S.
Department of Agriculture. The Government has certain rights in the invention.

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 p-hydroxyphenyl pyruvic acid dioxygenase enzyme.

Backqround Of The Invention The chloroplasts of higher plants contain manyunique, 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 (~-tocopherol or ~-toc), plastoquinones (PQ), 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 plastid; carotenoids are tetraterpenoids synthesized by a separate plastid-localized pathway.
Plastoquinone (PQ) often accounts for up to 50~
of the total plastidic quinone pool in green tissues.
The primary function of PQ is as a fundamental component of the photosynthetic electron transport chain, acting as an electron carrier between CA 0224~003 1998-07-28 W097/27285 PCT~US97/01384 photosystem II and the cytochrome b6~ 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 lg25, and the compound responsible for Vitamin E
activity was first identified as ~-tocopherol in 1936.
~-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., ToxicoloqY 23:147-169, 1993; Hess, Anti-oxidants in Hiqher 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 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 Enzym., 213A, 3-13, 1992). In addition to their many functions in plants, 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.

CA 02i4~003 1998-07-28 W 097/27285 PCT~US97/01384 Liebler, et al., Toxicoloqy 23:147-169, 1993; Krinsky, J. Nutr. 119:123-126, 1989.

Tocopherol and Plastoquinone Synthesis ~-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 solanylpyrophosphate, C20 and C45, respectively) coupled to a simultaneous decarboxylation by a phytyl/prenyl transferase to form the first true tocopherol and plastoquinone intermediates, 2-demethylphytylplastoquinol and 2-demethylplastoquinol-9, respectively. A single ring methylation occurs on 2-demethylplastoquinol to yield plastoquinol-9 (PQH2) which is then oxidized to pla~toquinone-9 (PQ). This oxidation is reversible and is the basis of electron transport by plastoquinone in the chloroplast.
The preferred route, as established in spinach, ~or ~-tocopherol formation from 2-demethylphytylplastoquinol appears to be 1) ring methylation of the intermediate, 2-~-demethylphytylplastoquinol, to yieldphytylplastoquinol, 2) cyclization to yield d-tocopherol and, finally, 3) a second ring methylation to yield ~-tocopherol. Ring methylation in both tocopherol and plastoquinone synthesis is carried out by a single enzyme that is specific for the site of methylation on the ring, but has CA 0224~003 1998-07-28 W 097/27285 PCTrUS97/~1384 relatively broad substrate speci~icity and accommodates both classes o~ ~uinone 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 ~-toc/PQ pathway have been localized to the inner chloroplast envelope by cell fractionation studies except for pOHPP dioxygenase and the tocopherol cyclase enzyme. Dif~iculties with cell ~ractionation 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 o~ 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 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 ~or bulk flow through the pathway, both because ~GA production is the first committed step in ~-toc/PQ
synthesis, and also because the reaction is essentially irreversible. There~ore modi~ying the levels of EGA by modifying pOHPP dioxygenase activity should have a direct impact on the total ~-toc/PQ
biosynthetic accumulation in plant tissues, and, as described below, because of the connection of PQ and carotenoid synthesis, should also affect carotenoid synthesi~ in plant tissues.

Carotenoid Biosynthesis; Q~;nones as Electron ~arriers In plants, carotenoids are synthesized and accumulate exclusively in plastids via the pathway shown on the le~t-hand side of Figure 1. The first CA 0224~003 1998-07-28 W 097/27285 PCT~US97/01384 committed step in carotenoid synthesis i8 the condensation of two molecules of the C20 hydrocarbon geranylgeranyl pyrophosphate (GGDP) by the enzyme phytoene synthase, to form the colorless C~0 hydrocarbon, phytoene. In oxygenic photosynthetic organisms (e.g. plants, algae, and cyanobacteria), phytoene undergoes two sequential desaturation reactions, catalyzed by phytoene desaturase, to produce ~-carotene through the intermediate phytofluene. Subsequently, ~-carotene undergoes two further desaturations, catalyzed by ~-carotene desaturase, to yield the red pigment lycopene.
Lycopene is cyclized to produce either ~-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 carotenoid pathway (phytoene synthase and phytoene desaturase) have been isolated and studied from a number of plant and bacterial sources in recent years.
S~n~m~nn, Eur. J. Biochem. 223:7-24, 1994. Phytoene desaturase has been the most intensively studied, both 2~ 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 desaturase enzymes have evolved by independent evolution: the crtI-type found in anoxygenic photosynthetic organisms (e.g. Rhodobacter and Erwinia), and the pds-type found in oxygenic photosynthetic organisms. Despite their differences 3~ in primary amino acid sequence, all phytoene desaturase enzymes contain a dinucleotide binding domain (FAD or NAD/NADP), which in Capsic~m~ ~nnrlm has CA 0224~003 1998-07-28 W097/2728S PCT~US97/01384 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 (e.g. Sulcotrione), which cause phytoene accumulation in treated tissues but unlike the well-studied pyridazone class (e.g. Norflorazon (NFZ)) do not directly affect the phytoene desaturase enzyme.
~ather, 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 PhYsiol. 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 nutrition, agriculture, and biochemical processes within plant cells, much remain~ 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.

CA 0224~003 1998-07-28 W 097/27285 PCT~US97/01384 SummarY o~ the Invention 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 se~uence sufficiently homologous to SEQ ID NO:l so as to code for the expression of Arabidopsis thaliana p-hydroxyphenyl pyruvic acid dioxygenase, and a geneticconstruct containing a DNA sequence sufficiently homologous to SEQ ID NO:l so as to code i~or the expression o~ Arabidopsis thaliana p-hydroxyphenyl pyruvic acid dioxygenase, together with a promoter located 5' to the DNA coding sequence and a 3' termination sequence.
In another embodiment, this invention provides a method of creating a transgenic plant in which the levels o~ the pOHPP dioxygenase enzyme are elevated sufficient such that production of plastoquinones, vitamin E, and carotenoids are modified.
It is an object of the present invention to genetically engineer higher plants to modi~y the production of plasto~uinones, vitamin E, and carotenoids.
It is another object o~ 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 of herbicides (i.e. sulcotrione).
It is another object of the present invention to provide a method for the preparation o~ the enzyme p-hydroxyphenyl pyruvic acid dioxygenase (pOHPP
dioxygenase), an enzyme which can be used to identify new pOHPPdioxygenase-inhibiting herbicides.
Other features and advantages of the invention will be apparent from the following description of the CA 0224~003 1998-07-28 W 097/27285 PCTrUS97/01384 preferred embodiments thereo~ and from the claims.

3rief Description of the Drawinqs Fig. 1 is a diagram of the pathways for synthesis of carotenoids, vitamin E (tocopherol3, 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 Descri~tion Of The Invention 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 identification, isolation, characterization and functional analysis of a higher plant pOHPP
dioxygenase cDNA, its role in ~-toc, PQ and carotenoid synthesis, and the use of this CDNA to modify pOHPP
dioxygenase acti~ity 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, CA 0224~003 1998-07-28 W O 97/27285 PCTrUS97/01384 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 identi~ied one genetic locus, designated pdsl (pd~= ~hytoene 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 indicates that pd61 defines a second gene product in addition to the phytoene desaturase enzyme, necessary for phytoene desaturation and hence carotenoid synthesis in higher plants. This gene product proved to 3~e pOHPP dioxygenase.
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 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 ~or 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 CA 0224~003 1998 07-28 W 097127285 PCTrUS97/01384 weeks, 80 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 manipulation.
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 ~or one gene, pdsl, whose mutation disrupts synthesis 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 1), that is directly required for the synthesis of plastoquinone and ~-tocopherol and indirectly ~or carotenoid synthesis.
In particular, the deduced function of the pdsl mutant and pOHPP dioxygenase enzyme are noted in Figure 2.
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 CA 0224~003 1998-07-28 W 097/27285 PCT~US97/01384 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, 6). 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 2). The Arabidopsi~
pOHPP dioxygenase gene/cDNA thus provides a ~asis for modifying the production of plastoquinones, ~-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 ~unctional 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 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 ~-toc, PQ and carotenoids.
~ levating pOHPP dioxygenase protein levels increases the amount of homogentisic acid (HGA) synthesized in plant tissues. Because HGA is the limiting precursor molecule for ~-toc and PQ synthesis (the end products of the pathway), increasing HGA
synthesis increases the levels of ~-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 CA 0224~003 l998-07-28 by increasing electron flow between photosystem II and photosystem I, because PQ i~ the primary electron transporter between the two photosystems. The increase in ~-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, W
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 required that a DNA sequence containing the pOHPP
dioxygenase coding sequence be combined with 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 (polyadenylation sequence) may also be added. Plant expression vectors, or plasmids constructed ~or 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 trans~er the constructs into plants. A sequence which codes for pO~PP dioxygenase includes, for example, SEQ ID NO:l, or versions of the designated sequence sufficient to effect coding for the expression of pOHPP dioxygenase. Commonly used methods of molecular biology well-~nown to those of skill in the art may be used to manipulate the DNA

CA 0224~003 1998-07-28 W097/27285 PCTrUS97/01384 sequences.
By "genetic construct" we mean any o~ a variety of ways of combining the protein-encoding sequences with a promoter se~uence (and termination ~equence, 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" o~
a protein-encoding sequence, while the termination sequence, if used, will be "downstream'l 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, promoter, and termination sequence, if added, may be combined with any other needed functional sequences and used without a vector.
The DNA sequence described by SEQ ID NO:1 is su~~icient to e~fect coding for the expression of pOHPP dioxygenase. However, it is envisioned that the above sequence could be truncated and still con~er the same properties. It is not known at present which speci~ic deletions would be successful, but it is 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 sequence and perform deletional analysis experiments to determine what portions of the designated sequence are essential to ef~ect coding ~or the expression of pOHPP dioxygenase. One could create a genetic construct with the candidate deletion mutations and per~orm experiments as described below in the Examples, to test whether such deletion mutation sequences ef~ect coding for the enzyme.
Expression of the enzyme activity indicates a CA 0224~003 1998-07-28 W 097127285 rCTAJS97/01384 successful deletion mutant or mutants. In this manner, one could determine which parts of the designated se~uence is essential for expression of the enzyme.
It i8 also known that the genetic code i8 degenerate, meaning that more than one codon, or set o~ 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 pOXPP dioxygenase sequences from other higher plants can be readily recovered.
Oligonucleotides can be made from the sequence set forth below to either hybridize against cDNA or genomic libraries or used for PCR amplification of homologous pOHPP dioxygenase sequences from other plants.
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 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 CA 0224~003 1998-07-28 W O 97/27285 PCT~US97101384 transgenic plant by the normal rules of Mendelian inheritance. Thus, once a genetic construct e~pressing 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 pO~PP dioxygenase present in the plant tissues.

EXAMPLES
I~olation of pd~1, a mutant defective in carotenoid synthe~is 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 (e.g. prior to production of ~-carotene) are lethal when grown in soil and the isolation of such mutations requires the design of 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, Phvsiol. 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 CA 0224~003 1998-07-28 W 097/27285 PCTrUS97/01384 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 ~or lethal pigm~ent 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 with the herbicide NFZ, an inhibitor o~ the phytoene desaturase enzyme (Figure 1) which has been reported to cause accumulation of phytoene in treated tissues.
Britton, Z. Naturforsch 34c:979-985, 1979. Figs. 3A-3E present the results of pigment analysis of wild-type, NFZ-wt, and pdsl tissues. A~breviations in Figs. 3A-3F are as follows: N, neoxanthin; V, violaxanthin; L, lutein; Cb, chlorophyll b; Ca, chlorophyll a; ~, ~-carotene.
Figure 3A shows Cl8 Reverse Phase ~PLC 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 analysi~ of the strongly absorbing 296nm peak at 33 minutes in NFZ-Wt tissue shows absorbance m~;m~ at 276, 286, and 298nm, indicative 35 of phytoene (Figure 3D). Figure 3C shows pigment analysis of tissue culture grown homozygous pdsl mutant plants. The low a~sorbance at 44Onm in Figures CA 0224~003 1998-07-28 W O 97/27285 PCT~US97/01384 3B and C demonst~ates 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 ~rom 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 A~laly8i8 For quantitative and qualitative carotenoid analysis, plant tissue is placed in a microfuge tu~e and ground with a micropestle in 200~1 of 80~ acetone.
120~1 of ethyl acetate is added and the mixture vortexed. 140~1 of water is added and the mixture centrifuged for 5 minutes. The carotenoid containing upper phase is then transferred to a fresh tube and vacuum dried in a Jouan ~C1010 Centrifugal Evaporator.
The dried extract is resuspended in ethyl acetate at a concentration o~ 0.5mg fresh weight of tissue per ~l and 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 Cl8 column, 25 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 CA 0224~003 1998-07-28 WO 97/27285 PCTrUS97/01384 relative 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.

Qll; nor~e analysi~3 Quinones were extracted from tissue using a method modified from that described in Bligh et al., Can. J. Biochem. Phvsiol. 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 was resuspended in methanol at a concentration of 10mg fresh weight per ml and immediately analyzed by HPLC.
Quinones were resolved by reversed-phase HPLC analysis on a LiChrosorb RP-8, 5 micron column, 25cm in length, (Alltech, San Jose, CA) using an isocratic solvent of 10~ H2O in Methanol for the first 14 minutes, at which time the solvent was switched to 100~ methanol for the r~m~n~er of the run (modified from the method described in Lichtenthaler, Handbook of Chromatoqraphy, CRC Press, 115-159, 1984). The flow rate was lml per minute for the duration. Peaks were identified based upon the retention time of known standards with detection at 280nm ~or ~-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 CA 0224~003 1998-07-28 W 097/27285 PCTrUS97/01384 collected, and submitted to the Southwest Environmental Health Science Center, Analytical Core laboratory ~or 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 ~rom sel~ing pdsl heterozygous plants. Prior to desiccation, Fl 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 (X2=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 ~p. Wetzel et al., Plant J. 6:161-175, 1994. To test this hypothesis, the pdsl mutation was mapped relative to visible markers. The pdsl mutation was ~ound to map to chromosome 1, approximately 7 cM ~rom disl toward clv2. Fr~n~m~nn et al., Plant J. 7:341-35Q, 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 ~or characterization o~ the pdsl mutant.

CA 0224~003 1998-07-28 W 097127285 PCT~US97/01384 Homozygous pd~1 mutants can be rescued ~y H~...~y~lti~ic Acid, an int~~~;ate in plastoquinone and tocopherol biosynthe~is AB described earlier, previous research suggesting a role for quinone~ 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 2). Albino pdsl homozygous plants were ~irst germinated on MS2 media and then transferred to MS2 media supplemented with 100~M 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 the pdsl mutation with homogentisic acid. Each profile represents pigments extracted from 10mg fresh weight of ti~sue. Abbreviations used in Figs. 5A-5C
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 pdsl + HGA tissue and wild-type tissue (Figures 5A and 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 dioxygenaBe~ becauBe pdsl mutants are not altered by growth on the substrate of this enzyme, pOHPP, but rather, are restored qualitatively to wild-type pigmentation by growth on the product of CA 0224~003 1998-07-28 W O 97/2728S PCT~US97/01384 this enzyme, HGA (refer to Figures 1 and 2). The complementation of pdsl with HGA also indicate~ 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.
HP~C analysi~ conclusively ~ trate~ that pdsl i~ a mutation in the pla~to~l; n~n~/ tocopherol biosynthetic pathway that al~o affects carotenoid ~ynthesis In addition to biochemical complementation of pdsl mutants, the plastoquinone/tocopherol pathway was also directly analyzed in pdsl tissue by utilizing C8 HPLC to resolve total lipid extracts and identify three separate classes of quinones: ubiquinone, plastoquinone, and ~-tocopherol (Vitamin E) (Figures 5 and 6). 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), making ubiquinone an ideal internal control in these analyses. Figure 6 shows the C8 HPLC analysis of lipid soluble extracts from N~Z-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 minu~esj, optical spectra, and mass spectra (resuIts not shown). NFZ-Wt tissue contained a peak (1) with a retention time of 13.5 minutes which was identified as ~-tocopherol based upon the retention time of a standard. However, optical spectroscopy and mass spectrometry demonstrated that peak 1 was composed of two major components: ~-tocopherol (la) and an CA 0224~003 1998-07-28 W 097/2728~ PCTrUS97/01384 unidentified compound (lb). The mass of ~-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. pd s 1 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 data demonstrate that this peak lacks ~-tocopherol (la) and is composed solely of the compound lb (data not shown). Therefore, homozygous pdsl plants accumulate ubiquinone but lac~ both plastoquinone and ~x-tocopherol. This i9 consistent with the pdsl mutation affecting pOHPP dioxygenase (refer to Figures 1 and 2), 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 2 5 Ara~idop5is 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 pd~l 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.

CA 0224~003 l998-07-28 W O 97/27285 PCTr~S97/01384 In an e~fort to ~urther understand t~e nature of this disease, pOHPP dioxygenase cDNAs have been cloned from several m~mm~l ian 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 ~80%;
in comparison, their identity to bacterial homologs is very low, less than 28%. By using m~mm~l ian 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
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 (~50% identity) to both the m~mm~l ian 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 sequences from mammals and bacteria identified three regions of high conservation, the highest being a 16 amino acid region near the carboxy end o~ pOHPP
dioxygenases that shows 62.5% identity across all phyla. Ruetschi et al., Eur. ~. 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 li~ely the pdsl locus.

CA 0224~003 1998-07-28 W 097/27285 PCTrUS97/01384 Isolation and Characterization of a f~ull length Ara~idopsi~pOHPP dio~yy~Llase cDNA
Utilizing the partial length Arabidop~is cDNA
probe, an Arabidopsis cDNA library was screened by nucleic acid hybridization ~or 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. pHPPl. 5 encodes a 446 amino acid protein (presented as SEQ ID NO:2), which is slightly larger in size than m~mm~l ian and bacterial pOHPP dioxygenases. pHPPl. 5 shows 34-40~
identity at the amino acid level to pOHPP dioxygenases from various m~mm~l s and bacteria. In comparing four 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 pOHPP
dioxygenases. Denoya et al., J. Bacteriol. 176:5312-5319, 1994. The pHPP1.5 coding region contains 52 of these 69 conserved amino acids.
D~ tration that pHPP1.5 encodes an active pO~PP
dioxygenase protein and complements the pd51 mutation In order to definitively demonstrate that pHPP1.5 is the gene product encoded by the pdsl locus and that it encodes a functional pOHPP dioxygenase protein, the pXPP1.5 cDNA was cloned into a plant transformation vector for molecular complementation experiments with the pd91. The full length wild-type pOHPP dioxygenase cDNA will be subcloned into a plant transformation vector driven by the Cauli~lower Mosaic Virus 35S
(CaMV) promoter and containing all necessary termination cassettes and selectable markers (Kanr).
The CaMV promoter is a strong constitutive promoter.
This single construct and the vector without the pHPP1.5 insert (as a control) will be used in vacuum infiltration transformation which uses whole soil CA 0224~003 1998-07-28 W 097/2728~ PCTrUS97/01384 grown 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 the pdsl mutation in retrospect by inspection of their sili~ues 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 (wild type pOHPP dioxygenase cDNA) will conclusively demonstrate complementation of the pdsl mutation with the pOHPP dioxygenase cDNA. To provide additional proof that pHPP1.5 is encoded by the pdsl locus, the pHPP1.5 cDNA has been mapped relative to the pd61 locus using recombinant inbred lines, as described in Lister et al., Plant J. 4:745-750, 1993. The pHPP1.5 cDNA mapped to the region of chromosome 1 containing the pd61 mutation (Figure 4). Finally, the pHPP1.5 cDNA will be overexpressed in E. coli and the activity of the protein determined.

CA 0224~003 1998-07-28 W 097127285 PCTnUS97/~1384 Modification of pOXPP 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 pHPPl.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).
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 resistance relative to normal plants.
Two di~ferent plant systems, Arabidopsis and tomato, are being used to demonstrate the effects of modified pOHPP dioxygenase in plant tissues.
Constitutive overexpression of pO~PP dioxygenase will be done in both plant systems utilizing the CaMV 35S
promoter and the p~PP1.5 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 ~ruit specific promoter derived from the tomato ~subunit CA 0224~003 1998-07-28 WO 97127285 PCT~US97/01384 gene, which is expressed specifically in developing, but not ripening tomato ~ruit. 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 quinone synthesis and the potential for manipulating chloroplastic homogentisic acid derived quinones (tocopherols and plastoquinones) and compounds that require quinones for their synthesis (carotenoids, etc) by increasing pOHPP dioxygenase activity.
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 o~ pOHPP mRNA determined by Northern blot analysis, pO~PP dioxygenase activity determined as described in Schulz et al., FEBS
318:162-166, 1993, and the effects on individual chloroplastic components o~ interest analyzed (tocopherols, plastoquinones and carotenoids). In green tissue containing constitutively expressing constructs this analysis can occur relatively soon after trans~ormants 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 HPL~, 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 absolute level o~ tocopherol will be quanti~ied by isotopic dilution with a known, "heavy carbon" tocopherol standard added at the start CA 0224~003 1998-07-28 W 097/2728S PCTrUS97/01384 of the extraction. Determination based on fresh weight of tissue can also be performed. Plastoquinone levels will be ~uantified 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 ~uantified 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.

Overexpression of pOHPP for in vitro Herbicide~5 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.

CA 02245003 l998-07-28 W 097/27285 PCTrUS97/01384 ~U~N~ LISTING
~l) GENERAL INFORMATION:
(i) APPLICANT: DellaPenna, Dean Norri~, Susan (ii) TITLE OF lNV~N~rlON: Cloned Plant P-Hydroxyphenyl Pyruvic Acid Dioxygena~e (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPuN~N~ ADDRESS:
IA~ ADDRESSEE: Quarles & Brady ~B~ STREET: PO Box 2113 ~C. CITY: Madison ~D STATE: WI
E~ CUUN'LKY: USA
~,FJ ZIP: 53701-2113 (v) COMPVTER READABLE FORM:
~A) MEDIUM TYPE: Floppy disk ~B) C~M~U'l~: IBM PC compatible ,C) OPERATING SYSTEM: PC-DOS/MS-DOS
2û ,D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) AlluRh~Y/AGENT INFORMATION:
(A) NAME: Seay, Nicholas J
(B) REGISTRATION NUMBER: 27,386 (C) REFERENCE/DOCKET NUMBER: 920214.90158 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 608-25}-5000 (B) TELEFAX: 608-251-9166 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 1519 base pairq ~B) TYPE: nucleic acid ) STR~N~ S double ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
4û (A) ORGANISM: Arabidopsis thaliana (vii) IMMEDIATE SOURCE:
~ (B) CLONE: pHPP1.5 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 37,,1374 CA 0224~003 1998-07-28 W 097/27285 PCTrUS97/01384 ~Xi) ~U~N~'~ DESC~IPTION: SEQ ID NO:l:

Met Gly His Gln Asn Ala Ala Val Ser Glu Asn Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys TCT GAT A~A TTC AAG GTT AAG CGC TTC CAT CAC ATC GAG TTC TGG TGC 198 Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Ary Arg Phe Ser Trp Gly Leu Gly Met AGA TTC TCC GCC A~A 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 Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro TAC TCT CCG TCT CTC TCC GCC GGA GAG ATT A~A CCG ACA ACC ACA GCT 390 Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro CCT ATC GTC CTC AAT GAA GCA GTT ACG ATC GCT GAG GTT A~A CTA TAC 582 Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr GGC GAT GTT GTT CTC CGA TAT GTT AGT TAC A~A GCA GAA GAT ACC GAA 630 Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu A~A TCC GAA TTC TTG CCA GGG TTC GAG CGT GTA GAG GAT GCG TCG TCG 678 Lys Ser Glu Phe Leu Pro Gly Phe Glu Ary Val Glu Asp Ala Ser Ser TTC CCA TTG GAT TAT GGT ATC CGG CGG CTT GAC CAC GCC GTG GGA A~C 726 Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly CA 0224~003 l998-07-28 WO 97/27285 PCTrUS97/01384 Phe His Gln Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gln Ile CAG ACG TAT TTG GAA CAT AAC GAA GGC GCA GGG CTA CAA CAT CTG GCT q66 Gln Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Leu Gln Hi~ Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gln A~n Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gln Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gln GGG ACG TTG CTT CAA ATC TTC ACA A~A CCA CTA GGT GAC AGG CCG ACG 1206 Gly Thr T eu T eu Glr. Ile Phe T~.r T ys Dro T eu Gly As~ Arg T'ro Thr ATA TTT ATA GAG ATA ATC CAG AGA GTA GGA TGC ATG ATG A~A GAT GAG 1254 Ile Phe Ile Glu Ile Ile Gln Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gln Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Ly~ Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gln Leu Val Gly *

TAATTAATGT AAAACTGTTT TATCTTATCA A~ACAATGTT ATACAACATC TCATTTAAAA 1464 ACGAGATCAA TCA~AAAATA CAATCTTA~A TTCA~AACCA Aa~LA~ AAAAA 1519 (2) INFORMATION FOR SEQ ID NO:2:
(i) ~u~:N~ CHARACTERISTICS:
IA) LENGTH: 446 amino acids B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein CA 0224~003 1998-07-28 W 097/27285 PCT~US97/01384 (xi) ~:~u~:~ DESCRIPTION: SEQ ID NO:2:
Met Gly His Gln Asn Ala Ala Val Ser Glu Asn Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr A6n Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala Ly8 Ser A6p Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly A~n Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gln Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg Lys Ser Gln Ile Gln Thr Tyr Leu Glu ~is Asn Glu Gly Ala Gly Leu Gln His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gln Asn Leu Ly8 Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gln Ile Lys Glu Cy5 Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gln Gly Thr Leu Leu Gln Ile Phe Thr Lys Pro Leu Gly Asp Arg Pro Thr Ile Phe Ile Glu Ile Ile Gln Arg Val Gly Cys Met Met Lys Asp Glu Glu Gly Lys Ala Tyr Gln Ser Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu iO 420 ~25 430 Glu Tyr Glu Lys Thr Leu Glu Ala Lys Gln Leu Val Gly *

Claims (17)

I claim:

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.

2. A vector containing the DNA sequence of claim 1.

3. A microbial host transformed by the vector of claim 2.

4. The DNA of claim 1, wherein the p-hydroxyphenyl pyruvic acid dioxygenase is from Arabidopsis thaliana.

5. 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:2.

8. A DNA plant gene expression construct comprising:
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 c. a 3' termination sequence effective in plant cells.

9. 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 p-hydroxyphenyl 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.

10. A bacteria containing the construction of Claim 9.

11. A tomato plant cell containing the construction of Claim 9.

12. An Arabidopsis plant cell containing the construction of Claim 9.

13. A transgenic tomato plant comprising in its genome a foreign genetic construction comprising, 5' to 3', 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 acid dioxygenase.

14. Seed of the tomato plant of claim 13.

15. Fruit of the tomato plant of claim 13.

16. The transgenic tomato plant of claim 11 wherein the DNA coding region encoding p-hydroxyphenyl pyruvic acid dioxygenase is that set forth in SEQ ID
NO:1.

17. 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 3', 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 (a) 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.